Distinct Subsets of Dendritic Cells Regulate the Pattern of Acute Xenograft Rejection and S

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儿童腺样体肥大的诊断与治疗

儿童腺样体肥大的诊断与治疗腺样体肥大是儿童常见的一类临床疾病，其起病隐匿，常表现为鼻塞、流涕、张口呼吸、打鼾等症状，严重者可能会引起阻塞性睡眠呼吸暂停以及心肺疾病危及生命。

然而由于儿童处于生长发育的重要时期，腺样体又是儿童时期重要的免疫器官，若处理不当将严重影响患儿的生长发育及生活质量，因此对于儿童腺样体肥大的诊断及治疗方式的选择非常重要。

本文就近年来关于该疾病的临床诊断及治疗等问题做如下综述。

1病因及发病机制腺样体（Adenoids）又被称为咽扁桃体(Pharyngeal tonsils)，为一群附着于鼻咽的后壁的淋巴组织。

婴儿出生后鼻咽部的淋巴组织随着年龄的增长而增生，一般在6岁达最大程度后逐渐退化。

腺样体为桔瓣状，有5~6条纵形沟裂，沟裂中易存留细菌。

儿童患流行性感冒、急性鼻炎及急性扁桃体炎等疾病时，沟裂中的细菌、病毒大量繁殖，刺激腺样体增生肥大，阻塞鼻腔诱发鼻炎鼻窦炎，鼻腔分泌物进步刺激腺样体使之继续增生[1]。

此外有研究表明儿童腺样体肥大的发病率与当地的大气污染具有重要的关系[2]。

自身免疫反应是引起腺样体增生肥大的重要因素。

有研究显示腺样体肥大与单纯急性扁桃体炎相比腺体重量和周长增加，腺样体表皮及滤泡中炎症细胞的数量增多[3]。

受炎症刺激后患儿血清及腺样体黏膜组织中IgA、IgG水平增高，表明在炎症刺激下腺样体发生自身免疫反应抵御外界刺激[4,5]。

Zelazowska-Rutkowska B等研究发现了单纯腺样体肿大与腺样体肿大伴分泌性中耳炎患者疫反应过程中的区别：与单纯腺样体肿大患者比较腺样体肿大伴分泌性中耳炎患者体内IL-5,TNF-α分泌增多而IL-8,IL-6,及IL-10分泌无明显变化[6]。

可见腺样体肥大伴分泌性中耳炎时免疫反应增强。

2临床表现儿童腺样体在炎症的反复刺激下发生病理性增生，增生的腺样体会堵塞上呼吸道引起鼻塞、流涕、打鼾、张口呼吸等一系列临床症状，长期张口呼吸会导致面部神经、肌肉以及软组织重排，颅骨发育畸形，面部缺乏表情，形成”腺样体面容”[7]。

树突细胞流式分类

树突细胞流式分类Tree dendritic cells (DCs) are a diverse group of immune cells that play a crucial role in initiating and regulating immune responses. 树突细胞（DCs）是一种多样化的免疫细胞群体，起着启动和调节免疫反应的关键作用。

They are responsible for capturing and processing antigens, presenting them to T cells, and coordinating the adaptive immune response. 它们负责捕捉和处理抗原，将其呈现给T细胞，并协调适应性免疫反应。

DCs are found in various tissues and organs throughout the body, where they act as sentinels, constantly surveying their environment for potential threats. DCs分布在全身各种组织和器官中，它们充当哨兵，不断监视周围环境的潜在威胁。

Flow cytometry is a powerful technique used for the identification and characterization of different cell types, including DCs. 流式细胞术是一种用于识别和表征不同细胞类型的强大技术，其中包括DCs。

By labeling cells with fluorescent markers that target specific surface molecules, flow cytometry allows researchers to analyze the expression of different proteins on individual cells. 通过用靶向特定表面分子的荧光标记细胞，流式细胞术允许研究人员分析单个细胞上不同蛋白质的表达。

粘膜免疫简介

Dimeric IgA binds to poly Ig receptor on
basal surface of epithelial cells
IgA-poly IgR complex endocytosed,
transported through cell and secreted into lumen - secretory IgA (sIgA)
泌尿生殖道 - 阴道，子宫，肾外分泌腺 - 唾液，胰脏，眼泪
Exocrine glands – saliva, pancreas, tears Breast
Importance of Mucosal Immunity 黏膜免疫的重要性
Protection vs pathogensrsensitivity to
Mechanical - Epithelial barrier trefoils + Mucus Humoral
- Stomach acid (pH <1) Mucin (antibacterial, antiviral) 体液 - 胃酸（pH值<1） Enzymes 酶粘蛋白（抗菌，抗病毒） Defensins (antibacterial peptides) 防御素（抗菌肽） Polyamines (spermine, spermidine)
Inhibitory macrophages, DC抑制巨噬细胞，DC Epithelial 上皮products,VIP血管活性肠肽 IL10, TGFb, PGE2
M Cells
Antigen
The M Cell “Pocket形成囊”
The M Cell “Pocket”
Priming and Redistribution of Intestinal T Cells

浆细胞样树突状细胞与肿瘤免疫

广东药科大学学报
ＪｏｕｒｎａｌｏｆＧｕａｎｇｄｏｎｇＰｈａｒｍａｃｅｕｔｉｃａｌＵｎｉｖｅｒｓｉｔｙＡｕｇ. ２０１８ꎬ３４(４)
浆细胞样树突状细胞与肿瘤免疫
陈桂思ꎬ王悦ꎬ吴婉文ꎬ邹海玲ꎬ沈晗
( 广东药科大学生命科学与生物制药学院ꎬ广东广州５１０００６)
摘要: 浆细胞样树突状细胞( ｐＤＣｓ) 是树突状细胞( ＤＣｓ) 的亚群之一ꎬ主要功能是分泌Ⅰ型干扰素ꎬ借此在抗病毒免疫中起着至关重要的作用ꎮ 而在肿瘤免疫方面ꎬ相比其他ＤＣｓ亚群ｐＤＣｓ受到的关注较少ꎮ 实际上ｐＤＣｓ桥接着先天免疫反应和适应性免疫反应ꎬ是肿瘤免疫治疗的潜在靶点ꎮ 本文对近年来ｐＤＣｓ的研究进展及其在肿瘤免疫中的作用进行综述ꎮ 关键词: 浆细胞样树突状细胞ꎻ Ⅰ型干扰素ꎻ 肿瘤免疫中图分类号: Ｒ３９２.１２文献标志码: Ａ文章编号:２０９６￣３６５３(２０１８)０４￣０５３３￣０５ＤＯＩ: １０.１６８０９ / ｊ.ｃｎｋｉ.２０９６－３６５３.２０１８０５１７０１
量Ⅰ型干扰素 ( Ｉｎｔｅｒｆｅｒｏｎꎬ ＩＦＮ) ꎬ 促进抗病毒免疫反应[２] ꎬ关于其参与肿瘤免疫的研究较少ꎮ
１ｐＤＣｓ的基本介绍
１.１ｐＤＣｓ的分化ｐＤＣｓ有两种来源ꎬ即髓系来源和淋巴系来源ꎮ 髓
系来源指的是由共同髓系祖细胞 ( ｃｏｍｍｏｎｍｙｅｌｏｉｄｐｒｏｇｅｎｉｔｏｒｓꎬＣＭＰｓ) 分化而来ꎬ淋巴系来源指的是由共同淋巴系祖细胞 ( ｃｏｍｍｏｎｌｙｍｐｈｏｉｄｐｒｏｇｅｎｉｔｏｒｓꎬＣＬＰｓ) 分化而来ꎮ ＣＭＰｓ和ＣＬＰｓ均起源于造血干细胞 ( ｈｅｍａｔｏｐｏｉｅｔｉｃｓｔｅｍｃｅｌｌｓꎬＨＳＣｓ) [３] ꎮ

Dendritic cells induce peripheral T cell unresponsiveness under steady state conditions in vivo

