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Regulatory T (T(reg)) cells are an immunosuppressive population that are required to maintain peripheral tolerance and prevent tissue damage from immunopathology, via anti-inflammatory cytokines, inhibitor receptors and metabolic disruption. Here we show that T(reg) cells acquire an effector-like state, yet remain stable and functional, when exposed to interferon gamma (IFNγ) during infection with lymphocytic choriomeningitis and influenza A virus. T(reg) cell-restricted deletion of the IFNγ receptor (encoded by Ifngr1), but not the interleukin 12 (IL12) receptor (encoded by Il12rb2), prevented T(H)1-like polarization (decreased expression of T-bet, CXC motif chemokine receptor 3 and IFNγ) and promoted T(H)2-like polarization (increased expression of GATA-3, CCR4 and IL4). T(H)1-like T(reg) cells limited CD8(+) T cell effector function, proliferation and memory formation during acute and chronic infection. These findings provide fundamental insights into how T(reg) cells sense inflammatory cues from the environment (such as IFNγ) during viral infection to provide guidance to the effector immune response. This regulatory circuit prevents prolonged immunoinflammatory responses and shapes the quality and quantity of the memory T cell response.

PDL1 is a protein that induces immunosuppression by binding to PD1 expressed on immune cells. In line with historical studies, we found that membrane-bound PD1 expression was largely restricted to immune cells; PD1 was not detectable at either the mRNA or protein level in peripheral neurons using single neuron qPCR, immunolabeling and flow cytometry. However, we observed widespread expression of PDL1 in both sensory and sympathetic neurons that could have important implications for patients receiving immunotherapies targeting this pathway that include unexpected autonomic and sensory related effects. While signaling pathways downstream of PD1 are well established, little to no information is available regarding the intracellular signaling downstream of membrane-bound PDL1 (also known as reverse signaling). Here, we administered soluble PD1 to engage neuronally expressed PDL1 and found that PD1 significantly reduced nocifensive behaviors evoked by algogenic capsaicin. We used calcium imaging to examine the underlying neural mechanism of this reduction and found that exogenous PD1 diminished TRPV1-dependent calcium transients in dissociated sensory neurons. Furthermore, we observed a reduction in membrane expression of TRPV1 following administration of PD1. Exogenous PD1 had no effect on pain-related behaviors in sensory neuron specific PDL1 knockout mice. These data indicate that neuronal PDL1 activation is sufficient to modulate sensitivity to noxious stimuli and as such, may be an important homeostatic mechanism for regulating acute nociception.

The spread of prion-like protein aggregates is believed to be a common driver of pathogenesis in many neurodegenerative diseases. Accumulated tangles of filamentous Tau protein are considered pathogenic lesions of Alzheimer’s disease (AD) and related Tauopathies, including progressive supranuclear palsy, and corticobasal degeneration. Tau pathologies in these illnesses exhibits a clear progressive and hierarchical spreading pattern that correlates with disease severity1, 2. Clinical observation combined with complementary experimental studies3, 4have shown that Tau preformed fibrils (PFF) are prion-like seeds that propagate pathology by entering cells and templating misfolding and aggregation of endogenous Tau. While several receptors of Tau are known, they are not specific to the fibrillar form of Tau. Moreover, the underlying cellular mechanisms of Tau PFF spreading remains poorly understood. Here, we show that the lymphocyte-activation gene 3 (Lag3) is a cell surface receptor that binds to PFF, but not monomer, of Tau. Deletion ofLag3or inhibition of Lag3 in primary cortical neurons significantly reduces the internalization of Tau PFF and subsequent Tau propagation and neuron-to-neuron transmission. Propagation of Tau pathology and behavioral deficits induced by injection of Tau PFF in the hippocampus and overlying cortex are attenuated in mice lackingLag3selectively in neurons. Our results identify neuronal Lag3 as a receptor of pathologic Tau in the brain, and for AD and related Tauopathies a therapeutic target.One Sentence SummaryLag3 is a neuronal receptor specific for Tau PFF, and is required for uptake, propagation and transmission of Tau pathology.

Supplementary Data from Systemic Immune Dysfunction in Cancer Patients Driven by IL6 Induction of LAG3 in Peripheral CD8+ T Cells

Many cancer patients do not develop a durable response to the current standard-of-care immunotherapies, despite substantial advances in targeting immune inhibitory receptors. A potential compounding issue, which may serve as an unappreciated, dominant resistance mechanism, is an inherent systemic immune dysfunction that is often associated with advanced cancer. Minimal response to inhibitory receptor (IR) blockade therapy and increased disease burden have been associated with peripheral CD8+ T-cell dysfunction, characterized by suboptimal T-cell proliferation and chronic expression of IRs (e.g., PD1 and LAG3). Here, we demonstrated that approximately a third of cancer patients analyzed in this study have peripheral CD8+ T cells that expressed robust intracellular LAG3 (LAG3IC), but not surface LAG3 (LAG3SUR) due to a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) cleavage. This is associated with poor disease prognosis and decreased CD8+ T-cell function, which could be partially reversed by anti-LAG3. Systemic immune dysfunction was restricted to CD8+ T cells, including, in some cases, a high percentage of peripheral naïve CD8+ T cells, and was driven by the cytokine IL6 via STAT3. These data suggest that additional studies are warranted to determine if the combination of increased LAG3IC in peripheral CD8+ T cells and elevated systemic IL6 can serve as predictive biomarkers and identify which cancer patients may benefit from LAG3 blockade.

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Many cancer patients do not develop a durable response to the current standard-of-care immunotherapies, despite substantial advances in targeting immune inhibitory receptors. A potential compounding issue, which may serve as an unappreciated, dominant resistance mechanism, is an inherent systemic immune dysfunction that is often associated with advanced cancer. Minimal response to inhibitory receptor (IR) blockade therapy and increased disease burden have been associated with peripheral CD8+ T-cell dysfunction, characterized by suboptimal T-cell proliferation and chronic expression of IRs (e.g., PD1 and LAG3). Here, we demonstrated that approximately a third of cancer patients analyzed in this study have peripheral CD8+ T cells that expressed robust intracellular LAG3 (LAG3IC), but not surface LAG3 (LAG3SUR) due to a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) cleavage. This is associated with poor disease prognosis and decreased CD8+ T-cell function, which could be partially reversed by anti-LAG3. Systemic immune dysfunction was restricted to CD8+ T cells, including, in some cases, a high percentage of peripheral naïve CD8+ T cells, and was driven by the cytokine IL6 via STAT3. These data suggest that additional studies are warranted to determine if the combination of increased LAG3IC in peripheral CD8+ T cells and elevated systemic IL6 can serve as predictive biomarkers and identify which cancer patients may benefit from LAG3 blockade.

