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EBV-induced gene 3 (Ebi3) is a β subunit within the IL-12 cytokine family that canonically binds to α subunits p19, p28, or p35 to form the heterodimeric cytokines IL-39, IL-27, and IL-35, respectively. In the last decade, the binding partners for Ebi3 have continued to expand to include IL-6 and the other IL-12 family β subunit p40, revealing the possibility that Ebi3 may be able to bind to other cytokines and have distinct functions. We first explored this possibility utilizing an in vivo mouse model of regulatory T cell-restricted deletions of the subunits composing the cytokine IL-35, p35, and Ebi3, and we observed a differential impact on CD8+ T cell inhibitory receptor expression despite comparable reduction in tumor growth. We then screened the ability of Ebi3 to bind to different cytokines with varying structural resemblance to the IL-12 family α subunits. These in vitro screens revealed extracellular binding of Ebi3 to both IFN-γ and IL-10. Ebi3 bound to IFN-γ and IL-10 abrogated signal transduction and downstream functions of both cytokines. Lastly, we validated that extracellular complex formation after mixing native proteins resulted in loss of function. These data suggest that secreted partnerless Ebi3 may bind to cytokines within the extracellular microenvironment and act as a cytokine sink, further expanding the potential immunological impact of Ebi3., (Copyright © 2024 by The American Association of Immunologists, Inc.)

Approaches to reverse or limit regulatory T cell (Treg) insufficiency are of great interest for development of immunotherapeutic treatments for autoimmune patients, including type 1 diabetes. Treg insufficiency is heavily implicated in the progression of autoimmune diabetes in the NOD mouse model and is characterized by defects in Treg numbers, development, and/or function. Utilizing a Treg-centric screen, we show that intraislet Tregs have a uniquely dysfunctional phenotype, hallmarked by an almost complete lack of neuropilin-1 (Nrp1), a cell surface receptor required to maintain Treg stability. Intraislet Nrp1- Tregs exhibit hallmark features of fragility, including reduced suppressive capacity, decreased CD73 and Helios, and increased Rorγt and Tbet. Intraislet Nrp1- Tregs also exhibit decreased Foxp3 expression on a per cell basis, suggesting that Nrp1 may also be required for long-term Treg stability. Mechanistically, Treg-restricted augmentation of Nrp1 expression limited the onset of autoimmune diabetes in NOD mice suggesting that Nrp1 critically impacts intraislet Treg function. Transcriptional analysis showed that Nrp1 restoration led to an increase in markers and pathways of TCR signaling, survival, and suppression, and when Nrp1 protein expression is examined by cellular indexing of transcriptomes and epitopes by sequencing, significant differences were observed between Nrp1+ and Nrp1- Tregs in all tissues, particularly in markers of Treg fragility. This translated into substantive differences between Nrp1+ and Nrp1- Tregs that afforded the former with a competitive advantage in the islets. Taken together, these data suggest that maintenance of Nrp1 expression and signaling on Tregs limits diabetes onset and may serve as a strategy to combat Treg insufficiency in autoimmune disease., (Copyright © 2024 by The American Association of Immunologists, Inc.)

