The gastrointestinal tract is a site of high immune challenge, as it must maintain a delicate balance between tolerating luminal contents and generating an immune response toward pathogens. CD4⁺ T cells are key in mediating the host protective and homeostatic responses. Yet, CD4⁺ T cells are also known to be the main drivers of inflammatory bowel disease (IBD) when this balance is perturbed. Many subsets of CD4⁺ T cells have been identified as players in perpetuating chronic intestinal inflammation. Over the last few decades, understanding of how each subset of Th cells plays a role has dramatically increased. Simultaneously, this has allowed development of therapeutic innovation targeting specific molecules rather than broad immunosuppressive agents. Here, we review the emerging evidence of how each subset functions in promoting and sustaining the chronic inflammation that characterizes IBD.

CD4 T cells play important roles in promoting protective immunity and autoimmune disease. A great deal of attention has been given to the differentiation and function of subsets of cytokine-producing CD4 T cells (i.e., Th1, Th2, and Th17 cells) in these settings. However, others have also observed the accumulation of granzyme-producing CD4 T cells in tumors and in autoimmune patients that are distinct from their cytokine-producing counterparts. Despite the relatively large numbers of granzyme-producing cells in diseased tissues, their roles in driving disease have remained enigmatic. This review will focus on the phenotype(s) and roles of granzyme-producing CD4 T cells in cancer and autoimmunity. We will also examine how granzyme-producing cells interact with current therapeutics and speculate how they may be targeted during disease.

IL-9–producing Th cells, termed Th9 cells, contribute to immunity against parasites and cancers but have detrimental roles in allergic disease and colitis. Th9 cells differentiate in response to IL-4 and TGF-β, but these signals are insufficient to drive Th9 differentiation in the absence of IL-2. IL-2–induced STAT5 activation is required for chromatin accessibility within Il9 enhancer and promoter regions and directly transactivates the Il9 locus. STAT5 also suppresses gene expression during Th9 cell development, but these roles are less well defined. In this study, we demonstrate that human allergy-associated Th9 cells exhibited a signature of STAT5-mediated gene repression that is associated with the silencing of a Th17-like transcriptional signature. In murine Th9 cell differentiation, blockade of IL-2/STAT5 signaling induced the expression of IL-17 and the Th17-associated transcription factor Rorγt. However, IL-2–deprived Th9 cells did not exhibit a significant Th17- or STAT3-associated transcriptional signature. Consistent with these observations, differentiation of IL-17–producing cells under these conditions was STAT3-independent but did require Rorγt and BATF. Furthermore, ectopic expression of Rorγt and BATF partially rescued IL-17 production in STAT3-deficient Th17 cells, highlighting the importance of these factors in this process. Although STAT3 was not required for the differentiation of IL-17–producing cells under IL-2–deprived Th9 conditions, their prolonged survival was STAT3-dependent, potentially explaining why STAT3-independent IL-17 production is not commonly observed in vivo. Together, our data suggest that IL-2/STAT5 signaling plays an important role in controlling the balance of a Th9 versus a Th17-like differentiation program in vitro and in allergic disease. Key Points Human Th9 cells exhibit a STAT5-mediated gene suppression signature. IL-2/STAT5 suppresses a STAT3-independent Th17-like phenotype in Th9 cells. IL-17 production by IL-2–deprived Th9 cells requires Rorγt and BATF.

Acute graft-versus-host disease (aGVHD) can occur after hematopoietic cell transplant in patients undergoing treatment for hematological malignancies or inborn errors. Although CD4 ⁺ T helper (Th) cells play a major role in aGVHD, the mechanisms by which they contribute, particularly within the intestines, have remained elusive. We have identified a potentially novel subset of Th cells that accumulated in the intestines and produced the serine protease granzyme A (GrA). GrA ⁺ Th cells were distinct from other Th lineages and exhibited a noncytolytic phenotype. In vitro, GrA ⁺ Th cells differentiated in the presence of IL-4, IL-6, and IL-21 and were transcriptionally unique from cells cultured with either IL-4 or the IL-6/IL-21 combination alone. In vivo, both STAT3 and STAT6 were required for GrA ⁺ Th cell differentiation and played roles in maintenance of the lineage identity. Importantly, GrA ⁺ Th cells promoted aGVHD-associated morbidity and mortality and contributed to crypt destruction within intestines but were not required for the beneficial graft-versus-leukemia effect. Our data indicate that GrA ⁺ Th cells represent a distinct Th subset and are critical mediators of aGVHD.

IL-9-producing CD4⁺ T helper cells, termed Th9 cells, differentiate from naïve precursor cells in response to a combination of cytokine and cell surface receptor signals that are elevated in inflamed tissues. After differentiation, Th9 cells accumulate in these tissues where they exacerbate allergic and intestinal disease or enhance anti-parasite and anti-tumor immunity. Previous work indicates that the differentiation of Th9 cells requires the inflammatory cytokines IL-4 and TGF-β and is also dependent of the T cell growth factor IL-2. While the roles of IL-4 and TGF-β-mediated signaling are relatively well understood, how IL-2 signaling contributes to Th9 cell differentiation outside of directly inducing the Il9 locus remains less clear. We show here that murine Th9 cells that differentiate in IL-2-limiting conditions exhibit reduced IL-9 production, diminished NF-kB activation and a reduced NF-kB-associated transcriptional signature, suggesting that IL-2 signaling is required for optimal NF-kB activation in Th9 cells. Interestingly, both IL-9 production and the NF-kB transcriptional signature could be rescued by addition of the NF-kB-activating cytokine IL-1β to IL-2-limiting cultures. IL-1β was unique among NF-kB-activating factors in its ability to rescue Th9 differentiation as IL-2 deprived Th9 cells selectively induced IL-1R expression and IL-1β/IL-1R1 signaling enhanced the sensitivity of Th9 cells to limiting amounts of IL-2 by suppressing expression of the Th9 inhibitory factor BCL6. These data shed new light on the intertwined nature of IL-2 and NF-kB signaling pathways in differentiating Th cells and elucidate the potential mechanisms that promote Th9 inflammatory function in IL-2-limiting conditions.

Tox is a member of the high mobility group (HMG)-Box transcription factors and plays important roles in thymic T cell development. Outside of the thymus, however, Tox is also highly expressed by CD8 and CD4 T cells in various states of activation and in settings of cancer and autoimmune disease. In CD4 T cells, Tox has been primarily studied in T follicular helper (TFH) cells where it, along with Tox2, promotes TFH differentiation by regulating key TFH-associated genes and suppressing CD4 cytotoxic T cell differentiation. However, the role of Tox in other T helper (Th) cell subtypes is less clear. Here, we show that Tox is expressed in several physiologically-activated Th subtypes and its ectopic expression enhances the in vitro differentiation of Th2 and T regulatory (Treg) cells. Tox overexpression in unpolarized Th cells also induced the expression of several genes involved in cell activation (Pdcd1), cellular trafficking (Ccl3, Ccl4, Xcl1) and suppressing inflammation (Il10) across multiple Th subtypes. We found that Tox binds the regulatory regions of these genes along with the transcription factors BATF, IRF4, and JunB and that Tox-induced expression of IL-10, but not PD-1, is BATF-dependent. Based on these data, we propose a model where Tox regulates Th cell chemotactic genes involved in facilitating dendritic cell-T cell interactions and aids in the resolution or prevention of inflammation through the production of IL-10.