TRAIL (Tnfsf10/TRAIL/CD253/Apo2L) is an important immune molecule that mediates apoptosis. TRAIL can play key roles in regulating cell death in the tumor and autoimmune microenvironments. However, dissecting TRAIL function remains difficult because of the lack of optimal models. We have now generated a conditional knockout (Tnfsf10L/L) for cell type–specific analysis of TRAIL function on C57BL/6, BALB/c, and NOD backgrounds. Previous studies have suggested a role for TRAIL in regulatory T cell (Treg)–mediated suppression. We generated mice with a Treg-restricted Tnfsf10 deletion and surprisingly found no impact on tumor growth in C57BL/6 and BALB/c tumor models. Furthermore, we found no difference in the suppressive capacity of Tnfsf10-deficient Tregs and no change in function or proliferation of T cells in tumors. We also assessed the role of TRAIL on Tregs in two autoimmune mouse models: the NOD mouse model of autoimmune diabetes and the myelin oligodendrocyte glycoprotein (MOG) C57BL/6 model of experimental autoimmune encephalomyelitis. We found that deletion of Tnfsf10 on Tregs had no effect on disease progression in either model. We conclude that Tregs do not appear to be dependent on TRAIL exclusively as a mechanism of suppression in both the tumor and autoimmune microenvironments, although it remains possible that TRAIL may contribute in combination with other mechanisms and/or in different disease settings. Our Tnfsf10 conditional knockout mouse should prove to be a useful tool for the dissection of TRAIL function on different cell populations in multiple mouse models of human disease.

Lymphocyte activation gene 3 protein (LAG3; CD223) is an inhibitory receptor that is highly upregulated on exhausted T cells in tumors and chronic viral infection. Consequently, LAG3 is now a major immunotherapeutic target for the treatment of cancer, and many mAbs against human (h) LAG3 (hLAG3) have been generated to block its inhibitory activity. However, little or no information is available on the epitopes they recognize. We selected a panel of seven therapeutic mAbs from the patent literature for detailed characterization. These mAbs were expressed as Fab or single-chain variable fragments and shown to bind hLAG3 with nanomolar affinities, as measured by biolayer interferometry. Using competitive binding assays, we found that the seven mAbs recognize four distinct epitopes on hLAG3. To localize the epitopes, we carried out epitope mapping using chimeras between hLAG3 and mouse LAG3. All seven mAbs are directed against the first Ig-like domain (D1) of hLAG3, despite their different origins. Three mAbs almost exclusively target a unique 30-residue loop of D1 that forms at least part of the putative binding site for MHC class II, whereas four mainly recognize D1 determinants outside this loop. However, because all the mAbs block binding of hLAG3 to MHC class II, each of the epitopes they recognize must at least partially overlap the MHC class II binding site.

Impaired chronic viral and tumor clearance has been attributed to CD8

The success of immunotherapy in oncology underscores the vital role of the immune system in cancer development. Regulatory T cells (Tregs) maintain a fine balance between autoimmunity and immune suppression. They have multiple roles in the tumor microenvironment (TME) but act particularly in suppressing T cell activation. This review focuses on the detrimental and sometimes beneficial roles of Tregs in tumors, our current understanding of recruitment and stabilization of Tregs within the TME, and current Treg-targeted therapeutics. Research identifying subpopulations of Tregs and their respective functions and interactions within the complex networks of the TME will be crucial to develop the next generation of immunotherapies. Through these advances, Treg-targeted immunotherapy could have important implications for the future of oncology.

Robust CD8+ T cell memory is essential for long-term protective immunity but is often compromised in cancer, where T cell exhaustion leads to loss of memory precursors. Immunotherapy via checkpoint blockade may not effectively reverse this defect, potentially underlying disease relapse. Here we report that mice with a CD8+ T cell–restricted neuropilin-1 (NRP1) deletion exhibited substantially enhanced protection from tumor rechallenge and sensitivity to anti-PD1 immunotherapy, despite unchanged primary tumor growth. Mechanistically, NRP1 cell-intrinsically limited the self-renewal of the CD44+PD1+TCF1+TIM3− progenitor exhausted T cells, which was associated with their reduced ability to induce c-Jun/AP-1 expression on T cell receptor restimulation, a mechanism that may contribute to terminal T cell exhaustion at the cost of memory differentiation in wild-type tumor-bearing hosts. These data indicate that blockade of NRP1, a unique ‘immune memory checkpoint’, may promote the development of long-lived tumor-specific Tmem that are essential for durable antitumor immunity. T cell exhaustion limits antitumor immune responses. Vignali and colleagues identify neuropilin-1 as a novel immune checkpoint that cell-intrinsically operates to limit memory cell formation and can be targeted to enhance antitumor responses.

Immunotherapy targeting coinhibitory receptors has been highly successful in treating a wide variety of malignancies; however, only a subset of patients exhibits durable responses. The first FDA-approved immunotherapeutics targeting coinhibitory receptors PD1 and CTLA4, alone or in combination, significantly improved survival but were also accompanied by substantial toxicity in combination. The third FDA-approved immune checkpoint inhibitor targets LAG3, a coinhibitory receptor expressed on activated CD4+ and CD8+ T cells, especially in settings of long-term antigenic stimulation, such as chronic viral infection or cancer. Mechanistically, LAG3 expression limits both the expansion of activated T cells and the size of the memory pool, suggesting that LAG3 may be a promising target for immunotherapy. Importantly, the mechanism(s) by which LAG3 contributes to CD8+ T-cell exhaustion may be distinct from those governed by PD1, indicating that the combination of anti-LAG3 and anti-PD1 may synergistically enhance antitumor immunity. Clinical studies evaluating the role of anti-LAG3 in combination with anti-PD1 are underway, and recent phase III trial results in metastatic melanoma demonstrate both the efficacy and safety of this combination. Further ongoing clinical trials are evaluating this combination across multiple tumor types and the adjuvant setting, with accompanying translational and biomarker-focused studies designed to elucidate the molecular pathways that lead to improved antitumor T-cell responses following dual blockade of PD1 and LAG3. Overall, LAG3 plays an important role in limiting T-cell activation and has now become part of the repertoire of combinatorial immunotherapeutics available for the treatment of metastatic melanoma.

While there have been extensive analyses characterizing cellular and humoral responses across the severity spectrum in COVID-19, predictors of outcomes within severe COVID-19 remain to be comprehensively elucidated. Recently, we identified divergent monocyte states as predictors of outcomes within severe COVID-19, but corresponding humoral profiles of risk have not been delineated. Furthermore, the nature of antibodies (Abs) directed against viral antigens beyond the spike protein or endemic coronavirus antigens and their associations with disease severity and outcomes remain poorly defined. We performed deep molecular profiling of Abs directed against a wide range of antigenic specificities in severe COVID-19 patients admitted to the ICU. The profiles consisted of canonical (S, RBD, N) and non-canonical (orf3a, orf8, nsp3, nps13 and M) antigenic specificities. Notably, multivariate machine learning (ML) models, generated using profiles of Abs directed against canonical or non-canonical antigens, were equally discriminative of recovery and mortality COVID-19 outcomes. In both ML models, survivors were associated with increased virus-specific IgA and IgG3 antibodies and with higher antigen-specific antibody galactosylation. Intriguingly, pre-pandemic healthy controls had cross-reactive Abs directed against nsp13 which is a conserved protein in other alpha and beta coronaviruses. Notably, higher levels of nsp13-specific IgA antibodies were associated with recovery in severe COVID-19. In keeping with these findings, a model built on Ab profiles for endemic coronavirus antigens was also predictive of COVID-19 outcome bifurcation, with higher levels of IgA and IgG3 antibodies against OC43 S and NL63 S being associated with survival. Our results suggest the importance of Abs targeting non-canonical SARS-CoV-2 antigens as well as those directed against endemic coronaviruses in favorable outcomes of severe COVID-19.

