CD28 Signaling Controls Metabolic Fitness of Pathogenic T Cells in Medium and Large Vessel Vasculitis.

BACKGROUND: In giant cell arteritis, vessel-wall infiltrating CD4 T cells and macrophages form tissue-destructive granulomatous infiltrates, and the artery responds with a maladaptive response to injury, leading to intramural neoangiogenesis, intimal hyperplasia, and luminal occlusion. Lesion-residing T cells receive local signals, which represent potential therapeutic targets. OBJECTIVES: The authors examined how CD28 signaling affects vasculitis induction and maintenance, and which pathogenic processes rely on CD28-mediated T-cell activation. METHODS: Vasculitis was induced by transferring peripheral blood mononuclear cells from giant cell arteritis patients into immunodeficient NSG mice engrafted with human arteries. Human artery-NSG chimeras were treated with anti-CD28 domain antibody or control antibody. Treatment effects and immunosuppressive mechanisms were examined in vivo and in vitro applying tissue transcriptome analysis, immunohistochemistry, flow cytometry, and immunometabolic analysis. RESULTS: Blocking CD28-dependent signaling markedly reduced tissue-infiltrating T cells and effectively suppressed vasculitis. Mechanistic studies implicated CD28 in activating AKT signaling, T-cell proliferation and differentiation of IFN-γ and IL-21-producing effector T cells. Blocking CD28 was immunosuppressive by disrupting T-cell metabolic fitness; specifically, the ability to utilize glucose. Expression of the glucose transporter Glut1 and of glycolytic enzymes as well as mitochondrial oxygen consumption were all highly sensitive to CD28 blockade. Also, induction and maintenance of CD4+CD103+ tissue-resident memory T cells, needed to replenish the vasculitic infiltrates, depended on CD28 signaling. CD28 blockade effectively suppressed vasculitis-associated remodeling of the vessel wall. CONCLUSIONS: CD28 stimulation provides a metabolic signal required for pathogenic effector functions in medium and large vessel vasculitis. Disease-associated glycolytic activity in wall-residing T-cell populations can be therapeutically targeted by blocking CD28 signaling.

Renal Cell Carcinoma (RCC) Tumors Display Large Expansion of Double Positive (DP) CD4+CD8+ T Cells With Expression of Exhaustion Markers.

Checkpoint inhibitors target the inhibitory receptors expressed by tumor-infiltrating T cells in order to reinvigorate an anti-tumor immune response. Therefore, understanding T cell composition and phenotype in human tumors is crucial. We analyzed by flow cytometry tumor-infiltrating lymphocytes (TILs) from two independent cohorts of patients with different cancer types, including RCC, lung, and colon cancer. In healthy donors, peripheral T cells are usually either CD4+ or CD8+ with a small percentage of CD4+ CD8+ DP cells (<5%). Compared to several other cancer types, including lung, and colorectal cancers, TILs from about a third of RCC patients showed an increased proportion of DP CD4+CD8+ T cells (>5%, reaching 30-50% of T cells in some patients). These DP T cells have an effector memory phenotype and express CD38, 4-1BB, and HLA-DR, suggesting antigen-driven expansion. In fact, TCR sequencing analysis revealed a high degree of clonality in DP T cells. Additionally, there were high levels of PD-1 and TIM-3 expression on DP T cells, which correlated with higher expression of PD-1 and TIM-3 in conventional single positive CD8 T cells from the same patients. These results suggest that DP T cells could be dysfunctional tumor-specific T cells with the potential to be reactivated by checkpoint inhibitors.

Circulating T Cell Subpopulations Correlate With Immune Responses at the Tumor Site and Clinical Response to PD1 Inhibition in Non-Small Cell Lung Cancer.

