CD40-CD154 Interaction Research Articles

Abstract PR002: Single-cell spatial analysis reveals microenvironmental features that contribute to immune discrepancies between adult and pediatric nasopharyngeal carcinomas

Abstract Background: Nasopharyngeal carcinoma (NPC) is an EBV-positive malignancy with high immune infiltrates but also high immunosuppressive activities. Pediatric NPC only constitutes 0.1-1% of the total NPC incidences and exhibits superior treatment responses and prognosis compared to adult NPC. Clinically, we consider that the differences in the tumor landscape between adult and pediatric NPC patients are the vital driver of such immune discrepancies. Therefore, we apply scRNA-seq and Visium spatial sequencing to primary pediatric NPC tissues and paired peripheral blood samples, and integrate our data with multiple independent adult NPC scRNA-seq cohorts. Furthermore, we establish a bioinformatics analysis pipeline that can calculate spatial cellular compositions and biological activities. Here, we demonstrate that low lipid metabolism, disrupted interactions between NPC cells and regulatory T cells (Tregs)/CD8+ T cells, and high infiltration of central stem memory T cells, contribute to more potent and enduring anti-tumor immunity in pediatric NPC patients, and these microenvironmental features might serve as new immunotherapeutic vulnerabilities. Method: 5' scRNA-seq coupled with TCR/BCR profiling was performed on 5 primary and treatment-naïve pediatric NPC tissues with 5 paired peripheral blood samples. Visium spatial-seq was performed on 4 paired pediatric and 7 adult NPC tissues. We incorporated our scRNA-seq cohort with three independent adult NPC cohorts. Our computational framework was integrated with existing analysis packages, including Seurat, Spacexr, and SpaCET, to analyze single-cell-referenced spatial data. The key results in our study were validated by multiplex IHC staining and flow cytometry on patient samples. Results: We established a multi-center NPC scRNA spatial cohort containing 503,021 cells from 69 samples, and 15,222 spatial spots from 11 samples. We developed a computational framework to calculate spatial co-localization and biological activities. We unveiled that lipid metabolism was elevated in the tumor-T cell core in adult NPC. Strong co-localizations between NPC cells and Tregs/CD8+ T cells were found in adult NPC, with spatially enriched CD70-CD27 and LGALS9-TIM3 interactions. This finding was corroborated by multiplex IHC staining of KRT19+ NPC cells, CD4+/FOXP3+ Tregs, and CD8+/PD-1/TIM-3+ exhausted T cells. We also identified and characterized novel IL7R+ central stem memory T cells in pediatric NPC tissues and blood, with strong resilience to immunosuppressive cues, including TGF-β, PEG2, and cyclic AMP, and could lead to enduring immunity and anti-PD-1/PD-L1 responses. Conclusion: We demonstrate that higher T-cell immunosuppression and exhaustion caused by aberrant lipid metabolism, tumor-T interactions, and a lower age-associated central stem memory T-cell pool, are vital drivers of immune discrepancies in adult and pediatric NPC patients. Thus, screening and targeting these molecular microenvironmental characteristics might help stratify immunotherapy responders and generate therapeutic benefits in NPC patients. Citation Format: Lanqi Gong, Yu Zhang, Grace Guan, Beibei Ru, Peng Jiang. Single-cell spatial analysis reveals microenvironmental features that contribute to immune discrepancies between adult and pediatric nasopharyngeal carcinomas [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr PR002.

A SINGLE‐CELL ATLAS OF DIFFUSE LARGE B CELL LYMPHOMA

Diffuse large B-cell lymphoma (DLBCL) is one of the most common yet aggressive types of B-cell lymphoma and remains incurable in 40% of patients. Herein, we profiled the transcriptomes of 94,324 cells from 17 DLBCLs and 3 control samples using single-cell RNA-sequencing technology, creating a comprehensive single-cell map for tumor and infiltrating immune cells for DLBCL. High degrees of inter- and intratumor heterogeneity were revealed, and we identified 73 gene expression programs expressed in malignant B cells. We further characterized 8 nonmalignant B-cell subclusters and 16 T-cell subclusters from the tumor microenvironment. Six myeloid subclusters were also identified, including the LAMP3+ DC subcluster enriched in ABC-DLBCL. More than 2000 likely cell-cell interactions were further predicted in DLBCL, illustrating a complex and highly dynamic DLBCL tumor microenvironment. Unique to B cell lymphomas, a strong costimulatory CD70-CD27 interaction was predicted between malignant cells and T cells. Furthermore, coinhibitory signals mediated by TIM-3 or TIGIT seemed to be the main driving force for T-cell exhaustion. Finally, we discovered that chronic hepatitis B virus infection may have a significant impact on tumor cell survival and immune evasion in DLBCL. Our results provide new insights into B-cell lymphomagenesis and may facilitate the design of targeted immunotherapy strategies. The research was funded by: Swedish Cancer Society, STINT, CIMED, Radiumhemmet, the Swedish Research Council, and the Knut and Alice Wallenberg Foundation, the Guangdong Enterprise Key Laboratory of Human Disease Genomics, the Shenzhen Key Laboratory of Single-Cell Omics Keywords: aggressive B-cell non-Hodgkin lymphoma, genomics, epigenomics, and other -omics, tumor biology and heterogeneity No conflicts of interests pertinent to the abstract.

