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CD8+ T cell proliferation and differentiation into effector and memory states are high-energy processes associated with changes in cellular metabolism. CD28-mediated costimulation of T cells activates the PI3K/AKT/mammalian target of rapamycin signaling pathway and induces eukaryotic translation initiation factor 4E–dependent translation through the derepression by 4E-BP1 and 4E-BP2. In this study, we demonstrate that 4E-BP1/2 proteins are required for optimum proliferation of mouse CD8+ T cells and the development of an antiviral effector function. We show that translation of genes encoding mitochondrial biogenesis is impaired in T cells derived from 4E-BP1/2–deficient mice. Our findings demonstrate an unanticipated role for 4E-BPs in regulating a metabolic program that is required for cell growth and biosynthesis during the early stages of CD8+ T cell expansion.

Immunotherapeutic strategies aimed at enhancing tumor cell killing by tumor-specific T cells hold great potential for reducing tumor burden and prolonging survival of cancer patients. Although many potential tumor antigens have been described, identifying relevant targets when designing anti-cancer vaccines or targeted cell therapies remains a challenge. To identify novel, potentially immunogenic candidate tumor antigens, we performed integrated tumor transcriptomic, seromic, and proteomic analyses of high grade serous ovarian cancer (HGSC) patient tumor samples. We identified tumor neo-antigens and over-expressed antigens using whole exome and RNA sequencing and examined these in relation to patient-matched auto-antibody repertoires. Focusing on MHC class I epitopes recognized by CD8+ T cells, HLA-binding epitopes were identified or predicted from the highly expressed, mutated, or auto-antibody target antigen, or MHC-associated peptides (MAPs). Recognition of candidate antigenic peptides was assessed within the tumor-infiltrating T lymphocyte (TIL) population expanded from each patient. Known tumor-associated antigens (TAA) and cancer/testis antigens (CTA) were commonly found in the auto-antibody and MAP repertoires and CD8+ TILs recognizing epitopes from these antigens were detected, although neither expression level nor the presence of auto-antibodies correlated with TIL recognition. Auto-antibodies against tumor-mutated antigens were found in most patients, however, no TIL recognition of the highest predicted affinity neo-epitopes was detected. Using high expression level, auto-antibody recognition, and epitope prediction algorithms, we identified epitopes in 5 novel antigens (MOB1A, SOCS3, TUBB, PRKAR1A, CCDC6) recognized by HGSC patient TILs. Furthermore, selection of epitopes from the MAP repertoire identified 5 additional targets commonly recognized by multiple patient TILs. We find that the repertoire of TIL specificities includes recognition of highly expressed and immunogenic self-antigens that are processed and presented by tumors. These results indicate an ongoing autoimmune response against a range of self-antigens targeted by HGSC TILs.

Responsiveness to PD-1 blockade depends on a cell subset known as Tpex cells, but how these cells are sustained is less understood. In this issue of Immunity, Dähling et al. show how dendritic cells form a niche for Tpex preservation.

High-grade serous ovarian cancer (HGSC), the principal cause of death from gynecologic malignancies in the world, has not significantly benefited from advances in cancer immunotherapy. Although HGSC infiltration by lymphocytes correlates with superior survival, the nature of antigens that can elicit anti-HGSC immune responses is unknown. The goal of this study was to establish the global landscape of HGSC tumor-specific antigens (TSA) using a mass spectrometry pipeline that interrogated all reading frames of all genomic regions. In 23 HGSC tumors, we identified 103 TSAs. Classic TSA discovery approaches focusing only on mutated exonic sequences would have uncovered only three of these TSAs. Other mutated TSAs resulted from out-of-frame exonic translation (n = 2) or from noncoding sequences (n = 7). One group of TSAs (n = 91) derived from aberrantly expressed unmutated genomic sequences, which were not expressed in normal tissues. These aberrantly expressed TSAs (aeTSA) originated primarily from nonexonic sequences, in particular intronic (29%) and intergenic (22%) sequences. Their expression was regulated at the transcriptional level by variations in gene copy number and DNA methylation. Although mutated TSAs were unique to individual tumors, aeTSAs were shared by a large proportion of HGSCs. Taking into account the frequency of aeTSA expression and HLA allele frequencies, we calculated that, in Caucasians, the median number of aeTSAs per tumor would be five. We conclude that, in view of their number and the fact that they are shared by many tumors, aeTSAs may be the most attractive targets for HGSC immunotherapy.

Innate and adaptive immune cells monitor, recognize, and eliminate transformed cells. Innate lymphoid cells (ILCs) are innate counterparts of T cells that play a key role in many facets of the immune response and have a profound impact on disease states, including cancer. ILCs regulate immune responses by responding and integrating a wide range of signals within the local microenvironment. As primarily tissue-resident cells, ILCs are ideally suited to sense malignant transformation and initiate anti-tumor immunity. However, as ILCs have been associated with anti-tumor and pro-tumor activities in established tumors, they could potentially have dual functions during carcinogenesis by promoting or suppressing the malignant outgrowth of premalignant lesions. Here we discuss emerging evidence that shows that ILCs can impact early tumor development by regulating immune responses against transformed cells, as well as the environmental cues that potentially induce ILC activation in premalignant lesions.

CD8+ T cells can be polarized into several different subsets as defined by the cytokines they produce and the transcription factors that govern their differentiation. Here, we identified the polarizing conditions to induce an IL22-producing CD8+ Tc22 subset, which is dependent on IL6 and the aryl hydrocarbon receptor transcription factor. Further characterization showed that this subset was highly cytolytic and expressed a distinct cytokine profile and transcriptome relative to other subsets. In addition, polarized Tc22 were able to control tumor growth as well as, if not better than, the traditional IFNγ-producing Tc1 subset. Tc22s were also found to infiltrate the tumors of human patients with ovarian cancer, comprising up to approximately 30% of expanded CD8+ tumor-infiltrating lymphocytes (TIL). Importantly, IL22 production in these CD8+ TILs correlated with improved recurrence-free survival. Given the antitumor properties of Tc22 cells, it may be prudent to polarize T cells to the Tc22 lineage when using chimeric antigen receptor (CAR)-T or T-cell receptor (TCR) transduction–based immunotherapies.

