CD40 Signaling Research Articles

Neurocomputational Method for Estimation of Balance of Resting-State Oscillatory Networks Associated With Personal Style of Emotional Regulation

CD95/Fas/APO-1 can trigger apoptotic as well as nonapoptotic pathways in immune cells. CD95 signaling in humans can be inhibited by several mechanisms, including mutations in the gene encoding CD95. CD95 mutations lead to autoimmune disorders, such as autoimmune lymphoproliferative syndrome (ALPS). Gaining further insight into the reported mutations of CD95 and resulting alterations of its signaling networks may provide further understanding of their presumed role in certain autoimmune diseases. For illustrative purposes and to better understand the potential outcomes of CD95 mutations, here we assign their positions to the recently determined 3D structures of human CD95. Based on this, we make certain predictions and speculate on the putative role of CD95 mutation defects in CD95-mediated signaling for certain autoimmune diseases.

Abstract MP223: CCR2 + Macrophages And T-cells Infiltrate The Murine Heart Following Immune Regulator Treatment With CD40

Immune checkpoint inhibitors have revolutionized cancer treatment but have been associated with severe adverse cardiac events including myocarditis. CD40 agonist (CD40ag) antibodies are immune regulators that have emerged as promising candidates with remarkable efficacy across tumors including those thought to be resistant to established ICIs. To investigate CD40 signaling and its potential for adverse cardiac events, C57BL/6J, CCR2-GFP, and CCR2-knockout mice were injected with CD40ag or isotype antibodies for 7 days. Flow cytometry showed a specific increase in CCR2 + macrophages that was independent of monocyte recruitment. There was no change in neutrophil recruitment and only a modest increase in dendritic cells was evident. We also observed a significant expansion of CD4 and CD8 T-cells that displayed an effector memory phenotype. Bulk cardiac tissue gene expression analysis (Figure A) revealed that CD40 activation upregulates multiple inflammatory cytokines (Ifng, Tnf, Il-12β) and chemokines (Ccl3, Ccl5, Ccl7, Cxcl9, Cxcl10), which are regulated by interferon gamma and coordinate the activation of APCs and T-cells. Finally, RNA in situ hybridization (Figure B-C) demonstrated increased levels of Cxcl9 (red) expression in cells consistent with CCR2 + (white) macrophages when comparing isotype control to treatment with CD40ag. These findings reveal that CD40 activation results in a robust expansion of CCR2 + macrophages and activation of T-cells within the heart, initiating a feed forward loop of activation that is mediated by interferon gamma and generates inflammatory cytotoxic mediators that may lead to myocardial injury.

Single-Cell RNA Sequencing Reveals the Migration of Osteoclasts in Giant Cell Tumor of Bone.

Giant cell tumor of bone (GCTB) is benign tumor that can cause significant osteolysis and bone destruction at the epiphysis of long bones. Osteoclasts are thought to be highly associated with osteolysis in GCTB. However, the migration of osteoclasts in GCTB remains unclear. A deeper understanding of the complex tumor microenvironment is required in order to delineate the migration of osteoclasts in GCTB. In this study, samples were isolated from one patient diagnosed with GCTB. Single-cell RNA sequencing (scRNA-seq) was used to detect the heterogeneity of GCTB. Multiplex immunofluorescence staining was used to evaluate the cell subtypes identified by scRNA-seq. A total of 8,033 cells were obtained from one patient diagnosed with GCTB, which were divided into eight major cell types as depicted by a single-cell transcriptional map. The osteoclasts were divided into three subsets, and their differentiation trajectory and migration status were further analyzed. Osteoclast migration may be regulated via a series of genes associated with cell migration. Furthermore, four signaling pathways (RANKL, PARs, CD137 and SMEA3 signaling pathway) were found to be highly associated with osteoclast migration. This comprehensive single-cell transcriptome analysis of GCTB identified a series of genes associated with cell migration as well as four major signaling pathways that were highly related to the migration of osteoclasts in GCTB. Our findings broaden the understanding of GCTB bionetworks and provides a theoretical basis for anti-osteolysis therapy against GCTB in the future.

A human CD137\xd7PD-L1 bispecific antibody promotes anti-tumor immunity via context-dependent T cell costimulation and checkpoint blockade

Immune checkpoint inhibitors demonstrate clinical activity in many tumor types, however, only a fraction of patients benefit. Combining CD137 agonists with these inhibitors increases anti-tumor activity preclinically, but attempts to translate these observations to the clinic have been hampered by systemic toxicity. Here we describe a human CD137xPD-L1 bispecific antibody, MCLA-145, identified through functional screening of agonist- and immune checkpoint inhibitor arm combinations. MCLA-145 potently activates T cells at sub-nanomolar concentrations, even under suppressive conditions, and enhances T cell priming, differentiation and memory recall responses. In vivo, MCLA-145 anti-tumor activity is superior to immune checkpoint inhibitor comparators and linked to recruitment and intra-tumor expansion of CD8 + T cells. No graft-versus-host-disease is observed in contrast to other antibodies inhibiting the PD-1 and PD-L1 pathway. Non-human primates treated with 100 mg/kg/week of MCLA-145 show no adverse effects. The conditional activation of CD137 signaling by MCLA-145, triggered by neighboring cells expressing >5000 copies of PD-L1, may provide both safety and potency advantages.

