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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.

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.

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

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.

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

Regulatory T cells (Tregs) are key immunosuppressive cells that promote tumor growth by hindering the effector immune response. Tregs 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, Tregs 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 Tregs 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 Tregs utilize to alter immune and non-immune cells outside and within the TME and discuss how these mechanisms collectively allow Tregs to create and promote a physical and biological barrier, resulting in an immune-excluded or limited tumor microenvironment.

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.

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.

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.

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

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

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

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.

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

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

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

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.

Inhibitory receptors are pivotal in controlling T cell homeostasis because of their intrinsic regulation of conventional effector T (Tconv) cell proliferation, viability and function. However, the role of Inhibitory receptors on regulatory T (Treg) cells remains obscure, as they could be required for suppressive activity and/or limit Treg cell function. We evaluated the role of Lymphocyte Activation Gene-3 (LAG3, CD223) on Treg cells by generating mice in which LAG3 is absent on the cell surface of Treg cells in a murine model of Type 1 Diabetes. Surprisingly, mice that lacked LAG3 expression on Treg cells exhibited reduced autoimmune diabetes, consistent with enhanced Treg cell proliferation and function. Whereas the transcriptional landscape of peripheral wild-type (WT) and Lag3-deficient Treg cells was largely comparable, substantial differences between intra-islet Treg cells were evident and involved a subset of genes and pathways that promote Treg cell maintenance and function. Consistent with these observations, Lag3-deficient Treg cells out-competed WT Treg cells in the islets but not in the periphery in co-transfer experiments due to enhanced IL2-Stat5 signaling and increased Eos expression. Our study suggests that LAG3 intrinsically limits Treg cell proliferation and function at inflammatory sites, promotes autoimmunity in a chronic autoimmune-prone environment and may contribute to Treg cell insufficiency in autoimmune disease.

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

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

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

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

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.

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

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

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

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

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

Apart from their role in humoral immunity, B cells can exhibit IL-10-dependent regulatory activity (Bregs). These regulatory subpopulations have been shown to inhibit inflammation and allograft rejection. However, our understanding of Bregs has been hampered by their rarity, lack of a specific marker, and poor insight into their induction and maintenance. We previously demonstrated that T cell immunoglobulin mucin domain-1 (TIM-1) identifies over 70% of IL-10-producing B cells, irrespective of other markers. We now show that TIM-1 is the primary receptor responsible for Breg induction by apoptotic cells (ACs). However, B cells that express a mutant form of TIM-1 lacking the mucin domain (TIM-1(Δmucin) ) exhibit decreased phosphatidylserine binding and are unable to produce IL-10 in response to ACs or by specific ligation with anti-TIM-1. TIM-1(Δmucin) mice also exhibit accelerated allograft rejection, which appears to be due in part to their defect in both baseline and induced IL-10(+) Bregs, since a single transfer of WT TIM-1(+) B cells can restore long-term graft survival. These data suggest that TIM-1 signaling plays a direct role in Breg maintenance and induction both under physiological conditions (in response to ACs) and in response to therapy through TIM-1 ligation. Moreover, they directly demonstrate that the mucin domain regulates TIM-1 signaling.

The CD3ε proline-rich sequence (PRS) binds to the cytosolic adaptor molecule Nck after TCR ligation. It has been proposed that this interaction is essential for immunological synapse formation and T cell activation. To assess the physiological importance of the CD3ε PRS, we have generated mice that lack this motif (CD3ε.PRSM). Pull-down experiments demonstrated the inability of Nck to bind to the CD3ε PRS in thymocytes from mutant mice after TCR ligation. Surprisingly, no differences were observed in the number and percentage of T cell subsets in the thymus and spleen, and there was no apparent defect in positive or negative selection. Furthermore, the proliferative response of CD3ε.PRSM T cells to staphylococcal enterotoxin B and anti-CD3 Ab was normal. TCR surface expression, constitutive internalization, and Ag-induced down-modulation were also normal. These data suggest that the interaction between the CD3ε PRS and Nck, or any other Src homology 3 domain-containing molecule, is not essential for T cell development and function.