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.

Type I interferons (IFN-Is) are central regulators of anti-tumor immunity and responses to immunotherapy, but they also drive the feedback inhibition underlying therapeutic resistance. In the present study, we developed a mass cytometry approach to quantify IFN-I-stimulated protein expression across immune cells and used multi-omics to uncover pre-therapy cellular states encoding responsiveness to inflammation. Analyzing peripheral blood cells from multiple cancer types revealed that differential responsiveness to IFN-Is before anti-programmed cell death protein 1 (PD1) treatment was highly predictive of long-term survival after therapy. Unexpectedly, IFN-I hyporesponsiveness efficiently predicted long-term survival, whereas high responsiveness to IFN-I was strongly associated with treatment failure and diminished survival time. Peripheral IFN-I responsive states were not associated with tumor inflammation, identifying a disconnect between systemic immune potential and 'cold' or 'hot' tumor states. Mechanistically, IFN-I responsiveness was epigenetically imprinted before therapy, poising cells for differential inflammatory responses and dysfunctional T cell effector programs. Thus, we identify physiological cell states with clinical importance that can predict success and long-term survival of PD1-blocking immunotherapy.

Cancers arising from germline DNA mismatch repair deficiency or polymerase proofreading deficiency (MMRD and PPD) in children harbour the highest mutational and microsatellite insertion–deletion (MS-indel) burden in humans. MMRD and PPD cancers are commonly lethal due to the inherent resistance to chemo-irradiation. Although immune checkpoint inhibitors (ICIs) have failed to benefit children in previous studies, we hypothesized that hypermutation caused by MMRD and PPD will improve outcomes following ICI treatment in these patients. Using an international consortium registry study, we report on the ICI treatment of 45 progressive or recurrent tumors from 38 patients. Durable objective responses were observed in most patients, culminating in a 3 year survival of 41.4%. High mutation burden predicted response for ultra-hypermutant cancers (>100 mutations per Mb) enriched for combined MMRD + PPD, while MS-indels predicted response in MMRD tumors with lower mutation burden (10–100 mutations per Mb). Furthermore, both mechanisms were associated with increased immune infiltration even in ‘immunologically cold’ tumors such as gliomas, contributing to the favorable response. Pseudo-progression (flare) was common and was associated with immune activation in the tumor microenvironment and systemically. Furthermore, patients with flare who continued ICI treatment achieved durable responses. This study demonstrates improved survival for patients with tumors not previously known to respond to ICI treatment, including central nervous system and synchronous cancers, and identifies the dual roles of mutation burden and MS-indels in predicting sustained response to immunotherapy., Hypermutation and microsatellite burden determine responses and long-term survival following PD-1 blockade in children and young adults with refractory cancers resulting from germline DNA replication repair deficiency.

Dendritic cells are sentinels of the immune system and represent a key cell in the activation of the adaptive immune response. Hypoxia-inducible factor 1 alpha (HIF-1α)–a crucial oxygen sensor stabilized during hypoxic conditions–has been shown to have both activating and inhibitory effects in immune cells in a context- and cell-dependent manner. Previous studies have demonstrated that in some immune cell types, HIF-1α serves a pro-inflammatory role. Genetic deletion of HIF-1α in macrophages has been reported to reduce their pro-inflammatory function. In contrast, loss of HIF-1α enhanced the pro-inflammatory activity of dendritic cells in a bacterial infection model. In this study, we aimed to further clarify the effects of HIF-1α in dendritic cells. Constitutive expression of HIF-1α resulted in diminished immunostimulatory capacity of dendritic cells in vivo, while conditional deletion of HIF-1α in dendritic cells enhanced their ability to induce a cytotoxic T cell response. HIF-1α-expressing dendritic cells demonstrated increased production of inhibitory mediators including IL-10, iNOS and VEGF, which correlated with their reduced capacity to drive effector CD8+ T cell function. Altogether, these data reveal that HIF-1α can promote the anti-inflammatory functions of dendritic cells and provides insight into dysfunctional immune responses in the context of HIF-1α activation.

Regulatory T cells (Treg) are an integral component of the adaptive immune system that negatively affect antitumor immunity. Here, we investigated the role of the E3 ubiquitin ligase casitas B-lineage lymphoma-b (Cbl-b) in establishing CD8+ T-cell resistance to Treg-mediated suppression to enhance antitumor immunity. Transcriptomic analyses suggested that Cbl-b regulates pathways associated with cytokine signaling and cellular proliferation. We showed that the hypersecretion of IFNγ by Cbl-b–deficient CD8+ T cells selectively attenuated CD8+ T-cell suppression by Tregs. Although IFNγ production by Cbl-b–deficient T cells contributed to phenotypic alterations in Tregs, the cytokine did not attenuate the suppressive function of Tregs. Instead, IFNγ had a profound effect on CD8+ T cells by directly upregulating interferon-stimulated genes and modulating T-cell activation. In murine models of adoptive T-cell therapy, Cbl-b–deficient T cells elicited superior antitumor immune response. Furthermore, Cbl-b–deficient CD8+ T cells were less susceptible to suppression by Tregs in the tumor through the effects of IFNγ. Collectively, this study demonstrates that the hypersecretion of IFNγ serves as a key mechanism by which Cbl-b–deficient CD8+ T cells are rendered resistant to Tregs. See related Spotlight by Wolf and Baier, p. 370.