Expression of the transmembrane protein Tim-3 is increased on dysregulated T cells undergoing chronic activation, including during chronic infection and in solid tumors. Thus, Tim-3 is generally thought of as an inhibitory protein. We and others previously reported that under some circumstances, Tim-3 exerts paradoxical costimulatory activity in T cells (and other cells), including enhancement of the phosphorylation of ribosomal S6 protein. Here, we examined the upstream signaling pathways that control Tim-3-mediated increases in phosphorylated S6 in T cells. We also defined the localization of Tim-3 relative to the T cell immune synapse and its effects on downstream signaling. Recruitment of Tim-3 to the immune synapse was mediated exclusively by the transmembrane domain, replacement of which impaired the ability of Tim-3 to costimulate T cell receptor (TCR)-dependent S6 phosphorylation. Furthermore, enforced localization of the Tim-3 cytoplasmic domain to the immune synapse in a chimeric antigen receptor still enabled T cell activation. Together, our findings are consistent with a model whereby Tim-3 enhances TCR-proximal signaling under acute conditions.

Regulatory T (Treg) cells play a crucial role in maintaining self‐tolerance and resolution of immune responses by employing multifaceted immunoregulatory mechanisms. However, Treg cells readily infiltrate into the tumor microenvironment (TME) and dampen anti‐tumor immune responses, thereby becoming a barrier to effective cancer immunotherapy. There has been a substantial expansion in the development of novel immunotherapies targeting various inhibitory receptors (IRs), such as CTLA4, PD1 and LAG3, but these approaches have mechanistically focused on the elicitation of anti‐tumor responses. However, enhanced inflammation in the TME could also play a detrimental role by facilitating the recruitment, stability and function of Treg cells by up‐regulating chemokines that promote Treg cell migration, and/or increasing inhibitory cytokine production. Furthermore, IR blockade may enhance Treg cell function and survival, thereby serving as a resistance mechanism against effective immunotherapy. Given that Treg cells are comprised of functionally and phenotypically heterogeneous sub‐populations that may alter their characteristics in a context‐dependent manner, it is critical to identify unique molecular pathways that are preferentially used by intratumoral Treg cells. In this review, we discuss markers that serve to identify certain Treg cell subsets, distinguished by chemokine receptors, IRs and cytokines that facilitate their migration, stability and function in the TME. We also discuss how these Treg cell subsets correlate with the clinical outcome of patients with various types of cancer and how they may serve as potential TME‐specific targets for novel cancer immunotherapies.

Regulatory T cells (Treg cells) maintain host self-tolerance but are a major barrier to effective cancer immunotherapy. Treg cells subvert beneficial anti-tumor immunity by modulating inhibitory receptor expression on tumor-infiltrating lymphocytes (TILs); however, the underlying mediators and mechanisms have remained elusive. Here, we found that the cytokines IL-10 and IL-35 (Ebi3-IL-12α heterodimer) were divergently expressed by Treg cell subpopulations in the tumor microenvironment (TME) and cooperatively promoted intratumoral T cell exhaustion by modulating several inhibitory receptor expression and exhaustion-associated transcriptomic signature of CD8+ TILs. While expression of BLIMP1 (encoded by Prdm1) was a common target, IL-10 and IL-35 differentially affected effector T cell versus memory T cell fates, respectively, highlighting their differential, partially overlapping but non-redundant regulation of anti-tumor immunity. Our results reveal previously unappreciated cooperative roles for Treg cell-derived IL-10 and IL-35 in promoting BLIMP1-dependent exhaustion of CD8+ TILs that limits effective anti-tumor immunity.

Pathologic α-synuclein (α-syn) spreads from cell-to-cell, in part, through binding to the lymphocyte-activation gene 3 (Lag3). Here we report that amyloid β precursor-like protein 1 (Aplp1) forms a complex with Lag3 that facilitates the binding, internalization, transmission, and toxicity of pathologic α-syn. Deletion of both Aplp1 and Lag3 eliminates the loss of dopaminergic neurons and the accompanying behavioral deficits induced by α-syn preformed fibrils (PFF). Anti-Lag3 prevents the internalization of α-syn PFF by disrupting the interaction of Aplp1 and Lag3, and blocks the neurodegeneration induced by α-syn PFF in vivo. The identification of Aplp1 and the interplay with Lag3 for α-syn PFF induced pathology advances our understanding of the molecular mechanism of cell-to-cell transmission of pathologic α-syn and provides additional targets for therapeutic strategies aimed at preventing neurodegeneration in Parkinson’s disease and related α-synucleinopathies.One Sentence SummaryAplp1 forms a complex with Lag3 that facilitates the binding, internalization, transmission, and toxicity of pathologic α-synuclein.Graphical AbstractAplp1 and the Aplp1-Lag3 complex facilitates transmission of pathologic α-synuclein.Aplp1 is a receptor that drives pathologic α-syn transmission, and genetic depletion of Aplp1 can significantly reduce the α-synuclein pathogenesis. Aplp1 and Lag3 forms an Aplp1-Lag3 complex that accounts for substantial binding of pathologic α-syn to cortical neurons. Together Aplp1 and Lag3 play a major role in pathologic α-syn internalization, transmission and toxicity. Double knockout of Aplp1 and Lag3 and or a Lag3 antibody that disrupts the Aplp1 and Lag3 complex almost completely blocks α-syn PFF-induced neurodegeneration.

Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 infection presents with varied clinical manifestations1, ranging from mild symptoms to acute respiratory distress syndrome (ARDS) with high mortality2,3. Despite extensive analyses, there remains an urgent need to delineate immune cell states that contribute to mortality in severe COVID-19. We performed high-dimensional cellular and molecular profiling of blood and respiratory samples from critically ill COVID-19 patients to define immune cell genomic states that are predictive of outcome in severe COVID-19 disease. Critically ill patients admitted to the intensive care unit (ICU) manifested increased frequencies of inflammatory monocytes and plasmablasts that were also associated with ARDS not due to COVID-19. Single-cell RNAseq (scRNAseq)-based deconvolution of genomic states of peripheral immune cells revealed distinct gene modules that were associated with COVID-19 outcome. Notably, monocytes exhibited bifurcated genomic states, with expression of a cytokine gene module exemplified by CCL4 (MIP-1β) associated with survival and an interferon signaling module associated with death. These gene modules were correlated with higher levels of MIP-1β and CXCL10 levels in plasma, respectively. Monocytes expressing genes reflective of these divergent modules were also detectable in endotracheal aspirates. Machine learning algorithms identified the distinctive monocyte modules as part of a multivariate peripheral immune system state that was predictive of COVID-19 mortality. Follow-up analysis of the monocyte modules on ICU day 5 was consistent with bifurcated states that correlated with distinct inflammatory cytokines. Our data suggests a pivotal role for monocytes and their specific inflammatory genomic states in contributing to mortality in life-threatening COVID-19 disease and may facilitate discovery of new diagnostics and therapeutics.

