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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.

The binding of PD-L1 to CD80 on antigen-presenting cells prevents PD-1 ligation on T cells. Therapeutic blockade of the cis-PD-L1–CD80 interaction liberates PD-L1 to bind to PD-1, inhibits autoreactive T cells and robustly alleviates autoimmune symptoms.

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.

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.

Foxp3+ regulatory T (Treg) cells expressing the interleukin (IL)-33 receptor ST2 mediate tissue repair in response to IL-33. Whether Treg cells also respond to the alarmin IL-33 to regulate specific aspects of the immune response is not known. Here we describe an unexpected function of ST2+ Treg cells in suppressing the innate immune response in the lung to environmental allergens without altering the adaptive immune response. Following allergen exposure, ST2+ Treg cells were activated by IL-33 to suppress IL-17-producing γδ T cells. ST2 signaling in Treg cells induced Ebi3, a component of the heterodimeric cytokine IL-35 that was required for Treg cell-mediated suppression of γδ T cells. This response resulted in fewer eosinophil-attracting chemokines and reduced eosinophil recruitment into the lung, which was beneficial to the host in reducing allergen-induced inflammation. Thus, we define a fundamental role for ST2+ Treg cells in the lung as a negative regulator of the early innate γδ T cell response to mucosal injury. Luster and colleagues show that Treg cells that reside in lung mucosa can respond to IL-33 upon allergen exposure and suppress innate cell responses. IL-33-activated ST2+ Treg cells secrete IL-35, which suppresses IL-17 production by γδ T cells and lessens eosinophil recruitment into the lung.

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.

β-Cell antigen recognition by autoreactive T cells is essential in type 1 diabetes (T1D) pathogenesis. Recently, insulin hybrid peptides (HIPs) were identified as strong agonists for CD4 diabetogenic T cells. Here, using BDC2.5 transgenic and NOD mice, we investigated T-cell recognition of the HIP2.5 epitope, which is a fusion of insulin C-peptide and chromogranin A (ChgA) fragments, and compared it with the WE14 and ChgA29–42 epitopes. We measured in situ two-dimensional affinity on individual live T cells from thymus, spleen, pancreatic lymph nodes, and islets before and after diabetes. Although preselection BDC2.5 thymocytes possess higher affinity than splenic BDC2.5 T cells for all three epitopes, peripheral splenic T cells maintained high affinity only to the HIP2.5 epitope. In polyclonal NOD mice, a high frequency (∼40%) of HIP2.5-specific islet T cells were identified at both prediabetic and diabetic stages comprising two distinct high- and low-affinity populations that differed in affinity by 100-fold. This high frequency of high- and low-affinity HIP2.5 T cells in the islets potentially represents a major risk factor in diabetes pathogenesis.

Pancreatic ductal adenocarcinoma (PDA) is an aggressive malignancy characterized by a paucity of tumor-proximal CD8+ T cells and resistance to immunotherapeutic interventions. Cancer-associated mechanisms that elicit CD8+ T-cell exclusion and resistance to immunotherapy are not well-known. Here, using a Kras- and p53-driven model of PDA, we describe a mechanism of action for the protumorigenic cytokine IL35 through STAT3 activation in CD8+ T cells. Distinct from its action on CD4+ T cells, IL35 signaling in gp130+CD8+ T cells activated the transcription factor STAT3, which antagonized intratumoral infiltration and effector function of CD8+ T cells via suppression of CXCR3, CCR5, and IFNγ expression. Inhibition of STAT3 signaling in tumor-educated CD8+ T cells improved PDA growth control upon adoptive transfer to tumor-bearing mice. We showed that activation of STAT3 in CD8+ T cells was driven by B cell– but not regulatory T cell–specific production of IL35. We also demonstrated that B cell–specific deletion of IL35 facilitated CD8+ T-cell activation independently of effector or regulatory CD4+ T cells and was sufficient to phenocopy therapeutic anti-IL35 blockade in overcoming resistance to anti–PD-1 immunotherapy. Finally, we identified a circulating IL35+ B-cell subset in patients with PDA and demonstrated that the presence of IL35+ cells predicted increased occurrence of phosphorylated (p)Stat3+CXCR3−CD8+ T cells in tumors and inversely correlated with a cytotoxic T-cell signature in patients. Together, these data identified B cell–mediated IL35/gp130/STAT3 signaling as an important direct link to CD8+ T-cell exclusion and immunotherapy resistance in PDA.