Nasopharyngeal carcinoma cells promote regulatory T cell development and suppressive activity via CD70-CD27 interaction

Despite the intense CD8+ T-cell infiltration in the tumor microenvironment of nasopharyngeal carcinoma, anti-PD-1 immunotherapy shows an unsatisfactory response rate in clinical trials, hindered by immunosuppressive signals. To understand how microenvironmental characteristics alter immune homeostasis and limit immunotherapy efficacy in nasopharyngeal carcinoma, here we establish a multi-center single-cell cohort based on public data, containing 357,206 cells from 50 patient samples. We reveal that nasopharyngeal carcinoma cells enhance development and suppressive activity of regulatory T cells via CD70-CD27 interaction. CD70 blocking reverts Treg-mediated suppression and thus reinvigorate CD8+ T-cell immunity. Anti-CD70+ anti-PD-1 therapy is evaluated in xenograft-derived organoids and humanized mice, exhibiting an improved tumor-killing efficacy. Mechanistically, CD70 knockout inhibits a collective lipid signaling network in CD4+ naïve and regulatory T cells involving mitochondrial integrity, cholesterol homeostasis, and fatty acid metabolism. Furthermore, ATAC-Seq delineates that CD70 is transcriptionally upregulated by NFKB2 via an Epstein-Barr virus-dependent epigenetic modification. Our findings identify CD70+ nasopharyngeal carcinoma cells as a metabolic switch that enforces the lipid-driven development, functional specialization and homeostasis of Tregs, leading to immune evasion. This study also demonstrates that CD70 blockade can act synergistically with anti-PD-1 treatment to reinvigorate T-cell immunity against nasopharyngeal carcinoma.

Selected Articles from This Issue

PD-1T tumor-infiltrating lymphocytes (TIL) are a dysfunctional T-cell population characterized by high PD-1 expression and tumor reactivity. To evaluate their potential as a biomarker for durable clinical benefit to PD-1 blockade, Hummelink and colleagues digitally quantified PD-1T TIL in 120 pretreatment samples from patients with advanced non-small cell lung cancer. PD-1T TIL showed a high sensitivity in predicting clinical benefit and survival to anti-PD-1 and were superior to PD-L1 or TLS as a biomarker. Importantly, the absence of PD-1T TIL reliably identified a patient group without benefit from PD-1 blockade, thereby providing a promising tool to reduce overtreatment.Immune checkpoint inhibitors (ICI) targeting programmed cell death protein 1 and its ligand (PD-1/PD-L1) have revolutionized therapeutic options for renal cell carcinoma (RCC). However, response rates to ICI vary widely (20–70%) in patients with RCC. Predicting responses using conventional tissue-based techniques evaluating PD-L1 expression is of limited value, owing to the dynamic and heterogenous nature of PD-L1 expression. Mulgaonkar and colleagues systematically examined the performance of immunoPET for PD-L1 imaging relative to immunohistochemistry in an RCC tumorgraft platform. This study demonstrates the promising potential of immunoPET for clinical real-time assessment of PD-L1 expression across tumor sites and heterogeneous intratumoral distribution.CSF1R inhibitor treatments have shown to be effective targeted therapies for TGCT. Trials are currently underway to study the effect of discontinuation and retreatment with CSF1R inhibitors. In this study, van IJzendoorn and colleagues investigate the tumor landscape in TGCT using single-cell sequencing and other molecular techniques. They show that the neoplastic cells are not targeted by CSF1R inhibitors, in contrast to what was previously postulated. The authors identify markers for the neoplastic cells and show the lack of an autocrine loop involving CSF1-CSF1R in the neoplastic cells. As a result, CSF1R inhibitor treatments are unlikely to affect the neoplastic cell population in TGCT as is currently assumed. Additionally, this work identifies two new pathways that could be targeted in future trials involving TGCT.The resistance of immunotherapy in renal cancer patients remains unclear despite the high infiltrations of CD8+T cells. Here, Benhamouda and colleagues show that baseline plasma soluble CD27 (sCD27) is associated with resistance to immunotherapy in renal cell carcinoma independently of clinical factors. sCD27 reflects the interaction between CD27 expressed by T lymphocytes and CD70 expressed by tumor cells, which associates with the apoptosis and dysfunction of T lymphocytes. Peripheral blood sCD27 could thus be considered as a marker of T-cell dysfunction due to the CD27-CD70 interaction in the tumor and predict the response to immunotherapy.

Plasma CD27, a Surrogate of the Intratumoral CD27-CD70 Interaction, Correlates with Immunotherapy Resistance in Renal Cell Carcinoma.