Physical Activity Moderates Inflammatory Gene Expression in Rheumatoid Arthritis

Background: While general population studies have shown associations between greater physical activity and lower inflammatory markers, effects of physical activity on systemic inflammation in immune-mediated inflammatory diseases such as rheumatoid arthritis (RA) are unknown. We examined whether physical activity associates with differential expression of inflammatory genes in RA. Methods: Data derived from an observational RA cohort. Physical activity was measured with the GT9X ActiGraph Link for 7 consecutive 24-hour periods. Clinical data and peripheral whole blood for RNA sequencing were collected. Physical activity groups were defined by the highest and lowest tertiles of moderate/vigorous activity (metabolic equivalent level ≥2.00). RNA extraction, library preparation, Illumina sequencing, and gene expression data processing were performed using established methods. Genes differentially expressed in the most versus least active groups were identified using DESeq2 with the Independent Hypothesis Weighting (IHW) multiple testing procedure and adjusting for sex, age, and race/ethnicity. Ingenuity Pathway Analysis (IPA) Canonical Pathway and Upstream Regulator Analyses were employed on differentially expressed genes with a P<0·1 ranked by log2-fold change to identify biological pathways affected by physical activity. Findings: 35 participants were included (mean age 56±12 years; 91% female; race/ethnicity 31% white, 9% African American, 9% Asian, 40% Hispanic). 767 genes were differentially expressed (padj<0.1) between high versus low activity groups. The high activity group exhibited downregulation of innate and adaptive immune signaling pathways implicated in RA pathogenesis including CD40, STAT3, TREM-1, IL-17a, IL-8, toll-like receptor and interferon signaling. Upstream cytokine activation states demonstrated lower TNF-alpha and interferon among individuals in the high vs. low activity group. Interpretation: RA patients who were more physically active had lower expression of genes involved in innate and adaptive immune signaling compared to those who were more sedentary, providing mechanistic evidence that physical activity may confer protective effects on RA disease outcomes. Funding: This work is supported by NIH/NIAMS R01 AR069616 and NIH/NHLBI K23HL138461-01A1. Additional support from the Rheumatology Research Foundation. Declaration of Interest: None to declare. Ethical Approval: The UCSF Institutional Review Board approved all procedures (UCSF IRB protocol #17-21790) and all participants provided written informed consent.

The Transcriptome of Paired Major and Minor Salivary Gland Tissue in Patients With Primary Sjögren's Syndrome.

BackgroundWhile all salivary glands (SGs) can be involved in primary Sjögren’s syndrome (pSS), their respective role in pathogenesis remains unclear. Our objective was to assess immunopathway activation in paired parotid and labial gland tissue from biopsy-positive and biopsy-negative pSS and non-SS sicca patients.MethodsParaffin-embedded, paired parotid and labial salivary gland tissue and peripheral blood mononuclear cells were obtained from 39 pSS and 20 non-SS sicca patients. RNA was extracted, complementary DNA libraries were prepared and sequenced. For analysis of differentially expressed genes (DEGs), patients were subdivided based on fulfillment of ACR-EULAR criteria and histopathology.ResultsWith principal component analysis, only biopsy-positive pSS could be separated from non-SS sicca patients based on SG gene expression. When comparing the transcriptome of biopsy-positive pSS and biopsy-negative non-SS sicca patients, 1235 and 624 DEGs (FDR<0.05, log2FC<-1 or >1) were identified for parotid and labial glands, respectively. The number of DEGs between biopsy-negative pSS and non-SS sicca patients was scarce. Overall, transcript expression levels correlated strongly between parotid and labial glands (R2 = 0.86, p-value<0.0001). Gene signatures present in both glands of biopsy-positive pSS patients included IFN-α signaling, IL-12/IL-18 signaling, CD3/CD28 T-cell activation, CD40 signaling in B-cells, DN2 B-cells, and FcRL4+ B-cells. Signature scores varied considerably amongst pSS patients.ConclusionTranscriptomes of paired major and minor SGs in pSS were overall comparable, although significant inter-individual heterogeneity in immunopathway activation existed. The SG transcriptome of biopsy-negative pSS was indistinguishable from non-SS sicca patients. Different patterns of SG immunopathway activation in pSS argue for personalized treatment approaches.

