Stem-like intestinal Th17 cells give rise to pathogenic effector T cells during autoimmunity.

While intestinal Th17 cells are critical for maintaining tissue homeostasis, recent studies have implicated their roles in the development of extra-intestinal autoimmune diseases including multiple sclerosis. However, the mechanisms by which tissue Th17 cells mediate these dichotomous functions remain unknown. Here, we characterized the heterogeneity, plasticity, and migratory phenotypes of tissue Th17 cells in vivo by combined fate mapping with profiling of the transcriptomes and TCR clonotypes of over 84,000 Th17 cells at homeostasis and during CNS autoimmune inflammation. Inter- and intra-organ single-cell analyses revealed a homeostatic, stem-like TCF1+ IL-17+ SLAMF6+ population that traffics to the intestine where it is maintained by the microbiota, providing a ready reservoir for the IL-23-driven generation of encephalitogenic GM-CSF+ IFN-γ+ CXCR6+ T cells. Our study defines a direct in vivo relationship between IL-17+ non-pathogenic and GM-CSF+ and IFN-γ+ pathogenic Th17 populations and provides a mechanism by which homeostatic intestinal Th17 cells direct extra-intestinal autoimmune disease.

A Human IgSF Cell-Surface Interactome Reveals a Complex Network of Protein-Protein Interactions.

Cell-surface protein-protein interactions (PPIs) mediate cell-cell communication, recognition, and responses. We executed an interactome screen of 564 human cell-surface and secreted proteins, most of which are immunoglobulin superfamily (IgSF) proteins, using a high-throughput, automated ELISA-based screening platform employing a pooled-protein strategy to test all 318,096 PPI combinations. Screen results, augmented by phylogenetic homology analysis, revealed ∼380 previously unreported PPIs. We validated a subset using surface plasmon resonance and cell binding assays. Observed PPIs reveal a large and complex network of interactions both within and across biological systems. We identified new PPIs for receptors with well-characterized ligands and binding partners for "orphan" receptors. New PPIs include proteins expressed on multiple cell types and involved in diverse processes including immune and nervous system development and function, differentiation/proliferation, metabolism, vascularization, and reproduction. These PPIs provide a resource for further biological investigation into their functional relevance and may offer new therapeutic drug targets.

SLAM receptors foster iNKT cell development by reducing TCR signal strength after positive selection.

Invariant natural killer T cells (iNKT cells) develop through an incompletely understood process that requires positive selection by CD4+CD8+ double-positive thymocytes and SLAM family receptors (SFRs). Here we found that SFRs promoted the development of iNKT cells by reducing the strength of the T cell antigen receptor (TCR) signal after positive selection. This effect improved the survival of iNKT cells and their responses to antigen. Loss of SFRs upregulated the expression of inhibitory receptors, including PD-1, on iNKT cells to mitigate the deleterious effect of SFR deficiency. The role of SFRs could be mimicked by expression of SLAMF6 alone in SFR-deficient mice, and this involved the adaptor SAP-kinase Fyn complex and the phosphatase SHP-1. Thus, SFRs foster iNKT cell development by attenuating TCR signal strength after positive selection.

PTPN2 regulates the generation of exhausted CD8+ T cell subpopulations and restrains tumor immunity.

CD8+ T cell exhaustion is a state of dysfunction acquired in chronic viral infection and cancer, characterized by the formation of Slamf6+ progenitor exhausted and Tim-3+ terminally exhausted subpopulations through unknown mechanisms. Here we establish the phosphatase PTPN2 as a new regulator of the differentiation of the terminally exhausted subpopulation that functions by attenuating type 1 interferon signaling. Deletion of Ptpn2 in CD8+ T cells increased the generation, proliferative capacity and cytotoxicity of Tim-3+ cells without altering Slamf6+ numbers during lymphocytic choriomeningitis virus clone 13 infection. Likewise, Ptpn2 deletion in CD8+ T cells enhanced Tim-3+ anti-tumor responses and improved tumor control. Deletion of Ptpn2 throughout the immune system resulted in MC38 tumor clearance and improved programmed cell death-1 checkpoint blockade responses to B16 tumors. Our results indicate that increasing the number of cytotoxic Tim-3+CD8+ T cells can promote effective anti-tumor immunity and implicate PTPN2 in immune cells as an attractive cancer immunotherapy target.

