CD40 Pathway Research Articles

Abstract IA-05: Immune health and intratumoral immune heterogeneity: Barriers to the efficacy of immunotherapy in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) has shown remarkable resistance to immunotherapy including immune checkpoint blockade, vaccines, and other immunomodulatory agents. In cancer, immunosurveillance can be regulated at both the priming and effector phases of an immune response. Based on this reasoning, we investigated a role for the CD40 pathway in mice and humans as a strategy to prime for tumor-reactive T cells. Our studies showed that systemic CD40 activation triggers productive innate immunosurveillance that sensitizes PDAC tumors to chemotherapy but is insufficient to provoke T cell dependent anti-tumor immunity. However, CD40 activation can restore the functional capacity of peritumoral T cells. Consistent with this, PDAC development associates with progressive loss in T cell priming capacity along with a deficiency in circulating dendritic cells. This biology observed in mouse models is mirrored in humans where patients with PDAC show a decrease in circulating dendritic cells and an increase in T cell dysfunction. To overcome these defects in T cell priming and deficiencies in immune health, we studied adoptive transfer of tumor-reactive chimeric antigen receptor (CAR)-modified autologous T cells in patients. Our findings show that the persistence of adoptively transferred T cells is markedly impaired in patients with PDAC compared to hematologic malignancies. In addition, CAR-T cells show limited anti-tumor activity in PDAC. This lack of efficacy has prompted our investigation into the biology of the tumor microenvironment which displays remarkable spatial heterogeneity in immune cell infiltrates. In our studies, we have identified phenotypically distinct cellular communities marked by weak (“cold”) and strong (“hot”) infiltration of T cells. We find that these distinct regions of PDAC tumors are orchestrated by unique immunoregulatory mechanisms with therapeutic implications. Overall, our findings demonstrate that immunological barriers residing both within and outside the tumor microenvironment have the potential to impair the efficacy of immunotherapy in PDAC. In addition, our data suggest that the immunogenicity of PDAC varies spatially throughout tumors raising the likelihood that multiple, distinct mechanisms of immune evasion will need to be circumvented for immunotherapy to produce benefit for most patients. Citation Format: Gregory Beatty. Immune health and intratumoral immune heterogeneity: Barriers to the efficacy of immunotherapy in pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr IA-05.

Newly characterized decidual Tim-3+ Treg cells are abundant during early pregnancy and driven by IL-27 coordinately with Gal-9 from trophoblasts.

What is the mechanism of Tim-3+ regulatory T (Treg)-cell accumulation in the decidua during early pregnancy and is its disruption associated with recurrent pregnancy loss (RPL)? IL-27 and Gal-9 secreted by trophoblasts activate the Tim-3 signaling pathway in CD4+ T cells and Treg cells and so promote accumulation of Tim-3+ Treg cells, the abnormal expression of IL-27 and Gal-9 is associated with impaired immunologic tolerance in RPL patients. Tim-3+ Treg cells are better suppressors of Teff cell proliferation, and display higher proliferative activity than Tim-3- Treg cells. Tim-3+ Treg cells are tissue-specific promoters of T-cell dysfunction in many tumors. These cells express a unique factor that influences and shapes the tumor microenvironment. The animal study included 80 normal pregnant mice. In human study, decidua tissues in the first trimester for flow cytometry analysis were collected from 32 normal pregnant women and 23 RPL patients. Placenta tissues for immunohistochemistry analysis were collected from 15 normal pregnant women. Placenta tissues for western blot analysis were collected from 5 normal pregnant women, 5 RPL patients and 5 women who have experienced one miscarriage. Blood samples for in vitro experiments were collected from 30 normal pregnant women. This study was performed between January 2017 and March 2019. In this study, we investigated the kinetics of Tim-3+ CD4+ T-cell accumulation, and the proportions of Tim-3+ Treg cells throughout murine pregnancies using flow cytometry. We compared Tim-3 expression on decidual CD4+ T cells and Treg cells during normal pregnancies with expression on the same cell populations in women suffering from RPL. IL-27 and Gal-9 transcription and protein expression in the placenta were determined by RT-PCR and western blot, respectively. An in vitro co-culture model consisting of peripheral CD4+ T cells and primary trophoblasts from early pregnancy was used to mimic the maternal-fetal environment. The percentage of Tim-3+ Treg cells present in mouse uteri fluctuates as gestation proceeds but does not change in the spleen. Levels of Tim3+ Treg cells in uteri peaked at pregnancy Day 6.5 (E 6.5), then progressively diminished, and fell to non-pregnant levels by E18.5. In pregnant mice, Tim-3+ Treg cells constituted 40-70% of Treg cells in uteri but were present at much lower abundance in spleens. About 60% of decidual Treg cells were Tim-3 positive at E6.5. Of these decidual Tim3+ Treg cells, nearly 90% were PD-1 positive. However, only about 16% of Tim3- Treg cells expressed PD-1. Blocking the Tim-3 signaling pathway decreased the proportion of Treg cells and led to embryo resorption. Moreover, much lower Tim-3 expression was observed on CD4+ T cells and Treg cells in women who had suffered from RPL at 6-9 gestational weeks compared with those who had normal pregnancies at matched gestations. In a normal pregnancy, Tim-3 expression on decidual CD4+ T cells is induced initially by IL-27. Then Gal-9-Tim-3 interaction promotes differentiation of decidual Tim-3+ CD4+ T cells into Treg cells. IL-27 and Gal-9 cooperatively induced Tim-3+ Treg cells in vitro. N/A. We did not investigate the kinetics of human decidual Tim-3+ CD4+ T and Tim-3+ Treg cell populations throughout pregnancy due to limited availability of second and third trimester decidua. In addition, functional suppressive data on the decidual Tim-3+ Treg cells are lacking due to limited and low quantities of these cells in decidua. These findings might have therapeutic clinical implications in RPL. This study was supported by research grants from the National Natural Science Foundation of China (No. 81871186) and National Key Research & Developmental Program of China (2018YFC1003900, 2018YFC1003904). The authors declare no conflict of interest.