Abstract 2368: miR-29-TRAF4 axis is a novel regulator of CD40 signaling in malignant B cells

Abstract The synchronous activation of BCR and CD40 signaling via B-T cell interactions is required for proliferation of normal (Luo et al, 2018) and some malignant B cells, especially in chronic lymphocytic leukemia (CLL) and follicular lymphoma (FL). In CLL/FL cells, proliferation occurs mainly in lymph nodes, but not in bone marrow or peripheral blood which lack access to proper B-T cell interactions. Here we have analyzed the mRNA and miRNAs profile in the proliferative intraclonal CLL cell subpopulation that has recently exited lymph node niches (CXCR4dimCD5bright cells; Calissano et al,2011; Pavlasova et al,2016) to reveal molecules potentially participating in synchronous regulation of CD40 and BCR pathway. This has identified 36 miRNAs and 1370 mRNAs differentially expressed in CLL cells exiting lymph nodes as compared to resting non-proliferative CLL cells (CXCR4brightCD5dim cells). Next, we overlapped the 36 miRNAs with their predicted target mRNAs with putative function in CD40/BCR (TargetScan, KEGG) which were anti-correlated to at least one miRNA (miRNAs typically decrease mRNA stability). This revealed among others anti-correlation of lower miR-29 with higher TRAF4 levels in immune niches, which was validated in paired lymph node biopsies vs peripheral blood CLL cells (P&amp;lt;0.01). The negative regulation of TRAF4 by miR-29 was confirmed by transfection of primary CLL and MEC1 cells with synthetic miR-29, miR-29 inhibitor and by a luciferase assay with binding site from TRAF4 3'UTR. Several other miR-29 targets involved in other pathways have been identified as additional targets. TRAF4 is an understudied member of a protein family regulating receptor-induced immune cell activation, however, its role in CD40 pathway or its interactome is unknown. Co-immunoprecipitation and mass spectrometry in B cells pre- and post- stimulation by CD40 ligand revealed TRAF4 interaction with members of the CD40 pathway and a novel function in positive regulation of CD40-induced NFκB activity. In B cells, TRAF4 silencing or its over-expression affected IKKα/β phosphorylation following CD40 ligation or co-culture with activated T cells (all P&amp;lt;0.01). Notably, BCR inhibition lead in vivo to miR-29 upregulation by interfering with its negative regulator MYC induced by BCR. This leads to TRAF4 repression and drastically impairs CD40 signaling (P&amp;lt;0.001), which at least partially explains the observed anti-proliferative effects of BCR inhibitors. Altogether, we have described a novel MYC-miR-29-TRAF4 axis that regulates CD40 signaling in B cells, and acts to synchronize BCR activation with CD40 pathway. Supported by: The ERC under the European Union's Horizon 2020 research and innovation program (grant agreement No 802644), MH CZ, grant nr. NV18-03-00054 and NU20-03-00292. All rights reserved. Czech Science Foundation (20-02566S), MEYS CZ under the project CEITEC 2020 (LQ1601), MH CZ-DRO (FNBr,65269705) and MUNI/A/1595/2020. Citation Format: Sonali Sharma, Vaclav Seda, Eva Vojackova, Gabriela Mladonicka Pavlasova, Daniel Filip, Laura Ondrisova, Veronika Sandova, Lenka Kostalova, Pedro F. Zeni, Marek Borsky, Sarka Pospisilova, Medhat Shehata, Laura Z. Rassenti, Ulrich Jaeger, Michael Doubek, Matthew S. Davids, Jennifer R. Brown, Jiri Mayer, Thomas J. Kipps, Marek Mraz. miR-29-TRAF4 axis is a novel regulator of CD40 signaling in malignant B cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2368.

Abstract 1567: IL-15 and anti-PD-1 augment the abscopal efficacy of agonistic intratumoral anti-CD40 in the TRAMP-C2 murine tumor model

Abstract IL-15 promotes activation and maintenance of natural killer (NK) and CD8+ T-effector memory (TEM) cells making it a potential immunotherapeutic agent for the treatment of cancer. However, high doses of γc cytokines lead to the paralysis/depression of naïve CD4 but not CD8 T-cells that is mediated through transient expression of suppressor of cytokine signaling 3 (SOCS3). Thus, the out-of-normal order addition of γc cytokines is associated with generation of “helpless” tumor antigen nonspecific CD8 T cells. The role of CD4 helper T cells can be alternatively mediated by CD40 signaling through the addition of agonistic anti-CD40 antibodies. However, parenteral administration of an agonistic anti-CD40 antibody is associated with unacceptable toxicity that can be avoided by intratumoral administration. Intratumoral immunotherapy that aims at generating a potent low toxic priming of antitumor immunity uses the tumor as its own vaccine. We investigated the combination of IL-15 with an intratumoral anti-CD40 monoclonal antibody using a dosing schedule based on our previous study in the TRAMP-C2 murine prostate cancer model. Here we demonstrated that given intratumorally anti-CD40 had abscopal efficacy, and that the combination of IL-15 with the intratumoral anti-CD40 showed an augmented immune response with an increase in the number of tumor specific tetramer positive CD8+ T-cells. Furthermore, anti-PD-1 antibody was additive to the anti-CD40 IL-15 combination. These studies support the initiation of a clinical trial in patients with cancer involving IL-15 in association with intratumoral optimized anti-CD40. Citation Format: Wei Chen. IL-15 and anti-PD-1 augment the abscopal efficacy of agonistic intratumoral anti-CD40 in the TRAMP-C2 murine tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1567.