Molecular mechanisms of lineage decisions in metabolite-specific T cells.

Mucosal-associated invariant T cells (MAIT cells) recognize the microbial metabolite 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU) presented by the MHC class Ib molecule, MR1. MAIT cells acquire effector functions during thymic development, but the mechanisms involved are unclear. Here we used single-cell RNA-sequencing to characterize the developmental path of 5-OP-RU-specific thymocytes. In addition to the known MAIT1 and MAIT17 effector subsets selected on bone-marrow-derived hematopoietic cells, we identified 5-OP-RU-specific thymocytes that were selected on thymic epithelial cells and differentiated into CD44- naive T cells. MAIT cell positive selection required signaling through the adapter, SAP, that controlled the expression of the transcription factor, ZBTB16. Pseudotemporal ordering of single cells revealed transcriptional trajectories of 5-OP-RU-specific thymocytes selected on either thymic epithelial cells or hematopoietic cells. The resulting model illustrates T cell lineage decisions.

The Self-Specific Activation Receptor SLAM Family Is Critical for NK Cell Education.

NK cell education, a term describing a process for NK cell acquisition of functional competence, is primarily achieved by self-MHC-I-specific inhibitory receptors. In this study, we have demonstrated that SLAM family receptors (SFRs) redundantly expressed on hematopoietic cells function as self-specific activation receptors critical for NK cell education. To overcome gene redundancy, we generated mice simultaneously lacking seven SFRs, revealing that NK-cell-mediated rejection of semi-allogeneic hematopoietic cells largely depended on the presence of SFRs on target cells. This stimulatory effect was determined by the presence of SFR-coupled adaptors; however, SFR-deficient mice displayed enhanced reactivity to hematopoietic cells. These findings demonstrate that SFRs endow NK cells with an ability to kill hematopoietic cells during the effector phase; however, the sustained engagement of SFRs can desensitize NK cell responses during an education process. Therefore, self-specific activating ligands may be "tolerogens" for NK cells, akin to self-antigens that induce T cell tolerance.

Targeted deletion of CD244 on monocytes promotes differentiation into anti-tumorigenic macrophages and potentiates PD-L1 blockade in melanoma.

BACKGROUND: In the myeloid compartment of the tumor microenvironment, CD244 signaling has been implicated in immunosuppressive phenotype of monocytes. However, the precise molecular mechanism and contribution of CD244 to tumor immunity in monocytes/macrophages remains elusive due to the co-existing lymphoid cells expressing CD244. METHODS: To directly assess the role of CD244 in tumor-associated macrophages, monocyte-lineage-specific CD244-deficient mice were generated using cre-lox recombination and challenged with B16F10 melanoma. The phenotype and function of tumor-infiltrating macrophages along with antigen-specific CD8 T cells were analyzed by flow cytometry and single cell RNA sequencing data analysis, and the molecular mechanism underlying anti-tumorigenic macrophage differentiation, antigen presentation, phagocytosis was investigated ex vivo. Finally, the clinical feasibility of CD244-negative monocytes as a therapeutic modality in melanoma was confirmed by adoptive transfer experiments. RESULTS: CD244fl/flLysMcre mice demonstrated a significant reduction in tumor volume (61% relative to that of the CD244fl/fl control group) 14 days after tumor implantation. Within tumor mass, CD244fl/flLysMcre mice also showed higher percentages of Ly6Clow macrophages, along with elevated gp100+IFN-γ+ CD8 T cells. Flow cytometry and RNA sequencing data demonstrated that ER stress resulted in increased CD244 expression on monocytes. This, in turn, impeded the generation of anti-tumorigenic Ly6Clow macrophages, phagocytosis and MHC-I antigen presentation by suppressing autophagy pathways. Combining anti-PD-L1 antibody with CD244-/- bone marrow-derived macrophages markedly improved tumor rejection compared to the anti-PD-L1 antibody alone or in combination with wild-type macrophages. Consistent with the murine data, transcriptome analysis of human melanoma tissue single-cell RNA-sequencing dataset revealed close association between CD244 and the inhibition of macrophage maturation and function. Furthermore, the presence of CD244-negative monocytes/macrophages significantly increased patient survival in primary and metastatic tumors. CONCLUSION: Our study highlights the novel role of CD244 on monocytes/macrophages in restraining anti-tumorigenic macrophage generation and tumor antigen-specific T cell response in melanoma. Importantly, our findings suggest that CD244-deficient macrophages could potentially be used as a therapeutic agent in combination with immune checkpoint inhibitors. Furthermore, CD244 expression in monocyte-lineage cells serve as a prognostic marker in cancer patients.

