Role of Tim4 in the regulation of ABCA1+ adipose tissue macrophages and post-prandial cholesterol levels

Dyslipidemia is a main driver of cardiovascular diseases. The ability of macrophages to scavenge excess lipids implicate them as mediators in this process and understanding the mechanisms underlying macrophage lipid metabolism is key to the development of new treatments. Here, we investigated how adipose tissue macrophages regulate post-prandial cholesterol transport. Single-cell RNA sequencing and protected bone marrow chimeras demonstrated that ingestion of lipids led to specific transcriptional activation of a population of resident macrophages expressing Lyve1, Tim4, and ABCA1. Blocking the phosphatidylserine receptor Tim4 inhibited lysosomal activation and the release of post-prandial high density lipoprotein cholesterol following a high fat meal. Both effects were recapitulated by chloroquine, an inhibitor of lysosomal function. Moreover, clodronate-mediated cell-depletion implicated Tim4+ resident adipose tissue macrophages in this process. Thus, these data indicate that Tim4 is a key regulator of post-prandial cholesterol transport and adipose tissue macrophage function and may represent a novel pathway to treat dyslipidemia.

The Phosphatidylserine Receptor TIM-1 Enhances Authentic Chikungunya Virus Cell Entry

Chikungunya virus (CHIKV) is a re-emerging, mosquito-transmitted, enveloped positive stranded RNA virus. Chikungunya fever is characterized by acute and chronic debilitating arthritis. Although multiple host factors have been shown to enhance CHIKV infection, the molecular mechanisms of cell entry and entry factors remain poorly understood. The phosphatidylserine-dependent receptors, T-cell immunoglobulin and mucin domain 1 (TIM-1) and Axl receptor tyrosine kinase (Axl), are transmembrane proteins that can serve as entry factors for enveloped viruses. Previous studies used pseudoviruses to delineate the role of TIM-1 and Axl in CHIKV entry. Conversely, here, we use the authentic CHIKV and cells ectopically expressing TIM-1 or Axl and demonstrate a role for TIM-1 in CHIKV infection. To further characterize TIM-1-dependent CHIKV infection, we generated cells expressing domain mutants of TIM-1. We show that point mutations in the phosphatidylserine binding site of TIM-1 lead to reduced binding, entry, and infection of CHIKV. Ectopic expression of TIM-1 renders immortalized keratinocytes permissive to CHIKV, whereas silencing of endogenously expressed TIM-1 in human hepatoma cells reduces CHIKV infection. Altogether, our findings indicate that, unlike Axl, TIM-1 readily promotes the productive entry of authentic CHIKV into target cells.

TIM4 expression by dendritic cells mediates uptake of tumor-associated antigens and anti-tumor responses

Acquisition of cell-associated tumor antigens by type 1 dendritic cells (cDC1) is essential to induce and sustain tumor specific CD8+ T cells via cross-presentation. Here we show that capture and engulfment of cell associated antigens by tissue resident lung cDC1 is inhibited during progression of mouse lung tumors. Mechanistically, loss of phagocytosis is linked to tumor-mediated downregulation of the phosphatidylserine receptor TIM4, that is highly expressed in normal lung resident cDC1. TIM4 receptor blockade and conditional cDC1 deletion impair activation of tumor specific CD8+ T cells and promote tumor progression. In human lung adenocarcinomas, TIM4 transcripts increase the prognostic value of a cDC1 signature and predict responses to PD-1 treatment. Thus, TIM4 on lung resident cDC1 contributes to immune surveillance and its expression is suppressed in advanced tumors.

