Expression Of B7 Research Articles

Comprehensive understanding of B7 family in gastric cancer: expression profile, association with clinicopathological parameters and downstream targets

Objectives: B7 family members were identified as co-stimulators or co-inhibitors of the immune response and played important roles in cancer immunotherapy; however, their dysregulation in gastric cancer is still unclear.Methods: Data were obtained from TCGA and GTEX database. B7 mutations, association with DNA methylation and affected proteins were analyzed in cBioportal. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and Gene Ontology (GO) project was studied by DAVID to find the downstream signaling pathway and important metabolic process, respectively. Protein-protein interaction network was analyzed in STRING and Cytoscape. A total of 160 paired specimens in tissue microarray from patients with gastric cancer were used to detect the expression levels of seven B7 family members via immunohistochemical analysis.Results: Bioinformatics studies revealed dysregulation of B7 members in gastric cancer. Gene and protein alteration were found in B7 family members. Furthermore, DNA methylation and gene alteration may be both involved in B7 member dysregulation in gastric cancer. Importantly, the high expression of B7-H6 is associated with good overall patient survival. B7 family members primarily affect the EGFR tyrosine kinase inhibitor resistance signaling pathway in gastric cancer and TP53 may be an important target of the family. The low expression of B7-1 and high expression of B7-H3 and B7-H7 were validated by IHC staining.Conclusions: Our results provide insight into B7 family member expression in gastric cancer and stress their importance in stomach tumorigenesis, which may be beneficial for designing future cancer treatments.

The effect of Moringa oleifera Lam leaf extract fermented by Lactobacillus plantarum on the expression of B220+ and CD11b+ cells in mice infected with Salmonella typhi

Defense against pathogens is essential for survival and it is controlled by innate and adaptive immunity. Phagocytosis is a complex mechanism that involved the activation of macrophages and its cytokines, stimulated T cells, then activation of B cells. Moringa oleifera plants are thought to have immunostimulatory properties due to its complex nutrients and phenolic acids and flavonoids contents as phytochemicals. Lactobacillus plantarum is able to produce lactic acid as the final product to reform carbohydrates, hydrogen peroxide, and bacteriocin as antimicrobial substances, which lead to inhibit pathogenic bacteria. This experiment aims to evaluate the CD11b+ and B220+ population after Salmonella typhi infection with/without administration of M.oleifera leaves extract. Salmonella typhi was labeled with carboxyfluorescein succinimidyl ester (CFSE) to determine phagocytosis activity. This experiment used was a complete randomized factorial pattern design. Mice were divided into two groups, namely the without treatment group and treatment group mice (fermented red M. oleifera leaves extract dose 84 mg/kg BW) and infected by Salmonella typhi. Data analysis was confirmed with the one-way ANOVA test followed by Tukey test (p<0.05). The results showed that fermented red M. oleifera leaves extract can increase the number of CD11b+and B220+ cells at the time of 2 hours and 4 hours.

High expression of B7-H3 on stromal cells defines tumor and stromal compartments in epithelial ovarian cancer and is associated with limited immune activation