The role of signaling lymphocyte activation molecule family receptors in hematologic malignancies.

PURPOSE OF REVIEW: In this review, we provide an overview of the current understanding of SLAM-family receptors in hematologic malignancies. We highlighted their contribution to the disease pathogenesis and targeting strategies to improve therapeutic outcomes. RECENT FINDINGS: Emerging studies have reported the tumor-promoting role of SLAM-family receptors in various hematologic malignancies, including chronic lymphocytic leukemia, acute myeloid leukemia, and multiple myeloma. Specifically, they regulate the interaction between malignant cells and the tumor microenvironment to promote apoptosis resistance, therapeutic resistance, impairment of antitumor and tumor progression. SUMMARY: SLAM-family receptors promote the progression of hematologic malignancies by regulating the interaction between malignant cells and the tumor microenvironment. This provides the rationale that SLAM-targeted therapies are appealing strategies to enhance therapeutic outcomes in patients.

2B4 inhibits the apoptosis of natural killer cells through phosphorylated extracellular signal-related kinase/B-cell lymphoma 2 signal pathway.

BACKGROUND AIMS: Decades after the identification of natural killer (NK) cells as potential effector cells against malignantly transformed cells, an increasing amount of research suggests that NK cells are a prospective choice of immunocytes for cancer immunotherapy in addition to T lymphocytes for cancer immunotherapy. Recent studies have led to a breakthrough in the combination of hematopoietic stem-cell transplantation with allogeneic NK cells infusion for the treatment of malignant tumors. However, the short lifespan of NK cells in patients is the major impediment, limiting their efficacy. Therefore, prolonging the survival of NK cells will promote the application of NK-cell immunotherapy. As we have known, NK cells use a "missing-self" mechanism to lyse target cells and exert their functions through a wide array of activating, co-stimulatory and inhibitory receptors. Our previous study has suggested that CD244 (2B4), one of the co-stimulatory receptors, can improve the function of chimeric antigen receptor NK cells. However, the underlying mechanism of how 2B4 engages in the function of NK cells requires further investigation. Overall, we established a feeder cell with the expression of CD48, the ligand of 2B4, to investigate the function of 2B4-CD48 axis in NK cells, and meanwhile, to explore whether the newly generated feeder cell can improve the function of ex vivo-expanded NK cells. METHODS: First, K562 cells overexpressing 4-1BBL and membrane-bound IL-21 (mbIL-21) were constructed (K562-41BBL-mbIL-21) and were sorted to generate the single clone. These widely used feeder cells (K562-41BBL-mbIL-21) were named as Basic Feeder hereinafter. Based on the Basic feeder, CD48 was overexpressed and named as CD48 Feeder. Then, the genetically modified feeder cells were used to expand primary NK cells from peripheral blood or umbilical cord blood. In vitro experiments were performed to compare proliferation ability, cytotoxicity, survival and activation/inhibition phenotypes of NK cells stimulated via different feeder cells. K562 cells were injected into nude mice subcutaneously with tail vein injection of NK cells from different feeder system for the detection of NK in vivo persistence and function. RESULTS: Compared with Basic Feeders, CD48 Feeders can promote the proliferation of primary NK cells from peripheral blood and umbilical cord blood and reduce NK cell apoptosis by activating the p-ERK/BCL2 pathway both in vitro and in vivo without affecting overall phenotypes. Furthermore, NK cells expanded via CD48 Feeders showed stronger anti-tumor capability and infiltration ability into the tumor microenvironment. CONCLUSIONS: In this preclinical study, the engagement of the 2B4-CD48 axis can inhibit the apoptosis of NK cells through the p-ERK/BCL2 signal pathway, leading to an improvement in therapeutic efficiency.