CD70 is a costimulatory molecule known to activate CD27-expressing T cells. CD27-CD70 interaction leads to the release of soluble CD27 (sCD27). Clear-cell renal cell carcinoma (ccRCC) expresses the highest levels of CD70 among all solid tumors; however, the clinical consequences of CD70 expression remain unclear. Tumor tissue from 25 patients with ccRCC was assessed for the expression of CD27 and CD70 in situ using multiplex immunofluorescence. CD27+ T-cell phenotypes in tumors were analyzed by flow cytometry and their gene expression profile were analyzed by single-cell RNA sequencing then confirmed with public data. Baseline sCD27 was measured in 81 patients with renal cell carcinoma (RCC) treated with immunotherapy (35 for training cohort and 46 for validation cohort). In the tumor microenvironment, CD27+ T cells interacted with CD70-expressing tumor cells. Compared with CD27- T cells, CD27+ T cells exhibited an apoptotic and dysfunctional signature. In patients with RCC, the intratumoral CD27-CD70 interaction was significantly correlated with the plasma sCD27 concentration. High sCD27 levels predicted poor overall survival in patients with RCC treated with anti-programmed cell death protein 1 in both the training and validation cohorts but not in patients treated with antiangiogenic therapy. In conclusion, we demonstrated that sCD27, a surrogate marker of T-cell dysfunction, is a predictive biomarker of resistance to immunotherapy in RCC. Given the frequent expression of CD70 and CD27 in solid tumors, our findings may be extended to other tumors.

Abstract 6127: Single-cell spatial transcriptome sequencing characterizes CD70+ immune-resilient nasopharyngeal carcinoma cells as a vital mediator of Treg-induced suppression

Abstract Background: The microenvironment of nasopharyngeal carcinoma (NPC) has an intense and heterogenous immune infiltration, due to chronic EBV infection and locoregional lymphoid structures. Phase II clinical trials have shown that the response rate of NPC patients to PD-1 inhibitors is only 20%, indicating the current immunotherapies are not optimal due to insufficient understanding of the tumor-immune interaction. In this study, we have applied an integrated transcriptome sequencing analysis to decipher immune dynamics, and have characterized a new target that can inhibit Treg-mediated suppression in NPC, and provided critical translational evidence to the precision immunotherapy. Methods: We established an integrated NPC single-cell sequencing cohort containing 189,750 T cells from 50 patient samples. Spatial transcriptome sequencing was performed on frozen samples from eight NPC patients. The whole blood was collected from healthy donors and PBMCs were isolated using density gradient centrifugation. T cells were magnetically isolated from PBMCs. The humanized mouse model was established by tail-vein injection of CD3/CD28 activated PBMCs. Results: Single-cell sequencing revealed the enrichment of suppressive Tregs in the NPC microenvironment. We found that the C666-1 cell line facilitated Treg differentiation and activation. Spatial sequencing identified CD70-CD27 interaction was upregulated between NPC cells and suppressive Tregs, indicating its role in regulating Treg function. CD70 was predominantly expressed on NPC cells and correlated to worse prognosis. CD70-knockout (KO) in C666-1 inhibited Treg differentiation. Decreased Treg activities further enhanced the cytotoxicity in CD8+ effector T cells, leading to higher tumor apoptosis. Cusatuzumab also generated a comparable inhibitory effect in Treg-mediated suppression. In the humanized mouse model, CD70-KO and inhibition shrank the size of subcutaneous tumors by enhancing T cell immunity. Conclusion: The enrichment and activation Tregs makes the NPC microenvironment highly immunosuppressive, which might jeopardize the clinical efficacy of anti-PD1 therapies in NPC patients. In this study, we revealed that CD70+ NPC cells were highly resilient to immune surveillance since they were capable of modulating Treg differentiation and activation via the CD70-CD27 interaction to counteract CD8+ T cells. Blocking CD70 has been shown to effectively induce tumor apoptosis by alleviating Treg-mediated suppression. The therapeutic efficacy and safety of the anti-CD70 therapy were tested in the humanized mouse model. These findings suggested that the anti-CD70 therapy exhibited a promising efficacy to kill NPC cells by deteriorating the immunosuppressive microenvironment, and it might synergistically enhance the efficacy of anti-PD1 therapies. Citation Format: Lanqi Gong, Jie Luo, Yuma Yang, Yu Zhang, Xin-Yuan Guan. Single-cell spatial transcriptome sequencing characterizes CD70+ immune-resilient nasopharyngeal carcinoma cells as a vital mediator of Treg-induced suppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6127.

A single-cell atlas of diffuse large B cell lymphoma.

Diffuse large B cell lymphoma (DLBCL) is one of the most common yet aggressive types of B cell lymphoma and remains incurable in 40% of patients. Herein, we profile the transcriptomes of 94,324 cells from 17 DLBCLs and 3 control samples using single-cell RNA sequencing. Altogether, 73 gene expression programs are identified in malignant cells, demonstrating high intra- and intertumor heterogeneity. Furthermore, 2,754 pairs of suggestive cell-cell interactions are predicted, indicating a complex and highly dynamic tumor microenvironment. Especially for B cell lymphomas, a strong costimulatory CD70-CD27 interaction is predicted between malignant and Tcells. Furthermore, coinhibitory signals mediated by TIM3 or TIGIT seem to be the main driving force for Tcell exhaustion. Finally, we find that chronic hepatitis B virus infection may have a significant impact on tumor cell survival and immune evasion in DLBCL. Our results provide insights into B cell lymphomagenesis and may facilitate the design of targeted immunotherapy strategies.