PurposeTo evaluate whether administration of the oral DNA hypomethylating agent CC-486 enhances the poor response rate of immunologically ‘cold’ solid tumors to immune checkpoint inhibitor durvalumab.Experimental designPD-L1/PD-1 inhibitor naïve patients with advanced microsatellite stable colorectal cancer; platinum resistant ovarian cancer; and estrogen receptor positive, HER2 negative breast cancer were enrolled in this single-institution, investigator-initiated trial. Two 28 day regimens, regimen A (CC-486 300 mg QD Days 1–14 (cycles 1–3 only) in combination with durvalumab 1500 mg intravenous day 15) and regimen B (CC-486 100 mg QD days 1–21 (cycle 1 and beyond), vitamin C 500 mg once a day continuously and durvalumab 1500 mg intravenous day 15) were investigated. Patients underwent paired tumor biopsies and serial peripheral blood mononuclear cells (PBMCs) collection for immune-profiling, transcriptomic and epigenomic analyzes.ResultsA total of 28 patients were enrolled, 19 patients treated on regimen A and 9 on regimen B. The combination of CC-486 and durvalumab was tolerable. Regimen B, with a lower dose of CC-486 extended over a longer treatment course, showed less grade 3/4 adverse effects. Global LINE-1 methylation assessment of serial PBMCs and genome-wide DNA methylation profile in paired tumor biopsies demonstrated minimal changes in global methylation in both regimens. The lack of robust tumor DNA demethylation was accompanied by an absence of the expected ‘viral mimicry’ inflammatory response, and consequently, no clinical responses were observed. The disease control rate was 7.1%. The median progression-free survival was 1.9 months (95% CI 1.5 to 2.3) and median overall survival was 5 months (95% CI 4.5 to 10).ConclusionsThe evaluated treatment schedules of CC-486 in combination with durvalumab did not demonstrate robust pharmacodynamic or clinical activity in selected immunologically cold solid tumors. Lessons learned from this biomarker-rich study should inform continued drug development efforts using these agents.Trial registration numberNCT02811497.

The differentiation of human hematopoietic stem cells into CD8 T cells can be achieved in vitro with the OP9-DL4 system. We considered that in the absence of medullary thymic epithelial cells, which serve to restrict the breath of the T-cell receptor (TCR) repertoire by expressing tissue-restricted antigens, a distinct repertoire would be generated in vitro. To test this notion, we compared the TCR-Vα/Vβ (TRAV/TRBV) gene usage of major histocompatibility complex-restricted antigen (MART-1)-specific T cells generated in vitro to that from ex vivo naive T cells and tumor-infiltrating lymphocytes (TILs) using high-throughput DNA sequencing. In contrast to naive T cells and TILs, which showed the expected narrow TRAV repertoire, in vitro-generated MART-1-specific T cells used almost all TRAV gene families and displayed unique CDR3 lengths. Our work demonstrates that the OP9-DL4 system supports the creation of a broad antigen-specific TCR repertoire, suggesting that T cells generated in vitro may undergo a different set of selection events that otherwise constrains the TCR repertoire of thymus-derived T cells.

Adoptive cell therapy using autologous tumor-infiltrating lymphocytes (TIL) has shown significant clinical benefit, but is limited by toxicities due to a requirement for post-infusion interleukin-2 (IL-2), for which high dose is standard. To assess a modified TIL protocol using lower dose IL-2, we performed a single institution phase II protocol in unresectable, metastatic melanoma. The primary endpoint was response rate. Secondary endpoints were safety and assessment of immune correlates following TIL infusion. Twelve metastatic melanoma patients were treated with non-myeloablative lymphodepleting chemotherapy, TIL, and low-dose subcutaneous IL-2 (125,000 IU/kg/day, maximum 9-10 doses over 2 weeks). All but one patient had previously progressed after treatment with immune checkpoint inhibitors. No unexpected adverse events were observed, and patients received an average of 6.8 doses of IL-2. By RECIST v1.1, two patients experienced a partial response, one patient had an unconfirmed partial response, and six had stable disease. Biomarker assessment confirmed an increase in IL-15 levels following lymphodepleting chemotherapy as expected and a lack of peripheral regulatory T-cell expansion following protocol treatment. Interrogation of the TIL infusion product and monitoring of the peripheral blood following infusion suggested engraftment of TIL. In one responding patient, a population of T cells expressing a T-cell receptor Vβ chain that was dominant in the infusion product was present at a high percentage in peripheral blood more than 2 years after TIL infusion. This study shows that this protocol of low-dose IL-2 following adoptive cell transfer of TIL is feasible and clinically active. (ClinicalTrials.gov identifier NCT01883323.).

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.

