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Type 1 diabetes results from defects in immune self-tolerance that lead to inflammatory infiltrate in pancreatic islets, beta cell dysfunction and T cell-mediated killing of beta cells. Although therapies that broadly inhibit immunity show promise to mitigate autoinflammatory damage caused by effector T cells, these are unlikely to permanently reset tolerance or promote regeneration of the already diminished pool of beta cells. An emerging concept is that certain populations of immune cells may have the capacity to both promote tolerance and support the restoration of beta cells by supporting proliferation, differentiation and/or regeneration. Here we will highlight three immune cell types-macrophages, regulatory T cells and innate lymphoid cells-for which there is evidence of dual roles of immune regulation and tissue regeneration. We explore how findings in this area from other fields might be extrapolated to type 1 diabetes and highlight recent discoveries in the context of type 1 diabetes. We also discuss technological advances that are supporting this area of research and contextualise new therapeutic avenues to consider for type 1 diabetes.

The linear model of Th cell lineage commitment is being revised due to reports that mature Th cells can trans-differentiate into alternate lineages. This ability of Th cells to reprogram is thought to be regulated by epigenetic mechanisms that control expression of transcription factors characteristic of opposing lineages. It is unclear, however, to what extent this new model of Th cell plasticity holds true in human Th cell subsets that develop under physiological conditions in vivo. We isolated in vivo-differentiated human Th1 and Th17 cells, as well as intermediate Th1/17 cells, and identified distinct epigenetic signatures at cytokine (IFNG and IL17A) and transcription factor (TBX21, RORC, and RORA) loci. We also examined the phenotypic and epigenetic stability of human Th17 cells exposed to Th1-polarizing conditions and found that although they could upregulate TBX21 and IFN-γ, this occurred without loss of IL-17 or RORC expression, and resulted in cells with a Th1/17 phenotype. Similarly, Th1 cells could upregulate IL-17 upon enforced expression of RORC2, but did not lose expression of IFN-γ or TBX21. Despite alterations in expression of these signature genes, epigenetic modifications were remarkably stable aside from the acquisition of active histone methylation marks at cytokine gene promoters. The limited capacity of human Th17 and Th1 cells to undergo complete lineage conversion suggests that the bipotent Th1/17 cells may arise from Th1 and/or Th17 cells. These data also question the broad applicability of the new model of Th cell lineage plasticity to in vivo-polarized human Th cell subsets.

Simple Summary Targeting the inhibitory receptors expressed by immune cells has revolutionized the clinical management of cancer patients. Initially developed to enhance T cell responses, recent investigations have demonstrated that innate lymphoid cells (ILC) also express many of these checkpoint molecules and could therefore be impacted by checkpoint blockade-based therapies. The diversity of the innate lymphoid cell family and their critical role in maintaining tissue homeostasis has drawn broad interest from the field to investigate their function in cancer. Here, we discuss recent findings highlighting the diversity of ILC in tissues and their capacity to migrate into organs upon inflammatory challenge. We further provide a comprehensive overview of the current knowledge on immune checkpoint (IC) expression on ILC, focusing on their therapeutic potential and capacity to modulate anti-tumor immune response. Abstract Immune checkpoints (IC) are broadly characterized as inhibitory pathways that tightly regulate the activation of the immune system. These molecular “brakes” are centrally involved in the maintenance of immune self-tolerance and represent a key mechanism in avoiding autoimmunity and tissue destruction. Antibody-based therapies target these inhibitory molecules on T cells to improve their cytotoxic function, with unprecedented clinical efficacies for a number of malignancies. Many of these ICs are also expressed on innate lymphoid cells (ILC), drawing interest from the field to understand their function, impact for anti-tumor immunity and potential for immunotherapy. In this review, we highlight ILC specificities at different tissue sites and their migration potential upon inflammatory challenge. We further summarize the current understanding of IC molecules on ILC and discuss potential strategies for ILC modulation as part of a greater anti-cancer armamentarium.

