TIMs, TAMs, and PS- antibody targeting: implications for cancer immunotherapy.
ABSTRACT: Immunotherapy for cancer is making impressive strides at improving survival of a subset of cancer patients. To increase the breadth of patients that benefit from immunotherapy, new strategies that combat the immunosuppressive microenvironment of tumors are needed. Phosphatidylserine (PS) signaling is exploited by tumors to enhance tumor immune evasion and thus strategies to inhibit PS-mediated immune suppression have potential to increase the efficacy of immunotherapy. PS is a membrane lipid that flips to the outer surface of the cell membrane during apoptosis and/or cell stress. Externalized PS can drive efferocytosis or engage PS receptors (PSRs) to promote local immune suppression. In the tumor microenvironment (TME) PS-mediated immune suppression is often termed apoptotic mimicry. Monoclonal antibodies (mAbs) targeting PS or PSRs have been developed and are in preclinical and clinical testing. The TIM (T-cell/transmembrane, immunoglobulin, and mucin) and TAM (Tyro3, AXL, and MerTK) family of receptors are PSRs that have been shown to drive PS-mediated immune suppression in tumors. This review will highlight the development of mAbs targeting PS, TIM-3 and the TAM receptors. Video Abstract.
Project description:The interaction of the host immune system with tumor cells in the tissue microenvironment is essential in understanding tumor immunity and development of successful cancer immunotherapy. The presence of lymphocytes in tumors is highly correlated with an improved outcome. T cells have a set of cell surface receptors termed immune checkpoints that when activated suppress T cell function. Upregulation of immune checkpoint receptors such as programmed cell death 1 (PD-1) and cytotoxic T lymphocyte associated protein 4 (CTLA-4) occurs during T cell activation in an effort to prevent damage from an excessive immune response. Immune checkpoint inhibitors allow the adaptive immune system to respond to tumors more effectively. There has been clinical success in different types of cancer blocking immune checkpoint receptors such as PD-1 and CTLA. However, relapse has occurred. The innate and acquired/therapy induced resistance to treatment has been encountered. Aberrant cellular signal transduction is a major contributing factor to resistance to immunotherapy. Combination therapies with other co-inhibitory immune checkpoints such as TIM-3, LAG3 and VISTA are currently being tested to overcome resistance to cancer immunotherapy. Expression of TIM-3 has been associated with resistance to PD-1 blockade and combined blockade of TIM-3 and PD-1 has demonstrated improved responses in preclinical models. LAG3 blockade has the potential to increase the responsiveness of cytotoxic T-cells to tumors. Furthermore, tumors that were found to express VISTA had an increased rate of growth due to the T cell suppression. The growing understanding of the inhibitory immune checkpoints' ligand biology, signaling mechanisms, and T-cell suppression in the tumor microenvironment continues to fuel preclinical and clinical advancements in design, testing, and approval of agents that block checkpoint molecules. Our review seeks to bridge fundamental regulatory mechanisms across inhibitory immune checkpoint receptors that are of great importance in resistance to cancer immunotherapy. We will summarize the biology of different checkpoint molecules, highlight the effect of individual checkpoint inhibition as anti-tumor therapies, and outline the literatures that explore mechanisms of resistance to individual checkpoint inhibition pathways.
Project description:The T cell immunoglobulin mucin (TIM) proteins regulate T cell activation and tolerance. Here we showed that TIM-4 is expressed on human and mouse macrophages and dendritic cells, and both TIM-4 and TIM-1 specifically bound phosphatidylserine (PS) on the surface of apoptotic cells but not any other phospholipid tested. TIM-4(+) peritoneal macrophages, TIM-1(+) kidney cells, and TIM-4- or TIM-1-transfected cells efficiently phagocytosed apoptotic cells, and phagocytosis could be blocked by TIM-4 or TIM-1 monoclonal antibodies. Mutations in the unique cavity of TIM-4 eliminated PS binding and phagocytosis. TIM-4 mAbs that blocked PS binding and phagocytosis mapped to epitopes in this binding cavity. These results show that TIM-4 and TIM-1 are immunologically restricted members of the group of receptors whose recognition of PS is critical for the efficient clearance of apoptotic cells and prevention of autoimmunity.