BackgroundB7-H3 and B7-H4 are highly expressed by many human malignancies making them attractive immunotherapeutic targets. However, their expression patterns and immune contexts in epithelial ovarian cancer have not been well characterized.MethodsWe used flow cytometry, immunohistochemistry, and genomic analyses to determine the patterns of B7-H3, B7-H4, and PD-L1 expression by tumor, stromal, and immune cells in the ovarian tumor microenvironment (TME). We analyzed immune cell frequency and expression of PD-1, TIM3, LAG3, ICOS, TIA-1, granzyme B, 2B4, CD107a, and GITR on T cells; CD20, CD22, IgD, BTLA, and CD27 on B cells; CD16 on monocytes; and B7-H3, B7-H4, PD-L1, PD-L2, ICOSL, CD40, CD86, and CLEC9a on antigen-presenting cells by flow cytometry. We determined intratumoral cellular location of immune cells using immunohistochemistry. We compared differences in immune infiltration in tumors with low or high tumor-to-stroma ratio and in tumors from the same or unrelated patients.ResultsOn non-immune cells, B7-H4 expression was restricted to tumor cells whereas B7-H3 was expressed by both tumor and stromal cells. Stromal cells of the ovarian TME expressed high levels of B7-H3 compared to tumor cells. We used this differential expression to assess the tumor-to-stroma ratio of ovarian tumors and found that high tumor-to-stroma ratio was associated with increased expression of CD16 by monocytes, increased frequencies of PD-1high CD8+ T cells, increased PD-L1 expression by APCs, and decreased CLEC9a expression by APCs. We found that expression of PD-L1 or CD86 on APCs and the proportion of PD-1high CD4+ T cells were strongly correlated on immune cells from tumors within the same patient, whereas expression of CD40 and ICOSL on APCs and the proportion of PD-1high CD8+ T cells were not.ConclusionsThis study provides insight into the expression patterns of B7-H3 and B7-H4 in the ovarian TME. Further, we demonstrate an association between the tumor-to-stroma ratio and the phenotype of tumor-infiltrating immune cells. We also find that some but not all immune parameters show consistency between peritoneal metastatic sites. These data have implications for the design of immunotherapies targeting these B7 molecules in epithelial ovarian cancer.

Evaluation of T-Cell Compartment By Complex Multiparameter Flow Cytometry Reveals Distinct Patterns of T-Cell Exhaustion in DLBCL, FL and HL Patients

Background: Immune checkpoint (IC) blockade has revolutionized the treatment of chemo-refractory solid tumors and has demonstrated promising results for the treatment of resistant blood cancers, including lymphoma. However, clinical response to PD1 blockade depends on the subtype of lymphoma, with durable responses observed in 70 % of Hodgkin lymphoma (HL) patients versus only 36 to 40% of Diffuse Large B-Cell Lymphoma (DLBCL) and Follicular Lymphoma (FL) patients(1-3). The biological aspects that hinder the efficacy of immunotherapy in DLBCL and FL patient are not well-known. Primary tumor resistance to PD1 blockade could be driven by inherent genomic alterations or by ineffective anti-tumor T cell responses. ICs expressed at the surface of tumor-infiltrating-lymphocytes (TILs) use distinct suppressive mechanisms to enforce T-cell dysfunction. The collective expression of ICs defines multiple subsets of exhausted T cells during cancer(4). Since multiple of these ICs collaborate to repress anti-tumor T cell functions, we hypothesized that TILs from DLBCL and FL patients express high levels of ICs and present a more advanced exhausted phenotype compared to TILs from HL patients. Methods: We studied lymphoma samples collected at diagnosis and frozen as a viable cell suspension from 57 patients with HL, FL or DLBCL. We performed an extended immunophenotype of TILs through flow cytometry using maturation markers, exhaustion markers, homing and chemokine receptors. The results were analyzed with FlowJo. Boolean gating was used to assess IC co-expression. Statistical analyses were performed using Prism. Results: Our data indicated that CD8 TILs from DLBCL patients express higher levels of classical ICs such as PD1 (1059 vs 1015 and 648), Tim3 (1747 vs 868 and 918,) and Lag3 (2603 vs 1013 and 1031) compared to FL and HL patients (p-value &lt; 0.01), while the expression of 2B4, BTLA and CD160 were similar. Boolean analysis revealed that DLBCL and FL had a higher frequency of CD8T cells that co-expressed 6-7 ICs, compared to HL, suggesting a more advanced state of exhaustion (19% and 26% vs 14%, p-value &lt; 0.05). This correlated with an increased frequency of PD-1highCD39high T cells in DLBCL and FL patients compared to HL (32% and 24% vs 14%, p-value &lt; 0.005), which correlated with increased frequency of EOMEShigh cells in both DLBCL and FL (84% and 76% vs 55%, p-value &lt; 0.005). These markers identify populations of cells with phenotypic and transcriptomic profiles of terminal exhaustion with reduced cytokine production, proliferative capacity and persistence (5, 6). In contrast, stem-like exhausted CD8 T cells are defined by intermediate expression of PD-1 and expression of the chemokine receptor CXCR5, and are driven by the transcription factor TCF-1(7, 8). We observed that DLBCL exhibited significantly less progenitor Tim3-/CXCR5+ cells compared to FL and HL (27% vs 52% and 40%, p-value &lt; 0.01). Further, the frequency and mean fluorescent intensity of TCF-1 was significantly reduced in DLBCL compared to FL and HL (43% / 495 ± 64 vs 70% / 752 ± 42 and 61% / 670 ± 74, p-value &lt; 0.01). This data correlated with lower frequency of CD27+ CD8 T cells in DLBCL vs FL and HL (82% vs 96% and 96%, p-value &lt; 0.01). Conclusion: Collectively, our data indicate that TILs from DLBCL and FL patients express a whole array of ICs that have non-redundant suppressive functions. Further, these patients present subsets of cells with increased functional exhaustion, subsets demonstrated to be unresponsive to PD-1 blockade(7). Further, DLBCL patients lack protective stem-like T-cell subsets which were recently demonstrated to correlate with clinical outcome(9). These results enlighten our knowledge of the cellular defects precluding potent anti-tumor T cell functions in DLBCL and FL and propose novel targets for immunotherapies in PD-1-unresponsive lymphomas. Research funding: This work is supported by the Canadian Institutes of Health Research (PJT-155996), Canadian Cancer Society and Cole Foundation (grant #705478).