J. Exp. Med. The Rockefeller University Press • 0022-1007/2001/09/769/11 $5.00Volume 194, Number 6,September 17, 2001769–779/cgi/content/full/194/6/769769Dendritic Cells Induce Peripheral T Cell Unresponsiveness Under Steady State Conditions In VivoDaniel Hawiger, 1 Kayo Inaba, 3, 5 Y air Dorsett, 1 Ming Guo, 1 Karsten Mahnke, 3 Miguel Rivera, 3 Jeffrey V . Ravetch, 4 3 1, 21 Laboratory of Molecular Immunology,2 Howard Hughes Medical Institute,3 Laboratory of CellularPhysiology and Immunology, and 4 Laboratory of Molecular Genetics and Immunology, The Rockefeller University, New Y ork, NY 100215 Laboratory of Immunobiology, Graduate School of Biostudies, Kyoto University,Kyoto 606-8502, JapanAbstractDendritic cells (DCs) have the capacity to initiate immune responses, but it has been postulated that they may also be involved in inducing peripheral tolerance. To examine the function of DCs in the steady state we devised an antigen delivery system targeting these specialized anti-gen presenting cells in vivo using a monoclonal antibody to a DC-restricted endocytic recep-tor, DEC-205. Our experiments show that this route of antigen delivery to DCs is several or-ders of magnitude more efficient than free peptide in complete Freund’s adjuvant (CFA) in inducing T cell activation and cell division. However, T cells activated by antigen delivered to DCs are not polarized to produce T helper type 1 cytokine interferon ␥ and the activation re-sponse is not sustained. Within 7 d the number of antigen-specific T cells is severely reduced,and the residual T cells become unresponsive to systemic challenge with antigen in CFA.Coinjection of the DC-targeted antigen and anti-CD40 agonistic antibody changes the out-come from tolerance to prolonged T cell activation and immunity. We conclude that in the absence of additional stimuli DCs induce transient antigen-specific T cell activation followed by T cell deletion and unresponsiveness.Key words:antigen delivery • DEC 205 • dendritic cells • peripheral T cell tolerance • CD40IntroductionDendritic cells (DCs) * are uniquely potent inducers of pri-mary immune responses in vitro and in vivo (1, 2). In tissue culture experiments, DCs are typically two orders of mag-nitude more effective as APCs than B cells or macrophages (3, 4). In addition, purified antigen-bearing DCs injected into mice or humans migrate to lymphoid tissues and effi-ciently induce specific immune responses (5–7). Likewise,DCs migrate from peripheral tissues to lymphoid organs during contact allergy (8, 9) and transplantation (10), two of the most powerful known stimuli of T cell immunity in vivo. Based on these and similar experiments, it has beenproposed that the principal function of DCs is to initiate T cell–mediated immunity (1). However, nearly all of these experiments involved DC purification or culture in vitro , or some perturbations in vivo that induce major alterations in DC maturation and function. Thus, the physiologic function of DCs in the steady state has not been deter-mined (6, 11).There is indirect evidence from a number of different laboratories suggesting that DCs may play a role in main-taining peripheral tolerance (summarized in reference 12).For example, injection of mice with 33D1, a rat mono-clonal antibody to an unknown DC antigen, appeared to induce T cell unresponsiveness to the rat IgG (13). How-ever, the specificity of antigen delivery was uncertain and the relevant T cell responses could not be analyzed di-rectly. In addition, peripheral tolerance to ovalbumin and hemagglutinin expressed in pancreatic islets was found to be induced by bone marrow–derived APCs (14–16), butAddress correspondence to M.C. Nussenzweig, Department of Molecular Immunology/HHMI, RRB Rm. 470, Box 220, 1230 York Ave., New York, NY 10021. Phone: 212-327-8067; Fax: 212-327-8370; E-mail:nussen@* Abbreviations used in this paper:CFSE, 5-(6)-carboxyfluorescein diace-tate succinimidyl diester; DC, dendritic cell; H EL, hen egg lysozyme;MMR, macrophage mannose receptor.the identity of these antigen presenting cells has not been determined (17).Materials and MethodsMice.6–8-wk-old females were used in all experiments andwere maintained under specific pathogen free conditions.B10.BR, B6.SJL (CD45.1), and B6/MRL (Fas lpr) mice werepurchased from The Jackson Laboratory. 3A9 transgenic micewere maintained by crossing with B10.BR mice. To obtainCD45.1 3A9 or 3A9/lpr T cells, B6.SJL or B6/MRL mice werecrossed extensively with 3A9 mice and tested for CD45.1 and I-A k,by flow cytometry. Fas lpr mutation was tested by PCR. Micewere injected subcutaneously with peptide in CFA and subcuta-neously or intravenously with chimeric antibodies. All experi-ments with mice were performed in accordance with NationalInstitutes of Health guidelines.Flow Cytometry and Antibodies Used for Staining.CD4- (L3T4),MH C II- (10-3.6), CD11c- (H L3), CD11c- (H L3), B220-(RA3-6B2), or CD3- (145-2C11), CD80(B7-1)-(16-10A1) I-A k-(10-3.6) CD45.1- (A20), Il-2- (JES6-5H4), IFN-␥- (XMG1.2), CD40- (HM40-3-FITC), CD86(B7-2)- (GL1) specific antibod-ies were from BD PharMingen. Rat IgG-PE(goat anti–rat IgG)specific antibody was from Serotec. 3A9 T cell receptor (1G12)–specific antibody was a gift from Dr. Emil Unanue, WashingtonUniversity, St. Louis, MO (18).For visualization of rat IgGs on surface of mononuclear cells,lymphoid cells were purified from peripheral LNs 14 h after anti-body injection and stained with anti–rat IgG-RPE(goat anti–ratIgG-RPE; Serotec) to visualize surface bound NLDC145 andGL117 antibodies. The cells were then incubated in mouse serumto block nonspecific binding and stained with FITC anti-CD11c(HL3), or -B220 (RA3-6B2), or -CD3 (145-2C11).For intracellular cytokine staining, lymphocytes were stimu-lated in vitro for 4 h with leukocyte activation cocktail (BDPharMingen) according to the manufacturer’s manual. Cellswere fixed and permeabilized using cytofix/cytoperm bufferfrom BD PharMingen.Immunohistology.Popliteal LNs were removed from antibodyinjected mice and 5-␮m cryosections (Microm; ZEISS) were prepared. Tissue specimens were fixed in acetone (5 min, room temperature [RT]) air dried, and stained in a moist chamber. The injected antibodies were detected by incubating the sections with streptavidin Cy3 or streptavidin-FITC (Jackson Immuno-tech). In double labeling experiments, the PE-conjugated anti-bodies were added for additional 30 min. Specimens were exam-ined using a fluorescence microscope and confocal optical sections of ف0.3-␮m thickness were generated using deconvolu-tion software (Metamorph).Constructing and Production of Hybrid Antibodies.Total RNAwas prepared from NLDC-145 (19) and GLII7 (gift of R.J. Hodes,National Institutes of H ealth, Bethesda, MD) hybridomas (bothrat IgG2a) using Trizol (GIBCO BRL). Full-length IgcDNAs were produced with 5Ј-RACE PCR kit (GIBCO BRL) using primers specific for 3Ј-ends of rat IgG2a and Ig kappa. The V regions were cloned in frame with mouse Ig kappa constant re-gions and IgG1 constant regions carrying mutations that interfere with FcR binding (20). DNA coding for hen egg lysozyme (HEL) peptide 46–61 with spacing residues on both sides was added to the C terminus of the heavy chain using synthetic oligonucle-otides. Gene specific primers for cloning of rat IgG2a and Ig kappa: 3Ј-ATAGTTTAGCGGCCGCGATATCTCACTAA-CACTCATTCCTGTTGAAGCT; 3Ј-ATAGTTTAGCGGC-CGCTCACTAGCTAGCTTTACCAGGAGAGTGGGAGAG-ACTCTTCT; HEL peptide fragment construction: 5Ј-CTAGC-GACATGGCCAAGAAGGAGACAGTCTGGAGGCTCGAG-GAGTTCGGTAGGTTCACAAACAGGAAC; 5Ј-ACAGACG-TAGCACAGACTATGGTATTCTCCAGATTAACAGCAG-GTATTATGACGGTAGGACATGATAGGC; 3Ј-GCTGTA-CCGGTTCTTCCTCTGTCAGACCTCCGAGCTCCTCAA-GCCATCCAAGTGTTTGTCCTTGTGTCTG; 3Ј-CCATC-GTGTCTGATACCATAAGAGGTCTAATTGTCGTCCATA ATACTGCCATCCTGTACTATCCGCCGG.Hybrid antibodies were transiently expressed in 293 cells after transfection using calcium-phosphate. Cells were grown in se-rum-free DMEM supplemented with Nutridoma SP (Boeh-ringer). Antibodies were purified on Protein G columns (Amer-sham Pharmacia Biotech). The concentrations of purified antibodies were determined by ELISA using goat anti–mouse IgG1 (Jackson Immunotech).Cell Culture and Proliferation Assays.Pooled axillary, brachial, inguinal, and popliteal LNs were dissociated in 5% FCS RPMI and incubated in presence of collagenase (Boehringer) and EDTA as described (21). For antigen presentation CD19ϩ and CD11cϩcells were purified using microbeads coupled to anti-mouse CD11c or CD19 IgG (Miltenyi Biotec) and irradiated with 1,500 rad. CD4 T cells were purified by depletion using rat antibodies supernatants specific for mouse: CD8 (TIB 211), B220 (RA3-6B2), MHC II (M5/114, TIB 120), F4/80 (F4/80), and magnetic beads coupled to anti–rat IgG (Dynal). In antigen loading experi-ments the isolated presenting cells from each experimental group were cultured in 96-well plates with 2 ϫ 105 purified 3A9 CD4ϩT cells. Cultures were maintained for 48 h with [3H]thymidine (1 ␮Ci) added for the last 6 h. The results were calculated as a ra-tio of proliferation in experimental groups to a PBS control group. The proliferation in PBS controls ranged from 500 to 2,000 cpm.For T cell proliferation assays in adoptive transfer recipients, 9 ϫ 104 of the same irradiated CD11cϩ cells isolated from spleens of wild-type B10.BR mice were cultured in 96-well plates with 3 ϫ 105 T cells from each experimental group. Synthetic HEL pep-tide, at final concentration of 100 ␮g/ml, was added to half of the cultures. Cultures were maintained for 24 h with [3H]thymidine (1 ␮Ci/ml) added for the last 6 h. Response to HEL peptide was determined by subtracting background (no HEL peptide added) proliferation from proliferation in the presence of HEL peptide. Proliferation index was calculated as the ratio of the response to H EL peptide in a given experimental group to the response to HEL of T cells from a PBS-injected control. Proliferation in PBS groups ranged from 4,000–8,000 cpm in the presence of peptide and the response to H EL peptide in these PBS controls was 1,000–3,000 counts above the background. Synthetic HEL 46-61 peptide was provided by the Howard Hughes Medical Institute Keck Biotechnology Resource Center.Adoptive Transfer.CD4 cells from 3A9 mice were enriched by depletion as described above, washed 3ϫ with PBS, and 5 ϫ106 cells injected intravenously per mouse. Alternatively, before depletion total cells were labeled with 2␮M 5-(6)-carboxyfluo-rescein diacetate succinimidyl diester (CFSE) in 5% FCS RPMI (Molecular Probes) at 37ЊC for 20 min and washed twice.ResultsTo examine the function of DCs in vivo, we devised a means of delivering antigens to DCs in situ. We used NLDC145 (19), a monoclonal antibody specific for DEC-770Dendritic Cells Induce Peripheral T Cell Tolerance in the Steady State205, an endocytic receptor that is a member of a family ofmultilectin receptors including the macrophage mannosereceptor (MMR) (22, 23). Like MMR, DEC-205 displaysan NH2-terminal cysteine-rich domain, a fibronectin typeII domain, and multiple C-type lectin domains (22). How-ever, the tissue distribution of DEC-205 and the MMRdiffer in that DEC-205 is highly expressed by DCs withinthe T cell areas of lymphoid tissues, particularly on CD8ϩDCs that have been implicated in cross-priming (24),whereas the MMR is expressed by some tissue macro-phages (25, 26). We chose DEC-205 for targeting antigensto DCs because the cytoplasmic domain of DEC-205 or-chestrates a distinct endocytic pathway that enhances anti-gen presentation (23). DEC-205 recycles through late en-dosomes or lysosomes rich in MH C II, and antigensdelivered to these compartments by DEC-205 are effi-ciently processed and presented to T cells (23).To determine whether the NLDC145 antibody targetsDCs in vivo, we injected mice subcutaneously with puri-fied NLDC145 or GL117, a nonspecific isotype-matchedrat monoclonal antibody control, and visualized the in-jected antibody in tissue sections 24 h after injection,NLDC145 was found localized to scattered large dendriticprofiles in the T cell areas of LNs and spleen while uptakeof control GL117 was undetectable (Fig. 1 A, left and mid-dle). This pattern was similar to the pattern found when theantibody was applied to sections directly (Fig. 1 A, right).The NLDC145-targeted cells were negative for B220 andCD4, markers for B cells and T cells, respectively, but pos-itive for characteristic DC markers including MH C IIand CD11c (Fig. 1 B). Thus, subcutaneously injectedNLDC145 targets specifically to CD11cϩMHC IIϩ DCs inlymphoid tissues in vivo.To further characterize the lymphoid cells that were tar-geted by NLDC145 in vivo, we stained lymphoid cell sus-pensions from antibody injected mice with anti–rat Ig andexamined the cells by multiparameter flow cytometry (Fig.1 C). High levels of injected NLDC145 were found on thesurface of most CD11cϩ DCs but not on the surface ofB220ϩ B cells or CD3ϩ T cells (Fig. 1 C). We concludethat when NLDC145 is injected into mice it binds effi-ciently and directly to DCs but not to other lymphoid cells.To deliver antigens to DCs in vivo, we produced fusionproteins with amino acids 46–61 of H EL added to theCOOH terminus of cloned NLDC145 (␣DEC/HEL) and GL117 (GL117/H EL) control antibody (Fig. 1 D). Tominimize antibody binding to Fc (FcR) receptors and fur-ther ensure the specificity of antigen targeting,the ratIgG2a constant regions of the original antibodies were re-placed with mouse IgG1 constant regions that carry pointmutations interfering with FcR binding (20). The hybridantibodies and control Igs without the terminal HEL pep-tide (␣DEC and GL117) were produced by transient trans-fection in 293 cells (Fig. 1 E).To determine whether antigens delivered by ␣DEC/ H EL were processed by DCs in vivo, we injected mice with the hybrid antibodies and controls and tested CD11cϩ DCs, CD19ϩ B cells and CD11cϪCD19Ϫ mono-nuclear cells for their capacity to present HEL peptide to naive H EL-specific T cells from 3A9 TCR transgenic mice (27). DCs isolated from antibody-injected mice ex-pressed levels of CD80 and MHC II similar to those found on PBS controls and thus showed no signs of increased maturation, in contrast to what occurs when DCs are stim-ulated with microbial products like bacterial LPS and CpG deoxyoligonucleotides (28, 29; Fig. 2 A). Nevertheless DCs from mice injected with ␣DEC/HEL induced strong T cell proliferative responses, whereas DCs isolated from PBS-injected mice or mice injected with the control anti-bodies had no effect (Fig. 2 B). DC isolated 3 d after␣DEC/HEL injection showed reduced antigen-presenting activity (data not shown). In contrast to DCs, B cells and bulk CD11cϪCD19Ϫ mononuclear cells purified from the same mice showed little antigen-presenting activity (Fig. 2 B). We conclude that antigens can be selectively and effi-ciently delivered to DC by ␣DEC/HEL in vivo, and the targeted DCs successfully process and load the peptides onto MHC II.As DC isolation leads to activation, we performed adoptive transfer experiments with H EL-specific trans-genic T cells to follow the response of these T cells to oth-erwise unmanipulated, antigen-targeted DCs in vivo. CD4ϩ3A9 T cells were transferred into B10.BR recipi-ents and 24 h later hybrid antibodies were injected subcu-taneously. To measure T cell responses, CD4ϩ cells were isolated from the draining LNs of the injected mice and cultured in vitro in the presence or absence of added HEL peptide. T cell responses were measured by [3H]thymidine incorporation and are shown as proliferation indices nor-malized to the PBS control (this index facilitates compari-son between experiments, see Materials and Methods). In addition to ␣DEC/H EL, GL117/H EL, ␣DEC, and GL117 antibodies, we included 100 ␮g of HEL peptide in CFA as a positive control.As described in previous reports (30, 31), CD4ϩ T cells isolated 2 d after challenge with 100 ␮g of HEL peptide in CFA showed strong proliferative responses to antigen when compared with PBS controls (Fig. 3 A). Similar re-sponses were obtained from mice injected with as little as 0.2 ␮g of ␣DEC/HEL (i.e.,ف4 ng peptide per mouse)but not from mice injected with up to 1 ␮g of ␣DEC, GL117, or GL117/H EL controls (Fig. 3 A, and not shown). We conclude that antigen delivered to DCs in vivo by ␣DEC/ HEL efficiently induces activation of specific T cells.To determine whether antigen delivered to DCs in vivo induces persistent T cell activation, we measured T cell re-sponses to antigen 7 d after the administration of ␣DEC/ H EL. CD4 T cells continued to show heightened re-sponses to antigen when purified from LNs 7 d after injec-tion with 100 ␮g of HEL peptide in CFA (30, 31; Fig. 3 B). In contrast, T cells isolated from mice 7 d after injec-tion with ␣DEC/H EL were no longer activated when compared with PBS controls (Fig. 3 B). Thus, T cell acti-vation by antigen delivered to DCs by ␣DEC/HEL in vivo is transient, readily detected at 2 but not 7 d. This transient activation resembles the CD4 T cell response to large doses771Hawiger et al.772Dendritic Cells Induce Peripheral T Cell Tolerance in the Steady StateFigure 1.NLDC-145 targets DCs in vivo. (A) Biotinylated NLDC-145 (scNLDC145, left) or rat IgG (scRatIgG, middle) was injected into the hind footpads (50 ␮g/footpad) and inguinal LNs harvested 24 h later. Sections were stained with Streptavidin Cy3. Control sections from uninjected mice were stained using biotinylated NLDC145 and streptavidin Cy3 (NLDC145, right). (B) Two-color immunofluorescense. Mice were injected with bio-tinylated NLDC145 as in panel A. Sections were stained with streptavidin FITC (green) and PE-labeled antibodies (red) to B220 as indicated. Specimens were analyzed by deconvolution microscopy. Double labeling is indicated by the yellow color. (C) FACS ® analysis of lymphoid cells 14 h after injection with NLDC145 and control GL117 antibody. Histograms show staining with anti–rat IgG on gated populations of CD11c ϩ DCs, B220ϩ B cells, and CD3ϩ T cells. (D) Diagrammatic representation of hybrid antibodies. (E) Hybrid antibodies. GL117, GL117/HEL, ␣DEC, and ␣DEC/HEL antibodies analyzed by PAGE under reducing conditions, molecular weights in kD are indicated.773Hawiger et al.of peptide in the absence of adjuvant, or the response to self-antigens presented by bone marrow–derived antigen-presenting cells in the periphery (15, 16, 30–32). To deter-mine whether the absence of persistent T cell activation in mice injected with ␣DEC/HEL is due to clearance of the injected antigen, multiple doses of ␣DEC/HEL were ad-ministered. Repeated injection of ␣DEC/HEL at 3-d in-tervals failed to induce prolonged T cell activation (Fig. 3C). In addition, after 7 or 20 d, T cells initially activated by ␣DEC/HEL could not be reactivated when the mice were challenged with 100 ␮g of HEL peptide in CFA (Fig. 3 D).In contrast, comparable numbers of 3A9 T cells found in PBS-injected controls mounted a vigorous response to challenge with H EL peptide in CFA (compare Figs. 3 D and 4 C). Thus, the transient nature of the T cell response in mice injected with ␣DEC/HEL is not due to a lack of antigen, and T cells initially activated by DCs under physi-ologic conditions are unresponsive to subsequent challenge with antigen even in the presence of strong adjuvants.Absence of persistent T cell responses could be due to DC deletion, T cell deletion, or induction of T cell anergy.To assess DC function in mice receiving multiple doses of ␣DEC/HEL, we isolated DCs from these mice and moni-tored presentation to 3A9 T cells in vitro (Fig. 3 E). DCs from mice injected with two doses of antibody showed the same T cell stimulatory activity as DCs isolated from mice receiving a single injection of ␣DEC/HEL (Fig. 3 E). In addition, the transfer of antigen specific T cells into␣DEC/HEL recipients did not alter the ability of the iso-lated DCs to stimulate 3A9 T cells in vitro. Thus, the tran-sient nature of the T cell response to DC-targeted antigens in vivo is not the result of a lack of antigen-bearing DCs.To examine the fate of 3A9 T cells after exposure to an-tigen presented by DCs in vivo , we performed adoptive transfer experiments with CD45.1ϩ 3A9 T cells labeled with CFSE, a reporter dye for cell division. As described previously, T cells challenged with peptide in CFA divide,upregulate CD69 but not CD25, and produce IL-2 and IFN-␥ but not IL-4 or IL-10. These cells are therefore considered to be Th1 polarized (30, 31; Fig. 4, A and B,and not shown). A burst of cell division and increase of CD69 but not CD25 expression was also seen after injec-tion with 0.2 ␮g ␣DEC/HEL but not with GL117/HEL.Only clonotype positive CD4 cells showed these effects (Fig. 4, A and C, and not shown). However, 3A9 cells acti-vated by antigen presented on ␣DEC/HEL targeted DCs produced only IL-2 but not IFN-␥, IL-4, or IL-10 at the time of the assay and thus were not polarized to Th1 or Th2 phenotype 3 d after antigen challenge. (Fig. 4 B, and not shown). Therefore, 3A9 cells proliferate in response to ␣DEC/HEL targeted DCs in vivo, but the T cells do not produce a normal effector cell cytokine profile.Although there was persistent expansion of 3A9 T cells in regional LNs and spleen 7 and 20 d after challenge with H EL peptide in CFA (Fig. 4 C, spleen not shown), few3A9 T cells survived in the LNs or spleen after exposure toFigure 2.DCs process and present antigen delivered by hybrid antibodies. (A) MHC II and CD80 expression on DCs is not altered by multiple injec-tions of ␣DEC/HEL and 3A9 T cells. B10.BR mice transferred with 3A9 T cells and controls were injected subcutaneously in the footpads with 0.2 ␮g ␣DEC/HEL or PBS either at 8 d (␣DEC/HEL) or at 1 and 8 d (␣DEC/HELX2) after transfer (similar results were obtained by intravenous injection of chimeric antibodies, data not shown). 24 h after the last ␣DEC/HEL injection, DCs were purified from peripheral LNs and analyzed by flow cytometry for expression of CD80 and MHC II. Dotted lines in histograms indicate PBS control. (B) ␣DEC/HEL delivers HEL peptide to DCs in vivo. B10.BR mice were injected subcutaneously into footpads with 0.3 ␮g of ␣DEC/HEL or GL117/HEL or ␣DEC or PBS as indicated. CD11c ϩ, CD19ϩ, and CD11c ϪCD19Ϫ cells were isolated from draining LNs 24 h after antibody injection and assayed for antigen processing and presentation to purified 3A9T cells in vitro. T cell proliferation was measured by [3H]thymidine incorporation and is expressed as a proliferation index relative to PBS controls. The results are means of triplicate cultures from one of four similar experiments.774Dendritic Cells Induce Peripheral T Cell Tolerance in the Steady Stateantigen delivered by ␣DEC/HEL. Surviving cells appeared to be anergic as they could not be stimulated in vivo by HEL peptide in CFA (see Fig 3 D). The loss of 3A9 T cells was Fas independent as it also occurred with 3A9/lpr T cells (Fig. 4 C, and not shown). Thus, the initial expansion of T cells in response to antigen presented by DCs in vivo is not sustained, and most of the initial responding T cells disappear from lymphoid organs by day 7. These cells are either deleted or persist in extravascular sites (33). If they do persist outside lymphoid tissues they must be anergic,because they cannot be activated by further exposure to an-tigen, including peptide in CFA (Fig. 3 D).DCs can be stimulated to increase their antigen present-ing activity and their immunogenic potential by exposureto bacterial products or CD40L (34–36), a TNF family member expressed on activated CD4 T cells, platelets, and mast cells (37). To determine whether the combination of costimulators and antigen delivery to DCs produces persis-tent T cell activation, mice were injected with ␣DEC/HEL and the agonistic anti-CD40 antibody FGK 45 (38).In contrast to ␣DEC/H EL, the combination of ␣DEC/HEL and FGK 45 induced persistent T cell activation (Fig.5 B). The level of T cell activation seen with ␣DEC/HEL and FGK 45 at day 7 was comparable to ␣DEC/HEL at day 2 or H EL peptide in CFA at day 2 and 7 (compare Figs. 3 B and 5 B). To determine whether anti-CD40treatment altered 3A9 T cell numbers in ␣DEC/HEL-treated mice, we performed adoptive transfer experimentsFigure 3.In vivo activation of CD4ϩ T cells by ␣DEC/HEL. In all experiments, 3A9 T cells were transferred into B10.BR mice, and the recipients were injected subcutaneously in the footpads with antibodies in PBS or 100 ␮g of HEL peptide in CFA 24 h after T cell transfer as indicated. T cell pro-liferation was measured by [3H]thymidine incorporation and is expressed as a proliferation index relative to PBS controls. (A) T cells are efficiently acti-vated by antigen delivered by ␣DEC/HEL. 48 h after challenge with antigen, CD4 T cells were isolated from peripheral LNs and cultured in vitro with irradiated B10.BR CD11c ϩ cells in the presence or absence of HEL peptide. (B) CD4ϩ T cells are only transiently activated by antigen (␣DEC/HEL 0.2 ␮g) delivered to DCs in vivo. CD4ϩ cells were purified from peripheral LNs 2 or 7 d after challenge with antigen and cultured with irradiated CD11c ϩ cells in the presence or absence of HEL peptide. (C) Failure to induce persistent T cell activation with multiple injections of ␣DEC/HEL. 3A9cells were transferred into B10.BR mice and recipients were injected with ␣DEC/HEL (0.2 ␮g/mouse) once (on day 9 or 2 before analysis) or multiple times (days 9, 6, and 2 before analysis). Assay for T cell activation was as above. (D) T cells initially activated by ␣DEC/HEL show diminished response to rechallenge with HEL peptide in CFA. Recipients were initially injected with either ␣DEC/HEL (0.2 ␮g), GL117/HEL(0.2 ␮g), or PBS and rechal-lenged 7 or 20 d later with 100 ␮g of HEL peptide in CFA or with PBS. CD4ϩ cells were purified from peripheral LNs (or spleens, not shown) 2 d after the rechallenge and cultured with irradiated CD11c ϩ cells in the presence or absence of HEL peptide. Assay for T cell activation was as above. (E) An-tigen loading of DCs with ␣DEC/HEL. B10.BR mice with or without transferred 3A9 T cells, were injected subcutaneously with 0.2 ␮g ␣DEC/HEL or PBS either at 8 d (␣DEC/HEL) or at 1 and 8 d (␣DEC/HELX2) after transfer. Antigen loading was measured 1 d after the last dose of ␣DEC/HEL by purifying CD11c ϩ DCs from peripheral LNs and culturing with purified 3A9 T cells. The results are means of triplicate cultures from one of three similar experiments.775Hawiger et al.with CD45.1 allotype-marked T cells and assayed by flow cytometry. Whereas FGK 45 alone showed no effect on the number of 3A9 T cells in LNs at day 7, the combina-tion of FGK 45 and ␣DEC/HEL induced persistent ف8–10-fold expansion of 3A9 T cells, an increase similar to that seen with HEL peptide in CFA at day 7 (Figs. 5 A and 4).We conclude that persistent T cell responses can be in-duced by antigen delivered to DCs in vivo if an additional activation signal such as CD40 ligation is provided.To determine if CD40 ligation induced detectable phe-notypic changes on DCs in our system, we analyzed DCs from mice transferred with 3A9 cells and injected with FGK 45 and ␣DEC/HEL. Consistent with work by others we found that those DCs upregulated their surface expres-sion of CD40 and CD86 (39; Fig. 5 C). This increase was more pronounced in the presence of antigen-specific T cells suggesting a positive feedback mechanism between ac-tivated DCs and T cells (Fig. 5 C).DiscussionTargeting Antigens to DCs In Situ through DEC-205.Our results establish that antigens can be selectively deliv-ered to DCs in vivo via the DEC-205 adsorptive endocy-tosis receptor. DEC-205, originally identified as an antigen recognized by the monoclonal antibody NLDC-145, offers several advantages as a receptor that will mediate antigen targeting to DCs in situ for purposes of antigen presenta-tion. DEC-205 is expressed in abundance on DCs in the T cell area (19, 40) and antibodies bound to DEC-205 are ef-ficiently internalized and delivered to antigen processing compartments (22, 23). When compared with the MMR, a closely related receptor, DEC-205 was at least 30 times more effective in antigen delivery to processing compart-ments (23). In vivo, anti–DEC-205 monoclonal antibody targets to DCs very efficiently, a dose of Ͻ1 ␮g of antibody(20 ng of HEL peptide) leading to presentation by DCs thatFigure 4.CD4ϩ T cells divide in re-sponse to antigen presented by DCs in vivo, produce IL-2 but not IFN-␥, and are then rapidly deleted. (A) CFSE labeled CD45.1ϩ 3A9 T cells were transferred into B10.BR and 24 h later, the recipients were injected subcutaneously in the foot-pads with ␣DEC/HEL (0.2 ␮g), GL117/H EL (0.2 ␮g), H EL peptide in CFA, or PBS. CD4ϩ T cells were purified by neg-ative selection from regional LNs 3 d after challenge with antigen and analyzed by flow cytometry. The plots show staining with 1G12 anti-3A9 and CFSE intensity on gated populations of CD4ϩCD45.1ϩcells. The numbers indicate the percent-age of CFSE high (undivided) and CFSE low (divided) CD4ϩ T cells. The results are from one of two similar experiments.(B) T cells produce IL-2 but not IFN-␥ inresponse to antigens presented on DCs under physiological conditions. 3A9 cells were transferred into B10.BR mice and 24 h later the recipients were injected subcutaneously in the footpads with ␣DEC/HEL (0.2 ␮g), GL117/HEL (0.2 ␮g), HEL peptide in CFA. CD4ϩ. Histograms show staining with anti–IL-2 and anti–IFN-␥ on gated populations of 3A9ϩCD4ϩ cells. The thick lines indicate PBS control. (C) Same as in panel A but analysis performed 7 or 20 d after antigen administration.。