Anti-C1q autoantibodies from systemic lupus erythematosus patients enhance CD40-CD154-mediated inflammation in peripheral blood mononuclear cells in vitro.

ObjectivesSystemic lupus erythematosus (SLE) is a clinically heterogeneous autoimmune disease with complex pathogenic mechanisms. Complement C1q has been shown to play a major role in SLE, and autoantibodies against C1q (anti‐C1q) are strongly associated with SLE disease activity and severe lupus nephritis suggesting a pathogenic role for anti‐C1q. Whereas C1q alone has anti‐inflammatory effects on human monocytes and macrophages, C1q/anti‐C1q complexes favor a pro‐inflammatory phenotype. This study aimed to elucidate the inflammatory effects of anti‐C1q on peripheral blood mononuclear cells (PBMCs).MethodsIsolated monocytes, isolated T cells and bulk PBMCs of healthy donors with or without concomitant T cell activation were exposed to C1q or complexes of C1q and SLE patient‐derived anti‐C1q (C1q/anti‐C1q). Functional consequences of C1q/anti‐C1q on cells were assessed by determining cytokine secretion, monocyte surface marker expression, T cell activation and proliferation.ResultsExposure of isolated T cells to C1q or C1q/anti‐C1q did not affect their activation and proliferation. However, unspecific T cell activation in PBMCs in the presence of C1q/anti‐C1q resulted in increased TNF, IFN‐γ and IL‐10 secretion compared with C1q alone. Co‐culture and inhibition experiments showed that the inflammatory effect of C1q/anti‐C1q on PBMCs was due to a direct CD40–CD154 interaction between activated T cells and C1q/anti‐C1q‐primed monocytes. The CD40‐mediated inflammatory reaction of monocytes involves TRAF6 and JAK3‐STAT5 signalling.ConclusionIn conclusion, C1q/anti‐C1q have a pro‐inflammatory effect on monocytes that depends on T cell activation and CD40–CD154 signalling. This signalling pathway could serve as a therapeutic target for anti‐C1q‐mediated inflammation.

Functional role of CD40 and CD154 costimulatory signals in IgZ-mediated immunity against bacterial infection

CD40 and CD154 are one of the best-characterized costimulatory molecules essential for adaptive immunity, which extensively involved in T and B cell activation, IgM Ab production, isotype class switching, germinal center formation and affinity maturation. However, the functionality of CD40 and CD154 in IgZ-mediated immunity remains limited. In this study, we explored the regulatory role of Cd40-Cd154 interaction in IgZ-mediated antibacterial immunity in zebrafish. The results showed that the IgZ-mediated antibacterial response can be significantly induced in response to A. hydrophila infection. The percentage of Cd40+IgZ+ B cells and the production of IgZ Ab were substantially increased upon A. hydrophila stimulation, but these reactions were markedly declined in Cd154 blockade fish by administering anti-Cd154 Ab or recombinant sCd40-Ig protein, accompanied with the impairment of the vaccine-initiated IgZ-mediated immunoprotection of fish against A. hydrophila infection. These observations suggested the essential role of Cd40-Cd154 interaction in IgZ-mediated bacterial immunity. Notably, the Cd40 and Cd154 costimulatory signals are required for a TD antigen-induced IgZ immunity, but are not indispensable for a TI antigen-induced IgZ immune response. These findings indicated the differential role of Cd40-Cd154 interaction in bacterial TD and TI antigen-induced IgZ immunity, which suggested the existence of diverse regulatory mechanisms underlying IgZ-mediated antibacterial immune reactions. To our knowledge, this is the first report to show the functional role of Cd40-Cd154 costimulatory signaling pathway in IgZ-mediated immune defense against bacterial infection. We hope this study will improve the current understanding of the coevolution between the IgZ/IgT immunoglobins and CD40/CD154 costimulatory molecules.