BackgroundCombining immunotherapy and antiangiogenic agents is a promising treatment strategy in endometrial cancer. To date, no biomarkers for response have been identified and data on post-immunotherapy progression are lacking. We explored the combination of a checkpoint inhibitor (nivolumab) and an antiangiogenic agent (cabozantinib) in immunotherapy-naïve endometrial cancer and in patients whose disease progressed on previous immunotherapy with baseline biopsy for immune profiling.Patients and methodsIn this phase II trial (ClinicalTrials.gov NCT03367741, registered December 11, 2017), women with recurrent endometrial cancer were randomized 2:1 to nivolumab with cabozantinib (Arm A) or nivolumab alone (Arm B). The primary endpoint was Response Evaluation Criteria in Solid Tumors-defined progression-free survival (PFS). Patients with carcinosarcoma or prior immune checkpoint inhibitor received combination treatment (Arm C). Baseline biopsy and serial peripheral blood mononuclear cell (PBMC) samples were analyzed and associations between patient outcome and immune data from cytometry by time of flight (CyTOF) and PBMCs were explored.ResultsMedian PFS was 5.3 (90% CI 3.5 to 9.2) months in Arm A (n=36) and 1.9 (90% CI 1.6 to 3.4) months in Arm B (n=18) (HR=0.59, 90% CI 0.35 to 0.98; log-rank p=0.09, meeting the prespecified statistical significance criteria). The most common treatment-related adverse events in Arm A were diarrhea (50%) and elevated liver enzymes (aspartate aminotransferase 47%, alanine aminotransferase 42%). In-depth baseline CyTOF analysis across treatment arms (n=40) identified 35 immune-cell subsets. Among immunotherapy-pretreated patients in Arm C, non-progressors had significantly higher proportions of activated tissue-resident (CD103+CD69+) ɣδ T cells than progressors (adjusted p=0.009).ConclusionsAdding cabozantinib to nivolumab significantly improved outcomes in heavily pretreated endometrial cancer. A subgroup of immunotherapy-pretreated patients identified by baseline immune profile and potentially benefiting from combination with antiangiogenics requires further investigation.

The aryl hydrocarbon receptor (AhR) is a sensor of products of tryptophan metabolism and a potent modulator of immunity. Here, we examined the impact of AhR in tumor-associated macrophage (TAM) function in pancreatic ductal adenocarcinoma (PDAC). TAMs exhibited high AhR activity and Ahr-deficient macrophages developed an inflammatory phenotype. Deletion of Ahr in myeloid cells or pharmacologic inhibition of AhR reduced PDAC growth, improved efficacy of immune checkpoint blockade, and increased intra-tumoral frequencies of IFNγ(+)CD8(+) T cells. Macrophage tryptophan metabolism was not required for this effect. Rather, macrophage AhR activity was dependent on Lactobacillus metabolization of dietary tryptophan to indoles. Removal of dietary tryptophan reduced TAM AhR activity and promoted intra-tumoral accumulation of TNFα(+)IFNγ(+)CD8(+) T cells; provision of dietary indoles blocked this effect. In patients with PDAC, high AHR expression associated with rapid disease progression and mortality, as well as with an immune-suppressive TAM phenotype, suggesting conservation of this regulatory axis in human disease.

The decision between T cell activation and tolerance is governed by the spatial and temporal integration of diverse molecular signals and events occurring downstream of TCR and costimulatory or coinhibitory receptor engagement. The PI3K–protein kinase B (PKB; also known as Akt) signaling pathway is a central axis in mediating proximal signaling events of TCR and CD28 engagement in T cells. Perturbation of the PI3K–PKB pathway, or the loss of negative regulators of T cell activation, such as the E3 ubiquitin ligase Cbl-b, have been reported to lead to increased susceptibility to autoimmunity. In this study, we further examined the molecular pathway linking PKB and Cbl-b in murine models. Our data show that the protein kinase GSK-3, one of the first targets identified for PKB, catalyzes two previously unreported phosphorylation events at Ser476 and Ser480 of Cbl-b. GSK-3 inactivation by PKB abrogates phosphorylation of Cbl-b at these two sites and results in reduced Cbl-b protein levels. We further show that constitutive activation of PKB in vivo results in a loss of tolerance that is mediated through the downregulation of Cbl-b. Altogether, these data indicate that the PI3K–PKB–GSK-3 pathway is a novel regulatory axis that is important for controlling the decision between T cell activation and tolerance via Cbl-b.

Notch activation, which is associated with basal-like breast cancer (BLBC), normally directs tissue patterning, suggesting that it may shape the tumor microenvironment. Here, we show that Notch in tumor cells regulates the expression of two powerful proinflammatory cytokines, IL1β and CCL2, and the recruitment of tumor-associated macrophages (TAM). Notch also regulates TGFβ-mediated activation of tumor cells by TAMs, closing a Notch-dependent paracrine signaling loop between these two cell types. We use a mouse model in which Notch can be regulated in spontaneous mammary carcinoma to confirm that IL1β and CCL2 production, and macrophage recruitment are Notch-dependent. In human disease, expression array analyses demonstrate a striking association between Notch activation, IL1β and CCL2 production, macrophage infiltration, and BLBC. These findings place Notch at the nexus of a vicious cycle of macrophage infiltration and amplified cytokine secretion and provide immunotherapeutic opportunities in BLBC. Significance: BLBC is aggressive and has an unmet need for effective targeted treatment. Our data highlight immunotherapeutic opportunities in Notch-activated BLBC. Effective IL1β and CCL2 antagonists are currently in clinical review to treat benign inflammatory disease, and their transition to the cancer clinic could have a rapid impact. Cancer Discov; 7(11); 1320–35. ©2017 AACR. This article is highlighted in the In This Issue feature, p. 1201

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

Regulatory T cells are integral to the regulation of autoimmune and anti-tumor immune responses. However, several studies have suggested that changes in T cell signaling networks can result in T cells that are resistant to the suppressive effects of regulatory T cells. Here, we investigated the role of Cbl-b, an E3 ubiquitin ligase, in establishing resistance to Treg-mediated suppression. We found that the absence of Cbl-b, a negative regulator of multiple TCR signaling pathways, rendered T cells impartial to Treg suppression by regulating cytokine networks leading to improved anti-tumor immunity despite the presence of Treg cells in the tumor. Specifically, Cbl-b KO CD4+FoxP3− T cells hyper-produced IL-2 and together with IL-2 Rα upregulation served as an essential mechanism to escape suppression by Treg cells. Furthermore, we report that IL-2 serves as the central molecule required for cytokine-induced Treg resistance. Collectively our data emphasize the role of IL-2 as a key mechanism that renders CD4+ T cells resistant to the inhibitory effects of Treg cells.

