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GPR81 is a G-protein-coupled receptor for lactate, which is upregulated in breast cancer and plays an autocrine role to promote tumor growth by tumor cell-derived lactate. Here we asked whether lactate has any paracrine role via activation of GPR81 in cells present in tumor microenvironment to help tumor growth. First, we showed that deletion of Gpr81 suppresses breast cancer growth in a constitutive breast cancer mouse model (MMTV-PyMT-Tg). We then used a syngeneic transplant model by monitoring tumor growth from a mouse breast cancer cell line (AT-3, Gpr81-negative) implanted in mammary fat pad of wild-type mice and Gpr81-null mice. Tumor growth was suppressed in Gpr81-null mice compared with wild-type mice. There were more tumor-infiltrating T cells and MHCIIhi-immune cells in tumors from Gpr81-null mice compared with tumors from wild-type mice. RNA-seq analysis of tumors indicated involvement of immune cells and antigen presentation in Gpr81-dependent tumor growth. Antigen-presenting dendritic cells expressed Gpr81 and activation of this receptor by lactate suppressed cell-surface presentation of MHCII. Activation of Gpr81 in dendritic cells was associated with decreased cAMP, IL-6 and IL-12. These findings suggest that tumor cell-derived lactate activates GPR81 in dendritic cells and prevents presentation of tumor-specific antigens to other immune cells. This paracrine mechanism is complementary to the recently discovered autocrine mechanism in which lactate induces PD-L1 in tumor cells via activation of GPR81 in tumor cells, thus providing an effective means for tumor cells to evade immune system. As such, blockade of GPR81 signaling could boost cancer immunotherapy.

HLA-DQ8, a genetic risk factor in type I diabetes (T1D), presents hybrid insulin peptides (HIPs) to autoreactive CD4+ T cells. The abundance of spliced peptides binding to HLA-DQ8 and how they are subsequently recognised by the autoreactive T cell repertoire is unknown. Here we report, the HIP ( GQV E LGGG NAV E VLK), derived from splicing of insulin and islet amyloid polypeptides, generates a preferred peptide-binding motif for HLA-DQ8. HLA-DQ8-HIP tetramer + T cells from the peripheral blood of a T1D patient are characterised by repeated TRBV5 usage, which matches the TCR bias of CD4+ T cells reactive to the HIP peptide isolated from the pancreatic islets of a patient with T1D. The crystal structure of three TRBV5+ TCR-HLA-DQ8-HIP complexes shows that the TRBV5 -encoded TCR β-chain forms a common landing pad on the HLA-DQ8 molecule. The N- and C-termini of the HIP is recognised predominantly by the TCR α-chain and TCR β-chain, respectively, in all three TCR ternary complexes. Accordingly, TRBV5 + TCR recognition of HIP peptides might occur via a ‘polarised’ mechanism, whereby each chain within the αβTCR heterodimer recognises distinct origins of the spliced peptide presented by HLA-DQ8. Epitopes formed by fusion of more than one self peptide, such as proinsulin and other β cell proteins, can result in the formation of non-self hybrid peptides that can potentially trigger autoimmune responses. Here the authors show how TRBV5 + T cell receptors are geared towards recognition of HLA-DQ8 bound hybrid peptides in patients with type 1 diabetes.

Pore-forming toxins (PFTs) are used by both the immune system and by pathogens to disrupt cell membranes. Cells attempt to repair this disruption in various ways, but the exact mechanism(s) that cells use are not fully understood, nor agreed upon. Current models for membrane repair include (1) patch formation (e.g., fusion of internal vesicles with plasma membrane defects), (2) endocytosis of the pores, and (3) shedding of the pores by blebbing from the cell membrane. In this study, we sought to determine the specific mechanism(s) that cells use to resist three different cholesterol-dependent PFTs: Streptolysin O, Perfringolysin O, and Intermedilysin. We found that all three toxins were shed from cells by blebbing from the cell membrane on extracellular microvesicles (MVs). Unique among the cells studied, we found that macrophages were 10 times more resistant to the toxins, yet they shed significantly smaller vesicles than the other cells. To examine the mechanism of shedding, we tested whether toxins with engineered defects in pore formation or oligomerization were shed. We found that oligomerization was necessary and sufficient for membrane shedding, suggesting that calcium influx and patch formation were not required for shedding. However, pore formation enhanced shedding, suggesting that calcium influx and patch formation enhance repair. In contrast, monomeric toxins were endocytosed. These data indicate that cells use two interrelated mechanisms of membrane repair: lipid-dependent MV shedding, which we term ‘intrinsic repair’, and patch formation by intracellular organelles. Endocytosis may act after membrane repair is complete by removing inactivated and monomeric toxins from the cell surface.