Potential of Sea-CD70 for the Treatment of Myeloid Leukemia

CD70 (CD27L) is a member of the tumor necrosis factor superfamily transiently expressed on activated adaptive and innate immune cells. Engagement of CD70 to the co-stimulatory receptor CD27, which is present on adaptive immune cells, promotes antigen-specific T cell responses. Both CD70 and CD27 have been reported to be aberrantly expressed on AML and MDS blasts, but are absent on normal hematopoietic progenitor cells, making them appealing therapeutic targets. CD70-CD27 interactions, working through auto- and/or paracrine signaling, can induce a stem-like signature and stimulate the proliferation of leukemic blasts. In addition, the CD70-CD27 interaction drives inflammatory cytokine production from both CD27 and CD70 pathways, resulting in cytokine production from leukemic blasts and the myeloid cells. Inflammatory signaling is a critical component of the crosstalk between hematopoietic stem and progenitor cells and their microenvironment during infection or hematopoietic stress. However, chronic inflammation can suppress normal hematopoietic stem cell function and eventually lead to bone marrow failure. Similarly, preleukemic and leukemic cell' interactions with the bone marrow microenvironment can induce secretion of pro-inflammatory factors, which in turn, could suppress normal blood cell formation. Therefore, blockade of the CD70-CD27 inflammatory signal pathway could help restore normal hematopoiesis, limit immune evasion, and increase the immune directed responses in MDS and AML. SEA-CD70 is a humanized, nonfucosylated monoclonal antibody targeting CD70. SEA-CD70 retains the ability to block CD27 mediated signaling and binds significantly better than the fucosylated parent to the FcγRIIIa receptor. SEA-CD70 mediates potent target killing through Fc-dependent antibody effector functions, including antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). SEA-CD70 can also disrupt CD70-CD27 signaling and could block cellular proliferation and survival of blast cells in vitro. SEA-CD70 may also inhibit the inflammatory signals originating from the interaction between the tumor microenvironment and leukemic blasts. In vivo, SEA-CD70 delayed tumor growth and increased survival in subcutaneous AML xenograft mouse tumor models both as a single agent and when combined with standard of care (SOC) agents. Overall, our data highlight the therapeutic potential of SEA-CD70 for the treatment of AML and MDS patients. Disclosures Diolaiti: Seattle Genetics:Current Employment, Current equity holder in publicly-traded company.Haass:Seattle Genetics:Current Employment, Current equity holder in publicly-traded company.Ulrich:Seattle Genetics:Current Employment, Current equity holder in publicly-traded company.Chen:Seattlke Genetics:Current Employment, Current equity holder in publicly-traded company.Ho:Seattle Genetics:Current Employment, Current equity holder in publicly-traded company.Gardai:Seattle genetics:Current Employment, Current equity holder in publicly-traded company.

Injection of CD40 DNA vaccine ameliorates the autoimmune pathology of non-obese diabetic mice with Sjögren’s syndrome

BackgroundOverexpression of CD40 has been reported in patients with primary Sjögren’s syndrome (pSS). The increased CD40 expression promote autoimmune response and enhance inflammation in pSS. The aim of this study is to block CD40-CD154 interaction with CD40 DNA vaccine to slow the disease progression of SS in non-obese diabetic (NOD) mice. MethodsFemale NOD mice were treated with CD40 DNA vaccine, empty vector and normal saline. The salivary flow rate was measured, whereas lymphocytes infiltration in the salivary glands was assessed by histopathology. Expression of CD40 and B220 in salivary were examined by immunohistochemistry. Splenic lymphocyte phenotypes were analyzed by flow cytometry. CD40, IL-1β, TNF-α and IL-6 levels in the salivary glands were detected by PCR. Serum anti-CD40 antibody was measured by ELISA. Serum anti-nuclear antibody (ANA) was monitored by immunofluorescence. ResultsNOD mice treated with CD40 DNA vaccine showed higher levels of anti-CD40 antibody compared with the controls. The expression of CD40 in the salivary glands of NOD mice in CD40 DNA vaccine group was decreased. The infiltration of lymphocytes was reduced in the salivary glands and saliva secretion was increased in the treatment group. The expression level of TNF-α and IL-6 in salivary glands were declined. The splenic dendritic cell and plasma cell populations were reduced and the level of ANA was decreased in NOD mice with CD40 DNA vaccine treatment. ConclusionsCD40 DNA vaccine inhibits the immune response and reduce inflammation in epithelial tissues SS in non-obese diabetic (NOD) mice, suggesting that CD40 DNA vaccine could be a new therapeutic approach in treatment of pSS.

CD70 expression determines the therapeutic efficacy of expanded human regulatory T cells

Regulatory T cells (Tregs) are critical mediators of immune homeostasis. The co-stimulatory molecule CD27 is a marker of highly suppressive Tregs, although the role of the CD27-CD70 receptor-ligand interaction in Tregs is not clear. Here we show that after prolonged in vitro stimulation, a significant proportion of human Tregs gain stable CD70 expression while losing CD27. The expression of CD70 in expanded Tregs is associated with a profound loss of regulatory function and an unusual ability to provide CD70-directed co-stimulation to TCR-activated conventional T cells. Genetic deletion of CD70 or its blockade prevents Tregs from delivering this co-stimulatory signal, thus maintaining their regulatory activity. High resolution targeted single-cell RNA sequencing of human peripheral blood confirms the presence of CD27−CD70+ Treg cells. These findings have important implications for Treg-based clinical studies where cells are expanded over extended periods in order to achieve sufficient treatment doses.

CD11b is a novel alternate receptor for CD154 during alloimmunity.