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

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.

Despite the success of immune checkpoint blockade therapy, few strategies sufficiently overcome immunosuppression within the tumor microenvironment (TME). Targeting regulatory T cells (T(regs)) is challenging, because perturbing intratumoral T(reg) function must be specific enough to avoid systemic inflammatory side effects. Thus, no T(reg)-targeted agents have proven both safe and efficacious in patients with cancer. Neuropilin-1 (NRP1) is recognized for its role in supporting intratumoral T(reg) function while being dispensable for peripheral homeostasis. Nonetheless, little is known about the biology of human NRP1 T(regs) and the signals that regulate NRP1 expression. Here, we report that NRP1 is preferentially expressed on intratumoral T(regs) across six distinct cancer types compared to healthy donor peripheral blood [peripheral blood lymphocyte (PBL)] and site-matched, noncancer tissue. Furthermore, NRP1(+) T(reg) prevalence is associated with reduced progression-free survival in head and neck cancer. Human NRP1(+) T(regs) have broad activation programs and elevated suppressive function. Unlike mouse T(regs), we demonstrate that NRP1 identifies a transient activation state of human T(regs) driven by continuous T cell receptor (TCR) signaling through the mitogen-activated protein kinase pathway and interleukin-2 exposure. The prevalence of NRP1(+) T(regs) in patient PBL correlates with the intratumoral abundance of NRP1(+) T(regs) and may indicate higher disease burden. These findings support further clinical evaluation of NRP1 as a suitable therapeutic target to enhance antitumor immunity by inhibiting T(reg) function in the TME.

Description, HHi reverses the CA-MSC driven tumor immune cell exclusion restoring the response to ICI., We investigated the impact of cancer-associated mesenchymal stem cells (CA-MSCs) on ovarian tumor immunity. In patient samples, CA-MSC presence inversely correlates with the presence of intratumoral CD8+ T cells. Using an immune “hot” mouse ovarian cancer model, we found that CA-MSCs drive CD8+ T cell tumor immune exclusion and reduce response to anti–PD-L1 immune checkpoint inhibitor (ICI) via secretion of numerous chemokines (Ccl2, Cx3cl1, and Tgf-β1), which recruit immune-suppressive CD14+Ly6C+Cx3cr1+ monocytic cells and polarize macrophages to an immune suppressive Ccr2hiF4/80+Cx3cr1+CD206+ phenotype. Both monocytes and macrophages express high levels of transforming growth factor β–induced (Tgfbi) protein, which suppresses NK cell activity. Hedgehog inhibitor (HHi) therapy reversed CA-MSC effects, reducing myeloid cell presence and expression of Tgfbi, increasing intratumoral NK cell numbers, and restoring response to ICI therapy. Thus, CA-MSCs regulate antitumor immunity, and CA-MSC hedgehog signaling is an important target for cancer immunotherapy.

Summary: 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 TregEbi3WT C57BL/6 reporter mice and identify IL-35 producers by expression of Ebi3TdTom gene reporter plus Ebi3 and p35 proteins. Curiously, both subunits of IL-35 are displayed on the surface of tolerogen-specific Foxp3+ and Foxp3neg (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. : Sullivan et al. show that while many factors and cytokines contribute to primary immunosuppression, EV-associated IL-35 uniquely promotes “infectious” tolerance not only by inducing IL-35 production in non-Treg cells but also by causing an immunosuppressive phenotype in EV-acquiring T and B cells, leading to secondary suppression of immune responses. Keywords: IL-35, extracellular vesicles, cytokines, tolerance, Treg, tetraspanin, Ebi3, p35, CD81

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.