General control nonderepressible 2 (GCN2) is an environmental sensor controlling transcription and translation in response to nutrient availability. Although GCN2 is a putative therapeutic target for immuno-oncology, its role in shaping the immune response to tumors is poorly understood. Here, we used mass cytometry, transcriptomics, and transcription factor-binding analysis to determine the functional impact of GCN2 on the myeloid phenotype and immune responses in melanoma. We found that myeloid-lineage deletion of GCN2 drives a shift in the phenotype of tumor-associated macrophages and myeloid-derived suppressor cells (MDSCs) that promotes antitumor immunity. Time-of-flight mass cytometry (CyTOF) and single-cell RNA sequencing showed that this was due to changes in the immune microenvironment with increased proinflammatory activation of macrophages and MDSCs and interferon-γ expression in intratumoral CD8+ T cells. Mechanistically, GCN2 altered myeloid function by promoting increased translation of the transcription factor CREB-2/ATF4, which was required for maturation and polarization of macrophages and MDSCs in both mice and humans, whereas targeting Atf4 by small interfering RNA knockdown reduced tumor growth. Last, analysis of patients with cutaneous melanoma showed that GCN2-dependent transcriptional signatures correlated with macrophage polarization, T cell infiltrates, and overall survival. Thus, these data reveal a previously unknown dependence of tumors on myeloid GCN2 signals for protection from immune attack.

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.

The paracaspase Malt1 is a key regulator of canonical NF-κB activation downstream of multiple receptors in both immune and nonimmune cells. Genetic disruption of Malt1 protease function in mice and MALT1 mutations in humans results in reduced regulatory T cells and a progressive multiorgan inflammatory pathology. In this study, we evaluated the altered immune homeostasis and autoimmune disease in Malt1 protease-deficient (Malt1PD) mice and the Ags driving disease manifestations. Our data indicate that B cell activation and IgG1/IgE production is triggered by microbial and dietary Ags preferentially in lymphoid organs draining mucosal barriers, likely as a result of dysregulated mucosal immune homeostasis. Conversely, the disease was driven by a polyclonal T cell population directed against self-antigens. Characterization of the Malt1PD T cell compartment revealed expansion of T effector memory cells and concomitant loss of a CD4+ T cell population that phenotypically resembles anergic T cells. Therefore, we propose that the compromised regulatory T cell compartment in Malt1PD animals prevents the efficient maintenance of anergy and supports the progressive expansion of pathogenic, IFN-γ–producing T cells. Overall, our data revealed a crucial role of the Malt1 protease for the maintenance of intestinal and systemic immune homeostasis, which might provide insights into the mechanisms underlying IPEX-related diseases associated with mutations in MALT1.

The tumor microenvironment is shaped by interactions between tumor cells, stroma, and immune cells. T cells are a critical component of the immunological anti-tumor response and most immunotherapeutic approaches in cancer aim to augment T cell responses. Recently, innate lymphoid cells (ILCs) have been added to the list of cell populations that influence T cell responses within the tumor microenvironment. ILCs were shown to modulate T cell functions through a variety of mechanisms. However, the impact of ILC and T cell interactions on disease outcome appears to be contextual as their phenotype and function is strongly influenced by the local tissue microenvironment. Here we focus on recent advances in understanding the mechanisms of ILCs in regulating T cell responses in inflammatory diseases and cancer.

Maternal immune tolerance against paternal alloreactive antigens expressed by the fetus is essential in the development of successful pregnancies. Disturbances of immunoregulatory mechanisms by genetic modifications or viral infections results in aberrant placental development, intrauterine growth restriction (IUGR), and fetal loss. Mechanisms of immune regulation during pregnancy has not been fully elucidated. Our lab and others were the first to uncover a novel subset of regulatory innate lymphoid cells (ILCregs) that suppressed immune responses in chronic viral infections, organ transplant rejection, and cancer. In this study, we identified a subset of decidual NK1.1+ ILC1s in healthy murine pregnancies that expressed surface markers and intracellular cytokines reminiscent of an ILCreg phenotype. Interestingly, these characteristics were more abundant early in gestation compared to mid-late gestation. Furthermore, depletion of NK1.1+ ILC1s using neutralizing antibodies induced fetal destruction in allogeneic (C57BL/6J female X BALB/c male), but not in syngeneic pregnancies (C57BL/6J female X C57BL/6J male). This highlights the importance of allogenicity in fetal destruction in the absence of NK1.1+ ILC1s. Decidual ILC1s have been shown to promote trophoblast invasion, placental angiogenesis, and in utero fetal growth during pregnancy. However, we are one of the first groups to thoroughly investigate potential immunosuppressive role of decidual ILC1s to prevent immune-mediated fetal destruction. Further elucidation of immune tolerance mechanisms during pregnancy may uncover novel therapeutic targets for pregnancy-related complications such as preeclampsia and spontaneous miscarriages.