Background B7-H3 and B7-H4 are highly expressed by many human malignancies making them attractive immunotherapeutic targets. However, their expression patterns and immune contexts in epithelial ovarian cancer have not been well characterized. Methods We used flow cytometry, immunohistochemistry, and genomic analyses to determine the patterns of B7-H3, B7-H4, and PD-L1 expression by tumor, stromal, and immune cells in the ovarian tumor microenvironment (TME). We analyzed immune cell frequency and expression of PD-1, TIM3, LAG3, ICOS, TIA-1, granzyme B, 2B4, CD107a, and GITR on T cells; CD20, CD22, IgD, BTLA, and CD27 on B cells; CD16 on monocytes; and B7-H3, B7-H4, PD-L1, PD-L2, ICOSL, CD40, CD86, and CLEC9a on antigen-presenting cells by flow cytometry. We determined intratumoral cellular location of immune cells using immunohistochemistry. We compared differences in immune infiltration in tumors with low or high tumor-to-stroma ratio and in tumors from the same or unrelated patients. Results On non-immune cells, B7-H4 expression was restricted to tumor cells whereas B7-H3 was expressed by both tumor and stromal cells. Stromal cells of the ovarian TME expressed high levels of B7-H3 compared to tumor cells. We used this differential expression to assess the tumor-to-stroma ratio of ovarian tumors and found that high tumor-to-stroma ratio was associated with increased expression of CD16 by monocytes, increased frequencies of PD-1high CD8+ T cells, increased PD-L1 expression by APCs, and decreased CLEC9a expression by APCs. We found that expression of PD-L1 or CD86 on APCs and the proportion of PD-1high CD4+ T cells were strongly correlated on immune cells from tumors within the same patient, whereas expression of CD40 and ICOSL on APCs and the proportion of PD-1high CD8+ T cells were not. Conclusions This study provides insight into the expression patterns of B7-H3 and B7-H4 in the ovarian TME. Further, we demonstrate an association between the tumor-to-stroma ratio and the phenotype of tumor-infiltrating immune cells. We also find that some but not all immune parameters show consistency between peritoneal metastatic sites. These data have implications for the design of immunotherapies targeting these B7 molecules in epithelial ovarian cancer.

Innate lymphoid cells (ILCs) are increasingly being recognized for their ability to impact both innate and adaptive immune cells in diverse contexts. ILCs have been observed in all secondary lymphoid tissues, in addition to being tissue-resident innate lymphocytes. In these locations, ILCs are poised to interact with various immune cells at different stages of an immune response. While the heterogeneity and plasticity of ILCs has complicated their study, their association with immune dysregulation in a wide range of pathologies highlights their importance to human health and disease. Notably, in addition to promoting inflammatory immune responses, populations of ILCs have been shown to inhibit immune responses through a variety of mechanisms. The reports of ILC-mediated regulation of immune responses have differed in terms of the phenotype of the regulatory ILC populations, and their mechanism of action. Yet the ability to modulate immune responses appears to be an important function of ILCs. As our understanding of this family of lymphocytes evolves, delineating the factors that dictate whether ILCs orchestrate inflammatory immune responses or suppresses these responses will be important for understanding various disease mechanisms. Here we focus on recent reports that examine how ILCs regulate immunity in different contexts.

Elucidating key factors that regulate immune-mediated pathology in vivo is critical for developing improved strategies to treat autoimmune disease and cancer. NK cells can exhibit regulatory functions against CD8+ T cells following viral infection. Here we show that while low doses of lymphocytic choriomeningitis virus (LCMV-WE) can readily induce strong CD8+ T cell responses and diabetes in mice expressing the LCMV glycoprotein on β-islet cells (RIP-GP mice), hyperglycemia does not occur after infection with higher doses of LCMV. High-dose LCMV infection induced an impaired CD8+ T cell response, which coincided with increased NK cell activity during early time points following infection. Notably, we observed increased NKp46 expression on NK cells during infection with higher doses, which resulted in an NK cell dependent suppression of T cells. Accordingly, depletion with antibodies specific for NK1.1 as well as NKp46 deficiency (Ncr1gfp/gfp mice) could restore CD8+ T cell immunity and permitted the induction of diabetes even following infection of RIP-GP mice with high-dose LCMV. Therefore, we identify conditions where innate lymphoid cells can play a regulatory role and interfere with CD8+ T cell mediated tissue specific pathology using an NKp46 dependent mechanism. CA extern