A chimeric antigen receptor-modified T-cell therapy recipient developed severe coronavirus disease 2019, intractable RNAemia, and viral replication lasting >2 months. Premortem endotracheal aspirate contained >2 × 1010 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA copies/mL and infectious virus. Deep sequencing revealed multiple sequence variants consistent with intrahost virus evolution. SARS-CoV-2 humoral and cell-mediated immunity were minimal. Prolonged transmission from immunosuppressed patients is possible.

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.

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.

The dimeric cytokine IL-12 is important in the control of various infections but also contributes to the pathology of certain diseases making it a potential target for therapy. However, its specific inhibition with antibodies is complicated by the fact that its two subunits are present in other cytokines: p40 in IL-23 and p35 in IL-35. This has led to erroneous conclusions like the alleged implication of IL-12 in experimental autoimmune encephalomyelitis (EAE). Here, we report the development of a mouse anti-mouse IL-12 vaccine and the production of monoclonal antibodies (mAbs) that do not react with p40 or p35 (in IL-35) but specifically recognize and functionally inhibit the IL-12 heterodimer. Using one of these mAbs, MM12A1.6, that strongly inhibited IFN-γ production and LPS-induced septic shock after viral infection, we demonstrate the critical role played by IL-12 in the rejection of male skin graft by female C57BL/6 syngeneic recipients and in the clearance of an immunogenic mastocytoma tumor variant by DBA/2 mice, but not in a parent to F1 immune aggression model nor in MOG-induced EAE, which was clearly prevented by anti-p40 mAb C17.8. Given this selective inhibition of IL-12, these mAbs provide new options for reassessing IL-12 function in vivo.

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.

SummaryColorectal cancer (CRC) is a common and deadly disease, and patients with metastatic tumors often fail to respond to therapy. While select members of the microbiome are associated with improved anti-tumor immunity, mechanistic understanding of how the microbiome provides a benefit is lacking. We show that modification of the CRC-associated microbiome with a single immunogenic commensal bacteria can alter T cell differentiation, inhibit tumor growth, and increase survival. Microbiome-driven control of CRC required the formation of colonic tertiary lymphoid structures (TLS) and increased infiltration of the tumor with cytotoxic immune cells. In the context of CRC, CD4+ T cells specific to the newly introduced commensals differentiated into T follicular helper cells and were necessary for the formation of TLS, immune infiltration of the tumor, and control over CRC. Thus, modification of the intestinal T cell response by the microbiome can be used to augment anti-tumor immunity in colorectal cancer.

Current immunotherapy paradigms aim to reinvigorate CD8+ T cells, but the contribution of humoral immunity to antitumor immunity remains understudied. Here, we demonstrate that in head and neck squamous cell carcinoma (HNSCC) caused by human papillomavirus infection (HPV+), patients have transcriptional signatures of germinal center (GC) tumor infiltrating B cells (TIL-Bs) and spatial organization of immune cells consistent with tertiary lymphoid structures (TLS) with GCs, both of which correlate with favorable outcome. GC TIL-Bs in HPV+ HNSCC are characterized by distinct waves of gene expression consistent with dark zone, light zone and a transitional state of GC B cells. Semaphorin 4a expression is enhanced on GC TIL-Bs present in TLS of HPV+ HNSCC and during the differentiation of TIL-Bs. Our study suggests that therapeutics to enhance TIL-B responses in HNSCC should be prioritized in future studies to determine if they can complement current T cell mediated immunotherapies., Recent studies have highlighted the importance of B cells and tertiary lymphoid structures (TLS) in the modulation of anti-tumor immune responses. Here, the authors characterize how HPV status influences the phenotype of tumor infiltrating B cells in patients with head and neck squamous cell carcinoma and demonstrate that TLS with germinal centres are associated with better survival.

