23ME-00610, a genetically informed, first-in-class antibody targeting CD200R1 to enhance antitumor T cell function.

Immune checkpoint inhibition (ICI) has revolutionized cancer treatment; however, only a subset of patients benefit long term. Therefore, methods for identification of novel checkpoint targets and development of therapeutic interventions against them remain a critical challenge. Analysis of human genetics has the potential to inform more successful drug target discovery. We used genome-wide association studies of the 23andMe genetic and health survey database to identify an immuno-oncology signature in which genetic variants are associated with opposing effects on risk for cancer and immune diseases. This signature identified multiple pathway genes mapping to the immune checkpoint comprising CD200, its receptor CD200R1, and the downstream adapter protein DOK2. We confirmed that CD200R1 is elevated on tumor-infiltrating immune cells isolated from cancer patients compared to the matching peripheral blood mononuclear cells. We developed a humanized, effectorless IgG1 antibody (23ME-00610) that bound human CD200R1 with high affinity (KD <0.1 nM), blocked CD200 binding, and inhibited recruitment of DOK2. 23ME-00610 induced T-cell cytokine production and enhanced T cell-mediated tumor cell killing in vitro. Blockade of the CD200:CD200R1 immune checkpoint inhibited tumor growth and engaged immune activation pathways in an S91 tumor cell model of melanoma in mice.

TRAF3 enhances TCR signaling by regulating the inhibitors Csk and PTPN22.

The adaptor protein TNF receptor associated factor (TRAF) 3 is required for effective TCR signaling and normal T cell effector functions, and associates with the CD3/CD28 complex upon activation. To determine how TRAF3 promotes proximal TCR signaling, we studied TRAF3-deficient mouse and human T cells, which showed a marked reduction in activating phosphorylation of the TCR-associated kinase Lck. The impact of TRAF3 on this very early signaling event led to the hypothesis that TRAF3 restrains one or both of two known inhibitors of Lck, C-terminal Src kinase (Csk) and protein tyrosine phosphatase N22 (PTPN22). TRAF3 associated with Csk, promoting the dissociation of Csk from the plasma membrane. TRAF3 also associated with and regulated the TCR/CD28 induced localization of PTPN22. Loss of TRAF3 resulted in increased amounts of both Csk and PTPN22 in T cell membrane fractions and decreased association of PTPN22 with Csk. These findings identify a new role for T cell TRAF3 in promoting T cell activation, by regulating localization and functions of early TCR signaling inhibitors.

The adaptor protein TRAF3 inhibits interleukin-6 receptor signaling in B cells to limit plasma cell development.

Tumor necrosis factor receptor-associated factor 3 (TRAF3) is an adaptor protein that inhibits signaling by CD40 and by the receptor for B cell-activating factor (BAFF) and negatively regulates homeostatic B cell survival. Loss-of-function mutations in TRAF3 are associated with human B cell malignancies, in particular multiple myeloma. The cytokine interleukin-6 (IL-6) supports the differentiation and survival of normal and neoplastic plasma cells. We found that mice with a deficiency in TRAF3 specifically in B cells (B-Traf3(-/-) mice) had about twice as many plasma cells as did their littermate controls. TRAF3-deficient B cells had enhanced responsiveness to IL-6, and genetic loss of IL-6 in B-Traf3(-/-) mice restored their plasma cell numbers to normal. TRAF3 inhibited IL-6 receptor (IL-6R)-mediated signaling by facilitating the association of PTPN22 (a nonreceptor protein tyrosine phosphatase) with the kinase Janus-activated kinase 1 (Jak1), which in turn blocked phosphorylation of the transcription factor STAT3 (signal transducer and activator of transcription 3). Consistent with these results, the number of plasma cells in the PTPN22-deficient mice was increased compared to that in the wild-type mice. Our findings identify TRAF3 and PTPN22 as inhibitors of IL-6R signaling in B cells and reveal a previously uncharacterized role for TRAF3 in the regulation of plasma cell differentiation.

Regulatory role of CD40 in obesity-induced insulin resistance.