Grape juice attenuates left ventricular hypertrophy in dyslipidemic mice.

ObjectiveWe evaluated the effects of grape juice (Vitis labrusca L.) on dyslipidemia, resistance to insulin, and left ventricular hypertrophy (LVH) in mice homozygous for the absence of the LDL receptor gene (LDLr -/-) under a hyperlipidemic diet.MethodologyWe divided 30 male mice (3 months old) into three groups (n = 10); the HL group was fed a high-fat diet, the HLU group received a high-fat diet and 2 g/kg/day of grape juice, and the HLS group was fed a high-fat diet and simvastatin (20 mg/kg/day). We assessed the blood pressure profile of the mice. We also determined the levels of C-reactive protein (CRP) and lipid profile, glycemic and insulinemic profiles, and calculated the HOMA-IR. Cardiomyocyte hypertrophy, interstitial collagen deposit, and the expression of CD40 ligand (CD40L) and metalloproteinases 2 and 9 were assessed immunohistologically.ResultsAfter 60 days, the mice treated with grape juice showed similar results as those of the group treated with simvastatin. The use of grape fruit attenuated dyslipidemia and insulin resistance and significantly increased the levels of high cholesterol density lipoproteins (HDLc). The antioxidant potential of phenolic compounds associated with the increase in HDLc levels in the mice of the HLU group prevented the development of LVH and arterial hypertension since it inhibited the inflammatory response induced by the CD40 pathway and its ligand CD40L. Consequently, there was a lower expression of MMP-2 and MMP-9 and lower serum levels of CRP.ConclusionGrape juice has a hypolipidemic and cardiac protective potential, presenting a similar effect as that of simvastatin through a direct antioxidant action of phenolic compounds, or indirectly, via antioxidant action and anti-inflammatory activity of the HDLc. These results suggest that grape juice is a functional food possessing a high potential to prevent cardiovascular diseases.

Differential gene expression of tumor-infiltrating CD8+ T cells in advanced versus early-stage colorectal cancer and identification of a gene signature of poor prognosis

BackgroundCytotoxic CD8+ T cell-mediated response is the most important arm of adaptive immunity, which dictates the capacity of the host immune response in eradicating tumor cells. Due to tumor intrinsic...

Abstract 5178: MEDI7526: a novel bispecific antibody that activates the CD40 pathway and down-regulates cell surface PD-L1 expression