Innate-like T cells bind to non-peptide antigens in a TCR-dependent manner and are not MHC-restricted. This subpopulation of T cells consists of mucosal associated invariant T cells (MAIT), natural killer T cells (NKT), and gamma delta (γδ) T cells. Innate-like T cells can recognize microbially-derived antigens and interact with gut commensals to help maintain intestinal homeostasis. Additionally, they are implicated in tumour immunity as producers of cytokines and cytotoxic molecules. Thus, innate-like T cells may serve as an intermediary between the gut microbiota and tumor development. Using syngeneic mouse tumour models and flow cytometric analysis, we aim to evaluate the relationships between gut microbiota modulation, tumour growth, checkpoint inhibitor response, and innate-like T cell phenotype. Gut dysbiosis was induced using broad spectrum oral antibiotics two weeks prior to subcutaneous injection of MC38 colon adenocarcinoma cells. Mice with palpable tumours were treated twice with anti-PD-L1 during the experiment. Compared to vehicle control, tumor size reduction in response to anti-PD-L1 was not significantly altered with antibiotic treatment. The following observations were noted in antibiotic-treated mice. The frequency of γδ T cells was decreased in the tumour, blood, and spleen. MAIT, NKT, and γδ T cells in the spleen had lower expression of tissue localization-associated markers, CD103 and CXCR6. MAIT cell activation state, characterized by CD25 and CD69 expression, was also elevated in the spleen. These findings suggest that changes to the immune landscape of tumor-bearing mice due to antibiotic-driven dysbiosis is characterized by alterations in innate-like T cell abundance and phenotype.

Antigen recognition by T cells bearing αβ T cell receptors (TCRs) is restricted by major histocompatibility complex (MHC). However, how antigens are recognized by T cells bearing γδ TCRs remains unclear. Although γδ T cells can recognize nonclassical MHC, it is generally thought that recognition of antigens is not MHC restricted. Here, we took advantage of an in vitro system to generate antigen-specific human T cells and show that melanoma-associated antigens, MART-1 and gp100, can be recognized by γδ T cells in an MHC-restricted fashion. Cloning and transferring of MART-1-specific γδ TCRs restored the specific recognition of the initial antigen MHC/peptide reactivity and conferred antigen-specific functional responses. A crystal structure of a MART-1-specific γδ TCR, together with MHC/peptide, revealed distinctive but similar docking properties to those previously reported for αβ TCRs, recognizing MART-1 on HLA-A*0201. Our work shows that antigen-specific and MHC-restricted γδ T cells can be generated in vitro and that MART-1-specific γδ T cells can also be found and cloned from the naive repertoire. These findings reveal that classical MHC-restricted human γδ TCRs exist in the periphery and have the potential to be used in developing of new TCR-based immunotherapeutic approaches.

Very little is known about the conformational properties of the MHC molecules that are able to signal positive selection of a given TCR. To try to understand these parameters and to determine whether these requirements are shared with interactions during negative selection and antigen recognition, we have studied selection and antigen recognition of a transgenic TCR (specific for lymphocytic choriomeningitls virus glycoprotein and H-2Db) in the context of two Db mutants, H-2bm13 and H-2bm14. The data showed that the transgenic TCR was not positively selected by the H-2bm14 haplotype but, interestingly, enhanced positive selection was seen in H-2bm13 mice. The transgenic TCR could not be negatively selected In H-2bm13animals persistently infected with the virus (neonatal virus carrier mice), nor could the transgenic TCR be activated by H-2bm13 infected cells in vivo or in vitro. These experiments show that although a TCR may be selected by a mutant MHC molecule, the corresponding viral antigen cannot be recognized in the context of the mutant MHC molecule, as Judged by both negative selection and T cell reactivity in vivo and in vitro. The ‘enhanced' positive selection occurring in the context of Dbm13 suggests that a different conformation of the MHC molecule is able to select the same TCR and also that various TCR-ligand avidities may permit positive selection

Dysfunctional T cells can render the immune system unable to eliminate infections and cancer. Therapeutic targeting of the surface receptors that inhibit T cell function has begun to show remarkable success in clinical trials. In this Review, we discuss the potential mechanisms of action of the clinical agents that target two of these receptors, programmed cell death protein 1 (PD1) and lymphocyte activation gene 3 protein (LAG3). We also suggest correlative studies that may define the predominant mechanisms of action and identify predictive biomarkers.

2537 Background: Adoptive transfer of T cell receptor (TCR) gene-engineered T cells can induce durable anti-cancer responses. Post-infusion cytokine release syndrome (CRS) has been associated with clinical utility. Pre-infusion lymphodepletion (LD) may influence CRS, graft persistence, and clinical responses. While the optimal LD regimen is not yet defined, most include both cyclophosphamide (CY) and fludarabine (FLU). TBI-1301 is a novel gene therapy produced by engineering autologous lymphocytes to express an NY-ESO-1-specific TCR using a retrovirus vector that encodes siRNA to silence endogenous TCR. Since less intensive LD may be sufficient with the use of this novel vector, we are conducting a study where patients are treated with TBI-1301 following LD with CY alone. Methods: Eligibility includes informed consent, HLA-A*02:01 or A*02:06 haplotype, and NY-ESO-1 expression by immunohistochemistry. Eligible patients undergo harvest of PBMC which are then processed locally to generate engineered TBI-1301 cells. The study design is to infuse 5x109 cells (day 0) to patients following LD with CY (750 mg/m2 on day -3 and -2). Endpoints include safety, efficacy, and biological correlates for persistence of NY-ESO-1-specific T cells post infusion. Results: Thus far, 9 patients have been treated, and 8 have received the target dose. To date, 8 patients are evaluable for response and toxicity, and no DLTs have been observed. Despite LD with CY alone, all 4 patients with synovial sarcoma and 1 with melanoma experienced clinical and laboratory evidence of grade 1-2 CRS with increased CRP, ferritin, and IL-6 levels. CRS resolved spontaneously in all but one patient who required tocilizumab due to grade 2 nausea/vomiting. Two subjects experienced grade 3 tumor-associated pain. Other treatment-associated grade 3 or 4 toxicities included neutropenia and hypophosphatemia. Best overall response by RECIST is as follows: 2 partial responses, 5 stable disease, and 1 progressive disease. Biomarker analysis demonstrates persistence of transferred TBI-1301 cells, > 100 days in some patients. Conclusions: TBI-1301 appears to be safe and to possess anti-tumor activity. Despite LD with CY alone, grade 1-2 CRS is induced. Additional cohorts to this study will examine the role of repeat infusions to enhance anti-tumor activity. Clinical trial information: NCT02869217.