Antagonism of the CD154/CD40 pathway is a highly effective means of inducing long-term graft survival in preclinical models. Using a fully allogeneic murine transplant model, we found that CD154 blockade was more effective in prolonging graft survival than was CD40 blockade, raising the possibility that CD154 binds a second receptor. To test this, we queried the impact of CD154 antagonism in the absence of CD40. Data indicated that anti-CD154 functioned to reduce graft-infiltrating CD8+ T cells in both WT and CD40-/- hosts. Because it has recently been reported that CD154 can ligate CD11b, we addressed the impact of blocking CD154-CD11b interactions during transplantation. We utilized a specific peptide antagonist that prevents CD154 binding of CD11b but has no effect on CD154-CD40 interactions. CD154:CD11b antagonism significantly increased the efficacy of anti-CD40 in prolonging allograft survival as compared to anti-CD40 plus control peptide. Mechanistically, CD154:CD11b antagonism functioned to reduce the frequency of graft-infiltrating CD8+ T cells and innate immune cells. These data therefore demonstrate that blocking CD154 interactions with both CD40 and CD11b is required for optimal inhibition of alloimmunity and provide an explanation for why CD40 blockers may be less efficacious than anti-CD154 reagents for the inhibition of allograft rejection.

CD70-mediated CD27 expression downregulation contributed to the regulatory B10 cell impairment in rheumatoid arthritis

Regulatory B10 cells have been shown to exhibit impaired functions in autoimmune diseases. However, the underlying mechanism is still obscure. In the present study, we aimed to understand the regulatory characteristics of regulatory B10 cells and how these cells are involved in the development of rheumatoid arthritis (RA). Here, we chose CD19+CD24hiCD27+ as the phenotype of regulatory B10 cells. We found that the frequencies of CD19+CD24hiCD27+ regulatory B10 cells were decreased and that their IL-10-producing function was impaired in patients with RA compared with healthy controls (HCs). The impairment in CD19+CD24hiCD27+ B10 cells was partially attributed to the decreased expression of CD27 induced by the upregulated CD70 expression on CD19 + B cells and CD4 + T cells. The proportion of CD19+CD24hiCD27+ regulatory B10 cells could be restored by blocking the CD70-CD27 interaction with an anti-CD70 antibody. Furthermore, the CD70-CD27 interaction significantly elevated IL-10 expression and might compensate for the decreased number of CD19+CD24hiCD27+ B cells. Hence, the CD70-CD27 interaction might play a critical role in the numerical and functional impairments of regulatory B10 cells, thus contributing to RA pathogenesis. In conclusion, the change in CD19+CD24hiCD27+ regulatory B10 cells in RA was only a consequence, not the cause, of RA development, but the increased expression of CD70 might be the culprit.

Assessment of the anti-CD40 antibody iscalimab in patients with primary Sjögren's syndrome: a multicentre, randomised, double-blind, placebo-controlled, proof-of-concept study.

Primary Sjögren's syndrome is an autoimmune disease that presents as dryness of the mouth and eyes due to impairment of the exocrine glands. To our knowledge, no systemic therapies for primary Sjögren's syndrome have shown efficacy. CD40-CD154-mediated T cell-B cell interactions in primary Sjögren's syndrome contribute to aberrant lymphocyte activation in inflamed tissue, leading to sialadenitis and other tissue injury. Therefore, we investigated the safety and preliminary efficacy of iscalimab (CFZ533), a novel anti-CD40 monoclonal antibody, in patients with primary Sjögren's syndrome. This multicentre, randomised, double-blind, placebo-controlled, proof-of-concept study took place at ten investigational sites across Europe (UK, n=4; Germany, Switzerland, and Hungary, n=1 each) and the USA (n=3). Eligible patients were aged 18-75 years and fulfilled the 2002 American European consensus group diagnostic classification criteria for primary Sjögren's syndrome. In the double-blind phase of the trial, patients were randomly assigned (2:1) via computer-generated unique randomisation numbers to receive subcutaneous iscalimab (3 mg/kg) or placebo at weeks 0, 2, 4, and 8 (cohort 1) or intravenous iscalimab (10 mg/kg) or placebo at weeks 0, 2, 4, and 8 (cohort 2). Randomisation was stratified according to baseline intake of oral corticosteroids. At week 12, patients in both cohorts received open-label iscalimab (same dose and route) for 12 weeks. The primary objectives of the study were to assess the safety, tolerability, and efficacy of multiple doses of iscalimab in the two sequential dose cohorts. Safety and tolerability were assessed by adverse events and efficacy of iscalimab versus placebo was assessed by clinical disease activity, as measured by the change in European League Against Rheumatism Sjögren's syndrome disease activity index (ESSDAI) score after 12 weeks of treatment. Analyses were done on a per-protocol basis. The trial was registered with ClinicalTrials.gov, NCT02291029. Between Oct 22, 2014, and June 28, 2016, we assessed 82 patients for eligibility (25 for cohort 1 and 57 for cohort 2). 38 patients were excluded because of ineligibility. In cohort 1, 12 patients were randomly assigned to receive either 3 mg/kg doses of iscalimab (n=8) or placebo (n=4), and in cohort 2, 32 patients were randomly assigned to receive either intravenous 10 mg/kg doses of iscalimab (n=21) or placebo (n=11). Adverse events were similar between iscalimab treatment groups and placebo groups, with adverse events occurring in all patients in cohort 1, and in 52% and 64% of the iscalimab and placebo groups, respectively, in cohort 2. Two serious adverse events were reported (one case of bacterial conjunctivitis in cohort 1 and one case of atrial fibrillation in cohort 2), which were unrelated to treatment with iscalimab. Intravenous treatment with iscalimab resulted in a mean reduction of 5·21 points (95% CI 0·96-9·46; one-sided p=0·0090) in ESSDAI score compared with placebo. There was no signficiant difference in ESSDAI score between subcutaneous iscalimab and placebo. To our knowledge, this is the first randomised, placebo-controlled proof-of-concept study of a new investigational drug for primary Sjögren's syndrome that indicates preliminary efficacy. Our data suggest a role of CD40-CD154 interactions in primary Sjögren's syndrome pathology and the therapeutic potential for CD40 blockade in this disease should be investigated further. Novartis Pharma.

