Antigen-presenting Cell Function Research Articles

Abstract P037: Interleukin-10 is a dominant and reversible mechanism of immune evasion in human colorectal cancer liver metastasis

Abstract Background: Colorectal cancer liver metastasis (CRLM) causes major morbidity and mortality. Improved systemic therapies are crucial. We previously reported that macrophages infiltrated CRLM and expressed the immunosuppressive cytokine interleukin-10 (IL-10). In patient-derived CRLM tumor slice cultures, we found that a neutralizing antibody against IL-10 (anti-IL-10) caused tumor apoptosis. Here, we investigated the efficacy and immune-dependent mechanisms of IL-10 blockade in a larger cohort of CRLM patients. Methods: Tumor specimens were obtained from consenting patients at the time of surgery and cut into 250mm-thick tumor slice cultures as previously described. Tumor slices were treated with control or neutralizing antibodies against programmed cell death protein 1 (PD-1, EH12.1), IL-10 (JES3-9D7), IL-10 receptor alpha (IL-10RA, 3F9), major histocompatibility complex (MHC) class I (G46-2.6) or class II (Tu39). After 4-6 days of treatment, tumor apoptosis was evaluated by cleaved caspase-3 (CC3) immunohistochemistry (IHC) or terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). Tumors were evaluated by IHC and in situ hybridization (ISH) for immune markers. Student's t-test, paired t-test, or 1-way ANOVA was used as indicated. p < 0.05 was defined as significant. Results: We generated tumor slice cultures from 34 unique CRLM patients, 76% (26/34) of whom received preoperative chemotherapy, and all of whom had either microsatellite stable tumors (76%, 26/34) or unknown microsatellite status. Anti-PD-1 did not generate tumor apoptosis (n = 4 patients' tumors). In contrast, anti-IL-10 caused nearly 2-fold increased tumor apoptosis compared with control, in the majority of 34 CRLM patients' tumors evaluated (median 50.1% versus 27.4% apoptotic cells, p < 0.0001). IL-10 receptor blockade generated a similar but non-significant increase in apoptosis. Tumors treated with anti-IL-10 demonstrated increased frequency of CD8+ T cells (median 17.8% versus 7.0% of total cells, n = 5 patients' tumors, p = 0.02) and a non-significant increase in activated PD-1+CD3+ T cells. IL-10 blockade also increased tumor IFNG expression (median 2.9 versus 2.3 counts per total cell number, n = 6 patients' tumors, p < 0.05). Macrophage frequency did not increase, but MHC class II expression nearly doubled (median 19.5% versus 11.5% of total cells, n = 5 patients' tumors, p = 0.02). To test which immune cells were required for anti-IL-10 effect, we treated human CRLM slices with anti-IL-10 +/- blocking antibodies against MHC class I or II. In the 3 of 4 patients' tumors that responded to anti-IL-10, the effect was nearly completely reversed by aMHC-I or aMHC-II (p = 0.0005). Conclusion: IL-10 is a dominant and reversible mechanism of immune evasion in human CRLM. IL-10 blockade nearly doubled tumor apoptosis in a heterogeneous cohort of CRLM patients' tumors, through a mechanism that required intact CD8+ T cell and antigen-presenting cell function. IL-10 is a compelling immunotherapeutic target for CRLM. Citation Format: Teresa S. Kim, Kevin Sullivan, Xiuyun Jiang, Cynthia Hsu, Kevin Labadie, Karan Kohli, Heidi Kenerson, Sara Daniel, Arezou Abbasi, Raymond Yeung, Venu Pillarisetty. Interleukin-10 is a dominant and reversible mechanism of immune evasion in human colorectal cancer liver metastasis [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P037.

Impairment of antigen-presenting function of peripheral γδ T cells in patients with sepsis

Impairment of antigen-presenting functions is a key mechanism contributing to sepsis-induced immunosuppression. Recently, γδ T cells have been demonstrated as professional antigen-presenting cells (APCs); however, their role in sepsis remains unknown. In this in vitro study, the APC function of human peripheral γδ T cells was assessed using samples collected from 42 patients with sepsis and 27 age-matched healthy controls. The APC-related markers HLA-DR, CD27, CD80, and CCR7 on fresh γδT cells were significantly higher in patients with sepsis compared with matched controls; however, they responded poorly to 4-hydroxy-3-methyl-2-butenyl pyrophosphate (HMBPP) stimulation, characterized by the deactivation of these APC markers and impaired proliferation. Furthermore, the adhesion function of γδ T cells, essential for antigen presentation, was greatly reduced in patients with sepsis; for instance, in co-cultures with green fluorescent protein-expressing Escherichia coli, HMBPP-activated γδT cells from healthy individuals adhered to E. coli efficiently, whereas no such phenomenon was observed with respect to γδT cells from patients with sepsis. In line with these results, in co-cultures with isolated CD4+ αβ T cells, HMBPP-activated γδT cells of healthy individuals promoted the efficient proliferation of CD4+ αβ T cells, whereas γδT cells from patients with sepsis did not do so. In conclusion, our findings show that the antigen-presenting function of γδT cells is severely impaired in patients with sepsis and the mechanisms behind need further study.