Excessive nutrient intake in obesity triggers the accumulation of various types of immune cells in adipose tissue, particularly visceral adipose tissue (VAT). This can result in chronic inflammation which disrupts insulin effects on adipocytes and muscle cells and culminates in development of insulin resistance. The interplay between immune cells and adipose tissue is a key event for the development of insulin resistance that precedes type 2 diabetes. CD40, a well-documented costimulatory receptor, is required for efficient systemic adaptive immune responses. However, we and other groups recently showed that CD40 unexpectedly ameliorates inflammation in VAT and accordingly attenuates obesity-induced insulin resistance. Specifically, although CD40 is typically considered to play its principal immune roles on B lymphocytes and myeloid cells, we found that CD40(+)CD8(+) T lymphocytes were major contributors to the protective effect. This unexpected inhibitory role of CD40 on CD8(+) T cell activation in VAT may reflect unique features of this microenvironment. Additional knowledge gaps include whether CD40 also plays roles in mucosal immunity that control the homeostasis of gut microbiota, and human metabolic diseases. Potential therapeutic approaches, including stimulating CD40 signaling and/or manipulating specific CD40 signaling pathways in the VAT microenvironment, may open new avenues for treatment of obesity-induced insulin resistance, and prevention of type 2 diabetes.

Roles of TRAF3 in T cells: many surprises.

Tumor necrosis factor receptor (TNFR)-associated factor 3 (TRAF3) is broadly involved in different receptor-mediated signaling pathways. Considerable progress was made recently in understanding the role of TRAF3 in T cell biology. Here we review these new findings about how TRAF3 participates in T cell development and function. The different roles of TRAF3 in distinct immune cells are also compared. That TRAF3 is required for T cell effector functions, and invariant Natural Killer T cell function and development, was unexpected. Another surprising finding is that TRAF3 normally restrains regulatory T cell development. It is now clear that TRAF3 regulates signaling to T cells not only through costimulatory members of the TNFR superfamily, but also through the T cell receptor complex, and cytokine receptors. The diverse roles it plays support the multifaceted nature of this molecule. How TRAF3 mediates integration of different signaling cascades is an important topic for future study.

The adaptor TRAF3 restrains the lineage determination of thymic regulatory T cells by modulating signaling via the receptor for IL-2.

The number of Foxp3+ regulatory T cells (Treg cells) must be tightly controlled for efficient suppression of autoimmunity with no impairment of normal immune responses. Here we found that the adaptor TRAF3 was intrinsically required for restraining the lineage determination of thymic Treg cells. T cell-specific deficiency in TRAF3 resulted in a two- to threefold greater frequency of Treg cells, due to the more efficient transition of precursors of Treg cells into Foxp3+ Treg cells. TRAF3 dampened interleukin 2 (IL-2) signaling by facilitating recruitment of the tyrosine phosphatase TCPTP to the IL-2 receptor complex, which resulted in dephosphorylation of the signaling molecules Jak1 and Jak3 and negative regulation of signaling via Jak and the transcription factor STAT5. Our results identify a role for TRAF3 as an important negative regulator of signaling via the IL-2 receptor that affects the development of Treg cells.

TRAF3 regulates homeostasis of CD8+ central memory T cells.

Our laboratory reported previously that TNF receptor associated factor 3 (TRAF3) is a positive regulator of TCR signaling and T cell function. In the current study, we present new findings that reveal differential roles for TRAF3 in the regulation of CD4+ and CD8(+) T cells. In response to TCR stimulation in vitro, TRAF3 has greater impact in CD4(+) T cells than in CD8+ T cells. However, T cell-specific TRAF3 deficient mice (CD4Cre TRAF3(fl°x)/(fl°x); T-TRAF3(-/-)) have a greater number of CD4(+)CD44(hi) effector/memory T cells than littermate control (LMC) mice, possibly due to an inefficient suppressive effect of TRAF3 deficient Foxp3+ regulatory T cells. In contrast, CD8(+)CD44(hi)CD62L(hi) central memory (Tcm) cells are markedly reduced in T-TRAF3(-/-) mice in comparison to LMC mice, although CD8(+)CD44(hi)CD62L(l°w) effector memory T (Tem) cells and naïve T cells (CD8(+)CD44(l°w)CD62L(hi)) do not show significant differences in number. Importantly, TRAF3-deficient Tcm cells exhibit defective homeostasis due to impaired IL-15 signaling. These results indicate that the involvement of TRAF3 in IL-15 mediated signaling to T cells plays a previously unappreciated and critical role in CD8(+) Tcm cell regulation and maintenance.