Regulatory T cells (Tregs) are an immunosuppressive cell population that inhibits immune cell function to maintain peripheral tolerance and homeostasis. We have previously shown that Treg-- response to interferon gamma (IFNγ) is required for response to cancer immunotherapy. We aimed to broaden these findings and determine the impact of Treg response to IFNγ and the IFNγ-inducing cytokine IL12, during viral infection. Mice with Treg-restricted deletion of the IFNγ receptor (IFNGR1, Ifngr1L/LFoxp3Cre-YFP) or the IL12 receptor β2 subunit (IL12Rβ2, Il12b2L/LFoxp3Cre-YFP), were infected with the acute (Armstrong), or chronic (Clone 13), strain of lymphocytic choriomeningitis virus (LCMV). Surprisingly, Treg response to IFNγ but not IL12 induced helper T cell 1 (TH1)-like polarization (expression of T-bet, CXCR3 and IFNγ) of Tregs in mice with chronic LCMV infection. Importantly, this effector-like TH1 Treg state was required for adequate suppression of effector T cells during LCMV infection. Additionally, during LCMV infection, Treg-restricted deletion of IFNGR1 not only prevented TH1-like polarization but also reverted these Tregs to a TH2-like state (expression of GATA-3, CCR4 and IL-4). scRNAseq of viral antigen-specific CD8+ T cells from Clone 13 infected mice showed that Ifngr1-deficient Tregs favored CD8+ T cell memory. These findings were confirmed in an Armstrong/Listeria monocytogenes-GP33 memory model. Further, Ifngr1 deletion from Tregs enhanced response to CD8+ T cell-mediated vaccination when challenged with Clone 13. These findings provide fundamental insight on how Tregs sense inflammatory cues from the environment (IFNγ) during viral infection to prevent overt tissue damage and shape the memory response. Supported by grants from NIH (F32 CA247004-01, T32 CA082084, P01 AI108545, R35 CA263850, R01 CA203689, R01 DK130897, P30 DK120531, P30 CA047904, R01 HL107380, R01 CA206517, R01 AI138504)

Regulatory T cells (T

LAG3 is an inhibitory receptor that is highly expressed on exhausted T cells. Although LAG3-targeting immunotherapeutics are currently in clinical trials, how LAG3 inhibits T cell function remains unclear. Here, we show that LAG3 moved to the immunological synapse and associated with the T cell receptor (TCR)-CD3 complex in CD4

CD4

Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works Mechanisms of resistance to cancer immunotherapy remain poorly understood. Lymphocyte activation gene–3 (LAG3) signaling is regulated by a disintegrin and metalloprotease domain-containing protein–10 (ADAM10)–and ADAM17-mediated cell surface shedding. Here, we show that mice expressing a metalloprotease-resistant, noncleavable LAG3 mutant (LAG3NC) are resistant to PD1 blockade and fail to mount an effective antitumor immune response. Expression of LAG3NC intrinsically perturbs CD4+ T conventional cells (Tconvs), limiting their capacity to provide CD8+ T cell help. Furthermore, the translational relevance for these observations is highlighted with an inverse correlation between high LAG3 and low ADAM10 expression on CD4+ Tconvs in the peripheral blood of patients with head and neck squamous cell carcinoma, which corresponded with poor prognosis. This correlation was also observed in a cohort of patients with skin cancers and was associated with increased disease progression after standard-of-care immunotherapy. These data suggest that subtle changes in LAG3 inhibitory receptor signaling can act as a resistance mechanism with a substantive effect on patient responsiveness to immunotherapy., NIH (Grant EB022433)

Regulatory T cells (Tregs) are an immunosuppressive subpopulation of CD4+ T cells that are endowed with potent suppressive activity and function to limit immune activation and maintain homeostasis. These cells are identified by the hallmark transcription factor FOXP3 and the high-affinity interleukin-2 (IL-2) receptor chain CD25. Tregs can be recruited to and persist within the tumor microenvironment (TME), acting as a potent barrier to effective antitumor immunity. This chapter will discuss [i] the history and hallmarks of Tregs; [ii] the recruitment, development, and persistence of Tregs within the TME; [iii] Treg function within TME; asnd [iv] the therapeutic targeting of Tregs in the clinic. This chapter will conclude with a discussion of likely trends and future directions.

Expression of the transmembrane protein Tim-3 is increased on dysregulated T cells undergoing chronic T cell activation, including in chronic infection and solid tumors. We and others previously reported that Tim-3 exerts apparently paradoxical co-stimulatory activity in T cells (and other cells), including enhancement of ribosomal S6 protein phosphorylation (pS6). Here we examined the upstream signaling pathways that control Tim3-mediated increases in pS6 in T cells. We have also defined the localization of Tim-3 relative to the T cell immune synapse and impacts on downstream signaling. Recruitment of Tim-3 to the immune synapse was mediated exclusively by the transmembrane domain, replacement of which impaired Tim-3 co-stimulation of pS6. Strikingly, enforced localization of the Tim-3 cytoplasmic domain to the immune synapse in the context of a chimeric antigen receptor still allowed for robust T cell activation. Our findings are consistent with a model whereby Tim-3 enhances TCR-proximal signaling under acute conditions.One Sentence SummaryHere we define elements of signaling and localization associated with Tim-3 co-stimulatory function in T cells.

Despite extensive analyses, there remains an urgent need to delineate immune cell states that contribute to mortality in critically ill Coronavirus disease 2019 (COVID-19) patients. Here, we present high-dimensional profiling of blood and respiratory samples in severe COVID-19 patients to examine the association between cell-linked molecular features and mortality outcomes. Peripheral transcriptional profiles by single-cell RNAseq based deconvolution of immune states are associated with COVID-19 mortality. Further, persistently high levels of an interferon signaling module in monocytes over time leads to subsequent concerted upregulation of inflammatory cytokines. SARS-CoV-2 infected myeloid cells in the lower respiratory tract upregulate CXCL10, leading to a higher risk of death. Our analysis suggests a pivotal role for viral infected myeloid cells and protracted interferon signaling in severe COVID-19., Graphical Abstract, Cillo et al identify transcriptional profiles in peripheral blood that are associated with mortality in critically ill COVID-19 patients. Inflammatory monocyte signatures are correlated with CXCL10 in plasma and precede upregulation of inflammatory cytokines in blood. SARS-CoV-2-infected macrophages in the respiratory tract expressed CXCL10, linking peripheral and lung immune profiles.