Agents targeting the PD1-PDL1 axis have transformed cancer therapy. Factors that influence clinical response to PD1-PDL1 inhibitors include tumor mutational burden, immune infiltration of the tumor, and local PDL1 expression. To identify peripheral correlates of the anti-tumor immune response in the absence of checkpoint blockade, we performed a retrospective study of circulating T cell subpopulations and matched tumor gene expression in melanoma and non-small cell lung cancer (NSCLC) patients. Notably, both melanoma and NSCLC patients whose tumors exhibited increased inflammatory gene transcripts presented high CD4+ and CD8+ central memory T cell (CM) to effector T cell (Eff) ratios in blood. Consequently, we evaluated CM/Eff T cell ratios in a second cohort of NSCLC. The data showed that high CM/Eff T cell ratios correlated with increased tumor PDL1 expression. Furthermore, of the 22 patients within this NSCLC cohort who received nivolumab, those with high CM/Eff T cell ratios, had longer progression-free survival (PFS) (median survival: 91 vs. 215 days). These findings show that by providing a window into the state of the immune system, peripheral T cell subpopulations inform about the state of the anti-tumor immune response and identify potential blood biomarkers of clinical response to checkpoint inhibitors in melanoma and NSCLC.

B cells from African American lupus patients exhibit an activated phenotype.

Systemic lupus erythematosus (SLE) is a complex systemic autoimmune disease driven by both innate and adaptive immune cells. African Americans tend to present with more severe disease at an earlier age compared with patients of European ancestry. In order to better understand the immunological differences between African American and European American patients, we analyzed the frequencies of B cell subsets and the expression of B cell activation markers from a total of 68 SLE patients and 69 normal healthy volunteers. We found that B cells expressing the activation markers CD86, CD80, PD1, and CD40L, as well as CD19+CD27-IgD- double-negative B cells, were enriched in African American patients vs. patients of European ancestry. In addition to increased expression of CD40L, surface levels of CD40 on B cells were lower, suggesting the engagement of the CD40 pathway. In vitro experiments confirmed that CD40L expressed by B cells could lead to CD40 activation and internalization on adjacent B cells. To conclude, these results indicate that, compared with European American patients, African American SLE patients present with a particularly active B cell component, possibly via the activation of the CD40/CD40L pathway. These data may help guide the development of novel therapies.

Functional Antagonism of Human CD40 Achieved by Targeting a Unique Species-Specific Epitope.

Current clinical anti-CD40 biologic agents include both antagonist molecules for the treatment of autoimmune diseases and agonist molecules for immuno-oncology, yet the relationship between CD40 epitope and these opposing biological outcomes is not well defined. This report describes the identification of potent antagonist domain antibodies (dAbs) that bind to a novel human CD40-specific epitope that is divergent in the CD40 of nonhuman primates. A similarly selected anti-cynomolgus CD40 dAb recognizing the homologous epitope is also a potent antagonist. Mutagenesis, biochemical, and X-ray crystallography studies demonstrate that the epitope is distinct from that of CD40 agonists. Both the human-specific and cynomolgus-specific molecules remain pure antagonists even when formatted as bivalent Fc-fusion proteins, making this an attractive therapeutic format for targeting hCD40 in autoimmune indications.

Advances in CTLA-4-Ig-mediated modulation of inflammatory cell and immune response activation in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a multifactorial and polygenic immune-mediated disease, the pathogenesis of which involves different cell types. T and B lymphocytes, macrophages, endothelial cells, fibroblasts and osteoclasts have all been implicated in mediating the production of autoantibodies, proinflammatory cytokines and ultimately bone erosions. Cytotoxic T lymphocyte-associated antigen 4 immunoglobulin fusion protein (CTLA-4-Ig, abatacept) is a unique biologic agent targeting the co-stimulatory molecules CD80/CD86, and is indicated for the treatment of moderate-to-severe RA in patients who have had an inadequate response to one or more disease-modifying anti-rheumatic drugs, including methotrexate or anti-tumor necrosis factor agents. There is a growing body of evidence that, through selective modulation of the CD80/CD86 co-stimulatory molecules expressed by a variety of activated cell types, CTLA-4-Ig may inhibit the pathogenic RA process at several levels, both directly and indirectly. Here, we provide an overview of recent mechanistic studies of the action of CTLA-4-Ig on different cell types involved in mediating inflammation and joint damage in RA.

Azaxanthene based selective glucocorticoid receptor modulators: design, synthesis, and pharmacological evaluation of (S)-4-(5-(1-((1,3,4-thiadiazol-2-yl)amino)-2-methyl-1-oxopropan-2-yl)-5H-chromeno[2,3-b]pyridin-2-yl)-2-fluoro-N,N-dimethylbenzamide (BMS-776532) and its methylene homologue (BMS-791826).