Heme-Oxygenase-1 Attenuates Oxidative Functions of Antigen Presenting Cells and Promotes Regulatory T Cell Differentiation during Fasciola hepatica Infection

Fasciola hepatica is a fluke that infects livestock and humans causing fasciolosis, a zoonotic disease of increasing importance due to its worldwide distribution and high economic losses. The parasite regulates the host immune system by inducing a strong Th2 and regulatory T (Treg) cell immune response through mechanisms that might involve the expression or activity of heme-oxygenase-1 (HO-1), the rate-limiting enzyme in the catabolism of free heme that also has immunoregulatory and antioxidant properties. In this paper, we show that F. hepatica-infected mice upregulate HO-1 on peritoneal antigen-presenting cells (APC), which produce decreased levels of both reactive oxygen and nitrogen species (ROS/RNS). The presence of these cells was associated with increased levels of regulatory T cells (Tregs). Blocking the IL-10 receptor (IL-10R) during parasite infection demonstrated that the presence of splenic Tregs and peritoneal APC expressing HO-1 were both dependent on IL-10 activity. Furthermore, IL-10R neutralization as well as pharmacological treatment with the HO-1 inhibitor SnPP protected mice from parasite infection and allowed peritoneal APC to produce significantly higher ROS/RNS levels than those detected in cells from infected control mice. Finally, parasite infection carried out in gp91phox knockout mice with inactive NADPH oxidase was associated with decreased levels of peritoneal HO-1+ cells and splenic Tregs, and partially protected mice from the hepatic damage induced by the parasite, revealing the complexity of the molecular mechanisms involving ROS production that participate in the complex pathology induced by this helminth. Altogether, these results contribute to the elucidation of the immunoregulatory and antioxidant role of HO-1 induced by F. hepatica in the host, providing alternative checkpoints that might control fasciolosis.

Costimulation of γδTCR and TLR7/8 promotes Vδ2 T-cell antitumor activity by modulating mTOR pathway and APC function

BackgroundGamma delta (γδ) T cells are attractive effector cells for cancer immunotherapy. Vδ2 T cells expanded by zoledronic acid (ZOL) are the most commonly used γδ T cells for adoptive...

Multi-Tissue Characterization of GILZ Expression in Dendritic Cell Subsets at Steady State and in Inflammatory Contexts.

Dendritic cells (DCs) are key players in the control of tolerance and immunity. Glucocorticoids (GCs) are known to regulate DC function by promoting their tolerogenic differentiation through the induction of inhibitory ligands, cytokines, and enzymes. The GC-induced effects in DCs were shown to critically depend on increased expression of the Glucocorticoid-Induced Leucine Zipper protein (GILZ). GILZ expression levels were further shown to control antigen-presenting cell function, as well as T-cell priming capacity of DCs. However, the pattern of GILZ expression in DC subsets across tissues remains poorly described, as well as the modulation of its expression levels in different pathological settings. To fill in this knowledge gap, we conducted an exhaustive analysis of GILZ relative expression levels in DC subsets from various tissues using multiparametric flow cytometry. This study was performed at steady state, in the context of acute as well as chronic skin inflammation, and in a model of cancer. Our results show the heterogeneity of GILZ expression among DC subsets as well as the complexity of its modulation, that varies in a cell subset- and context-specific manner. Considering the contribution of GILZ in the control of DC functions and its potential as an immune checkpoint in cancer settings, these results are of high relevance for optimal GILZ targeting in therapeutic strategies.

Regulatory T Cells: Regulation of Identity and Function.

