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Sex differences in glioblastoma (GBM) incidence and outcome are well recognized, and emerging evidence suggests that these extend to genetic/epigenetic and cellular differences, including immune responses. However, the mechanisms driving immunological sex differences are not fully understood. Using GBM models, we demonstrate that T cells play a critical role in driving GBM sex differences. Male mice exhibited accelerated tumor growth, with decreased T cell infiltration and increased T cell exhaustion. Furthermore, a higher frequency of progenitor exhausted T cells was found in males, with improved responsiveness to anti-PD1 treatment. Bone marrow chimera and adoptive transfer models indicated that T cell-mediated tumor control was predominantly regulated in a cell-intrinsic manner, which was further corroborated by in vitro exhaustion assays. Moreover, increased T cell exhaustion was observed in male GBM patients. These findings demonstrate sex-specific pre-determined behavior of T cells is critical in inducing sex differences in GBM progression and immunotherapy response.Statement of significanceImmunotherapies in GBM patients have been unsuccessful due to a variety of factors including the highly immunosuppressive tumor microenvironment in GBM. This study demonstrates that sex-specific T cell behaviors are predominantly intrinsically regulated, further suggesting sex-specific approaches can be leveraged to potentially improve therapeutic efficacy of immunotherapy in GBM.

Reperfusion of organ allografts induces a potent inflammatory response that directs rapid memory T cell, neutrophil, and macrophage graft infiltration and their activation to express functions mediating graft tissue injury. The role of cardiac allograft IL-1 receptor (IL-1R) signaling in this early inflammation and the downstream primary alloimmune response was investigated. When compared with complete MHC-mismatched wild-type cardiac allografts, IL-1R−/− allografts had marked decreases in endogenous memory CD8 T cell and neutrophil infiltration and expression of proinflammatory mediators at early times after transplant, whereas endogenous memory CD4 T cell and macrophage infiltration was not decreased. IL-1R−/− allograft recipients also had marked decreases in de novo donor-reactive CD8, but not CD4, T cell development to IFN-γ–producing cells. CD8 T cell–mediated rejection of IL-1R−/− cardiac allografts took 3 wk longer than wild-type allografts. Cardiac allografts from reciprocal bone marrow reconstituted IL-1R−/−/wild-type chimeric donors indicated that IL-1R signaling on graft nonhematopoietic-derived, but not bone marrow–derived, cells is required for the potent donor-reactive memory and primary CD8 T cell alloimmune responses observed in response to wild-type allografts. These studies implicate IL-1R–mediated signals by allograft parenchymal cells in generating the stimuli-provoking development and elicitation of optimal alloimmune responses to the grafts.

Recent advances in immunosuppressive regimens have decreased acute cellular rejection (ACR) rates and improved intestinal transplant (ITx) recipient survival. We investigated the role of myeloid-derived suppressor cells (MDSCs) in ITx. We identified MDSCs as CD33(+)CD11b(+)lineage(CD3/CD56/CD19)(−)HLA-DR(−/low) cells with 3 subsets, CD14(−)CD15(−) (e-MDSC), CD14(+)CD15(−) (M-MDSC), and CD14(−)CD15(+) (PMN-MDSC), in peripheral blood mononuclear cells (PBMCs) and mononuclear cells in the grafted intestinal mucosa. Total MDSC numbers increased in PBMCs following ITx; among MDSC subsets, M-MDSC numbers were maintained at high level after 2 months following ITx. The MDSC numbers decreased in ITx recipients suffering from ACR. MDSC numbers were positively correlated with serum IL-6 levels and the glucocorticoid administration index. IL-6 and methylprednisolone enhanced the differentiation of bone marrow cells (BMCs) to MDSCs in vitro. M-MDSCs and e-MDSCs expressed CCR1, -2, and -3, e-MDSCs and PMN-MDSCs expressed CXCR2, and intestinal grafts expressed the corresponding chemokine ligands following ITx. Of note, the percentage of MDSCs among intestinal mucosal CD45(+) cells increased after ITx. A novel in vitro assay demonstrated that MDSCs suppressed donor-reactive T-cell-mediated destruction of donor intestinal epithelial organoids. Taken together, our results suggest that MDSCs accumulate in the recipient PBMCs and the grafted intestinal mucosa in ITx, and may regulate ACR.