B7-H4, an immune suppressive member of the B7 family, is highly expressed in a wide variety of human malignancies making it an attractive immunotherapeutic target. However, the association between B7-H4 expression in the tumor microenvironment and the immune infiltrate has not been comprehensively examined. To evaluate the immune tumor microenvironment, we analyzed epithelial ovarian tumors from 28 patients using flow cytometry, immunohistochemistry, functional, and genomic analyses. We determined B7-H4 expression patterns and compared the immune infiltrates of tumors with high and low surface expression of B7-H4. Frequencies and phenotypes of tumor and immune cells were determined using multiple flow cytometry panels. Immunohistochemistry was used to analyze cellular infiltration and location. Publicly available datasets were interrogated to determine intratumoral cytokine and chemokine expression. We found that B7-H4 was predominantly expressed by tumor cells in the epithelial ovarian tumor microenvironment. Surface expression of B7-H4 on tumor cells was correlated with higher levels of infiltrating mature antigen-presenting cells. Further, expression of CXCL17, a monocyte and dendritic cell chemoattractant, correlated strongly with B7-H4 expression. T cells expressed activation markers, but T cells expressing a combination of markers associated with T cell activation/exhaustion phenotype were not prevalent. Overall, our data suggest that B7-H4 is associated with a pro-inflammatory tumor microenvironment.

Antitumor T cells are subject to multiple mechanisms of negative regulation1–3. Recent findings that innate lymphoid cells (ILCs) regulate adaptive T cell responses4–6 led us to examine the regulatory potential of ILCs in the context of cancer. We identified a unique ILC population that inhibits tumor-infiltrating lymphocytes (TILs) from high-grade serous tumors, defined their suppressive capacity in vitro, and performed a comprehensive analysis of their phenotype. Notably, the presence of this CD56+CD3− population in TIL cultures was associated with reduced T cell numbers, and further functional studies demonstrated that this population suppressed TIL expansion and altered TIL cytokine production. Transcriptome analysis and phenotypic characterization determined that regulatory CD56+CD3− cells exhibit low cytotoxic activity, produce IL-22, and have an expression profile that overlaps with those of natural killer (NK) cells and other ILCs. NKp46 was highly expressed by these cells, and addition of anti-NKp46 antibodies to TIL cultures abrogated the ability of these regulatory ILCs to suppress T cell expansion. Notably, the presence of these regulatory ILCs in TIL cultures corresponded with a striking reduction in the time to disease recurrence. These studies demonstrate that a previously uncharacterized ILC population regulates the activity and expansion of tumor-associated T cells.

CD8(+) T cell functions are critical for preventing chronic viral infections by eliminating infected cells. For healthy immune responses beneficial destruction of infected cells must be balanced against immunopathology resulting from collateral damage to tissues. These processes are regulated by factors controlling CD8(+) T cell function which are still incompletely understood. Here we show that the interferon regulatory factor 4 (IRF4) and its cooperating binding partner B cell activating transcription factor (BATF) are necessary for sustained CD8(+) T cell effector function. Although Irf4( / ) CD8(+) T cells were initially capable of proliferation IRF4 deficiency resulted in limited CD8(+) T cell responses after infection with the lymphocytic choriomeningitis virus. Consequently Irf4( / ) mice established chronic infections but were protected from fatal immunopathology. Absence of BATF also resulted in reduced CD8(+) T cell function limited immunopathology and promotion of viral persistence. These data identify the transcription factors IRF4 and BATF as major regulators of antiviral cytotoxic T cell immunity.Cell Death and Differentiation advance online publication 14 February 2014; doi:10.1038/cdd.2014.19.

Natural killer (NK) cells are important mediators of the immune response against microbial pathogens and tumors. There is growing evidence from mouse and human studies that, NK cells exhibit immunoregulatory functions and can limit T cell immunity. NK cell regulatory activity has been demonstrated in a variety of disease models including chronic viral infection, autoimmunity, and transplantation. Depending on the nature of the immune challenge, NK cells use different strategies to limit T cell function, including via cytokines, interactions with NK receptors NKG2D and NKp46, or by perforin-mediated T cell death. Future work should address whether specific subsets of NK cells inhibit T cell responses, and how NK cells acquire immunosuppressive functions.

