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Exciting progress in the field of cancer immunotherapy has renewed the urgency of the need for basic studies of immunoregulation in both adaptive cell lineages and innate cell lineages. Here we found a central role for major histocompatibility complex (MHC) class I in controlling the phagocytic function of macrophages. Our results demonstrated that expression of the common MHC class I component β 2 -microglobulin (β2M) by cancer cells directly protected them from phagocytosis. We further showed that this protection was mediated by the inhibitory receptor LILRB1, whose expression was upregulated on the surface of macrophages, including tumor-associated macrophages. Disruption of either MHC class I or LILRB1 potentiated phagocytosis of tumor cells both in vitro and in vivo , which defines the MHC class I–LILRB1 signaling axis as an important regulator of the effector function of innate immune cells, a potential biomarker for therapeutic response to agents directed against the signal-regulatory protein CD47 and a potential target of anti-cancer immunotherapy. Host cells display ‘don’t eat me’ signals to protect themselves from phagocytosis. Maute and colleagues identify a novel ‘don’t eat me’ system based on recognition of MHC class I by the phagocyte-expressed inhibitory molecule LILRB1.

Macrophage-mediated programmed cell removal (PrCR) is a process essential for the clearance of unwanted (damaged, dysfunctional, aged, or harmful) cells. The detection and recognition of appropriate target cells by macrophages is a critical step for successful PrCR, but its molecular mechanisms have not been delineated. Here using the models of tissue turnover, cancer immunosurveillance, and hematopoietic stem cells, we show that unwanted cells such as aging neutrophils and living cancer cells are susceptible to “labeling” by secreted calreticulin (CRT) from macrophages, enabling their clearance through PrCR. Importantly, we identified asialoglycans on the target cells to which CRT binds to regulate PrCR, and the availability of such CRT-binding sites on cancer cells correlated with the prognosis of patients in various malignancies. Our study reveals a general mechanism of target cell recognition by macrophages, which is the key for the removal of unwanted cells by PrCR in physiological and pathophysiological processes. Macrophage-mediated programmed cell removal (PrCR) allows clearance of living cells. Here the authors show that, in mouse models, activated macrophages create an “eat me” signal via calreticulin secretion on neutrophils during peritonitis and on cancer cells, determining in both cases clearance by PrCR.

Prolonged exposure of CD8 + T cells to antigenic stimulation, as in chronic viral infections, leads to a state of diminished function termed exhaustion. We now demonstrate that even during exhaustion there is a subset of functional CD8 + T cells defined by surface expression of SIRPα, a protein not previously reported on lymphocytes. On SIRPα + CD8 + T cells, expression of co-inhibitory receptors is counterbalanced by expression of co-stimulatory receptors and it is only SIRPα + cells that actively proliferate, transcribe IFNγ and show cytolytic activity. Furthermore, target cells that express the ligand for SIRPα, CD47, are more susceptible to CD8 + T cell-killing in vivo. SIRPα + CD8 + T cells are evident in mice infected with Friend retrovirus, LCMV Clone 13, and in patients with chronic HCV infections. Furthermore, therapeutic blockade of PD-L1 to reinvigorate CD8 + T cells during chronic infection expands the cytotoxic subset of SIRPα + CD8 + T cells. SIRPa is most commonly known as a phagocytosis inhibitory receptor expressed by myeloid cells. Here the authors show SIRPa is expressed on a subset of CD8+ T cells with higher proliferative and effector activity during the chronic phase of the immune response to viral infection.

Multiplex imaging technologies are now routinely capable of measuring more than 40 antibody-labeled parameters in single cells. However, lateral spillage of signals in densely packed tissues presents an obstacle to the assignment of high-dimensional spatial features to individual cells for accurate cell-type annotation. We devised a method to correct for lateral spillage of cell surface markers between adjacent cells termed REinforcement Dynamic Spillover EliminAtion (REDSEA). The use of REDSEA decreased contaminating signals from neighboring cells. It improved the recovery of marker signals across both isotopic (i.e., Multiplexed Ion Beam Imaging) and immunofluorescent (i.e., Cyclic Immunofluorescence) multiplexed images resulting in a marked improvement in cell-type classification.