Structurally novel 5H-chromeno[2,3-b]pyridine (azaxanthene) selective glucocorticoid receptor (GR) modulators have been identified. A screening paradigm utilizing cellular assays of GR-mediated transrepression of proinflammatory transcription factors and transactivation of GR-dependent genes combined with three physiologically relevant assays of cytokine induction in human whole blood has allowed for the identification of high affinity, selective GR ligands that display a broad range of pharmacological profiles. Agonist efficacy in reporter assays can be tuned by halogenation of a pendent phenyl ring and correlates well with efficacy for cytokine inhibition in human whole blood. A hypothetical binding mode is proposed, invoking an expanded ligand binding pocket resembling that of arylpyrazole-bound GR structures. Two compounds of close structural similarity (35 and 37; BMS-776532 and BMS-791826, respectively) have been found to maintain distinct and consistent levels of partial agonist efficacy across several assays, displaying anti-inflammatory activity comparable to that of prednisolone 2 in suppressing cytokine production in whole blood and in rodent models of acute and chronic inflammation.

Abatacept limits breach of self-tolerance in a murine model of arthritis via effects on the generation of T follicular helper cells.

Abatacept modulates CD28-mediated T cell costimulation and is efficacious in the treatment of rheumatoid arthritis (RA). Its mechanism of action has not been fully elucidated but will likely reveal critical pathologic pathways in RA. We show that abatacept substantially modulated Ag-specific T and B cell responses in vivo. Ag-specific T cell proliferation was reduced, and the acquisition of an activated phenotype, characterized by upregulation of CD69, OX40, ICOS, and programmed death-1 and downregulation of CD62L, was suppressed. Furthermore, abatacept suppressed the production of inflammatory cytokines, such as IFN-gamma and IL-17. These effects were associated with a failure of Ag-specific T cells to acquire the CXCR5(+)ICOS(+) T follicular helper cell phenotype. This, in turn, led to a failure of these cells to enter B cell follicles, resulting in reduced specific Ab responses, despite normal B cell clonal expansion. To test the pathologic significance of this, we used a novel model of RA associated with breach of self-tolerance to self-Ag and demonstrated that abatacept prevented the emergence of self-reactivity. Thus, CD28-dependent signaling is required for optimal T follicular helper cell maturation and expansion, and its inhibition prevents loss of self-tolerance in a model of articular pathology. Thus, we provide a novel mode of action for abatacept with profound implications for its potential usefulness in early inflammatory arthropathies associated with autoantibody expression.

Abatacept does not induce direct gene expression changes in antigen-presenting cells.

BACKGROUND: It has been proposed that ligation of CD80 and CD86 induces reverse signaling into antigen-presenting cells. In this study, we tested the ability of abatacept, a soluble human fusion protein comprising the extracellular domain of cytotoxic T lymphocyte antigen 4 and a fragment of the Fc domain of IgG(1), to activate antigen-presenting cells by measuring changes in global transcriptional responses. METHODS: Affymetrix chips were used to measure gene expression levels using mRNA isolated from immature and mature human dendritic cells and a B cell line following 6 h of treatment with abatacept. RESULTS: In contrast to robust transcriptional responses induced by the control treatment phorbol-12-myristate-13-acetate, abatacept induced minimal gene changes in three different populations of antigen-presenting cells. Furthermore, no gene changes were observed in response to belatacept, a modified version of abatacept that binds with higher avidity to CD80 and CD86. CONCLUSIONS: We conclude that reverse signaling in antigen-presenting cells is unlikely to occur in response to either abatacept or belatacept, thereby supporting the modulation of CD28 signaling on T cells as the main mechanism of action for these therapeutics.

Abatacept binds to the Fc receptor CD64 but does not mediate complement-dependent cytotoxicity or antibody-dependent cellular cytotoxicity.