T regulatory cells suppress a variety of immune responses to self-antigens and play a role in peripheral tolerance maintenance by limiting autoimmune disorders, and other pathological immune responses such as limiting immune reactivity to oncoprotein encoded antigens. Forkhead box P3 (FOXP3) expression is required for Treg stability and affects functional activity. Mutations in the master regulator FOXP3 and related components have been linked to autoimmune diseases in humans, such as IPEX, and a scurfy-like phenotype in mice. Several lines of evidence indicate that Treg use a variety of immunosuppressive mechanisms to limit an immune response by targeting effector cells, including secretion of immunoregulatory cytokines, granzyme/perforin-mediated cell cytolysis, metabolic perturbation, directing the maturation and function of antigen-presenting cells (APC) and secretion of extracellular vesicles for the development of immunological tolerance. In this review, several regulatory mechanisms have been highlighted and discussed.

Antigen Presenting Cells Contribute to Persistent Immune Activation Despite Antiretroviral Therapy Initiation During Hyperacute HIV-1 Infection.

Human immunodeficiency virus (HIV)-induced changes in immune cells during the acute phase of infection can cause irreversible immunological damage and predict the rate of disease progression. Antiretroviral therapy (ART) remains the most effective strategy for successful immune restoration in immunocompromised people living with HIV and the earlier ART is initiated after infection, the better the long-term clinical outcomes. Here we explored the effect of ART on peripheral antigen presenting cell (APC) phenotype and function in women with HIV-1 subtype C infection who initiated ART in the hyperacute phase (before peak viremia) or during chronic infection. Peripheral blood mononuclear cells obtained longitudinally from study participants were used for immunophenotyping and functional analysis of monocytes and dendritic cells (DCs) using multiparametric flow cytometry and matched plasma was used for measurement of inflammatory markers IL-6 and soluble CD14 (sCD14) by enzyme-linked immunosorbent assay. HIV infection was associated with expansion of monocyte and plasmacytoid DC (pDC) frequencies and perturbation of monocyte subsets compared to uninfected persons despite antiretroviral treatment during hyperacute infection. Expression of activation marker CD69 on monocytes and pDCs in early treated HIV was similar to uninfected individuals. However, despite early ART, HIV infection was associated with elevation of plasma IL-6 and sCD14 levels which correlated with monocyte activation. Furthermore, HIV infection with or without early ART was associated with downmodulation of the co-stimulatory molecule CD86. Notably, early ART was associated with preserved toll-like receptor (TLR)-induced IFN-α responses of pDCs. Overall, this data provides evidence of the beneficial impact of ART initiated in hyperacute infection in preservation of APC functional cytokine production activity; but also highlights persistent inflammation facilitated by monocyte activation even after prolonged viral suppression and suggests the need for therapeutic interventions that target residual immune activation.

MHC class II in renal tubules plays an essential role in renal fibrosis.

Immunomodulation is considered a potential therapeutic approach for chronic kidney disease (CKD). Although it has been previously reported that CD4+ T cells contribute to the development of renal fibrosis, the role of MHC class II (MHCII) in the development of renal fibrosis remains largely unknown. The present study reports that the expression of MHCII molecules in renal cortical tubules is upregulated in mouse renal fibrosis models generated by unilateral ureter obstruction (UUO) and folic acid (FA). Proximal tubule epithelial cells (PTECs) are functional antigen-presenting cells that promote the proliferation of CD4+ T cells in an MHCII-dependent manner. PTECs from mice with renal fibrosis had a stronger ability to induce T cell proliferation and cytokine production than control cells. Global or renal tubule-specific ablation of H2-Ab1 significantly alleviated renal fibrosis following UUO or FA treatment. Renal expression of profibrotic genes showed a consistent reduction in H2-Ab1 gene-deficient mouse lines. Moreover, there was a marked increase in renal tissue CD4+ T cells after UUO or FA treatment and a significant decrease following renal tubule-specific ablation of H2-Ab1. Furthermore, renal tubule-specific H2-Ab1 gene knockout mice exhibited higher proportions of regulatory T cells (Tregs) and lower proportions of Th2 cells in the UUO- or FA-treated kidneys. Finally, Immunohistochemistry (IHC) studies showed increased renal expression of MHCII and the profibrotic gene α smooth muscle actin (α-SMA) in CKD patients. Together, our human and mouse data demonstrate that renal tubular MHCII plays an important role in the pathogenesis of renal fibrosis.