BackgroundTargeting PD1 with monoclonal antibodies has yielded clinical success across a variety of tumor types, yet overcoming inhibitory receptor (IR)-mediated tolerance is essential to improve immunotherapeutic responses. LAG3 co-expresses with PD1 on CD8+ tumor-infiltrating T cells (TIL), signifying a highly exhausted phenotype, and dual PD1/LAG3 blockade in C57BL/6 mice enhances antitumor immunity. As CD8+ TIL is the dominant LAG3-expressing TIL population, it is hypothesized that PD1 and LAG3 synergizes to limit CD8+ TIL function controlling antitumor immunity.MethodsTo understand the cellular and mechanistic basis for PD1/LAG3 synergy, conditional knock-in mice ”surgically dissect” Pdcd1 and/or Lag3 floxed alleles restricted to CD8+ T cells expressing E8ICre.GFP. These mice were crossed with the Pmel transgene to assess PD1 and/or LAG3-sufficient or deficient gp100-specific CD8+ T cell populations. CD8+ Pmel cells were isolated and adoptively transferred into C57BL/6 mice harboring a B16-gp100-overexpressing tumor to observe therapeutic benefit, or to assess T cell functionality within the tumor.ResultsWhile little therapeutic benefit was observed with a prophylactic adoptive transfer of wild-type CD8+ Pmel cells into mice which then received B16-gp100 tumor, there was reduced tumor growth in mice receiving PD1-deficient CD8+ Pmel cells, which was further enhanced in mice receiving PD1/LAG3-deficient CD8+ Pmel cells with long-term tumor-free survival. Likewise, a therapeutic administration of PD1/LAG3-deficient CD8+ Pmel cells into mice once the tumor has been established showed an initial therapeutic benefit, with enhanced survival, that was not demonstrated with adoptive transfer of PD1 or LAG3-deficient, or wild-type, counterparts. Each PD1 and/or LAG3-sufficient or deficient CD8+ Pmel mice were differentially congenically marked to assess each of the four genotypes that can be adoptively transferred into the same host as a ”quad transfer” system. Recovery of these populations within the tumor show that the PD1/LAG3-deficient CD8+ Pmel cells out-compete PD1 or LAG3-deficient, or wild-type, counterparts due to enhanced proliferation (Ki67/BrdU). Furthermore, PD1/LAG3-deficient CD8+ T cells were more functional with increased IFNg and GzmB release observed by flow cytometry.ConclusionsOverall PD1 and LAG3 limit anti-tumor immune effects as removal of both IRs on a gp100 antigen-specific CD8+ T cell population results in reduced B16-gp100 tumor growth and enhanced survival in an adoptive transfer model, as a result of enhanced CD8+ TIL functionality and proliferation. These results provide striking evidence that the development of anti-LAG3 agents in the clinic would yield improved responses with anti-PD1.

Robust CD8

Regulatory T cells (T

Summary Following antigen-driven expansion in lymph node, transforming growth factor-β (TGFβ) is required for differentiation of skin-recruited CD8+ T cell effectors into epidermal resident memory T (Trm) cells and their epidermal persistence. We found that the source of TGFβ -supporting Trm cells was autocrine. In addition, antigen-specific Trm cells that encountered cognate antigen in the skin, and bystander Trm cells that did not, both displayed long-term persistence in the epidermis under steady-state conditions. However, when the active-TGFβ was limited or when new T cell clones were recruited into the epidermis, antigen-specific Trm cells were more efficiently retained than bystander Trm cells. Genetically enforced TGFβR signaling allowed bystander Trm cells to persist in the epidermis as efficiently as antigen-specific Trm cells in both contexts. Thus, competition between T cells for active TGFβ represents an unappreciated selective pressure that promotes the accumulation and persistence of antigen-specific Trm cells in the epidermal niche.

Summary Regulatory T (Treg) cells pose a major barrier to effective anti-tumor immunity. Although Treg cell depletion enhances tumor rejection, the ensuing autoimmune sequelae limits its utility in the clinic and highlights the need for limiting Treg cell activity within the tumor microenvironment. Interleukin-35 (IL-35) is a Treg cell-secreted cytokine that inhibits T cell proliferation and function. Using an IL-35 reporter mouse, we observed substantial enrichment of IL-35 + Treg cells in tumors. Neutralization with an IL-35-specific antibody or Treg cell-restricted deletion of IL-35 production limited tumor growth in multiple murine models of human cancer. Limiting intratumoral IL-35 enhanced T cell proliferation, effector function, antigen-specific responses, and long-term T cell memory. Treg cell-derived IL-35 promoted the expression of multiple inhibitory receptors (PD1, TIM3, LAG3), thereby facilitating intratumoral T cell exhaustion. These findings reveal previously unappreciated roles for IL-35 in limiting anti-tumor immunity and contributing to T cell dysfunction in the tumor microenvironment.

Checkpoint blockade immunotherapy established a new paradigm in cancer treatment: for certain patients curative treatment requires immune reinvigoration. Despite this monumental advance, only 20%–30% of patients achieve an objective response to standard of care immunotherapy, necessitating the consideration of alternative targets. Optimal strategies will not only stimulate CD8+T cells, but concomitantly modulate immunosuppressive cells in the tumor microenvironment (TME), most notably regulatory T cells (Tregcells). In this context, the immunoregulatory receptor Neuropilin-1 (NRP1) is garnering renewed attention as it reinforces intratumoral Tregcell function amidst inflammation in the TME. Loss of NRP1 on Tregcells in mouse models restores antitumor immunity without sacrificing peripheral tolerance. Enrichment of NRP1+Tregcells is observed in patients across multiple malignancies with cancer, both intratumorally and in peripheral sites. Thus, targeting NRP1 may safely undermine intratumoral Tregcell fitness, permitting enhanced inflammatory responses with existing immunotherapies. Furthermore, NRP1 has been recently found to modulate tumor-specific CD8+T cell responses. Emerging data suggest that NRP1 restricts CD8+T cell reinvigoration in response to checkpoint inhibitors, and more importantly, acts as a barrier to the long-term durability of CD8+T cell-mediated tumor immunosurveillance. These novel and distinct regulatory mechanisms present an exciting therapeutic opportunity. This review will discuss the growing literature on NRP1-mediated immune modulation which provides a strong rationale for categorizing NRP1 as both a key checkpoint in the TME as well as an immunotherapeutic target with promise either alone or in combination with current standard of care therapeutic regimens.

We have shown that murine regulatory T cells (Tregs) produce the cytokine interferon gamma (IFNγ) during anti-PD1 immunotherapy and that IFNγ-driven Treg dysfunction is necessary for tumor clearance by anti-PD1. However, it is unknown how Treg production of IFNγ dictates response to cancer immunotherapy. It has been shown that IL12-induced production of IFNγ is required for response to anti-PD1 therapy. However, it has yet to be determined whether Tregs are a key responder to IL12. Thus, elucidating the interplay of IL12, IFNγ and Tregs in the tumor microenvironment (TME) is essential for understanding the mechanistic events required for response to immunotherapy. Our lab has generated two novel murine models that allow for Treg-restricted deletion of Ifng (IfngL/LFoxp3Cre-YFP) or Il12rb2 (Il12rb2L/LFoxp3Cre-YFP). These murine models were used to assess the contribution of Treg-derived IFNγ and response to IL12 on the growth of various syngeneic tumor models and response to checkpoint blockade, vaccination and tumor-specific antibodies. Surprisingly, mice with Ifng-deficient Tregs were completely resistant to anti-PD1 immunotherapy. Preliminary experiments suggest that IFNγ production by Tregs is an early requirement to produce an effector-like Treg state in order to mediate an effective anti-tumor response. Mice with Il12rb2-deficient Tregs had diminished therapeutic response to anti-PD1. These data suggest that IFNγ producing Tregs are required to shift the balance from an immunosuppressive TME to one that favors the reinvigoration of the anti-tumor response generated by cancer immunotherapies. Future studies will determine the mechanisms of resistance and whether IFNγ+ Tregs predict response to immunotherapy.