International audience; The developmental origins of memory T cells remain incompletely understood. During the expansion phase of acute viral infection, we identified a distinct subset of virus-specific CD8 + T cells that possessed distinct characteristics including expression of CD62L, T cell factor 1 (TCF-1), and Eomesodermin; relative quiescence; expression of activation markers; and features of limited effector differentiation. These cells were a quantitatively minor subpopulation of the TCF-1 + pool and exhibited self-renewal, heightened DNA damage surveillance activity, and preferential long-term recall capacity. Despite features of memory and somewhat restrained proliferation during the expansion phase, this subset displayed evidence of stronger TCR signaling than other responding CD8 + T cells, coupled with elevated expression of multiple inhibitory receptors including programmed cell death 1 (PD-1), lymphocyte activating gene 3 (LAG-3), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), CD5, and CD160. Genetic ablation of PD-1 and LAG-3 compromised the formation of this CD62L hi TCF-1 + subset and subsequent CD8 + T cell memory. Although central memory phenotype CD8 + T cells were formed in the absence of these cells, subsequent memory CD8 + T cell recall responses were compromised. Together, these results identify an impor tant link between genome integrity maintenance and CD8 + T cell memory. Moreover, the data indicate a role for inhibitory receptors in preserving key memory CD8 + T cell precursors during initial activation and differentiation. Identification of this rare subpopulation within the memory CD8 + T cell precursor pool may help reconcile models of the developmental origin of long-term CD8 + T cell memory.

Background Regulatory T (Treg) cells are vital for preventing autoimmunity but are a major barrier to robust cancer immunity as the tumor microenvironment (TME) recruits and promotes their function. The deregulated cellular metabolism of tumor cells leads to a metabolite-depleted, hypoxic, and acidic TME. While the TME impairs the effector function of highly glycolytic tumor infiltrating CD8 T cells, Treg cell suppressive function is maintained. Further, studies of in vitro induced and ex vivo Treg cells reveal a distinct metabolic profile compared to effector T cells. Thus, it may be that the altered metabolic landscape of the TME and the increased activity of intratumoral Treg cells are linked. Methods Flow cytometry, isotopic flux analysis, Foxp3 driven Cre-lox, glucose tracers, Seahorse extracellular flux analysis, RNA sequencing. Results Here we show Treg cells display heterogeneity in terms of their glucose metabolism and can engage an alternative metabolic pathway to maintain their high suppressive function and proliferation within the TME and other tissues. Tissue derived Treg cells (both at the steady state and under inflammatory conditions) show broad heterogeneity in their ability to take up glucose. However, glucose uptake correlates with poorer suppressive function and long-term functional stability, and culture of Treg cells in high glucose conditions decreased suppressive function. Treg cells under low glucose conditions upregulate genes associated with the uptake and metabolism of the glycolytic end-product lactic acid. Treg cells withstand high lactate conditions, and lactate treatment prevents the destabilizing effects of high glucose culture. Treg cells utilize lactate within the TCA cycle and generate phosphoenolpyruvate (PEP), a critical intermediate that can fuel intratumoral Treg cell proliferation in vivo. Using mice with a Treg cell-restricted deletion of lactate transporter Slc16a1 (MCT1) we show MCT1 is dispensable for peripheral Treg cell function but required intratumorally, resulting in slowed tumor growth and prolonged survival. Conclusions These data support a model in which Treg cells are metabolically flexible such that they can utilize ‘alternative’ metabolites present in the TME to maintain their suppressive identity. Further, our studies support the notion that tumors avoid immune destruction not only by depriving effector T cells of essential nutrients, but also by metabolically supporting regulatory T cells.