BackgroundRed blood cells circulate for ~120 days in humans and ~50 days in mice before they are phagocytosed by macrophages in the splenic red pulp at the end of their lifespans.1 2 The red blood cells are protected from non-specific premature destruction by macrophages by their expression of CD47, a ‘don’t eat me’ signal that binds to macrophage expressed SIRPa to inhibit phagocytosis.3 However, as monoclonal antibodies have been developed to block the CD47-SIRPa interaction for cancer immunotherapy, it has been necessary to overcome the toxicity associated with the off-target destruction of red blood cells.4 It was found that a small loading dose of CD47 antibody confers to red blood cells protection against much greater subsequent dosing.5 Here we identify the mechanism of this protection in mice and show that it can be leveraged therapeutically against autoimmune anemia and thrombocytopenia (figure 1a-b).MethodsFemale C57/B6 and BALB/c mice were treated with a pre-clinical murine CD47 antibody [clone MIAP410] or an isotype control antibody. Flow cytometry was used to quantify CD47 expression by red blood cells and their progenitors from murine blood and bone marrow at various timepoints after CD47 antibody therapy and to quantify the expression of the four principal antibody-binding Fc-gamma receptors (FcgR) in myeloid and NK cell populations from the bone marrow, spleen, liver, and peritoneum of these mice. Fluorescently-labeled red blood cells were infused into mice to measure their survival in the circulation and phagocytosis by red pulp macrophages.ResultsWe find that a loading dose of CD47 antibody protects red blood cells by impairing antibody binding and phagocytosis by red pulp macrophages. We begin by demonstrating that CD47 antibody induces FcgR-mediated pruning of erythroid CD47, with global concomitant FcgR loss by reticuloendothelial myeloid populations. We then show that CD47 antibody therapy impairs red pulp macrophage phagocytosis of red blood cells. Finally, we show that CD47 antibody therapy protects red blood cells and platelets from antibody-mediated destruction and thus may have therapeutic potential for autoimmune hemolytic anemia (AIHA) and immune thrombocytopenia (ITP).ConclusionsWe demonstrate that CD47 antibody therapy prevents macrophage-mediated destruction of red blood cells and platelets in mice, both by reducing myeloid FcgR expression organism-wide and by directly impairing macrophage phagocytosis. We then show that this mechanism can be leveraged therapeutically to protect mice from antibody-mediated anemia and thrombocytopenia, and thus has therapeutic potential for these disorders.ReferencesBratosin D, Mazurier J, Tissier JP, et al. Cellular and molecular mechanisms of senescent red blood cell phagocytosis by macrophages. A review. Biochimie. 1998;80(2):173–195.de Back DZ, Kostova EB, van Kraaij M, van den Berg TK, van Bruggen R. Of macrophages and red blood cells; a complex love story. Front Physiol. 2014;5:9.Oldenborg, P.A. et al. Role of CD47 as a marker of self on red blood cells. Science 2000; 288, 2051–2054.Willingham, S. B. et al. The CD47-signal regulatory protein alpha (SIRPa) interaction is a therapeutic target for human solid tumors. Natl Acad. Sci. USA 2012; 109, 6662–6667.Sikic BI, et al. First-in-Human, First-in-Class Phase I Trial of the Anti-CD47 Antibody Hu5F9-G4 in Patients With Advanced Cancers. J Clin Oncol 2019; 37(12):946–953.Ethics ApprovalAll mouse experiments adhered to the ethical care guidelines set forth by the Stanford University Administrative Panel on Laboratory Animal Care (APLAC); protocol #26270.Abstract 506 Figure 1(A) Quantitation of mouse in vivo platelet survival after CD47 antibody therapy (+) [***P<0.0001, unpaired two-tailed t-test] and (B) of opsonized RBC phagocytosis after CD47 antibody therapy (+), n=5 mice [***P<0.0001, unpaired two-tailed t-test]