Relatlimab (rela; anti-LAG-3) plus nivolumab (nivo; anti-PD-1) is safe and effective for treatment of advanced melanoma. We designed a trial (NCT03743766) where advanced melanoma patients received rela, nivo, or rela+nivo to interrogate the immunologic mechanisms of rela+nivo. Analysis of biospecimens from this ongoing trial demonstrated that rela+nivo led to enhanced capacity for CD8 + T cell receptor signaling and altered CD8 + T cell differentiation, leading to heightened cytotoxicity despite the retention of an exhaustion profile. Co-expression of cytotoxic and exhaustion signatures was driven by PRDM1, BATF, ETV7, and TOX. Effector function was upregulated in clonally expanded CD8 + T cells that emerged after rela+nivo. A rela+nivo intratumoral CD8 + T cell signature was associated with a favorable prognosis. This intratumoral rela+nivo signature was validated in peripheral blood as an elevated frequency of CD38 + TIM3 + CD8 + T cells. Overall, we demonstrated that cytotoxicity can be enhanced despite the retention of exhaustion signatures, which will inform future therapeutic strategies., Competing Interests: Declaration of interests A.R.C. is a consultant for AboundBio. J.M.K. is on consulting or scientific advisory boards of Ankyra Therapeutics, Axio Research LLC, Bristol Myers Squibb, Cancer Network, CytomX Therapeutics, DermTech, iOnctura, Iovance Biotherapeutics, IQVIA, Istari Oncology, Jazz Pharmaceuticals Inc., Lytix Biopharma AS, Magnolia Innovation LLC, Merck, Natera Inc., Novartix Pharmaceuticals, OncoCyte Corporation, PathAI Inc., Pfizer Inc., Piper Sandler & Co., PyrOjas Corporation, Regeneron Pharmaceuticals, Replimune Inc., Scopus BioPharma Inc., Takeda, and Valar Labs. J.M.K. received research trial support to institution from Amgen Inc., Bristol Myers Sqiubb, Checkmate Pharmaceuticals, Harbour BioMed, Immvira Pharma Co., Immunocore Ltd., Iovance Biotherapeutics, Lion Biotechnologies Inc., Lytix Biopharma AS, Novartis Pharmaceuticals, Takeda, and Verastem Inc. T.C.B. is a consultant for Galvanize Therapeutics, Tallac Therapeutics, Mestag Therapeutics, Attivare Therapeutics, and Kalivir Therapeutics, and is on the scientific advisory board of Tabby Therapeutics. D.A.A.V. is a cofounder and stockholder of Novasenta, Potenza, Tizona, and Trishula; is a stockholder of Oncorus and Werewolf; has patents licensed and royalties with BMS and Novasenta; is a scientific advisory board member of Tizona, Werewolf, F-Star, Bicara, Apeximmune, and T7/Imreg Bio; and is a consultant for BMS, Incyte, G1 Therapeutics, Inzen Therapeutics, Regeneron, and Avidity Partners., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Overcoming immune-mediated resistance to PD-1 blockade remains a major clinical challenge. Enhanced efficacy has been demonstrated in melanoma patients with combined nivolumab (anti-PD-1) and relatlimab (anti-LAG-3) treatment, the first in its class to be FDA approved. However, how these two inhibitory receptors synergize to hinder anti-tumor immunity remains unknown. Here, we show that CD8 + T cells deficient in both PD-1 and LAG-3, in contrast to CD8 + T cells lacking either receptor, mediate enhanced tumor clearance and long-term survival in mouse models of melanoma. PD-1- and LAG-3-deficient CD8 + T cells were transcriptionally distinct, with broad TCR clonality and enrichment of effector-like and interferon-responsive genes, resulting in enhanced IFN-γ release indicative of functionality. LAG-3 and PD-1 combined to drive T cell exhaustion, playing a dominant role in modulating TOX expression. Mechanistically, autocrine, cell-intrinsic IFN-γ signaling was required for PD-1- and LAG-3-deficient CD8 + T cells to enhance anti-tumor immunity, providing insight into how combinatorial targeting of LAG-3 and PD-1 enhances efficacy., Competing Interests: Declaration of interests D.A.A.V. and C.J.W. have patents covering LAG-3, with others pending, and are entitled to a share of net income generated from licensing of these patent rights for commercial development. D.A.A.V. is cofounder and stock holder of Novasenta, Potenza, Tizona, and Trishula; stock holder of Oncorus and Werewolf; has patents licensed and royalties from BMS and Novasenta; is a scientific advisory board member at Tizona, Werewolf, F-Star, Bicara, Apeximmune, and T7/Imreg Bio; is a consultant for BMS, Incyte, Regeneron, Ono Pharma, Peptone, and Avidity Partners; and receives funding from BMS and Novasenta. E.J.W. is an advisor for Danger Bio, Marengo, Janssen, New Limit, Pluto Immunotherapeutics Related Sciences, Rubius Therapeutics, Santa Ana Bio, Synthekine, and Surface Oncology. E.J.W. is a founder of and holds stock in Surface Oncology, Danger Bio, and Arsenal Biosciences. A.H.S. has patents/pending royalties on the PD-1 pathway from Roche and Novartis and has research funding from IOME, AbbVie, and Taiwan Bio unrelated to the submitted work. A.H.S. serves on advisory boards for Selecta, Elpiscience, Monopteros, Bicara, Fibrogen, IOME, BioEntre, Corner, and Alixia Therapeutics. She also is on scientific advisory boards for the Massachusetts General Cancer Center, Program in Cellular and Molecular Medicine at Boston Children’s Hospital, Human Oncology and Pathogenesis Program at Memorial Sloan Kettering Cancer Center, Johns Hopkins Bloomberg Kimmel Institute for Cancer Immunotherapy, Gladstone Institute, Glaxo Smith Kline, Amgen, and Janssen. She is an academic editor for the Journal of Experimental Medicine. A.R.C. is a consultant for Abound Bio., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Lymphocyte activation gene 3 (LAG3) is an inhibitory receptor that plays a critical role in controlling T cell tolerance and autoimmunity and is a major immunotherapeutic target. LAG3 is expressed on the cell surface as a homodimer but the functional relevance of this is unknown. In this study, we show that the association between the TCR/CD3 complex and a murine LAG3 mutant that cannot dimerize is perturbed in CD8+ T cells. We also show that LAG3 dimerization is required for optimal inhibitory function in a B16-gp100 tumor model. Finally, we demonstrate that a therapeutic LAG3 Ab, C9B7W, which does not block LAG3 interaction with its cognate ligand MHC class II, disrupts LAG3 dimerization and its association with the TCR/CD3 complex. These studies highlight the functional importance of LAG3 dimerization and offer additional approaches to therapeutically target LAG3., (Copyright © 2024 by The American Association of Immunologists, Inc.)