OBJECTIVE: To assess the ability of abatacept to mediate complement-dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC) of antigen-presenting cells, and to characterize the binding of abatacept to the 3 Fc receptor classes. METHODS: CDC was measured in vitro using rabbit, baby rabbit, guinea pig, or human complement with human B cell line PM-LCL as the target. ADCC was also measured with PM-LCL target cells, but with human peripheral blood mononuclear cells from 12 healthy blood donors as effectors. Fc receptor binding was analyzed in vitro by flow cytometry and surface plasmon resonance (SPR). RESULTS: In contrast to unmodified CTLA4-Ig, abatacept did not mediate CDC or ADCC of target B cells. While abatacept was found to bind its target receptor, CD80/86, it did not appreciably bind the low-affinity Fc receptors CD16 and CD32 as measured by flow cytometry and SPR. Abatacept was found to minimally bind the high-affinity Fc receptor CD69 as measured by flow cytometry and SPR with a Kd of 3 X 10-7 M as measured by SPR. CONCLUSION: Abatacept does not mediate CDC or ADCC of target B cells in vitro and has limited Fc receptor binding. These data support the concept that abatacept therapeutic activity is primarily due to the binding to CD80/86 through the CTLA4 extracellular domain and not through activities mediated by the modified Fc domain.

Abatacept treatment does not exacerbate chronic Mycobacterium tuberculosis infection in mice.

OBJECTIVE: Treatment of rheumatoid arthritis and other autoimmune disorders with anti-tumor necrosis factor (anti-TNF) agents is associated with an increased risk of reactivation of latent Mycobacterium tuberculosis. While the mechanism of action of abatacept is fundamentally different from that of anti-TNF therapies, its effect on the protective response to latent tuberculosis is not known. We undertook this study to determine the effect of abatacept treatment in a murine model of chronic M tuberculosis infection. METHODS: Chronic M tuberculosis infection was established in C57BL/6 mice. Four months after infection, mice were treated for up to 16 weeks with abatacept, anti-murine TNF antibody, or vehicle. The primary end point was survival; body weight, bacterial load, histologic features, interferon-gamma (IFNgamma) production by T cells, and cellular infiltration were also assessed. RESULTS: Abatacept- and vehicle-treated groups both maintained control of M tuberculosis infection, with 100% survival after 16 weeks of treatment. These 2 groups had no significant differences in body weight, no clinically relevant differences in bacterial load in the lungs, lymph nodes, or spleen, and no differences in the mean percentage of total or activated T cells, macrophages, neutrophils, or B cells, or in IFNgamma production in the lung or lymph nodes. In contrast, 100% mortality was seen in the anti-TNF antibody-treated group by week 9, with significant body weight loss and increased bacterial load in the lungs, lymph nodes, and spleen. Furthermore, the anti-TNF antibody-treated group had increased pathology consistent with the exacerbation of M tuberculosis infection. CONCLUSION: Abatacept did not impair the ability of mice to control a chronic M tuberculosis infection. In contrast, mice treated with anti-TNF therapy showed increased pathology and bacterial load, with 100% mortality by week 9. The clinical significance of these findings has not yet been determined.

In vivo administration of 15-deoxyspergulin inhibits antigen-presenting cell stimulation of T cells and NF-kappaB activation.

15-Deoxyspergulin (DSG), a synthetic derivative of spergulin, was initially characterized for its antibiotic and antitumor effects. Recent studies have described the immunosuppressive properties of this molecule, but its mechanism of action is not clearly understood. In the study reported here, mice were treated in vivo with DSG prior to the measurement of IL-2 production and proliferation in an in vitro antigen presentation assay. At suboptimal antigen concentrations, elicited peritoneal macrophages or percoll isolated B cells from DSG-treated mice showed a 50-96% reduction in their ability to present chicken ovalbumin (cOva), cOva peptide, or superantigen (SAg) to MHC class II-matched antigen-specific primary T cells. No significant changes could be found in the cell surface expression of CD80, CD86, MHC I, MHC II, CD18, CD11b, CD40, CD25, and CD54 in antigen-presenting cells (APC) from control or DSG-treated animals. Activation with SAg of macrophages or splenocytes from DSG-treated mice revealed that there was a significant reduction in nuclear NF-kappaB levels compared to cells from untreated animals. Additionally, analysis of cytokines showed that production of TNF-alpha and IL-1beta was inhibited in cultures where macrophages from DSG-mice were used to present cOva to T cells. These results indicate that the effects of DSG in mice are not simply due to altered antigen processing or from any marked changes in cell surface antigen expression. Rather, the immunosuppression may arise from alterations in the release of one or more soluble factor from DSG-treated APC, which prevents effective antigen presentation and T cell activation.