Increased SLAMF7+CD8+ T cells are associated with the pathogenesis of experimental autoimmune pancreatitis in mice.

BACKGROUND: IgG4-related autoimmune pancreatitis (AIP) is considered to be a T cell-mediated autoimmune disease. However, CD8+ T cells have only received brief mention, and have yet to be completely studied. The study aimed to investigate the expression of signaling lymphocytic activation molecule family 7 (SLAMF7) on CD8+ T cells and the features of SLAMF7+CD8+ T cells in MRL/Mp mice with AIP. METHODS: A murine model of AIP was established by intraperitoneal injection with polyinosinic:polycytidylic acid (poly I:C) for 8 weeks. Dexamethasone treatment was daily administrated for the last 2 weeks during a 6-week course of poly I:C. SLAMF7 expression on CD8+ T cells in the spleen and pancreas was detected by flow cytometry. Granzyme B (GZMB) and cytokines including IFN-γ, TNF-α, and IL-2, were monitored in an in vitro T cell activation assay. Dexamethasone suppression assays were performed to downregulate SLAMF7 expression on T cells upon T cell receptor stimulation. RESULTS: AIP in MRL/Mp mice was induced by repeated intraperitoneal administration of poly I:C and CD8+ T cells were increased in the inflamed pancreas. SLAMF7+CD8+ T cells were elevated in the spleen and pancreas of AIP mice. SLAMF7+CD8+ T subsets produced more GZMB, IFN-γ, TNF-α and IL-2 than SLAMF7-CD8+ T subsets. Dexamethasone treatment ameliorated pancreatic inflammatory and fibrosis of AIP. Dexamethasone could downregulate SLAMF7+CD8+ T cells and reduce GZMB, IFN-γ and TNF-α levels both in vitro and in vivo. CONCLUSIONS: Increased SLAMF7+CD8+ T cells exhibit enhanced cytotoxicity and cytokines secretion capacity, which may be involved in the pathogenesis of AIP.

SLAMF7 Signaling Reprograms T Cells toward Exhaustion in the Tumor Microenvironment.

T cell exhaustion represents one of the most pervasive strategies tumors employ to circumvent the immune system. Although repetitive, cognate TCR signaling is recognized as the primary driving force behind this phenomenon, and it remains unknown what other forces drive T cell exhaustion in the tumor microenvironment (TME). In this study, we show that activation of the self-ligand SLAMF7 immune receptor on T cells induced STAT1 and STAT3 phosphorylation, expression of multiple inhibitory receptors, and transcription factors associated with T cell exhaustion. Analysis of The Cancer Genome Atlas revealed that SLAMF7 transcript levels were strongly correlated with various inhibitory receptors and that high SLAMF7 expression was indicative of poor survival in clear cell renal cell carcinoma (ccRCC). Targeted reanalysis of a CyTOF dataset, which profiled the TME in 73 ccRCC patients, revealed cell-type-specific SLAMF7 expression patterns, strong correlations between exhausted T cells and SLAMF7+ tumor-associated macrophages (TAMs), and a unique subset of SLAMF7highCD38high TAMs. These SLAMF7highCD38high TAMs showed the strongest correlations with exhausted T cells and were an independent prognostic factor in ccRCC. Confirmatory ex vivo coculture studies validated that SLAMF7-SLAMF7 interactions between murine TAMs and CD8+ T cells induce expression of multiple inhibitory receptors. Finally, mice lacking SLAMF7 show restricted growth of B16-F10 tumors, and CD8+ T cells from these mice express less PD-1 and TOX and exhibited an impaired ability to progress through the exhaustion developmental trajectory to terminal exhaustion. These findings suggest that SLAMF7 might play an important role in modulating T cell function in the TME.