Regulatory T (Treg) cells are crucial for immune homeostasis but they also contribute to tumor immune evasion by promoting a suppressive tumor microenvironment (TME). Mice with Treg cell-restricted Neuropilin 1 deficiency show tumor resistance while maintaining peripheral immune homeostasis, thereby providing a controlled system to interrogate the impact of intratumoral Treg cells on the TME. Using this and other genetic models, we showed that Treg cells shaped the transcriptional landscape across multiple tumor-infiltrating immune cell types. Treg cells suppressed CD8(+) T cell secretion of interferon-γ (IFNγ), which would otherwise block the activation of sterol regulatory elementbinding protein 1 (SREBP1)-mediated fatty acid synthesis in immunosuppressive (M2-like) tumor-associated macrophages (TAMs). Thus, Treg cells indirectly but selectively sustained M2-like TAM metabolic fitness, mitochondrial integrity and survival. SREBP1 inhibition augmented the efficacy of immune checkpoint blockade, suggesting that targeting Treg cells or their modulation of lipid metabolism in M2-like TAMs could improve cancer immunotherapy.

The T cell receptor (TCR)-CD3 complex is composed of a genetically diverse αβ TCR heterodimer associated noncovalently with the invariant CD3 dimers CD3ϵγ, CD3ϵδ, and CD3ζζ. The TCR mediates peptide-MHC recognition, whereas the CD3 molecules transduce activation signals to the T cell. Although much is known about downstream T cell signaling pathways, the mechanism whereby TCR engagement by peptide-MHC initiates signaling is poorly understood. A key to solving this problem is defining the spatial organization of the TCR-CD3 complex and the interactions between its subunits. We have applied solution NMR methods to identify the docking site for CD3 on the β chain of a human autoimmune TCR. We demonstrate a low affinity but highly specific interaction between the extracellular domains of CD3 and the TCR constant β (Cβ) domain that requires both CD3ϵγ and CD3ϵδ subunits. The mainly hydrophilic docking site, comprising 9-11 solvent-accessible Cβ residues, is relatively small (∼400 Å(2)), consistent with the weak interaction between TCR and CD3 extracellular domains, and devoid of glycosylation sites. The docking site is centered on the αA and αB helices of Cβ, which are located at the base of the TCR. This positions CD3ϵγ and CD3ϵδ between the TCR and the T cell membrane, permitting us to distinguish among several possible models of TCR-CD3 association. We further correlate structural results from NMR with mutational data on TCR-CD3 interactions from cell-based assays.

Summary Regulatory T (Treg) cells are crucial for immune homeostasis, but they also contribute to tumor immune evasion by promoting a suppressive tumor microenvironment (TME). Mice with Treg cell-restricted Neuropilin-1 deficiency show tumor resistance while maintaining peripheral immune homeostasis, thereby providing a controlled system to interrogate the impact of intratumoral Treg cells on the TME. Using this and other genetic models, we showed that Treg cells shaped the transcriptional landscape across multiple tumor-infiltrating immune cell types. Treg cells suppressed CD8+ T cell secretion of interferon-γ (IFNγ), which would otherwise block the activation of sterol regulatory element-binding protein 1 (SREBP1)-mediated fatty acid synthesis in immunosuppressive (M2-like) tumor-associated macrophages (TAMs). Thus, Treg cells indirectly but selectively sustained M2-like TAM metabolic fitness, mitochondrial integrity, and survival. SREBP1 inhibition augmented the efficacy of immune checkpoint blockade, suggesting that targeting Treg cells or their modulation of lipid metabolism in M2-like TAMs could improve cancer immunotherapy.

Neuropilin-1 (Nrp1) on regulatory T (Treg) cells is shown to interact with semaphorin-4a (Sema4a) to promote a program of Treg-cell stability and survival, in part through PTEN-mediated modulation of Akt signalling; Nrp1-deficient Treg cells can maintain immune homeostasis but fail to suppress in inflammatory sites, such as tumours, providing an attractive immunotherapeutic target for the treatment of cancers. Regulatory T cells (Treg) constitute a barrier to effective anti-tumour immunity. Their depletion can induce reduction and clearance of many tumours, but as the cells perform an important balancing role in the immune system, depletion also results in unchecked autoimmunity and death. This paper describes an interaction between semaphorin-4a — an activator for T-cell-mediated immunity — and the neuropilin receptor Nrp1 on Treg cells that is required for Treg cells to limit anti-tumour immune responses and to cure established inflammatory colitis, but is dispensable for suppression of autoimmunity and maintenance of immune homeostasis. It remains to be determined whether it is feasible to limit tumour growth by targeting Treg cells without unleashing autoimmunity. The two biological activities may be inseparable, but this work points to ways in which this important system can be further characterized. Regulatory T cells (Treg cells) have a crucial role in the immune system by preventing autoimmunity, limiting immunopathology, and maintaining immune homeostasis1. However, they also represent a major barrier to effective anti-tumour immunity and sterilizing immunity to chronic viral infections1. The transcription factor Foxp3 has a major role in the development and programming of Treg cells2,3. The relative stability of Treg cells at inflammatory disease sites has been a highly contentious subject4,5,6. There is considerable interest in identifying pathways that control the stability of Treg cells as many immune-mediated diseases are characterized by either exacerbated or limited Treg-cell function. Here we show that the immune-cell-expressed ligand semaphorin-4a (Sema4a) and the Treg-cell-expressed receptor neuropilin-1 (Nrp1) interact both in vitro, to potentiate Treg-cell function and survival, and in vivo, at inflammatory sites. Using mice with a Treg-cell-restricted deletion of Nrp1, we show that Nrp1 is dispensable for suppression of autoimmunity and maintenance of immune homeostasis, but is required by Treg cells to limit anti-tumour immune responses and to cure established inflammatory colitis. Sema4a ligation of Nrp1 restrained Akt phosphorylation cellularly and at the immunologic synapse by phosphatase and tensin homologue (PTEN), which increased nuclear localization of the transcription factor Foxo3a. The Nrp1-induced transcriptome promoted Treg-cell stability by enhancing quiescence and survival factors while inhibiting programs that promote differentiation. Importantly, this Nrp1-dependent molecular program is evident in intra-tumoral Treg cells. Our data support a model in which Treg-cell stability can be subverted in certain inflammatory sites, but is maintained by a Sema4a–Nrp1 axis, highlighting this pathway as a potential therapeutic target that could limit Treg-cell-mediated tumour-induced tolerance without inducing autoimmunity.