Abstract 1852: A novel PD1-CD40 bispecific antibody YH008 induces potent anti-tumor activity in vivo by PD1 dependent activation of CD40 signaling

Abstract Immunotherapies that target PD1/PDL1 pathway have marked a new era of cancer therapy. Combinatorial treatment of inhibiting PD1/PDL1 signaling and other immunomodulating agents has been widely tested and validated as a better strategy compared to single agent treatment. CD40 (TNFRSF5) is a member of the TNFR super family, a key T cell co-stimulatory signal critical for B-cell maturation and immunoglobulin class switching. Biocytogen developed a novel first in class PD1-CD40 bispecific antibody (YH008) that agonizes CD40 conditionally upon PD1 binding. In vitro study data shows YH008 can block the interaction of PD1 to PDL1 and induced dose dependent NFAT activation. YH008 was able to activate human CD40 on reporter cells in the presence of PD1-expressing jurkat cells in a dose dependent manner without relying on FcγR expressing cells. This feature is beneficial for conditionally activation of CD40+ cells in tumor microenvironment and tumor-draining lymph nodes, where tumor specific PD1+ T cells are enriched. In vivo study result shows that YH008 demonstrated robust anti-tumor efficacy against MC38 and B16F10 tumors in PD1/CD40 double humanized mice (B-hPD1/hCD40). In hCD40 mice which has no human PD1 engagement, YH008 did not show anti-tumor activity against MC38 tumor. Furthermore, YH008 did not elevate liver enzymes of MC38 bearingB-hPD1/hCD40 mice at dose as high as 26 mg/kg, demonstrating improved liver safety profile than selicrelumab. Taken together, these data demonstrated that YH008 is a promising new immunotherapeutic candidate in cancer treatment. Citation Format: Baihong Liu, Li Hui, Chonghui Liu, Zhihong Li, Fangxia Pan, Benny Yang, Qingcong Lin. A novel PD1-CD40 bispecific antibody YH008 induces potent anti-tumor activity in vivo by PD1 dependent activation of CD40 signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1852.

RNA Sequencing Reveals Mechanisms Underlying Modulation of Hodgkin Lymphoma Cells and Tumor Microenvironment By the PI3K δ/γ Inhibitor, RP6530