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) interacts 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 deepens our insight about molecular mechanisms 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., (© 2024. The Author(s).)

The spread of prion-like protein aggregates is a common driver of pathogenesis in various neurodegenerative diseases, including Alzheimer's disease (AD) and related Tauopathies. Tau pathologies exhibit a clear progressive spreading pattern that correlates with disease severity. Clinical observation combined with complementary experimental studies has 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 cell surface receptors of Tau are known, they are not specific to the fibrillar form of Tau. Moreover, the underlying cellular mechanisms of Tau PFF spreading remain poorly understood. Here, it is shown that the lymphocyte-activation gene 3 (Lag3) is a cell surface receptor that binds to PFF but not the monomer of Tau. Deletion of Lag3 or 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 lacking Lag3 selectively in neurons. These results identify neuronal Lag3 as a receptor of pathologic Tau in the brain，and for AD and related Tauopathies, a therapeutic target., (© 2023 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

Human regulatory T cells (T regs ) are crucial regulators of tissue repair, autoimmune diseases, and cancer. However, it is challenging to inhibit the suppressive function of T regs for cancer therapy without affecting immune homeostasis. Identifying pathways that may distinguish tumor-restricted T regs is important, yet the transcriptional programs that control intratumoral T reg gene expression, and that are distinct from T regs in healthy tissues, remain largely unknown. We profiled single-cell transcriptomes of CD4 + T cells in tumors and peripheral blood from patients with head and neck squamous cell carcinomas (HNSCC) and those in nontumor tonsil tissues and peripheral blood from healthy donors. We identified a subpopulation of activated T regs expressing multiple tumor necrosis factor receptor (TNFR) genes (TNFR + T regs ) that is highly enriched in the tumor microenvironment (TME) compared with nontumor tissue and the periphery. TNFR + T regs are associated with worse prognosis in HNSCC and across multiple solid tumor types. Mechanistically, the transcription factor BATF is a central component of a gene regulatory network that governs key aspects of TNFR + T regs . CRISPR-Cas9-mediated BATF knockout in human activated T regs in conjunction with bulk RNA sequencing, immunophenotyping, and in vitro functional assays corroborated the central role of BATF in limiting excessive activation and promoting the survival of human activated T regs . Last, we identified a suite of surface molecules reflective of the BATF-driven transcriptional network on intratumoral T regs in patients with HNSCC. These findings uncover a primary transcriptional regulator of highly suppressive intratumoral T regs , highlighting potential opportunities for therapeutic intervention in cancer without affecting immune homeostasis.

Lymphocyte activation gene 3 (LAG-3) is an inhibitory receptor that is highly expressed by exhausted T cells. LAG-3 is a promising immunotherapeutic target, with more than 20 LAG-3-targeting therapeutics in clinical trials and a fixed-dose combination of anti-LAG-3 and anti-PD-1 now approved to treat unresectable or metastatic melanoma. Although LAG-3 is widely recognized as a potent inhibitory receptor, important questions regarding its biology and mechanism of action remain. In this Perspective, we focus on gaps in the understanding of LAG-3 biology and discuss the five biggest topics of current debate and focus regarding LAG-3, including its ligands, signaling and mechanism of action, its cell-specific functions, its importance in different disease settings, and the development of novel therapeutics., (© 2023. Springer Nature America, Inc.)

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., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

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., (©2022 American Association for Cancer Research.)

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., Competing Interests: Declaration of interests D.A.A.V. is cofounder and stockholder of Novasenta, Potenza, Tizona, and Trishula; stockholder of Oncorus, Werewolf, and Apeximmune; has patents licensed and royalties from Astellas, BMS, Novasenta; scientific advisory board member of Tizona, Werewolf, F-Star, Bicara, Apeximmune, and T7/Imreg Bio; is a consultant for Astellas, BMS, Almirall, Incyte, G1 Therapeutics, and Inzen Therapeutics; and obtained research funding from BMS, Astellas, and Novasenta. T.C.B. receives research funding for Alkermes and Pfizer; is a consultant for Walking Fish Therapeutics, iTeos Therapeutics, and BeSpoke Therapeutics. The remaining authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: JLS: Research Funding— Cygnal Therapeutics. DAAV: cofounder and stock holder – Novasenta, Potenza, Tizona, Trishula; stock holder – Oncorus, Werewolf, Apeximmune; patents licensed and royalties - Astellas, Bristol Myers Squibb, Novasenta; scientific advisory board member - Tizona, Werewolf, F-Star, Bicara, Apeximmune; consultant - Astellas, Bristol Myers Squibb, Almirall, Incyte, G1 Therapeutics; research funding – Bristol Myers Squibb, Astellas and Novasenta.., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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., (Copyright © 2022 by The American Association of Immunologists, Inc.)

