De novo identification of CD4+ T cell epitopes.

CD4+ T cells recognize peptide antigens presented on class II major histocompatibility complex (MHC-II) molecules to carry out their function. The remarkable diversity of T cell receptor sequences and lack of antigen discovery approaches for MHC-II make profiling the specificities of CD4+ T cells challenging. We have expanded our platform of signaling and antigen-presenting bifunctional receptors to encode MHC-II molecules presenting covalently linked peptides (SABR-IIs) for CD4+ T cell antigen discovery. SABR-IIs can present epitopes to CD4+ T cells and induce signaling upon their recognition, allowing a readable output. Furthermore, the SABR-II design is modular in signaling and deployment to T cells and B cells. Here, we demonstrate that SABR-IIs libraries presenting endogenous and non-contiguous epitopes can be used for antigen discovery in the context of type 1 diabetes. SABR-II libraries provide a rapid, flexible, scalable and versatile approach for de novo identification of CD4+ T cell ligands from single-cell RNA sequencing data using experimental and computational approaches.

Autoreactive CD8+ T cells are restrained by an exhaustion-like program that is maintained by LAG3.

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.

People critically ill with COVID-19 exhibit peripheral immune profiles predictive of mortality and reflective of SARS-CoV-2 lung viral burden.

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.

Understanding adverse events of immunotherapy: A mechanistic perspective.

The treatment of many cancers has been revolutionized by immune checkpoint blockade (ICB) as a standard-of-care therapeutic. Despite many successes, a large proportion of patients treated with ICB agents experience immune-related adverse events (irAEs) in the form of clinical autoimmunity, ranging from mild to life threatening, that can limit cancer treatment. A mechanistic understanding of these irAEs is required to better treat or prevent irAEs and to predict those patients who are susceptible to irAEs. We propose several mechanisms that may contribute to the generation of irAEs: (1) preexisting susceptibility to autoimmunity, (2) aberrant presentation of "self" by the tumor, and (3) loss of tolerance driven by the tumor or tissue microenvironment.

Bifurcated monocyte states are predictive of mortality in severe COVID-19.

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-1ß) associated with survival and an interferon signaling module associated with death. These gene modules were correlated with higher levels of MIP-1ß 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.

Metabolic support of tumour-infiltrating regulatory T cells by lactic acid.

Regulatory T (Treg) cells, although vital for immune homeostasis, also represent a major barrier to anti-cancer immunity, as the tumour microenvironment (TME) promotes the recruitment, differentiation and activity of these cells1,2. Tumour cells show deregulated metabolism, leading to a metabolite-depleted, hypoxic and acidic TME3, which places infiltrating effector T cells in competition with the tumour for metabolites and impairs their function4-6. At the same time, Treg cells maintain a strong suppression of effector T cells within the TME7,8. As previous studies suggested that Treg cells possess a distinct metabolic profile from effector T cells9-11, we hypothesized that the altered metabolic landscape of the TME and increased activity of intratumoral Treg cells are linked. Here we show that Treg cells display broad heterogeneity in their metabolism of glucose within normal and transformed tissues, and can engage an alternative metabolic pathway to maintain suppressive function and proliferation. Glucose uptake correlates with poorer suppressive function and long-term instability, and high-glucose conditions impair the function and stability of Treg cells in vitro. Treg cells instead upregulate pathways involved in the metabolism of the glycolytic by-product lactic acid. Treg cells withstand high-lactate conditions, and treatment with lactate prevents the destabilizing effects of high-glucose conditions, generating intermediates necessary for proliferation. Deletion of MCT1-a lactate transporter-in Treg cells reveals that lactate uptake is dispensable for the function of peripheral Treg cells but required intratumorally, resulting in slowed tumour growth and an increased response to immunotherapy. Thus, Treg cells are metabolically flexible: they can use 'alternative' metabolites in the TME to maintain their suppressive identity. Further, our results suggest that tumours avoid destruction by not only depriving effector T cells of nutrients, but also metabolically supporting regulatory populations.

Regulatory T Cell-Derived TRAIL Is Not Required for Peripheral Tolerance.

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 (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.

Inhibitory receptor agonists: the future of autoimmune disease therapeutics?

Central and peripheral tolerance both contribute to protection against autoimmunity. The pathogenesis of autoimmunity, however, can result from critical deficits or limitations in peripheral and/or central tolerance mechanisms, presenting an opportunity for therapeutic intervention. Recent advances highlight the substantial impact of inhibitory receptors (IRs), which mediate peripheral tolerance, in autoimmunity. Deletion and blockade studies in mice, IR disruption in humans, and correlation with positive disease outcomes all highlight potential clinical benefits of enhancing IR signaling (agonism)-specifically CTLA4, PD1, LAG3, TIM3 and TIGIT-to treat autoimmune disease. Although critical questions remain, IR agonists represent an unappreciated and untapped opportunity for the treatment of autoimmune and inflammatory diseases.

PIK3IP1/TrIP restricts activation of T cells through inhibition of PI3K/Akt.

Phosphatidylinositol-3 kinases (PI3Ks) modulate cellular growth, proliferation, and survival; dysregulation of the PI3K pathway can lead to autoimmune disease and cancer. PIK3IP1 (or transmembrane inhibitor of PI3K [TrIP]) is a putative transmembrane regulator of PI3K. TrIP contains an extracellular kringle domain and an intracellular domain with homology to the inter-SH2 domain of the PI3K regulatory subunit p85, but the mechanism of TrIP function is poorly understood. We show that both the kringle and p85-like domains are necessary for TrIP inhibition of PI3K and that TrIP is down-modulated from the surface of T cells during T cell activation. In addition, we present evidence that the kringle domain may modulate TrIP function by mediating oligomerization. Using an inducible knockout mouse model, we show that TrIP-deficient T cells exhibit more robust activation and can mediate clearance of Listeria monocytogenes infection faster than WT mice. Thus, TrIP is a negative regulator of T cell activation and may represent a novel target for immune modulation.