INTRODUCTION: Aberrant activation of the phosphoinositide 3-kinase (PI3K) pathway plays a central role in Hodgkin Lymphoma (HL) pathogenesis. The pleiotropic activities of the δ and γ isoforms of PI3K in regulating the interactions of Hodgkin/Reed-Sternberg (HRS) cells with the tumor microenvironment suggest thatPI3K inhibitors might exert a therapeutic potential in the treatment of HL. The dual PI3K δ/γ inhibitor RP6530 was proven to affect HL tumor cells and to exert immunosuppressive activities. However, a genome-wide search for molecular mediators of RP6530 is largely unexplored in HL. The present study was therefore aimed at evaluating the anti-lymphoma functions and the underlying genes affected by RP6530 in HL cell lines.METHODS: We identified differentially expressed genes (DEGs) in two HL cell lines (L-540 and KM-H2) treated with RP6530 for 6 and 24 h by next generation RNA sequencing (RNA-Seq). The expression level of some of these genes was verified in HL cell lines using real time-PCR. Ingenuity Pathway Analysis (IPA) software was used to assign the DEGs to distinct canonical pathways and to predict the biological functions of the transcriptional changes. Gene silencing experiments were used to investigate and validate potential predictive biomarkers of response to RP6530 treatment. Additionally, serum levels of Thymus- and activation-regulated chemokine (TARC/CCL17) were analyzed by commercially available sandwich enzyme-linked immunosorbent assay (ELISA) in RP6530-treated HL patients enrolled in a phase 1 study.RESULTS: RNA-Seq analysis following treatment of L-540 cells for 6 h caused an upregulation of 1544 and downregulation of 839 genes. After a 24 h treatment period, 625 genes remained upregulated while the number of genes that were downregulated increased to 1362 (FDR ≤ 0.1). In KM-H2 cells, treatment with RP6530 for 6 or 24 h upregulated 137 and 980 genes while downregulating 139 and 833 genes, respectively (FDR ≤ 0.1). We further classified modulated genes according to their canonical pathways using the IPA suite. The most significantly upregulated pathways detected in both L-540 and KM-H2 cell lines included apoptosis, p53, death receptor signaling and G1/S cell cycle checkpoint regulation. In contrast, PI3K/AKT, ERK/MAPK, JAK/Stat, CD40, IL-4, IL-6, IL-10 and iNOS signaling were downregulated. We also identified 118 downregulated genes shared between the two cell lines that were associated with glycolysis, HIF1, chemokine and cytokine signaling. Upon RP6530 incubation, RNA-seq analysis showed a strikingly reduced expression of Pyruvate kinase muscle isozyme M2 (PKM2), which is a limiting glycolytic enzyme that catalyzes the final step in glycolysis. Here we show that lactic acid produced by tumor cells, as a by-product of glycolysis, has a critical function in inducing the M2-like polarization of tumor-associated macrophages (TAMs). Furthermore, we demonstrated that this effect was mediated by PKM2 and was inhibited after RP6530 treatment.We also found that the lactate-induced expression of M2 markers by macrophages, i.e., CCL-17 and CCL-22, were markedly inhibited both by RP6530 [-30% and -50% (mean of HL cells), respectively] and PKM2 silencing [-50% and -30% (mean of HL cells), respectively]. However, RP6530 increased the expression of M1 markers, switching the activation of macrophages from an immunosuppressive M2-like phenotype to an inflammatory M1-like state. PKM2 pathway therefore appears to be a critical determinant of RP6530-mediated inhibition of M2 macrophage polarization. Finally, RP6530 reduced the expression of key inflammation- and immunity-related genes as well as cytokines/chemokines and angiogenic factors (including, TARC/CCL17, CCL22, CCL5, IL-7, IL-13, and VEGF) known to be secreted by HRS cells to promote a pro-tumoral microenvironment. As the chemokine TARC/CCL17 is considered a biomarker of disease activity in HL patients, we observed a severe reduction of serum TARC/CCL17 levels in RP6530-treated HL patients that achieved CR or PR, but not in PD patients. Collectively, these findings suggest that RP6530 affects a mechanism of communication between HL tumor cells and tumor microenvironment.CONCLUSIONS: Results demonstrate that RP6530 can affect both HL tumor cells and HL tumor microenvironment, suggesting that a novel therapeutic opportunity may be achievable for the treatment of HL patients. DisclosuresViswanadha:Incozen Therapeutics: Employment. Vakkalanka:Rhizen Pharmaceuticals S A: Employment, Equity Ownership. Santoro:Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees. Carlo-Stella:Boehringer Ingelheim: Consultancy, Honoraria; Rhizen Pharmaceuticals: Research Funding; ADC Therapeutics: Research Funding.

Cell-specific and divergent roles of the CD40L-CD40 axis in atherosclerotic vascular disease

Atherosclerosis is a major underlying cause of cardiovascular disease. Previous studies showed that inhibition of the co-stimulatory CD40 ligand (CD40L)-CD40 signaling axis profoundly attenuates atherosclerosis. As CD40L exerts multiple functions depending on the cell-cell interactions involved, we sought to investigate the function of the most relevant CD40L-expressing cell types in atherosclerosis: T cells and platelets. Atherosclerosis-prone mice with a CD40L-deficiency in CD4+ T cells display impaired Th1 polarization, as reflected by reduced interferon-γ production, and smaller atherosclerotic plaques containing fewer T-cells, smaller necrotic cores, an increased number of smooth muscle cells and thicker fibrous caps. Mice with a corresponding CD40-deficiency in CD11c+ dendritic cells phenocopy these findings, suggesting that the T cell-dendritic cell CD40L-CD40 axis is crucial in atherogenesis. Accordingly, sCD40L/sCD40 and interferon-γ concentrations in carotid plaques and plasma are positively correlated in patients with cerebrovascular disease. Platelet-specific deficiency of CD40L does not affect atherogenesis but ameliorates atherothrombosis. Our results establish divergent and cell-specific roles of CD40L-CD40 in atherosclerosis, which has implications for therapeutic strategies targeting this pathway.

Dynamic regulation of B cell complement signaling is integral to germinal center responses.

B cell maturation within germinal centers (GCs) generates diversified B cell pools and high-affinity B cell antigen receptors (BCRs) for pathogen clearance. Increased receptor affinity is achieved by iterative cycles of T cell-dependent, affinity-based B cell positive selection and clonal expansion by incompletely understood mechanisms. Here, we found that as part of a physiologic program, GC B cells repressed expression of decay-accelerating factor (DAF/CD55) and other complement C3-convertase regulators via Bcl-6, but increased C5b-9 inhibitor (CD59) expression. These changes permitted C3 cleavage on GC B cell surfaces, without membrane attack complex formation, and activated C3a-receptor and C5a-receptor signals required for positive selection. Genetic disruption of this pathway in antigen-activated B cells, by conditional transgenic DAF overexpression or deletion of C3a and C5a receptors, limited mTOR activity in response to BCR-CD40 signaling, causing premature GC collapse and impaired affinity maturation. These results reveal that coordinated shifts in complement regulation within the GC provide crucial signals underlying GC B cell positive selection.