Pathologically expanded peripheral T helper cell subset drives B cells in rheumatoid arthritis.

CD4+ T cells are central mediators of autoimmune pathology; however, defining their key effector functions in specific autoimmune diseases remains challenging. Pathogenic CD4+ T cells within affected tissues may be identified by expression of markers of recent activation. Here we use mass cytometry to analyse activated T cells in joint tissue from patients with rheumatoid arthritis, a chronic immune-mediated arthritis that affects up to 1% of the population. This approach revealed a markedly expanded population of PD-1hiCXCR5-CD4+ T cells in synovium of patients with rheumatoid arthritis. However, these cells are not exhausted, despite high PD-1 expression. Rather, using multidimensional cytometry, transcriptomics, and functional assays, we define a population of PD-1hiCXCR5- 'peripheral helper' T (TPH) cells that express factors enabling B-cell help, including IL-21, CXCL13, ICOS, and MAF. Like PD-1hiCXCR5+ T follicular helper cells, TPH cells induce plasma cell differentiation in vitro through IL-21 secretion and SLAMF5 interaction (refs 3, 4). However, global transcriptomics highlight differences between TPH cells and T follicular helper cells, including altered expression of BCL6 and BLIMP1 and unique expression of chemokine receptors that direct migration to inflamed sites, such as CCR2, CX3CR1, and CCR5, in TPH cells. TPH cells appear to be uniquely poised to promote B-cell responses and antibody production within pathologically inflamed non-lymphoid tissues.

SLAMF7 is critical for phagocytosis of haematopoietic tumour cells via Mac-1 integrin.

Cancer cells elude anti-tumour immunity through multiple mechanisms, including upregulated expression of ligands for inhibitory immune checkpoint receptors. Phagocytosis by macrophages plays a critical role in cancer control. Therapeutic blockade of signal regulatory protein (SIRP)-α, an inhibitory receptor on macrophages, or of its ligand CD47 expressed on tumour cells, improves tumour cell elimination in vitro and in vivo, suggesting that blockade of the SIRPα-CD47 checkpoint could be useful in treating human cancer. However, the pro-phagocytic receptor(s) responsible for tumour cell phagocytosis is(are) largely unknown. Here we find that macrophages are much more efficient at phagocytosis of haematopoietic tumour cells, compared with non-haematopoietic tumour cells, in response to SIRPα-CD47 blockade. Using a mouse lacking the signalling lymphocytic activation molecule (SLAM) family of homotypic haematopoietic cell-specific receptors, we determined that phagocytosis of haematopoietic tumour cells during SIRPα-CD47 blockade was strictly dependent on SLAM family receptors in vitro and in vivo. In both mouse and human cells, this function required a single SLAM family member, SLAMF7 (also known as CRACC, CS1, CD319), expressed on macrophages and tumour cell targets. In contrast to most SLAM receptor functions, SLAMF7-mediated phagocytosis was independent of signalling lymphocyte activation molecule-associated protein (SAP) adaptors. Instead, it depended on the ability of SLAMF7 to interact with integrin Mac-1 (refs 18, 19, 20) and utilize signals involving immunoreceptor tyrosine-based activation motifs. These findings elucidate the mechanism by which macrophages engulf and destroy haematopoietic tumour cells. They also reveal a novel SAP adaptor-independent function for a SLAM receptor. Lastly, they suggest that patients with tumours expressing SLAMF7 are more likely to respond to SIRPα-CD47 blockade therapy.