The epigenetic immunomodulator, HBI-8000, enhances the response and reverses resistance to checkpoint inhibitors

BackgroundTreatment with immune checkpoint inhibitors (ICIs) targeting CTLA-4 and the PD-1/PD-L1 axis is effective against many cancer types. However, due in part to unresponsiveness or acquired resistance, not all patients experience a durable response to ICIs. HBI-8000 is a novel, orally bioavailable class I selective histone deacetylase inhibitor that directly modifies antitumor activity by inducing apoptosis, cell cycle arrest, and resensitization to apoptotic stimuli in adult T cell lymphoma patients. We hypothesized that HBI-8000 functions as an epigenetic immunomodulator to reprogram the tumor microenvironment from immunologically cold (nonresponsive) to hot (responsive).MethodMice bearing syngeneic tumors (MC38 and CT26 murine colon carcinoma and A20 B-cell lymphoma were treated daily with HBI-8000 (orally), alone or in combination with PD-1, PD-1 L, or CTLA-4 antibodies. MC38 tumors were also analyzed in nanoString gene expression analysis.ResultsHBI-8000 augmented the activity of ICI antibodies targeting either PD-1, PD-L1 or CTLA-4, and significantly increased tumor regression (p < 0.05) in the above models. Gene expression analysis of the treated MC38 tumors revealed significant changes in mRNA expression of immune checkpoints, with enhanced dendritic cell and antigen-presenting cell functions, and modulation of MHC class I and II molecules.ConclusionsThese findings suggest that HBI-8000 mediates epigenetic modifications in the tumor microenvironment, leading to improved efficacy of ICIs, and provide strong rationale for combination therapies with ICIs and HBI-8000 in the clinical setting.PrecisAs an HDACi, HBI-8000 plays an important role in priming the immune system in the tumor microenvironment. The current preclinical data further justifies testing combination of HBI-8000 and ICIs in the clinic.

Monophosphoryl lipid A directly regulates Th1 cytokine production in human CD4+ T-cells through Toll-like receptor 2 and 4

BackgroundThe monophosphoryl lipid A (MPLA) is a detoxified LPS derivative and an emerging safe immune adjuvant in human vaccine development. The adjuvant MPLA promotes antigen-presenting cell (APC) function and preferentially induces a Th1 response following vaccination. However, the mechanism by which the MPLA detoxicates and exerts its adjuvants effect on T-cell, particualrly the Th1 response is unknown. AimsWe assessed the direct effects of MPLA on murine and human CD4+ T-cell proliferation and the profile of cytokine production ex vivo. ResultsWe report that CD4+ T-cells only express functional TLR2 and TLR4 when activated by TCR stimulation, in particularly in the presence of IFNα. The activated T cells thereafter can respond directly to MPLA. MPLA does not affect T-cell proliferation in human T cells, but can induce a balanced Th1 cytokine profile in CD4+ T-cells by reducing the production of Th1 cytokines and enhancing the production of the regulatory cytokine IL-10. The MPLA-mediated regulatory effect on activated CD4+ T-cells is TLR2 and TLR4 dependent and can be abolished by the lipid A blocker polymyxin B. ConclusionThese data provide evidence, at least in part, for the safe induction of an appropriate level of Th1 response by adjuvant MPLA in human vaccine development.

Ewing Sarcoma-Derived Extracellular Vesicles Impair Dendritic Cell Maturation and Function.

Ewing sarcoma (EwS) is an aggressive pediatric cancer of bone and soft tissues characterized by scant T cell infiltration and predominance of immunosuppressive myeloid cells. Given the important roles of extracellular vesicles (EVs) in cancer-host crosstalk, we hypothesized that EVs secreted by EwS tumors target myeloid cells and promote immunosuppressive phenotypes. Here, EVs were purified from EwS and fibroblast cell lines and exhibited characteristics of small EVs, including size (100–170 nm) and exosome markers CD63, CD81, and TSG101. Treatment of healthy donor-derived CD33+ and CD14+ myeloid cells with EwS EVs but not with fibroblast EVs induced pro-inflammatory cytokine release, including IL-6, IL-8, and TNF. Furthermore, EwS EVs impaired differentiation of these cells towards monocytic-derived dendritic cells (moDCs), as evidenced by reduced expression of co-stimulatory molecules CD80, CD86 and HLA-DR. Whole transcriptome analysis revealed activation of gene expression programs associated with immunosuppressive phenotypes and pro-inflammatory responses. Functionally, moDCs differentiated in the presence of EwS EVs inhibited CD4+ and CD8+ T cell proliferation as well as IFNγ release, while inducing secretion of IL-10 and IL-6. Therefore, EwS EVs may promote a local and systemic pro-inflammatory environment and weaken adaptive immunity by impairing the differentiation and function of antigen-presenting cells.

Nanocages displaying SIRP gamma clusters combined with prophagocytic stimulus of phagocytes potentiate anti-tumor immunity.