Project description:Phosphatidylserine (PS) receptors enhance infection of many enveloped viruses through virion-associated PS binding that is termed apoptotic mimicry. Here we show that this broadly shared uptake mechanism is utilized by SARS-CoV-2 in cells that express low surface levels of ACE2. Expression of members of the TIM (TIM-1 and TIM-4) and TAM (AXL) families of PS receptors enhance SARS-CoV-2 binding to cells, facilitate internalization of fluorescently-labeled virions and increase ACE2-dependent infection of SARS-CoV-2; however, PS receptors alone did not mediate infection. We were unable to detect direct interactions of the PS receptor AXL with purified SARS-CoV-2 spike, contrary to a previous report. Instead, our studies indicate that the PS receptors interact with PS on the surface of SARS-CoV-2 virions. In support of this, we demonstrate that: 1) significant quantities of PS are located on the outer leaflet of SARS-CoV-2 virions, 2) PS liposomes, but not phosphatidylcholine liposomes, reduced entry of VSV/Spike pseudovirions and 3) an established mutant of TIM-1 which does not bind to PS is unable to facilitate entry of SARS-CoV-2. As AXL is an abundant PS receptor on a number of airway lines, we evaluated small molecule inhibitors of AXL signaling such as bemcentinib for their ability to inhibit SARS-CoV-2 infection. Bemcentinib robustly inhibited virus infection of Vero E6 cells as well as multiple human lung cell lines that expressed AXL. This inhibition correlated well with inhibitors that block endosomal acidification and cathepsin activity, consistent with AXL-mediated uptake of SARS-CoV-2 into the endosomal compartment. We extended our observations to the related betacoronavirus mouse hepatitis virus (MHV), showing that inhibition or ablation of AXL reduces MHV infection of murine cells. In total, our findings provide evidence that PS receptors facilitate infection of the pandemic coronavirus SARS-CoV-2 and suggest that inhibition of the PS receptor AXL has therapeutic potential against SARS-CoV-2.
Project description:The immune checkpoint blockade (ICB) immunotherapy has prolonged overall survival for cancer patients but the response rates are low. The resistance to ICB is likely due to compensatory upregulation of additional immune inhibitory molecules. In this study, we first systematically examined Tim-3 expression in immune cells in mouse tumors and found that Tim-3 was specifically up-regulated in a large number of Treg, conventional CD4<sup>+</sup>, CD8<sup>+</sup> T cells, dendritic cell 1 (DC1), and macrophage 1 (M1) in the tumor microenvironment (TME). Interestingly, Tim-3<sup>+</sup> T cells in the TME were phenotypically effector but not "exhausted" T cells because Tim-3<sup>+</sup> PD-1<sup>+</sup> CD8<sup>+</sup> T cells had a higher number of mitochondria, greater levels of glycolysis, and higher tumor-specific cytolytic activities compared to Tim-3<sup>-</sup> PD-1<sup>-</sup> CD8<sup>+</sup> T cells. The combination treatment with Tim-3 and PD-1 mAbs resulted in a synergistic antitumor activity but also increased the expression of Lag-3 and GITR in TIL, demonstrating cross-regulation between multiple checkpoint molecules. Furthermore, we found that the antitumor efficacy with triple combination of Tim-3, PD-1, and Lag3 mAbs was much greater than any two antibodies. Mechanistically, we demonstrated that simultaneous targeting of Tim-3, PD-1, and Lag-3 cooperatively increased the levels of granzyme B and tumor-specific cytolytic activities of CD8<sup>+</sup> TIL. Our data indicate that multiple checkpoint molecules are coordinately upregulated to inhibit the function of hyperactivated T cells in the TME and requirement for the simultaneous blockade of PD-1, Tim-3 and Lag3 for cancer treatment.
Project description:Dengue viruses (DVs) are responsible for the most medically relevant arboviral diseases. However, the molecular interactions mediating DV entry are poorly understood. We determined that TIM and TAM proteins, two receptor families that mediate the phosphatidylserine (PtdSer)-dependent phagocytic removal of apoptotic cells, serve as DV entry factors. Cells poorly susceptible to DV are robustly infected after ectopic expression of TIM or TAM receptors. Conversely, DV infection of susceptible cells is inhibited by anti-TIM or anti-TAM antibodies or knockdown of TIM and TAM expression. TIM receptors facilitate DV entry by directly interacting with virion-associated PtdSer. TAM-mediated infection relies on indirect DV recognition, in which the TAM ligand Gas6 acts as a bridging molecule by binding to PtdSer within the virion. This dual mode of virus recognition by TIM and TAM receptors reveals how DVs usurp the apoptotic cell clearance pathway for infectious entry.