Contact hypersensitivity (CHS) is a T cell response to hapten skin challenge of sensitized individuals proposed to be mediated by hapten-primed CD8 cytolytic T cells. Effector CD8 T cell recruitment into hapten challenge sites to elicit CHS requires prior CXCL1- and CXCL2-mediated neutrophil infiltration into the site. We investigated whether neutrophil activities directing hapten-primed CD8 T cell skin infiltration in response to 2,4-dinitro-1-fluorobenzene (DNFB) required Fas ligand (FasL) and perforin expression. Although DNFB sensitization of gld/perforin−/− mice induced hapten-specific CD8 T cells producing IFN-γ and IL-17, these T cells did not infiltrate the DNFB challenge site to elicit CHS but did infiltrate the challenge site and elicit CHS when transferred to hapten-challenged naive wild-type recipients. Hapten-primed wild-type CD8 T cells, however, did not elicit CHS when transferred to naive gld/perforin−/− recipients. Wild-type bone marrow neutrophils expressed FasL and perforin, and when transferred to sensitized gld/perforin−/− mice, they restored hapten-primed CD8 T cell infiltration into the challenge site and CHS. The FasL/perforin-mediated activity of wild-type neutrophils induced the expression of T cell chemoattractants, CCL1, CCL2, and CCL5, within the hapten-challenged skin. These results indicate FasL/perforin-independent functions of hapten-primed CD8 T cells in CHS and identify new functions for neutrophils in regulating effector CD8 T cell recruitment and immune responses in the skin.

Contact hypersensitivity (CHS) is a CD8 T cell-mediated response to hapten skin sensitization and challenge. The points at which IL-1R signaling is required during this complex, multistep immune response have not been clearly delineated. The role of IL-1R signaling during 2, 4 dinitro-1-fluorobenezene (DNFB) sensitization to induce hapten-specific CD8 effector T cells and in the trafficking of the effector T cells to the DNFB challenge site to elicit the response were investigated using IL-1R deficient mice. DNFB-sensitized IL-1R−/− mice had low CHS responses to hapten challenge that were caused in part by marked decreases in hapten-specific CD8 T cell development to IL-17– and IFN-γ–producing cells during sensitization. Hapten-primed wild type CD8 T cell transfer to naive IL-1R−/− mice did not result in T cell activation in response to hapten challenge, indicating a need for IL-1R signaling for the localization or activation, or both, of the CD8 T cells at the challenge site. Decreased CD8 T cell priming in sensitized IL-1R−/− mice was associated with marked decreases in hapten-presenting dendritic cell migration from the sensitized skin to draining lymph nodes. Transfer of hapten-presenting dendritic cells from wild type donors to naive IL-1R−/− mice resulted in decreased numbers of the dendritic cells in the draining lymph nodes and decreased priming of hapten-specific CD8 T cells compared with dendritic cell transfer to naive wild type recipients. These results indicate that IL-1R signaling is required at multiple steps during the course of sensitization and challenge to elicit CHS.