In 2016 Delong et al. discovered a new type of neoepitope formed by the fusion of two unrelated peptide fragments. Remarkably these neoepitopes, called hybrid insulin peptides, or HIPs, are recognized by pathogenic CD4 + T cells in the NOD mouse and human pancreatic islet-infiltrating T cells in people with type 1 diabetes. Current data implicates CD4 + T-cell responses to HIPs in the immune pathogenesis of human T1D. Because of their role in the immune pathogenesis of human T1D it is important to identify new HIPs that are recognized by CD4 + T cells in people at risk of, or with, T1D. A detailed knowledge of T1D-associated HIPs will allow HIPs to be used in assays to monitor changes in T cell mediated beta-cell autoimmunity. They will also provide new targets for antigen-specific therapies for T1D. However, because HIPs are formed by the fusion of two unrelated peptides there are an enormous number of potential HIPs which makes it technically challenging to identify them. Here we review the discovery of HIPs, how they form and discuss approaches to identifying new HIPs relevant to the immune pathogenesis of human type 1 diabetes.

Vasculogenesis and angiogenesis are process of formation of blood vessels. Blood vessels are evolved to distribute nutrients and oxygen to distant organs. These vessels are crucial for growth and repair of wounded tissue. During tumor condition there occurs imbalance in the growth of blood vessels which leads to neo-angiogenesis. Neo-angiogenesis is major perpetrator behind the establishment of tumor. Tumor cells secrete pro-angiogenic factor VEGFA which binds to VEGFR2 present over surface of endothelial cells and triggers formation of new blood vessels. To inhibit tumor-angiogenesis, a physiologically-safe small molecule inhibitor was screened which can potentially interact with kinase domain of VEGFR2 and inhibit its activity. Molecular-docking module and biochemical analysis identified andrographolide as one of the best docking molecules that binds to ATP-binding pocket of VEGFR2 and inhibits its kinase activity. Thus, for a more radical approach towards safe VEGFR2 inhibitor, andrographolide was repurposed to inhibit tumor-angiogenesis and reduce tumor burden.

Lung cancer is the leading cause of cancer-related deaths worldwide. Despite decades of research, the treatment options for lung cancer patients remain inadequate, either to offer a cure or even a substantial survival advantage owing to its intrinsic resistance to chemotherapy. Our results propose the effectiveness of capsaicin in down-regulating VEGF expression in non-small cell lung carcinoma (NSCLC) cells in hypoxic environment. Capsaicin-treatment re-activated p53-SMAR1 positive feed-back loop in these cells to persuade p53-mediated HIF-1α degradation and SMAR1-induced repression of Cox-2 expression that restrained HIF-1α nuclear localization. Such signal-modulations consequently down regulated VEGF expression to thwart endothelial cell migration and network formation, pre-requisites of angiogenesis in tumor micro-environment. The above results advocate the candidature of capsaicin in exclusively targeting angiogenesis by down-regulating VEGF in tumor cells to achieve more efficient and cogent therapy of resistant NSCLC.

Antigen-driven T-cell proliferation is often measured using fluorescent dye dilution assays, such as the CFSE-based proliferation assay. Dye dilution assays have been powerful tools to detect human CD4 + T-cell responses, particularly against autoantigens. However, it is not known how many cells within the proliferating population are specific for the stimulating antigen. Here we determined the frequency of CD4 + T cells specific for the stimulating antigen within the antigen-responsive population of CFSE-based proliferation assays. We compared CD4 + T-cell responses to a type 1 diabetes autoantigen (proinsulin C-peptide) and to a vaccine antigen (tetanus toxoid). The TCRs expressed by antigen-responsive CD4 + T cells were sequenced, and their antigen specificity was tested functionally by expressing them in a reporter T-cell line. Responses to C-peptide were weak, but detectable, in PBMC from individuals with T1D, whereas responses to tetanus toxoid were much stronger. The frequency of antigen-specific CD4 + T cells correlated with the strength of the response to antigen in the proliferation assay. However, antigen-specific CD4 + T cells were rare among antigen-responsive CD4 + T cells. For C-peptide, an average frequency of 7.5% (1%–11%, n = 4) of antigen-responsive CD4 + T cells were confirmed to be antigen specific. In the tetanus-toxoid-stimulated cultures, on average, 45% (16%–78%, n = 5) of the antigen-responsive CD4 + T cells were tetanus toxoid specific. These data show that antigen-specific CD4 + T cells are a minority of the cells that proliferate in response to antigen and have important implications for in vitro CD4 + T-cell proliferation assays.