Hemolysis-associated phosphatidylserine exposure promotes polyclonal plasmablast differentiation

Antimalarial antibody responses are essential for mediating the clearance of Plasmodium parasite–infected RBCs from infected hosts. However, the rapid appearance of large numbers of plasmablasts in Plasmodium-infected hosts can suppress the development and function of durable humoral immunity. Here, we identify that the formation of plasmablast populations in Plasmodium-infected mice is mechanistically linked to both hemolysis-induced exposure of phosphatidylserine on damaged RBCs and inflammatory cues. We also show that virus and Trypanosoma infections known to trigger hemolytic anemia and high-grade inflammation also induce exuberant plasmablast responses. The induction of hemolysis or administration of RBC membrane ghosts increases plasmablast differentiation. The phosphatidylserine receptor Axl is critical for optimal plasmablast formation, and blocking phosphatidylserine limits plasmablast expansions and reduces Plasmodium parasite burden in vivo. Our findings support that strategies aimed at modulating polyclonal B cell activation and phosphatidylserine exposure may improve immune responses against Plasmodium parasites and potentially other infectious diseases that are associated with anemia.

524 Tim-4+ resident macrophages impair anti-tumor immunity in the serous body cavities By sequestering viable and cytotoxic CD8+ T cells expressing high levels of phosphatidylserine

BackgroundMalignant pleural effusions and peritoneal carcinomatosis are associated with poor outcomes in patients with cancer.1–3 Macrophages in these serous body cavities express the phosphatidylserine receptor Tim-4.4–8 Prior reports demonstrated that Tim-4 abrogation is associated with improved anti-tumor activity.9–11 Whether macrophages expressing Tim-4 contribute to immunosuppression in the serous body cavities has not been previously investigated.MethodsWe retrospectively annotated sites of metastases in 500 patients with lung cancer and assessed for clinical outcomes. Utilizing a combination of flow cytometry, immunohistochemistry, and antibody biodistribution assays, we surveyed for Tim-4 expression across various tissues and cell types. We performed flow cytometry on 55 consecutive pleural and peritoneal effusions from patients with lung cancer. We utilized murine models of peritoneal carcinomatosis to determine whether Tim-4 abrogation could enhance the anti-tumor efficacy of anti-PD-1 therapy. We characterized CD8+ T cells with high levels of phosphatidylserine (PShigh) with flow cytometry, cytotoxicity assays, and paired single cell RNA and TCR sequencing. Confocal microscopy was utilized to visualize interactions between Tim-4+ macrophages and PShigh CD8+ T cells.ResultsMetastatic disease involvement of the pleural or peritoneal cavity was associated with reduced response rate and progression-free and overall survival. We demonstrate that Tim-4 is highly expressed on pleural and peritoneal macrophages and other select resident macrophages, but not on monocytes, tumor-associated macrophages, or tumor cells in mice and humans. High levels of Tim-4 on macrophages from fluid biospecimens is associated with reduced levels CD39+ CD8+ T cells, which comprise the tumor-reactive portion of CD8+ T lymphocytes. In order to further elucidate the mechanism of Tim-4+ macrophage-mediated immunosuppression, we established a murine model of peritoneal carcinomatosis with MC38 and CT26 colon carcinoma. Genetic or pharmacologic abrogation of Tim-4 improved the efficacy of anti-PD-1 therapy and was associated with enhanced CD39+ CD8+ T cell numbers. In parallel, we observed in mice and humans that CD8+ T cell activation results in PS upregulation despite not undergoing cell death. PShigh CD8+ T cells expressed genes associated with cytotoxicity, activation/exhaustion, and proliferation, and mediated greater cytotoxicity. Mechanistic studies revealed that Tim-4 mediates sequestration of PShigh CD8+ T cells by macrophages which subsequently impedes CD8+ T cell cytotoxicity of tumor cells.Abstract 524 Figure 1After activation by antigen-presenting cells in the lymph nodes, viable CD8+ T cells express high levels of phosphatidylserine, which coincides with a highly proliferative and cytotoxic state. As they migrate towards tumors cells in the serous body cavities, they are sequestered by Tim-4+ resident macrophages which impede their anti-tumor cytotoxicity. Tim-4 abrogation can alleviate this sequestration and enhance anti-tumor immunityConclusionsWe demonstrate that Tim-4+ resident macrophages impair anti-tumor CD8+ T cell immunity in the serous body cavities and Tim-4 blockade represents on a novel therapeutic strategy to overcome resistance to immune checkpoint blockade (figure 1).Ethics ApprovalThe retrospective clinical analysis was approved by Memorial Sloan Kettering Cancer Center IRB #16-1566. The human biospecimen analyses were approved by Memorial Sloan Kettering Cancer Center IRB #06-107 and 14-091.ReferencesPorcel JM, et al., Clinical features and survival of lung cancer patients with pleural effusions. Respirology 2015;20:654–659.Donnenberg AD, Luketich JD, Dhupar R, Donnenberg VS. Treatment of malignant pleural effusions: the case for localized immunotherapy. J Immunother Cancer 2019;7:110.Morano WF, et al., Intraperitoneal immunotherapy: historical perspectives and modern therapy. Cancer Gene Ther 2016;23:373–381.Bain CC, et al., Long-lived self-renewing bone marrow-derived macrophages displace embryo-derived cells to inhabit adult serous cavities. Nat Commun 2016;7:ncomms11852.Wong K, et al., Phosphatidylserine receptor Tim-4 is essential for the maintenance of the homeostatic state of resident peritoneal macrophages. Proc Natl Acad Sci U S A 2010;107:8712–8717.Miyanishi M, et al., Identification of Tim4 as a phosphatidylserine receptor. Nature 2007;450:435–439.Rodriguez-Manzanet R, et al. T and B cell hyperactivity and autoimmunity associated with niche-specific defects in apoptotic body clearance in TIM-4-deficient mice. Proc Natl Acad Sci U S A 2010;107:8706–8711.Kobayashi N, et al. TIM-1 and TIM-4 glycoproteins bind phosphatidylserine and mediate uptake of apoptotic cells. Immunity 2007;27:927–940.LD Cunha et al. LC3-Associated phagocytosis in myeloid cells promotes tumor immune tolerance. Cell 2018;175:429–441 e416.Baghdadi M, et al, TIM-4 glycoprotein-mediated degradation of dying tumor cells by autophagy leads to reduced antigen presentation and increased immune tolerance. Immunity 2013;39:1070–1081.Baghdadi M, et al. Combined blockade of TIM-3 and TIM-4 augments cancer vaccine efficacy against established melanomas. Cancer Immunol Immunother 2013;62:629–637.