Abstract CD40 stimulation on antigen-presenting cells (APCs) promotes activation of CD8(+) cytotoxic T cells and several CD40 agonistic antibodies have shown promising anti-tumor activities in initial clinical trials. However, agonistic CD40 stimulation also leads to PD-L1 upregulation on APCs, thus creating a negative feedback loop to dampen the anti-tumor immune response. We hypothesized that the combination of PD-L1 antagonism with CD40 agonists would achieve stronger antitumor responses than with anti-CD40 alone. Here we report a novel bispecific antibody MEDI7526, which is composed of an anti-PD-L1 antibody, two trimer forms of CD40 ligand (CD40L) fusion protein (FP) and a human IgG4p Fc. MEDI7526 has been designed to stimulate the CD40 pathway and suppress PD-L1 function to improve T cell activation and potentially overcome the limitations associated with conventional CD40 agonists. The binding characteristics and functional activity of MEDI7526 were analyzed in vitro using flow cytometry and cell-based reporter assays. Internalization of MEDI7526, CD40, and PD-L1 were examined by flow cytometry and immunofluorescence microscopy. Effects of MEDI7526 on cytokine production from human PBMC were analyzed with MSD multiplex assay. Anti-tumor efficacy was determined by a murine surrogate version of MEDI7526 in a B16F10 syngeneic model. We confirmed that MEDI7526 can bind CD40 and PD-L1 simultaneously. MEDI7526 induced robust activation of CD40 signaling and triggered fast internalization of CD40. Interestingly, MEDI7526 also triggered rapid and robust down-regulation of PD-L1 on the cell surface of multiple types of cells that express CD40, including THP1, MDA-MB-231 and human monocytes, whereas PD-L1 expression was not affected by the treatment of either anti-PD-L1 antibody or CD40L FP. Prolonged treatment of MEDI7526 on MDA-MB-231 cells induced sustained down-regulation of PD-L1 for up to 4 days. When THP1 cells were transiently treated with MEDI7526, cell surface CD40 returned after 24 hours but levels of PD-L1 on the cell membrane remained low, indicating re-expression of cell surface CD40 and PD-L1 following distinct pathways. We demonstrated that internalized PD-L1 undergoes lysosome-dependent, proteasome-independent degradation, resulting in sustained down-regulation of PD-L1 from cell surface. Moreover, MEDI7526 induced robust IFNγ production in antigen recall assays, and the murine surrogate of MEDI7526 effectively inhibited tumor growth in the B16F10 syngeneic model. These studies demonstrate that MEDI7526 drives the internalization and degradation of PD-L1 and exhibits robust in vitro and in vivo activities. Building this novel bispecific molecule brings two molecules together, resulting in novel mechanisms of action that are different from combinations of parental reagents. MEDI7526, through its unique function of simultaneously stimulating the CD40 pathway and down-regulating PD-L1 expression, represents a promising therapeutic approach against cancer. Citation Format: Yaya Wang, Sean Turman, Deepali Malhotra, Ryan Gilbreth, Yue Wang. MEDI7526: a novel bispecific antibody that activates the CD40 pathway and down-regulates cell surface PD-L1 expression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5178.

Abstract 2267: MEDI5083, a novel CD40L-Fc fusion protein, activates the CD40 pathway on antigen-presenting cells and promotes robust anti-tumor immune responses in both hot and cold murine tumor models

Abstract Background: While diverse strategies may boost anti-tumor immunity, of significant interest are therapeutic approaches that are efficacious in tumors with high and low immune cell infiltration. Targeting CD40 with its natural ligand (CD40L) augments the immune-stimulatory function of antigen-presenting cells and enhances T cell priming, making this an attractive means of promoting anti-tumor responses. MEDI5083 is a novel fusion protein containing a hexameric recombinant human CD40L structure covalently linked to human IgG4p Fc. Here, we present preclinical functional characterization of MEDI5083. Methods MEDI5083 functionality was first established using several in vitro approaches with human cell lines and primary human cells. To examine anti-tumor activity in vivo, we constructed a murine surrogate (mCD40L-Fc) with similar physical and target-binding properties to those of MEDI5083. The effects of mCD40L-Fc on immune cell activation and tumor growth were determined in the immunologically cold B16F10 model of melanoma and the immunologically hot CT26 model of colon carcinoma. Results: MEDI5083 specifically bound to human CD40, triggered its internalization, and robustly stimulated CD40 signaling as measured by NF-κB activation in Ramos and THP-1 cells. MEDI5083 potently upregulated expression of MHCII and the co-stimulatory molecules CD80 and CD86 on primary human B cells and myeloid cells, while inducing secretion of pro-inflammatory cytokines. MEDI5083 also enhanced secretion of IFN-γ by memory CD8+ T cells in an antigen-recall assay. In the B16F10 model, repeated mCD40L-Fc dosing significantly decreased tumor growth. Consistent with enhancing immune cell activation and T cell priming, mCD40L-Fc treatment elevated serum levels of IL-12 and IFN-γ, and increased numbers of tumor-infiltrating CD8+ T cells. While this model responds poorly to checkpoint blockade, co-administration of mCD40L-Fc with anti-PD-L1 antibody demonstrated additivity, consistent with PD-1/PD-L1-mediated regulation of mCD40L-Fc-driven adaptive immunity. Repeated dosing of mCD40L-Fc in the CT26 model inhibited tumor growth. Treatment was associated with diminished tumor-infiltrating Treg numbers, increased CD8+ T cell:Treg ratios, and phenotypic changes in tumor-infiltrating immune cells. Tumor supernatants exhibited increased IFN-γ, consistent with enhanced anti-tumor immunity. Rechallenge of mice with complete tumor regression confirmed generation of durable memory. Conclusions: We have demonstrated that stimulation of CD40 signaling with MEDI5083 or mCD40L-Fc leads to robust activation of immune cells and enhanced adaptive immune responses. In vivo, this translates to enhanced control of both poorly- and well-infiltrated syngeneic tumors and generation of durable memory in mice with complete tumor regression. Citation Format: Deepali Malhotra, Sean Turman, Kelly McGlinchey, Yaya Wang, Yue Wang. MEDI5083, a novel CD40L-Fc fusion protein, activates the CD40 pathway on antigen-presenting cells and promotes robust anti-tumor immune responses in both hot and cold murine tumor models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2267.