Ovarian cancer and triple-negative breast cancer are among the most lethal diseases affecting women, with few targeted therapies and high rates of metastasis. Cancer cells are capable of evading clearance by macrophages through the overexpression of anti-phagocytic surface proteins called ‘don’t eat me’ signals—including CD47 1 , programmed cell death ligand 1 (PD-L1) 2 and the beta-2 microglobulin subunit of the major histocompatibility class I complex (B2M) 3 . Monoclonal antibodies that antagonize the interaction of ‘don’t eat me’ signals with their macrophage-expressed receptors have demonstrated therapeutic potential in several cancers 4 , 5 . However, variability in the magnitude and durability of the response to these agents has suggested the presence of additional, as yet unknown ‘don’t eat me’ signals. Here we show that CD24 can be the dominant innate immune checkpoint in ovarian cancer and breast cancer, and is a promising target for cancer immunotherapy. We demonstrate a role for tumour-expressed CD24 in promoting immune evasion through its interaction with the inhibitory receptor sialic-acid-binding Ig-like lectin 10 (Siglec-10), which is expressed by tumour-associated macrophages. We find that many tumours overexpress CD24 and that tumour-associated macrophages express high levels of Siglec-10. Genetic ablation of either CD24 or Siglec-10, as well as blockade of the CD24–Siglec-10 interaction using monoclonal antibodies, robustly augment the phagocytosis of all CD24-expressing human tumours that we tested. Genetic ablation and therapeutic blockade of CD24 resulted in a macrophage-dependent reduction of tumour growth in vivo and an increase in survival time. These data reveal CD24 as a highly expressed, anti-phagocytic signal in several cancers and demonstrate the therapeutic potential for CD24 blockade in cancer immunotherapy. CD24 interacts with the tumour-associated-macrophage receptor Siglec-10 to inhibit the macrophage-mediated clearance of cancer cells, revealing a new ‘don’t eat me’ signal as a potential target for cancer immunotherapy.

Red blood cells (erythrocytes, RBCs) are common to almost all vertebrate life on earth. In humans, RBCs circulate for approximately 120 days prior to their phago-cytic destruction by macrophages. RBCs are protected from premature destruction by their expression of the surface protein CD47, which interacts with the macrophage surface receptor SIRPa to inhibit phagocytosis. CD47 is expressed by all cells, and is overexpressed in many cancers. Antibody-mediated blockade of the CD47-SIRPa interaction overcomes this resistance, promoting macrophage-mediated destruction of cancer cells. This strategy works both by disrupting the anti-phagocytic CD47-SIRPa interaction and by engaging pro-phagocytic macrophage-expressed Fc gamma receptors (FcgRs). While a number of CD47-blocking antibodies are now in clinical trials for cancer immunotherapy, one question remains: how are patients able to tolerate CD47 antibody, given that it sensitizes their RBCs to phagocytic destruction? We find that CD47 antibody therapy induces myeloid FcgR-mediated pruning of RBC CD47 in mice, with global concomitant myeloid FcgR loss and splenic myeloid phagocytic inhibition, which protect antibody-bound and CD47-deficient RBCs from phagocytosis. Our findings provide a mechanism for the surprising tolerability of CD47 blocking antibodies and a mechanism by which RBC-binding antibodies may protect against autoimmune destruction of circulating cells like RBCs and platelets.

Cell-free RNA from liquid biopsies can be analyzed to determine disease tissue of origin. We extend this concept to identify cell types of origin using the Tabula Sapiens transcriptomic cell atlas as well as individual tissue transcriptomic cell atlases in combination with the Human Protein Atlas RNA consensus dataset. We define cell type signature scores, which allow the inference of cell types that contribute to cell-free RNA for a variety of diseases. Cell types affected by various diseases are inferred from cell-free RNA.