Donor Ag-reactive CD4 and CD8 T cell production of IFN-γ is a principal effector mechanism promoting tissue injury during allograft rejection. The CXCR3-binding chemokines CXCL9 and CXCL10 recruit donor-reactive T cells to the allograft, but their role during the priming of donor-reactive T cells to effector function is unknown. Using a murine model of MHC-mismatched cardiac transplantation, we investigated the influence of CXCL9 and CXCL10 during donor-reactive T cell priming. In allograft recipient spleens, CXCL9 and CXCL10 were expressed as early as 24 h posttransplant and increased with similar kinetics, concurrently with CXCR3 expression on T cells. CXCL9, but not CXCL10, expression required NK cell production of IFN-γ. The absence of CXCL9 in donor allografts, recipients, or both significantly decreased the frequency of donor-reactive CD8 T cells producing IFN-γ and increased the frequency of donor-reactive CD8 T cells producing IL-17A. In contrast, the absence of CXCL10 increased the frequency of IFN-γ–producing CD8 T cells in a CXCL9-dependent manner. These data provide novel evidence that donor-reactive CD8 T cells use the CXCR3 chemokine axis as a costimulation pathway during priming to allografts where CXCL9 promotes the development of IFN-γ–producing CD8 T cells, and CXCL10 antagonizes this skewing.

Effector CD8 T cell recruitment into the skin in response to antigen challenge requires prior CXCL1/KC-directed neutrophil infiltration. Mechanisms inducing CXCL1 production and the dynamics of neutrophil-CD8 T cell interactions during elicitation of antigen-specific responses in the skin were investigated. CXCL1 and CXCL2/MIP-2 were produced within 3–6 hours of antigen challenge at 10-fold higher levels in skin challenge sites of antigen-sensitized vs. non-sensitized mice. In the challenge sites of sensitized mice this production decreased at 6–9 hours post-challenge to near the levels observed in skin challenge sites of non-sensitized mice but rose to a second peak 12 hours after challenge. The elevated early neutrophil chemoattractant production at 3–6 hours after skin challenge of sensitized animals required both IFN-γ and IL-17, produced by distinct populations of antigen-primed CD8 T cells in response to antigen challenge. Although induced by the antigen-primed CD8 T cells, the early CXCL1 and CXCL2 production was accompanied by neutrophil but not CD8 T cell infiltration into the skin antigen challenge site. Infiltration of the CD8 T cells into the challenge site was not observed until 18–24 hours after challenge. These results demonstrate an intricate series of early interactions between antigen-specific and innate immune components that regulate the sequential infiltration of neutrophils and then effector T cells into the skin to mediate an immune response.

Green tea polyphenols (GrTP), the active ingredient of green tea, may have immunosuppressive properties, but whether and how GrTP affect transplant-reactive T cells is unknown. To address this, we tested the effects of GrTP on in vitro and in vivo transplant-reactive T cell immunity. GrTP inhibited IFNgamma secretion by cultured monoclonal T cells and by alloreactive T cells in mixed lymphocyte reactions. Oral GrTP significantly prolonged minor antigen-disparate skin graft survival and decreased the frequency of donor-reactive interferon gamma-producing T cells in recipient secondary lymphoid organs compared to controls. In contrast to other hypothesized actions, oral GrTP did not alter dendritic cell trafficking to lymph nodes or affect metalloproteinase activity in the graft. This is the first report of an immunosuppressive effect of GrTP on transplant-reactive T cell immunity. The results suggest that oral intake of green tea could act as an adjunctive therapy for prevention of transplant rejection in humans.

The expression and function of ICAM-1 are critical components in the initiation and elicitation of many T cell-mediated responses. Whether ICAM-1 expression is required on the T cells or on the APC during T cell priming remains unclear. To address this issue in alloantigen-specific T cell activation, the priming and function of T cells in response to heart allografts from MHC-mismatched wild-type vs ICAM-1−/− donors were tested. Wild-type C57BL/6 (H-2b) heart allografts were rejected by A/J (H-2a) recipients on days 7–9, whereas B6.ICAM-1−/− allografts survived until days 18–23 post-transplant. On day 7 post-transplant, infiltrating macrophages and CD4+ and CD8+ T cells in the ICAM-1−/− allografts were 20–30% those observed in the wild-type allografts. ELISPOT analyses indicated that the number of alloantigen-specific T cells producing IFN-γ from recipients of ICAM-1-deficient grafts was 60% lower than that from recipients of wild-type allografts. On day 16 post-transplant, these numbers did not markedly increase in ICAM-1-deficient allograft recipients. Consistent with the reduced priming of alloreactive T cells, isolated dendritic cells from ICAM-1−/− mice stimulated allogeneic T cell proliferation poorly compared with wild-type dendritic cells. When A/J mice were primed with wild-type dendritic cells and then received wild-type or ICAM-1-deficient heart allografts 3 days later, the primed recipients rejected the wild-type and ICAM-1−/− allografts on days 5–6 post-transplant. These results indicate that optimal priming of alloreactive T cells requires allograft expression of ICAM-1, but, once primed, recipient T cell infiltration into the allograft is independent of graft ICAM-1 expression.