Effect of Avelumab to Immune Response in AML: A Phase I Study of Avelumab in Combination with Decitabine As First Line Treatment of Unfit Patients

Introduction: Treatment of AML remains challenging. Decitabine is a hypomethylating agent (HMA) commonly used for treating AML. However, its efficacy is limited. Recent success targeting immune checkpoints offers great promise and several agents blocking the PD-1 pathway have been FDA approved for cancer therapy. It has been demonstrated that HMA enhances PD-1 pathway in MDS and AML patients, providing a strong rationale for combining HMA and PD-1 inhibition in AML treatment. Avelumab is a PD-L1 antibody that blocks the interaction between PD-L1 and PD-1. It has been FDA approved for treating Merkel cell and urothelial carcinoma and in combination with axitinib for renal cell carcinoma. This phase I study aimed to investigate the safety of avelumab combining with decitabine in AML. In addition, extensive correlative studies were performed to define the effect of avelumab on the immune response. Methods: This is a single arm, open label phase I study to evaluate safety and tolerability of avelumab in combination with decitabine in patients with untreated AML, who are unfit for intensive chemotherapy. Decitabine was given at 20mg/m2 IV day 1-5, every 28 days. Avelumab was given at 10mg/kg IV day 1, every 14 days. Each treatment cycle was 28 days. Treatment length was up to one year or until progression. In the initial stage, classic "3+3" design was applied to determine safety. Patients were evaluated for DLTs in cycle 1. In the expansion stage, a cohort of 9 additional patients using the same regimen was planned. For correlative studies, blood and bone marrow samples were collected prior to and post initiation of avelumab. Flow cytometry-based assays for immune phenotype and functions were performed. Results: Seven patients have been treated on protocol. Median age was 71 (62-78). Most patients (6/7) carried adverse cytogenetics per ELN 2017 risk stratification. Median number of treatment cycles was 2 (range 1-12). No DLT was detected. The most frequent TEAEs (any grade or cause) were anorexia (86%), fatigue (71%), edema (71%), pleural effusion (71%), febrile neutropenia (57%) and respiratory failure (57%). Two treatment-related TRAEs were observed: fever (14%) and pneumonitis (14%). TRAEs led to treatment discontinuation in 1 patient (14%). The trial was closed before accrual met for the best interest of patients due to the recent FDA approval of venetoclax, a novel treatment for the same patient population. Among the 7 patients accrued, 1 patient achieved CR, 3 patients stable disease, 1 had progression of disease; response could not be assessed in 2 patients due to death before response assessment. To investigate the effect of avelumab on immune response, we conducted complex immune assays using blood samples collected from each patient prior to vs. 1 month post treatment. We observed no alteration in the frequency of each immune component (NK, NKT, B cells, DCs, Monocytes, CD4 T cells, CD8 T cells, and Treg) upon avelumab and decitabine treatment. However, there was an increase in effector memory differentiation while decreased terminal differentiated subsets in CD8 T cells. We next performed comprehensive phenotypic and functional analysis of CD8 T cells. Data of the first accrued 6 patients are shown here (Figure 1); the data from patient 7 is underway and will be integrated into the presentation at the meeting. We observed a strong trend of up-regulation of activation markers and co-stimulatory receptors (CD69, CD226, CD28, ICOS and 4-1BB) on CD8 T cells post treatment of avelumab and decitabine. In contrast, the expression of inhibitory molecules, including TIGIT, TIM-3, CD160, LAG-3, 2B4 and BTLA, were trending down. Consistently, CD8 T cell function was enhanced in majority of patients, manifested by higher expression of granzyme B, perforin and Ki67, as well as more cytokine release (IFN-γ and TNF-α) upon in vitro TCR engagement. Importantly, avelumab is likely the major contributor for the positive regulatory effect as studies on samples from patients who received decitabine alone did not show the same trend. Conclusion: In this phase I study, we demonstrated that avelumab in combination with decitabine as first line treatment of unfit patients with AML is safe and tolerable. CD8 T cell response trends up upon avelumab treatment. Our study strongly supports the addition of avelumab to AML treatment with a potential to improve anti-leukemia immunity. Disclosures Zheng: Pfizer: Research Funding. Claxton:Daiichi Sankyo Co. and Ambit Biosciences Corp, Astellas Pharma, Novartis Pharmaceuticals, Incyte Corporation, Cyclacel Pharmaceuticals, Inc, Celegene Corporation, Medimmune, Inc, Merck Sharp &amp; Dohme Corp., Gilead Sciences, Inc.: Research Funding. Naik:Celgene: Other: Advisory board.