Antigen-presenting cells (APCs), including macrophages and dendritic cells (DCs), play a crucial role in bridging innate and adaptive immunity; thereby, innate immune checkpoint blockade-based therapy is an attractive approach for the induction of sustainable tumor-specific immunity. The interaction between the cluster of differentiation 47 (CD47) on tumor and signal-regulatory protein alpha (SIRPα) on phagocytic cells inhibits the phagocytic function of APCs, acting as a "don't eat me" signal. Accordingly, CD47 blockade is known to increase tumor cell phagocytosis, eliciting tumor-specific CD8+ T-cell immunity. Here, we introduced a nature-derived nanocage to deliver SIRPγ for blocking of antiphagocytic signaling through binding to CD47 and combined it with prophagocytic stimuli using a metabolic reprogramming reagent for APCs (CpG-oligodeoxynucleotides). Upon delivering the clustered SIRPγ variant, the nanocage showed enhanced CD47 binding profiles on tumor cells, thereby promoting active engulfment by phagocytes. Moreover, combination with CpG potentiated the prophagocytic ability, leading to the establishment of antitumorigenic surroundings. This combination treatment could competently inhibit tumor growth by invigorating APCs and CD8+ T-cells in TMEs in B16F10 orthotopic tumor models, known to be resistant to CD47-targeting therapeutics. Collectively, enhanced delivery of an innate immune checkpoint antagonist with metabolic modulation stimuli of immune cells could be a promising strategy for arousing immune responses against cancer.

Research advances in the role of the glucocorticoid-induced tumor necrosis factor receptor-related protein and its ligand in liver diseases

Glucocorticoid-induced tumor necrosis factor receptor (GITR) is a member of the tumor necrosis factor receptor superfamily, and after it specifically binds to GITR ligand (GITRL), the downstream signals mediated by them can not only serve as a costimulatory molecule to promote the proliferation and cytokine secretion of effector T cells, but also affect the proliferation of regulatory T cells and inhibit the function of effector T cells, thereby regulating the inflammatory response and tumor cell-killing effect of effector T cells. GITRL is mainly expressed in antigen- presenting cells and has an intracellular domain, and the binding of GITRL to GITR can affect the function of antigen-presenting cells via receptor/ligand reverse signaling. In liver diseases, GITRL/GITR signal is not only involved in immune rejection after liver transplantation and gene therapy, but also associated with the formation of tumor immune microenvironment and the immune escape of tumor. Further studies are needed to explore the role of GITRL/GITR in the development and progression of other liver diseases.

Protective Role of Collectin 11 in a Mouse Model of Rheumatoid Arthritis.

Collectin 11 (CL-11) is a soluble C-type lectin, a mediator of innate immunity. Its role in autoimmune disorders is unknown. We undertook this study to determine the role of CL-11 in a mouse model of rheumatoid arthritis (RA). A murine collagen-induced arthritis (CIA) model was used and combined two approaches, including gene deletion of Colec11 and treatment with recombinant CL-11 (rCL-11). Joint inflammation and tissue destruction, circulating levels of inflammatory cytokines, and adaptive immune responses were assessed in mice with CIA. Splenic CD11c+ cells were used to examine the influence of CL-11 on antigen-presenting cell (APC) function. Serum CL-11 levels in RA patients were also examined. Colec11-/- mice developed more severe arthritis than wild-type mice, as determined by disease incidence, clinical arthritis scores, and histopathology (P < 0.05). Disease severity was associated with significantly enhanced APC activation, Th1/Th17 responses, pathogenic IgG2a production and joint inflammation, as well as elevated circulating levels of inflammatory cytokines. In vitro analysis of CD11c+ cells revealed that CL-11 is critical for suppression of APC activation and function. Pharmacologic treatment of mice with rCL-11 reduced the severity of CIA in mice. Analysis of human blood samples revealed that serum CL-11 levels were lower in RA patients (n = 51) compared to healthy controls (n = 53). Reduction in serum CL-11 was inversely associated with the Disease Activity Score in 28 joints, erythrocyte sedimentation rate, and C-reactive protein level (P < 0.05). Our findings demonstrate a novel role of CL-11 in protection against RA, suggesting that the underlying mechanism involves suppression of APC activation and subsequent T cell responses.