During growth in the host, tumor cells are subjected to the stresses of innate and adaptive immunity (immunoediting), which provoke epigenetic changes in the tumor and increase tumor resistance to these immune responses. Our recent studies in methylcholanthrene-induced fibrosarcomas have indicated the appearance and rapid growth of tumor variants deficient in producing the T cell chemoattractant chemokine CXCL9/Mig, an important component of antitumor immunity. In the current report, we demonstrate that highly tumorigenic Mig-deficient tumor variants arise in both cutaneous fibrosarcoma and melanoma as a result of immune stress imposed by IFN-γ and T cells. The consequence of the loss of tumor-derived Mig expression is the increased resistance of Mig-deficient tumors to T cell-mediated immunity, which promotes the accelerated growth of these tumor variants. Remarkably, the ability of Mig-deficient tumor cells to express another CXCR3 ligand, CXCL10/IFN-γ–inducible protein, does not compensate for the absent antitumor functions of Mig, suggesting a nonredundant role for this chemokine in the suppression of tumor growth. To our knowledge, these studies report for the first time that IFN-γ–mediated stress leads to the loss of specific chemokine expression by tumor cells, which in turn promotes tumor growth and evasion of the immune response.

Contact hypersensitivity is a CD8 T cell-mediated response to hapten sensitization and challenge of the skin. Effector CD8 T cell recruitment into the skin parenchyma to elicit the response to hapten challenge requires prior CXCL1/KC-directed neutrophil infiltration within 3–6 h after challenge and is dependent on IFN-γ and IL-17 produced by the hapten-primed CD8 T cells. Mechanisms directing hapten-primed CD8 T cell localization and activation in the Ag challenge site to induce this early CXCL1 production in response to 2,4-dinitrofluorobenzene were investigated. Both TNF-α and IL-17, but not IFN-γ, mRNA was detectable within 1 h of hapten challenge of sensitized mice and increased thereafter. Expression of ICAM-1 was observed by 1 h after challenge of sensitized and nonsensitized mice and was dependent on TNF-α. The induction of IL-17, IFN-γ, and CXCL1 in the challenge site was not observed when ICAM-1 was absent or neutralized by specific Ab. During the elicitation of the contact hypersensitivity response, endothelial cells expressed ICAM-1 and produced CXCL1 suggesting this as the site of CD8 T cell localization and activation. Endothelial cells isolated from challenged skin of naive and sensitized mice had acquired the hapten and the ability to activate hapten-primed CD8 T cell cytokine production. These results indicate that hapten application to the skin of sensitized animals initiates an inflammatory response promoting hapten-primed CD8 T cell localization to the challenge site through TNF-α–induced ICAM-1 expression and CD8 T cell activation to produce IFN-γ and IL-17 through endothelial cell presentation of hapten.