Hyperglycemia Activates the CD27-CD70 Axis on Human Peripheral Blood Mononuclear Cells

Obesity is now strongly associated with chronic low-grade inflammation that, without intervention, contributes to the development of pre-diabetes and eventually Type 2 diabetes. While the exact mechanism that inflammation plays in the pathogenesis from obesity to Type 2 diabetes is unclear, activated immune cells and pro-inflammatory cytokines have been found in the adipose tissue of Type 2 diabetics, implicating their role in the disease process. The CD27-CD70 pathway provides a crucial inflammatory co-stimulatory signal, with CD70 being expressed on activated antigen presenting cells, and CD27 expressed on lymphocytes. While the CD27-CD70 axis is being explored in other models of chronic inflammation, such as rheumatoid arthritis and colitis, the role played in Type 2 diabetes remains unknown. Here we report the downregulation of CD27 on CD4 T cells when co-cultured with dendritic cells primed in increasing concentrations of glucose, indicating an effector phenotype of these T cells. Importantly, we also highlight that CD70 is concurrently upregulated on dendritic cells primed in high concentrations of glucose, resulting in increased production of IFN-γ and TNF-α by the CD70 expressing dendritic cells, when compared to dendritic cells primed in a lower concentration of glucose. These results reveal a novel role for CD27-CD70 interactions in the pathogenesis of Type 2 diabetes and provide support for future investigations into this pathway. Additionally, CD27 could be analyzed as a way to further stratify pre-diabetic patients and guide diagnosticians towards the most efficient therapy.

First-in-human clinical trial to assess pharmacokinetics, pharmacodynamics, safety, and tolerability of iscalimab, an anti-CD40 monoclonal antibody.

Iscalimab is a fully human, CD40 pathway blocking, nondepleting monoclonal antibody being developed as an immunosuppressive agent. We describe a first-in-human, randomized, double-blind, placebo-controlled study investigating the safety, tolerability, pharmacokinetics, and pharmacodynamics of iscalimab in healthy subjects and rheumatoid arthritis patients. Healthy subjects (n=56) received single doses of intravenous iscalimab (0.03, 0.1, 0.3, 1, or 3mg/kg), or subcutaneous iscalimab (3mg/kg), or placebo. Rheumatoid arthritis patients (n=20) received single doses of intravenous iscalimab (10 or 30mg/kg) or placebo. Iscalimab exhibited target-mediated drug disposition resulting in dose-dependent and nonlinear pharmacokinetics. Complete (≥90%) CD40 receptor occupancy on whole blood B cells was observed at plasma concentrations >0.3-0.4µg/mL. In subjects receiving 3mg/kg iscalimab, antibody responses to keyhole limpet hemocyanin were transiently suppressed. CD40 occupancy by iscalimab prevented ex vivo human rCD154-induced expression of CD69 on B cells in whole blood. All doses were generally safe and well tolerated, with no clinically relevant changes in any safety parameters, including no evidence of thromboembolic events. Iscalimab appears to be a promising blocker of the CD40-CD154 costimulatory pathway with potential use in transplantation and other autoimmune diseases.

Identification and Characterization of Post-activated B Cells in Systemic Autoimmune Diseases.

Autoimmune diseases (AID) such as systemic lupus erythematosus (SLE), primary Sjögren's syndrome (pSS), and rheumatoid arthritis (RA) are chronic inflammatory diseases in which abnormalities of B cell function play a central role. Although it is widely accepted that autoimmune B cells are hyperactive in vivo, a full understanding of their functional status in AID has not been delineated. Here, we present a detailed analysis of the functional capabilities of AID B cells and dissect the mechanisms underlying altered B cell function. Upon BCR activation, decreased spleen tyrosine kinase (Syk) and Bruton's tyrosine kinase (Btk) phosphorylation was noted in AID memory B cells combined with constitutive co-localization of CD22 and protein tyrosine phosphatase (PTP) non-receptor type 6 (SHP-1) along with hyporesponsiveness to TLR9 signaling, a Syk-dependent response. Similar BCR hyporesponsiveness was also noted specifically in SLE CD27− B cells together with increased PTP activities and increased transcripts for PTPN2, PTPN11, PTPN22, PTPRC, and PTPRO in SLE B cells. Additional studies revealed that repetitive BCR stimulation of normal B cells can induce BCR hyporesponsiveness and that tissue-resident memory B cells from AID patients also exhibited decreased responsiveness immediately ex vivo, suggesting that the hyporesponsive status can be acquired by repeated exposure to autoantigen(s) in vivo. Functional studies to overcome B cell hyporesponsiveness revealed that CD40 co-stimulation increased BCR signaling, induced proliferation, and downregulated PTP expression (PTPN2, PTPN22, and receptor-type PTPs). The data support the conclusion that hyporesponsiveness of AID and especially SLE B cells results from chronic in vivo stimulation through the BCR without T cell help mediated by CD40–CD154 interaction and is manifested by decreased phosphorylation of BCR-related proximal signaling molecules and increased PTPs. The hyporesponsiveness of AID B cells is similar to a form of functional anergy.