Dendritic cell (DC) vaccines offer a robust platform for the development of cancer vaccines, but their effectiveness is thought to be limited by T regulatory cells (Tregs). Recombinant adenoviruses (RAdV) have been used successfully to engineer tumor antigen expression in DCs, but the impact of virus transduction on susceptibility to suppression by Tregs is unknown. We investigated the functional consequences of exposure to adenovirus on interactions between human monocyte-derived DCs and Tregs. Since the development of Tregs is linked to that of pro-inflammatory Th17 cells, the role of Th17 cells and IL-17-producing Tregs in the context of DC-based immunotherapies was also investigated. We found that Tregs potently suppressed the co-stimulatory capacity of RAdV-transduced DCs, regardless of whether the DCs were maturated by inflammatory cytokines or by exposure to Th1 or Th17 cells. Furthermore, exposure of Tregs to RAdV-exposed DCs increased IL-17 production and suppressive capacity, and correlated with enhanced secretion of IL-1β and IL-6 by DCs. The findings that DCs exposed to RAdV are suppressed by Tregs, promote Treg plasticity, and enhance Treg suppression indicates that strategies to limit Tregs will be required to enhance the efficacy of such DC-based immunotherapies.

Th17 cells are proinflammatory cells associated with many immune-mediated diseases. Major factors limiting the study of human Th17 cells are the lack of an accepted method for their in vitro differentiation or for isolation of a homogenous population of Th17 cells that do not cosecrete IFN-γ. To overcome these hurdles, we established a novel method to isolate in vivo differentiated Th17 cells from peripheral blood by sorting CD161+CCR4+CCR6+CXCR3−CD4+ T cells. The resulting cells produce high levels of IL-17 but not IFN-γ, express high levels of retinoic acid-related orphan receptor variant 2, and maintain this phenotype upon expansion. Ex vivo Th17 cells exhibit a low cytotoxic potential and are hyporesponsive to polyclonal anti-CD3/anti-CD28 stimulation. Importantly, ex vivo Th17 cells were susceptible to suppression by both naive and memory regulatory T cells (Tregs), which inhibited production of IL-17, IL-22, and CXCL8. Moreover, Tregs suppressed the antifibrotic effects of Th17 cells in a wound-healing model. These findings provide new tools for the study of normal and pathological functions of bona fide Th17 cells in humans. They also provide new insight into the cross-talk between Th17 cells and immune and nonimmune cells, and they establish the paradigm that adoptive Treg-based therapies may effectively limit Th17-mediated inflammation.

OTHER ARTICLES PUBLISHED IN THIS MINI-REVIEW SERIES ON Th17 CELLS Induction of interleukin-17 production by regulatory T cells. Clin Exp Immunol 2009; doi:10.1111/j.1365-2249.2009.04038.x Are T helper 17 cells really pathogenic in autoimmunity? Clin Exp Immunol 2009; doi:10.1111/j.1365-2249.2009.04039.x CD4+ T helper cells: functional plasticity and differential sensitivity to regulatory T cell-mediated regulation. Clin Exp Immunol 2009; doi:10.1111/j.1365-2249.2009.04040.x Development of mouse and human T helper 17 cells. Clin Exp Immunol 2009; doi:10.1111/j.1365-2249.2009.04041.xSummaryT helper (Th) cell have a central role in modulating immune responses. While Th1 and Th2 cells have long been known to regulate cellular and humoral immunity, Th17 cells have been identified only recently as a Th lineage that regulates inflammation via production of distinct cytokines such as interleukin (IL)-17. There is growing evidence that Th17 cells are pathological in many human diseases, leading to intense interest in defining their origins, functions and developing strategies to block their pathological effects. The cytokines that regulate Th17 differentiation have been the focus of much debate, due primarily to inconsistent findings from studies in humans. Evidence from human disease suggests that their in vivo development is driven by specialized antigen-presenting cells. Knowledge of how Th17 cells interact with other immune cells is limited, but recent data suggest that Th17 cells may not be subject to strict cellular regulation by T regulatory cells. Notably, Th17 cells and T regulatory cells appear to share common developmental pathways and both cell types retain significant plasticity. Herein, we will discuss the molecular and cellular regulation of Th17 cells with an emphasis on studies in humans.