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., (©2022 American Association for Cancer Research.)

While there have been extensive analyses characterizing cellular and humoral responses across the severity spectrum in COVID-19, outcome predictors within severe COVID-19 remain less comprehensively elucidated. Furthermore, properties of antibodies (Abs) directed against viral antigens beyond spike and their associations with disease outcomes remain poorly defined. We perform deep molecular profiling of Abs directed against a wide range of antigenic specificities in severe COVID-19 patients. The profiles included canonical (spike [S], receptor-binding domain [RBD], and nucleocapsid [N]) and non-canonical (orf3a, orf8, nsp3, nsp13, and membrane [M]) antigenic specificities. Notably, multivariate Ab profiles directed against canonical or non-canonical antigens are equally discriminative of survival in severe COVID-19. Intriguingly, pre-pandemic healthy controls have cross-reactive Abs directed against nsp13, a protein conserved across coronaviruses. Consistent with these findings, a model built on Ab profiles for endemic coronavirus antigens also predicts COVID-19 outcome. Our results suggest the importance of studying Abs targeting non-canonical severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and endemic coronavirus antigens in COVID-19., Competing Interests: Declaration of interests J.D. is a consultant for SeromYx Systems., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Impaired chronic viral and tumor clearance has been attributed to CD8 + T cell exhaustion, a differentiation state in which T cells have reduced and altered effector function that can be partially reversed upon blockade of inhibitory receptors. The role of the exhaustion program and transcriptional networks that control CD8 + T cell function and fate in autoimmunity is not clear. Here we show that intra-islet CD8 + T cells phenotypically, transcriptionally, epigenetically and metabolically possess features of canonically exhausted T cells, yet maintain important differences. This 'restrained' phenotype can be perturbed and disease accelerated by CD8 + T cell-restricted deletion of the inhibitory receptor lymphocyte activating gene 3 (LAG3). Mechanistically, LAG3-deficient CD8 + T cells have enhanced effector-like functions, trafficking to the islets, and have a diminished exhausted phenotype, highlighting a physiological role for an exhaustion program in limiting autoimmunity and implicating LAG3 as a target for autoimmune therapy., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

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 + and CD8 + T cells, in the absence of binding to major histocompatibility complex class II-its canonical ligand. Mechanistically, a phylogenetically conserved, acidic, tandem glutamic acid-proline repeat in the LAG3 cytoplasmic tail lowered the pH at the immune synapse and caused dissociation of the tyrosine kinase Lck from the CD4 or CD8 co-receptor, which resulted in a loss of co-receptor-TCR signaling and limited T cell activation. These observations indicated that LAG3 functioned as a signal disruptor in a major histocompatibility complex class II-independent manner, and provide insight into the mechanism of action of LAG3-targeting immunotherapies., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Cancer immunotherapy offers substantive benefit to patients with various tumour types, in some cases leading to complete tumour clearance. However, many patients do not respond to immunotherapy, galvanizing the field to define the mechanisms of pre-existing and acquired resistance. Interferon-γ (IFNγ) is a cytokine that has both protumour and antitumour activities, suggesting that it may serve as a nexus for responsiveness to immunotherapy. Many cancer immunotherapies and chemotherapies induce IFNγ production by various cell types, including activated T cells and natural killer cells. Patients resistant to these therapies commonly have molecular aberrations in the IFNγ signalling pathway or express resistance molecules driven by IFNγ. Given that all nucleated cells can respond to IFNγ, the functional consequences of IFNγ production need to be carefully dissected on a cell-by-cell basis. Here, we review the cells that produce IFNγ and the different effects of IFNγ in the tumour microenvironment, highlighting the pleiotropic nature of this multifunctional and abundant cytokine., (© 2021. Springer Nature Limited.)

Despite extensive analyses, there remains an urgent need to delineate immune cell states that contribute to mortality in people critically ill with COVID-19. Here, we present high-dimensional profiling of blood and respiratory samples from people with severe COVID-19 to examine the association between cell-linked molecular features and mortality outcomes. Peripheral transcriptional profiles by single-cell RNA sequencing (RNA-seq)-based deconvolution of immune states are associated with COVID-19 mortality. Further, persistently high levels of an interferon signaling module in monocytes over time lead 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., Competing Interests: D.A.A.V.: cofounder and stockholder for Novasenta, Tizona, Trishula, Potenza; stockholder for Oncorus, Werewolf, Apeximmune; patents licensed and royalties for Astellas, BMS, Novasenta; scientific advisory board member for Tizona, Werewolf, F-Star, Bicara, and Apeximmune; consultant for Astellas, BMS, Almirall, Incyte, G1 Therapeutics; research funding for BMS, Astellas, and Novasenta. G.D.K.: research funding for Karius, Inc. T.C.B: research funding for Alkermes and Pfizer; consultant for Walking Fish Therapeutics, iTeos Therapeutics, and BeSpoke Therapeutics. The remaining authors declare no competing interests., (© 2021 The Author(s).)