免疫细胞学英语

IntroductionImmunocytology, a specialized branch of cellular biology, delves into the intricate world of immune cells, their structure, function, and interactions within the complex network of the immune system. These cells, often referred to as leukocytes or white blood cells, play a pivotal role in defending our bodies against a myriad of pathogens, foreign substances, and even aberrant cells that arise from within. This essay provides a comprehensive, high-quality analysis of immunocytology, examining various aspects of immune cells, including their classification, development, activation mechanisms, effector functions, and the emerging therapeutic applications that harness their power.Classification and Development of Immune CellsThe immune system is composed of a diverse array of cell types, each with distinct roles and characteristics. Broadly, immune cells can be classified into two main categories: innate immune cells and adaptive immune cells. Innate immune cells, such as neutrophils, monocytes/macrophages, dendritic cells (DCs), natural killer (NK) cells, and mast cells, provide the first line of defense against invading pathogens. They recognize conserved pathogen-associated molecular patterns (PAMPs) via pattern recognition receptors (PRRs) and respond rapidly but non-specifically.In contrast, adaptive immune cells, comprising B cells and T cells, offer a highly specific, long-lasting defense. B cells produce antibodies, while T cells execute cytotoxic or helper functions depending on their subsets (CD4+ T helper cells, CD8+ cytotoxic T cells, regulatory T cells, etc.). The development of these immune cells occurs primarily in the bone marrow (for B cells and myeloid cells) and the thymus (for T cells). A tightly regulated process involving hematopoietic stem cell (HSC) differentiation, gene rearrangements, positive and negative selection, and maturation ensures the generation of a diverse and self-tolerant immune repertoire.Activation Mechanisms and Signal TransductionThe activation of immune cells is a finely orchestrated process triggeredby the recognition of antigens or danger signals. For innate immune cells, PRR engagement initiates signaling cascades involving adaptor proteins like MyD88 and TRIF, leading to the activation of transcription factors such as NF-κB and IRF3/7, which drive the expression of pro-inflammatory cytokines, chemokines, and antimicrobial peptides.Adaptive immune cells, particularly T and B cells, require antigen recognition through their unique antigen receptors (TCR for T cells, BCR for B cells). This interaction, when accompanied by appropriate co-stimulatory signals, activates intracellular signaling pathways involving kinases such as Lck, Zap70, and PI3K, ultimately leading to the activation of transcription factors like NF-κB, AP-1, and NFAT. These transcription factors orchestrate the expression of genes involved in cell proliferation, differentiation, and effector function.Effector Functions of Immune CellsInnate immune cells execute various effector functions to combat infections. Neutrophils phagocytose and kill pathogens through the release of reactive oxygen species (ROS) and granule contents. Monocytes/macrophages display similar phagocytic abilities and also present antigens to T cells, produce inflammatory cytokines, and participate in tissue repair. DCs are professional antigen-presenting cells (APCs) that capture, process, and present antigens to naïve T cells, initiating adaptive immune responses. NK cells directly eliminate virus-infected or transformed cells without prior sensitization, relying on the balance of activating and inhibitory receptors interacting with cell surface ligands.Adaptive immune cells contribute to immunity through antibody production and cell-mediated responses. B cells differentiate into plasma cells that secrete antibodies, which neutralize pathogens, opsonize them for enhanced phagocytosis, or activate complement. T cells, upon activation, differentiate into effector subsets: CD4+ T helper cells (Th1, Th2, Th17, Tfh, etc.) that provide help to other immune cells, and CD8+ cytotoxic T cells that directlykill infected or transformed cells. Regulatory T cells (Tregs) maintain immune homeostasis by suppressing excessive immune responses and preventing autoimmunity.Emerging Therapeutic ApplicationsRecent advances in immunocytology have paved the way for innovative therapeutic strategies targeting immune cells. Cancer immunotherapy, for instance, has revolutionized cancer treatment, with approaches such as immune checkpoint inhibitors (e.g., anti-PD-1, anti-CTLA-4 antibodies) that unleash the cytotoxic potential of T cells suppressed by tumor microenvironment. Chimeric antigen receptor (CAR)-T cell therapy involves engineering patient's T cells to express CARs, enabling targeted recognition and destruction of tumor cells. Additionally, adoptive transfer of ex vivo expanded or genetically modified NK cells is being explored for cancer therapy due to their inherent ability to recognize and kill malignant cells.In autoimmune diseases and transplant rejection, therapies targeting immune cells aim to suppress pathogenic immune responses. These include the use of monoclonal antibodies against pro-inflammatory cytokines or their receptors, T cell-depleting agents, and Treg-based therapies. Moreover, modulation of innate immune cells, particularly DCs, through targeted delivery of antigens or immunomodulatory molecules, holds promise for the induction of tolerance in autoimmune and allergic disorders.ConclusionImmunocytology offers a rich tapestry of knowledge, elucidating the complexities of immune cells and their integral role in maintaining host defense. From the classification and development of these cells to the intricate mechanisms governing their activation and effector functions, understanding immunocytology is crucial for both fundamental biological insights and translational applications. The ongoing advancements in this field continue to fuel the development of novel therapeutic strategies that harness the power of immune cells, transforming the landscape of modern medicine in the fight againstinfectious diseases, cancer, and autoimmune disorders.。

再生障碍性贫血患者外周血树突状细胞亚群分析

ｏｆＣＤ１ｌｃＤＣｗｅｒｅｓｉｇｎｉｆｉｃａｎｔｌｙｉｎｃｒｅａｓｅｄｃｏｍｐａｒｉｎｇｗｉｔｈｔｈａｔｏｆｈｅａｌｔｈｙｃｏｎｔｒｏｌｓ（Ｐ＜０．０５），ｂｕｔｔｈｅｌｅｖｅｌｏｆｔｈｅＣＤ１２３ＤＣｗａｓｎｏｔｃｈａｎｇｅｄｓｉｇｎｉｆｉｃａｎｔｌｙ（Ｐ＞Ｏ．０５）．ＣｏｎｃｌｕｓｉｏｎＴｈｅｐｒｅｄｏｍｉｎａｎｃｅｏｆＣＤ１ｌｃＤＣｉｎｐｅｒｉｐｈｅｒａｌｂｌｏｏｄｏｆＡＡｐａｔｉｅｎｔｓｍａｙｂｅｕｌｔｉ— ｍａｔｅｌｙｆａｖｏｒｔｏｔｈｅＴｈｌ—ｄｏｍｉｎａｔｅｄｉｍｍｕｎｉｔｙ．ｗｈｉｃｈｍａｙｐｌａｙａｎｉｍｐｏｒｔａｎｔｒｏｌｅｉｎｔｈｅｐａｔｈｏｇｅｎｅｓｉｓｏｆＡＡ．
ｗｉｔｈＮａｎｊｉｎｇＵｎｉｖｅｒｓｉｔｙｏｆＣｈｉｎｅｓｅＭｅｄｉｃｉｎｅ，Ｎａｎｊｉｎｇ２１００２９，Ｃｈｉｎａ）［Ａｂｓｔｒａｃｔ］ＯｂｊｅｃｔｉｖｅＴｏｍｅａｓｕｒｅｔｈｅｄｅｎｄｒｉｔｉｃｃｅｌｌ（ＤＣ）ｓｕｂｓｅｔｓｏｆｐｅｒｉｐｈｅｒａｌｂｌｏｏｄｉｎｐａｔｉｅｎｔｓｗｉｔｈａｐｌａｓｔｉｃａｎｅｍｉａ（ＡＡ），ａｎｄｅｘｐｌｏｒｅｔｈｅｓｉｇｎｉｆｉｃａｎｃｅ．ＭｅｔｈｏｄｓＴｈｅＤＣ１／ＣＤ１ｌｃａｎｄＤＣ２／ＣＤ１２３ｓｕｂｓｅｔｓｆｒｏｍ１４ＡＡｐａｔｉｅｎｔｓａｎｄ１４ｈｅａｌｔｈｙｃｏｎｔｒｏｌｓｗｅｒｅｍｅａｓｕｒｅｄｂｙｆｌｏｗｃｙｔｏｍｅｔｒｙｗｉｔｈｔｈｒｅｅ—ｃｏｌｏｒｉｍｍｕｎｏｆｌｕｏｒｅｓｃｅｎｔｓｔａｉｎｉｎｇ．ＲｅｓｕｌｔｓＩｎＡＡｐａｔｉｅｎｔｓｐｅｒｉｐｈｅｒａｌｂｌｏｏｄ，ｔｈｅｒａｔｅｓ