Ovarian cancer and triple-negative breast cancer (TNBC) are among the most lethal diseases affecting women, with few targeted therapies and high rates of metastasis. Here we show that CD24 can be the dominant innate immune checkpoint in ovarian cancer and breast cancer, and is a new, promising target for cancer immunotherapy. Cancer cells are capable of evading clearance by macrophages through the overexpression of anti-phagocytic surface proteins, called “don’t eat me” signals, including CD471, programmed cell death ligand 1 (PD-L1)2, and the beta-2 microglobulin subunit of the major histocompatibility class I complex (B2M)3. Monoclonal antibodies which antagonize the interaction of “don’t eat me” signals with their macrophage-expressed receptors have demonstrated therapeutic potential in several cancers4–5. However, variability in the magnitude and durability of the response to these agents has suggested the presence of additional, as yet unknown, “don’t eat me” signals. Here we demonstrate a novel role for tumor-expressed CD24 in promoting immune evasion through its interaction with the inhibitory receptor, Sialic Acid Binding Ig Like Lectin 10 (Siglec-10), expressed by tumor-associated macrophages (TAMs). We observe that many tumors overexpress CD24 and that TAMs express high levels of Siglec-10. Both genetic ablation of CD24 or Siglec-10, and monoclonal antibody blockade of the CD24–Siglec-10 interaction, robustly augment the phagocytosis of all CD24-expressing human tumors tested. Genetic ablation as well as therapeutic blockade of CD24 resulted in a macrophage-dependent reduction of tumor growth and extension of survival, in vivo. These data highlight CD24 as a highly-expressed, anti-phagocytic signal in several cancers and demonstrate the therapeutic potential for CD24-blockade as cancer immunotherapy.

Immune responses to infectious agents are initiated when a pathogen or its components bind to pattern recognition receptors (PRRs). PRR binding sets off a cascade of events that activates immune responses. We now show that, in addition to activating immune responses, PRR signaling also initiates an immunosuppressive response, probably to limit inflammation. The importance of the current findings is that blockade of immunomodulatory signaling, which is mediated by the upregulation of the CD47 molecule, can lead to enhanced immune responses to any pathogen that triggers PRR signaling. Since most or all pathogens trigger PRRs, CD47 blockade could be used to speed up and strengthen both innate and adaptive immune responses when medically indicated. Such immunotherapy could be done without a requirement for knowing the HLA type of the individual, the specific antigens of the pathogen, or, in the case of bacterial infections, the antimicrobial resistance profile. It is well understood that the adaptive immune response to infectious agents includes a modulating suppressive component as well as an activating component. We now show that the very early innate response also has an immunosuppressive component. Infected cells upregulate the CD47 “don’t eat me” signal, which slows the phagocytic uptake of dying and viable cells as well as downstream antigen-presenting cell (APC) functions. A CD47 mimic that acts as an essential virulence factor is encoded by all poxviruses, but CD47 expression on infected cells was found to be upregulated even by pathogens, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), that encode no mimic. CD47 upregulation was revealed to be a host response induced by the stimulation of both endosomal and cytosolic pathogen recognition receptors (PRRs). Furthermore, proinflammatory cytokines, including those found in the plasma of hepatitis C patients, upregulated CD47 on uninfected dendritic cells, thereby linking innate modulation with downstream adaptive immune responses. Indeed, results from antibody-mediated CD47 blockade experiments as well as CD47 knockout mice revealed an immunosuppressive role for CD47 during infections with lymphocytic choriomeningitis virus and Mycobacterium tuberculosis . Since CD47 blockade operates at the level of pattern recognition receptors rather than at a pathogen or antigen-specific level, these findings identify CD47 as a novel potential immunotherapeutic target for the enhancement of immune responses to a broad range of infectious agents. IMPORTANCE Immune responses to infectious agents are initiated when a pathogen or its components bind to pattern recognition receptors (PRRs). PRR binding sets off a cascade of events that activates immune responses. We now show that, in addition to activating immune responses, PRR signaling also initiates an immunosuppressive response, probably to limit inflammation. The importance of the current findings is that blockade of immunomodulatory signaling, which is mediated by the upregulation of the CD47 molecule, can lead to enhanced immune responses to any pathogen that triggers PRR signaling. Since most or all pathogens trigger PRRs, CD47 blockade could be used to speed up and strengthen both innate and adaptive immune responses when medically indicated. Such immunotherapy could be done without a requirement for knowing the HLA type of the individual, the specific antigens of the pathogen, or, in the case of bacterial infections, the antimicrobial resistance profile.