Project description:Co-signaling receptors for the T cell receptor (TCR) are important therapeutic targets, with blockade of co-inhibitory receptors such as PD-1 now central in immuno-oncology. Advancing additional therapeutic immune modulation approaches requires understanding ligand regulation of other co-signaling receptors. One poorly understood potential therapeutic target is TIM-3 (T cell immunoglobulin and mucin domain containing-3). Which of TIM-3's several proposed regulatory ligands is/are relevant for signaling is unclear, and different studies have reported TIM-3 as a co-inhibitory or co-stimulatory receptor in T cells. Here, we show that TIM-3 promotes NF-κB signaling and IL-2 secretion following TCR stimulation in Jurkat cells, and that this activity is regulated by binding to phosphatidylserine (PS). TIM-3 signaling is stimulated by PS exposed constitutively in cultured Jurkat cells, and can be blocked by mutating the PS-binding site or by occluding this site with an antibody. We also find that TIM-3 signaling alters CD28 phosphorylation. Our findings clarify the importance of PS as a functional TIM-3 ligand, and may inform the future exploitation of TIM-3 as a therapeutic target.
Project description:Immune checkpoint blockade (ICB) has been a remarkable clinical advance for cancer; however, the majority of patients do not respond to ICB therapy. We show that metastatic disease in the pleural and peritoneal cavities is associated with poor clinical outcomes after ICB therapy. Cavity-resident macrophages express high levels of Tim-4, a receptor for phosphatidylserine (PS), and this is associated with reduced numbers of CD8<sup>+</sup> T cells with tumor-reactive features in pleural effusions and peritoneal ascites from patients with cancer. We mechanistically demonstrate that viable and cytotoxic anti-tumor CD8<sup>+</sup> T cells upregulate PS and this renders them susceptible to sequestration away from tumor targets and proliferation suppression by Tim-4<sup>+</sup> macrophages. Tim-4 blockade abrogates this sequestration and proliferation suppression and enhances anti-tumor efficacy in models of anti-PD-1 therapy and adoptive T cell therapy in mice. Thus, Tim-4<sup>+</sup> cavity-resident macrophages limit the efficacy of immunotherapies in these microenvironments.
Project description:Secretion and exchange of biomolecules by extracellular vesicles (EVs) are crucial in intercellular communication and enable cells to adapt to alterations in their microenvironment. EVs are involved in a variety of cellular processes under physiological conditions as well as in pathological settings. In particular, they exert profound effects on the innate immune system, and thereby are also capable of modulating adaptive immunity. The mechanisms underlying their interaction with their recipient cells, particularly their preferential association with monocytes and granulocytes in the circulation, however, remain to be further clarified. Surface molecules exposed on EVs are likely to mediate immune recognition and EV uptake by their recipient cells. Here, we investigated the involvement of Tyro3, Axl, and Mer (TAM) tyrosine kinase receptors and of integrin CD11b in the binding of platelet-derived EVs, constituting the large majority of circulating EVs, to immune cells in the circulation. Flow cytometry and Western Blotting demonstrated a differential expression of TAM receptors and CD11b on monocytes, granulocytes, and lymphocytes, as well as on monocyte subsets. Of the TAM receptors, only Axl and Mer were detected at low levels on monocytes and granulocytes, but not on lymphocytes. Likewise, CD11b was present on circulating monocytes and granulocytes, but remained undetectable on lymphocytes. Differentiation of monocytes into classical, intermediate, and non-classical monocyte subsets revealed distinct expression patterns of Mer and activated CD11b. Co-incubation of isolated monocytes and granulocytes with platelet-derived EVs showed that the binding of EVs to immune cells was dependent on Ca<sup>++</sup>. Our data do not support a particular role for TAM receptors or for activated CD11b in the association of platelet-derived EVs with monocytes and granulocytes in the circulation, as anti-TAM antibodies did not interfere with EV binding to isolated immune cells, as binding was not dependent on the presence of TIM4 acting synergistically with TAM receptors, and as neither low levels of Gas6, required as a linker between phosphatidylserine (PS) on the EV surface and TAM receptors on immune cells, nor masking of PS on the EV surface did interfere with EV binding.
Project description:Immune suppression in the tumor microenvironment (TME) is a central obstacle to effective immunotherapy. Tumor-associated macrophages (TAMs) are key components of the TME. Although TAMs have been viewed as an ideal target of intervention to steer immunity in cancer treatment, the approach has been hampered by the lack of knowledge of how TAM plasticity is controlled by cell intrinsic factors. VentX is a homeobox protein implicated in proliferation and differentiation of human hematopoietic and immune cells. Using clinical samples obtained from cancer patients, we find that VentX expression is drastically reduced in TAMs. We show here that VentX promotes M1 differentiation of TAMs, and that VentX-regulated TAMs, in turn, revert immune suppression at the TME. Using a NSG mouse model of human colon cancers, we demonstrate that VentX regulates TAM function in tumorigenesis in vivo. Our findings suggest a mechanism underlying immune suppression at TME and potential applications of VentX-regulated TAMs in cancer immunotherapy.