Chemokines, including monokine induced by interferon-gamma (Mig/CXCL9), are produced both in allografts and during the direct T-cell infiltration that mediates graft rejection. Neither the specific production nor contribution of allograft donor versus recipient Mig in allograft rejection is currently known. C57BL/6 mice with a targeted deletion in the Mig gene were used as both skin allograft donors and recipients in a class II major histocompatibility complex-mismatched graft model to test the requirement for donor- versus recipient-derived Mig for acute rejection. B6.Mig(-/-) allografts had a 10-day prolonged survival in B6.H-2(bm12) recipients when compared with wild-type C57BL/6 allograft donors, and B6.H-2(bm12) skin allografts had a 5-day prolonged survival in B6.Mig(-/-) versus wild-type recipients. Transplantation of B6.Mig(-/-) skin grafts onto B6.H-2(bm12).Mig(-/-) recipients resulted in further prolonged allograft survival with more than 30% of the grafts surviving longer than 60 days. Prolonged allograft survival was also associated with delayed cellular infiltration into grafts but not with altered T-cell proliferative responses to donor stimulators. Immunohistochemical staining of allograft sections indicated that Mig is produced by both donor- and recipient-derived sources, but Mig from each of these sources appeared in different areas of the allograft tissue. These results therefore demonstrate the synergy of donor- and recipient-derived Mig in promoting T-cell infiltration into allografts.

Contact hypersensitivity (CHS) is a CD8+ T cell-mediated, inflammatory response to hapten sensitization and challenge of the skin. During sensitization, the magnitude and duration of hapten-specific CD8+ T cell expansion in the skin-draining lymph nodes (LN) are restricted by CD4+CD25+ T regulatory cells (Treg). The regulation of hapten-specific CD8+ T cell priming in Class II MHC-deficient (MHC–/–) mice was investigated. Although hapten-specific CD8+ T cell priming and CHS responses were elevated in Class II MHC–/– versus wild-type mice, presensitization depletion of CD4+ or CD25+ cells in Class II MHC–/– mice further increased CD8+ T cell priming and the elicited CHS response. Flow cytometry analyses of LN cells from Class II MHC–/– mice revealed a population of CD4+ T cells with a majority expressing CD25. Forkhead box p3 mRNA was expressed in LN cells from Class II MHC–/– and was reduced to background levels by depletion of CD4+ or CD25+ cells. Isolated CD4+CD25+ T cells from wild-type and Class II MHC–/– mice limited in vitro proliferation of alloantigen- and hapten-specific T cells to antigen-presenting stimulator cells. These results identify functional CD4+CD25+ Treg in Class II MHC–/– mice, which restrict hapten-specific CD8+ T cell priming and the magnitude of CHS responses.

Interleukin (IL)-2 functions to promote, as well as down-regulate, expansion of antigen-reactive CD4+ and CD8+ T cells, but the role of IL-2 in hapten-specific CD8+ T cell priming for contact hypersensitivity (CHS) responses remains untested. Using enzyme-linked immunospot to enumerate numbers of hapten-specific CD4+ and CD8+ T cells producing IL-2 in hapten-sensitized mice, the number of IL-2-producing CD8+ T cells was tenfold that of CD4+ T cells. Hapten-primed D4+ T cells produced low amounts of IL-2 during culture with hapten-presenting Langerhans cells, whereas production by hapten-primed CD8+ T cells was fivefold greater. CD8+ T cells did not express CD25 during hapten priming, but treatment with anti-IL-2 or anti-CD25 monoclonal antibodies during hapten sensitization increased hapten-specific effector CD8+ T cells as well as the magnitude and duration of the CHS response. These results indicate that CD8+ T cells are the primary source of IL-2 and that this IL-2 is required for the function of a population of CD4+CD25+ T cells to restrict the development of the hapten-reactive effector CD8+ T cells that mediate CHS responses.