Tumor necrosis factor alpha (TNF-alpha) is a potent, pleiotrophic cytokine, which is proinflammatory but can also suppress T lymphocyte function. In chronic inflammatory disease such as rheumatoid arthritis, exposure of T cells to TNF-alpha alters their ability to mount a response by modulating the T cell receptor (TCR) signaling pathway, but the mechanisms involved remain obscure. Here, we investigated the specific role of TNF receptor 1 (TNFR1) signaling in the modulation of the TCR signaling pathway. We observed a down-regulation of the intracellular calcium ([Ca(2+)](i)) signal in Jurkat T cells after just 30 min exposure to TNF-alpha, and maximum suppression was reached after 3 h. This effect was transient, and signals returned to normal after 12 h. This depression of [Ca(2+)](i) was also observed in human CD4+ T lymphocytes. The change in Ca(2+) signal was related to a decrease in the plasma membrane Ca(2+) influx, which was apparent even when the TCR signal was bypassed using thapsigargin to induce a Ca(2+) influx. The role of TNF-alpha-induced activation of the sphingolipid cascade in this pathway was examined. The engagement of TNFR1 by TNF-alpha led to a time-dependent increase in acid sphingomyelinase (SMase; ASM) activity, corresponding with a decrease in cellular sphingomyelin. In parallel, there was an increase in cellular ceramide, which correlated directly with the decrease in the magnitude of the Ca(2+) response to phytohemagglutinin. Exogenous addition of SMase or ceramide mimicked the effects of TNFR1 signals on Ca(2+) responses in Jurkat T cells. Direct evidence for the activation of ASM in this pathway was provided by complete abrogation of the TNF-alpha-induced inhibition of the Ca(2+) influx in an ASM-deficient murine T cell line (OT-II(+/+)ASM(-/-)). This potent ability of TNF-alpha to rapidly modulate the TCR Ca(2+) signal via TNFR1-induced ASM activation can explain its suppressive effect on T cell function. This TNFR1 signaling pathway may play a role as an important regulator of T cell responses.

Lymphocyte homeostasis is a central biological process that is tightly regulated. However, its molecular and cellular control is poorly understood. We show that aged mice deficient in lymphocyte activation gene 3 (LAG-3), an MHC class II binding CD4 homologue, have twice as many T cells as wild-type controls. CD4+ and CD8+ LAG-3-deficient T cells showed enhanced homeostatic expansion in lymphopenic hosts, which was abrogated by ectopic expression of wild-type LAG-3, but not by a signaling-defective mutant. In addition, in vivo treatment with anti-LAG-3 mAb resulted in enhanced T cell expansion to a level comparable to that in LAG-3-deficient cells. This deregulation of T cell homeostasis also resulted in the expansion of multiple cell types, including B cells, macrophages, granulocytes, and dendritic cells. Lastly, regulatory T cells were dependent on LAG-3 for their optimal control of T cell homeostasis. Our data suggest that LAG-3 negatively regulates T cell homeostasis by regulatory T cell-dependent and independent mechanisms.

Lymphocyte activation gene (LAG)-3 (CD223) is a CD4-related activation-induced cell surface molecule that binds to MHC class II molecules with high affinity and negatively regulates T cell expansion and homeostasis. In this study, we show that LAG-3 inhibits CD4-dependent, but not CD4-independent, T cell function via its cytoplasmic domain. Although high affinity interaction with MHC class II molecules is essential for LAG-3 function, tailless LAG-3 does not compete with CD4 for ligand binding. A single lysine residue (K468) within a conserved “KIEELE” motif is essential for interaction with downstream signaling molecules. These data provide insight into the mechanism of action of this important T cell regulatory molecule.