免疫学经典Janeway's Immunobiology Antigen Presentation to T Lymphocytes

1Vertebrate adaptive immune cells possess two types of antigen receptors: the immunoglobulins that serve as antigen receptors on B cells, and the T-cell receptors. While immunoglobulins can recognize native antigens, T cells rec-ognize only antigens that are displayed by MHC complexes on cell surfaces. The conventional α:β T cells recognize antigens as peptide:MHC complexes (see Section 4-13). The peptides recognized by α:β T cells can be derived from the normal turnover of self proteins, from intracellular pathogens, such as viruses, or from products of pathogens taken up from the extracellular fluid. Various tolerance mechanisms normally prevent self peptides from initiating an immune response; when these mechanisms fail, self peptides can become the target of autoimmune responses, as discussed in Chapter 15. Other classes of T cells, such as MAIT cells and γ:δ T cells (see Sections 4-18 and 4-20), rec-ognize different types of surface molecules whose expression may indicate infection or cellular stress.The first part of this chapter describes the cellular pathways used by various types of cells to generate peptide:MHC complexes recognized by α:β T cells. This process participates in adaptive immunity in at least two different ways. In somatic cells, peptide:MHC complexes can signal the presence of an intra-cellular pathogen for elimination by armed effector T cells. In dendritic cells, which may not themselves be infected, peptide:MHC complexes serve to acti-vate antigen-specific effector T cells. We will also introduce mechanisms by which certain pathogens defeat adaptive immunity by blocking the produc-tion of peptide:MHC complexes.The second part of this chapter focuses on the MHC class I and II genes and their tremendous variability. The MHC molecules are encoded within a large cluster of genes that were first identified by their powerful effects on the immune response to transplanted tissues and were therefore called the major histocompatibility complex (MHC). There are several different MHC mole-cules in each class, and each of their genes is highly polymorphic, with many variants present in the population. MHC polymorphism has a profound effect on antigen recognition by T cells, and the combination of multiple genes and polymorphism greatly extends the range of peptides that can be presented to T cells in each individual and in populations as a whole, thus enabling indi-viduals to respond to the wide range of potential pathogens they will encoun-ter. The MHC also contains genes other than those for the MHC molecules; some of these genes are involved in the processing of antigens to produce pep-tide:MHC complexes.The last part of the chapter discusses the ligands for unconventional classes of T cells. We will examine a group of proteins similar to MHC class I mole-cules that have limited polymorphism, some encoded within the MHC and others encoded outside the MHC. These so-called nonclassical MHC class I proteins serve various functions, some acting as ligands for γ:δ T-cell receptors and MAIT cells, or as ligands for NKG2D expressed by T cells and NK cells. In addition, we will introduce a special subset of α:β T cells known as invariant NKT cells that recognize microbial lipid antigens presented by these proteins.Antigen Presentation to T Lymphocytes6IN THIS CHAPTERThe generation of α:β T -cell receptor ligands.The major histocompatibility complex and its function.Generation of ligands forunconventional T -cell subsets.©Garland Science. Preview Content from Janeway's Immunobiology, Ninth Edition.For more information, contact science@.©Garland Science. Preview Content from Janeway's Immunobiology, Ninth Edition.For more information, contact science@.2Chapter 6: Antigen Presentation to T LymphocytesThe generation of α:β T-cell receptor ligands.The protective function of T cells depends on their recognition of cells har-boring intracellular pathogens or that have internalized their products. As wesaw in Chapter 4, the ligand recognized by an α:β T-cell receptor is a peptidebound to an MHC molecule and displayed on a cell surface. The generation ofpeptides from native proteins is commonly referred to as antigen processing,while peptide display at the cell surface by the MHC molecule is referred to asantigen presentation. We have already described the structure of MHC mole-cules and seen how they bind peptide antigens in a cleft, or groove, on theirouter surface (see Sections 4-13 to 4-16). We will now look at how peptides aregenerated from the proteins derived from pathogens and how they are loadedonto MHC class I or MHC class II molecules.6-1Antigen presentation functions both in arming effector T cellsand in triggering their effector functions to attack pathogen-infected cells.The processing and presentation of pathogen-derived antigens has two distinctpurposes: inducing the development of armed effector T cells, and triggeringthe effector functions of these armed cells at sites of infection. MHC class Imolecules bind peptides that are recognized by CD8 T cells, and MHC class IImolecules bind peptides that are recognized by CD4 T cells, a pattern of rec-ognition determined by specific binding of the CD8 or CD4 molecules to therespective MHC molecules (see Section 4-18). The importance of this specific-ity of recognition lies in the different distributions of MHC class I and class IImolecules on cells throughout the body. Nearly all somatic cells (except redblood cells) express MHC class I molecules. Consequently, the CD8 T cell isprimarily responsible for pathogen surveillance and cytolysis of somatic cells.Also called cytotoxic T cells, their function is to kill the cells they recognize.CD8 T cells are therefore an important mechanism in eliminating sources ofnew viral particles and bacteria that live only in the cytosol, and thus freeingthe host from infection.By contrast, MHC class II molecules are expressed primarily only on cells ofthe immune system, and particularly by dendritic cells, macrophages, and Bcells. Thymic cortical epithelial cells and activated, but not naive, T cells canexpress MHC class II molecules, which can also be induced on many cells inresponse to the cytokine IFN-γ. Thus, CD4 T cells can recognize their cognateantigens during their development in the thymus, on a limited set of ‘profes-sional’ antigen-presenting cells, and on other somatic cells under specificinflammatory conditions. Effector CD4 T cells comprise several subsets withdifferent activities that help eliminate the pathogens. Importantly, naive CD8and CD4 T cells can become armed effector cells only after encountering theircognate antigen once it has been processed and presented by activated den-dritic cells.In considering antigen processing, it is important to distinguish between thevarious cellular compartments from which antigens can be derived (Fig. 6.1).These compartments, which are separated by membranes, include the cytosoland the various vesicular compartments involved in endocytosis and secre-tion. Peptides derived from the cytosol are transported into the endoplasmicreticulum and directly loaded onto newly synthesized MHC class I moleculeson the same cell for recognition by T cells, as we will discuss below in greaterdetail. Because viruses and some bacteria replicate in the cytosol or in thecontiguous nuclear compartment, peptides from their components can beloaded onto MHC class I molecules by this process (Fig. 6.2, first upper panel).3The generation of α:β T -cell receptor ligands.This pathway of recognition is sometimes referred to as direct presenta-tion , and can identify both somatic and immune cells that are infected by a pathogen.Certain pathogenic bacteria and protozoan parasites survive ingestion by macrophages and are able to replicate inside the intracellular vesicles of the endosomal–lysosomal system (Fig. 6.2, second panel). Other pathogenic bacteria proliferate outside cells, and can be internalized, along with their toxic products, by phagocytosis, receptor-mediated endocytosis, or macro-pinocytosis into endosomes and lysosomes, where they are broken down by digestive enzymes. For example, receptor-mediated endocytosis by B cells can efficiently internalize extracellular antigens through B-cell receptors (Fig. 6.2, third panel). Virus particles and parasite antigens in extracellular fluids can also be taken up by these routes and degraded, and their peptides presented to T cells.Some pathogens may infect somatic cells but not directly infect phagocytes such as dendritic cells. In this case, dendritic cells must acquire antigens from exogenous sources in order to process and present antigens to T cells. For example, to eliminate a virus that infects only epithelial cells, activation of CD8 T cells will require that dendritic cells load MHC class I molecules with peptides derived from viral proteins taken up from virally infected cells. This exogenous pathway of loading MHC class I molecules is called cross- presentation , and is carried out very efficiently by some specialized types of dendritic cells (Fig. 6.3). The activation of naive T cells by this pathway is called cross-priming.Fig. 6.1 There are two categories of major intracellular compartments, separated by membranes. One compartment is the cytosol, which communicates with the nucleus via pores in the nuclear membrane. The other is the vesicular system, which comprises the endoplasmic reticulum, Golgi apparatus, endosomes, lysosomes, and other intracellular vesicles. The vesicular system can be thought of as being continuous with the extracellular fluid. Secretory vesicles bud off from the endoplasmic reticulum and are transported viafusion with Golgi membranes to move vesicular contents out of the cell. Extracellular material is taken up by endocytosis or phagocytosis into endosomes or phagosomes, respectively. The fusion of incoming and outgoing vesicles is important both for pathogen destruction in cells such as neutrophils and for antigen presentation. Autophagosomes surroundcomponents in the cytosol and deliver them to lysosomes in a process known as autophagy.Fig. 6.2 Cells become targets of T -cell recognition by acquiring antigens from either the cytosolic or the vesicular compartments. Top, first panel: viruses and some bacteria replicate in the cytosolic compartment. Their antigens are presented by MHC class I molecules to activate killing by cytotoxic CD8 T cells. Second panel: other bacteria and some parasites are taken up into endosomes, usually by specialized phagocytic cells such as macrophages. Here they are killed and degraded, or in some cases are able to survive andproliferate within the vesicle. Their antigens are presented by MHC class II molecules to activate cytokine production by CD4 T cells. Third panel: proteins derived from extracellular pathogens may bind to cell-surface receptors and enter thevesicular system by endocytosis, illustrated here for antigens bound by the surface immunoglobulin of B cells. These antigens are presented by MHC class II molecules to CD4 helper T cells, which can then stimulate the B cells to produce antibody.©Garland Science. Preview Content from Janeway's Immunobiology, Ninth Edition.For more information, contact science@.4Chapter 6: Antigen Presentation to T LymphocytesFor loading peptides onto MHC class II molecules, dendritic cells, macro-phages, and B cells are able to capture exogenous proteins via endocytic ves-icles and through specific cell-surface receptors. For B cells, this process of antigen capture can include the B-cell receptor. The peptides that are derived from these proteins are loaded onto MHC class II molecules in specially mod-ified endocytic compartments in these antigen-presenting cells, which we will discuss in more detail later. In dendritic cells, this pathway operates to activate naive CD4 T cells to become effector T cells. Macrophages take up particulate material by phagocytosis and so mainly present pathogen-derived peptides on MHC class II molecules. In macrophages, such antigen presentation may be used to indicate the presence of a pathogen within its vesicular compartment. Effector CD4 T cells, on recognizing antigen, produce cytokines that can acti-vate the macrophage to destroy the pathogen. Some intravesicular pathogens have adapted to resist intracellular killing, and the macrophages in which they live require these cytokines to kill the pathogen: this is one of the roles of the T H 1 subset of CD4 T cells. Other CD4 T cell subsets have roles in regulating other aspects of the immune response, and some CD4 T cells even have cyto-toxic activity. In B cells, antigen presentation may serve to recruit help from CD4 T cells that recognize the same protein antigen as the B cell. By efficiently endocytosing a specific antigen via their surface immunoglobulin and pre-senting the antigen-derived peptides on MHC class II molecules, B cells can activate CD4 T cells that will in turn serve as helper T cells for the production of antibodies against that antigen.Beyond the presentation of exogenous proteins, MHC class II molecules can also be loaded with peptides derived from cytosolic proteins by a ubiquitous pathway of autophagy , in which cytoplasmic proteins are delivered into the endocytic system for degradation in lysosomes (Fig. 6.4). This pathway can serve in the presentation of self-cytosolic proteins for the induction of toler-ance to self antigens, and also as a means for presenting antigens from patho-gens, such as herpes simplex virus, that have accessed the cell’s cytosol.6-2Peptides are generated from ubiquitinated proteins in the cytosol by the proteasome.Proteins in cells are continually being degraded and replaced with newly syn-thesized proteins. Much cytosolic protein degradation is carried out by a large, multicatalytic protease complex called the proteasome (Fig. 6.5). A typical proteasome is composed of one 20S catalytic core and two 19S regulatory caps , one at each end; both the core and the caps are multisubunit complexes of proteins. The 20S core is a large cylindrical complex of some 28 subunits, arranged in four stacked rings of seven subunits each around a hollow core. The two outer rings are composed of seven distinct α subunits and are noncat-alytic. The two inner rings of the 20S proteasome core are composed of seven distinct β subunits. The constitutively expressed proteolytic subunits are β1, β2, and β5, which form the catalytic chamber. The 19S regulator is composed of a base containing nine subunits that binds directly to the αring of the 20SFig. 6.3 Cross-presentation of extracellular antigens on MHC class I molecules by dendritic cells. Certain subsets of dendritic cells are efficient in capturing exogenous proteins and loading peptides derived from them onto MHC class I molecules. There is evidence that several cellular pathways may be involved. One route may involve thetranslocation of ingested proteins from the phagolysosome into the cytosol for degradation by the proteasome, with the resultant peptides then passing through TAP (see Section 6-3) into the endoplasmic reticulum, where they load onto MHC class I molecules in the usual way. Another route may involve direct transport of antigens from the phagolysosome into a vesicular loading compartment—without passage through the cytosol—where peptides are allowed to be bound to mature MHC class I molecules.Fig. 6.4 Autophagy pathways can deliver cytosolic antigens for presentation by MHC class IImolecules. In the process of autophagy, portions of the cytoplasm are taken into autophagosomes, specialized vesicles that are fused with endocytic vesicles and eventually with lysosomes, where the contents are catabolized. Some of the resulting peptides of this process can be bound to MHC class II molecules and presented on the cell surface. In dendritic cells and macrophages, this can occur in the absence of activation, so that immature dendritic cells may express self peptides in a tolerogenic context, rather than inducing T -cell responses to self antigens.©Garland Science. Preview Content from Janeway's Immunobiology, Ninth Edition.For more information, contact science@.5 The generation of α:β T-cell receptor ligands.core particle and a lid that has up to 10 different subunits. The association of the 20S core with a 19S cap requires ATP as well as the ATPase activity of many of the caps’ subunits. One of the 19S caps binds and delivers proteins into the proteasome, while the other keeps them from exiting prematurely.Proteins in the cytosol are tagged for degradation via the ubiquitin–proteasome system (UPS). This begins with the attachment of a chain of several ubiquitin molecules to the target protein, a process called ubiquitination. First, a lysine residue on the targeted protein is chemically linked to the glycine at the carboxy terminus of one ubiquitin molecule. Ubiquitin chains are then formed by linking the lysine at residue 48 (K48) of the first ubiquitin to the carboxy-terminal glycine of a second ubiquitin, and so on until at least 4 ubiquitin molecules are bound. This K48-linked type of ubiquitin chain is recognized by the 19S cap of the proteasome, which then unfolds the tagged protein so that it can be introduced into the proteasome’s catalytic core. There the protein chain is degraded with a general lack of sequence specificity into short peptides, which are subsequently released into the cytosol. The general degradative functions of the proteasome have been co-opted for antigen presentation, so that MHC molecules have evolved to work with the peptides that the proteasome can produce.Various lines of evidence implicate the proteasome in the production of pep-tide ligands for MHC class I molecules. Experimentally tagging proteins with ubiquitin results in more efficient presentation of their peptides by MHC class I molecules, and inhibitors of the proteolytic activity of the proteasome inhibit antigen presentation by MHC class I molecules. Whether the proteas-ome is the only cytosolic protease capable of generating peptides for transport into the endoplasmic reticulum is not known.The constitutive β1, β2, and β5 subunits of the catalytic chamber are sometimes replaced by three alternative catalytic subunits that are induced by interferons. These induced subunits are called β1i (or LMP2), β2i (or MECL-1), and β5i (or LMP7). Both β1i and β5i are encoded by the PSMB9 and PSMB8 genes, which are located in the MHC locus, whereas β2i is encoded by PSMB10 outside the MHC locus. Thus, the proteasome can exist both as both a constitutive proteasome present in all cells and as the immunoproteasome, which is present in cells stimulated with interferons. MHC class I proteins are also induced by interferons. The replacement of the β subunits by their interferon-inducible counterparts alters the enzymatic specificity of the proteasome such that there is increased cleavage of polypeptides after hydrophobic residues, and decreased cleavage after acidic residues. This produces peptides with carboxy-terminal residues that are preferred anchor residues for binding to most MHC class I molecules (see Chapter 4) and are also the preferred structures for transport by TAP.Another substitution for a β subunit in the catalytic chamber has been found to occur in cells in the thymus. Epithelial cells of the thymic cortex (cTECs) express a unique β subunit, called β5t, that is encoded by PSMB11. In cTECs, β5t becomes a component of the proteasome in association with β1i and β2i,and this specialized type of proteasome is called the thymoproteasome. Mice lacking expression of β5t have reduced numbers of CD8 T cells, indicating that the peptide:MHC complexes produced by the thymoproteasome are impor-tant in CD8 T-cell development in the thymus.Interferon-γ (IFN-γ) can further increase the production of antigenic pep-tides by inducing expression of the PA28 proteasome-activator complex that binds to the proteasome. PA28 is a six- or seven-membered ring composed of two proteins, PA28α and PA28β, both of which are induced by IFN-γ. A PA28 ring, which can bind to either end of the 20S proteasome core in place of the 19S regulatory cap, acts to increase the rate at which peptides are released (Fig. 6.6). In addition to simply providing more peptides, the increased rate ofFig. 6.5Cytosolic proteins are degraded by the ubiquitin–proteasome system into short peptides. The proteasome is composed of a 20S catalytic core, which consists of four multisubunit rings (see text), and two 19S regulatory caps on either end. Proteins (orange) that are targeted become covalently tagged with K48-linked polyubiquitin chains (yellow) through the actions of various E3 ligases. The 19S regulatory cap recognizes polyubiquitin and draws the tagged protein inside the catalytic chamber; there, the protein is degraded, giving rise to small peptide fragments that are released back into the cytoplasm.©Garland Science. Preview Content from Janeway's Immunobiology, Ninth Edition.For more information, contact science@.6Chapter 6: Antigen Presentation to T Lymphocytesflow allows potentially antigenic peptides to escape additional processing that might destroy their antigenicity.Translation of self or pathogen-derived mRNAs in the cytoplasm generates not only properly folded proteins but also a significant quantity—possibly up to 30%—of peptides and proteins that are known as defective ribosomal prod-ucts (DRiPs). These include peptides translated from introns in improperly spliced mRNAs, translations of frameshifts, and improperly folded proteins, which are tagged by ubiquitin for rapid degradation by the proteasome. This seemingly wasteful process provides another source of peptides and ensures that both self proteins and proteins derived from pathogens generate abun-dant peptide substrates for eventual presentation by MHC class I proteins.6-3Peptides from the cytosol are transported by TAP into the endoplasmic reticulum and further processed before binding to MHC class I molecules.6-4 Newly synthesized MHC class I molecules are retained in the endoplasmic reticulum until they bind a peptide.6-5 Dendritic cells use cross-presentation to present exogenous proteins on MHC class I molecules to prime CD8 T cells.6-6Peptide:MHC class II complexes are generated in acidified endocytic vesicles from proteins obtained through endocytosis, phagocytosis, and autophagy.ααββa bccatalytic chamberPA28PA28Fig. 6.6 The PA28 proteasome activator binds to either end of the proteasome. Panel a: in this side view cross-section, the heptamer rings of the PA28 proteasome activator (yellow) interact with the α subunits (pink) at either end of the core proteasome (the β subunits that make up the catalytic cavity of the core are in blue). Within this region is the α-annulus (green), a narrow ringlike opening that is normally blocked by other parts of the α subunits (shown in red). Panel b: a close-up view from the top, looking into the α-annulus without PA28 bound. Panel c: with the same perspective, the binding of PA28 to the proteasome changes the conformation of the α subunits, moving those parts of the molecule that block the α-annulus, and opening the end of the cylinder. For simplicity, PA28 is not shown. Structures courtesy of F . Whitby.©Garland Science. Preview Content from Janeway's Immunobiology, Ninth Edition.For more information, contact science@.7The generation of α:β T -cell receptor ligands.6-7 The invariant chain directs newly synthesized MHC class II molecules to acidified intracellular vesicles.6-8 The MHC class II-like molecules HLA-DM and HLA-DO regulate exchange of CLIP for other peptides.6-9Cessation of antigen processing occurs in dendritic cells after their activation through reduced expression of the MARCH-1 E3 ligase.Summary.The major histocompatibility complex and its function.6-10 Many proteins involved in antigen processing andpresentation are encoded by genes within the MHC.6-11 The protein products of MHC class I and class II genes arehighly polymorphic.6-12 MHC polymorphism affects antigen recognition by T cells byinfluencing both peptide binding and the contacts between T -cell receptor and MHC molecule.6-13 Alloreactive T cells recognizing nonself MHC molecules arevery abundant.6-14 Many T cells respond to superantigens.6-15 MHC polymorphism extends the range of antigens to whichthe immune system can respond.Summary.Generation of ligands for unconventional T-cell subsets.6-16 A variety of genes with specialized functions in immunity arealso encoded in the MHC.6-17 Specialized MHC class I molecules act as ligands for theactivation and inhibition of NK cells and unconventional T -cell subsets.6-18 Members of the CD1 family of MHC class I-like moleculespresent microbial lipids to invariant NKT cells.6-19 The nonclassical MHC class I molecule MR1 presentsmicrobial folate metabolites to MAIT cells.6-20γ:δ T cells can recognize a variety of diverse ligands.©Garland Science. Preview Content from Janeway's Immunobiology, Ninth Edition.For more information, contact science@.©Garland Science. Preview Content from Janeway's Immunobiology, Ninth Edition.For more information, contact science@.8Chapter 6: Antigen Presentation to T LymphocytesSummary.Summary to Chapter 6.T-cell receptors on conventional α:β T cells recognize peptides bound to MHCmolecules. In the absence of infection, MHC molecules are occupied by selfpeptides, which do not normally provoke a T-cell response, because of var-ious tolerance mechanisms. But during infections, pathogen-derived pep-tides become bound to MHC molecules and are displayed on the cell surface,where they can be recognized by T cells that have been previously activatedand armed for the specific peptide:MHC complex. Naive T cells become acti-vated when they encounter their specific antigen presented on activated den-dritic cells. MHC class I molecules in most cells bind to peptides derived fromproteins that have been synthesized and then degraded in the cytosol. Somedendritic cells can obtain and process exogenous antigens and present themon MHC class I molecules.This process of cross-presentation is important forpriming CD8 T cells to many viral infections.Through assembly with the invariant chain (Ii), MHC class II molecules bindpeptides derived from proteins degraded in endocytic vesicles, but they canalso acquire self antigens through autophagy. Stable peptides are bound aftera process of peptide editing in the endocytic compartment involving HLA-DMand HLA-DO. CD8 T cells recognize peptide:MHC class I complexes and areactivated to kill cells displaying foreign peptides derived from cytosolic path-ogens, such as viruses. CD4 T cells recognize peptide:MHC class II complexesand are specialized to activate other immune effector cells, for example, B cellsor macrophages, to act against the foreign antigens or pathogens that theyhave taken up.For each class of MHC molecule, there are several genes arranged in clusterswithin a larger region known as the major histocompatibility complex (MHC).Within the MHC, the genes for the MHC molecules are closely linked to genesinvolved in the degradation of proteins into peptides, the formation of thecomplex of peptide and MHC molecule, and the transport of these complexesto the cell surface. Because the several different genes for the MHC class I andclass II molecules are highly polymorphic and are expressed in a codominantfashion, each individual expresses a number of different MHC class I andclass II molecules. Each different MHC molecule can bind stably to a rangeof different peptides, and thus the MHC repertoire of each individual can rec-ognize and bind many different peptide antigens. Because the T-cell receptorbinds a combined peptide:MHC ligand, T cells show MHC-restricted antigenrecognition, such that a given T cell is specific for a particular peptide boundto a particular MHC molecule.Unconventional T-cell subsets include iNKT cells, MAIT cells, and γ:δ T cells,which recognize nonpeptide ligands of various types. Some CD1 moleculesbind self lipids and pathogen-derived lipid molecules and present them toiNKT cells. MAIT cells recognize vitamin metabolites that are specific to bac-teria and yeast and that are presented by MR1. γ:δ T cells are activated by adiverse array of ligands, including MHC class Ib molecules and EPCR, thatare induced by infection or cellular stress. These T-cell subsets function in thetransitional area between innate and adaptive immunity, relying on a reper-toire of receptors produced by somatic gene rearrangement but recognizingligands in a manner somewhat similar to the way PAMPs are recognized byTLRs and other fully innate receptors.。

淋巴细胞亚群和细胞因子的区别

淋巴细胞亚群和细胞因子的区别英文回答：Lymphocyte Subsets and Cytokines are two important components of the immune system that play different roles in the body's defense mechanisms.Lymphocyte Subsets:Lymphocytes are a type of white blood cells that are crucial for immune responses. They are divided into two main subsets: B cells and T cells.B cells: B cells are responsible for producing antibodies, which are proteins that can recognize and bind to specific antigens. When an antigen enters the body, B cells are activated and differentiate into plasma cells, which secrete large amounts of antibodies. B cells are mainly involved in the humoral immune response, which targets extracellular pathogens.T cells: T cells are involved in cell-mediated immunity, which targets infected cells. There are several subtypes of T cells, including helper T cells (CD4+), cytotoxic T cells (CD8+), and regulatory T cells (Tregs).Helper T cells: Helper T cells play a crucial role in coordinating immune responses. They help activate B cells, cytotoxic T cells, and macrophages, and also secrete cytokines to regulate the immune response.Cytotoxic T cells: Cytotoxic T cells are responsiblefor killing infected cells. They recognize and bind to antigens presented on the surface of infected cells, and release cytotoxic molecules to induce cell death.Regulatory T cells: Regulatory T cells are involved in suppressing immune responses to prevent excessive inflammation and autoimmune reactions.Cellular Subsets:Different subsets of lymphocytes can have distinct functions and characteristics. For example, within the Tcell population, there are Th1 cells, Th2 cells, Th17 cells, and Tregs. Each subset has a specific cytokine profile and performs different functions in immune responses.Th1 cells: Th1 cells secrete cytokines such asinterferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α), which promote cell-mediated immunity and enhance the activity of macrophages and cytotoxic T cells.Th2 cells: Th2 cells secrete cytokines such as interleukin-4 (IL-4), interleukin-5 (IL-5), andinterleukin-13 (IL-13), which are involved in the humoral immune response and promote the production of antibodies by B cells.Th17 cells: Th17 cells secrete cytokines such as interleukin-17 (IL-17) and interleukin-22 (IL-22), whichare important for the defense against extracellularbacteria and fungi.Tregs: Regulatory T cells, as mentioned earlier, play a role in suppressing immune responses and maintaining immune tolerance. They secrete cytokines such as interleukin-10 (IL-10) and transforming growth factor-beta (TGF-β) to suppress the activity of other immune cells.In summary, lymphocyte subsets refer to different types of lymphocytes, such as B cells and T cells, while cytokines are small proteins secreted by immune cells that regulate immune responses. Lymphocyte subsets havedifferent functions, and subsets of T cells can secrete specific cytokines to modulate immune responses.中文回答：淋巴细胞亚群和细胞因子是免疫系统中的两个重要组成部分，它们在身体的防御机制中扮演不同的角色。

脓毒症过程中树突状细胞的研究进展

ｏｆＧｅｎｅｒａｌＳｕｒｇｅｒｙ，五ｎｌｉｎｇＨｏｓｐｉｔａｌ，Ｎａ彬ｎｇ２１０００２，Ｊｉａｎｇｓｕ，Ｃｈｉｎａ）
ｏｆｍｏｒｔａｌｉｔｙｉｎｃｒｉｔｉｃａｌｌｙｉｌｌｐａｔｉｅｎｔｓ．Ｓｔｕｄｉｅｓｉｎｄｉｃａｔｅｔｈａｔｉｍｍｕｎｅ
Ａｂｓｔｒａｃｔ：
Ｓｅｐｓｉｓｉｓｔｈｅｌｅａｄｉｎｇ
Ｉ）Ｃ的改变对脓毒症预后的意义实验研究和临床观察均证明脓毒症过程中ＤＣｓ
来提高ＩＬ．１２和ＴＮＦ．仪的生成量¨３｜。国内也有报道Ｆｈ３Ｌ可促进多器官功能障碍综合征（ｍｕｌｔｉｐｌｅ
ｇａｎｄｙｓｆｕｎｃｔｉｏｎ
ｏｒ－
的上述改变对于判断患者的预后有重要意义。临床数据显示因脓毒症死亡的患者外周血ＤＣ数目明显低于存活的脓毒症患者，且这种外周血ＤＣ数目的变化与患者的ＳＡＰＳＩＩ评分密切相关¨６ｌ。动物实验的结果进一步证实ＤＣ的改变对脓毒症预后有很大影响。Ｓｃｕｍｐｉａ等¨引给培育出的基因工程小鼠，注射白喉毒素后，ＣＤｌｌｃ阳性细胞（即ＤＣｓ）消失，行盲肠结扎穿孔术后注射白喉毒素后的小鼠死亡率明显高于对照组。但在注射白喉毒素的同时注射正常小鼠骨髓来源的ＤＣ则可改变这一结果。另一项研究中，小鼠在盲肠结肠穿孔术后予以抗生素治疗，待其存活到第１５ｄ时，予以注射曲霉菌孢子，４ｄ内小鼠全部死亡。但注射补充正常小鼠或假手术组小鼠骨髓的ＤＣ，小鼠可继续存活ｍＪ。这证明脓毒症炎症高峰过后，正常ＤＣ的补充有助于机体抵御再次感染的侵袭。
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医学研究生学报
２００９年６月
第２２卷
产生的同时也诱发抗炎介质产生，调控最初的炎症反应，以避免炎症反应的放大。炎症反应的转归取决于上述２类物质的平衡，任何一方的过度均可造成炎症反应失控。促炎与抗炎反应在经历相持和交替制衡后往往抗炎机制获得优势，从而导致脓毒症患者中后期常出现免疫功能下降【４】。对其机制进一步探求也就成为了脓毒症研究的热点。近年来研究发现，ＤＣ在这一变化过程中发挥了重要作用。２树突状细胞是机体免疫系统的重要组成部分ＤＣ根据功能状态可分为非成熟态和成熟态。非成熟态ＤＣ吞噬能力较强，但抗原提呈和活化Ｔ细胞的能力较弱；成熟态ＤＣ吞噬抗原能力大为减弱，但具有很强的抗原提呈和激活Ｔ细胞的能力。白骨髓干细胞分化来的ＤＣ，以非成熟态随血流分布于全身各处（其中以皮肤和胃肠道比例最高）¨１。非成熟态ＤＣ持续捕获抗原，监视内外环境变化。一旦有外源性抗原出现或有组织损伤，非成熟态ＤＣ摄取抗原后进人成熟过程，在消化处理抗原同时，细胞表面的趋化因子受体发生改变，并在趋化因子的引导下迁移至淋巴器官的Ｔ细胞区，同时细胞表面ＣＤ８０、ＣＤ８６等共刺激信号上调，将抗原以ＭＨＣ复合物的形式提呈于特异性Ｔ细胞，激活Ｔ细胞，启动免疫防御反应。ＤＣ的成熟过程就是连接非特异性免疫和特异性免疫的桥梁，任一环节的改变都可能损害机体的免疫防御能力旧Ｊ。ＤＣ对Ｔ细胞的作用不仅是激活，还可诱导Ｔ细胞失活甚至凋亡，这由ＤＣ所处的功能状态决定，而其功能状态又受其所摄取抗原种类、所处环境等影响。ＤＣ通过与幼稚型Ｔ细胞相互作用，生成的细胞因子影响Ｔ细胞的分化方向＂，８Ｊ。因此，鉴于ＤＣ在机体免疫系统中的枢纽地位，可推测其对诊断和治疗脓毒症具有重要意义。３脓毒症过程中树突状细胞大量丢失近年来的几项研究均发现在脓毒症过程中ＤＣ大量丢失。２００３年，Ｔｉｎｓｌｅｙ等归１首先报道行盲肠结扎穿孔（ＣＬＰ）法制备的脓毒症小鼠，术后１２ｈ出现脾ＤＣ丢失，且以滤泡状树突状细胞（ＦＤＣ）的丢失最为明显。其后，Ｄｉｎｇ等¨刚的研究也证实了这一点，并发现除脾外，ＣＬＰ小鼠的腹膜、肠系膜淋巴结等处的ＤＣ也大量丢失。这一现象几乎与机体免疫