Skin but not vascularized cardiac allografts from B6.H-2bm12 mice are acutely rejected by C57BL/6 recipients in response to the single class II MHC disparity. The underlying mechanisms preventing acute rejection of B6.H-2bm12 heart allografts by C57BL/6 recipients were investigated. B6.H-2bm12 heart allografts induced low levels of alloreactive effector T cell priming in C57BL/6 recipients, and this priming was accompanied by low-level cellular infiltration into the allograft that quickly resolved. Recipients with long-term-surviving heart allografts were unable to reject B6.H-2bm12 skin allografts, suggesting potential down-regulatory mechanisms induced by the cardiac allografts. Depletion of CD25+ cells from C57BL/6 recipients resulted in 15-fold increases in alloreactive T cell priming and in acute rejection of B6.H-2bm12 heart grafts. Similarly, reconstitution of B6.Rag−/− recipients with wild-type C57BL/6 splenocytes resulted in acute rejection of B6.H-2bm12 heart grafts only if CD25+ cells were depleted. These results indicate that acute rejection of single class II MHC-disparate B6.H-2bm12 heart allografts by C57BL/6 recipients is inhibited by the emergence of CD25+ regulatory cells that restrict the clonal expansion of alloreactive T cells.

The 2-5A/RNase L system is a regulated RNA decay pathway that mediates some of the antiviral and tumor suppressor activities of the interferons. Previously, we demonstrated that RNase L-null mice have increased susceptibility to viral infections and are partially deficient in induced and spontaneous apoptosis. To determine if RNase L functions in cellular, as well as innate, immunity, skin allograft rejection and contact hypersensitivity (CHS) experiments were performed in RNase L+/+ and RNase L-/- mice. Although no consistent alterations in CHS were found, we did observe a delay of 5 days in the acute rejection of class II major histocompatibility complex (MHC) disparate skin allografts in mice lacking RNase L. Accordingly, histologic examinations of the allografts harvested from RNase L-/- mice revealed a dramatic reduction in inflammatory infiltrates, suggesting a delay in T-cell priming or a deficiency in immune cell trafficking. Results consistent with a proinflammatory role for RNase L extend the known functions of the 2-5A/RNase L system beyond innate immunity into some, but not all, types of cellular immunity.

Contact hypersensitivity (CHS) is a CD8 T cell-mediated response to cutaneous sensitization and subsequent challenge with hapten. Hapten-primed effector CD8 T cell recruitment into the challenged skin to mediate CHS requires prior CXCL1/KC and CXCL2/MIP-2 directed neutrophil infiltration into the challenge site. The mechanisms inducing neutrophil chemoattractant production and the dynamics of neutrophil-CD8 T cell infiltration into the challenge site during the elicitation of CHS to dinitrofluorobenzene (DNFB) were investigated. Following DNFB challenge, CXCL1/KC and CXCL2/MIP-2 were produced within 3–6 h at 10-fold higher levels in sensitized vs. non-sensitized mice. This early chemokine production was dependent on hapten-specific CD8 T cells. At 6 h post-challenge of sensitized but not naïve mice, vascular endothelial cells (EC) in the challenge site stained positively for CXCL1/KC protein as well as for hapten. Although DNFB-primed CD8 T cells induced early CXCL1/KC production, the effector T cells did not infiltrate the parenchymal tissue of the challenge site at 6 h post-challenge but did 12 h later. In contrast, neutrophils infiltrated the challenge site at 6 h post-challenge and this was dependent on hapten-primed CD8 T cell induction of CXCL1/KC production. In vitro, CD8 T cells also stimulated hapten-presenting EC to produce KC. These results indicate hapten challenge to elicit CHS results in EC presentation of hapten to primed CD8 T cells. The T cells stimulate EC production of neutrophil chemoattractants that direct the neutrophil recruitment required for the subsequent infiltration of the effector CD8 T cells to mediate the allergic response.