The balance between inflammatory and anti-inflammatory immune responses is maintained through immunoregulatory cell populations and immunosuppressive cytokines. Interleukin-35 (IL-35), an inhibitory cytokine that belongs to the IL-12 family, is capable of potently suppressing T cell proliferation and inducing IL-35-producing induced regulatory T cells (iTr35) to limit inflammatory responses. Over the past decade, a growing number of studies have indicated that IL-35 plays an important role in controlling immune-related disorders, including autoimmune diseases, infectious diseases, and cancer. In this review, we summarize the current knowledge about the biology of IL-35 and its contribution in different diseases, and we discuss the potential of and barriers to harnessing IL-35 as a clinical biomarker or immunotherapy.

CD4 + T cells share common developmental pathways with CD8 + T cells, and upon maturation, CD4 + T conventional T (Tconv) cells lack phenotypic markers that distinguish these cells from FoxP3 + T regulatory cells. We developed a tamoxifen-inducible ThPOK CreERT2.hCD2 line with Frt sites inserted on either side of the CreERT2-hCD2 cassette, and a Foxp3 Ametrine-FlpO strain, expressing Ametrine and FlpO in Foxp3 + cells. Breeding these mice resulted in a CD4conv iCreERT2-hCD2 line that allows for the specific manipulation of a gene in CD4 + Tconv cells. As FlpO removes the CreERT2-hCD2 cassette, CD4 + Treg cells are spared from Cre activity, which we refer to as allele conditioning. Comparison with an E8I iCreERT2.GFP mouse that enables inducible targeting of CD8 + T cells, and deletion of two inhibitory receptors, PD-1 and LAG-3, in a melanoma model, support the fidelity of these lines. These engineered mouse strains present a resource for the temporal manipulation of genes in CD4 + T cells and CD4 + Tconv cells., Competing Interests: Declaration of interests D.A.A.V. is a cofounder and stockholder in Novasenta, Tizona, Trishula, and Potenza; a stockholder in Oncorus, Werewolf, and Apeximmune; has patents licensed and royalties in Astellas and BMS; is a scientific advisory board member of Tizona, Werewolf, F-Star, Bicara, and Apeximmune; a consultant to Astellas, BMS, Almirall, Incyte, G1 Therapeutics; and has received research funding from BMS, Astellas, and Novasenta. A.H.S. has patents/pending royalties on the PD-1 pathway from Roche and Novartis. A.H.S. is on the advisory boards of Surface Oncology, Elstar, SQZ Biotechnologies, Elpiscience, Selecta, Bicara, and Monopteros; consults for Novartis; and has received research funding from Novartis, Roche, Quark, Merck, and AbbVie that is unrelated to this project. E.J.W. has consulting agreements with and/or is on the scientific advisory boards of Merck, Elstar, Janssen, Related Sciences, Synthekine, and Surface Oncology; is a founder of Surface Oncology and Arsenal Biosciences; and has a patent licensing agreement on the PD-1 pathway with Roche/Genentech., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Rationale: There is an urgent need for improved understanding of the mechanisms and clinical characteristics of acute respiratory distress syndrome (ARDS) due to coronavirus disease (COVID-19). Objectives: To compare key demographic and physiologic parameters, biomarkers, and clinical outcomes of COVID-19 ARDS and ARDS secondary to direct lung injury from other etiologies of pneumonia. Methods: We enrolled 27 patients with COVID-19 ARDS in a prospective, observational cohort study and compared them with a historical, pre-COVID-19 cohort of patients with viral ARDS ( n = 14), bacterial ARDS ( n = 21), and ARDS due to culture-negative pneumonia ( n = 30). We recorded clinical demographics; measured respiratory mechanical parameters; collected serial peripheral blood specimens for measurement of plasma interleukin (IL)-6, IL-8, and IL-10; and followed patients prospectively for patient-centered outcomes. We conducted between-group comparisons with nonparametric tests and analyzed time-to-event outcomes with Kaplan-Meier and Cox proportional hazards models. Results: Patients with COVID-19 ARDS had higher body mass index and were more likely to be Black, or residents of skilled nursing facilities, compared with those with non-COVID-19 ARDS ( P < 0.05). Patients with COVID-19 had lower delivered minute ventilation compared with bacterial and culture-negative ARDS ( post hoc P < 0.01) but not compared with viral ARDS. We found no differences in static compliance, hypoxemic indices, or carbon dioxide clearance between groups. Patients with COVID-19 had lower IL-6 levels compared with bacterial and culture-negative ARDS at early time points after intubation but no differences in IL-6 levels compared with viral ARDS. Patients with COVID-19 had longer duration of mechanical ventilation but similar 60-day mortality in both unadjusted and adjusted analyses. Conclusions: COVID-19 ARDS bears several similarities to viral ARDS but demonstrates lower minute ventilation and lower systemic levels of IL-6 compared with bacterial and culture-negative ARDS. COVID-19 ARDS was associated with longer dependence on mechanical ventilation compared with non-COVID-19 ARDS. Such detectable differences of COVID-19 do not merit deviation from evidence-based management of ARDS but suggest priorities for clinical research to better characterize and treat this new clinical entity.