Implications of Immune Checkpoint Expression During Aging in HIV-Infected People on Antiretroviral Therapy.

Immune checkpoint molecules (ICMs) regulate T cell responses. In chronic viral infections and cancer, where antigens can persistently stimulate the immune system, ICMs can serve as a barrier to effective immune responses. The role of ICMs in the setting of systemic low-grade inflammation as in aging and antiretroviral therapy (ART)-suppressed HIV infection is not known. In this study, we made use of stored samples from the FLORAH cohort of HIV-infected ART-suppressed adults (age range 19-77 years.) and age-matched HIV-uninfected controls. We measured the expression levels of ICMs: PD-1, LAG-3, TIGIT, TIM-3, and 2B4 on resting CD4 and CD8 T cells and maturation subsets. To determine how expression of these molecules can affect T cell function, we stimulated peripheral blood mononuclear cell with HIV Gag or p09/H1N1 antigen and performed intracellular cytokine staining by multiparameter flow cytometry. ICMs were expressed at higher levels in CD8 compared with CD4. PD-1 was the only molecule that remained significantly higher in HIV-infected individuals compared with controls. LAG-3 expression increased with age in CD4 and CD8 T cells. 2B4 expression on CD8 T cells was negatively associated with IL-2 production but showed no effect on CD4 T cell function. TIM-3 expression was negatively associated with IL-21 production in CD4 and CD8 T cells and also negatively correlated with flu vaccine responses in HIV-negative individuals. Taken altogether, this study demonstrates the marked variation in ICM expression in T cells among adults and sheds light on the biology of these molecules and their effects on antigen-specific T cell functions. Overall, our results point to TIM-3 as a potential biomarker for immune function in HIV+ individuals on ART.