Abstract 1073: A tumor-targeted CD40 agonistic DARPin® molecule leading to antitumor activity with limited systemic toxicity

Abstract CD40 is a member of the tumor necrosis factor receptor (TNFR) superfamily which can activate both innate and adaptive tumor immunity, making it an attractive target for cancer immunotherapy. Systemic administration of agonistic anti-CD40 antibodies has shown signs of activity in cancer patients, but dose-limiting toxicity has prevented the exploration of the full dose range and possibly has impaired clinical efficacy. To overcome this issue, we developed a multispecific DARPin® therapeutic candidate, MP0317, intended to activate CD40 receptor locally, rather than systemically, in the tumor microenvironment through cross-linking with fibroblast activation protein (FAP) that is abundantly present in the stroma of many solid tumors, but has limited expression in healthy adult tissues. Using primary human 1) B cells and 2) dendritic cells, MP0317 activated the CD40 pathway in vitro. Upregulation of CD80, CD86, CD69 and IL12 occurred exclusively in the presence of FAP-positive, but not with FAP-negative cells, confirming a mechanism of action strictly dependent on FAP-mediated cross-linking. A surrogate mouse-specific FAP x CD40 DARPin® molecule (mFAP x mCD40) was generated and tested in murine cell-based in vitro assays showing comparable results as MP0317 with a strict FAP-dependent activation of CD40. The mFAP x mCD40 was also active in vivo and inhibited substantially the progression of FAP-positive tumors. Moreover, in contrast to an anti-mouse CD40 antibody (clone FGK45), the antitumor activity of mFAP x mCD40 was neither associated with elevated blood cytokine levels nor with hepatotoxicity, both of which manifest as dose limiting toxicities of some of the clinical CD40 activating antibodies. These data support a mode of action, both in vitro and in vivo, that is dependent on FAP-mediated crosslinking of CD40 receptor resulting in a tumor-localized CD40 activation without peripheral or off-tumor organ toxicity. In conclusion, we have designed a tumor-targeted CD40 agonistic DARPin® therapeutic candidate that is able to activate the CD40 receptor locally in FAP-positive tumors producing impressive antitumor activity in the absence of systemic toxicity. Citation Format: Nicolo Rigamonti, Sarah Jetzer, Niels Van der Velden, Omar Abdelmotaleb, Sophie Barsin, Jonas Schwestermann, Susanne Mangold, Yvonne Kaufmann, Ralph Bessey, Andrea Valeri, Heidi Poulet, Christof Zitt, Pamela A. Trail, Victor Levitsky, Niina Veitonmäki, Clara Metz. A tumor-targeted CD40 agonistic DARPin® molecule leading to antitumor activity with limited systemic toxicity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1073.

Rosuvastatin Alleviates Intestinal Injury by Down-Regulating the CD40 Pathway in the Intestines of Rats Following Traumatic Brain Injury.

Statins have been reported to suppress CD40 expression and nuclear factor (NF)-κB activation, which are both up-regulated in the intestines following traumatic brain injury (TBI)-induced intestinal injury. In this study, we aimed to investigate the effects of the statin rosuvastatin on post-TBI jejunal injury in rats, focusing on potential mechanisms involving the CD40/NF-κB signaling pathway. The jejunal CD40 expression was determined by western blotting. The DNA-binding activity of NF-κB was assessed by electrophoretic mobility shift assays (EMSAs). The tumor necrosis factor (TNF)-α and interleukin (IL)-1β levels were assessed by enzyme-linked immunosorbent assays (ELISAs). The severity of the jejunal mucosal injury was assessed by hematoxylin and eosin (HE) staining and histopathological evaluation. We found that the post-TBI upregulation of both CD40 expression and NF-κB activity in the jejunal tissues were significantly inhibited by rosuvastatin, while the post-TBI expression of TNF-α and IL-1β was significantly suppressed by rosuvastatin. In addition, rosuvastatin significantly ameliorated TBI-induced effects on the villus height, crypt depth, and villous surface area. Rosuvastatin suppressed TBI-induced intestinal injury in rats, which may be associated with the blockade of the CD40/NF-κB pathway.