SLAMF1 is expressed and secreted by hepatocytes and the liver in nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is one of the most prevalent forms of chronic liver disease in the United States and worldwide. Nonalcoholic steatohepatitis (NASH), the most advanced form of NAFLD, is characterized by hepatic steatosis associated with inflammation and hepatocyte death. No treatments are currently available for NASH other than lifestyle changes, and the disease lacks specific biomarkers. The signaling lymphocytic activation molecule family 1 (SLAMF1) protein is a self-ligand receptor that plays a role in orchestrating an immune response to some pathogens and cancers. We found that livers from humans and mice with NASH showed a more prominent immunohistochemistry staining for SLAMF1 than non-NASH controls. Furthermore, SLAMF1 levels are significantly increased in NASH plasma samples from mice and humans compared with their respective controls. In mice, the levels of SLAMF1 correlated significantly with the severity of the NASH phenotype. To test whether SLAMF 1 is expressed by hepatocytes, HepG2 cells and primary murine hepatocytes were treated with palmitic acid (PA) to induce a state of lipotoxicity mimicking NASH. We found that PA treatments of HepG2 cells and primary hepatocytes lead to significant increases in SLAMF1 levels. The downregulation of SLAMF1 in HepG2 cells improved the cell viability and reduced cytotoxicity. The in vivo data using mouse and human NASH samples suggests a potential role for this protein as a noninvasive biomarker for NASH. The in vitro data suggest a role for SLAMF1 as a potential therapeutic target to prevent hepatocyte death in response to lipotoxicity.NEW & NOTEWORTHY This study identified for the first time SLAMF1 as a mediator of hepatocyte death in nonalcoholic fatty liver disease (NASH) and as a marker of NASH in humans. There are no pharmacological treatments available for NASH, and diagnostic tools are limited to invasive liver biopsies. Therefore, since SLAMF1 levels correlate with disease progression and SLAMF1 mediates cytotoxic effects, this protein can be used as a therapeutic target and a clinical biomarker of NASH.

SLAMF7 modulates B cells and adaptive immunity to regulate susceptibility to CNS autoimmunity.

BACKGROUND: Multiple sclerosis (MS) is a chronic, debilitating condition characterized by CNS autoimmunity stemming from a complex etiology involving both environmental and genetic factors. Our current understanding of MS points to dysregulation of the immune system as the pathogenic culprit, however, it remains unknown as to how the many genes associated with increased susceptibility to MS are involved. One such gene linked to MS susceptibility and known to regulate immune function is the self-ligand immune cell receptor SLAMF7. METHODS: We subjected WT and SLAMF7-/- mice to multiple EAE models, compared disease severity, and comprehensively profiled the CNS immune landscape of these mice. We identified all SLAMF7-expressing CNS immune cells and compared the entire CNS immune niche between genotypes. We performed deep phenotyping and in vitro functional studies of B and T cells via spectral cytometry and BioPlex assays. Adoptive transfer studies involving the transfer of WT and SLAMF7-/- B cells into B cell-deficient mice (µMT) were also performed. Finally, B-T cell co-culture studies were performed, and a comparative cell-cell interaction network derived from scRNA-seq data of SLAMF7+ vs. SLAMF7- human CSF immune cells was constructed. RESULTS: We found SLAMF7-/- mice to be more susceptible to EAE compared to WT mice and found SLAMF7 to be expressed on numerous CNS immune cell subsets. Absence of SLAMF7 did not grossly alter the CNS immune landscape, but allowed for altered immune cell subset infiltration during EAE in a model-dependent manner. Global lack of SLAMF7 expression increased myeloid cell activation states along with augmented T cell anti-MOG immunity. B cell profiling studies revealed increased activation states of specific plasma and B cell subsets in SLAMF7-/- mice during EAE, and functional co-culture studies determined that SLAMF7-/- B cells induce exaggerated T cell activation. Adoptive transfer studies revealed that the increased susceptibility of SLAMF7-/- mice to EAE is partly B cell dependent and reconstruction of the human CSF SLAMF7-interactome found B cells to be critical to cell-cell communication between SLAMF7-expressing cells. CONCLUSIONS: Our studies have identified novel roles for SLAMF7 in CNS immune regulation and B cell function, and illuminate underpinnings of the genetic association between SLAMF7 and MS.

