The phosphatidylserine receptor has essential functions during embryogenesis but not in apoptotic cell removal

BACKGROUND: Phagocytosis of apoptotic cells is fundamental to animal development, immune function and cellular homeostasis. The phosphatidylserine receptor (Ptdsr) on phagocytes has been implicated in the recognition and engulfment of apoptotic cells and in anti-inflammatory signaling. To determine the biological function of the phosphatidylserine receptor in vivo, we inactivated the Ptdsr gene in the mouse. RESULTS: Ablation of Ptdsr function in mice causes perinatal lethality, growth retardation and a delay in terminal differentiation of the kidney, intestine, liver and lungs during embryogenesis. Moreover, eye development can be severely disturbed, ranging from defects in retinal differentiation to complete unilateral or bilateral absence of eyes. Ptdsr (-/-) mice with anophthalmia develop novel lesions, with induction of ectopic retinal-pigmented epithelium in nasal cavities. A comprehensive investigation of apoptotic cell clearance in vivo and in vitro demonstrated that engulfment of apoptotic cells was normal in Ptdsr knockout mice, but Ptdsr-deficient macrophages were impaired in pro- and anti-inflammatory cytokine signaling after stimulation with apoptotic cells or with lipopolysaccharide. CONCLUSION: Ptdsr is essential for the development and differentiation of multiple organs during embryogenesis but not for apoptotic cell removal. Ptdsr may thus have a novel, unexpected developmental function as an important differentiation-promoting gene. Moreover, Ptdsr is not required for apoptotic cell clearance by macrophages but seems to be necessary for the regulation of macrophage cytokine responses. These results clearly contradict the current view that the phosphatidylserine receptor primarily functions in apoptotic cell clearance

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

The scavenger receptor CD36 plays a role in cytokine-induced macrophage fusion

Multinucleated giant cells, characteristic of granulomatous infections, originate from the fusion of macrophages. Using an antibody screening strategy we found that the scavenger receptor CD36 participates in macrophage fusion induced by the cytokines IL-4 and GM-CSF. Our results demonstrate that exposure of phosphatidylserine on the cell surface and lipid recognition by CD36 are required for cytokine-induced fusion of macrophages. We also show that CD36 acts in a heterotypic manner during giant-cell formation and that the formation of osteoclasts is independent of CD36. The discovery of molecules involved in the formation of multinucleated giant cells will enable us to determine their functional significance. Furthermore, our results suggest that lipid capture by cell surface receptors may be a general feature of cell fusion

Alternative Activation of Macrophages: Concepts and Prospects

Macrophages of the body represent a widely dispersed, versatile, and highly responsive homeostatic and defense system. They contribute to innate as well as adaptive immunity to infection, mediating trophic as well as injurious interactions with their local and systemic environment. The concept of macrophage activation has evolved over recent decades, from relatively simple paradigms to bewildering complexity. This chapter will review the background to present understanding of alternative activation of macrophages, consider its relevance to health and disease, and suggest questions for future studies

1alpha,25-Dihydroxyvitamin D3 is a potent suppressor of interferon gamma-mediated macrophage activation.

1alpha,25-Dihydroxyvitamin D3 (1alpha,25(OH)2D3), the activated vitamin D3 hormone, is a key regulator of calcium homeostasis and thereby indispensable for bone metabolism. In addition, 1alpha,25(OH)2D3 is known to mediate predominantly immunosuppressive responses in vitro and in vivo. It has been demonstrated that macrophages can produce 1alpha,25(OH)2D3 on activation with interferon gamma (IFN-gamma), although little is understood about the biologic significance of this response. We show here that 1alpha,25(OH)2D3 can selectively suppress key effector functions of IFN-gamma-activated macrophages. Among these are the suppression of listericidal activity, the inhibition of phagocyte oxidase-mediated oxidative burst, and the suppression of important IFN-gamma-induced genes, including Ccl5, Cxcl10, Cxcl9, Irf2, Fcgr1, Fcgr3, and Tlr2. The deactivation of IFN-gamma-stimulated macrophages is dependent on a functional vitamin D receptor and 1alpha,25(OH)2D3 acts specifically on IFN-gamma-activated macrophages, whereas the steroid has no effects on resting macrophages. Therefore, the 1alpha,25(OH)2D3-mediated suppression of macrophage functions is distinct from previously described macrophage deactivation mechanisms. In conclusion, our data indicate that the production of 1alpha,25(OH)2D3 by IFN-gamma-stimulated macrophages might be an important negative feedback mechanism to control innate and inflammatory responses of activated macrophages

The activity of myeloid cell-specific VHH immunotoxins is target-, epitope-, subset- and organ dependent

The central role of myeloid cells in driving autoimmune diseases and cancer has raised interest in manipulating their function or depleting them for therapeutic benefits. To achieve this, antibodies are used to antagonize differentiation, survival and polarization signals or to kill target cells, for example in the form of antibody-drug conjugates (ADC). The action of ADC in vivo can be hard to predict based on target expression pattern alone. The biology of the targeted receptor as well as its interplay with the ADC can have drastic effects on cell apoptosis versus survival. Here we investigated the efficacy of CD11b or Ly-6C/Ly-6G-specific variable fragments of camelid heavy chain-only antibodies (VHH) conjugated to Pseudomonas exotoxin A to deplete myeloid cells in vitro and in vivo. Our data highlight striking differences in cell killing in vivo, depending on the cell subset and organs targeted, but not antigen expression level or VHH affinity. We observed striking differences in depletion efficiency of monocytes versus granulocytes in mice. Despite similar binding of Ly-6C/Ly-6G-specific VHH immunotoxin to granulocytes and monocytes, granulocytes were significantly more sensitive than monocytes to immunotoxins treatment. Our results illustrate the need of early, thorough in vivo characterization of ADC candidates