Genome-wide association study in a Korean population identifies six novel susceptibility loci for rheumatoid arthritis

ObjectiveGenome-wide association studies (GWAS) in rheumatoid arthritis (RA) have discovered over 100 RA loci, explaining patient-relevant RA pathogenesis but showing a large fraction of missing heritability. As a continuous effort,...

Local reinfusion of B10 cells is effective in the treatment of pustular psoriasis.

Psoriasis is a common chronic skin disease characterized by epidermal proliferation and inflammation. Pustular psoriasis (PP) is one of the most serious and refractory. The number, differentiation, and function of B10 cells in patients with PP were analyzed, and the relationship between B10 cells and PP, an autoimmune disease, was explored. We established an Imiquimod psoriasis mouse model and subcutaneously injected B10 cells as treatment. We found that the proportion of B10 cells in the peripheral blood of patients with PP was lower than that of the normal controls. However, the number of B10 precursor cells increased. B10 cells in the peripheral blood may be mobilized to effector sites, such as the skin. In patients with PP, B10 cells do not display evident developmental disorders under the CD40 and TLR9 pathways. Normal human B10 cells were found to inhibit the secretion of IFN-γ and TNF-α by lymphocytes significantly. Whereas the function of B10 cells in patients with PP is impaired, and the inhibition is not apparent. Treatment with a B10 cells injection displays a certain therapeutic effect on PP. This study enriched the etiology and pathogenesis of PP. It provides a foundation for cell therapy for the treatment of autoimmune diseases.

Disease Activity-Associated Alteration of mRNA m5 C Methylation in CD4+ T Cells of Systemic Lupus Erythematosus.

Epigenetic processes including RNA methylation, post-translational modifications, and non-coding RNA expression have been associated with the heritable risks of systemic lupus erythematosus (SLE). In this study, we aimed to explore the dysregulated expression of 5-methylcytosine (m5C) in CD4+ T cells from patients with SLE and the potential function of affected mRNAs in SLE pathogenesis. mRNA methylation profiles were ascertained through chromatography-coupled triple quadrupole mass spectrometry in CD4+ T cells from two pools of patients with SLE exhibiting stable activity, two pools with moderate-to-major activity, and two pools of healthy controls (HCs). Simultaneously, mRNA methylation profiles and expression profiling were performed using RNA-Bis-Seq and RNA-Seq, respectively. Integrated mRNA methylation and mRNA expression bioinformatics analysis was comprehensively performed. mRNA methyltransferase NSUN2 expression was validated in CD4+ T cells from 27 patients with SLE and 28 HCs using real-time polymerase chain reaction and western blot analyses. Hypomethylated-mRNA profiles of NSUN2-knockdown HeLa cells and of CD4+ T cells of patients with SLE were jointly analyzed using bioinformatics. Eleven methylation modifications (including elevated Am, 3′OMeA, m1A, and m6A and decreased Ψ, m3C, m1G, m5U, and t6A levels) were detected in CD4+ T cells of patients with SLE. Additionally, decreased m5C levels, albeit increased number of m5C-containing mRNAs, were observed in CD4+ T cells of patients with SLE compared with that in CD4+ T cells of HCs. m5C site distribution in mRNA transcripts was highly conserved and enriched in mRNA translation initiation sites. In particular, hypermethylated m5C or/and significantly up-regulated genes in SLE were significantly involved in immune-related and inflammatory pathways, including immune system, cytokine signaling pathway, and interferon signaling. Compared to that in HCs, NSUN2 expression was significantly lower in SLE CD4+ T cells. Notably, hypomethylated m5C genes in SLE and in NSUN2-knockdown HeLa cells revealed linkage between eukaryotic translation elongation and termination, and mRNA metabolism. Our study identified novel aberrant m5C mRNAs relevant to critical immune pathways in CD4+ T cells from patients with SLE. These data provide valuable perspectives for future studies of the multifunctionality and post-transcriptional significance of mRNA m5C modification in SLE.

Identification and Analysis of Dysfunctional Genes and Pathways in CD8+ T Cells of Non-Small Cell Lung Cancer Based on RNA Sequencing.

Lung cancer, the most common of malignant tumors, is typically of the non-small cell (NSCLC) type. T-cell-based immunotherapies are a promising and powerful approach to treating NSCLCs. To characterize the CD8+ T cells of non-small cell lung cancer, we re-analyzed the published RNA-Seq gene expression profiles of 36 CD8+ T cell isolated from tumor (TIL) samples and 32 adjacent uninvolved lung (NTIL) samples. With an advanced Monte Carlo method of feature selection, we identified the CD8+ TIL specific expression patterns. These patterns revealed the key dysfunctional genes and pathways in CD8+ TIL and shed light on the molecular mechanisms of immunity and use of immunotherapy.