S1PR1介导的IFNAR1降解可以调节浆细胞样树突状细胞α-干扰素的自动扩增／信号放大(外文翻译)

S1PR1-mediated IFNAR1 degradation modulates plasmacytoiddendritic cell interferon-α autoamplification由S1PR1介导的IFNAR1降解可以调节浆细胞样树突状细胞α-干扰素的自动扩增／信号放大摘要：Blunting immunopathology without abolishing host defense is the foundation for safe and effective modulation of infectious and autoimmune diseases.没有废除宿主防御机制的免疫病理钝化是安全、有效调节传染病和自身免疫性疾病的基础。

Sphingosine 1-phosphate receptor 1 (S1PR1) agonists are effective in treating infectious and multiple autoimmune pathologies; however, mechanisms underlying their clinical efficacy are yet to be fully elucidated.1-磷酸-鞘氨醇受体1（S1PR1）促效药对于治疗传染病和多种自身免疫性疾病是有效的，然而，其临床疗效的具体机制尚未被完全阐明。

Here, we uncover an unexpected mechanism of convergence between S1PR1 and interferon alpha receptor 1 (IFNAR1) signaling pathways.在本研究中，我们意外发现S1PR1与α-干扰素受体1（IFNAR1）信号通路之间的趋同／聚集机制。

Activation of S1PR1 signaling by pharmacological tools or endogenous ligand sphingosine-1 phosphate (S1P) inhibits type 1 IFN responses that exacerbate numerous pathogenic conditions.通过药理作用或内源性配体1-磷酸-鞘氨醇（S1P）发出信号激活S1PR1可以抑制1型干扰素应答，这将提供大量致病条件。

activation of dendritic cells

(Brucella b 1,2 Cyclic Glucan Is an Activator of Human and Mouse Dendritic Cells)
lipoprotein
• 19-kDa lipoprotein from M. tuberculosis, as well as synthetic lipopeptides, induced DC maturation. The resulting mature DC had increased cell surface expression of MHC-II, CD80, CD83, CD86, CD54, and CD58, suggesting that the lipopeptide alone is sufficient to induce maturation events. • （Microbial Lipopeptides Stimulate Dendritic Cell Maturation Via Toll-Like Receptor 2）
Cylic Glucans
The Brucella cyclic glucans showed neither toxicity nor immunogenicity compared to LPS and triggered antigen-specific CD8+ T cell responses in vivo. These cyclic glucans also enhanced antigen-specific CD4+ and CD8+ T cell responses including crosspresentation by different human DC subsets. Brucella b 1,2 cyclic glucans increased the memory CD4+ T cell responses of blood mononuclear cells exposed to recombinant fusion proteins composed of anti-CD40 antibody and antigens from both hepatitis C virus and Mycobacterium tuberculosis.

Immune cells in experimental acute kidney injury

Nephrology Division, Department of Medicine, Samsung Medical Centre, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, 81 Irwon-Ro Gangnam-gu,Seoul 135-710, South Korea (H.R.J.). Ross Building,Room 965, Nephrology Division, Department of Medicine, Johns Hopkins University School of Medicine, 720 Rutland Avenue, Baltimore, MD 21205, USA (H.R.). Correspondence to: H.R.hrabb1@ Immune cells in experimental acutekidney injuryHye Ryoun Jang and Hamid RabbAbstract | Acute kidney injury (AKI) prolongs hospital stay and increases mortality in various clinical settings. Ischaemia–reperfusion injury (IRI), nephrotoxic agents and infection leading to sepsis are among the major causes of AKI. Inflammatory responses substantially contribute to the overall renal damage in AKI. Both innate and adaptive immune systems are involved in the inflammatory process occurring in post-ischaemic AKI. Proinflammatory damage-associated molecular patterns, hypoxia-inducible factors, adhesion molecules, dysfunction of the renal vascular endothelium, chemokines, cytokines and Toll-like receptors are involvedin the activation and recruitment of immune cells into injured kidneys. Immune cells of both the innate and adaptive immune systems, such as neutrophils, dendritic cells, macrophages and lymphocytes contributeto the pathogenesis of renal injury after IRI, and some of their subpopulations also participate in the repair process. These immune cells are also involved in the pathogenesis of nephrotoxic AKI. Experimental studies of immune cells in AKI have resulted in improved understanding of the immune mechanisms underlyingAKI and will be the foundation for development of novel diagnostic and therapeutic targets. This Review describes what is currently known about the function of the immune system in the pathogenesis and repairof ischaemic and nephrotoxic AKI.Jang, H. R. & Rabb, H. Nat. Rev. Nephrol. 11, 88–101 (2015); published online 21 October 2014; doi:10.1038/nrneph.2014.180 IntroductionDespite remarkable advances in modern medicine, acutekidney injury (AKI) still remains a challenging conditionthat lacks specific tools for its early diagnosis and treat-ment. AKI worsens the overall clinical course of affectedpatients by causing uraemia, acid–base or electrolytedisturbances, and volume overload. The incidence ofAKI has been reported to be as high as 5% of hospital-ized patients or 30% of critically ill patients.1 The risk ofchronic kidney disease and end-stage renal disease is sub-stantially increased in patients with AKI.2 Most patientswith AKI are diagnosed when injury is already estab-lished and, therefore, only conservative treatment includ-ing fluid therapy and dialysis is available. To improve theclinical outcome of AKI, novel diagnostic and therapeu-tic strat e gies need to be developed. Understanding thepathophysi o logy of AKI is, therefore, the cornerstone ofexploration of novel diagnostic and therapeutic strategies.Experimental models of AKI can be divided into severalcategories depending on the induction method (Figure 1).In models of septic AKI, the initial immune responseagainst foreign antigens and innate triggers causes acomplex secondary inflammatory response that facili-tates renal injury.3 Non-septic and septic AKI are known tohave very different pathophysiological features. Septic AKIis a systemic manifestation of sepsis following exposureto foreign antigens such as bacteria or viruses; detaileddiscussion of septic AKI is beyond the scope of this review.Immune mechanisms were not expected to have animportant role in models of aseptic AKI, but numer-ous studies conducted over the past two decades haverevealed that inflammatory processes mediated by theimmune system are crucial in mediating renal injury.3Immune mechanisms involved in the pathogenesis ofrenal injury have been studied most extensively in modelsof ischaemic AKI employing cold or warm ischaemia.Both types of ischaemia occur during organ transplanta-tion; cold ischaemia starts when the organ is cooled withcold perfusion solution after procurement, and lasts untilthe temperature of the organ reaches the physiologic tem-perature. Thereafter, warm ischaemia begins, and endswhen perfusion is restored after completion of surgicalanastomosis. Thus, two distinct periods of warm ischae-mia occur in the transplantation setting—during organretrieval and implantation.4 Interestingly, the nephro t oxi-city induced by cisplatin, a chemotherapeutic agent, hasmany pathophysiological features that overlap with thoseof ischaemia–reperfusion injury (IRI).Both innate and adaptive immune systems are directlyinvolved in the pathogenesis of ischaemic AKI. Variouscellular and humoral immune system components con-tribute to AKI, some of which are also thought to beinvolved in the repair process following IRI.5,6 The healthykidney produces hormones that influence the immunesystem, such as vitamin D (calcitriol) and erythropoi-etin,7 and the renal tubular epithelium expresses Toll-likereceptors (TLRs), which critically contribute to activationof the complement system and recruitment of immune Competing interestsThe authors declare no competing interests.REVIEWScells in response to inflammatory stimuli.8,9 Several types of resident immune cells, such as dendritic cells, macro-phages, mast cells and lymphocytes are homeostati-cally maintained in the normal kidney, although these cells constitute a small population.10–13 Under normal conditions, the renal mononuclear phagocytes mainly comprise macrophages located in the renal medulla and capsule and renal dendritic cells found in the tubulo-interstitium.10,11,14 In mice, renal dendritic cells show a specific CD11c +CD11b +EMR1(F4/80)+CX 3CR1 (CX 3C-chemokine receptor)+CD8–CD205– phenotype, and have a similar transcriptome as dendritic cells residing in other nonlymphoid tissues.15,16 Dendritic cells are recruited to the kidney by a CX 3CR1–CX 3CL1 (CX 3C-chemokine ligand 1, also known as fractalkine) chemokine pair,17 and have an important role in local injury or infection. Dendritic cells not only function as a potent source of other factors, such as neutrophil-recruiting chemokines and cytokines,12,18but also present antigens to T cells.Intrarenal macrophages exert homeostatic functions by phagocytosis of antigens in the kidney and undergo pheno t ypic changes that enable them to participate in both inflammatory and anti-inflammatory processes.14 Both dendritic cells and macrophages contribute substan-tially to homeostasis and regulation of immune responses (as resident renal mononuclear phagocytes) in the normal kidney. Mast cells also reside in the tubulointerstitium and mediate pathogenic processes in crescentic and other forms of glomerulonephritis. However, the exact roles of dendritic cells, macrophages and mast cells in the normal kidney are yet to be elucidated.19–21 Lymphocytes, including both T cells and B cells, have been found in normal mouse kidneys even after extensive exsanguin-ation and perfusion.22 Intrarenal resident T cells show distinctly different phenotypes from T cells in spleen and blood; those from normal mouse kidneys contain an increased percentage of CD3+CD4–CD8– double-negative T cells. Intrarenal T cells also show a high proportion of activated, effector and memory phenotypes, whereas a small percentage of regulatory T cells and natural killer (NK) T cells exist in perfused and exsanguinated mouse kidney.22In this Review, we describe how immune cells partici-pate in the pathogenesis of AKI, focusing on ischaemic and nephrotoxic AKI. Immune system function in septic AKI is only outlined in this article, because the pathophysi-ology of septic AKI includes both immune responses to various foreign antigens and secondary systemic inflam-matory responses, which are distinctly d ifferent to the immune responses that occur in aseptic AKI.Aseptic ischaemic AKIRobust inflammatory responses mediated by the immune system start during the initial ischaemic insult and accel-erate upon reperfusion of the post-ischaemic kidney. However, post-ischaemic kidneys are not only targets of the immune system, but can also interact with systemic immune factors to recruit and activate immune cells. The mechanisms underlying activation and recruitment of immune cells in the post-ischaemic kidney involve proinflammatory damage-associated molecular pat-terns (DAMPs) in conjunction with hypoxia-inducible factors (HIFs) and adhesion molecules. These initiators of the inflammatory process cause permeability dysfunc-tion of the renal vascular endothelium and are associated with the release of proinflammatory chemokines and cytokines, and activation of TLRs (Figure 2). Various immune cells of both the innate and adaptive immune systems also have critical functions in the pathogenesis of renal injury following IRI (Tables 1 and 2).DAMPs, HIFs and adhesion moleculesDAMPs normally exist in the intracellular compart-ment and are concealed from the immune system by the plasma membrane.23,24 Proinflammatory DAMPs are released or exposed following hypoxic or anoxic cell injury, after which they can activate the innate immune system.25 Uric acid and nonmethylated CpG-rich DNA are DAMPs that contribute to the inflammation inducedFigure 1 | Experimental models of AKI. Models of AKI can be broadly categorized according to whether foreign antigens are involved (aseptic or septic AKI). Each category can be subdivided according to the method used to induce AKI. Ischaemic AKI is induced by ischaemia–reperfusion injury and by the type of ischaemia (warm or cold). Nephrotoxic AKI is induced by nephrotoxic agents, such as cisplatin. Abbreviation: AKI, acute kidney injury.REVIEWSby cell death.26,27 Among several DAMPs with intrinsic proinflammatory activity, IL-1α28 might have an impor-tant role in the recruitment of neutrophils in the post-ischaemic kidney. The induction of heat shock protein (HSP)27, one of the DAMPs in renal tubular cells, attenuated necrosis in vitro.27 However, in vivo, systemic up-regulation of HSP27 worsened renal injury by exacer-bating inflammation in post-ischaemic kidneys,29 which suggests that HSP27 recruits circulating immune cells. Overall intrarenal inflammatory processes, including the recruitment of immune cells, can also be triggered by the recognition of altered or injured cell structures and decreased expression of anti-inflammatory factors on injured cells.30Intra-renal activation of HIFs occurs in tubular, inter-stitial and endothelial cells following IRI. Upregulation of HIF-1α occurs within 1 h and is sustained up to 7 days, and induces the infiltration of macrophages fol-lowing IRI.31 Cobalt, an inhibitor of HIF-1α degrada-tion, showed renoprotective effects in post-ischaemic kidneys of rats, which was attributed to attenuation of macrophage infiltration.32 Preconditioning treatment resulting in activation of HIF improved both short-term and l ong-term renal outcomes after IRI in rats.33 Upregulation of adhesion molecules also substantially contributes to recruitment of immune cells into the post-ischaemic kidney. The expression of intercellular adhesion molecule-1 (ICAM-1, also known as CD54) is augmented within 1 h after IRI,32 and anti-ICAM-1 anti-bodies have renoprotective effects in normal mice, but not in neutrophil-depleted mice.34 Subsequent studies found that other adhesion molecules (P-selectin and E-selectin) affect the infiltration of immune cells and have important roles in the pathogenesis of renal IRI.35Renal vascular dysfunctionMechanical interruption of renal vascular endothelial integrity caused by IRI, and the consequent increase in vascular permeability, is another factor that facilitates infiltration of immune cells into the post-ischaemic kidney.36,37 Endothelial cell dysfunction is thought to con-tribute to the failure of blood to reperfuse an ischaemic area after removal of any physical obstruction (termed the ‘no-reflow’ phenomenon) in post-ischaemic kidneys. One study found that endothelial cell transfer attenu-ated renal injury in a rat model of renal IRI.36 Increased micro v ascular permeability after IRI was also attenuated in mice deficient in CD3+ T cells, suggesting that mol-ecules such as sphingosine-1-phosphate (S1P, a major regulator of both immune system and vascular function) and immune system components such as T cells are also mediators of increased vascular permeability after IRI.38Figure 2 | Major effector cells of both innate and adaptive immune systems contribute to the establishment of renal injury in ischaemic AKI. An immune response is initiated in post-ischaemic kidneys by resident immune cells and is potentiated by a rapid influx of immune cells through the disrupted endothelium. TLRs, adhesion molecules and DAMPs released from dying cells facilitate the recruitment and activation of various immune cells including neutrophils, macrophages, dendritic cells, NK cells, T cells and B cells during the early injury phase. Activation of the complement system and increased production of proinflammatory cytokines and chemokines are important promoters of leucocyte infiltration into the post-ischaemic kidney. Major effector cells of the innate immune system, such as macrophages, dendritic cells and NK cells are involved in the pathogenesis of renal injury after IRI. T cells, the major effector cells of the adaptive immune system, also substantially contribute to the development of renal injury from the early to late injury phase. Plasma cells seem to participate in the tubular damage process during the late injury phase. Abbreviations: AKI, acute kidney injury; DAMPs, damage-associated molecular patterns; HIF, hypoxia-inducible factor; IRI, ischaemia–reperfusion injury; NK, natural killer;TLR, Toll-like receptor; TREG cell, regulatory T cell. Modified with permission from Elsevier © Jang, H. R. & Rabb, H. Theinnate immune response in ischemic acute kidney injury. Clin. Immunol. 130, 41–50 (2009).Cytokines and chemokinesCytokines and chemokines are crucial mediators that regulate the infiltration of immune cells into post- ischaemic kidneys. Cytokine production is facilitated in the post-ischaemic kidney through interaction between cytokines and the transcriptional response induced directly by hypoxia. Intrarenal activation of transcrip-tion factors such as nuclear factor κB (NF-κB), heat shock factor protein 1 and HIF-1α occurs after IRI39,40 and stimulates the synthesis of a cascade of proinflam-matory cytokines, such as IL-1, IL-6 and tumour necro-sis factor (TNF).35,41,42 Splenectomy attenuated renal IRI by decreasing systemic production of inflammatory cytokines, including TNF, in rats.43 Chemokines are also direct mediators of chemotaxis and activation of immune cells: specifically, they guide neutrophils and proinflammatory (M1) macrophages to the injury site.44,45 Previous studies showed that IL-8 (also known as C-X-C motif chemokine ligand 8, or CXCL8) induced neutrophil recruitment into the post-ischaemic kidney.46,47 The aug-mented expression of CXC receptor 3 (CXCR3) follow-ing IRI orchestrates recruitment of T helper type 1 (TH1) cells into the post-ischaemic kidney because this receptor is predominantly expressed on activated TH1 cells.48 The infiltration and activation of macrophages following IRI are enhanced by C-C motif chemokine2 (also known as monocyte chemo a ttractant protein 1, or MCP-1) via C-C chemokine receptor type 2 (CCR2) signalling49 and C-X3-C motif chemokine receptor 1 (CX3CR1, also known as fractalkine receptor) signalling, which regulates the infiltration and phenotype change of macrophages, and affects renal interstitial fibrosis.50REVIEWSTLRsTLR expression on renal tubular epithelial cells is an important contributor to the recruitment and activa-tion of immune cells, especially effector cells of the innate immune system. TLR2 and TLR4 are expressed on normal renal tubular epithelial cells and their expres-sion further increases after IRI.8,9,51 DAMPs such as histones or high-mobility-group protein B1 released from necrotic tubules activate TLRs on dendritic cells or macrophages and inflammasomes in the cytosol to trigger the secretion of proinflammatory cytokines and chemokines in the post-ischaemic kidney.51–55NeutrophilsNeutrophils are important effector cells of the innate immune system that phagocytose pathogens and par-ticles, generate reactive oxygen and nitrogen species, and release antimicrobial peptides. Neutrophil infil-tration has been detected in post-ischaemic mouse kidneys 56,57 and in biopsy samples from patients with early AKI.58,59 Neutrophils were, therefore, expected to have an i mportant role in the pathogenesis of renal injury f ollowing IRI.IL-17 produced by neutrophils regulates IFN-γ-mediated neutrophil migration into the post-ischaemic kidney,60 and warm ischaemia promotes neutrophil trafficking into the post-ischaemic kidney in mice.61 However, the precise role and kinetics of neutrophil trafficking into the post-ischaemic kidney after IRI remain controversial, despite many studies focusing on the role of neutrophils in renal IRI. In one study, renal injury was attenuated by inhibition of neutrophil infiltration or activity in rats,34 whereas others failed to find a protective effect of neutrophil blockade or deple-tion.62,63 Many factors that affect neutrophil infiltration or activation, including neutrophil elastase, tissue type plasminogen activator, hepatocyte growth factor and CD44 expression contribute to renal injury following IRI.64–67 Treatments that target several adhesion mol-ecules involved in migration of neutrophils (as well as other leucocytes), such as selectins, ICAM-1, and CD11a–CD18 (integrin αL β2, also known as lympho-cyte function-associated antigen-1, LFA-1), exert partial protection in the post-ischaemic kidney in rodents.34,57,63 A phase I clinical trial of ICAM-1-blocking antibodies showed a reduced rate of delayed graft function fol-lowing kidney transplantation in the treated group.68 However, a randomized controlled trial of anti-ICAM-1 monoclonal antibody in recipients of cadaveric renal transplants failed to show a reduction in the rate of delayed graft function or acute rejection.69 Blockade of platelet-activating factor (PAF), which facilitates neutrophil adherence to the endothelium, also had a protective effect in a rat model of cold IRI.70Despite conflicting results reported thus far, neutro-phils are likely to participate in the induction of renal injury, by obstructing the renal microvasculature and secreting oxygen free radicals and proteases. It is likely that neutrophils have a much less important role in renal IRI than they do in cardiac or skeletal muscle IRI.59,71,72MacrophagesMacrophages were expected to have an important function in immune-mediated renal injury because these cells function as both effector cells and antigen- presenting cells, thereby connecting the innate and adaptive immune systems. Activated macrophages exert potent phagocytic activity and release several impor-tant cytokines, such as IL-1, IL-6, IL-8, IL-12 and TNF. Although the resident macrophages in normal kidneys are few, their number markedly increases in post- ischaemic kidneys (especially in the outer medulla), soon after IRI.73 Monocytes adhere to the vasa recta 2 h after reperfusion, and most macrophage recruitment occurs around post-capillary venules in the outer medulla.74 IRI facilitated endothelial damage and modifications of heparin sulphate proteoglycans in the microvascular basement membrane, which promoted their binding to L-selectin, as well as induction of MCP-1. These changes induced the early influx of monocytes and macrophages into the post-ischaemic kidney.75Macrophage influx upon reperfusion of the post-ischaemic kidney seems to facilitate the inflammatory cascade through secretion of cytokines, recruitment of neutrophils and induction of apoptosis, which contribute to the establishment of renal injury. Systemic depletion of monocytes and macrophages using liposomal clodronate attenuated early renal injury in a mouse model of renal IRI.76 Although IL-18 was suggested as a key mediator ofmacrophage influx in the pathogenesis of IRI, a study of liposomal clodronate treatment in wild-type and caspase- 1-knockout mice revealed that macrophages are not the source of the injurious IL-18 in ischaemic AKI.77 Although macrophages do have a role in injury occurring in the early phase of IRI,76,78 the augmented production of haem oxygenase 1 by infiltrated macrophages has been associated with the protective effects of statins in AKI.79Macrophages are also suspected to have a role in renal repair following IRI (Figure 3). In one study, post- ischaemic kidneys of mice with knockout of osteopontin (a macrophage chemoattractant) had fewer infiltrating macrophages and less fibrosis than did post-ischaemic kidneys of wild-type mice.80 A few reports show that macrophages influence the development of renal fibro-sis during the recovery phase of IRI, which supports the concept that macrophages have an adverse effect on the repair of post-ischaemic kidneys.81,82 However, macrophage-specific deletion of transforming growth factor (TGF)-β1 did not halt the process of renal fibrosis following severe IRI.83 Colony-stimulating factor-1 pro-motes renal repair and attenuates interstitial fibrosis by inducing the expression of insulin-like growth factor-1 and anti-inflammatory genes in macrophages.84 One well-designed study showed that macrophages promote the renal repair process by switching from a proinflamma-tory M1 phenotype characterized by expression of indu-cible nitric oxide synthase, to an anti-inflammatory (M2)Figure 3 | Immune modulation during the repair phase of ischaemic AKI is a key factor in determining the outcome of AKI. T REG cells, B cells and macrophages have substantial roles in determining whether repair results in tubular regeneration or atrophy and interstitial fibrosis. T REG cells and M2 macrophages have important roles in tubular regeneration, whereas B cells enhance tubular atrophy and suppress tubular regeneration. Humoral factors, such as proinflammatory or anti-inflammatory cytokines and chemokines, also change the intrarenal microenvironment and affect phenotype switching of macrophages. The exact mechanisms by which these immune processes regulate tubular atrophy or regeneration are not yet known. Abbreviations: AKI, acute kidney injury; IL-10, interleukin-10; TGF-β, transforming growth factor β; T REG cells, regulatory T cells. Modified with permission from Elsevier © Jang, H. R. & Rabb, H. The innate immune response in ischemic acute kidney injury. Clin. Immunol . 130, 41–50 (2009).REVIEWSphenotype characterized by expression of arginase-1 and the mannose receptor.85 This report suggests that macrophages have a complex role in both IRI-induced i nflammation and the subsequent repair process. Switching of macrophages to an anti-inflammatory M2 phenotype seems to be induced by changes in the intrarenal microenvironment as well as by the phago-cytic uptake of apoptotic neutrophils by macrophages during the injury phase in post-ischaemic kidneys.45,86 In mouse models of renal IRI and selective proximal tubule injury induced by diphtheria toxin, the increased number of M2 phenotype macrophages resulted mainly from in situ proliferation of resident renal macrophages. Furthermore, genetic or pharmacological inhibition of macrophage colony-stimulating factor 1 (CSF-1) signal-ling blocked intrarenal proliferation of macrophages and dendritic cells, reduced M2 polarization, and inhibited renal recovery.87 Treatment of macrophages with netrin 1 suppressed the inflammatory response by inducing conversion to an M2 phenotype, which protected the kidney against subsequent IRI.88 IL-1 receptor-associated kinases (IRAKs) are involved in the IL-1 receptor–TLR–Myd88-dependent activation of NF-κB and are impor-tant regulators of macrophage phenotype polarization.89 IRAK-M selectively inhibits IRAK-4–mediated phos-phorylation of TNF receptor–associated factor 6, which is an essential step in this signalling pathway in mono-cytes and macrophages.90 IRAK-M induction during the recovery phase after renal IRI facilitates renal recovery by suppressing M1-macrophage-dependent renal inflam-mation, whereas IRAK-M inhibition (achieved by loss-of-function mutations or transient exposure to bacterial DNA) halted the repair process and induced persistent macrophage-related renal inflammation.91Renal dendritic cellsThe basic function of dendritic cells is the presentation of antigens to T cells; thus, they act as messengers between the innate and adaptive immune systems. The results of several studies show that dendritic cells participate in ischaemic AKI. In a rat model of transplant-induced IRI, recipient leucocytes that expressed MHC class II antigens were trafficked into the transplanted kidney despite no signs of acute rejection, and some of them were identified as dendritic cells.92 The number of renal dendritic cells and their expression of MHC class II antigens increased after IRI.9 A subsequent study revealed that the population of resident dendritic cells predominantly consists of TNF-secreting cells in the early phase of AKI following IRI.93 Furthermore, binding of dendritic cells to the endo t helium and their migration seem to be facilitated during the initial inflammatory response following IRI.94 Trafficking of immature myeloid dendritic cells into the transplanted kidneys is also increased following IRI, resulting in an increased ratio of myeloid to plasmacytoid dendritic cells that might predispose to delayed graft function and acute rejection.95 In a study of syngeneic kidney transplantation from wild-type rats to transgenic rats expressing green fluorescent protein, cold IRI was associated with loss of graft-specific dendritic cells and progressive recruitment of host dendritic cells and T cells.96 Contrary to previous studies, this report suggested that renal resident dendritic cells might have protective regulatory functions in the post-ischaemic kidney.LymphocytesLymphocytes are key cells of the adaptive immune system. Lymphocytes were not expected to contribute to post-ischaemic AKI, given the traditional concept that lymphocytes respond to alloantigens or self-antigens in a delayed fashion. However, many studies performed during the past decade have revealed the substantial role of a diverse subset of lymphocytes in post-ischaemic and nephrotoxic AKI.Natural killer cellsNK cells are a class of large, granular, cytotoxic lym-phocytes that lack T-cell and B-cell receptors. They kill infected cells directly and produce a variety of cytokines, including IFN-γ and TNF. NK cells were expected to have a role in inducing renal injury following IRI because in other organs, they secrete cytokines that facilitate the inflammatory process and activate macrophages and neutrophils.97,98 So far, few reports exist on the role of NK cells in AKI. NK cells were reported to contribute directly to renal injury following IRI by killing tubular epithelial cells, and in the same report, depletion of NK cells attenuated renal injury after IRI both functionally and structurally.99 A subsequent study by the same team reported that osteopontin expressed on renal tubular epithelial cells can directly activate NK cells to mediate apoptosis of tubular epithelial cells, and can also regulate chemotaxis of NK cells to the tubular epithelium.100CD4+ and CD8+ T cellsSeveral research teams have reported that T cells, particu-larly CD4+ T cells, contribute both directly and indirectly to the establishment of renal injury in the early phase of IRI.74,101–105 T-cell-targeted medications such as tacroli-mus and mycophenolate mofetil substantially attenuated early renal injury following IRI.106,107 Blockade of the T-cell CD28–B7 co-stimulatory pathway with CTLA-4–Ig (a recombinant fusion protein containing CTLA-4, a structural homologue of CD28, fused to an IgG1heavy chain), also substantially reduced early renal injury after cold IRI.108 Furthermore, CTLA-4–Ig treatment on the day of cold IRI and during the first week after cold IRI decreased proteinuria in uninephrectomized rats (a model of chronic, progressive proteinuria).109 Direct evidence of the pathophysiological role of T cells in ischaemic AKI was demonstrated in a mouse model of warm IRI.101 In this study, CD4,CD8 double-knockout mice were largely protected from early renal injury, and their T cells showed a twofold increase in adherence to renal tubular epithelial cells in vitro after hypoxia and reoxygenation. Another T-cell-knockout mouse strain, athymic Foxn1nu/nu mice, was also protected from IRI. Adoptive transfer of T cells into these mice restored renal injury following IRI, demonstrating that T-cell deficiency conferred renal protection from IRI.102 CD4-knockoutREVIEWS。