Conditioning treatment with CD27 Ab enhances expansion and antitumor activity of adoptively transferred T cells in mice

Cyclophosphamide plus fludarabine (C/F) are currently used to improve the expansion and effectiveness of adoptive cell therapy (ACT). However, these chemotherapeutics cause pan-leukopenia and adverse events, suggesting that safer and more effective conditioning treatments are needed to improve ACT outcomes. Previously, we reported that varlilumab, a CD27-targeting antibody, mediates Treg -preferential T cell depletion, CD8-T cell dominant costimulation, and systemic immune activation in hCD27 transgenic mice and cancer patients. We reasoned that the activities induced by varlilumab may provide an effective conditioning regimen for ACT. Varlilumab pretreatment of hCD27+/+mCD27 − /− mice resulted in prominent proliferation of transferred T cells isolated from wild-type mice. These studies uncovered a critical role for CD27 signaling for the expansion of transferred T cells, as transfer of T cells from CD27 deficient mice or treatment with a CD70 blocking antibody greatly reduced their proliferation. In this model, varlilumab depletes endogenous hCD27+/+ T cells and blocks their subsequent access to CD70, allowing for more CD70 costimulation available to the mCD27+/+ transferred T cells. CD27-targeted depletion led to a greater expansion of transferred T cells compared to C/F conditioning and resulted in longer median survival and more cures than C/F conditioning in the E.G7 tumor model receiving OT-I cell therapy. We propose that translation of this work could be achieved through engineering of T cells for ACT to abrogate varlilumab binding but preserve CD70 ligation. Thus, varlilumab could be an option to chemotherapy as a conditioning regimen for ACT.

A phase 1 dose-escalation study of a PD-L1xCD27 bispecific antibody CDX-527 in patients with advanced malignancies.

2585 Background: CDX-527 is a bispecific antibody (BsAb) targeting PD-L1 and CD27 that is designed to block immune checkpoint PD-L1/PD-1 interactions while providing immune costimulation through CD27 signaling. CD27 is a key immunostimulatory molecule that enhances T cell activation, effector function, and survival. Combining anti-PD-L1 and anti-CD27 mAbs synergize in preclinical studies, activating complementary cytotoxic and proliferative gene expression profiles, respectively. Clinical studies demonstrated the safety and biological activity of combining varlilumab, an agonist anti-CD27 mAb, with nivolumab or atezolizumab, along with modest clinical activity of the combinations. CDX-527 is a novel human BsAb containing a neutralizing, high affinity IgG1k PD-L1 mAb and the single chain Fv fragment (scFv) of an agonist anti-CD27 mAb genetically attached to the C-terminus of each heavy chain, thereby making CDX-527 bivalent for each target. Pre-clinical studies demonstrated enhanced T cell activation by CDX-527 and anti-tumor activity of a surrogate bispecific compared to individual mAb combinations. Methods: CDX527-01 is a phase 1 first-in-human, open-label, multi-center, dose-escalation (DE) and expansion study evaluating safety, pharmacokinetics (PK), pharmacodynamics (PD), and clinical activity of CDX-527 in patient with advanced solid tumors that have progressed on standard-of-care therapy. The primary study objective is to characterize the safety and tolerability of CDX-527. CDX-527 is administered intravenously Q2W with doses ranging from 0.03 mg/kg up to 10.0 mg/kg or until the maximum tolerated dose. The first 2 cohorts of the DE phase initiate with single patients and subsequent DE cohorts will be conducted in 3+3 manner. Tumor-specific expansion cohorts may be enrolled to further characterize the safety, PK, PD, and efficacy of CDX-527. Tumor assessments are performed Q8W by the investigator per iRECIST. Biomarker assessments include characterizing the effects on peripheral blood immune cells and cytokines, and for the expansion cohorts, the impact of CDX-527 on the tumor microenvironment in paired tumor biopsies. Results: To date, 8 patients have received CDX-527 in doses ranging from 0.03 mg/kg to 1 mg/kg and 3 are still on treatment. There has been no drug related SAEs, DLTs or discontinuations due to an AE. Most common treatment related AEs were influenza-like illness, fatigue, and arthralgia (all at 25%). All drug related AEs have been grade 1 or 2. Conclusions: Preliminary results indicate that the novel anti-PD-L1xCD27 bispecific antibody CDX-527 up to and including the 1 mg/kg dose level has been well tolerated. Additional data will be presented, including the safety profile at higher dose levels along with clinical activity, as well as PK and PD data. Clinical trial information: NCT04440943.