Abstract 4055: Late-differentiated effector neoantigen-specific CD8+ T cells are enriched in non-small cell lung cancer patients responding to atezolizumab treatment

Abstract There is strong evidence that immunotherapy-mediated tumor rejection can be driven by the reinvigoration of tumor-specific CD8+ T cells recognizing neoantigens derived from tumor somatic mutations (citations). Thus, it is possible that the relative abundance and/or characteristics of these tumor-reactive, mutation-specific CD8+ T cells can be used as predictive biomarkers of response to immunotherapy. However, only a fraction of these potential neoantigens are usually immunogenic and in addition, these tumor-reactive, mutation-specific CD8+ cells are present only at low frequencies in blood, making it difficult to reliably identify these effector cells. Here, mass cytometry and highly-multiplexed combinatorial tetramer staining together with cellular barcoding was used to profile immune cells in longitudinally collected PBMCs from 14 non-small cell lung cancer (NSCLC) patients treated with anti-PD-L1 (atezolizumab) antibody to compare patients with objective response (n=8) and progressive disease (n=6). Although no significant phenotypic differences were detected in bulk CD8+ T cells, greater insight was gained from a parallel analysis using highly multiplexed peptide-MHC multimer staining to screen and profile antigen-specific T cells. A longitudinal analysis was performed using peripheral blood CD8+ T cells for 800 candidate tumor neoantigens and 73 known viral-derived control peptides across all patient samples. In addition to virus antigen-specific T cells, a total of 20 different neoantigen-specific T cell populations were detected and their high dimensional profiles were compared. We found that neoantigen-specific T cells were more frequently detected in responding patients and their phenotypes were almost entirely distinct from non-responding patients. Neoantigen-specific T cells from responding patients showed a differentiated effector phenotype with high expression of KLRG1, 2B4 (CD244) and CD57, similar to CD8+ T cells associated with CMV and some types of EBV infection. In contrast, more memory-like phenotypic profiles, with high CD27 and CD127 expression, were observed for neoantigen-specific CD8+ T cells from patients with progressive disease. In addition to the utility of this approach for the ex vivo identification, characterization, and longitudinal tracking of rare tumor-specific T cells, this study supports further research into assessing whether the presence of late-differentiated neoantigen-specific T cells could be used as a predictor of response to checkpoint blockade. Citation Format: Mahesh Yadav, Michael Fehlings, Suchit Jhunjhunwala, Bill O'Gorman, Priti Hegde, Leesun Kim, Alessandra Nardin, Susan Flynn, Hermi Sumatoh, Marcus Ballinger, David Shames, Boon Heng Lee, Evan Newell. Late-differentiated effector neoantigen-specific CD8+ T cells are enriched in non-small cell lung cancer patients responding to atezolizumab treatment [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 4055.

CD8+ T cells retain protective functions despite sustained inhibitory receptor expression during Epstein-Barr virus infection in vivo.

Epstein Barr virus (EBV) is one of the most ubiquitous human pathogens in the world, persistently infecting more than 90% of the adult human population. It drives some of the strongest human CD8+ T cell responses, which can be observed during symptomatic primary infection known as infectious mononucleosis (IM). Despite high viral loads and prolonged CD8+ T cell stimulation during IM, EBV enters latency and is under lifelong immune control in most individuals that experience this disease. We investigated whether changes in T cell function, as frequently characterized by PD-1 up-regulation, occur during IM due to the prolonged exposure to high antigen levels. We readily detected the expansion of PD-1 positive CD8+ T cells together with high frequencies of Tim-3, 2B4, and KLRG1 expression during IM and in mice with reconstituted human immune system components (huNSG mice) that had been infected with a high dose of EBV. These PD-1 positive CD8+ T cells, however, retained proliferation, cytokine production, and cytotoxic abilities. Multiple subsets of CD8+ T cells expanded during EBV infection, including PD-1+Tim-3+KLRG1+ cells that express CXCR5 and TCF-1 germinal center homing and memory markers, and may also contain BATF3. Moreover, blocking the PD-1 axis compromised EBV specific immune control and resulted in virus-associated lymphomagenesis. Finally, PD-1+, Tim-3+, and KLRG1+ CD8+ T cell expansion coincided with declining viral loads during low dose EBV infection. These findings suggest that EBV infection primes PD-1 positive CD8+ T cell populations that rely on this receptor axis for the efficient immune control of this ubiquitous human tumor virus.