CD40-mediated maintenance of immune homeostasis in the adipose tissue microenvironment.

Chronic inflammation in visceral adipose tissue is considered a key element for induction of insulin resistance in obesity. CD40 is required for efficient systemic adaptive immune responses and is implicated in various inflammatory conditions. However, its role in modulating immunity in the microanatomical niches of adipose tissue remains largely undefined. Here, we show that, in contrast to its well-documented costimulatory effects, CD40 regulates development of insulin resistance in a diet-induced obesity (DIO) mouse model by ameliorating local inflammation in adipose tissues. CD40 deficiency (CD40KO) resulted in greater body weight gain, more severe inflammation in epididymal adipose tissue (EAT), and aggravated insulin resistance in response to DIO. Interestingly, we found that CD40KO CD8(+) T lymphocytes were major contributors to exacerbated insulin resistance. Specifically, CD8(+) T cells in EAT of DIO CD40KO mice produced elevated chemokines and proinflammatory cytokines and were critical for macrophage recruitment. These results indicate that CD40 plays distinct roles in different tissues and, unexpectedly, plays an important role in maintaining immune homeostasis in EAT. Further study of how CD40 promotes maintenance of healthy metabolism could contribute to better understanding of and ability to therapeutically manipulate the increasing health problem of obesity and insulin resistance.

Roles for TNF-receptor associated factor 3 (TRAF3) in lymphocyte functions.

TRAF3 is an adapter protein that serves and regulates the functions of several types of receptors, located both inside the cell and at the plasma membrane. These include members of the TNF receptor superfamily (TNFR-SF), toll-like receptors (TLR), and cytokine receptors. It has become increasingly evident that the roles and functions of TRAF3 are highly context-dependent. TRAF3 can serve distinct roles for different receptors in the same cell, and also has highly cell-type-dependent functions. This review focuses upon the current state of knowledge regarding how TRAF3 regulates the biology and effector functions of B and T lymphocytes, two major cell types of the adaptive immune response in which TRAF3 has markedly distinct roles.

TNF receptor associated factor 3 plays a key role in development and function of invariant natural killer T cells.

TCR signaling is a prerequisite for early stage development of invariant natural killer T (iNKT) cells, whereas IL-15 signaling is required for expansion and maturation at later stages. In this study, we show that TNF receptor associated factor 3 (TRAF3) plays a critical role in the transition between these two distinct signaling pathways and developmental stages. TRAF3-deficient iNKT cells in CD4(Cre)TRAF3(flox/flox) (T-TRAF3(-/-)) mice exhibit defective up-regulation of T-bet and CD122, two critical molecules for IL-15 signaling, and as a consequence, IL-15-mediated iNKT cell proliferation and survival are impaired. Consistently, development of iNKT cells in T-TRAF3(-/-) mice shows a major defect at developmental stages 2 and 3, but not stages 0 and 1. We further demonstrated that defective T-bet up-regulation occurring during the stage 1 to stage 2 transition results from reduced TCR signaling in TRAF3(-/-) iNKT cells. In addition, mature TRAF3(-/-) iNKT cells displayed defective cytokine responses upon TCR stimulation. Collectively, our results reveal that by modulating the relative strength of TCR signaling, TRAF3 is an important regulator of iNKT cell development and functions.

IFN-γ receptor deficiency prevents diabetes induction by diabetogenic CD4+, but not CD8+, T cells.