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., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Regulatory T cells (T regs ) are key immunosuppressive cells that promote tumor growth by hindering the effector immune response. T regs utilize multiple suppressive mechanisms to inhibit pro-inflammatory responses within the tumor microenvironment (TME) by inhibition of effector function and immune cell migration, secretion of inhibitory cytokines, metabolic disruption and promotion of metastasis. In turn, T regs are being targeted in the clinic either alone or in combination with other immunotherapies, in efforts to overcome the immunosuppressive TME and increase anti-tumor effects. However, it is now appreciated that T regs not only suppress cells intratumorally via direct engagement, but also serve as key interactors in the peritumor, stroma, vasculature and lymphatics to limit anti-tumor immune responses prior to tumor infiltration. We will review the suppressive mechanisms that T regs utilize to alter immune and non-immune cells outside and within the TME and discuss how these mechanisms collectively allow T regs to create and promote a physical and biological barrier, resulting in an immune-excluded or limited tumor microenvironment., Competing Interests: DV: cofounder and stockholder – Novasenta, Tizona and Trishula; stockholder – Oncorus, Werewolf and Apeximmune; patents licensed and royalties - Astellas, BMS; scientific advisory board member - Tizona, Werewolf, F-Star, Bicara, Apeximmune; consultant - Astellas, BMS, Incyte, Almirall, G1 Therapeutics; research funding – Novasenta, BMS and Astellas. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Scott, Gocher, Workman and Vignali.)

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 ( Tnfsf10 L/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 (T reg )-mediated suppression. We generated mice with a T reg -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 T regs and no change in function or proliferation of T cells in tumors. We also assessed the role of TRAIL on T regs 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 T regs had no effect on disease progression in either model. We conclude that T regs 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., (Copyright © 2021 The Authors.)

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., Competing Interests: Declaration of Interests D.W.G. is a consultant and equity holder of Indalo Therapeutics, which provided compound CWHM-12. All other authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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 T mem that are essential for durable antitumor immunity.

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 (LAG3 NC ) are resistant to PD1 blockade and fail to mount an effective antitumor immune response. Expression of LAG3 NC intrinsically perturbs CD4 + T conventional cells (T convs ), 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 + T convs 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., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

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 (T reg cells). In this context, the immunoregulatory receptor Neuropilin-1 (NRP1) is garnering renewed attention as it reinforces intratumoral T reg cell function amidst inflammation in the TME. Loss of NRP1 on T reg cells in mouse models restores antitumor immunity without sacrificing peripheral tolerance. Enrichment of NRP1 + T reg cells is observed in patients across multiple malignancies with cancer, both intratumorally and in peripheral sites. Thus, targeting NRP1 may safely undermine intratumoral T reg cell 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., Competing Interests: Competing interests: DAV has submitted patents covering NRP1 that are licensed or pending and is entitled to a share in net income generated from licensing of these patent rights for commercial development., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Interleukin-35 (IL-35) is an immunosuppressive cytokine composed of Epstein-Barr-virus-induced protein 3 (Ebi3) and IL-12α chain (p35) subunits, yet the forms that IL-35 assume and its role in peripheral tolerance remain elusive. We induce CBA-specific, IL-35-producing T regulatory (Treg) cells in Treg Ebi3WT C57BL/6 reporter mice and identify IL-35 producers by expression of Ebi3 TdTom gene reporter plus Ebi3 and p35 proteins. Curiously, both subunits of IL-35 are displayed on the surface of tolerogen-specific Foxp3 + and Foxp3 neg (iTr35) T cells. Furthermore, IL-35 producers, although rare, secrete Ebi3 and p35 on extracellular vesicles (EVs) targeting a 25- to 100-fold higher number of T and B lymphocytes, causing them to acquire surface IL-35. This surface IL-35 is absent when EV production is inhibited or if Ebi3 is genetically deleted in Treg cells. The unique ability of EVs to coat bystander lymphocytes with IL-35, promoting exhaustion in, and secondary suppression by, non-Treg cells identifies a novel mechanism of infectious tolerance., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Regulatory T cells (T regs ) 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. T regs 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 T regs ; [ii] the recruitment, development, and persistence of T regs within the TME; [iii] T reg function within TME; asnd [iv] the therapeutic targeting of T regs in the clinic. This chapter will conclude with a discussion of likely trends and future directions.