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.

The activation of T cells is a tightly regulated process that has evolved to maximize protective immune responses to pathogens while minimizing damage to self-tissues. A delicate balance of cell-intrinsic, costimulatory, and transcriptional pathways as well as micro-environmental cues such as local cytokines controls the magnitude and nature of T-cell responses in vivo. The discovery of functional small noncoding RNAs called micro-RNAs (miRNAs) has introduced new mechanisms that contribute to the regulation of protein translation and cellular responses to stimuli. miRNAs are short (approximately 22 bp) RNA species, which bind to mRNAs and suppress translation. Due to their short length and imperfect base pairing requirements, each miRNA has the potential to regulate various pathways through the translational inhibition of multiple mRNAs. The human and mouse genomes each encode hundreds of miRNAs, and studying the function of miRNAs has led to the realization that they play important roles in diverse biological processes from development and cancer to immunity. This review focuses on the function of mir-155 in T cells and the impact of this miRNA on autoimmunity, tumor immunity, and pathogen-induced immunity.

Differentiation of CD8 single-positive (SP) T cells is predicated by the ability of lymphocyte progenitors to integrate multiple signaling cues provided by the thymic microenvironment. In the thymus and the OP9-DL1 system for T cell development, Notch signals are required for progenitors to commit to the T cell lineage and necessary for their progression to the CD4+CD8+ double-positive (DP) stage of T cell development. However, it remains unclear whether Notch is a prerequisite for the differentiation of DP cells to the CD8 SP stage of development. In this study, we demonstrate that Notch receptor–ligand interactions allow for efficient differentiation and selection of conventional CD8 T cells from bone marrow–derived hematopoietic stem cells. However, bone marrow–derived hematopoietic stem cells isolated from Itk−/−Rlk−/− mice gave rise to T cells with decreased IFN-γ production, but gained the ability to produce IL-17. We further reveal that positive and negative selection in vitro are constrained by peptide–MHC class I expressed on OP9 cells. Finally, using an MHC class I–restricted TCR-transgenic model, we show that the commitment of DP precursors to the CD8 T cell lineage is dependent on Notch signaling. Our findings further establish the requirement for Notch receptor–ligand interactions throughout T cell differentiation, including the final step of CD8 SP selection.

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.

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.

Recent studies have begun to define the role of micro-RNAs in regulating the immune response. Micro-RNA155 (mir-155) has been shown to play a role in germinal center formation, T cell inflammation, and regulatory T cell development. In this study, we evaluated the role of mir-155 in cytotoxic T cell function. We report in this study that mice lacking mir-155 have impaired CD8+ T cell responses to infections with lymphocytic choriomeningitis virus and the intracellular bacteria Listeria monocytogenes. We show by a series of adoptive transfer studies that the impaired CD8+ T cell response to L. monocytogenes is T cell intrinsic. In addition, we observed that CD8+ T cells lacking mir-155 have impaired activation of the prosurvival Akt pathway after TCR cross-linking. These data suggest that mir-155 may be a good target for therapies aimed at modulating immune responses.

Cluster of differentiation (CD)8(+) T cells are like a double edged sword during chronic viral infections because they not only promote virus elimination but also induce virus-mediated immunopathology. Elevated levels of reactive oxygen species (ROS) have been reported during virus infections. However, the role of ROS in T-cell-mediated immunopathology remains unclear. Here we used the murine lymphocytic choriomeningitis virus to explore the role of ROS during the processes of virus elimination and induction of immunopathology. We found that virus infection led to elevated levels of ROS producing granulocytes and macrophages in virus-infected liver and spleen tissues that were triggered by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Lack of the regulatory subunit p47phox of the NADPH oxidase diminished ROS production in these cells. While CD8(+) T cells exhibited ROS production that was independent of NADPH oxidase expression, survival and T-cell function was elevated in p47phox-deficient (Ncf1(-/-)) mice. In the absence of p47phox, enhanced T-cell immunity promoted virus elimination and blunted corresponding immunopathology. In conclusion, we find that NADPH-mediated production of ROS critically impairs the immune response, impacting elimination of virus and outcome of liver cell damage.

Immunotherapy is a promising means to fight cancer, prompting a steady increase in clinical trials and correlative laboratory studies in this field. As antitumor T cells play central roles in immunity against malignant diseases, most immunotherapeutic protocols aim to induce and/or strengthen their function. Various treatment strategies have elicited encouraging clinical responses; however, major challenges have been uncovered that should be addressed in order to fully exploit the potential of immunotherapy. Here, we outline pitfalls for the mobilization of antitumor T cells and offer solutions to improve their therapeutic efficacy. We provide a critical perspective on the main methodologies used to characterize T-cell responses to cancer therapies, with a focus on discrepancies between T-cell attributes measured in vitro and protective responses in vivo. This review altogether provides recommendations to optimize the design of future clinical trials and highlights important considerations for the proficient analysis of clinical specimens available for research.