Interferon regulatory factor 5 expression in myeloid cells and T cells negatively impacts the outcome of visceral leishmaniasis

Abstract Visceral leishmaniasis (VL) is a potentially fatal disease caused by Leishmania donovani and/or L. infantum. VL is characterized by hepatosplenomegaly, immune suppression, and chronic inflammation. We have previously reported that the transcription factor IRF-5 largely contributes to the induction of inflammation and the development of splenomegaly in mice infected with L. donovani. Moreover, Irf5−/− mice only develop limited IFNγ+ CD4 T responses and are more susceptible to infection. However, the cellular source of IRF-5 responsible for these effects is yet unknown. IRF-5 is constitutively expressed in several cells, such as macrophages, B cells, and dendritic cells. We have recently reported that CD4 T cells also express IRF-5 during chronic VL. Here, we investigate IRF-5 function in myeloid and CD4 T cells during VL, using cell-specific knockout mice. First, we show that IRF-5 expression in CD11c+ cells is required for the generation of splenomegaly and leads to increased disease susceptibility. We also demonstrate that IRF-5 expression in CD11c+ cells is not crucial for the development of Th1 responses. Moreover, we demonstrate that IRF-5 activation in Th1 cells results in cell death during chronic L. donovani infection. Triggering of TLR7 by apoptotic cell material promotes IRF-5 activation in Th1 cells. This results in the upregulation of death receptor 5 and caspase 8, making IFNγ+ CD4 T cells more prone to cell death. Because chronic inflammation and tissue disruption are common characteristics of persistent infections, the TLR7- IRF-5 pathway in CD4 T cells may represent a novel mechanism aimed at protecting tissues against sustained inflammation.

Engineered Type-1 Regulatory T Cells as Cellular Therapy for Treatment of Immune Mediated Diseases

Abstract Type 1 regulatory cells (Tr1) are a promising cellular product for suppression of effector T cells in immune mediated diseases, including graft-versus-host-disease (GvHD) in allogeneic hematopoietic stem cell transplantation (allo-HSCT) (Roncarolo et al. Immunity 2018). We have developed an in vitro protocol to produce Tr1 cells by lentiviral transduction of the human IL10 with a constitutive promoter into human CD4+ T cells (Locafaro et al. Molecular Therapy 2017). These engineered Tr1 cells, called LV-10, acquire a characteristic Tr1 cytokine profile (IL-10 and IFN-g high, IL-4 low and expression of intracellular perforin and granzyme B). In vitro, LV-10 cells suppress the proliferation of responder CD4+ T cells upon activation by allogeneic dendritic cells. LV-10 cells also degranulate in response to and kill myeloid cells, including myeloid blasts from patients with acute myeloid leukemia, through a granzyme B- and perforin-dependent mechanism. Interestingly, the ability to degranulate and kill myeloid cells is not present when LV-10 are activated and expanded with anti-CD3 and anti-CD28 coated beads, suggesting that signals beyond TCR, CD28, and IL-10 receptor pathway activation are necessary to reprogram LV-10 cells into cytotoxic cells. In vivo, LV-10 cells injected into NSG mice do not induce xeno-GvHD, in contrast to control CD4+ T cells. In addition, LV-10 cells suppress CD4-induced xeno-GvHD and prevent expansion of myeloid leukemic cells. Experiments are ongoing to compare the potency and in vivo survival of allogeneic vs autologous LV-10 cells. These findings demonstrate the promise of using LV-10 to treat immune mediated diseases, including GvHD in AML patients receiving allo-HSCT.

Intestinal CD14+ Macrophages Protect CD4+ T Cells From Activation-induced Cell Death via Exosomal Membrane TNF in Crohn's Disease.

Sustained activation of CD4+ T cells plays important roles in the pathogenesis of Crohn's disease [CD]. Under physiologic conditions, activated T cells can be timely eliminated by a process termed activation-induced cell death [AICD], restraining T cell over-activation and preventing immunological destruction. We inquired whether defective AICD might explain CD4+ T cell over-activation in CD and investigated the underlying mechanisms. CD14+ macrophages [Mφ] and CD4+ T cells were isolated from intestinal tissues or peripheral blood of controls and CD patients. An ex vivo evaluation system was employed to simulate AICD and cell apoptosis was measured by flow cytometry. CD4+ T cells from CD patients fail to undergo AICD in the ex vivo system. Specifically, proinflammatory type 1 helper T [Th1] and type 17 helper T [Th17] cells, rather than immunosuppressive regulatory T [Treg] cells evade AICD in CD. CD14+ Mφ in the intestinal inflammatory microenvironment of CD promote AICD resistance in CD4+ T cells via a cell-to-cell contact-independent manner. Mechanistically, CD14+ Mφ released exosomes express membrane tumour necrosis factor [TNF] which engages TNFR2 on CD4+ T cells and triggers NF-κB signalling, thereby causing AICD resistance. Importantly, clinically applicable anti-TNF antibodies effectively blocked exosomal membrane TNF-induced CD4+ T cell AICD resistance. CD14+ Mφ participate in CD pathogenesis by inducing AICD resistance through release of exosomal membrane TNF to activate the TNFR2/NF-κB pathway in CD4+ T cells. These results present new insights into CD pathogenesis and extend mechanistic understanding of anti-TNF agents. CD14+ Mφ in the intestinal microenvironment of CD patients maintain the sustained activation of CD4+ T cells through exosomal membrane TNF to induce apoptosis resistance via TNFR2/NF-κB signalling, which could be effectively blocked by clinically applicable anti-TNF agents.