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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., (© 2019 John Wiley & Sons Ltd.)

Regulatory T cells (T reg cells) maintain host self-tolerance but are a major barrier to effective cancer immunotherapy. T reg 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 T reg 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 T reg cell-derived IL-10 and IL-35 in promoting BLIMP1-dependent exhaustion of CD8 + TILs that limits effective anti-tumor immunity.

Immune checkpoint therapy has revolutionized cancer treatment by blocking inhibitory pathways in T cells that limits the an effective anti-tumor immune response. Therapeutics targeting CTLA-4 and PD1/PDL1 have progressed to first line therapy in multiple tumor types with some patients exhibiting tumor regression or remission. However, the majority of patients do not benefit from checkpoint therapy emphasizing the need for alternative therapeutic options. Lymphocyte Activation Gene 3 (LAG3) or CD223 is expressed on multiple cell types including CD4 + and CD8 + T cells, and T regs , and is required for optimal T cell regulation and homeostasis. Persistent antigen-stimulation in cancer or chronic infection leads to chronic LAG3 expression, promoting T cell exhaustion. Targeting LAG3 along with PD1 facilitates T cell reinvigoration. A substantial amount of pre-clinical data and mechanistic analysis has led to LAG3 being the third checkpoint to be targeted in the clinic with nearly a dozen therapeutics under investigation. In this review, we will discuss the structure, function and role of LAG3 in murine and human models of disease, including autoimmune and inflammatory diseases, chronic viral and parasitic infections, and cancer, emphasizing new advances in the development of LAG3-targeting immunotherapies for cancer that are currently in clinical trials., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Regulatory T cells (T regs ) are a barrier to anti-tumor immunity. Neuropilin-1 (Nrp1) is required to maintain intratumoral T reg stability and function but is dispensable for peripheral immune tolerance. T reg -restricted Nrp1 deletion results in profound tumor resistance due to T reg functional fragility. Thus, identifying the basis for Nrp1 dependency and the key drivers of T reg fragility could help to improve immunotherapy for human cancer. We show that a high percentage of intratumoral NRP1 + T regs correlates with poor prognosis in melanoma and head and neck squamous cell carcinoma. Using a mouse model of melanoma where Nrp1-deficient (Nrp1 -/- ) and wild-type (Nrp1 +/+ ) T regs can be assessed in a competitive environment, we find that a high proportion of intratumoral Nrp1 -/- T regs produce interferon-γ (IFNγ), which drives the fragility of surrounding wild-type T regs , boosts anti-tumor immunity, and facilitates tumor clearance. We also show that IFNγ-induced T reg fragility is required for response to anti-PD1, suggesting that cancer therapies promoting T reg fragility may be efficacious., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Inhibitory receptors (IRs) are pivotal in controlling T cell homeostasis because of their intrinsic regulation of conventional effector T (T conv ) cell proliferation, viability, and function. However, the role of IRs on regulatory T cells (T regs ) remains obscure because they could be required for suppressive activity and/or limit T reg function. We evaluated the role of lymphocyte activation gene 3 (LAG3; CD223) on T regs by generating mice in which LAG3 is absent on the cell surface of T regs in a murine model of type 1 diabetes. Unexpectedly, mice that lacked LAG3 expression on T regs exhibited reduced autoimmune diabetes, consistent with enhanced T reg proliferation and function. Whereas the transcriptional landscape of peripheral wild-type (WT) and Lag3 -deficient T regs was largely comparable, substantial differences between intra-islet T regs were evident and involved a subset of genes and pathways that promote T reg maintenance and function. Consistent with these observations, Lag3 -deficient T regs outcompeted WT T regs in the islets but not in the periphery in cotransfer experiments because of enhanced interleukin-2-signal transducer and activator of transcription 5 signaling and increased Eos expression. Our study suggests that LAG3 intrinsically limits T reg proliferation and function at inflammatory sites, promotes autoimmunity in a chronic autoimmune-prone environment, and may contribute to T reg insufficiency in autoimmune disease., (Copyright © 2017, American Association for the Advancement of Science.)