CD84 Links T Cell and Platelet Activity in Cerebral Thrombo-Inflammation in Acute Stroke.

RATIONALE: Ischemic stroke is a leading cause of morbidity and mortality worldwide. Recanalization of the occluded vessel is essential but not sufficient to guarantee brain salvage. Experimental and clinical data suggest that infarcts often develop further due to a thromboinflammatory process critically involving platelets and T cells, but the underlying mechanisms are unknown. OBJECTIVE: We aimed to determine the role of CD (cluster of differentiation)-84 in acute ischemic stroke after recanalization and to dissect the underlying molecular thromboinflammatory mechanisms. METHODS AND RESULTS: Here, we show that mice lacking CD84-a homophilic immunoreceptor of the SLAM (signaling lymphocyte activation molecule) family-on either platelets or T cells displayed reduced cerebral CD4+ T-cell infiltration and thrombotic activity following experimental stroke resulting in reduced neurological damage. In vitro, platelet-derived soluble CD84 enhanced motility of wild-type but not of Cd84-/- CD4+ T cells suggesting homophilic CD84 interactions to drive this process. Clinically, human arterial blood directly sampled from the ischemic cerebral circulation indicated local shedding of platelet CD84. Moreover, high platelet CD84 expression levels were associated with poor outcome in patients with stroke. CONCLUSIONS: These results establish CD84 as a critical pathogenic effector and thus a potential pharmacological target in ischemic stroke.

Critical Role for SLAM/SAP Signaling in the Thymic Developmental Programming of IL-17- and IFN-γ-Producing γδ T Cells.

During thymic development, mouse γδ T cells commit to either an IFN-γ- or an IL-17-producing phenotype through mechanisms that remain unclear. In this study, we investigated the extent to which the SLAM/SAP signaling pathway regulates the functional programming of γδ T cells. Characterization of SLAM family receptor expression revealed that thymic γδ T cell subsets were each marked by distinct coexpression profiles of SLAMF1, SLAMF4, and SLAMF6. In the thymus, Vγ1 and Vγ4 T cells that exhibited an SLAMF1+SLAMF6+ double positive phenotype were largely contained within immature CD24+CD73- and CD24+CD73+ subsets, whereas SLAMF1 single positive, SLAMF6 single positive, or SLAMF1SLAMF6 double negative cells were found within mature CD24-CD73+ and CD24-CD73- subsets. In the periphery, SLAMF1 and SLAMF6 expression distinguished IL-17- and IFN-γ-producing γδ T cells, respectively. Disruption of SLAM family receptor signaling through deletion of SAP resulted in impaired thymic Vγ1 and Vγ4 T cell maturation at the CD24+CD73-SLAMF1+SLAMF6+ double positive stage that was associated with a decreased frequency of CD44+RORγt+ γδ T cells. Impaired development was in turn associated with decreased γδ T cell IL-17 and IFN-γ production in the thymus as well as in peripheral tissues. The role for SAP was subset-specific, as Vγ1Vδ6.3, Vγ4, Vγ5, but not Vγ6 subsets were SAP-dependent. Together, these data suggest that the SLAM/SAP signaling pathway plays a larger role in γδ T cell development than previously appreciated and represents a critical checkpoint in the functional programming of both IL-17- and IFN-γ-producing γδ T cell subsets.