Despite intense efforts to design immunotherapeutic strategies, the success of cancer immunotherapy remains extremely limited. Major factors limiting immune responses to tumors include insufficient activation of tumor-reactive effector T cells, poor intra-tumor recruitment of effector T cells, and/or suppression of their functions within the tumor. One mediator involved in effector T cell activation and recruitment to inflammatory sites is the T cell chemoattractant chemokine CXCL9/Mig. Our recent studies in murine models of aggressive skin cancer (cutaneous sarcoma and melanoma) have indicated that these tumors cease the production of CXCL9/Mig as a result of immunoediting mediated by T cells and IFN-g. The consequence of Mig deficiency in the tumor microenvironment is the increased resistance of immunoedited Mig-deficient tumor variants to T cell-mediated immunity. Here we demonstrated that inducing constitutive expression of Mig within Mig-deficient tumors by delivery of Mig-encoding retroviral vector promotes protective anti-tumor immune responses and results in rejection of cutaneous tumors. This modulation of intra-tumor Mig expression induced potent tumor-specific CD4 and CD8 T cell-mediated responses capable of complete tumor elimination and subsequent protection to tumor re-challenge. Furthermore, the delivery of Mig-overexpressing tumor cells as a vaccine into mice with established cutaneous tumors induced systemic anti-tumor immune responses that suppressed growth of established tumors. This therapeutic effect correlated with the presence of dendritic cells (DC) in the tumor-draining lymph nodes (TDLN) that were very potent in the activation of tumor antigen-specific IFN-g producing T cells. These results suggest that besides the recruitment of effector T cells into the tumor, tumor-derived Mig enhances activation of these T cells possibly by affecting tumor-infiltrating DC that acquire tumor antigens and then migrate into TDLN to prime tumor-specific effector T cells. Therefore, Mig might be a potent component of vaccines aimed to boost anti-tumor immunity in patients with aggressive skin cancers deficient in the expression of this chemokine. Citation Format: Anton Gorbachev, Danielle Kish, Robert Fairchild. Induction of CXCL9/Mig expression in the tumor microenvironment promotes protective antitumor immune responses. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2831. doi:10.1158/1538-7445.AM2013-2831

Contact hypersensitivity (CHS) is a CD8 T cell mediated immune response to skin sensitization and challenge with hapten. In wild-type (WT) mice, hapten-primed CD8 T cell populations producing IFN-g and IL-17 are required for elicitation of CHS. This study tested the role of the IFN-g receptor (IFN-gR) in CHS to 2,4-dinitrofluorobenzene (DNFB). The magnitude of responses in sensitized IFN-gR-/- and WT mice were similar 24 hr after challenge but were markedly extended in duration in the IFN-gR-/- mice. CD8 T cell depletion abrogated CHS in both WT and IFN-gR-/- mice. Sensitization of WT mice induced high numbers of CD8 T cells producing IFN-g and IL-17 that were detected on day 5 but were reduced to naïve levels by day 8. In contrast, sensitization of IFN-gR-/- mice induced 2.5 more IFN-g producing and 5 fold more IL-17 producing CD8 T cells on day 5 and these levels were only slightly decreased on day 8. These increases were associated with increased cellular infiltration into the hapten challenged skin site of sensitized IFN-gR-/- vs. WT mice through 48 hr post-challenge. Treatment of sensitized IFN-gR-/- mice with anti-IL-17 but not anti-IFN-g mAb during challenge attenuated the CHS response. Thus the absence of IFN-g receptor signaling dysregulates CD8 T cell priming for CHS, increasing the development of hapten-specific CD8 T cells producing IL-17 which mediate CHS without the need for IFN-g.