Th17 cells are defined by their capacity to produce IL-17, and are important mediators of inflammation and autoimmunity. Human Th17 cells express high levels of the retinoic acid-related orphan receptor variant 2 (RORC2), but it is currently unclear whether expression of this transcription factor alone is sufficient to recapitulate all the known properties of Th17 cells. We used lentivirus-mediated transduction to investigate the role of RORC2 in defining aspects of the human Th17 cell lineage. Expression of RORC2 induced production of IL-17A, IL-22, IL-6 and TNF-alpha, a Th17-cell-associated chemokine receptor profile and upregulation of CD161. RORC2-transduced T cells were hypo-responsive to TCR-mediated stimulation, a property shared with ex vivo Th17 cells and overcome by addition of exogenous IL-2 or IL-15. Co-culture experiments revealed that RORC2-expressing cells were partially resistant to Treg cells since production of IL-17 and proliferation were not suppressed. Evidence that IL-17 stimulates CD4(+) T cells to produce IL-2 and proliferate suggested that the resistance of Th17 cells to Treg-mediated suppression may be partly attributed to IL-17 itself. These findings demonstrate that expression of RORC2 in T cells has functional consequences beyond altering cytokine production and provides insight into the factors regulating the development of human Th17 cells.

The development of a healthy adaptive immune system requires a T cell repertoire diverse enough to recognize foreign antigen while limiting recognition of self-antigens to avoid autoimmunity. However, this idea gives rise to many questions. If all lymphocytes that recognize and respond to self-antigens are eliminated, will the remaining repertoire be sufficient to respond to the array of unknown pathogens? And if all self-recognizing T cells are tolerized, how does autoimmunity and immunosurveillance occur? The T cell repertoire needs to be capable of the precarious balance between tolerance and immunity to allow for specific recognition and clearance of dangerous pathogens and transformed cells, while remaining tolerant to normal self-tissues, commensal bacteria, and food and environmental antigens. To strike this balance, there are many regulatory checkpoints in the development of the immune system and in the maintenance of immune homeostasis. This chapter will outline T cell selection, mechanisms of tolerance, and how dysregulation of these processes may lead to autoimmunity.

CD4+FOXP3+ regulatory T cells are essential for immune tolerance, and murine studies suggest that their dysfunction can lead to type 1 diabetes (T1D). Human studies assessing regulatory T cell dysfunction in T1D have relied on analysis of FOXP3-expressing cells. Recently, distinct subsets of CD4+FOXP3+ T cells with differing function were identified. Notably, CD45RA−CD25intFOXP3low T cells lack suppressive function and secrete the proinflammatory cytokine IL-17. Therefore, we evaluated whether the relative fractions of CD4+FOXP3+ subsets are altered in new-onset T1D subjects. We report that children with new-onset T1D have an increased proportion of CD45RA−CD25intFOXP3low cells that are not suppressive and secrete significantly more IL-17 than other FOXP3+ subsets. Moreover, these T1D subjects had a higher proportion of both CD4+ and CD8+ T cells that secrete IL-17. The bias toward IL-17–secreting T cells in T1D suggests a role for this proinflammatory cytokine in the pathogenesis of disease.

We have previously shown that human Th17 lymphocytes are characterized by the selective expression of IL-23 receptor (IL-23R), CCR6, CD161, and the transcription factor retinoic acid-related orphan receptor C (RORC), and originate from a CD161(+)CD4(+) naive T-cell precursor in response to the combined activity of IL-1β and IL-23. We show here that not only CD4(+)TCRαβ(+), but also CD8(+)TCRαβ(+), CD4(-)CD8(-) TCRαβ(+), and CD4(-)CD8(-) TCRγδ(+) circulating lymphocytes that produce IL-17 express the distinctive marker CD161 on their surface. In addition, we demonstrate that CD161 expression identifies CD8(+) and CD4(-)CD8(-) umbilical cord blood T cells that already express RORC and IL-23R mRNA and that can be induced to differentiate into IL-17-producing cells in the presence of IL-1β and IL-23. Finally, we provide evidence that umbilical cord blood naive CD4(+)CD161(-) T cells, upon lentivirus-mediated transduction with RORC2 can acquire the ability to express IL-23R, IL-1RI, and CD161, as well as to produce IL-17. Taken together, these data allow to conclude that T-cell subsets able to produce IL-17, as well as precursors of IL-17-producing T cells, exhibit surface expression of CD161, and that this feature is at least in part RORC2-dependent.

One of the defining features of the majority of FOXP3(+) Tregs is their inability to produce typical T-cell-derived cytokines. Little is known, however, about their capacity to produce chemokines. As Tregs are constitutively present in, and rapidly traffic to, non-lymphoid tissues, we hypothesized that they may produce chemokines to direct the composition of cells that infiltrate inflamed tissues. Surprisingly, we found that Tregs produce high amounts of CXCL8 (IL-8), a potent neutrophil chemoattractant. Tregs also produced other CC and CXC family chemokines, including CCL2-5, CCL7, and CXCL10. Whereas ectopic expression of FOXP3 suppressed cytokine production, it significantly induced CXCL8. Moreover, supernatants from Tregs attracted neutrophils via a CXCL8-dependent mechanism. These data provide the first evidence that although classical Tregs are defined by their lack of proinflammatory cytokine production, they secrete significant quantities of chemokines and thus may have an unappreciated role in directing the recruitment of immune cells.