Targeting the CD40-CD154 Signaling Pathway for Treatment of Autoimmune Arthritis.

Full activation of T lymphocytes requires signals from both T cell receptors and costimulatory molecules. In addition to CD28, several T cell molecules could deliver costimulatory signals, including CD154, which primarily interacts with CD40 on B-cells. CD40 is a critical molecule regulating several B-cell functions, such as antibody production, germinal center formation and cellular proliferation. Upregulated expression of CD40 and CD154 occurs in immune effector cells and non-immune cells in different autoimmune diseases. In addition, therapeutic benefits have been observed by blocking the CD40-CD154 interaction in animals with collagen-induced arthritis. Given the therapeutic success of the biologics abatacept, which blocks CD28 costimulation, and rituximab, which deletes B cells in the treatment of autoimmune arthritis, the inhibition of the CD40-CD154 axis has two advantages, namely, attenuating CD154-mediated T cell costimulation and suppressing CD40-mediated B-cell stimulation. Furthermore, blockade of the CD40-CD154 interaction drives the conversion of CD4+ T cells to regulatory T cells that mediate immunosuppression. Currently, several biological products targeting the CD40-CD154 axis have been developed and are undergoing early phase clinical trials with encouraging success in several autoimmune disorders, including autoimmune arthritis. This review addresses the roles of the CD40-CD154 axis in the pathogenesis of autoimmune arthritis and its potential as a therapeutic target.

Abstract 2321: Dual-function of CD27-CD70 costimulatory signal in CAR T cell therapy

Abstract Purpose: To investigate the function of co-stimulatory molecule CD27 on the antitumor efficacy of chimeric antigen receptor (CAR) T cells. Experimental Design: IL13 receptor α2 (IL13Rα2)-targeted CAR T cell products from glioblastoma (GBM) patients were characterized for CD27 surface expression and proliferation potential against recursive tumor challenge. Isolated CD27+ and CD27- fractions of CAR T cells were then evaluated for their antitumor potency against orthotopic GBM xenografts, as well as their molecular characteristics before and after tumor stimulation. Further, T cells were engineered to express a CAR construct together with a constitutively-expressed CD27 to investigate the role of continuous CD27 costimulation on CAR T cell maintenance and effector potency. Results: CD27 expression on CAR T cell products was correlated with their proliferation capacity against recursive tumor stimulation. Isolated CD27+ CAR T cells outperformed CD27- and unsorted cells in mediating long-term tumor eradication. CD27+ CAR T cells exhibited memory-associated genetic signatures, and were less exhausted following in vitro and in vivo tumor stimulation, both phenotypically and functionally. Moreover, CD27 also sensitized CAR T cells to target low antigen-expressing tumors. When targeting tumor cells expressing CD70 (CD27 ligand), CD27 on CAR T cells became rapidly down-regulated, suggesting a transient CD27-CD70 interaction. The advantageous effector function of CD27+ CAR T cells was diminished by blocking the CD27-CD70 interaction with CD70-targeting antibodies. CD70 is also expressed on CAR T cells, displaying a mutually exclusive expression pattern with CD27. Extended in vitro culture resulted in a phenotypic switch of CAR T cells from CD27+CD70- to CD27-CD70+. Surprisingly, constitutive expression of CD27 (CD27-cons) greatly impaired the effector activity of CAR T cells. These cells were able to eliminate tumor cells in vitro, but failed to mediate potent antitumor effect in vivo. Further characterization showed an early exhaustion phenotype for CD27-cons CAR T cells during in vitro expansion. Additionally, CD27-cons CAR T cells were also prone to activation-induced T cell apoptosis especially against high antigen-expressing targets. Conclusion: These studies demonstrate a dual-function of CD27 costimulation for CAR T cell antitumor activity. In the enriched CD27+ CAR T cell subset, the transient CD27-CD70 interaction with tumor cells resulted in enhanced antitumor potency. By contrast, constitutively expressed CD27 drove CAR T cell exhaustion and induced apoptosis. These results illustrate the critical role of CD27 in mediating potent CAR T cell therapeutic efficacy, and suggest the potential of CD27 as a marker for the functional quality of CAR T cell products. Citation Format: Dongrui Wang, Alfonso Brito, Darya Alizadeh, Renate Starr, Brenda Aguilar, Behnam Badie, Stephen J. Forman, Christine E. Brown. Dual-function of CD27-CD70 costimulatory signal in CAR T cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2321.