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N E W SCancer immunotherapy—which trains the body’s own immune system to fight tumors—has made medical headlines in the last few years, with analysts projecting that it could give rise to treatments worth $35 billion a year over the next decade. For the most part, cancer immunotherapy has relied on the power of T cells. Now, another class of immune cells—known as natural killer (NK) cells—that can function to kill cancer is nearing its big break. NK cells may hold the potential to kill off cancer cells without damaging healthy tissues or risking the T cell–driven inflammatory cytokine storm that can accompany other immunotherapies. But a complicated assortment of protein receptors that control their function makes NK cells unwieldy and unpredictable.The wider adoption of a technology called CyTOF—short for cytometry by time of flight–is now making NK cell research more tractable, allowing scientists to classify and describe NK cell subsets at a level never before possible. Using this technology, Catherine Blish and Peter Parham, two immunologists at Stanford University School of Medicine, recently described 30,000 unique NK cell subsets found among 22 healthy individuals (Sci. Transl. Med. 5, 208ra145, 2013). And that may just scratchthe surface; statistical extrapolation suggests there may be three times as many NK cell subsets within the study population alone. NK cells contain varying combinations of activating and repressing protein receptors that both define the subsets and control their function. Repressive receptors, for example, prevent autoimmunity by recognizing familiar healthy tissues. Meanwhile, activating receptors prompt NK cells to attack infected, damaged or transformed cells. The standard method used to analyze which receptors are expressed on NK cells, called flow cytometry, relies on monoclonal antibodies linked to fluorescing compounds. These antibodies each attach to specific receptors on a given NK cell and the fluorescent compounds emit different wavelengths of light as the cell passes through a laser. To avoid overlap between wavelengths, researchers use a maximum of ten to twelve antibodies for each sample they run.As a post-doc studying the role NK cells have in controlling HIV , Blish realized that she would need to analyze more than a handful of receptors to generate meaningful data from the small blood samples she had saved from a study of HIV-positive Kenyan sex workers. Three years ago, when Blish openedher own lab at Stanford, where immunologist G ary Nolan had been championing the use of CyTOF to study other immune cells since 2010, she saw that this new technology easily fit the bill for NK cells.Although CyTOF relies on the same monoclonal antibodies as regular flow cytometry, it tags them with one of 80–100 different stable metal isotopes, instead of with fluorescent compounds. A technique called mass spectrometry, which vaporizes cells into particles that can be separated on the basis of mass and ionic charge, distinguishes between the stable isotopes. As a result, CyTOF avoids the spectral overlap problem that plagues flow cytometry. In practice, immunologists have so far used up to about 50 markers at one time. Blish’s group used 42, which was enough to reveal that each individual carries an average of 18,000 distinct NK cell subsets. “Now you have a tool that could tell you everything about the problem and opportunity of NK cells,” Nolan says.CyTOF takes offSince the technology was introduced in 2009, several companies and at least 12 institutions besides Stanford have set up their own machines. Ruth Montgomery,Cataloging immune cells for immunotherapyBy Amanda B. Keenern p g© 2015 N a t u r e A m e r i c a , I n c . A l l r i g h t s r e s e r v e d .N E W Sa rheumatologist who directs the CyTOF facility that began operating in 2014 at the Yale School of Medicine in New Haven, Connecticut, often finds herself recommending the technique to colleagues during hallway conversations. “When you realize how much more information you’re getting with CyTOF, it’s hard to be satisfied with only flow cytometry,” she says.But the power to look at 40–50 parameters on each cell at once presents a huge bioinformatics challenge. Computational biologists like Dana Pe’er at Columbia University in New York are taking on this challenge and helping immunologists interpret their CyTOF data. Pe’er’s lab developed an algorithm called viSNE that allows researchers to visualize their multi-dimensional CyTOF data as a two-dimensional image with easily recognizable patterns. “We’re very good at thinking about things in three dimensions,” says Pe’er. “Imagine thinking about them in 40.” For example, by incorporating data about dozens of receptors and other proteins on the surfaces of bone marrow cells, viSNE plots leukemic cells as distinguishable clumps that scientists can home in on for further study (Nat. Biotech. 