Emerging evidence indicates that the pathogenesis of Parkinson's disease (PD) may be due to cell-to-cell transmission of misfolded preformed fibrils (PFF) of α-synuclein (α-syn). The mechanism by which α-syn PFF spreads from neuron to neuron is not known. Here, we show that LAG3 (lymphocyte-activation gene 3) binds α-syn PFF with high affinity (dissociation constant = 77 nanomolar), whereas the α-syn monomer exhibited minimal binding. α-Syn-biotin PFF binding to LAG3 initiated α-syn PFF endocytosis, transmission, and toxicity. Lack of LAG3 substantially delayed α-syn PFF-induced loss of dopamine neurons, as well as biochemical and behavioral deficits in vivo. The identification of LAG3 as a receptor that binds α-syn PFF provides a target for developing therapeutics designed to slow the progression of PD and related α-synucleinopathies., (Copyright © 2016, American Association for the Advancement of Science.)

Regulatory T (Treg ) cells play a crucial role in maintaining peripheral tolerance and preventing autoimmunity. However, they also represent a major barrier to effective antitumor immunity and immunotherapy. Consequently, there has been considerable interest in developing approaches that can selectively or preferentially target Treg cells in tumors, while not impacting their capacity to maintain peripheral immune homeostasis. In this review, we describe our current understanding of the mechanisms underlying the recruitment, expansion, and suppressive activity of tumor-associated Treg cells, and discuss the approaches used and the challenges encountered in the immunotherapeutic targeting of Treg cells. In addition, we summarize the primary clinical targets and some emerging data on exciting new potential Treg cell-restricted targets. We propose that discovering and understanding mechanisms that are preferentially used by Treg cells within the tumor microenvironment will lead to strategies that selectively target Treg cell-mediated suppression of antitumor immunity while maintaining peripheral immune tolerance., (© 2016 Federation of European Biochemical Societies.)

Background: The influence of donor-side regulation toward recipient antigens on graft outcome is poorly understood., Methods: Because this influence might be due in part to the accumulation of tissue-resident memory T cells in the donor organ, we used a standard murine tolerization model (donor-specific transfusion plus CD40L blockade) to determine the kinetics of development and peripheralization of allospecific regulatory T cell in lymphoid tissues and liver, a secondary lymphoid organ used in transplantation., Results: We found that donor-specific transfusion and CD40L blockade leads to a progressive and sustained T regulatory allospecific response. The cytokines IL10, TGFβ, and IL35 all contributed to the regulatory phenomenon as determined by trans vivo delayed hypersensitivity assay. Unexpectedly, an early and transient self-specific regulatory response was found as well. Using double reporter mice (forkhead box p 3 [Foxp3]-yellow fluorescent protein, Epstein-Barr virus-induced gene 3 [Ebi3]-TdTomRed), we found an increase in Foxp3+CD25+ regulatory T (Treg) cells paralleling the regulatory response. The Ebi3+ CD4 T cells (IL35-producing) were mainly classic Treg cells (Foxp3+CD25+), whereas TGFβ+ CD4 T cells are mostly Foxp3-negative, suggesting 2 different CD4 Treg cell subsets. Liver-resident TGFβ+ CD4 T cells appeared more rapidly than Ebi3-producing T cells, whereas at later timepoints, the Ebi3 response predominated both in lymphoid tissues and liver., Conclusions: The timing of appearance of donor organ resident Treg cell subsets should be considered in experiments testing the role of bidirectional regulation in transplant tolerance.

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Environmental factors including drugs, mineral oils and heavy metals such as lead, gold and mercury are triggers of autoimmune diseases in animal models or even in occupationally exposed humans. After exposure to subtoxic levels of mercury (Hg), genetically susceptible strains of mice develop an autoimmune disease characterized by the production of highly specific anti-nucleolar autoantibodies, hyperglobulinemia and nephritis. However, mice can be tolerized to the disease by a single low dose administration of Hg. Lymphocyte Activation Gene-3 (LAG-3) is a CD4-related, MHC-class II binding molecule expressed on activated T cells and NK cells which maintains lymphocyte homeostatic balance via various inhibitory mechanisms. In our model, administration of anti-LAG-3 monoclonal antibody broke tolerance to Hg resulting in autoantibody production and an increase in serum IgE level. In addition, LAG-3-deficient B6.SJL mice not only had increased susceptibility to Hg-induced autoimmunity but were also unresponsive to tolerance induction. Conversely, adoptive transfer of wild-type CD4(+) T cells was able to partially rescue LAG-3-deficient mice from the autoimmune disease. Further, in LAG-3-deficient mice, mercury elicited higher amounts of IL-6, IL-4 and IFN-γ, cytokines known to play a critical role in mercury-induced autoimmunity. Therefore, we conclude that LAG-3 exerts an important regulatory effect on autoimmunity elicited by a common environmental pollutant.

Regulatory T cells (Treg cells) have a crucial role in the immune system by preventing autoimmunity, limiting immunopathology, and maintaining immune homeostasis. However, they also represent a major barrier to effective anti-tumour immunity and sterilizing immunity to chronic viral infections. The transcription factor Foxp3 has a major role in the development and programming of Treg cells. The relative stability of Treg cells at inflammatory disease sites has been a highly contentious subject. 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.