IFN-γ is generally believed to be important in the autoimmune pathogenesis of type 1 diabetes (T1D). However, the development of spontaneous ß-cell autoimmunity is unaffected in NOD mice lacking expression of IFN-γ or the IFN-γ receptor (IFNγR), bringing into question the role IFN-γ has in T1D. In the current study, an adoptive transfer model was employed to define the contribution of IFN-γ in CD4(+) versus CD8(+) T cell-mediated ß-cell autoimmunity. NOD.scid mice lacking expression of the IFNγR ß chain (NOD.scid.IFNγRB(null)) developed diabetes following transfer of ß cell-specific CD8(+) T cells alone. In contrast, ß cell-specific CD4(+) T cells alone failed to induce diabetes despite significant infiltration of the islets in NOD.scid.IFNγRB(null) recipients. The lack of pathogenicity of CD4(+) T-cell effectors was due to the resistance of IFNγR-deficient ß cells to inflammatory cytokine-induced cell death. On the other hand, CD4(+) T cells indirectly promoted ß-cell destruction by providing help to CD8(+) T cells in NOD.scid.IFNγRB(null) recipients. These results demonstrate that IFN-γR may play a key role in CD4(+) T cell-mediated ß-cell destruction.

Long-term remission of diabetes in NOD mice is induced by nondepleting anti-CD4 and anti-CD8 antibodies.

Residual ß-cells found at the time of clinical onset of type 1 diabetes are sufficient to control hyperglycemia if rescued from ongoing autoimmune destruction. The challenge, however, is to develop an immunotherapy that not only selectively suppresses the diabetogenic response and efficiently reverses diabetes, but also establishes long-term ß-cell-specific tolerance to maintain remission. In the current study, we show that a short course of nondepleting antibodies (Abs) specific for the CD4 and CD8 coreceptors rapidly reversed clinical disease in recent-onset diabetic NOD mice. Once established, remission was maintained indefinitely and immunity to foreign antigens unimpaired. Induction of remission involved selective T-cell purging of the pancreas and draining pancreatic lymph nodes and upregulation of transforming growth factor (TGF)-ß1 by pancreas-resident antigen-presenting cells. Neutralization of TGF-ß blocked the induction of remission. In contrast, maintenance of remission was associated with tissue-specific immunoregulatory T cells. These findings demonstrate that the use of nondepleting Ab specific for CD4 and CD8 is a robust approach to establish long-term ß-cell-specific T-cell tolerance at the onset of clinical diabetes.

Roles of tumor necrosis factor receptor associated factor 3 (TRAF3) and TRAF5 in immune cell functions.

A large and diverse group of receptors utilizes the family of cytoplasmic signaling proteins known as tumor necrosis factor receptor (TNFR)-associated factors (TRAFs). In recent years, there has been a resurgence of interest and exploration of the roles played by TRAF3 and TRAF5 in cellular regulation, particularly in cells of the immune system, the cell types of focus in this review. This work has revealed that TRAF3 and TRAF5 can play diverse roles for different receptors even in the same cell type, as well as distinct roles in different cell types. Evidence indicates that TRAF3 and TRAF5 play important roles beyond the TNFR-superfamily (SF) and viral mimics of its members, mediating certain innate immune receptor and cytokine receptor signals, and most recently, signals delivered by the T-cell receptor (TCR) signaling complex. Additionally, much research has demonstrated the importance of TRAF3-mediated cellular regulation via its cytoplasmic interactions with additional signaling proteins. In particular, we discuss below evidence for the participation by TRAF3 in a number of the regulatory post-translational modifications involving ubiquitin that are important in various signaling pathways.

beta cell-specific CD4+ T cell clonotypes in peripheral blood and the pancreatic islets are distinct.

Type 1 diabetes is an autoimmune disease mediated by beta cell-specific CD4(+) and CD8(+) T cells. Tracking beta cell-specific T cells is one approach to monitor the diabetogenic response in at risk or diabetic individuals. Such analyses, however, are limited to PBL because T cells infiltrating the pancreatic islets are normally inaccessible. A key issue is whether peripheral beta cell-specific T cells accurately reflect those cells infiltrating the target tissue. We investigated the properties of CD4(+) T cells specific for a mimetic epitope recognized by the BDC2.5 clonotypic TCR in NOD mice. Soluble IA(g7)-Ig (sIA(g7)-Ig) multimer complexes covalently linked to a mimetic BDC peptide (sIA(g7)-mBDC) were used to identify or isolate CD4(+) T cells from PBL and the islets of NOD mice. A temporal increase in sIA(g7)-mBDC binding (g7-mBDC(+)) T cells corresponding with the progression of beta cell autoimmunity was detected in both PBL and islets in NOD female mice. In contrast to T cells in PBL, however, the majority of islet g7-mBDC(+) T cells exhibited a type 1 phenotype, and mediated diabetes upon transfer into NOD.scid recipients. TCR-beta and CDR-beta gene usage of single islet-infiltrating g7-mBDC(+) CD4(+) T cells from individual NOD mice showed a restricted repertoire dominated by one or two clones typically expressing TCR beta-chain variable TRBV-15. In contrast, a distinct and diverse TCR repertoire was detected for PBL-derived g7-mBDC(+) T cells. These results demonstrate that PBL and islet CD4(+) T cells specific for a given beta cell epitope can differ regarding pathogenicity and TCR repertoire.