Background Current FDA-approved immunotherapies aim to reinvigorate CD8+ T cells, but the contribution of the humoral arm of the immune response in human cancer remains poorly understood. B cells within tissues can mediate anti-tumor immunity and regulate immune responses by presenting antigen and producing tumor-specific antibodies and immunomodulatory cytokines. Head and neck squamous cell carcinoma (HNSCC) can be induced by human papillomavirus (HPV+) and carcinogens such as tobacco and alcohol (HPV-), and the immune infiltrate is quite distinct in the two etiologies, in particular, increased B cells in HPV+ HNSCC patients. Further, increased B cells in HNSCC patients correlate with improved patient survival. Our study seeks to differentiate B cell phenotype, function and location in HPV+ and HPV- HNSCC to identify putative B cell-centric immunotherapeutic targets. Methods We utilized a multi-level approach to clearly categorize B cells in HNSCC patients. Single cell RNA sequencing (scRNAseq) was performed on CD45+ tumor infiltrating lymphocytes (TIL) from HPV+ and HPV- HNSCC patients. HNSCC TIL and PBL were stained via spectral cytometry (Cytek Aurora,25 parameters) for unbiased analysis of B cell subsets via computational spectral unmixing. Paraffin embedded slides from HNSCC primary tumors were utilized for multispectral immunofluorescence (mIF) to identify tertiary lymphoid structures (TLS) and identify differences in HPV+ and HPV- disease. Results We demonstrated distinct trajectories for B cells in HPV+ and HPV- disease. HPV- HNSCC tumors mainly contained memory B cells and plasma cells, while the B cells in HPV+ HNSCC were naive and germinal center (GC). Further, we quantified B cells and CD4+ T cells in TLS, and germinal center-like TLS were associated with improved outcome in HPV+ disease. We also observed that transcriptional and protein expression of Semaphorin A (SEMA4a) was restricted to GC B cells and increased on GC B cells in HNSCC patients compared to healthy tonsils. Additionally, we identified distinct waves of gene expression in GC B cells in HNSCC tumors, ultimately revealing a novel transitional state for GC B cells in the tumor microenvironment (TME). Conclusions Understanding B cell function in human cancers and how different TMEs influence B cells and TLS are important for devising novel therapeutic options for cancer patients. Ultimately, development of therapeutics to enhance B cell responses in the TME should be prioritized as a compliment to T-cell mediated therapies.

Background Resistance to the current generation of immunotherapies is mediated by complex relations between stromal, cancer and immune cells found within the tumor microenvironment (TME). Development of more efficacious drugs is predicated on improved understanding of these multi-spatial interactions. With emergence of new immune checkpoint receptor (ICR)-targeting therapies, a better understanding of topological expression of immune checkpoint ligand (ICL) on suppressive cell types in the TME may allow for improved strategies to treat cancer patients. Methods Single cell RNA sequencing (scRNAseq) was performed from head and neck squamous cell carcinoma (HNSCC) specimens (n=18) with matched blood from treatment-naive patients. Immune and non-immune cells were enriched from tumor cell suspensions. Novel transcriptomic cell-to-cell interactions were predicted between heterogeneous cell populations. Histologic inflammation was corroborated with scRNAseq and multiplex flow cytometry. Cell type-specific PD-L1 contributions within the TME were quantified using multispectral imaging. Results Major cell type clusters (immune, epithelial, fibroblast and endothelial cells) were identified. Expression patterns for PD-1, TIGIT, LAG-3 and TIM-3 ligands were evaluated on these suppressive TME cell types. By modeling receptor-ligand interactions between CD8+ T cells and the rest of the major TME cell types, CD8+ T cells were predicted to form more ICR-ICL interactions with tumor-associated macrophages (TAMs) than with any other cell type. With focus on LGALS9/galectin-9 and CD274/PD-L1, flow cytometric analyses validated the scRNAseq observation that both ligands were expressed on TAMs from both inflamed and non-inflamed tumors. Furthermore, flow cytometry and multispectral imaging analyses implicated macrophages as one of the major contributors of CD274/PD-L1 within the TME. Conclusions Our data suggest that in the setting of HNSCC, TAMs are one of the major contributors of ICL in the HNSCC TME. Strategies that selective target this immunosuppressive population may be necessary to break tolerance to PD-1-targeting therapies. Ethics Approval The study was approved by the UPMC Hillman Cancer’s Ethics Board, approval number 99-069.