肿瘤相关巨噬细胞

肿瘤相关巨噬细胞研究进展免疫细胞浸润是肿瘤恶化的标志之一，在所有免疫细胞中，肿瘤相关巨噬细胞是免疫系统和肿瘤的相互作用过程中最关键的调控中心。

肿瘤相关巨噬细胞是实体肿瘤中大量存在的一种白细胞1，在大多数人类肿瘤中，肿瘤相关巨噬细胞的浸润和肿瘤相关巨噬细胞相关基因的上调表达严重影响肿瘤的预后和治疗效果2, 3。

本文将对肿瘤相关巨噬细胞的促肿瘤功能、肿瘤相关巨噬细胞功能的调控以及针对肿瘤相关巨噬细胞的肿瘤免疫治疗进展加以综述。

1. 肿瘤相关巨噬细胞的促肿瘤功能：肿瘤相关巨噬细胞可以通过免疫和非免疫过程促进肿瘤的生长，它们可以大量分泌促血管新生因子，如血管内皮生长因子VEGF，后者促进肿瘤的血管生成4和血源性细胞转移过程5。

此外，肿瘤相关巨噬细胞可以通过抑制抗肿瘤免疫反应达到促肿瘤的效果。

如肿瘤相关巨噬细胞产生免疫抑制因子IL-10、TGFb、PGE2等，其中IL-10通过抑制化疗引起的抗肿瘤免疫，显著抑制了该抗肿瘤治疗的效果6。

2. 肿瘤相关巨噬细胞功能的调控：大多数情况下，肿瘤相关巨噬细胞主要来自于血液中的单核细胞，通过CCL2趋化因子被招募到肿瘤部位7。

最近的报道证明，肿瘤巨噬细胞的主要来源是CCR2+单核细胞，后者占据了肿瘤内40%的CD45+细胞8。

在到达肿瘤病灶的过程中，单核细胞发生一系列变化，而到达不同组织的肿瘤后，形成的肿瘤相关巨噬细胞类群在表型和功能上也各不相同9，调控这一过程的信号主要来自于肿瘤和组织两部分。

其中，组织中免疫系统来源的促炎因子至关重要，后者可以通过驯化肿瘤相关巨噬细胞的促肿瘤功能，调控肿瘤的发展。

例如肿瘤相关巨噬细胞产生的IFNr可以上调免疫抑制酶NOS2和IDO的表达，达到免疫抑制的效果，进而促进肿瘤发生10, 11。

此外，肾细胞癌病人体内的促炎因子IL-1b也能赋予单核细胞促肿瘤的功能，达到促肿瘤的效果12。

最后，肿瘤细胞代谢产生的信号也能直接调控肿瘤相关巨噬细胞的功能，肿瘤微环境中存在大量乳酸，这些乳酸诱上调了肿瘤相关巨噬细胞细胞中促肿瘤基因Vegf和Arg1的表达13。

树突状细胞与免疫耐受

树突状细胞与免疫耐受【关键词】骨髓Steinman和Cohn于1973年从小鼠外周淋巴组织中分离出一种新的细胞，同其他单核白细胞一样，这种细胞拥有丰硕的线粒体、各类内涵体，核膜外有异染色质。

但其细胞表面具有树突样或伪足样突起，因此而得名为树突状细胞（dendritic cell，DC）［1］。

在初期的实验中，人们发现脾脏来源的DC刺激同种异体混合淋巴细胞反映（mixed lymphocyte reaction，MLR）的能力明显强于淋巴细胞和巨噬细胞［2］。

其后的研究表明成熟的DC能够聚集在T淋巴细胞周围，激发自体混合淋巴细胞反映，但其强度弱于前一种MLR。

这一特性可以将DC与其他脾脏细胞群区分开［3］。

随着这些研究的慢慢深切，人们逐渐熟悉到在取得性免疫中，DC是最重要，功能最壮大的抗原递呈细胞（antigen-presenting cell，APC）。

其独特的功能在于DC是唯一能活化naiveT淋巴细胞的APC［4］。

1 DC的来源、散布及分类初期的研究表明DC是骨髓来源［5］。

其发育中的DC前体自骨髓向血中迁移［6］。

DC可分为髓样DC和淋巴样DC。

淋巴样DC主要散布于淋巴结、脾脏、黏膜相关组织中的淋巴滤泡生发中心，主要与B淋巴细胞功能有关；髓样细胞主要散布于T细胞富含区，与TC细胞功能有关。

按其散布部位可分为：（1）Langhams细胞，主要散布在皮肤黏膜。

（2）间质性DC，主要散布于心、肺、肝、肾、甲状腺、胃肠道、膀胱等处。

（3）树突状DC，主要散布于脾脏、淋巴结和胸腺等淋巴器官的T细胞富含区。

（4）外周血DC和淋巴DC，主要散布于外周血和输入淋巴管。

以前人们将这4种DC视为独立的细胞群体，最近几年研究发现它们不过是一类细胞处于不同分化经受阶段或不同部位算了［7］。

DC还可以按其表达的细胞表面标志物细分为不同的种群。

在鼠淋巴组织中DC的最明显标志为CD11c［8］。

但是由于它们所散布的脏器或所处的发育阶段不同，它们能够表达淋巴样标志CD4和CD8α，或髓源性标志CD11b和F4/80，或在DC群表达相对较少的DEC205和33D1［6，8，9］。

巨噬细胞极化英文

巨噬细胞极化英文Macrophage PolarizationMacrophage polarization is the biological process by which macrophages, a type of white blood cells, specialize into two major subsets: M1 and M2. Depending on the signalsit receives, a single cell can switch between either subset, allowing them to adapt to different situations and contribute to a variety of physiological functions. In the context of innate immunity, M1 macrophages (also known as classical macrophages) are specialized for pro-inflammatory activity and defending against bacterial and viral infections, while M2 macrophages (also known as alternative macrophages) are more involved in wound healing, tissue remodeling and other forms of homeostatic repair.Studies have revealed that the polarization of macrophages is regulated by a variety of environmental signals. This includes hormones and growth factors secreted by other cells, as well as extracellular matrices, microbial products, and other chemical or physical signals. The type of signal received will in turn induce the expression of various genes, resulting in distinct morphological, metabolic and functional characteristics that distinguish one subset from the other.M1 macrophages are generally considered to be pro-inflammatory and are characterized by high levels of nitric oxide production, phagocytosis, expression of cytokines like interleukin-1 (IL-1) and tumor necrosis factor (TNF), and the secretion of other inflammatory mediators. Their primary duty is to clear invading pathogens and initiate an immuneresponse. On the other hand, M2 macrophages are considered anti-inflammatory and are responsible for dampening down this response when it is no longer needed. They have increased expression of scavenger receptors and exhibit enhanced production of anti-inflammatory cytokines like interleukin-10 (IL-10).In addition to their role in innate immunity, macrophage polarization is known to influence a number of other processes such as cancer, cardiovascular disease, and tissue regeneration. By selectively activating distinct subsets of macrophages, it is possible to modulate their activity and promote beneficial outcomes within the body. As such, macrophage polarization has become an important area of research for its potential therapeutic applications.。

MDSCs与肿瘤免疫逃逸

中国肿瘤生物治疗杂志　htt p://www .bi other .orgChin J Cancer B i other,Aug .2009,Vol .16,No .4DO I:10.3872/j .iss n.10072385X .2009.04.001・述评・MD SCs 与肿瘤免疫逃逸刘秋燕,曹雪涛(第二军医大学免疫学研究所,医学免疫学国家重点实验室,上海　200433) 刘秋燕,博士,副教授,中国免疫学会和中国抗癌协会会员。

1988年毕业于河北医科大学预防医学系(本科),2001年获免疫学硕士学位,2004年获免疫学博士学位,2006年完成浙江大学免疫学博士后研究。

2007年1月通过人才引进至第二军医大学免疫学研究所工作,任肿瘤免疫实验室负责人。

主要从事肿瘤免疫逃逸及其相关机制的研究,以第一作者在国内外刊物发表论文20多篇,其中SC I 期刊论文10篇,包括J I mm unol 2篇、J M ol M ed 1篇、M ol I mm unol 2篇、BB RC 3篇等。

作为课题负责人承担了多项国家级研究课题,并参与多项国家级、省部级科研项目,参加编写教材3部。

E 2mail:lqy1969@yahoo .com.cn 曹雪涛,博士,教授,博士生导师,中国工程院院士。

现任第二军医大学副校长和免疫学研究所所长,医学免疫学国家重点实验室主任,中国免疫学会理事长,国家重点基础研究发展计划(973)项目首席科学家,兼任《中国肿瘤生物治疗杂志》主编以及Journa l of I mm u 2nology,Jou rnal of B iological Che m istry 、European Journal of I mm unology 、Gene Therapy 、Cancer I mm unology I mm unotherapy 、Cancer S cience 等SC I 收录杂志编委。

急性淋巴细胞白血病治疗现状

急性淋巴细胞白血病治疗现状（作者:___________单位: ___________邮编: ___________）【摘要】急性淋巴细胞白血病（ALL）是一组异质性疾病，采用现代治疗策略，成人ALL初治完全缓解率已显著提高，但预后仍不理想。

ALL患者可能的高危因素的确定，有助于预测治疗结局并指导整体治疗策略的制订。

联合化疗结合造血干细胞移植，单克隆抗体及分子靶向治疗有望进一步改善ALL疗效。

【关键词】急性淋巴细胞白血病；预后因素；治疗；成人急性淋巴细胞白血病（ acute lymphoblastic leukemia, ALL）是一组生物学及预后不同的异质性疾病。

近20年来随诊断技术（特别是分子生物学技术）的提高，临床上根据细胞表型和遗传学特征做出诊断,识别预后不同的亚型；通过对临床预后因素判断认识的提高和化疗方案的改进，使ALL治疗效果得以稳步改善；根据其危险度调整治疗策略,使治疗更加个体化和高效低毒。