The Intracellular Domain of CD40 is a Potent Costimulatory Element in Chimeric Antigen Receptors.

The costimulatory domains incorporated into second-generation and third-generation chimeric antigen receptors (CARs) strongly influence CAR-T-cell function. Here, we explored second-generation and third-generation CARs harboring the signaling domain of the CD40 receptor as a new costimulatory element in comparison with similar CARs carrying the 4-1BB domain. In CARs of both generations, CD40 was more potent than 4-1BB in triggering the NF-κB signaling pathway. In human T cells from 2 donors, CD40 was comparable to 4-1BB in upregulating costimulatory and activation markers, inducing proinflammatory cytokine secretion and mediating target cell killing. Interestingly, differences in the response pattern of T cells from the 2 donors with respect to CD40 and 4-1BB were evident. We conclude that in human T cells, the CD40 signaling domain is a potent costimulatory element in both second-generation and third-generation CARs.

CD137 Signaling Is Critical in Fungal Clearance during Systemic Candida albicans Infection.

Invasive fungal infections by Candida albicans frequently cause mortality in immunocompromised patients. Neutrophils are particularly important for fungal clearance during systemic C. albican infection, yet little has been known regarding which surface receptor controls neutrophils’ antifungal activities. CD137, which is encoded by Tnfrsf9, belongs to the tumor necrosis receptor superfamily and has been shown to regulate neutrophils in Gram-positive bacterial infection. Here, we used genetic and immunological tools to probe the involvement of neutrophil CD137 signaling in innate defense mechanisms against systemic C. albicans infection. We first found that Tnfrsf9−/− mice were susceptible to C. albicans infection, whereas injection of anti-CD137 agonistic antibody protected the host from infection, suggesting that CD137 signaling is indispensable for innate immunity against C. albicans infection. Priming of isolated neutrophils with anti-CD137 antibody promoted their phagocytic and fungicidal activities through phospholipase C. In addition, injection of anti-CD137 antibody significantly augmented restriction of fungal growth in Tnfrsf9−/− mice that received wild-type (WT) neutrophils. In conclusion, our results demonstrate that CD137 signaling contributes to defense mechanisms against systemic C. albicans infection by promoting rapid fungal clearance.

Activation of CD137 signaling promotes macrophage apoptosis dependent on p38 MAPK pathway-mediated mitochondrial fission.

CD137 signaling is an essential factor in cell fate and atherosclerosis. An increase in the number of apoptotic macrophages accelerates atherosclerotic development involving mitochondrial dynamics.However, the role of CD137 signaling in macrophage apoptosis and changes in mitochondria has not been demonstrated clearly. Here, we used ApoE-/- mice as a model of atherosclerotic plaques. Mouse agonist anti-CD137 L and inhibitory anti-CD137 antibody were used to activate or block the CD137 signaling, respectively. Treatment of ApoE-/- mice with agonist anti-CD137 L promoted the formation of necrotic cores and macrophage apoptosis in plaques. Further, activation of CD137 signaling caused macrophage apoptosis in vitro, with upregulation of caspase-9 and caspase-3 expression and an increase in the Bax/Bcl-2 ratio. Meanwhile, CD137 signaling promoted mitochondrial fission observed by mitochondrial fragmentation. Interestingly, inhibition of mitochondrial dynamin-related protein 1 (Drp1) using Mdivi-1 reduced the expression of pro-apoptotic proteins and the amounts of apoptotic macrophages induced by CD137 signaling. Finally, we also found that the p38 MAPK pathway activated by CD137 signaling increased the expression of Drp1 expression and number of mitochondrial fragmented structures. Inhibition of the p38 MAPK pathway by SB203580 attenuated mitochondrial dysfunction through reducing mitochondrial membrane potential loss, cytochrome c release, and mitochondrial reactive oxygen species (ROS) generation. Overall, we identify a novel mechanism whereby CD137 signaling induces macrophage apoptosis through promoting mitochondrial fission dependent on the p38 MAPK pathway.

MiR-29 modulates CD40 signaling in chronic lymphocytic leukemia by targeting TRAF4: an axis affected by BCR inhibitors