Transient silencing of transforming growth factor‐β1 expression restores nitric oxide generation in diabetic hematopoietic stem/progenitor cells: Role of thrombospondin‐1.

Adult hematopoietic stem/progenitor cells (HSPCs) participate in the cardiovascular repair by stimulating regeneration and angiogenic functions of endothelium. Long‐term diabetes is associated with impaired vasoprotective functions of HSPCs. Transforming growth factor β (TGFβ1) is pleiotropic regulator of HSPC functions. Previous studies have shown that transient silencing of TGFβ1 expression improves in vivo migratory functions of diabetic HSPCs partly by restoring nitric oxide (NO) generation. This study tested the hypothesis that restoration of NO by TGFβ1‐silencing is mediated by thrombospondin‐1 (TSP1)/CD47 pathway. TSP1 is known to attenuate NO generation via inhibition of endothelial nitric oxide synthase (eNOS). HSPCs were isolated from peripheral blood samples obtained from either male or female healthy (n=25) or diabetic (both type 1 and type 2) (n=27) individuals (50–80 years of age) by immunomagnetic enrichment. TGFβ1 expression was transiently blocked by using TGFβ1‐antisense delivered in the form of phosphorodiamidate morpholino oligomer (PMO‐TGFβ1). TGFβ1 and TSP1 gene expressions were determined in HSPCs treated with either PMO‐control or PMO‐TGFβ1. NO generation induced by stromal‐derived factor‐1α (SDF) was determined by DAF‐FM flow cytometry. Diabetic cells have higher expression of TGFβ1 or TSP1 compared to that observed in cells derived from healthy individuals (n=10). Treatment with TGFβ1‐PMO decreased the expression of TGFβ1, which was associated with decreased TSP1 expression. CD47 expression is similar in healthy or diabetic individuals. NO generation by SDF is attenuated in diabetic compared to healthy cells (P<0.01, n=5). PMO‐TGFβ1‐treated diabetic cells showed increased generation of NO in response to SDF compared to PMO‐control‐treatment (P<0.01, n=5). Future studies will determine the effect of TSP1 on SDF/NO generation in cells with or without CD47‐siRNA, to further support the hypothesis. Collectively, these studies indicate the involvement of TGFβ1/TSP1/CD47/eNOS pathway in diabetic impairment of NO generation however involvement of other pathways cannot be ruled out.Support or Funding InformationThis study is partly supported by funding from NIH National Institute of Aging (AG056681).

Five Treatment Strategies to Suppress Donor Specific Antibodies: Highlights from a Decade of Research Experience in a Mouse Model of Allo-Sensitization

Alloantibody-mediated rejection and difficulty in desensitization in highly sensitized patients remain problematic in organ transplantation. For over 12 years, our laboratory has been carrying out an HLA antibody desensitization program to develop strategies for suppressing donor specific antibodies (DSA). A mouse model of allo-sensitization involving a C57BL mouse as recipient of a skin graft (SG) from a C57BL- tg-HLA.A2 mouse is used to study monotherapy or combinational therapies. Drugs tested include (1) anti-CD20 (B-cell depletion); (2) Ibrutinib (Bruton's tyrosine kinase inhibitor, BTKi); (3) Tofacitinib (Janus Kinase Inhibitor, JAKi); (4) anti-IL6 receptor and anti-IL6 (interleukin 6 pathway blockage) and (5) CTLA4Ig (abatacept, CD28 pathway manipulation). DSA suppression was observed in each of the monotherapies studied. BTK inhibitor ibrutinib and JAK antagonist tofacitinib showed mild but significant reduction in de novo DSA. Administration of anti-CD20 significantly reduced levels of DSA IgG (p<0.01 vs. control), depleting >90% B220+/CD5- B-cells. Anti-IL6 receptor antibody exhibited significant suppression on de novo and recall DSA, which is associated with an increase in Treg cell population. In comparison, anti-IL6 antibody showed DSA suppression only in a 3-dose regimen, not in a 15-dose treatment group. Serum IL-6 concentrations in the 15-dose group increased by 188-fold at day 14, indicating that anti-IL-6 antibodies cause profound disturbance in IL-6 homeostasis. CTLA4Ig, by interrupting CD28/B7 costimulatory pathway is a potent inhibitor of de novo alloantibody responses and a moderate attenuator of recall alloantibody response. The differential effects of CTLA4Ig in de novo versus recall responses indicate that CTLA4Ig may not only interrupt naïve T cell activation and subsequent T-dependent B cell maturation, but also mitigate recall B cell responses by Targeting B7-1 (CD80) expressed on Plasma Cells. A combined therapy using anti-CD20, CTLA4Ig and anti-IL6r resulted in strongest DSA reduction in both de novo and recall responses. Our data demonstrate that a maximal suppression can be achieved by combined therapies targeting multiple pathways critical of CD4 T-cell activation, B/plasma cell activation and antibody production.