31, 545–552, 2013). Next, Pe’er plans to roll out a program called Phenograph that distinguishes statistically likely subsets of cells from CyTOF data based on receptor combinations.Blish is ready to move beyond subsets and find out how all of those NK cell receptor combinations relate to cell function. Afterall, she says, diversity in nature is costly. “If NK cells have only one or two functions, why wouldn’t they all be alike?” She suspects there may be groups of NK subsets that are functionally similar and tailored to expertly handle different sorts of pathogens. She hopes to eventually unravel those groups and find out which are best at killing HIV . In the meantime, Blish’s group is performing a longitudinal study at Stanford’s Human Immune Monitoring Center on 12 healthy people over the course of six months to answer some more basic questions. For example, she wants to learn whether individuals retain the same NK cell repertoire over time and whether those with high NK cell diversity maintain it. Blish and Parham’s initial data showed that repressive receptors are controlled by genetics, but that activating receptors are highly influenced by environmental factors—factors her lab is now trying to identify.Infusion of curiosity Todd Fehniger, an oncologist at Washington University School of Medicine in St. Louis, Missouri, says he a nticipates an explosion of CyTOF studies exploring how things like infection and cancer alter NK cell repertoires. CyTOF has already become an important tool for researchers trying to propagate NK cells in the lab that are trained to kill cancer once infused into patients. Laurence Cooper, a pediatric oncologist at MD Anderson Cancer Center in Houston, Texas says there are some receptors and characteristics to look for in NK cells derived to attack cancer cells; “we have a shot on goal with a few things,” he says. The complexity of CyTOF data will help researchers construct a more detailed profile of NK cells used for cancer immunotherapy. Cooper is one of the minds behind a new therapy for pediatric medulloblastoma and other brain cancers that involves activating patient's own NK cells and injecting them back in through a vessel that carries fluid to the brain. The New Opportunity Advancing Hope protocol will be tested in a phase 1 trial beginning this month.So far, NK cell therapy has been most successful in patients with refractory acutemyeloid leukemia (AML); in a 2005 study, about 30% of these patients experienced remission after receiving an infusion of donor NK cells (Blood 105, 3051–3057, 2005). That statistic has held up in other clinical studies, but it’s still not clear why some people respond and some don’t. To resolve this question, both Fehniger and Parham have established collaborations with Jeffrey Miller, an oncologist who has been at the forefront of NK cell immunotherapy research over the last ten years. Part of the problem, Miller says, has been a lack of understanding about how NK cells change after they’ve been infused. Fehniger plans to use CyTOF to fill some of those gaps. He is currently six months into a phase 1 trial that will infuse highly active donor-derived NK cells into the blood of 24 people with AML. In collaboration with Miller, he is saving cells before and after infusion for CyTOF analysis and plans to search for links between expansion of certain cell subsets and patient outcomes.Stem cell transplant serves as another avenue for using NK cells to fight cancer. As donor stem cells rebuild the immunesystem, new NK cells unfamiliar with the host have a better chance to attack cancer cells. Scientists have linked certain NK cell receptor genotypes to a reduced incidence of relapse in AML patients for the first three years after stem cell transplant (Blood 116, 2411–2419, 2010), but Fehniger says that is just a small snapshot of what is possible. CyTOF, he says, could help researchers track how not just one receptor, but entireNK cell subsets affecttransplant outcomes. Along these lines,Parham is using CyTOFto follow NK cells as they repopulate in AMLpatients in an effort todefine what a ‘good’ NK cell repertoire looks like when a patient responds well to stem cell transplant. “You have to know all of the details before you can simplify things with intelligence,” he says.All of these researchers are working to generate a profile of an NK cell population with expert cancer-killing skills in hopes of developing methods to engineer NK cells that mimic that expert population. With the right technology in place, this goal could soon become a reality and elevate the immunotherapeutic status of the humble NK cell.Amanda B. Keener is a former news intern at Nature Medicine in New York and currently a freelance writer in Winston-Salem, North Carolina.“Now you have a tool that can tell you everything about the problem and opportunity of NK cells.”Subset surveyor: Catherine Blish.C a t h e r i n e B l i s h© 2015 N a t u r e A m e r i c a , I n c . A l l r i g h t s r e s e r v e d .。