Cholesterol Induces CD8+ T Cell Exhaustion in the Tumor Microenvironment

Tumor-infiltrating Tcells often lose their effector function; however, the mechanisms are incompletely understood. We report that cholesterol in the tumor microenvironment induces CD8+ Tcell expression of immune checkpoints and exhaustion.Tumor tissues enriched with cholesterol and cholesterol content in tumor-infiltrating CD8+ Tcells were positively and progressively associated with upregulated Tcell expression of PD-1, 2B4, TIM-3, and LAG-3. Adoptively transferred CD8+ Tcells acquired cholesterol, expressed high levels of immune checkpoints, and became exhausted upon entering a tumor. Tumor culture supernatant or cholesterol induced immune checkpoint expression by increasing endoplasmic reticulum (ER) stress in CD8+ Tcells. Consequently, the ER stress sensor XBP1 was activated and regulated PD-1 and 2B4 transcription. Inhibiting XBP1 or reducing cholesterol in CD8+ Tcells effectively restored antitumor activity. This study reveals a mechanism underlying Tcell exhaustion and suggests a new strategy for restoring Tcell function by reducing cholesterol to enhance Tcell-based immunotherapy.

Expression Regulation of Hox Genes by Perfluorochemicals in Mouse Liver

Homeobox (Hox) genes encode homeodomain proteins, which play important roles in the development and morphological diversification of plants and animals. Perfluorochemicals (PFCs), which are the persistent environmental pollutants and frequently detected in human blood, can interfere with animal development. PFCs produce numerous hepatic adverse effects, such as increased peroxisomes, hepatomegaly and altered lipid metabolism, primarily via activation of peroxisome proliferator‐activated receptors (PPARs) and/or constitutive androstane receptor (CAR). No study has reported that PFCs can regulate the expression of Hox genes. This study was designed to determine the expression regulation of Hox and paraHox genes by perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), and perfluorodecanoic acid (PFDA) in mouse liver. A single dose of 0.1 mmole/kg PFOA, PFNA and PFDA was intraperitoneally administered to adult male wild‐type, and age‐matched male PPARα‐null and male CAR‐null mice (n=6/treatment). Four days later, mouse livers were collected and processed. Messenger RNA expression of Hox genes (including Hox‐a to ‐d subfamily members) and paraHox genes (including Gsx, Pdx, and Cdx gene family members) were determined. Our results showed that PFOA, PFNA, and PFDA all induced mRNA expression of Hoxb7, c5, d10 and Zeb2 in wild‐type mouse liver, but not in PPARα‐null mouse liver. We further showed that PFNA induced mRNA expression of Hoxa5, b7, c5, c8, and d10 in livers of wild‐type and CAR‐null mice, but not in PPARα‐null mouse liver, indicating a PPARα‐dependent mechanism. Furthermore, knocking‐out of PPARα or CAR in mouse liver decreased constitutive mRNA expression of Hoxc5. Wy‐14643, a classical PPARα agonist, induced mRNA expression of Hoxc5, but not of Hoxa5, b7 or d10 in wild‐type mouse liver. 1,4‐bis‐[2‐(3,5,‐dichloropyridyloxy)] benzene (TCPOBOP), a classical CAR agonist, also induced mRNA expression of Hoxc5, but not of Hoxa5, b7 or d10 in wild‐type mouse liver. Messenger RNA expression of studied paraHox genes were not apparently altered by PFCs in mouse liver. In conclusion, perfluorochemicals induced mRNA expression of certain Hox genes such as Hoxc5, maybe via activation of PPARα and/or CAR.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Association of increased B7 protein expression by infiltrating immune cells with progression of gastric carcinogenesis.