Necrotizing soft tissue infections are lethal polymicrobial infections. Two key microbes that cause necrotizing soft tissue infections are Streptococcus pyogenes and Clostridium perfringens . These pathogens evade innate immunity using multiple virulence factors, including cholesterol-dependent cytolysins (CDCs). CDCs are resisted by mammalian cells through the sequestration and shedding of pores during intrinsic membrane repair. One hypothesis is that vesicle shedding promotes immune evasion by concomitantly eliminating key signaling proteins present in cholesterol-rich microdomains. To test this hypothesis, murine macrophages were challenged with sublytic CDC doses. CDCs suppressed LPS or IFNγ-stimulated TNFα production and CD69 and CD86 surface expression. This suppression was cell intrinsic. Two membrane repair pathways, patch repair and intrinsic repair, might mediate TNFα suppression. However, patch repair did not correlate with TNFα suppression. Intrinsic repair partially contributed to macrophage dysfunction because TLR4 and the IFNγR were partially shed following CDC challenge. Intrinsic repair was not sufficient for suppression, because pore formation was also required. These findings suggest that even when CDCs fail to kill cells, they may impair innate immune signaling responses dependent on cholesterol-rich microdomains. This is one potential mechanism to explain the lethality of S. pyogenes and C. perfringens during necrotizing soft tissue infections.

CD8+ T‐regulatory (Treg) cells are emerging as crucial components of immune system. Previous studies have reported the presence of FOXP3+CD8+ Treg cells, similar to CD4+ Tregs, in cancer patients which produce high levels of the immunosuppressive cytokines, IL10 and TGFβ. At an early stage of tumor development, we have identified a subset of FOXP3−CD8+CD25+KIR+CD127− Treg‐like cells, which are IFNγ+. However, this early‐induced CD8+CD25+CD127− T‐cell subset is certainly distinct from the IFNγ+CD8+ T‐effector cells. These CD8+CD25+CD127− T cells express other FOXP3−CD8+ Treg cell signature markers, and can selectively suppress autoreactive HLA‐E+ TFH cells as well as tumor‐induced CD4+ Treg cells. In contrast to FOXP3+CD8+ Tregs, this subset does not inhibit effector T‐cell proliferation or their functions as they are HLA‐E−. Adoptive transfer of this early‐CD8+ Treg‐like subset restrained tumor growth and inhibited CD4+ Treg generation that impedes the immune surveillance and impairs cancer immunotherapy. At the late stage of tumor development, when CD4+ Treg cells dominate the tumor‐microenvironment, CD4+ Tregs mediate the clonal deletion of these tumor‐suppressive FOXP3−IFNγ+CD8+CD25+CD127− T cells and ensure tumor immune evasion. Our findings suggest that at an early stage of the tumor, this tumor‐induced IFNγ‐producing FOXP3−CD8+CD25+CD127− T‐cell subset can potentiate immune surveillance by targeting HLA‐E‐restricted CD4+ Treg cells while leaving the effector T‐cell population unaffected. Hence, manipulating their profile can open up a new avenue in cancer immunotherapy. In this study, a FOXP3−CD8+CD25+KIR+CD127− Treg‐like subset has been identified. This subset predominated at the early stage of the tumor and suppressed HLA‐E+ TFH cells. With the progression of the tumor, CD4+ Treg cells increase in number and selectively kill these IFN‐γ producing CD8+CD25+CD127− Treg‐like cells.

Abstract Type 1 diabetes (T1D) is an autoimmune disease that develops when T cells destroy the insulin-producing beta cells that reside in the pancreatic islets. Immune cells, including T cells, infiltrate the islets and gradually destroy the beta cells. Human islet-infiltrating CD4+ T cells recognize peptide epitopes derived from proinsulin, particularly C-peptide. Hybrid insulin peptides (HIPs) are neoepitopes formed by the fusion of two peptides derived from beta cell granule proteins and are known to be the targets of pathogenic CD4+ T cells in the non-obese diabetic (NOD) mouse and human islet-infiltrating CD4+ T cells. Proinsulin is widely recognized as a central antigen in T1D, but its role in forming HIPs is unclear. We developed a method to functionally screen TCRs derived from human islet-infiltrating CD4+ T cells and applied this to the identification of new proinsulin-derived HIPs. We generated a library of 4,488 candidate HIPs formed by fusion of proinsulin fragments and predicted to bind to HLA-DQ8. This library was screened against 109 islet-infiltrating CD4+ T cell receptors (TCRs) isolated from four organ donors who had T1D. We identified 13 unique HIPs recognized by nine different TCRs from two organ donors. HIP-specific T cell avatars responded specifically to a peptide extract from human islets. These new HIPs predominantly stimulated CD4+ T cell proliferation in peripheral blood mononuclear cells from individuals with T1D in contrast to HLA-matched controls. This is the first unbiased functional, islet-infiltrating T cell based, screen to identify proinsulin-derived HIPs. It has revealed many new HIPs and a central role of proinsulin C-peptide in their formation.