Forkhead box P3 (FOXP3) is currently thought to be the most specific marker for naturally occurring CD4(+)CD25(+) T regulatory cells (nTregs). In mice, expression of FoxP3 is strictly correlated with regulatory activity, whereas increasing evidence suggests that in humans, activated T effector cells (Teffs) may also express FOXP3. In order to better define the role of FOXP3 in human Teff cells, we investigated the intensity and kinetics of expression in ex vivo Teff cells, suppressed Teff cells and Teff cell lines. We found that all dividing Teff cells expressed FOXP3, but only transiently, and at levels that were significantly lower than those in suppressive nTregs. This temporary expression in Teff cells was insufficient to suppress expression of reported targets of FOXP3 repressor activity, including CD127, IL-2 and IFN-gamma, and was not correlated with induction of a nTreg phenotype. Thus expression of FOXP3 is a normal consequence of CD4(+) T cell activation and, in humans, it can no longer be used as an exclusive marker of nTregs. These data indicate that our current understanding of how FOXP3 contributes to immune tolerance in humans needs to be re-evaluated.

Acute graft versus host disease (aGVHD) following haematopoietic stem cell transplantation (HCT) occurs when donor T cells infused with the graft recognise and react to histo-incompatible recipient antigens causing tissue damage. Historically, the inflammatory response in aGVHD was attributed to alloreactive CD4+ T helper and CD8+ cytotoxic T cells and alterations in cytokine production. Recently, a new CD4+ T cell subset, characterised by IL-17 production has been identified. TH17 cells produce high levels of proinflammatory cytokines, including IL-17A, IL-17F, and IL-22, and have been implicated in solid organ rejection and more recently a number of murine studies suggest that Th17 cells play a role in the development of aGVHD. It is well known that FOXP3+ regulatory T cells (Tregs) are critical for the maintenance of self-tolerance, and control the immune response to alloantigens. Murine studies have shown that adoptive transfer of these cells can prevent acute GVHD whereas selective depletion leads to an increased severity. In humans, Tregs also appear to control acute GVHD as they occur at a lower frequency in the peripheral blood patients with aGVHD compared to patients without GVHD. These findings have led to active interest into the use of these cells to prevent or decrease GVHD following allogeneic HCT. It has been reported that in vitro, Th17 cells are resistant to Treg cell mediated suppression of proliferation and IL-17 production, suggesting that the effector functions of Th17 cells might not be susceptible to Treg-cell-mediated inhibition. If true, this would suggest that Treg-based therapies might not be effective at limiting Th17-cell-mediated tissue damage. However, there is currently no evidence regarding whether Treg cells affect the phenotype or function of Th17 cells in tissues. Understanding the interactions between suppressive Tregs and pro-inflammatory T effectors in tissues that are targets of aGVHD, such as the skin, is critical to better define the potential of Tregs as adoptive therapy for the prevention or treatment of aGVHD. In order to address this question, we developed two methods to generate human Th17 cells, one based on over-expression of RORC2 and the other on sorting CCR4+CCR6+CD4+ T cells. We found that ectopic expression of RORC induces a cytokine and chemokine receptor profile analogous to in vivo differentiated Th17 cells. Although expression of RORC2 made CD4+ T cells resistant to Treg-cell mediated suppression of proliferation and IL-17 production, production of IFN-g, TNF-a and IL-6 could be suppressed in these Th17-like cells. In order to further delineate the functional consequence of the interaction between Treg and Th17 cells in tissues we developed a modified the human skin explant model that involves culture of 4 mm punch biopsies of skin with ex vivo Th17 cells (CCR4+CCR6+CD4+ T cells), RORC2 transduced CD4+ T cells, or controls, in the presence or absence of Treg and grading the graft-versus-host reactivity (grades I–IV) histopathologically. Preliminary data suggest that Th17 cells cause significant tissue destruction in this skin explant model, and experiments are ongoing to determine whether Treg cells can counteract these effects.