Regulatory T cells (Tregs) are a barrier to anti-tumor immunity. Neuropilin-1 (Nrp1) is required to maintain intratumoral Treg stability and function but is dispensable for peripheral immune tolerance. Treg-restricted Nrp1 deletion results in profound tumor resistance due to Treg functional fragility. Thus, identifying the basis for Nrp1 dependency and the key drivers of Treg fragility could help to improve immunotherapy for human cancer. We show that a high percentage of intratumoral NRP1+ Tregs 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+/+) Tregs can be assessed in a competitive environment, we find that a high proportion of intratumoral Nrp1–/– Tregs produce interferon-γ (IFNγ), which drives the fragility of surrounding wild-type Tregs, boosts anti-tumor immunity, and facilitates tumor clearance. We also show that IFNγ-induced Treg fragility is required for response to anti-PD1, suggesting that cancer therapies promoting Treg fragility may be efficacious.

As tumors employ complementary overlapping and/or independent mechanisms to evade immune surveillance, many emerging cancer immunotherapies attempt to target multiple pathways to eradicate malignant cells. Although modulation of independent pathways by simultaneous administration of multiple immune modulators (e.g., checkpoint inhibitors, cytokines, and growth factors) has shown great promise, the clinical impact remains limited due to severe toxicity associated with high systemic levels of many of these drugs. Therefore, novel platforms for efficient delivery of multi-component therapies at lower effective doses would be enabling. Here, a drug delivery platform called immunomodulatory molecule delivery system (iMods), which provides sustained extracellular delivery of a checkpoint inhibitor (anti-PD-L1) and simultaneously, targeted intracellular delivery of a tumor antigen (OVA) along with adjuvant (poly(I:C)), and the indoleamine deoxygenase inhibitor 1-MT is described. In melanoma tumor-bearing mice, combinatorial delivery of these factors with iMods leads to regression of both treated and untreated (contralateral) melanoma tumors and 100% survival. These promising therapeutic outcomes are attributed to significantly enhanced ratios of anti-tumor CD8 T-cell/tumor-protective regulatory T-cell (Treg) in tumors and tumor draining lymph nodes. Overall, the iMods delivery platform described here represents a promising advance in multi-factor cancer immunotherapy.

Since dual specificity at the antibody active-site level involves new principles relative to monospecific antigen-antibody interactions and may be a general property of autoantibodies, it was important to further characterize such antibodies. Four lupus derived autoantibodies were studied to understand parameters and mechanisms involved in the participation of dual-specific antibody molecules in the formation of highly stable immune complexes. Because the dual-specific binding properties of selected lupus-related murine autoantibodies had been previously described using a solid-phase polystyrene-based ELISA, a conformational sensitive membrane based assay (CSI) was used on a comparative basis to further characterize NZB/NZW F1 murine monoclonal anti-DNA autoantibodies BV 04–01 (anti-ssDNA), BV 16–19 (anti-ssDNA), BV 17–45 (anti-dsDNA), and BV 16–13 (anti-dsDNA). All four monoclonal autoantibodies exhibited anti-IgG binding in the solid-phase ELISA. However in the CSI assay, only anti-dsDNA monoclonal autoantibodies BV 17-45 and BV 16-13 demonstrated anti-IgG binding, while anti-ssDNA autoantibodies BV 04–01 and BV 16–19 did not. Upon subjection to time-dependent thermal denaturation, with and without thiol reduction at 100°C in the CSI, the self-binding activities of BV 17–45 and BV 16–13 were abrogated demonstrating that the recognized IgG autoepitope(s) possessed conformational or discontinuous three-dimensional properties. The immunological implications of dual specificity are discussed on a structure–function basis and its correlation with formation of pathogenic immune complexes. © 1997 John Wiley & Sons, Ltd.

Bovine serum albumin (BSA) highly derivatized with fluorescein isothiocyanate (FITC, isomer I) served as a fluorescent enhancement substrate to measure protease activity. In the native globular BSA structure, the fluorescence of the lysine-conjugated fluorescein moieties was quenched 98%. Proteolytic digestion of highly derivatized BSA with Pronase® resulted in fluorescence enhancement of 4300%. Both α-chymotrypsin and proteinase K yielded lower but similar fluorescence enhancement values of 2880% and 2800%, respectively. Digestion of the fluorescein-BSA substrate with trypsin, which required basic amino acids for activity, showed fluorescence enhancement of 1480% reflecting the fluorescein-lysine thiocarbamyl linkage. When derivatized substrate was pretreated with a thiol-reducing agent prior to incubation with proteases, a relatively small increase in fluorescence was noted relative to the untreated substrate except in the case of Pronase. The minimum sensitivity of proteolytic activity, based on a comparison of untreated and reduced FITC25BSA was 32 × 10−6 units for 1 ng proteinase K, 1 × 10−3 units for 1 ng α-chymotrypsin and 10 × 10−3 units for Pronase and trypsin (1 ng each). The fluorescence enhancement assay was suited for sensitive intensity measurements or as an endpoint assay to detect protease activity.