Regulatory T cells (T

INTRODUCTION Parkinson’s disease (PD) is the second most common neurodegenerative disorder and leads to slowness of movement, tremor, rigidity, and, in the later stages of PD, cognitive impairment. Pathologically, PD is characterized by the accumulation of α-synuclein in Lewy bodies and neurites. There is degeneration of neurons throughout the nervous system, with the degeneration of dopamine neurons in the substantia nigra pars compacta leading to the major symptoms of PD. RATIONALE In the brains of PD patients, pathologic α-synuclein seems to spread from cell to cell via self-amplification, propagation, and transmission in a stereotypical and topographical pattern among neighboring cells and/or anatomically connected brain regions. The spread or transmission of pathologic α-synuclein is emerging as a potentially important driver of PD pathogenesis. The underlying mechanisms and molecular entities responsible for the transmission of pathologic α-synuclein from cell to cell are not known, but the entry of pathologic α-synuclein into neurons is thought to occur, in part, through an active clathrin-dependent endocytic process. RESULTS Using recombinant α-synuclein preformed fibrils (PFF) as a model system with which to study the transmission of misfolded α-synuclein from neuron to neuron, we screened a library encoding transmembrane proteins for α-synuclein-biotin PFF–binding candidates via detection with streptavidin-AP (alkaline phosphatase) staining. Three positive clones were identified that bind α-synuclein PFF and include lymphocyte-activation gene 3 (LAG3), neurexin 1β, and amyloid β precursor-like protein 1 (APLP1). Of these three transmembrane proteins, LAG3 demonstrated the highest ratio of selectivity for α-synuclein PFF over the α-synuclein monomer. α-Synuclein PFF bind to LAG3 in a saturable manner (dissociation constant = 77 nM), whereas the α-synuclein monomer does not bind to LAG3. Co-immunoprecipitation also suggests that pathological α-synuclein PFF specifically bind to LAG3. Tau PFF, β-amyloid oligomer, and β-amyloid PFF do not bind to LAG3, indicating that LAG3 is specific for α-synuclein PFF. The internalization of α-synuclein PFF involves LAG3 because deletion of LAG3 reduces the endocytosis of α-synuclein PFF. LAG3 colocalizes with the endosomal guanosine triphosphatases Rab5 and Rab7 and coendocytoses with pathologic α-synuclein. Neuron-to-neuron transmission of pathologic α-synuclein and the accompanying pathology and neurotoxicity is substantially attenuated by deletion of LAG3 or by antibodies to LAG3. The lack of LAG3 also substantially delayed α-synuclein PFF–induced loss of dopamine neurons, as well as biochemical and behavioral deficits in vivo. CONCLUSION We discovered that pathologic α-synuclein transmission and toxicity is initiated by binding to LAG3 and that neuron-to-neuron transmission of pathological α-synuclein involves the endocytosis of exogenous α-synuclein PFF by the engagement of LAG3 on neurons. Depletion of LAG3 or antibodies to LAG3 substantially reduces the pathology set in motion by the transmission of pathologic α-synuclein. The identification of LAG3 as an α-synuclein PFF–binding protein provides a new target for developing therapeutics designed to slow the progression of PD and related α-synucleinopathies. LAG3 deletion or antibodies to LAG3 delay α-synuclein PFF transmission. Compared with wild-type neurons, binding and endocytosis of α-synuclein PFF is dramatically reduced with antibodies to LAG3 or when LAG3 is deleted, resulting in delayed pathologic α-synuclein transmission and toxicity. Illustration credit: I-Hsun Wu

Preventing activation of diabetogenic T cells is critical for delaying type 1 diabetes onset. The inhibitory molecule lymphocyte activation gene 3 (LAG-3) and metalloprotease tumor necrosis factor-α converting enzyme (TACE) work together to regulate TH1 responses. The aim of this study was to determine if regulating redox using a catalytic antioxidant (CA) could modulate TACE-mediated LAG-3 shedding to impede diabetogenic T-cell activation and progression to disease. A combination of in vitro experiments and in vivo analyses using NOD mouse strains was conducted to test the effect of redox modulation on LAG-3 shedding, TACE enzymatic function, and disease onset. Systemic treatment of NOD mice significantly delayed type 1 diabetes onset. Disease prevention correlated with decreased activation, proliferation, and effector function of diabetogenic T cells; reduced insulin-specific T-cell frequency; and enhanced LAG-3+ cells. Redox modulation also affected TACE activation, diminishing LAG-3 cleavage. Furthermore, disease progression was monitored by measuring serum soluble LAG-3, which decreased in CA-treated mice. Therefore, affecting redox balance by CA treatment reduces the activation of diabetogenic T cells and impedes type 1 diabetes onset via decreasing T-cell effector function and LAG-3 cleavage. Moreover, soluble LAG-3 can serve as an early T-cell–specific biomarker for type 1 diabetes onset and immunomodulation.

Administration of a single dose of anti-CD40L mAb at the time of allogeneic BM transplantation tolerizes peripheral alloreactive T cells and permits establishment of mixed hematopoietic chimerism in mice. Once engrafted, mixed chimeras are systemically tolerant to donor Ags through a central deletion mechanism and will accept any donor organ indefinitely. We previously found that the PD-1/PD-L1 pathway is required for CD8 T-cell tolerance in this model. However, the cell population that must express PD-1 and the role of other inhibitory molecules were unknown. Here, we report that LAG-3 is required for long-term peripheral CD8 but not CD4 T-cell tolerance and that this requirement is CD8 cell-extrinsic. In contrast, adoptive transfer studies revealed a CD8 T cell–intrinsic requirement for CTLA4/B7.1/B7.2 and for PD-1 for CD8 T-cell tolerance induction. We also observed that both PD-L1 and PD-L2 are independently required on donor cells to achieve T-cell tolerance. Finally, we uncovered a requirement for TGF-β signaling into T cells to achieve peripheral CD8 but not CD4 T-cell tolerance in this in vivo system.