O1 Combinatorial CD8+ and PD-L1+ cell densities correlate with response and improved survival in non-small cell lung cancer (NSCLC) patients treated with durvalumab #### Sonja Althammer1, Keith Steele2, Marlon Rebelatto2, Tze Heng Tan1, Tobias Wiestler1, Guenter Schmidt1, Brandon Higgs2, Xia

Adoptive transfer of T cells with engineered T-cell receptor (TCR) genes that target tumor-specific antigens can mediate cancer regression. Accumulating evidence suggests that the clinical success of many immunotherapies is mediated by T-cells targeting mutated neoantigens unique to the patient. We hypothesized that the most frequent TCR clonotypes infiltrating the tumor were reactive against tumor antigens. To test this, we developed a multi-step strategy that involved TCRB deep sequencing of the CD8+PD-1+ T-cell subset, matching of TCRA-TCRB pairs by pairSEQ and single cell RT-PCR, followed by testing of the TCRs for tumor-antigen specificity. Analysis of 12 fresh metastatic melanomas revealed that in 11 samples, up to 5 tumor-reactive TCRs were present in the 5 most frequently occurring clonotypes, which included reactivity against neoantigens. These data demonstrate the feasibility of developing a rapid, personalized, TCR-gene therapy approach that targets the unique set of antigens presented by the autologous tumor without the need to identify their immunologic reactivity.

Accumulating evidence suggests that proteins tethered to the plasma membrane through glycosylphosphatidylinositol (GPI) anchors share common biological properties. In the present study we demonstrate that GPI-anchored proteins regulate T cell growth. Specifically, anti-TCR-induced proliferation was profoundly inhibited by co-immobilized mAb specific for Thy-1, CD48 and Ly6A/E. However, neither IL-2 production nor the effector function of cytotoxic T lymphocytes was impaired in these circumstances. Analysis of the IL-2 receptor (IL-2R) signaling pathway revealed that the association of IL-2R beta and gamma chains with the Janus kinases, JAK1 and JAK3, was not perturbed in the presence of mAb specific for GPI-linked proteins. However, in these conditions, IL-2-mediated recruitment of IL-2Ralpha, beta and gamma chains, resulting in the formation of the high-affinity hetero-trimeric IL-2R, was inhibited. The resulting phosphorylation of JAK1 and JAK3, indicative of their activation states, was correspondingly reduced. These results characterize a novel state of T cell physiology in which effector function is maintained, in the absence of clonal expansion. A physiological role for GPI-anchored proteins in the maintenance of cellular homeostasis and function is discussed.

CD45 is expressed on all B cells and has been reported to be essential for their B cell receptor mediated stimulation. The present study addressed T-cell-independent B cell responses in CD45-deficient mice using the glycoprotein (G) of vesicular stomatitis virus (VSV) as a model antigen. VSV-G exists in two forms exhibiting different degrees of repetitiveness. In mice, the two forms of VSV-G induce either a type 1 or a type 2 T-cell-independent B cell response. We found that CD45-deficient mice mounted T-cell-independent B cell responses to both forms of VSV-G. This demonstrates that the B cell receptor complex is able to generate a functional signal in the absence of CD45, provided the cross-linking stimulus is sufficiently strong.

The protooncogene Vav functions as a GDP/GTP exchange factor (GEF) for Rho-like small GTPases involved in cytoskeletal reorganization and cytokine production in T cells. Gene-targeted mice lacking Vav have a severe defect in positive and negative selection of T cell antigen receptor transgenic thymocytes in vivo, and vav−/− thymocytes are completely resistant to peptide-specific and anti-CD3/anti-CD28–mediated apoptosis. Vav acts upstream of mitochondrial pore opening and caspase activation. Biochemically, Vav regulates peptide-specific Ca2+ mobilization and actin polymerization. Peptide-specific cell death was blocked both by cytochalasin D inhibition of actin polymerization and by inhibition of protein kinase C (PKC). Activation of PKC with phorbol ester restored peptide-specific apoptosis in vav−/− thymocytes. Vav was found to bind constitutively to PKC-θ in thymocytes. Our results indicate that peptide-triggered thymocyte apoptosis is mediated via Vav activation, changes in the actin cytoskeleton, and subsequent activation of a PKC isoform.

Allogeneic bone marrow transplantation is frequently associated with graft-vs-host disease (GVHD). To understand the effector mechanisms of GVHD, we investigated the role of the TNF receptor p55 (TNFRp55), which is known to be important in inflammation and cytotoxicity. After the transplantation of allogeneic bone marrow and spleen cells to lethally irradiated mice, all wild-type recipients developed early lethal GVHD within 1 wk, whereas TNFRp55-deficient recipients had much reduced GVHD and survived for at least 3 wk. No defect in alloantigen presentation was found, since T cell proliferation and cytotoxicity were similar to allogeneic wild-type and TNFRp55-deficient stimulator and target cells. Also, TNF alpha release did not differ significantly between the two types of recipients. Therefore, early acute GVHD in wild-type mice was primarily due to TNFRp55-mediated tissue damage. Interestingly, lethal GVHD was not entirely dependent upon the TNFRp55. In experimental conditions using sublethal irradiation and high donor spleen cell numbers, TNFRp55-deficient recipient mice developed lethal GVHD with similar kinetics and frequency as the control mice. These data suggest that the effector mechanisms leading to organ damage in murine acute GVHD can be dissected in a cytokine pathway through the TNFRp55, as demonstrated here, and in a cellular pathway through direct interaction of cytotoxic lymphocytes with target tissues involving perforin and Fas/Fas ligand, as reported previously.

T cells play a central role in the elimination of both intracellular and extracellular pathogens. Expression of the αβ T cell receptor (TCR) heterodimer enables T cells to specifically recognize peptides presented by major histocompatibility complex (MHC) molecules and generate an immune response to those peptides. CD4+ helper T cells are restricted to MHC class II molecules, and produce cytokines to augment T and B cell responses. CD8+ T cells are restricted to MHC class I molecules, and are generally cytotoxic T lymphocytes (CTLs) whose effector function involves the lysis of infected cells. Broad issues in T cell biology include how mature T cells develop from bone marrow-derived precursors and how T cell activation is regulated. These questions and others have been addressed in many models, including murine TCR transgenic systems, in which the transgenic expression of TCR chains provides a clonal population of antigen-specific T cells that can be readily monitored.