Feasibility of platelet marker analysis in ischemic stroke patients and their association with one-year outcome. A pilot project within a subsample of the Stroke Induced Cardiac Failure in Mice and Men (SICFAIL) cohort study.

Patients with ischemic stroke (IS) are at increased risk of mortality and recurrent cerebro- or cardiovascular events. Determining prognosis after IS remains challenging but blood-based biomarkers might provide additional prognostic information. As platelets are crucially involved in the pathophysiology of vascular diseases, platelet surface proteins (PSP) are promising candidates as prognostic markers in the hyperacute stage. In this pilot study, feasibility of PSP analysis by flow cytometry (HMGB1, CD84, CXCR4, CXCR7, CD62p with and without ADP-stimulation, CD41, CD61, CD40, GPVI) was investigated in 99 (median 66 years, 67.5% male) acute IS patients admitted to Stroke Unit within a substudy of the Stroke-Induced Cardiac FAILure in mice and men (SICFAIL) cohort study. Association between PSP expression and unfavorable one-year outcome (cerebro- or cardiovascular event, all-cause mortality and care dependency defined as Barthel Index <60) was explored. PSP measurements were feasible. Several process- (e.g. temperatures, processing times) and patient-related factors (e.g. prestroke ischemic events, surgery, blood pressure, antiplatelet therapy) were identified to be potentially associated with PSP expression. Elevated CD40 levels above study population's median were associated with unfavorable outcome. Standardized conditions during blood draw and processing within the hyperacute stroke unit setting are required and patient-related characteristics must be considered for valid measurements of PSP.Trial registration: German Clinical Trials Register (DRKS00011615).

SLAMF9 regulates pDC homeostasis and function in health and disease.

SLAMF9 belongs to the conserved lymphocytic activation molecule family (SLAMF). Unlike other SLAMs, which have been extensively studied, the role of SLAMF9 in the immune system remained mostly unexplored. By generating CRISPR/Cas9 SLAMF9 knockout mice, we analyzed the role of this receptor in plasmacytoid dendritic cells (pDCs), which preferentially express the SLAMF9 transcript and protein. These cells display a unique capacity to produce type I IFN and bridge between innate and adaptive immune response. Analysis of pDCs in SLAMF9-/- mice revealed an increase of immature pDCs in the bone marrow and enhanced accumulation of pDCs in the lymph nodes. In the periphery, SLAMF9 deficiency resulted in lower levels of the transcription factor SpiB, elevation of pDC survival, and attenuated IFN-α and TNF-α production. To define the role of SLAMF9 during inflammation, pDCs lacking SLAMF9 were followed during induced experimental autoimmune encephalomyelitis. SLAMF9-/- mice demonstrated attenuated disease and delayed onset, accompanied by a prominent increase of immature pDCs in the lymph node, with a reduced costimulatory potential and enhanced infiltration of pDCs into the central nervous system. These results suggest the crucial role of SLAMF9 in pDC differentiation, homeostasis, and function in the steady state and during experimental autoimmune encephalomyelitis.

Evidence for the loss and recovery of SLAMF9 during human evolution: implications on Dollo's law.

Signaling lymphocyte activation molecule family member 9 (SLAMF9) is a cell surface protein of the CD2/SLAM family of leukocyte surface receptors. It is conserved throughout mammals and has roles in the initiation of inflammatory responses and regulation of plasmacytoid dendritic cell function. Through comparison of reference sequences encoding SLAMF9 in human, mouse, and primate sequences, we have determined that the SLAMF9 gene underwent successive mutation events, resulting in the loss of the protein and subsequent recovery of a less stable version. The mutations included a single base pair deletion in the second exon and a change in the splice acceptor site of that same exon. These changes would have had the effect of creating and later repairing a frameshift in the coding sequence. These events took place since the divergence of the human lineage from the chimpanzee-human last common ancestor and represent the first known case of the functional loss and recovery of a gene within the human lineage.