Background Colorectal cancer remains one of the most common and deadliest cancers worldwide and effective therapies are lacking. While immunotherapy has revolutionized treatment for many cancers, the overwhelming majority of colorectal cancer patients are non-responsive and the 5-year survival rate for advanced disease is Methods We utilized a carcinogen-induced mouse model of CRC and colonized half of the tumor-bearing mice with Helicobacter hepaticus (Hhep) 7 weeks post AOM. Tumor number was assessed 12 weeks post AOM. We isolated lymphocytes from the lamina propria, colonic epithelium, mesenteric lymph nodes, and tumor(s) to track the spatial and transcriptional Hhep-specific and endogenous immune responses during tumor progression through 5’ single cell RNAseq, flow cytometry, and immunofluorescence. In addition, we utilized 16S sequencing and FISH to track Hhep colonization, location within the colon, and its impact on the surrounding microbiome. Results We have found that rational modification of the microbiome of colon tumor-bearing mice through addition of a single bacteria, Hhep, led to tumor control or clearance and a significant survival advantage. Colonization led to the expansion of the lymphatic network and development of numerous peri- or intra-tumoral tertiary lymphoid structures (TLS) composed of Hhep-specific CD4 T follicular helper cells (TFH) as well as the bacteria itself. This led to an overall ‘heating’ of the tumor, wherein we saw an increase of CD4 T cell infiltration to the tumor core as well as an increase in CD103+ type 1 DC (cDC1) recruitment through increased chemokines such as CCL5 and XCL1. Hhep-specific TFH were both necessary and sufficient to drive TLS formation, increased immune invasion, and anti-tumor immunity. Conclusions We have shown that addition of a single bacteria, Hhep, leads to a reduction in CRC tumor burden or clearance through lymphatic expansion, TLS formation, and remodeling of the tumor microenvironment, and that Hhep-specific T cells are required for tumor control. These studies suggest that rational modification of the microbiome and microbiome-specific T cells can positively impact anti-tumor immunity and may represent a unique immunotherapeutic target to turn resistant tumors into responsive tumors.

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

The developmental origins of memory T cells remain incompletely understood. During the expansion phase of acute viral infection, we identified a distinct subset of virus-specific CD8(+) T cells that possessed distinct characteristics including expression of CD62L, TCF-1 and Eomes; relative quiescence; expression of activation markers; and features of limited effector differentiation. These cells were a quantitatively minor subpopulation of the TCF-1(+) pool and exhibited self-renewal, heightened DNA damage surveillance activity, and preferential long-term recall capacity. Despite features of memory and somewhat restrained proliferation during the expansion phase, this subset displayed evidence of stronger TCR signaling than other responding CD8(+) T cells, coupled with elevated expression of multiple inhibitory receptors including PD-1, LAG-3, CTLA-4, CD5, and CD160. Indeed, genetic ablation of PD-1 and LAG-3 compromised the formation of this CD62L(hi) TCF-1(+) subset and subsequent CD8(+) T cell memory. Although central memory phenotype CD8(+) T cells (T(CM)) were formed in the absence of these cells, subsequent memory CD8(+) T cell recall responses were compromised. Together, these results identify an important link between genome integrity maintenance and CD8(+) T cell memory. Moreover, the data indicate a role for inhibitory receptors in preserving key memory CD8(+) T cell precursors during initial activation and differentiation. Identification of this rare subpopulation within the memory CD8(+) T cell precursor pool may help reconcile models of the developmental origin of long-term CD8(+) T cell memory.