B-cell receptor (BCR) signaling and T-cell interactions play a pivotal role in chronic lymphocytic leukemia (CLL) pathogenesis and disease aggressiveness. CLL cells can use microRNAs (miRNAs) and their targets to modulate microenvironmental interactions in the lymph node niches. To identify miRNA expression changes in the CLL microenvironment, we performed complex profiling of short noncoding RNAs in this context by comparing CXCR4/CD5 intraclonal cell subpopulations (CXCR4dimCD5bright vs CXCR4brightCD5dim cells). This identified dozens of differentially expressed miRNAs, including several that have previously been shown to modulate BCR signaling (miR-155, miR-150, and miR-22) but also other candidates for a role in microenvironmental interactions. Notably, all 3 miR-29 family members (miR-29a, miR-29b, miR-29c) were consistently down-modulated in the immune niches, and lower miR-29(a/b/c) levels associated with an increased relative responsiveness of CLL cells to BCR ligation and significantly shorter overall survival of CLL patients. We identified tumor necrosis factor receptor–associated factor 4 (TRAF4) as a novel direct target of miR-29s and revealed that higher TRAF4 levels increase CLL responsiveness to CD40 activation and downstream nuclear factor-κB (NF-κB) signaling. In CLL, BCR represses miR-29 expression via MYC, allowing for concurrent TRAF4 upregulation and stronger CD40–NF-κB signaling. This regulatory loop is disrupted by BCR inhibitors (bruton tyrosine kinase [BTK] inhibitor ibrutinib or phosphatidylinositol 3-kinase [PI3K] inhibitor idelalisib). In summary, we showed for the first time that a miRNA-dependent mechanism acts to activate CD40 signaling/T-cell interactions in a CLL microenvironment and described a novel miR-29–TRAF4–CD40 signaling axis modulated by BCR activity.

CD40 intracellular TRAF binding domains modulate B cell activation and experimental autoimmune encephalomyelitis development

Abstract CD40 is a costimulatory surface protein expressed on multiple immune cells, including B cells, where it is required for their full activation. Upon interacting with CD40 ligand, CD40 initiates downstream signaling that culminates in activation of naïve B cells. Previous work from our lab revealed that cell autonomous expression of CD40 on B cells regulates their activity, including germinal center development and antibody class switching. Intracellular (IC) domains of the CD40 protein are crucial for signaling. Several of these domains are known to be associated with TNF receptor associated factor proteins (TRAFs) which facilitate further downstream signaling. These include a TRAF6 binding domain and a TRAF2/3 binding domain. We analyzed the CD40 IC domain requirements for CD40 signaling in B cells and for induction of experimental autoimmune encephalomyelitis (EAE). To do so, multiple mouse strains were generated with elimination of the putative TRAF6 or TRAF2/3 domains by CRISPR technology. Whereas eliminating the putative TRAF6 binding site (Domain 1) had little to no effect on B cell viability and in vitro activation by anti-CD40 antibody, eliminating the putative TRAF2/3 binding site (Domain 2) impaired both these functions. Similarly, generation of high affinity IgG in vivo was also dependent on Domain 2 but not Domain 1. In contrast, both Domain 1 and Domain 2 were required for full development of EAE. Interestingly, mixed BM chimeras revealed that the ameliorating effect of CD40 mutations on EAE was mediated by hematopoietic derived non-B cells. Our results indicate that the CD40 TRAF2/3 binding Domain 2 is critical for CD40 dependent B cell function, while both Domain 1 and Domain 2 expression by other APCs function in EAE induction.

CD40 signaling augments IL-10 expression and the tolerogenicity of IL-10-induced regulatory dendritic cells.

CD40 expressed on stimulatory dendritic cells (DC) provides an important accessory signal for induction of effector T cell responses. It is also expressed at lower levels on regulatory DC (DCreg), but there is little evidence that CD40 signaling contributes to the tolerogenic activity of these cells. Indeed, CD40 silencing within DCreg has been reported to induce T cell tolerance in multiple disease models, suggesting that CD40 is superfluous to DC-induced tolerance. We critically assessed whether CD40 does have a role in tolerance induced by IL-10-differentiated DC (DC10) by using DC10 generating from the bone marrow of wild-type (w.t.) or CD40-/- donor mice, or IL-10-complemented CD40-/- DC10 to treat asthmatic mice. Wild-type DC10 ablated the OVA-asthma phenotype via induction of Foxp3+ Treg responses, but CD40-/- DC10 had no discernible effects on primary facets of the phenotype (e.g., IL-5, IL-9, IL-13 levels, IgE & IgG1 antibodies; p>0.05) and were ≤40% effective in reversal of others. Foxp3+ T cells from the lungs of CD40-/- DC10-treated mice expressed reduced levels of a panel of six Treg-specific activation markers relative to Treg from w.t. DC10-treated mice. Coculture with effector T cells from asthmatic mice induced a marked upregulation of cell surface CD40 on w.t. DC10. While untreated CD40-/- and w.t. DC10 secreted equally low levels of IL-10, stimulation of w.t. DC10 with anti-CD40 for 72 h increased their expression of IL-10 by ≈250%, with no parallel induction of IL-12. Complementing IL-10 expression in CD40-/- DC10 by IL-10 mRNA transfection fully restored the cells’ abilities to suppress the asthma phenotype. In summary, CD40 signaling in DC10 contributes importantly to their expression of IL-10 and to a robust induction of tolerance, including activation of induced Treg.