生物学

Abbreviations: mAb, monoclonal antibody; phamide; DC. dendritic cells; CFA, complete vant; IFA, incomplete Freund’s adjuvant; TBI, * Corresponding author. Tel.: 212-327-8106; 8875. ’ Present address: Pharmaceutical Research Kirin Brewery Co., Ltd., l-2-2 Souja, Maebashi, Japan. 0022-1759/95/$09.50 0 1995 Elsevier SSDI 0022-1759(94)tigens are shared between different types of human leukocytes. In an effort to obtain new lineage-specific monoclonal antibodies, particularly antibodies to dendritic cells, we attempted to tolerize newborn mice to one type of leukocyte and then immunize the adults with another. We found that T cells, either unstimulated T cells or T blasts, were more effective at inducing neonatal tolerance than non-T cells or B cell lines. However, the tolerance that was achieved was not restricted to T cells, since we could not elicit from the tolerized mice a specific antibody response to a B cell line or to blood dendritic cells. Here we describe several efforts, all unsuccessful, to achieve cell specific immune responses in tolerant mice. The parameters we considered included the type of cell used to tolerize neonatal mice, the regimen of injections for inducing tolerance or eliciting immunity, and the use of several different adjuvants.
Keywords:
Monoclonal
antibody; Neonatal tolerance; Selective antibody response; Dendritic cell
1. Introduction
The potency of dendritic cells as antigen presenting cells for several T-dependent immune responses is a characteristic feature of human cells in culture, as it is in experimental animals
JOURNAL OF IMMUHOl.OGICAL METHODS
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Journal of Immunological Methods 181(1995) 115-124
Difficulties in obtaining monoclonal antibodies to subsets of human leukocytes, using neonatal tolerance induction in mice
cells, monocytes, and dendritic cells). To prepare T blasts (TBl), the purified T cells were stimulated with concanavalin A (Boehringer Mannheim; 3 pg/ml) and phorbol myristic acetate (Sigma, 5 ng/ml). After 3 days, the aggregates in the lectin stimulated cultures were treated with a-methyl mannoside (Sigma, 500 PM) for 1 h to release the TBl. The TBl were washed twice with RPM1 1640. E rosette-negative cells were used as a starting point to prepare enriched populations of monocytes, B cells, and dendritic cells as described (Freudenthal and Steinman, 1990; O’Doherty et al., 1993a). The human B cell line, Ket, was a gift from Dr. Janet S. Lee. 2.3. Neonatal tolerization and immunization For tolerization, cells were resuspended in RPM1 1640 culture medium at 2 X log/ml and injected in 100 ~1 volumes i.p. into C X D2 F2 neonates. For immunization, 5 X lo6 cells were injected i.p. Complete (CFA) and incomplete Freund’s adjuvant (IFA) were from Gibco and used as indicated; likewise for RIB1 adjuvant (Immunochemistry Research, Hamilton, MT). Tolerization and immunization schedules are described in the results section. 2.4. Preparation of monoclonal antibodies Spleen cells from mice that had been tolerized neonatally with one cell type, and then boosted as adults with another cell type, were fused with the SP2/0 myeloma cell line (five spleen cells/myeloma cell) using 50% PEG 1500 (Boehringer). The hybridomas were plated at 10’ spleen cells per flat bottomed microtest well, with either 2 x lo4 feeder peritoneal cells or with recombinant huIL-6 (Kirin, 5 ng/ml, specific activity 2 X 10’ U/ml). The culture supernatants were tested for reactivity to leukocytes by indirect immunolabeling (below) at days lo-14 after fusion. In most experiments with control mice, about 2/3 of the wells were positive for growth, and about 30% showed reactivity with human leukocytes. As presented in the Results, neonatally tolerized mice gave lower yields of growth and reactivity with human leukocytes.
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phamide to selectively deplete B cells that are reactive with shared antigens. While this method has had some success with certain neuronal antigens (Matthew and Sandrock, 1987; Ou et al., 19911, we found that the cyclophosphamide regimen could be nonspecifically suppressive (O’Doherty et al., 1993b). A second approach would be to inject a nonspecific cell type into neonatal mice with the hope of tolerizing the antibody response to shared antigens. Again there are reports of successful neonatal tolerance of mice to rodent neuronal antigens (Hockfield, 1987). In a preliminary study, we tolerized neonatal mice to human T cells and then showed that the adult mice made normal responses to sheep red blood cells (O’Doherty et al., 1993b). We therefore tried to extend these findings, i.e., to tolerize neonates to human T cells and then immunize these mice with other types of human leukocytes. However, we were unable to elicit specific antibody responses to either the B cell line, Ket, or to blood-derived dendritic cells. Here we describe these findings, as well as many efforts to develop a method for inducing neonatal tolerance in such a way that responses to cellspecific surface antigens are maintained.