Abstract 2774: Establishment of in vitro human T cell exhaustion model to enable high throughput target validation for T cell phenotype restoring

Abstract Immune surveillance was defined as three phases during tumorigenesis: elimination, equilibrium and escape. During the escape phase, cancer cell acquire a series of mechanism to evade the recognition by immune system, including reduce the function of antigen presenting cells, recruitment of suppressive cells, induce T cell exhaustion etc. The exhaustion phenotype of T cells were frequently observed in various cancer types and exhibit as reduced effector cytokine production, decrease in proliferation and cytolytic activity and overexpression of inhibitory receptors. Restoring T cell exhaustion phenotype become the inspiring filed for cancer therapy and PD1 blocking antibody further encouraging the concept of intervention on T cell exhausted phenotype in tumor microenvironment. In this study, we utilize human primary T cell to establish the in vitro model of T cell exhaustion. Subsequent functional study reveal that the induced exhausted T cell (Tex) display significantly weak response towards dendritic cell activation compare to normal T cell (Tnorm). We take advantage of the model to validate the function of PD1 antibody and demonstrate that blocking of PD1 could partially restore the function of Tex, which suggesting there are still other regulators involved in mediating T cell exhaustion beyond PD1. Citation Format: Tienan Wang, Jie Zhang, Zhijun Wang, Yingnian Li, Linghong Shi, Qing Lin. Establishment of in vitro human T cell exhaustion model to enable high throughput target validation for T cell phenotype restoring [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 2774.

The regulation of CD47-SIRPα signaling axis by microRNAs in combination with conventional cytotoxic drugs together with the help of nano-delivery: a choice for therapy?

CD47, a member of the immunoglobulin superfamily, is an important "Don't Eat-Me" signal in phagocytosis process [clearance of apoptotic cells] as well as a regulator of the adaptive immune response. The lower level of CD47 on the cell surface leads to the clearance of apoptotic cells. Dysregulation of CD47 plays a critical role in the development of disorders, particularly cancers. In cancers, recognition of CD47 overexpression on the surface of cancer cells by its receptor, SIRPα on the phagocytic cells, inhibits phagocytosis of cancer cells. Thus, blocking of CD47-SIRPα signaling axis might be as a promising therapeutic target, which promotes phagocytosis of cancer cells, antigen-presenting cell function as well as adaptive T cell-mediated anti-cancer immunity. In this respect, it has been reported that CD47 expression can be regulated by microRNAs (miRNAs). MiRNAs can regulate phagocytosis of macrophages apoptotic process, drug resistance, relapse of disease, radio-sensitivity, and suppress cell proliferation, migration, and invasion through post-transcriptional regulation of CD47-SIRPα signaling axis. Moreover, the regulation of CD47 expression by miRNAs and combination with conventional cytotoxic drugs together with the help of nano-delivery represent a valuable opportunity for effective cancer treatment. In this review, we review studies that evaluate the role of miRNAs in the regulation of CD47-SIRPα in disorders to achieve a novel preventive, diagnostic, and therapeutic strategy.Please confirm if the author names are presented accurately and in the correct sequence (given name, middle name/initial, family name). Also, kindly confirm the details in the metadata are correct. Confirmed.Journal standard instruction requires a structured abstract; however, none was provided. Please supply an Abstract with subsections..Not confirmed. This is a review article. According to submission guidelines: "The abstract should be presented divided into subheadings (unless it is a mini or full review article)". Kindly check and confirm whether the corresponding authors and mail ID are correctly identified. Confirmed.

A Novel Role for Histone Deacetylase 10 (HDAC10) in the Regulation of PD-L1 Expression and Immune Tolerance Mediated By Antigen Presenting Cells (APCs)