The inhibitory receptor Lymphocyte activation gene 3 (LAG3; CD223) plays a key role in regulating T cell activation and tolerance. LAG3 can be expressed on CD4+ FoxP3+ regulatory T cells (Tregs) and conventional effector T cells (Tcons). We found that LAG3 is highly expressed by CD4+ FoxP3+ Tregs in the CNS of mice with experimental autoimmune encephalitis, a mouse model of multiple sclerosis, leading us to examine LAG3 function on CD4+ FoxP3+ Treg in EAE. To study LAG3 function on Tregs in EAE, we crossed mice in which LAG3 is absent on the cell surface of CD4+ FoxP3+ Tregs with 2D2 T cell receptor (TCR) transgenic specific for myelin oligodendrocyte glycoprotein (MOG35-55). Surprisingly, 100% of the 2D2 TCR transgenic mice lacking LAG3 on Tregs developed spontaneous EAE. In marked contrast, none of the 2D2 control mice developed EAE. Similarly, non-TCR transgenic mice lacking LAG3 on Tregs developed more severe induced EAE (by immunization with MOG35-55 in CFA) compared to controls (max clinical score 3.5±0.3 in LAG3 deficient vs. 2.8±0.1 in control SEM). Histological analysis revealed significantly more lesions in both brain and spinal cord of LAG3 deficient mice (mean number in spinal cord 226.5 ± 10.57 LAG3 deficient vs. 51.4 ± 40.81 in controls SEM). Ex vivo analysis at the peak of disease showed that disease severity was accompanied by an increase in the pathogenicity of Tcons producing high levels of IFN-γ and granzyme B. Although no significant differences were observed in CNS infiltration of Tregs, LAG3 deficient Tregs produced IFN-γ and granzyme B, suggesting that they are no longer protective, but instead contribute to CNS disease. These findings point to a key role for LAG3 in controlling Treg function and maintenance of tolerance in the CNS.

The mechanism and stimulatory requirements of regulatory T cell (Treg)-mediated suppression are still unclear. To assess the requirement for Treg stimulation by cognate peptide:MHC, we used T cells from OTII and AND TCR transgenic mice that are specific for and restricted by distinct, noncrossreactive peptide:MHC combinations. This allowed us to independently activate Tregs and their conventional T cell (Tconv) targets. Surprisingly, we found that suppression can occur in the absence of peptide:MHC-mediated stimulation of Tregs. This suppression was Treg dependent and not due to cold target inhibition. Using Rag1−/− TCR transgenic T cells, we show that regulation of Tconv proliferation by heterogeneous Tregs is not due to alloreactivity or crossreactivity. Finally, using anti-TCR-Vβ8-coated microbeads and Vβ8− Tregs, we show that TCR stimulation-independent suppression can occur in the absence of APCs. These data suggest that Tregs may possess constitutive regulatory activity that can be mediated in the absence of cognate peptide:MHC-TCR stimulation.

Lymphocyte activation gene 3 (LAG-3) is a CD4-related, activation-induced cell surface molecule expressed by various lymphoid cell types and binds to MHC class II with high affinity. We have previously shown that LAG-3 negatively regulates the expansion of activated T cells and T cell homeostasis, and is required for maximal regulatory T cell function. In this study, we demonstrate for the first time that LAG-3 is also expressed on CD11clow/B220+/PDCA-1+ plasmacytoid dendritic cells (pDCs). Lag3 expression, as determined by real time PCR, was ∼10-fold greater in pDCs than in either regulatory T cells or activated T effector cells. Activated pDCs also generate ∼5 times more sLAG-3 than activated T cells. LAG-3-deficient pDCs proliferate and expand more than wild-type pDCs in vivo in response to the TLR9 ligand, CpG. However, the effect of LAG-3 appears to be selective as there was no effect of LAG-3 on the expression of MHC class II, TLR9, and chemokine receptors, or on cytokine production. Lastly, adoptive transfer of either Lag3+/+ or Lag3−/− T cells plus or minus Lag3+/+ or Lag3−/− pDCs defined a role for LAG-3 in controlling pDC homeostasis as well as highlighting the consequences of deregulated Lag3−/− pDCs on T cell homeostasis. This raised the possibility of homeostatic reciprocity between T cells and pDCs. Collectively, our data suggests that LAG-3 plays an important but selective cell intrinsic and cell extrinsic role in pDC biology, and may serve as a key functional marker for their study.

T cell exhaustion often occurs during chronic infection and prevents optimal viral control. The molecular pathways involved in T cell exhaustion remain poorly understood. Here we show that exhausted CD8+ T cells are subject to complex layers of negative regulation resulting from the coexpression of multiple inhibitory receptors. Exhausted CD8+ T cells expressed up to seven inhibitory receptors. Coexpression of multiple distinct inhibitory receptors was associated with greater T cell exhaustion and more severe infection. Regulation of T cell exhaustion by various inhibitory pathways was nonredundant, as blockade of the T cell inhibitory receptors PD-1 and LAG-3 simultaneously and synergistically improved T cell responses and diminished viral load in vivo. Thus, CD8+ T cell responses during chronic viral infections are regulated by complex patterns of coexpressed inhibitory receptors.

The T cell antigen receptor (TCR)-CD3 complex is unique in having ten cytoplasmic immunoreceptor tyrosine-based activation motifs (ITAMs). The physiological importance of this high TCR ITAM number is unclear. Here we generated 25 groups of mice expressing various combinations of wild-type and mutant ITAMs in TCR-CD3 complexes. Mice with fewer than seven wild-type CD3 ITAMs developed a lethal, multiorgan autoimmune disease caused by a breakdown in central rather than peripheral tolerance. Although there was a linear correlation between the number of wild-type CD3 ITAMs and T cell proliferation, cytokine production was unaffected by ITAM number. Thus, high ITAM number provides scalable signaling that can modulate proliferation yet ensure effective negative selection and prevention of autoimmunity.

Regulatory T cells suppress immune function by direct cell to cell contact and secretion of the cytokines IL-10 and TGF-β. In this paper, a novel member of the IL-12 cytokine family that contributes to Treg function is identified. Regulatory T (Treg) cells are a critical sub-population of CD4+ T cells that are essential for maintaining self tolerance and preventing autoimmunity1,2, for limiting chronic inflammatory diseases, such as asthma and inflammatory bowel disease3,4, and for regulating homeostatic lymphocyte expansion5. However, they also suppress natural immune responses to parasites6 and viruses7 as well as anti-tumour immunity induced by therapeutic vaccines8. Although the manipulation of Treg function is an important goal of immunotherapy, the molecules that mediate their suppressive activity remain largely unknown. Here we demonstrate that Epstein-Barr-virus-induced gene 3 (Ebi3, which encodes IL-27β) and interleukin-12 alpha (Il12a, which encodes IL-12α/p35) are highly expressed by mouse Foxp3+ (forkhead box P3) Treg cells but not by resting or activated effector CD4+ T (Teff) cells, and that an Ebi3–IL-12α heterodimer is constitutively secreted by Treg but not Teff cells. Both Ebi3 and Il12a messenger RNA are markedly upregulated in Treg cells co-cultured with Teff cells, thereby boosting Ebi3 and IL-12α production in trans. Treg-cell restriction of this cytokine occurs because Ebi3 is a downstream target of Foxp3, a transcription factor that is required for Treg-cell development and function. Ebi3–/– and Il12a–/– Treg cells have significantly reduced regulatory activity in vitro and fail to control homeostatic proliferation and to cure inflammatory bowel disease in vivo. Because these phenotypic characteristics are distinct from those of other IL-12 family members, this novel Ebi3–IL-12α heterodimeric cytokine has been designated interleukin-35 (IL-35). Ectopic expression of IL-35 confers regulatory activity on naive T cells, whereas recombinant IL-35 suppresses T-cell proliferation. Taken together, these data identify IL-35 as a novel inhibitory cytokine that may be specifically produced by Treg cells and is required for maximal suppressive activity.