B7 negative costimulatory molecules are a group of molecules associated with the occurrence, development, and therapy of cancers. Here, we aimed to determine the clinical significance of PD-L1, B7-H3, and B7-H4 and their expression in CD8 and CD68 positive cells at different stages of gastric carcinogenesis.We detected PD-L1, B7-H3, B7-H4, CD8, and CD68 expression in samples by immunohistochemical staining of 62 chronic superficial gastritis (CSG) samples, 72 chronic atrophic gastritis (CAG) samples, 68 low-grade intraepithelial neoplasia (LIN) samples, 65 high-grade intraepithelial neoplasia (HIN) samples obtained from gastroscopic biopsies and 50 gastric adenocarcinoma (GA) samples obtained from surgical resections. Then we statistically analyzed the expression differences and correlations.Our results indicated that B7 and CD68 expression on infiltrating immune cells was associated with disease progression. However, infiltration of CD8+ cells decreased with disease progression. B7-H3 expression was markedly enhanced at neoplasia and GA stages. B7-H3 in tumor cells was negatively correlated with CD8-expressing cells. Conversely, B7-H3 expression in tumor-infiltrating immune cells was positively correlated with CD68-expressing cells. B7-H4 expression was found in the cell membrane at the stages of gastritis and low-grade neoplasia and was gradually expressed in the cytoplasm at high-grade neoplasia and GA stages. High B7-H4 expression in infiltrating immune cells was also significantly associated with lower CD8-positive and higher CD68-positive cell densities.Increased B7 protein expression by infiltrating immune cells was associated with disease progression, and specifically, the level of B7-H3 expression and localization of B7-H4 expression differed significantly among different stages of gastric carcinogenesis.

A Subpopulation of Synovial Fibroblasts Leads to Osteochondrogenesis in a Mouse Model of Chronic Inflammatory Rheumatoid Arthritis.

Specific major histocompatibility complex (MHC) class II genes result in a high susceptibility to rheumatoid arthritis (RA), with co-stimulatory molecules working together with MHC class II during the progression of the disease. To elucidate the involvement of the B7.1 co-stimulatory molecule in RA, we analyzed the phenotype of B7.1 transgenic (named D1BC) mice and the sequential differentiation of synovial fibroblasts (SFs) by studying the expression of chondrogenic and osteogenic lineage markers together with lineage tracing experiment using B7.1 transgene in vivo. The B7.1 transgene was driven by a collagen type II (CII) promoter and enhancer in the D1BC mouse. A low-dose of bovine CII (bCII) was used to induce chronic articular inflammation with interstitial pneumonitis. Joint damage was analyzed by histopathological examination and computed tomography. B7.1 was expressed in articular cartilage and SFs of D1BC mice. Chronic inflammatory arthritis in the bCII-D1BC mouse shared common features with those found in patients with RA, such as pannus formation, bone destruction, osteoporosis, and joint ankylosis. A subpopulation of SFs (Runx2 +, Sox9 +, Col10a1 +, Osx+, and CX-) in the pannus was classified as osteochondrogenic lineage rather than mesenchymal stromal lineage. These cells underwent differentiation into osteogenic lineage via hypertrophic chondrocytes at the end of the chronic phase. The ectopic expression of B7.1 in chondrocytes and SFs leads to an increased susceptibility to chronic inflammatory arthritis and subsequent new bone formation, reminiscent of ankylosis. The regulation of cartilage remodeling in pannus tissue is an important consideration in the treatment of RA.

Unregulated antigen-presenting cell activation by T cells breaks self tolerance

T cells proliferate vigorously following acute depletion of CD4+ Foxp3+ T regulatory cells [natural Tregs (nTregs)] and also when naive T cells are transferred to syngeneic, nTreg-deficient Rag1-/- hosts. Here, using mice raised in an antigen-free (AF) environment, we show that proliferation in these two situations is directed to self ligands rather than food or commensal antigens. In both situations, the absence of nTregs elevates B7 expression on host dendritic cells (DCs) and enables a small subset of naive CD4 T cells with high self affinity to respond overtly to host DCs: bidirectional T/DC interaction ensues, leading to progressive DC activation and reciprocal strong proliferation of T cells accompanied by peripheral Treg (pTreg) formation. Likewise, high-affinity CD4 T cells proliferate vigorously and form pTregs when cultured with autologous DCs in vitro in the absence of nTregs: this anti-self response is MHCII/peptide dependent and elicited by the raised level of B7 on cultured DCs. The data support a model in which self tolerance is imposed via modulation of CD28 signaling and explains the pathological effects of superagonistic CD28 antibodies.