ALL治疗进展主要体现在临床预后因素判断认识的提高和化疗方案的改进。

1 预示临床预后的因素早年主要根据回顾性研究资料，观察生物学指标和临床特征来预测临床预后。

近20年，随分子生物学研究发展和治疗方法改进，部分评估预后的指标已取消，如男性患者预后不良的观点已被推翻。

目前对预示临床预后的因素有了更全面的了解，特别是从临床特征、细胞遗传学、免疫表型和基因表达方面指标的研究。

1.1 临床特征早在20世纪80年代就观察发现成人年龄愈大、白血病细胞计数愈高，则预后越差。

近几年研究发现，老年ALL患者其白血病细胞具有不同于年轻患者的一些生物学特征,如：CD34和经典多药耐药蛋白P糖蛋白（P-gp）常高表达。

最重要的是细胞遗传学特征不同,t（9;22）异常患者比例高达35%～50%,其白血病细胞对化疗药物敏感性减低,易出现耐药。

60岁以上患者诱导化疗完全缓解（CR）率仅35%～55%,中位生存期3～14个月。

淋巴细胞提取

淋巴细胞提取一实验目的：小鼠脾淋巴细胞提取二实验对象：B／C 小鼠三实验器材：1．试剂：淋巴细胞分离液、1640培养基2．器材：无菌培养皿、200目尼龙网（裁成90mm*90mm正方形，灭菌）、10mL玻璃注射器内活塞（灭菌）、不同规格的镊子、剪刀若干（灭菌）、细胞实验常用器材（离心管、移液管、加样枪、离心机）、75%乙醇、烧杯（无菌）、大头针、超净台四实验步骤：1、断头处死小鼠，浸入75%的乙醇中浸泡1-2分钟。

2、在超净台中小心剪开小鼠腹部外皮，用大头针固定，再剪开小鼠腹腔，用镊子摘下小鼠脾脏。

注意无菌操作。

3、参考图一，在35mm培养皿中放入4-5ml淋巴细胞分离液（使用前摇匀淋巴细胞分离液）。

用镊子固定尼龙网，然后用注射器活塞轻轻研磨小鼠脾脏，使得分散的单细胞透过尼龙网进入淋巴细胞分离液中。

（没研磨每一只脾脏话费的时间最好控制在5分钟之内，防止在研磨过程中液体挥发，使得密度与渗透压改变，影响分离效果）4、把悬有脾脏细胞的分离液立即转移到离心管中，离心前再覆盖上大约200μL的1640培养基。

