Establishment of engineered cell-based assays mediating LAG3 and PD1 immune suppression enables potency measurement of blocking antibodies and assessment of signal transduction.

LAG3 is an important regulator of T cell homeostasis and studies in mouse tumor models have demonstrated that simultaneously antagonizing LAG3 and PD1 can augment tumor-specific T cell responses and induce tumor rejection. The combined use of LAG3 antagonist antibodies with established anti-PD1 therapies is currently being evaluated in human clinical trials. A functional assay for human LAG3 was developed by co-culture of a Jurkat T-cell lymphoma line overexpressing LAG3 with a Raji B-cell lymphoma line in the presence of staphylococcal enterotoxins. Reversal of LAG3 repression was measured as an increase in IL-2 production or NFAT activation in response to treatment with MK-4280, an anti-human LAG3 antagonist antibody. Changes in cytokines, chemokines, and other mRNA transcripts were in agreement with published in vitro and in vivo models for LAG3 biology which highlights the physiological relevance of the Jurkat functional assay. Additional engineering of PD1 and PDL1 components into the LAG3 assay resulted in a bi-functional assay that is capable of inducing a 10-fold response to individual antibodies blocking either PD1 or LAG3. Importantly, when MK-4280 and pembrolizumab were combined to block both pathways, a synergistic 50-fold increase in response was observed.

Monoclonal antibody-mediated CD200 receptor signaling suppresses macrophage activation and tissue damage in experimental autoimmune uveoretinitis.

Macrophage responses are regulated by multiple secreted factors as well as by cell surface receptors, including the inhibitory signals resulting from ligation of myeloid CD200 receptors (CD200R) by the widely distributed CD200. In the absence of CD200, animals display increased susceptibility to autoimmunity and earlier onset aggressive autoimmune disease. In these current experiments, an agonist monoclonal rat anti-mouse CD200R (DX109) antibody delivered a negative signal to bone marrow-derived macrophages, which suppressed interferon (IFN)gamma-mediated nitric oxide (NO) and interleukin-6 production. Experimental autoimmune uveoretinitis (EAU) was used as a model of organ-specific autoimmunity in the eye, a tissue with extensive neuronal and endothelial CD200 expression. In mice lacking CD200 (CD200(-/-)), increased numbers of retina-infiltrating macrophages displaying heightened NO responses were observed during EAU. In addition, we aimed to suppress disease by maintaining tonic suppression of macrophage activation via CD200R. Systemically administered DX109 monoclonal antibody suppressed EAU despite maintained T-cell proliferation and IFNgamma production. Furthermore, locally administered DX109 monoclonal antibody resulted in an earlier resolution of disease. These experiments demonstrate that promoting CD200R-mediated signaling can successfully prevent full expression of IFNgamma-mediated macrophage activation and protect against tissue damage during autoimmune responses.

Regulation of myeloid cell function through the CD200 receptor.

Myeloid cells play pivotal roles in chronic inflammatory diseases through their broad proinflammatory, destructive, and remodeling capacities. CD200 is widely expressed on a variety of cell types, while the recently identified CD200R is expressed on myeloid cells and T cells. CD200 deletion in vivo results in myeloid cell dysregulation and enhanced susceptibility to autoimmune inflammation, suggesting that the CD200-CD200R interaction is involved in immune suppression. We demonstrate in this study that CD200R agonists suppress mouse and human myeloid cell function in vitro, and also define a dose relationship between receptor expression and cellular inhibition. IFN-gamma- and IL-17-stimulated cytokine secretion from mouse peritoneal macrophages was inhibited by CD200R engagement. Inhibitory effects were not universal, as LPS-stimulated responses were unaffected. Inhibition of U937 cell cytokine production correlated with CD200R expression levels, and inhibition was only observed in low CD200R expressing cells, if the CD200R agonists were further cross-linked. Tetanus toxoid-induced human PBMC IL-5 and IL-13 secretion was inhibited by CD200R agonists. This inhibition was dependent upon cross-linking the CD200R on monocytes, but not on cross-linking the CD200R on CD4+ T cells. In all, we provide direct evidence that the CD200-CD200R interaction controls monocyte/macrophage function in both murine and human systems, further supporting the potential clinical application of CD200R agonists for the treatment of chronic inflammatory diseases.

Recombinant CD200 protein does not bind activating proteins closely related to CD200 receptor.

CD200 (OX2) is a cell surface glycoprotein that interacts with a structurally related receptor (CD200R) expressed mainly on myeloid cells and is involved in regulation of macrophage and mast cell function. In mouse there are up to five genes related to CD200R with conflicting data as to whether they bind CD200. We show that mouse CD200 binds the inhibitory receptor CD200R with a comparable affinity (Kd = 4 microM) to those found for the rat and human CD200 CD200R interactions. CD200 gave negligible binding to the activating receptors, CD200RLa, CD200RLb, and CD200RLc, by direct analysis at the protein level using recombinant monomeric and dimeric fusion proteins or to CD200RLa and CD200RLb when expressed at the cell surface. An additional potential activating gene, CD200RLe, found in only some mouse strains also did not bind CD200. Thus, the CD200 receptor family consists of both activatory and inhibitory members like several other paired ligand receptors, such as signal regulatory protein, killer cell Ig-like receptor/KAR, LY49, dendritic cell immunoreceptor/dendritic cell immunoactivating receptor, and paired Ig-like type 2 receptor. Although the ligand for the inhibitory product is a widely distributed host protein, the ligands of the activating forms remain to be identified, and one possibility is that they are pathogen components.