Suppression of ongoing T cell-mediated autoimmunity by peptide-MHC class II dimer vaccination.

Tissue-specific autoimmune diseases such as type 1 diabetes (T1D) are characterized by T cell-driven pathology. Administration of autoantigenic peptides provides a strategy to selectively target the pathogenic T cell response. Indeed, treatment with beta cell peptides effectively prevents T1D in NOD mice. However, the efficacy of peptide immunotherapy generally wanes as beta cell autoimmunity progresses and islet inflammation increases. With the goal of enhancing the efficacy of peptide immunotherapy, soluble (s)IA(g7)-Ig dimers covalently linked to beta cell autoantigen-derived peptides were tested for the capacity to suppress late preclinical T1D. NOD female mice with established beta cell autoimmunity were vaccinated i.v. with a short course of sIA(g7)-Ig dimers tethered to peptides derived from glutamic acid decarboxylase (GAD)65 (sIA(g7)-pGAD65). Treatment with sIA(g7)-pGAD65 dimers and the equivalent of only approximately 7 microg of native peptide effectively blocked the progression of insulitis and the development of diabetes. Furthermore, suppression of T1D was dependent on beta cell-specific IL-10-secreting CD4+ T cells, although the frequency of GAD65-specific FoxP3-expressing CD4+ T cells was also increased in sIA(g7)-pGAD65 dimer vaccinated NOD mice. These results demonstrate that MHC class II-Ig dimer vaccination is a robust approach to suppress ongoing T cell-mediated autoimmunity, and may provide a superior strategy of adjuvant-free peptide-based immunotherapy to induce immunoregulatory T cells.

A novel role for c-Src and STAT3 in apoptotic cell-mediated MerTK-dependent immunoregulation of dendritic cells.

Dendritic cells (DCs) play an instrumental role in regulating tolerance to self-antigens and preventing autoimmunity. One mechanism by which "tolerogenic" DCs are established is through the inhibitory effects of apoptotic cells (ACs). Immature DCs encountering ACs are resistant to stimuli that activate and mature DCs. We have shown that the Mer receptor tyrosine kinase (MerTK) plays a key role in transducing inhibitory signals upon binding of ACs, which in turn involve the phosphatidylinositol 3-kinase (PI3K) pathway. Nevertheless, the molecular basis for AC-induced inhibition of DCs is ill defined. In the current study, the proximal signaling events induced by MerTK after AC binding were studied. AC treatment of bone marrow-derived or splenic DCs established a complex consisting of MerTK, the nonreceptor tyrosine kinase c-Src, the transcription factor STAT3, and PI3K. In contrast, AC treatment of DCs lacking MerTK expression failed to increase c-Src and STAT3 activation. In addition, the inhibitory effects of ACs were blocked by treating DCs with pharmacologic inhibitors or siRNA specific for c-Src and STAT3. These findings demonstrate that AC-induced inhibition of DCs requires MerTK-dependent activation of c-Src and STAT3, and provide evidence for novel roles for c-Src and STAT3 in the immunoregulation of DCs.

CD8(+) T cells specific for beta cells encounter their cognate antigens in the islets of NOD mice.