5、800g离心30分钟，注意离心设置较慢的加速度和减速度（如果有十档，一般设置在第三档）。

离心结束后淋巴细胞会漂浮上来，在1640覆盖层下面聚集，细胞分层如图二所示6、析出淋巴细胞层，再加入10mL1640培养基，250g离心10分钟。

倾倒上清液，加入3-5mL Lympho-SpotTM无血清培养基重悬，细胞计数。

五、注意事项：①离心前在细胞悬液上面加盖一层1640培养基，既有利于漂浮上来的淋巴细胞的聚集，又有利于下一步的吸取操作。

覆盖层不必太厚，2Μl足矣。

②如果实验者一次实验要处理很多只小鼠，需要注意两点：其一，每研磨一只小鼠，立即把脾细胞悬液从培养皿转入离心管中，注意盖严管盖，切不可敞口放在超净台中。

否则，液体挥发，密度与渗透压都会改变，严重影响分离效果。

其二，在所有的小鼠脾脏处理完后，统一再加1640覆盖层。

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Distinct Subsets of Dendritic Cells Regulate the Pattern of Acute Xenograft Rejection and Susceptibility toCyclosporine Therapy1Hao Wang,*†¶Jacqueline Arp,‡Xuyan Huang,‡Weihua Liu,§Siobhan Ramcharran,¶Jifu Jiang,‡Bertha Garcia,§Nobuyuki Kanai,‡Weiping Min,†Peta J.O’Connell,†‡¶and Robert Zhong2*†‡§¶We determined whether distinct subclasses of dendritic cells(DC)could polarize cytokine production and regulate the pattern of xenograft rejection.C57BL/6recipients,transplanted with Lewis rat hearts,exhibited a predominantly CD11c؉CD8␣؉splenic DC population and an intragraft cytokine proﬁle characteristic of a Th1-dominant response.In contrast,BALB/c recipients of Lewis rat heart xenografts displayed a predominantly CD11c؉CD8␣؊splenic DC population and IL-4intragraft expression characteristic of a Th2response.In addition,the CD11c؉IL-12؉splenic DC population in C57BL/6recipients was signiﬁcantly higher than that in BALB/c recipients.Adoptive transfer of syngeneic CD8␣؊bone marrow-derived DC shifted a Th1-dominant, slow cell-mediated rejection to a Th2-dominant,aggressive acute vascular rejection(AVR)in C57BL/6mice.This was associated with a cytokine shift from Th1to Th2in these mice.In contrast,transfer of CD8␣؉bone marrow-derived DC shifted AVR to cell-mediated rejection in BALB/c mice and signiﬁcantly prolonged graft survival time from6.0؎0.6days to14.2؎0.8days. CD8␣؉DC transfer rendered BALB/c mice susceptible to cyclosporine therapy,thereby facilitating long-term graft survival. Furthermore,CD8␣؉DC transfer in IL-12-deﬁcient mice reconstituted IL-12expression,induced Th1response,and attenuated AVR.Our data suggest that the pattern of acute xenogeneic rejection can be regulated by distinct DC subsets.The Journal of Immunology,2006,176:3525–3535.I t has been known that the recipient genetic background playsan import role to transplant Ags.We previously reported that C57BL/6(Th1-predisposition)and BALB/c(Th2-predispo-sition)mice had very different immune responses to Lewis rat heart xenografts(1).BALB/c mice rapidly rejected Lewis heart xenografts with aggressive acute vascular rejection(AVR),3while C57BL/6mice rejected the same xenografts with less aggressive cell-mediated rejection(CMR)(1).Furthermore,we found that cytokines play a key role in determining the pattern of xenograft rejection.Unlike allografts,Th1cytokines such as IFN-␥and IL-12are beneﬁcial to xenograft survival because of their negative effect on AVR.In recentﬁndings,we demonstrated that distinct cytokine proﬁles expressed by different mouse strains played an essential role in regulating the pattern of rejection and outcome of cyclosporin A(CsA)/rapamycin therapy in allotransplantation(2). These data suggest that the cytokine proﬁle appears to be a key factor in determining the pattern of rejection and susceptibility to immunosuppressive agents.The factors that regulate the polariza-tion of cytokine proﬁles in these two murine strains following xenotransplantation are not clear.In this study,we used the same xenotransplantation model to extend our previous observations(1) and to determine the factors that direct distinct immune responses to a xenoantigen.Dendritic cells(DC)are bone marrow(BM)-derived profes-sional APC with the unique ability to both initiate and regulate immune responses(3).DC provide a critical link between the in-nate and adaptive immune responses.In the human,type1IFN-producing cells,also known as plasmacytoid DC differentiate into a unique type of mature DC,which directly regulate the function of T cells and thus link innate and adaptive immune responses when they are exposed to an Ag(4).In mice,DC can be broadly subdivided into at least three subsets in most lymphoid and non-lymphoid tissues:CD8␣ϩDC(5,6),CD8␣ϪDC(5,6),and B220 DC(7–9).Although CD8␣ϩDC and CD8␣ϪDC are reported to be efﬁcient stimulators of T cell activation and proliferation,there is little evidence that B220DC are important for the initiation of adaptive immunity.B220DC are,however,the primary source of type1IFNs following exposure to viral Ags(7–10).DC provide T cells with Ag-speciﬁc information that reﬂects the nature of the pathogen or irritant and affected tissue(signal1).In addition,DC provide costimulatory signals(signal2)that are nec-essary for efﬁcient T cell priming.More recently,DC have been reported to provide a third signal that polarizes the Th response (11).CD8␣ϩDC have been shown to be the primary source of IL-12,sometimes referred to as the third signal necessary for T cell priming and a key determinant of type1T cell differentiation. Moreover,Ag-pulsed CD8␣ϩDC have been shown to sensitize naive T lymphocytes to induce a dominant Th1response.In contrast,*Multi-Organ Transplant Program,London Health Sciences Centre-University Hos-pital,London,Ontario,Canada;†Department of Surgery,University of Western On-tario,London,Ontario,Canada;‡Transplantation Group,Robarts Research Institute,London,Ontario,Canada;and§Department of Pathology and¶Department of Microbi-ology and Immunology,University of Western Ontario,London,Ontario,CanadaReceived for publication May20,2005.Accepted for publication January5,2006.The costs of publication of this article were defrayed in part by the payment of pagecharges.This article must therefore be hereby marked advertisement in accordancewith18U.S.C.Section1734solely to indicate this fact.1This study was supported by Canadian Institutes of Health Research and the Multi-Organ Transplant Program,London Health Sciences Centre.2Address correspondence and reprint requests to Dr.Robert Zhong,Department ofSurgery,London Health Sciences Centre-University Hospital,339Windermere Road,P.O.Box5339,London,Ontario,Canada N6A5A5.E-mail address:zzhong@uwo.ca3Abbreviations used in this paper:AVR,acute vascular rejection;BM,bone marrow;CMR,cell-mediated rejection;CsA,cyclosporin A;DC,dendritic cell;KO,knockout;POD,postoperative day;xAb,xenoreactive Ab.The Journal of Immunology Copyright©2006by The American Association of Immunologists,Inc.0022-1767/06/$02.00immunization with CD8␣ϪDC that typically produce lower levels of IL-12induce a predominant Th2response(12,13).In addition,se-lective expansion of either the CD8␣ϩor CD8␣ϪDC subset in vivo results in increased Ag-speciﬁc Ab titers with distinct isotype proﬁles (14).These data suggest that DC subsets initiate T cell responses and direct the type of adaptive immunity that ensues as a result of Th-polarizing cytokines.The nature of the Ag-presented mouse strain, maturation of DC,tissue of DC origin,and cytokines(12,15)are all signiﬁcant factors that impact the differentiation and polarization of Th cells.Because of the unique features of CD8␣ϩand CD8␣ϪDC in regulating T cell responses,we focused on evaluating these two subsets of DC in a mouse xenotransplantation model for this study. Until now,the role of DC subsets in the manipulation of im-mune responses following xenotransplantation has not been inves-tigated.As a result of the ubiquitous role of DC in controlling immunity,we hypothesized that DC subsets regulate the pattern of acute xenograft rejection,in part,through polarizing T cell cyto-kine proﬁles.In this study,we compared DC subsets,cytokine and Ig proﬁles,and graft rejection patterns in two well-deﬁned mouse strains,BALB/c and C57BL/6,that had received Lewis rat cardiac xenografts.Adoptive transfer of syngeneic CD8␣ϩor CD8␣ϪDC to BALB/c or C57BL/6mice was performed to determine whether transfer of a speciﬁc DC subset would change the pattern of xeno-graft rejection.We showed that transfer of CD8␣ϩBM-DC atten-uated AVR and prolonged graft survival time in BALB/c mice. Furthermore,adoptive transfer of CD8␣ϩDC was found to act synergistically with CsA and to protect xenografts once rejection was shifted from AVR to CMR.Materials and MethodsAnimals and immunosuppressive drugTwo-week-old Lewis rats(RT11)(25–30g)were used as heart donors. Male adult BALB/c(H-2d)mice,wild-type C57BL/6(H-2b)mice,and IL-12p40knockout(KO)C57BL/6mice(C57BL/6-IL-12btm1jm)(16), weighing25–30g,were used as recipients(The Jackson Laboratory). There were eight mice in each group.In the groups receiving immunosup-pression,the recipients were given daily s.c.injections of CsA(15mg/kg) from day0until the study end point or graft rejection.Animals were housed under conventional conditions at the Animal Care Facility of the University of Western Ontario and were cared for in accordance with the guidelines established by the Canadian Council on Animal Care(17). Heterotopic cardiac transplantationIntra-abdominal heterotopic cardiac transplantation was performed as pre-viously described by Corry et al.(18).Brieﬂy,a median sternotomy was performed on the donor,and the heart graft was slowly perfused in situ with1.0ml of cold heparinized Ringer’s lactate solution through the in-ferior vena cava and aorta before the superior vena cava and pulmonary veins were ligated and divided.The ascending aorta and pulmonary artery were transected and the graft was removed from the donor.The graft was then revascularized with end-to-side anastomoses between the donor’s pul-monary artery and the recipient’s inferior vena cava as well as the donor’s aorta and the recipient’s abdominal aorta using11-0nylon suture.The heartbeat of the graft was monitored daily by direct abdominal palpation. The degree of pulsation was scored as:A,beating strongly;B,noticeable decline in the intensity of pulsation;or C,complete cessation of pulsation. When the heartbeat was no longer palpable,the graft was removed for routine histology,immunohistochemistry,and RT-PCR.Serum samples were collected,and circulating anti-donor Abs were evaluated byﬂow cytometry.Graft histologyTissue samples were removed at necropsy andﬁxed in10%buffered form-aldehyde.Specimens were then embedded in parafﬁn,sectioned,and stained with either H&E or martius scarlet blue.Microscopic tissue sec-tions were examined,in a blinded fashion for severity of rejection by a pathologist(B.G).Criteria for graft rejection included the presence of vas-culitis,thrombosis,hemorrhage,and lymphocyte inﬁltration.These changes were scored as:0,no change;1,minimal change;2,mild change; 3,moderate change;or4,marked change.ImmunohistochemistryFour-micrometer sections were cut from tissue samples embedded in Tis-sue-Tek OCT gel(Skura Finetek,Torrance,CA),mounted on gelatin-coated glass microscope slides,and stained by a standard indirect avidin-biotin immunoperoxidase staining method using an Elite Vectastain ABC kit(Vector Laboratories).Specimens were stained for CD4ϩand CD8ϩcells with biotinylated rat anti-mouse CD4mAb(clone YTS191.1.2; Cedarlane Laboratories)or anti-mouse CD8mAb(clone53-6;BD Bio-sciences),respectively.Mouse IgG and IgM deposition in grafts was detected using biotin-conjugated goat anti-mouse-IgG and goat anti-mouse-IgM(Cedarlane laboratories).Intragraft IgG1and IgG2a deposition was detected using a biotin-conjugated rat anti-mouse IgG1mAb(clone A85-1;BD Biosciences)and rat anti-mouse IgG2a mAb(clone R19-15; BD Biosciences).Slides were washed with PBS between steps,and examined using light microscopy.Negative controls were performed by omitting the primary Abs.The sections of immunoperoxidase staining for intragraft IgG, IgG1,IgG2a and IgM deposition were graded from0to4ϩaccording to the staining intensity:0,negative;1ϩ,equivocal;2ϩ,weak staining;3ϩ,mod-erate staining;and4ϩ,very intensive staining.The sections of immunoper-oxidase staining for intragraft inﬁltration of CD4ϩand CD8ϩT cells were analyzed by counting the number of all positively stained cells in the whole section and dividing by the section area examined.Generation and puriﬁcation of BM-derived DCGeneration of DC from BM was performed as originally described by Inaba et al.(19)with modiﬁcations(20).Brieﬂy,BM cells wereﬂushed from the femurs and tibias of naive wild-type BALB/c and C57BL/6mice, washed,and cultured at a concentration of2ϫ106cells/well in24-well plates(Corning)in2ml of RPMI1640(Invitrogen Life Technologies) supplemented with10%FCS(Invitrogen Life Technologies),100U/ml penicillin,100␮g/ml streptomycin,50␮M2-ME(Invitrogen Life Tech-nologies),10ng/ml recombinant murine GM-CSF(PeproTech),and10 ng/ml IL-4(PeproTech).Nonadherent cells were removed after48h of culture,and fresh medium was added every48h.After6days of culture, the nonadherent cells(containing at least90%DC,as assessed by mor-phology and speciﬁc staining,with anti-CD11c mAb N418)were labeled with anti-mouse CD8␣mAb-conjugated superparamagnetic microBeads (Miltenyi Biotec),and CD11cϩCD8␣ϩand CD11cϩCD8␣Ϫcells were isolated and puriﬁed by passage through a magnetic column(21).Mouse BM has few CD8␣ϩDC when cultured as described.Thus,to obtain suf-ﬁcient puriﬁed CD8␣ϩBM-DC for adoptive transfer,eight naive BALB/c mice were required to culture6ϫ106CD8␣ϩDC and126ϫ106CD8␣ϪDC.Similarly,eight C57BL/6mice were required to generate12ϫ106 CD8␣ϩDC and65ϫ106CD8␣ϪDC in vitro.Adoptive DC transferThe CD11cϩCD8␣ϩand CD11cϩCD8␣ϪDC puriﬁed from syngeneic wild-type BALB/c and C57BL/6mice were adoptively transferred into wild-type BALB/c and wild-type or IL-12KO C57BL/6recipients,respec-tively,by i.v.injection.Each recipient received1ϫ107BM-DC.Eight animals were included in each group.One day after adoptive DC transfer, heart transplantation was performed in these recipient mice.Flow cytometryPhenotypic analysis of isolated or cultured DC was performed on a FACSCaliburﬂow cytometer(BD Biosciences).DC were labeled with FITC-conjugated anti-mouse CD11c mAb(BD Biosciences)and PE-conjugated anti-mouse CD8␣mAb(BD Biosciences).For detection of IL-12expression, cells were permeabilized with a cell permeabilization kit(Caltag Laboratories) and labeled with PE-conjugated anti-mouse IL-12mAb(BD Biosciences).All ﬂow cytometric analyses were performed using isotype-matched,irrelevant Ig as negative controls(Cedarlane Laboratories).The circulating anti-donor IgM and IgG(including IgG1and IgG2a isotypes)were evaluated in the recipient serum byﬂow cytometry(22,23). In brief,Lewis rat splenocytes were isolated and incubated at37°C for30 min with sera from all experimental groups.To stain for anti-donor IgG, IgG1,IgG2a,and IgM,the cells were washed and incubated with FITC-conjugated goat Ab speciﬁc for the Fc portion of mouse IgG or with PE-conjugated goat Ab speciﬁc for the␮-chain of mouse IgM(Jackson Im-munoResearch Laboratories),FITC-conjugated goat anti-mouse IgG1 (Caltag Laboratories),or with FITC-conjugated goat anti-mouse IgG2a (Caltag Laboratories).After1h of staining at4°C,the cells were washed with PBS,resuspended at5ϫ106/ml,and analyzed byﬂow cytometry. Data are expressed as mean channelﬂuorescence intensity(MFI),which represents the intensity of Ab binding.Naive sera from BALB/c,C57BL/6, or IL-12KO C57BL/6mice were used as negative controls.3526ROLE OF DC IN XENOGRAFT REJECTIONRT-PCRTo measure relative differences in cytokine transcript levels between car-diac transplants,we used a semiquantitative RT-PCR technique as previ-ously described(24).Total RNA was obtained from grafted or normal tissue using TRIzol Reagent(Invitrogen Life Technologies)according to the manufacturer’s speciﬁcations.Total RNA was reverse transcribed using aﬁrst-strand cDNA synthesis kit(Pharmacia).PCR ampliﬁcation was con-ducted in25-␮l reactions using PCR Supermix High Fidelity(10790-020; Invitrogen Life Technologies),which was prepared for multiple reactions. Each PCR consisted of1␮l of cDNA,100nM of each GAPDH primer,a reference gene,and200nM of each pair of speciﬁc primers(IFN-␥,sense AGC TCT GAG ACA ATG AAC GCT ACA C and antisense ACC TGT GGG TTG TTG ACC TCA AAC;IL-12,sense AAA CAG TGA ACC TCA CCT GTG ACA C and antisense TTC ATC TGC AAG TTC TTG GGC G;IL-2,sense ACA TTG ACA CTT GTG CTC CGT GTC and antisense TTG AGG GCT TGT TGA GAT GAT GCT;IL-4,sense AGC TAG TTG TCA TCC TGC TCT TC and antisense AGC ATG GTG GCT CAG TAC TACG;and IL-10,sense TGC TAT GCT GCC TGC TCT TAC TGA C and antisense AAT CAC TCT TCA CCT GCT CCA CTG).The PCR was ampliﬁed as follows:denaturation at95°C for3min followed by 8cycles at95°C for1min,annealing of the primers at60°C,at72°C for 1min,followed by30cycles at95°C for1min,annealing of primers at 50°C for1min,at72°C for2min,and aﬁnal extension at72°C for5min. After PCR ampliﬁcation,a standardized volume of each PCR product was subjected to electrophoresis using a3%agarose gel.The PCR product size of376bp is for IFN-␥,451bp for IL-12,417bp for IL-2,423bp for IL-4, and249bp for GAPDH.The cytokine PCR product was compared with the GAPDH PCR product as an internal control for the same cDNA using the same master mixture prepared concomitantly.In addition,to differentially compare cytokine transcription levels between experimental groups,we used densitometry to measure the ratio of each cytokine RT-PCR product compared with standardized and titratable levels of GAPDH. Statistical analysisData are generally reported as meanϮSD,unless otherwise speciﬁed. Xenograft survival among experimental groups was compared using the rank log test.Histological and immunohistologicalﬁndings of intragraft Ab deposition were analyzed using the ANOVA on rank.Immunohistological ﬁndings of intragraft cellular inﬁltration,ﬂow cytometric data,and RT-PCR data were analyzed using one-way ANOVA.Differences with p val-uesϽ0.05were considered signiﬁcant.ResultsBALB/c and C57BL/6mice acquire distinct DC subsets after xenograftingWe have previously demonstrated that Lewis rat hearts in BALB/c mice were rejected in6.0Ϯ0.6days,while the same grafts lasted up to20.6Ϯ4.9days in C57BL/6mice(1).To investigate the role of DC in regulating the immune response to xenoantigens,weﬁrst studied DC subsets in BALB/c and C57BL/6mice receiving Lewis rat cardiac transplants at a uniform time point,postoperative day (POD)6.We found that C57BL/6recipients transplanted with Lewis rat hearts displayed a predominant CD11cϩCD8␣ϩsplenic DC subset(Fig.1A),as evidenced by a high ratio of CD8␣ϩ: CD8␣ϪDC on POD6(Fig.1B).Conversely,BALB/c recipients exhibited a predominant CD11cϩCD8␣Ϫsplenic DC subset(Fig. 1A),evidenced by a low CD8␣ϩ:CD8␣ϪDC ratio on POD6(pϽ0.01,Fig.1B).As compared with naive animals,there was no change in the CD8␣ϩ:CD8␣ϪDC ratio in BALB/c mice after xenografting;however,a dramatic change in this ratio was ob-served in transplanted C57BL/6recipients(pϽ0.01;Fig.1B).We also found that the total number of CD11cϩDC in spleen was 18.3Ϯ1.1ϫ106and28.4Ϯ0.7ϫ106in BALB/c and C57BL/6 xenograft recipients,respectively.These DC contained 5.6Ϯ0.4ϫ106CD11cϩCD8␣ϩcells and12.7Ϯ 1.0ϫ106CD11cϩCD8␣Ϫcells in BALB/c mice,as well as20.6Ϯ0.9ϫ106CD11cϩCD8␣ϩcells and7.7Ϯ0.4ϫ106CD11cϩCD8␣Ϫcells in C57BL/6mice.To note,the number of splenic CD8␣ϩDC in C57BL/6mice was signiﬁcantly higher than that in BALB/c mice after xenografting(pϽ0.05).In addition,the splenic CD11cϩIL-12ϩcell population increased after transplantation in both C57BL/6and BALB/c recipients.Importantly,CD11cϩIL-12ϩcells were signiﬁcantly more abundant in C57BL/6recipients compared with BALB/c mice on POD6(pϽ0.01;Fig.1C).Fur-thermore,to determine whether the polarity of DC subsets isalso parison of distinct subsets of DC in BALB/c and C57BL/6mice on POD6.Splenocytes were isolated from naive or trans-planted BALB/c and C57BL/6mice on POD6as described in Materials and Methods.Phenotypic analysis of DC subtypes was performed using ﬂow cytometry.DC were stained by FITC-conjugated anti-mouse CD11c mAb and PE-conjugated anti-mouse CD8␣mAb or PE-conjugated anti-mouse IL-12mAb.The polarity of DC subsets in BALB/c and C57BL/6 recipients of xenografts is shown in a representative dot plot analysis(A). The ratio of splenic CD11cϩCD8␣ϩcells:CD11cϩCD8␣Ϫcells(B)as well as the expression of intracellular IL-12in splenic DC(C)were compared in these two strains of mice.Results are meanϮSD of six independent experiments.B,Naive C57BL/6mice vs naive or transplanted BALB/c mice:ء,pϽ0.05;transplanted C57BL/6recipients on POD6vs naive BALB/c or C57BL/6mice and transplanted BALB/c recipients at the same day:ءء,pϽ0.01.C,Transplanted BALB/c recipients vs naive BALB/c and C57BL/6mice:ء,pϽ0.01;transplanted C57BL/6recipients on POD6 vs transplanted BALB/c recipients at the same day:ءء,pϽ0.01.3527The Journal of Immunologyfound in lymph nodes,we measured the DC number in abdominal lymph nodes by ﬂow cytometry.We found that the similar polarity of DC subsets was found in abdominal lymph nodes,even though the total number of CD11c ϩDC in lymph nodes was signiﬁcantly less than that in spleens.For example,abdominal lymph nodes in BALB/c and C57BL/6xenograft recipients have total numbers of (0.11Ϯ0.015)ϫ106and (0.16Ϯ0.009)ϫ106CD11c ϩDC,respectively.These DC contained (0.037Ϯ0.004)ϫ106CD11c ϩCD8␣ϩcells and (0.069Ϯ0.013)ϫ106CD11c ϩCD8␣Ϫcells in BALB/c mice,as well as (0.11Ϯ0.012)ϫ106CD11c ϩCD8␣ϩcells and (0.047Ϯ0.004)ϫ106CD11c ϩCD8␣Ϫcells in C57BL/6mice.Interestingly,the number of CD8␣ϩDC in the lymph nodes of C57BL/6mice was signiﬁcantly higher than that in BALB/c mice after xenografting (p Ͻ0.05).These data indicate that these two mouse strains exhibit signiﬁcant differences in both cell number and ratio of DC subsets after receiving the same type of xenograft.Opposing cytokine proﬁles are present in BALB/c and C57BL/6recipient mice following Lewis rat cardiac xenografting To determine whether different DC populations would differen-tially affect the Th cytokine proﬁles following xenotransplantation,we compared BALB/c and C57BL/6intragraft cytokine mRNA expression on day 6after Lewis rat heart transplantation.We found that gene transcripts for Th1cytokines such as IFN-␥,IL-2,and IL-12were signiﬁcantly higher in C57BL/6recipients than those in BALB/c mice (p Ͻ0.01;Fig.2).In contrast,the Th2cytokine transcripts,as evidenced by IL-4and IL-10,were much higher in BALB/c mice than in C57BL/6mice (p Ͻ0.01;Fig.2).These data suggest that following exposure to xenoantigen,different DC subsets may play important roles in regulating Th1/Th2cytokine proﬁles.Adoptive transfer of CD8␣ϪDC shifts CMR to AVR through manipulation of cytokine proﬁles in C57BL/6recipients of Lewis rat heartsTo further determine whether a causal relationship exists between DC subsets and recipient immune responses to xenoantigen,we adoptively transferred syngeneic CD8␣Ϫor CD8␣ϩBM-DC (1ϫ107cells/mouse)wild-type,syngeneic C57BL/6recipients.One day after BM-DC transfer,C57BL/6recipients received Lewis rat heart transplants.To detect whether CD8␣ϪDC transfer would change the polarity of DC subsets in C57BL/6recipients,we sac-riﬁced three recipients 3days after DC transfer (e.g.,2days after heart xenografting)and did ﬁnd an ϳ30%increase in cell numbers of splenic CD11c ϩCD8␣Ϫcells in C57BL/6recipients as com-pared with the same recipients without DC transfer (data not shown).After CD8␣ϪDC transfer,graft survival time in C57BL/6mice was signiﬁcantly shortened from 20.6Ϯ4.9days to 9.5Ϯ0.6days (p Ͻ0.01;Table I).In contrast,CD8␣ϩDC transfer did not accelerate graft rejection in C57BL/6recipients.Moreover,there was no signiﬁcant difference in xenograft survival between CD8␣ϩDC-transferred and non-DC-transferred animals (18.4Ϯ1.3days vs 20.6Ϯ4.9days,Table I).Based on these observations,we used POD9as a uniform time point for comparison of immune changes in C57BL/6recipients for all studies described hereafter.At the end point of rejection (approximately POD9),trans-planted heart grafts from C57BL/6recipients infused with CD8␣ϪDC exhibited a typical AVR proﬁle with prominent interstitial hemorrhage,intravascular thrombosis (Fig.3Ac ),ﬁbrin deposition (Fig.3Af ),and a few inﬁltrating CD4ϩ(Fig.3,Ai and B )and CD8ϩ(Fig.3,Al and B )cells.In contrast,at the same time point,grafts in C57BL/6mice,with either no DC transfer or CD8␣ϩDC transfer,showed no evidence of AVR.Speciﬁcally,these mice had “clear”’vessels (Fig.3A,a,b,d,and e )and heavy CD4ϩ(Fig.3A,g and h,and B )and CD8ϩ(Fig.3A,j and k,and B )cell inﬁltration,a typical feature of CMR.Table I.Survival time and histopathology of cardiac xenografts aGroup (strain combinations)Treatment Mean Survival ϮSD (days)Histopathology at EndPoint1:Lewis rat 3C57BL/6None20.6Ϯ4.9AVR ϩCMR2:Lewis rat 3C57BL/6CD8␣ϪDC transfer 9.5Ϯ0.6a AVR 3:Lewis rat 3C57BL/6CD8␣ϩDC transfer18.4Ϯ1.3AVR ϩCMR4:Lewis rat 3BALB/c None6.0Ϯ0.6AVR 5:Lewis rat 3BALB/c CD8␣ϩDC transfer 14.2Ϯ0.8b CMR 6:Lewis rat 3BALB/c CD8␣ϪDC transfer5.8Ϯ0.5AVR 7:Lewis rat 3BALB/c CsA11.3Ϯ1.2AVR8:Lewis rat 3BALB/c CD8␣ϩDC transfer ϩCsA 80.6Ϯ7.0c AVR ϩCMR (mild)9:Lewis rat 3BALB/cCD8␣ϪDC transfer ϩCsA12.8Ϯ1.6AVR 10:Lewis rat 3IL-12KO C57BL/6None 6.2Ϯ1.0AVR 11:Lewis rat 3IL-12KO C57BL/6CD8␣ϩDC transfer 16.4Ϯ0.9d CMR 12:Lewis rat 3IL-12KO C57BL/6CD8␣ϪDC transfer 6.0Ϯ0.8AVRa Arrow,transplanted into:a ,p Ͻ0.01,compared with group 1;b ,p Ͻ0.01,compared with group 4;c ,p Ͻ0.01,compared with groups 5,7,and 9;d,p Ͻ0.01,compared with groups 10and12.FIGURE 2.Intragraft cytokine expression in BALB/c and C57BL/6mouse recipients on POD6.Heart grafts were harvested from transplanted recipients on POD6.Semiquantitative RT-PCR was performed for intra-graft mRNA expression of IL-2,IFN-␥,IL-12,IL-4,and IL-10.GAPDH was to ensure ampliﬁcation of comparable quantities of cDNA.Data shown are mean densitometry measurements for cytokine cDNA:GAPDH cDNA ratios.Results are mean ϮSD of six independent experiments.C57BL/6recipients on POD6vs BALB/c recipients at the same day:ء,p Ͻ0.01.3528ROLE OF DC IN XENOGRAFT REJECTIONIn addition,our study revealed that on POD9,adoptive CD8␣ϪDC transfer markedly increased intragraft IL-4and IL-10levels,but decreased IFN-␥,IL-2,and IL-12levels in C57BL/6recipients (Fig.4).In contrast,CD8␣ϩDC transfer failed to change the in-tragraft expression of Th1and Th2cytokines in C57BL/6recipi-ents (Fig.4).Combined,these results indicate that CD8␣ϪDC transfer promotes a predominant Th2response and facilitates AVR in xenotransplantation.Change of Ab levels is associated with AVR after transfer of CD8␣ϪDCTo determine whether the onset of AVR following adoptive trans-fer of CD8␣ϪDC is associated with changes in xenoreactive Abs (xAbs),we measured serum levels of anti-donor Abs by ﬂow cy-tometry on POD9.We found signiﬁcantly higher levels of anti-donor xAbs,primarily IgG,were induced in C57BL/6miceafterFIGURE 3.A,Histology and immunohistology for cellular inﬁltrates in cardiac xenografts of C57BL/6mice with or without BM-DC transfer.Lewis rat heart grafts were harvested on POD9.H&E-stained or mar-tius scarlet blue-stained parafﬁn sections of Lewis rat xenografts in C57BL/6mice with no DC transfer (a and d ),with CD8␣ϩDC transfer (b and e )or CD8␣ϪDC transfer (c and f ).Immunoperoxidase staining of frozen sections of CD4ϩcells (g,h,and i )and CD8ϩcells (j,k,and l )in C57BL/6mice with or without BM-DC transfer.Arrows indicate positive staining.B,The number of inﬁltrating CD4ϩand CD8ϩcells were determined in the xenografts by counting all positively labeled cells in the section and dividing by the area assessed (cells/mm 2).Results are mean ϮSD of six experiments.C57BL/6mice with CD8␣ϪDC transfer vs C57BL/6mice with no DC transfer or with CD8␣ϩDC transfer:ء,p Ͻ0.01.3529The Journal of ImmunologyCD8␣ϪDC transfer when compared with C57BL/6mice that had either CD8␣ϩDC transfer or no DC transfer (p Ͻ0.01,Fig.5A).In addition,serum levels of anti-donor IgM were slightly elevated in C57BL/6recipients following transplantation as compared with naive animals (p Ͻ0.05;Fig.5A );however,there was no signif-icant difference in IgM levels in these recipients with or without DC transfer (Fig.5A ).Consistent with the cytokine proﬁles measured in the grafts,serum IgG isotypes switched to a Th2-predominant pattern,indi-cated by a high IgG1:IgG2a ratio after adoptive transfer of CD8␣ϪDC in C57BL/6mice (p Ͻ0.01;Fig.5B ).Conversely,transfer of CD8␣ϩDC did not change IgG isoforms in these recipients.A low IgG1:IgG2a ratio,indicative of a Th1-predominant pattern,was shown in C57BL/6recipients with either CD8␣ϩDC transfer or no DC transfer (Fig.5B ).To further investigate the role of DC transfer in regulating xAb production,we investigated IgG,IgG1,IgG2a,and IgM deposition in grafts using immunostaining techniques.Table II shows that concordant with the circulating xAb levels,after CD8␣ϪDC trans-fer,high levels of anti-donor IgG deposition,predominantly IgG1,was present in grafts on POD9.At the same time point,C57BL/6mice with either CD8␣ϩDC transfer or no DC transfer were ob-served to have only mild xenograft deposition of anti-donor IgG with IgG2a-dominant Abs.Minimal IgM deposition was observed in all groups (Table II).These results indicate that Th2-promoting CD8␣ϪDC can override a Th1bias in C57BL/6mice and cause IgG subclass switching from Th1to Th2dominant,thereby accelerating AVR.Adoptive transfer of CD8␣ϩDC,but not CD8␣ϪDC attenuates AVR and prolongs xenograft survival in BALB/c miceTo conﬁrm the role of DC subsets in regulating the pattern of xenograft rejection,we adoptively transferred syngeneic CD8␣ϩDC or CD8␣ϪDC (1ϫ107cells/mouse)into BALB/c recipients 1day before Lewis heart transplantation.By sacriﬁcing three re-cipients 3days after DC transfer,we found ϳ30%increase of splenic CD11c ϩCD8␣ϩcells in BALB/c recipients as compared to those without DC transfer (data not shown).With CD8␣ϩDC transfer to BALB/c mice,heart xenograft survival time was sig-niﬁcantly prolonged from 6.0Ϯ0.6days to 14.2Ϯ0.8days (Table I).Accordingly,the graft rejection pattern had shifted from typical AVR (Fig.6Aa )to predominantly CMR (Fig.6A c).In particular,after CD8␣ϩDC transfer,grafts had clear vessels (Fig.6Ac )and massive CD4ϩ(Fig.6B )and CD8ϩ(Fig.6B )cell inﬁltration when compared with BALB/c recipients that had received either CD8␣ϪDC or no DC (p Ͻ0.01).Furthermore,adoptive CD8␣ϪDC transfer neither prolonged graft survival nor changed the rejection pattern of BALB/c mice.These heart xenografts were rejected with predominant AVR (Fig.6,Ae ,and B )at 5.8Ϯ0.5days (Table I).To further investigate the ability of distinct DC subsets to reg-ulate the cytokine proﬁle and Ab production,we analyzed intra-graft cytokine expression and xAb production in BALB/c recipi-ents transferred with DC subsets at the same time point,POD6.In comparison to BALB/c recipients receiving either CD8␣ϪDC transfer or no DC transfer,adoptive CD8␣ϩDC transferfacilitatedFIGURE 4.Intragraft cytokine expression in wild-type C57BL/6recip-ients with or without BM-DC transfer.Heart grafts were harvested from C57BL/6recipients 9days after xenografting.Semiquantitative RT-PCR was performed for intragraft mRNA expression of IL-2,IFN-␥,IL-12,IL-4,and IL-10.GAPDH was used to ensure ampliﬁcation of comparable quantities of cDNA.Data shown are mean densitometry measurements for cytokine cDNA:GAPDH cDNA ratios.Results are mean ϮSD of six ex-periments.C57BL/6mice with CD8␣ϪDC transfer vs C57BL/6mice with no DC transfer or with CD8␣ϩDC transfer:ء,p Ͻ0.01.FIGURE 5.The levels of anti-donor Abs (A )and the ratio of IgG1:IgG2a (B )in the sera of C57BL/6recipients with or without BM-DC trans-fer.Sera were collected from naive C57BL/6mice and from C57BL/6recipients 9days after xenografting.Serum levels of anti-donor total IgG and IgM,as well as the ratio of IgG1:IgG2a,were determined by ﬂow cytometry for comparison in different groups.Results are mean ϮSD of six independent experiments.A,C57BL/6mice with CD8␣ϪDC transfer vs naive C57BL/6mice and C57BL/6recipients with no DC transfer or with CD8␣ϩDC transfer:ء,p Ͻ0.01;C57BL/6mice with CD8␣ϪDC transfer vs naive C57BL/6mice:ءء,p Ͻ0.05.B,C57BL/6recipients with no DC transfer or with CD8␣ϩDC transfer vs naive C57BL/6mice:ء,p Ͻ0.05;C57BL/6mice with CD8␣ϪDC transfer vs naive C57BL/6mice and C57BL/6recipients with no DC transfer or with CD8␣ϩDC transfer:ءء,p Ͻ0.01.3530ROLE OF DC IN XENOGRAFT REJECTION。