The CD200 receptor is a novel and potent regulator of murine and human mast cell function.

CD200R is a member of the Ig supergene family that is primarily expressed on myeloid cells. Recent in vivo studies have suggested that CD200R is an inhibitory receptor capable of regulating the activation threshold of inflammatory immune responses. Here we provide definitive evidence that CD200R is expressed on mouse and human mast cells and that engagement of CD200R by agonist Abs or ligand results in a potent inhibition of mast cell degranulation and cytokine secretion responses. CD200R-mediated inhibition of FcepsilonRI activation was observed both in vitro and in vivo and did not require the coligation of CD200R to FcepsilonRI. Unlike the majority of myeloid inhibitory receptors, CD200R does not contain a phosphatase recruiting inhibitory motif (ITIM); therefore, we conclude that CD200R represents a novel and potent inhibitory receptor that can be targeted in vivo to regulate mast cell-dependent pathologies.

Molecular mechanisms of CD200 inhibition of mast cell activation.

CD200 and its receptor CD200R are both type I membrane glycoproteins that contain two Ig-like domains. Engagement of CD200R by CD200 inhibits activation of myeloid cells. Unlike the majority of immune inhibitory receptors, CD200R lacks an ITIM in the cytoplasmic domain. The molecular mechanism of CD200R inhibition of myeloid cell activation is unknown. In this study, we examined the CD200R signaling pathways that control degranulation of mouse bone marrow-derived mast cells. We found that upon ligand binding, CD200R is phosphorylated on tyrosine and subsequently binds to adapter proteins Dok1 and Dok2. Upon phosphorylation, Dok1 binds to SHIP and both Dok1 and Dok2 recruit RasGAP, which mediates the inhibition of the Ras/MAPK pathways. Activation of ERK, JNK, and p38 MAPK are all inhibited by CD200R engagement. The reduced activation of these MAPKs is responsible for the observed inhibition of mast cell degranulation and cytokine production. Similar signaling events were also observed upon CD200R engagement in mouse peritoneal cells. These data define a novel inhibitory pathway used by CD200R in modulating mast cell function and help to explain how engagement of this receptor in vivo regulates myeloid cell function.

Characterization of the CD200 receptor family in mice and humans and their interactions with CD200.

CD200 (OX2) is a broadly distributed cell surface glycoprotein that interacts with a structurally related receptor (CD200R) expressed on rodent myeloid cells and is involved in regulation of macrophage function. We report the first characterization of human CD200R (hCD200R) and define its binding characteristics to hCD200. We also report the identification of a closely related gene to hCD200R, designated hCD200RLa, and four mouse CD200R-related genes (termed mCD200RLa-d). CD200, CD200R, and CD200R-related genes were closely linked in humans and mice, suggesting that these genes arose by gene duplication. The distributions of the receptor genes were determined by quantitative RT-PCR, and protein expression was confirmed by a set of novel mAbs. The distribution of mouse and human CD200R was similar, with strongest labeling of macrophages and neutrophils, but also other leukocytes, including monocytes, mast cells, and T lymphocytes. Two mCD200 receptor-like family members, designated mCD200RLa and mCD200RLb, were shown to pair with the activatory adaptor protein, DAP12, suggesting that these receptors would transmit strong activating signals in contrast to the apparent inhibitory signal delivered by triggering the CD200R. Despite substantial sequence homology with mCD200R, mCD200RLa and mCD200RLb did not bind mCD200, and presently have unknown ligands. The CD200 receptor gene family resembles the signal regulatory proteins and killer Ig-related receptors in having receptor family members with potential activatory and inhibitory functions that may play important roles in immune regulation and balance. Because manipulation of the CD200-CD200R interaction affects the outcome of rodent disease models, targeting of this pathway may have therapeutic utility.

Ras-induced serine phosphorylation of the focal adhesion protein paxillin is mediated by the Raf-->MEK-->ERK pathway.

Cells transformed by Ras and Raf display dramatic alterations in cell morphology, adhesion, and intracellular architecture. Consequently, we investigated whether Ras or Raf might influence the behavior of proteins known to be involved in the assembly and integrity of focal adhesion complexes that play a crucial role in many of these processes. We identified Raf-induced serine phosphorylation of the adaptor protein paxillin in a variety of cell types. Raf-induced paxillin serine phosphorylation had no effect on paxillin tyrosine phosphorylation and occurred regardless of whether cells were attached or maintained in suspension. Two sites of serine phosphorylation--S126 and S130--were identified. Mutation of these serines to alanine, either alone or in combination, inhibited the ability of Raf to induce paxillin phosphorylation. These data indicate that paxillin is a target for phosphorylation downstream of the Ras-activated Raf-->MEK pathway. However, we have no evidence to suggest that ERK1/2 are the kinases responsible for Raf-induced paxillin phosphorylation. Furthermore, we did not detect any alterations in the binding of paxillin to a number of focal adhesion proteins following either activation of the Raf-->MEK-->ERK pathway or expression of the S126A/S130A form of paxillin in mammalian cells.