Antigen-presenting cells (APCs) play a central role in determining T-cell activation versus T-cell anergy in the context of tumor growth. The inflammatory status of APCs at the time of tumor antigen presentation has been proposed as explanation for the induction of such divergent T-cell outcomes. This overall inflammatory status of APCs can be regulated epigenetically.HDAC10 is a member of the class IIb family of HDACs that represses gene transcription independent of its deacetylase activity when tethered to a promoter. It is normally expressed in liver, kidney, pancreas and spleen and its overexpression has been shown to accelerate cancer development. While we previously demonstrated a role for HDAC6, the other member of the class IIb family, in control of APC inflammatory vs tolerogenic status (Cheng F et al, J Immunol 2014), little is known about the role (if any) of HDAC10 in regulating APC's function.Here we show for the first time that knockdown of HDAC10 in APCs, using shRNA specific for murine HDAC10 or non-target control (NT), resulted in the induction of inflammatory APCs displaying an 20-30% increased expression of MHC class II molecules and abrogation of PD-L1 gene transcription (4 fold decreased) and expression as compared to controls. To further confirm these results in vivo, HDAC10 knock out (KO) mice were used. These HDAC10 KO mice, as compared to wild type (WT) mice, reveal no gross macroscopic abnormalities in any lymphoid organs. Macrophages isolated from HDAC10 KO mice demonstrated increased expression of MHC II molecules and decreased expression of PD-L1, analogous to the shRNA data. Furthermore, in vitro co-culture of these macrophages with antigen-specific CD4+ T cells lead to effective priming of naïve CD4+ T-cells and restoration of the responsiveness of tolerant T-cells, as determined by their 3 fold increased production of IL-2 and IFN-g in response to cognate antigen. This demonstrates that APCs lacking HDAC10 are more immunogenic than control cells. Finally, to assess the role of HDAC10 in tumor immunity, we challenged HDAC10KO mice as well as WT control with murine mantle cell lymphoma (MCL) cells (FC-muMCL1 cells) given s.c. and measured tumor growth. A significant delay in MCL growth was observed in HDAC10KO mice as compared to WT controls (p<0.05). Studies are ongoing to assess the phenotype and function of APCs and T-cells isolated from these MCL-bearing mice.In summary, our studies have unveiled previously unknown immunoregulatory properties of HDAC10. In particular, its regulatory effects upon the expression of the tolerogenic molecule PD-L1, opens new frontiers for the understanding of PD-L1 regulation and points to disruption of the HDAC10/PD-L1 axis as a novel epigenetic target for cancer immunotherapy. DisclosuresSmith:Gilead: Research Funding; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Kite Pharma: Research Funding; Morphosys: Consultancy; Genentech: Membership on an entity's Board of Directors or advisory committees.

Patients with MDS Demonstrate Reduced Numbers and Impaired Differentiation of CD141Hi Conventional Dendritic Cells

Immunotherapeutic approaches to treat patients with myeloid cancers show considerable promise. Delineation of the immunologic milieu in these patients is necessary to develop rationally-designed strategies. We have previously reported a phase I trial in which intermediate/high-risk(I/HR) MDS patients were vaccinated against NY-ESO-1 tumor antigen (anti-DEC-205-NY-ESO-1 fusion protein with poly-ICLC adjuvant; CDX-1401) in combination with decitabine (20 mg/m2/day x 5 days x 4 cycles). Vaccination of MDS patients induced NY-ESO-1-specific immune responses but there was significant intra-patient variability in the quantity and quality of these responses. CDX-1401 targets multiple populations of antigen presenting cells (APCs) including CD141Hi conventional dendritic cells (cDCs). Compared to other APCs, CD141Hi cDCs express higher levels of DEC-205, an antigen uptake receptor, and TLR3, the poly-ICLC receptor. These cells regulate anti-tumor immune responses in solid tumor models and are necessary for the efficacy of checkpoint inhibitors. Thus, we hypothesized that the observed variations in immune response to vaccine resulted from quantitative and/or qualitative differences in CD141Hi cDCs in MDS. Overall, in MDS patients compared with healthy donors, there was a 9-fold decrease in the frequency of CD141Hi cDCs (0.001 ± 0.002% versus 0.009 ± 0.006% respectively; p < 0.001). In contrast with this finding, frequencies of the related CD1c+ cDC population were not different (0.18 ± 0.11% versus 0.27 ± 0.61% respectively; p = 0.12). Strikingly, MDS patients with the highest frequency of CD141Hi cDCs demonstrated a greater functional response to vaccination (both humoral and T-cell mediated). Expression of DEC-205 in cDCs from MDS patients was not lower than in those from healthy donors, suggesting that response to vaccination was not mediated by differential expression of the vaccine target. To further analyze gene expression in cDCs, we performed RNA-seq analysis of DCs isolated from healthy donors (n = 3) and a vaccine responsive MDS patient from the study. Principal component analysis showed that gene expression profiles were similar in CD141Hi and CD1c+ cDCs from MDS and normal patients, and further these profiles were distinct from plasmacytoid DCs and CD34+ progenitors. This suggests that for MDS patients with detectable CD141Hi populations, cDC function is intact. We expanded our observations to a larger cohort of untreated HR MDS patients and confirmed a selective decrease in the baseline number of bone marrow CD141Hi cDCs (n = 17; 0.002% ± 0.006%) compared to healthy donors (n = 5; 0.14 ± 0.01%; p < 0.001). We further characterized cDC progenitors in a subset of these patients using flow cytometry (n = 8). MDS patients demonstrated a population of bipotent cDC progenitors (early pre-DC) with an expected ratio of differentiation to committed CD1c+ cDC progenitors and terminally differentiated CD1c+ cDCs (p < 0.01). In contrast, the expected relationships between early pre-DCs and CD141Hi cDC committed progenitors or terminally differentiated CD141Hi cDCs was lost (p= 0.33 and 0.79 respectively). These data suggest a defect in the differentiation program for CD141Hi cDCs in MDS patients. We then hypothesized that decitabine treatment might promote a more normal cDC differentiation pattern. Using an in vitro model of cDC differentiation, we found that decitabine treatment increased differentiation of MDS CD34+ progenitors to CD1c+ cDCs by an average of 1.72 ± 0.96 fold (n = 3). However, we were unable to detect a similar effect on CD141Hi cDC differentiation. Since in vitro models do not reliably recapitulate the human environment, we analyzed serial bone marrow samples isolated from patients treated on study with decitabine and vaccination (n = 4). In these assays, 2/4 and 3/4 patients showed increased numbers of both CD141Hi and CD1c+ cDC at the end of study compared to pre-treatment. Together, these data suggest that CD141Hi cDCs are selectively reduced in patients with HR MDS and that decitabine treatment promotes cDC differentiation in vitro and in MDS patients. Optimal immunotherapeutic approaches for MDS patients may therefore benefit from strategies that address defects in APC differentiation and function. DisclosuresNo relevant conflicts of interest to declare.