Contact hypersensitivity (CHS) is a CD8 T cell-mediated response to skin sensitization and challenge with hapten. Hapten-primed CD8 T cell recruitment into the challenged skin to mediate CHS requires prior CXCL1/KC and CXCL2/MIP-2 directed neutrophil infiltration. Factors inducing KC and MIP-2 production in the skin challenge site during CHS to dinitrofluorobenzene (DNFB) were investigated. KC and MIP-2 were produced 3-6 h after challenge of DNFB sensitized, but not unsensitized, mice and required hapten-specific CD8 T cells. Hapten-primed CD8 T cells stimulated hapten-labeled endothelial cell (EC) cultures to produce KC that was blocked by either anti-IFN-g or anti-IL-17 mAb. Addition of rIFN-g plus rIL-17 stimulated cultured EC to produce KC. DNFB sensitization induced hapten-specific CD8 T cell populations producing IFN-g; and IL-17. Induction of KC and MIP-2 in the skin challenge site of sensitized wild type mice was accompanied by expression of IFN-g, IL-17, and IL-21 mRNA and all were absent following challenge of sensitized IFN-g-/- and IL-17-/- mice. Co-transfer of CD8 T cells from sensitized IFN-g-/- and IL-17-/- mice, but not each population alone, to naïve mice restored hapten challenge induced KC production. Activation of hapten-primed CD8 T cell populations producing IFN-g and IL-17 is required to induce production of the KC and MIP-2 required to initiate elicitation of CHS.

CD4+CD25+ T cells limit the expansion of effector CD8 T cells in skin draining lymph nodes (LN) during hapten sensitization for contact hypersensitivity (CHS) responses. This regulatory function results in decreased swelling responses to hapten challenge of the skin. Depletion of CD4 T cells prior to hapten sensitization results in increased numbers of hapten-primed effector CD8 T cells and CHS responses that are elevated in magnitude and extended in duration. These increased CHS responses are similar to those observed in sensitized and challenged class II MHC−/− mice. However, depletion of CD4 T cells in class II MHC−/− mice increased CHS responses to hapten sensitization and challenge when compared to untreated class II MHC−/− mice. ELISPOT analyses indicated increased hapten-specific CD8 T cell priming in LN of class II MHC−/− mice compared to the wild-type and these numbers increased significantly with anti-CD4 or anti-CD25 mAb treatment prior to sensitization. Flow cytometry analyses of LN cells from class II MHC−/− mice revealed a small but distinct population of CD4T cells and ≥50% expressed CD25. qRT-PCR analyses of RNA prepared from LN cells of class II MHC−/− and wild-type mice indicated the presence of FoxP3 expression that was absent when the animals were depleted of CD4 or CD25, but not NK1.1, cells. Purified CD4+ CD25+ T cells from class II MHC−/− and wild-type mice were equivalent in the ability to limit CD4+CD25− T cell proliferation in vitro. These results identify a CD4+ CD25+ regulatory T cell population in class II MHC−/− mice.

Contact hypersensitivity (CHS) is a CD8 T cell-mediated response to cutaneous sensitization and challenge with hapten. Our recent studies indicate that hapten-primed CD8 T cells induce CXCL1/KC production in response to hapten challenge, which directs subsequent neutrophil infiltration into the challenge site and these infiltrating neutrophils then recruit effector CD8 T cells into site to mediate the swelling response. Keratinocyte-hapten contact induces IL1 production, which in turn also stimulates CXCL1/KC production. This study investigated the role of IL1 in the elicitation of CHS. IL1R−/− mice had decreased priming of effector CD8 T cells and CHS swelling responses to DNFB sensitization and challenge than wild-type mice. Production of CXCL1/KC following challenge of sensitized IL1R−/− mice was near background levels. Flow cytometry analyses of cell suspensions prepared from the challenged skin revealed decreased neutrophil infiltration in IL1R−/− vs. wild-type mice. Transfer of wild-type hapten-primed CD8 T cells to naïve wild-type recipients induced CXCL1/KC production in response to hapten challenge whereas transfer to naive IL1R−/− mice did not. Transfer of CD8 T cells from sensitized IL1R−/− mice did not induce CXCL1/KC production in wild-type mice. Injection of LPS i.v. stimulated endothelial cells in the skin of the IL1R−/− mice to produce KC indicating the ability of the IL1R−/− endothelial cells to produce the chemokine. We hypothesize that IL1 signaling is required for optimal CD8 T cell priming to hapten as well as for hapten-primed CD8 T cell interaction with endothelial cells in the challenge site to induce CXCL1/KC production and the CHS response.