If biology did have a motto, it may well be ‘nothing is ever as simple as it seems,’ an idea which is exemplified by today’s scientific story. It comes to us from the Prlic Lab in the Vaccine and Infectious Disease Division at Fred Hutch, who spend their time thinking about T cells—how they function, how they interact with their environments, and how we can manipulate them to better human health. A recent publication from the lab, spearheaded by technician Alexis Taber and appearing in the Proceedings of the National Academy of Sciences, takes a closer look at the relationship between memory CD8 T cells and a particular signaling molecule called TGF-β.
First, a bit of background. You’re most likely familiar with T cells—the workhorses of your adaptive immune system. T cells come in different flavors commonly distinguished by their expression of different surface proteins. When you picture a T cell killing a cell displaying a immunogenic antigen (this could be a cell infected with a pathogen or a tumor cell, for instance), you’re most likely picturing a CD8+ T cell, also known as a cytotoxic T cell. CD8 T cells are further categorized into effector cells, which actively fight an immunological insult, and memory cells, which exist in a dormant state, ready to reactivate the next time your body faces the same pathogen again. To do their jobs, T cells have cytotoxic functions (they secrete factors which destroy target cells) as well as inflammatory functions (they secrete signals that recruit other immune cells into the fray, among other things). Importantly, T cells don’t function in a vacuum—they influence and are influenced by their environments, which include different combinations of interacting cell types and signaling molecules. While relatively less understood than cell-intrinsic features, T cell-environment interactions are emerging as a crucial determinant of adaptive immunity and are a major focus of therapeutic development.
This most recent study from the Prlic Lab blossomed from previous efforts in the lab aimed at identifying molecules that suppress the reactivation of CD8 T cells; among the most prominent hits from these studies was a signaling molecule called transforming growth factor beta (TGF-β). To be fair, this alone wasn’t a breakthrough: TGF-β is one of the most well-studied cytokines, with roles in many aspects of development and immunity—particularly suppressing immune responses in the tumor microenvironment. As Taber and colleagues took a closer look at the relationship between TGF-β and T cells, however, they noticed several enticing knowledge gaps. “TGF-β is generally regarded as a broadly immunosuppressive molecule,” notes Taber, “but its specific effects on memory CD8 T cell reactivation, which is an important immune mechanism in transmissible disease and cancer, are largely unknown.” Taber also noticed another interesting trend in the literature: much of what we do know about TGF-β’s effect on T cells comes from transgenic mice engineered to lack TGF-β receptors in specific T cell populations—in these cells, TGF-β signaling was practically completely silenced. While scoring high for physiological relevance, these approaches lack an ability to examine dose- and time-specific effects of TGF-β on T cells. As Taber puts it, “these mouse studies are powerful but modulate TGF-β in a binary fashion; we figured that a memory T cell in its natural habitat would encounter environments with different TGF-β concentrations at different times before or after activation, so we sought a system that could capture this nuance.”
To get at this question, the team implanted mice with CD8 T cells engineered to recognize a single antigen (chicken ovalbumin, the main constituent of egg whites), before exposing the mice to an ovalbumin-expressing virus and giving them time to mount an immune response and produce CD8 memory T cells. Taber then extracted these memory T cells, exposed them to a combination of a strong reactivating stimulus and various doses of TGF-β, and measured various functional parameters, which led to her first major discovery: rather than broadly suppressing the function of reactivated CD8 memory T cells, TGF-β preferentially suppressed their cytotoxic function while largely sparing their inflammatory function. Interestingly, this phenomenon was TGF-β dose-dependent and present in endogenous mouse and human CD8 memory T cells. As Taber notes, “this finding adds more nuance to the widely-held notion that TGF-β is a broad suppressor of T cell function—at least in CD8 memory T cells, it instead appears to specifically hamper cytotoxic functions while largely sparing their inflammatory capacity.”
So, if the dose of TGF-β matters, what about the strength of the activating signal, and the relative timing between the two? Here again, Taber was able to leverage the strengths of her ex vivo approach—giving the memory T cells different activating stimuli, she found that the strength of activating stimulus affected the sensitivity of the T cells to TGF-β. In particular, exposing the T cells to a mix of cytokines (which reactivates them using a pathway distinct from binding ovalbumin) made them less susceptible to TGF-β mediated cytotoxic suppression, even at high doses of TGF-β. By varying the order in which she reactivated the T cells and treated them with TGF-β, Taber made perhaps the most interesting discovery of the study—reactivation and TGF-β sensitivity can interact even when the cells are exposed to the signals at different times! TGF-β can still hamper cytotoxic function of T cells which had been reactivated hours earlier, and cells given a short ‘pulse/washout’ of TGF-β still showed suppressed cytotoxic functions (but intact inflammatory function) at least 24 hours later. “These results were exciting,” Taber explains, “because they really showed us how reactivation and TGF-β tune T cell cytotoxic functions in dose and time-dependent manners, which may more accurately reflect how this signaling occurs in an intact immune environment.”
To figure out how a brief pulse of TGF-β can affect T cell cytotoxicity many hours later, Taber and colleagues concluded their study by profiling the gene regulatory effects of TGF-β exposure on their mouse CD8 memory T cells. To make a long story short, this analysis revealed that—rather than simply suppressing immune-related genes across the board—a mere 2-hour exposure to TGF-β caused diverse changes to transcripts and genome accessibility of a large collection of genes. While the team was able to substantiate their previous finding that TGF-β suppressed cytotoxic but not inflammatory T cell function using this approach, they were surprised to find that TGF-β also transcriptionally upregulates a collection of genes important for T cell chemotaxis (movement towards or away from a chemical stimulus).
Besides providing valuable support for their earlier findings that TGF-β isn’t strictly immunosuppressive, these results suggest potential considerations in therapeutic targeting of TGF-β. As Taber puts it, “If you operate under the assumption that TGF-β is strictly immunosuppressive, you might conclude that inhibiting TGF-β would be strictly immune-boosting. Our results suggest that the relationship between T cell function and TGF-β is more complicated, which might mean that therapies targeting TGF-β may have unintended consequences for T cell chemotaxis, for instance.” The study also revealed that exposure to TGF-β greatly enhanced expression of CCR8, a chemokine receptor that is usually expressed on a T cell population that suppresses other T cells in tumors. Current clinical trial efforts aim to deplete these CCR8-expressing T cells to specifically eliminate suppressive T cells, but this study indicates that this could potentially also lead to the depletion of reactivated, tumor-specific memory CD8 T cells—an eventuality that is under ongoing investigation in the Prlic lab.
The spotlighted research was funded by the National Institutes of Health and utilized the Fred Hutch Flow Cytometry Shared Resource and the Genomics Core Lab of the Benaroya Research Institute.
Fred Hutch/University of Washington/Seattle Children’s Cancer Consortium members Dr. Martin Prlic and Dr. Shannon Oda contributed to this study.
Taber, A., Konecny, A., Oda, S. K., Scott-Browne, J., & Prlic, M. (2023). TGF-β broadly modifies rather than specifically suppresses reactivated memory CD8 T cells in a dose-dependent manner. Proceedings of the National Academy of Sciences, 120(48), e2313228120.
Science Spotlight writer David Sokolov is a graduate student in the Sullivan Lab at the Fred Hutch. He studies how cancer cells modify their metabolism to facilitate rapid proliferation and accommodate tumor-driving mitochondrial defects. He's originally from the east coast and has bachelors' and masters' degrees from West Virginia University. Outside of the lab, you'll find him enjoying the outdoors, playing music, or raising composting worms in his front yard.
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