Sufficiency of CD40 activation and immune checkpoint blockade for T cell priming and tumor immunity

Innate immune receptors such as toll-like receptors (TLRs) provide critical molecular links between innate cells and adaptive immune responses. Here, we studied the CD40 pathway as an alternative bridge between dendritic cells (DCs) and adaptive immunity in cancer. Using an experimental design free of chemo- or radiotherapy, we found CD40 activation with agonistic antibodies (⍺CD40) produced complete tumor regressions in a therapy-resistant pancreas cancer model, but only when combined with immune checkpoint blockade (ICB). This effect, unachievable with ICB alone, was independent of TLR, STING, or IFNAR pathways. Mechanistically, αCD40/ICB primed durable T cell responses, and efficacy required DCs and host expression of CD40. Moreover, ICB drove optimal generation of polyfunctional T cells in this "cold" tumor model, instead of rescuing T cell exhaustion. Thus, immunostimulation via αCD40 is sufficient to synergize with ICB for priming. Clinically, combination αCD40/ICB may extend efficacy in patients with "cold" and checkpoint-refractory tumors.

Sox12 enhances Fbw7-mediated ubiquitination and degradation of GATA3 in Th2 cells.

Allergic asthma that is caused by inhalation of house dust mites (HDMs) is mainly mediated by Th2 cells. Recently, the roles of Sox (SRY-related high-mobility-group (HMG)-box) family members in various immune responses have been investigated. However, the roles of Sox12, a member of the SoxC group, in Th2 cell differentiation and allergic airway inflammation, remain unknown. We showed that Sox12 mRNA was significantly increased during Th2 cell differentiation. In vivo, HDM-induced eosinophil infiltration into the lung and Th2 cell differentiation were exacerbated in Sox12-/- mice compared with those in control Sox12+/- mice. In vitro, Sox12-/- CD4+ T cells that were cultured under Th2 conditions had increased production of Th2 cytokines and GATA3 protein compared with those of control Sox12+/- CD4+ T cells. Importantly, forced expression of Sox12 decreased the protein levels of GATA3 in CD4+ T cells under Th2 conditions without affecting mRNA expression. Furthermore, Sox12 induced degradation of GATA3 through the proteasome pathway in CD4+ T cells. Consistently, Sox12 enhanced ubiquitination of GATA3, which was mediated by the E3 ligase Fbw7. Finally, we found that Fbw7 knockdown partly abrogated Sox12-mediated GATA3 suppression in CD4+ T cells. Taken together, these results suggest that Sox12 suppresses Th2 cell differentiation by accelerating Fbw7-mediated GATA3 degradation, and attenuates HDM-induced allergic inflammation.

CD28-Dependent CTLA-4 Expression Fine-Tunes the Activation of Human Th17 Cells.

SummaryPrevious work has demonstrated that Th17 memory cells but not Th1 cells are resistant to CD28/CTLA-4 blockade with CTLA-4 Ig, leading us to investigate the individual roles of the CD28 and CTLA-4 cosignaling pathways on Th1 versus Th17 cells. We found that selective CD28 blockade with a domain antibody (dAb) inhibited Th1 cells but surprisingly augmented Th17 responses. CD28 agonism resulted in a profound increase in CTLA-4 expression in Th17 cells as compared with Th1 cells. Consistent with these findings, inhibition of the CD28 signaling protein AKT revealed that CTLA-4 expression on Th17 cells was more significantly reduced by AKT inhibition relative to CTLA-4 expression on Th17 cells. Finally, we found that FOXO1 and FOXO3 overexpression restrained high expression of CTLA-4 on Th17 cells but not Th1 cells. This study demonstrates that the heterogeneity of the CD4+ T cell compartment has implications for the immunomodulation of pathologic T cell responses.