CD8(+) T cells play a key role in the initiation of insulitis. However, the site(s) where naive CD8(+) T cells encounter beta-cell antigens and the mechanism(s) by which beta-cell autoimmunity is initiated remain to be determined. In the current study, an adoptive transfer model was employed assessing the initial site of priming and the nature of antigen recognition by naive beta-cell-specific CD8(+) T cells. Temporal analysis demonstrated that unlike CD4(+) T cells that are primed in the draining pancreatic lymph nodes, initial proliferation of transferred CD8(+) T cells was detected in the islets. These results indicate that in our model, naive beta-cell-specific CD8(+) T cells encounter beta-cell antigens in the islets. Furthermore, ectopic expression of CD80 by beta cells accelerated the onset of insulitis mediated by beta-cell-specific CD8(+) T cells, but had no effect on CD4(+) T-cell-mediated diabetes, suggesting an antigenic interaction between beta cells and naive CD8(+) T cells. However, it remains to be determined whether the initiation of insulitis in spontaneous diabetes is the result of a cognate interaction between naive CD8(+) T cells and islet beta cells.

MerTK regulates thymic selection of autoreactive T cells.

T cell-mediated autoimmune diseases such as type 1 diabetes (T1D) are believed to be the result in part of inefficient negative selection of self-specific thymocytes. However, the events regulating thymic negative selection are not fully understood. In the current study, we demonstrate that nonobese diabetic (NOD) mice lacking expression of the Mer tyrosine kinase (MerTK) have reduced inflammation of the pancreatic islets and fail to develop diabetes. Furthermore, NOD mice deficient in MerTK expression (Mer(-/-)) exhibit a reduced frequency of beta cell-specific T cells independent of immunoregulatory effectors. The establishment of bone marrow chimeric mice demonstrated that the block in beta cell autoimmunity required hematopoietic-derived cells lacking MerTK expression. Notably, fetal thymic organ cultures and self-peptide administration showed increased thymic negative selection in Mer(-/-) mice. Finally, thymic dendritic cells (DC) prepared from Mer(-/-) mice exhibited an increased capacity to induce thymocyte apoptosis in a peptide-specific manner in vitro. These findings provide evidence for a unique mechanism involving MerTK-mediated regulation of thymocyte negative selection and thymic DC, and suggest a role for MerTK in contributing to beta cell autoimmunity.

Immunotherapy of type 1 diabetes.

Type 1 diabetes (T1D) is an autoimmune disease in which the insulin-producing beta cells are destroyed. Diabetic patients manage their hyperglycemia by daily insulin injections. However, insulin therapy is by no means a cure. Accordingly, a significant effort has been ongoing to develop immunotherapies that effectively prevent and/or treat T1D in the clinic. This review focuses on antigen- and antibody-based immunotherapies and discusses the respective strengths and weaknesses of these approaches.

MerTK is required for apoptotic cell-induced T cell tolerance.

Self-antigens expressed by apoptotic cells (ACs) may become targets for autoimmunity. Tolerance to these antigens is partly established by an ill-defined capacity of ACs to inhibit antigen-presenting cells such as dendritic cells (DCs). We present evidence that the receptor tyrosine kinase Mer (MerTK) has a key role in mediating AC-induced inhibition of DC activation/maturation. Pretreatment of DCs prepared from nonobese diabetic (NOD) mice with AC blocked secretion of proinflammatory cytokines, up-regulation of costimulatory molecule expression, and T cell activation. The effect of ACs on DCs was dependent on Gas6, which is a MerTK ligand. NOD DCs lacking MerTK expression (NOD.MerTK(KD/KD)) were resistant to AC-induced inhibition. Notably, autoimmune diabetes was exacerbated in NOD.MerTK(KD/KD) versus NOD mice expressing the transgenic BDC T cell receptor. In addition, beta cell-specific CD4(+) T cells adoptively transferred into NOD.MerTK(KD/KD) mice in which beta cell apoptosis was induced with streptozotocin exhibited increased expansion and differentiation into type 1 T cell effectors. In both models, the lack of MerTK expression was associated with an increased frequency of activated pancreatic CD11c(+)CD8alpha(+) DCs, which exhibited an enhanced T cell stimulatory capacity. These findings demonstrate that MerTK plays a critical role in regulating self-tolerance mediated between ACs, DCs, and T cells.