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.

Meeting abstracts Inhibitory receptors control immune responses preventing exacerbated T cell activation and the onset of autoimmunity; however, they also limit antitumor immunity. Enhanced co-expression of PD1 and LAG3 phenotypically mark functionally exhausted tumor-specific T cells, with dual

Inhibitory receptors on immune cells are pivotal regulators of immune escape in cancer. Among these inhibitory receptors, CTLA-4 (targeted clinically by ipilimumab) serves as a dominant off-switch while other receptors such as PD-1 and LAG-3 seem to serve more subtle rheostat functions. However, the extent of synergy and cooperative interactions between inhibitory pathways in cancer remain largely unexplored. Here, we reveal extensive coexpression of PD-1 and LAG-3 on tumor-infiltrating CD4+ and CD8+ T cells in three distinct transplantable tumors. Dual anti–LAG-3/anti–PD-1 antibody treatment cured most mice of established tumors that were largely resistant to single antibody treatment. Despite minimal immunopathologic sequelae in PD-1 and LAG-3 single knockout mice, dual knockout mice abrogated self-tolerance with resultant autoimmune infiltrates in multiple organs, leading to eventual lethality. However, Lag3−/−Pdcd1−/− mice showed markedly increased survival from and clearance of multiple transplantable tumors. Together, these results define a strong synergy between the PD-1 and LAG-3 inhibitory pathways in tolerance to both self and tumor antigens. In addition, they argue strongly that dual blockade of these molecules represents a promising combinatorial strategy for cancer. Cancer Res; 72(4); 917–27. ©2011 AACR.

Regulatory T cells (T(reg) cells) have a critical role in the maintenance of immunological self-tolerance. Here we show that treatment of naive human or mouse T cells with IL-35 induced a regulatory population, which we call 'iT(R)35 cells', that mediated suppression via IL-35 but not via the inhibitory cytokines IL-10 or transforming growth factor-β (TGF-β). We found that iT(R)35 cells did not express or require the transcription factor Foxp3, and were strongly suppressive and stable in vivo. T(reg) cells induced the generation of iT(R)35 cells in an IL-35- and IL-10-dependent manner in vitro and induced their generation in vivo under inflammatory conditions in intestines infected with Trichuris muris and within the tumor microenvironment (B16 melanoma and MC38 colorectal adenocarcinoma), where they contributed to the regulatory milieu. Thus, iT(R)35 cells constitute a key mediator of infectious tolerance and contribute to T(reg) cell-mediated tumor progression. Furthermore, iT(R)35 cells generated ex vivo might have therapeutic utility.

High titers of autoantibodies against glutamic acid decarboxylase (GAD) 65 are commonly observed in patients suffering from type 1 diabetes as well as stiff-person syndrome (SPS), a disorder that affects the CNS, and a variant of SPS, progressive encephalomyelitis with rigidity and myoclonus. Although there is a considerable amount of data focusing on the role of GAD65-specific CD4+ T cells in type 1 diabetes, little is known about their role in SPS. In this study, we show that mice possessing a monoclonal GAD65-specific CD4+ T cell population (4B5, PA19.9G11, or PA17.9G7) develop a lethal encephalomyelitis-like disease in the absence of any other T cells or B cells. GAD65-reactive CD4+ T cells were found throughout the CNS in direct concordance with GAD65 expression and activated microglia: proximal to the circumventricular organs at the interface between the brain parenchyma and the blood-brain barrier. In the presence of B cells, high titer anti-GAD65 autoantibodies were generated, but these had no effect on the incidence or severity of disease. In addition, GAD65-specific CD4+ T cells isolated from the brain were activated and produced IFN-γ. These findings suggest that GAD65-reactive CD4+ T cells alone mediate a lethal encephalomyelitis-like disease that may serve as a useful model to study GAD65-mediated diseases of the CNS.