Infertility Caused by Inefficient Apoptotic Germ Cell Clearance in Xkr8-Deficient Male Mice

ABSTRACT During spermatogenesis, up to 75% of germ cells in the testes undergo apoptosis and are cleared by Sertoli cells. X-linked XK blood group-related 8 (Xkr8) is a plasma membrane protein that scrambles phospholipids in response to apoptotic signals, exposing phosphatidylserine (PtdSer). Here, we found that Xkr8−/− male mice were infertile due to reduced sperm counts in their epididymides. Apoptotic stimuli could not induce PtdSer exposure in Xkr8−/− germ cells. Consistent with the hypothesis that PtdSer functions as an “eat-me” signal to phagocytes, cells expressing phosphatidylserine receptor TIM4 and MER tyrosine kinase receptor efficiently engulfed apoptotic wild-type male germ cells but not Xkr8−/− germ cells. Fluorescence and electron microscopy revealed Sertoli cells carrying engulfed and degenerated dead cells. However, many unengulfed apoptotic cells and residual bodies and much cell debris were present in Xkr8−/− testes and epididymides. These results indicate that Xkr8-mediated PtdSer exposure is essential for the clearance of apoptotic germ cells by Sertoli cells. There was no apparent inflammation in Xkr8−/− testes, suggesting that the unengulfed apoptotic cells may have undergone secondary necrosis, releasing noxious materials that affected the germ cells. Alternatively, failure to engulf the apoptotic germ cells may have caused the Sertoli cells to starve and lose their ability to support spermatogenesis.