COVID-19 Severity in Obesity: Leptin and Inflammatory Cytokine Interplay in the Link Between High Morbidity and Mortality.

Obesity is one of the foremost risk factors in coronavirus infection resulting in severe illness and mortality as the pandemic progresses. Obesity is a well-known predisposed chronic inflammatory condition. The dynamics of obesity and its impacts on immunity may change the disease severity of pneumonia, especially in acute respiratory distress syndrome, a primary cause of death from SARS-CoV-2 infection. The adipocytes of adipose tissue secret leptin in proportion to individuals’ body fat mass. An increase in circulating plasma leptin is a typical characteristic of obesity and correlates with a leptin-resistant state. Leptin is considered a pleiotropic molecule regulating appetite and immunity. In immunity, leptin functions as a cytokine and coordinates the host’s innate and adaptive responses by promoting the Th1 type of immune response. Leptin induced the proliferation and functions of antigen-presenting cells, monocytes, and T helper cells, subsequently influencing the pro-inflammatory cytokine secretion by these cells, such as TNF-α, IL-2, or IL-6. Leptin scarcity or resistance is linked with dysregulation of cytokine secretion leading to autoimmune disorders, inflammatory responses, and increased susceptibility towards infectious diseases. Therefore, leptin activity by leptin long-lasting super active antagonist’s dysregulation in patients with obesity might contribute to high mortality rates in these patients during SARS-CoV-2 infection. This review systematically discusses the interplay mechanism between leptin and inflammatory cytokines and their contribution to the fatal outcomes in COVID-19 patients with obesity.

Co-delivery of Phagocytosis Checkpoint Silencer and Stimulator of Interferon Genes Agonist for Synergetic Cancer Immunotherapy

Efficient capture and presentation of tumor antigens by antigen-presenting cells (APCs), especially dendritic cells (DCs), are crucial for activating the anti-tumor immunity. However, APCs are immunosuppressed in the tumor microenvironment, which hinders the tumor elimination. To reprogram APCs for inducing strong anti-tumor immunity, we report here a co-delivery immunotherapeutic strategy targeting the phagocytosis checkpoint (signal regulatory protein α, SIRPα) and stimulator of interferon genes (STING) of APCs to jointly enhance their ability of capturing and presenting tumor antigens. In brief, a small interfering RNA targeting SIRPα (siSIRPα) and a STING agonist (cGAMP) were co-delivered into APCs by the encapsulation into poly(ethylene glycol)-b-poly(lactide-co-glycolide)-based polymeric nanoparticles (NPsiSIRPα/cGAMP). siSIRPα-mediated SIRPα silence promoted APCs to actively capture tumor antigens by engulfing tumor cells. The cGAMP-stimulated STING signaling pathway further enhanced the functions of APCs, thereby increased the activation and expansion of CD8+ T cells. Using ovalbumin (OVA)-expressing melanoma as a model, we demonstrated that NPsiSIRPα/cGAMP stimulated the activation of OVA-specific CD8+ T cells and induced holistic anti-tumor immune responses by reversing the immunosuppressive phenotype of APCs. Collectively, this co-delivery strategy synergistically enhanced the functions of APCs and can be extended to the treatment of tumors with poor immunogenicity.