OBJECTIVE—Type 1 diabetes is mediated by T-cell entry into pancreatic islets and destruction of insulin-producing β-cells. The relative contribution of T-cells specific for different autoantigens is largely unknown because relatively few have been assessed in vivo.RESEARCH DESIGN AND METHODS—We generated mice possessing a monoclonal population of T-cells expressing 1 of 17 T-cell receptors (TCR) specific for either known autoantigens (GAD65, insulinoma-associated protein 2 (IA2), IA2β/phogrin, and insulin), unknown islet antigens, or control antigens on a NOD.scid background using retroviral-mediated stem cell gene transfer and 2A-linked multicistronic retroviral vectors (referred to herein as retrogenic [Rg] mice). The TCR Rg approach provides a mechanism by which T-cells with broad phenotypic differences can be directly compared.RESULTS—Neither GAD- nor IA2-specific TCRs mediated T-cell islet infiltration or diabetes even though T-cells developed in these Rg mice and responded to their cognate epitope. IA2β/phogrin and insulin-specific Rg T-cells produced variable levels of insulitis, with one TCR producing delayed diabetes. Three TCRs specific for unknown islet antigens produced a hierarchy of insulitogenic and diabetogenic potential (BDC-2.5 > NY4.1 > BDC-6.9), while a fourth (BDC-10.1) mediated dramatically accelerated disease, with all mice diabetic by day 33, well before full T-cell reconstitution (days 42–56). Remarkably, as few as 1,000 BDC-10.1 Rg T-cells caused rapid diabetes following adoptive transfer into NOD.scid mice.CONCLUSIONS—Our data show that relatively few autoantigen-specific TCRs can mediate islet infiltration and β-cell destruction on their own and that autoreactivity does not necessarily imply pathogenicity.

T-cell receptor (TCR) transgenic (Tg) mice have revolutionized our understanding of many aspects of T-cell biology. Whereas they provide an almost unlimited source of T cells with a single specificity, breeding them onto different backgrounds and/or new knockout/knock-in mouse models is often time-consuming (6 months to several years), which can make the process costly and can significantly delay research. This protocol describes a new method for expressing defined TCR-alpha and TCR-beta proteins from a single 2A peptide-linked multicistronic retroviral vector in mice, using retrovirus-mediated stem cell gene transfer. We refer to these as 'retrogenic' (Rg) mice ('retro' from retrovirus and 'genic' from Tg) to avoid confusion with traditional transgenic mice. We have successfully used this approach to express over 50 different TCRs on several different mouse backgrounds in as little as 6 weeks.

Tight control of T-cell proliferation and effector function is essential to ensure an effective but appropriate immune response. Here, we reveal that this is controlled by the metalloprotease-mediated cleavage of LAG-3, a negative regulatory protein expressed by all activated T cells. We show that LAG-3 cleavage is mediated by two transmembrane metalloproteases, ADAM10 and ADAM17, with the activity of both modulated by two distinct T-cell receptor (TCR) signaling-dependent mechanisms. ADAM10 mediates constitutive LAG-3 cleavage but increases approximately 12-fold following T-cell activation, whereas LAG-3 shedding by ADAM17 is induced by TCR signaling in a PKCtheta-dependent manner. LAG-3 must be cleaved from the cell surface to allow for normal T-cell activation as noncleavable LAG-3 mutants prevented proliferation and cytokine production. Lastly, ADAM10 knockdown reduced wild-type but not LAG-3(-/-) T-cell proliferation. These data demonstrate that LAG-3 must be cleaved to allow efficient T-cell proliferation and cytokine production and establish a novel paradigm in which T-cell expansion and function are regulated by metalloprotease cleavage with LAG-3 as its sole molecular target.