Pomalidomide increases immune surface marker expression and immune recognition of oncovirus-infected cells

ABSTRACTMost chronic viruses evade T-cell and natural killer (NK) immunity through downregulation of immune surface markers. Previously we showed that Pomalidomide (Pom) increases surface expression of major histocompatibility complex class I (MHC-I) in Kaposi sarcoma-associated herpesvirus-infected latent and lytic cells and restores ICAM-1 and B7-2 in latent cells. We explored the ability of Pom to increase immune surface marker expression in cells infected by other chronic viruses, including human T-cell leukemia virus type-1 (HTLV-1), Epstein-Barr virus (EBV), human papilloma virus (HPV), Merkel cell polyoma virus (MCV), and human immunodeficiency virus type-1 (HIV-1). Pom increased MHC-1, ICAM-1, and B7-2/CD86 in immortalized T-cell lines productively infected with HTLV-1 and also significantly increased their susceptibility to NK cell-mediated cytotoxicity. Pom enhancement of MHC-I and ICAM-1 in primary cells infected with HTLV-1 was abrogated by knockout of HTLV-1 orf-1. Pom increased expression of ICAM-1, B7-2 and MHC class I polypeptide related sequence A (MICA) surface expression in the EBV-infected Daudi cells and increased their T-cell activation and susceptibility to NK cells. Moreover, Pom increased expression of certain of these surface markers on Akata, Raji, and EBV lymphoblastic cell lines. The increased expression of immune surface markers in these virus-infected lines was generally associated with a decrease in IRF4 expression. By contrast, Pom treatment of HPV, MCV and HIV-1 infected cells did not increase these immune surface markers. Pom and related drugs may be clinically beneficial for the treatment of HTLV-1 and EBV-induced tumors by rendering infected cells more susceptible to both innate and adaptive host immune responses.

Inducible Correction of a RUNX1 Mutation of Human AML Causes a Switch of AML to B-ALL

Background: Point mutations of RUNX1 gene are frequently seen in myeloid malignancies such as myelodysplasia (MDS) and acute myeloid leukemia (AML), and are associated with unfavorable clinical outcomes. AML patients bearing RUNX1 mutations have a significantly lower complete remission (CR) rate compared with patients with wild-type RUNX1 (30% vs. 73%), whereas in contrast, the CR rate of ALL patients, in which RUNX1 is rarely mutated, are at 92%. It remains unclear whether RUNX1 mutations found in AML patients are causal to AML maintenance, and if RUNX1 mutations serve as a valid therapeutic target in the treatment of such therapy-resistant AML.Experimental Approach: The RUNX1 mutations are found frequently associated with MLL-partial tandem duplication (MLL-PTD) in AML. We have developed two complementary mouse models that express a MLL-PTDknock-in mutation together with a RUNX1 S291fsX300 mutation found in AML patients: in the first model RUNX1 S291fsX300 was introduced into the bone marrow cells of the MLL-PTD knock-in mice by retrovirus transduction, while in the second model the tetracycline-inducible RUNX1 S291fsX300 mutation was knock-in at the Collagen a1 locus and the mice was crossed to MLL-PTD knock-in. In the second model, the mutant RUNX1 protein is only expressed when the mice were fed with doxycycline containing food and the RUNX1 mutant is absent or “corrected” upon doxycycline withdrawn. BM cells of these mice, as well as WT and MLL-PTD controls, were transplanted to syngeneic mice and the recipients were tracked for disease development and progression at bi-monthly intervals. The doxycycline induction or withdraw was carried out after secondary transplant, and the expression of RUNX1 S291fsX300 was monitored by a built-in GFP reporter and verified by RT-PCR.Under tetracycline induction, the RUNX1 S291fsX300 and MLL-PTD double mutation bearing mice developed a spontaneously AML and had a survival time 6-10 months after transplantation, and they showed symptoms of MDS/AML or AML, including thrombocytopenia, anemia, leukocytosis, splenomegaly, abnormal BM and spleen cell morphologies. We transplanted the AML mouse bone marrow to secondary recipients, and observed them with or without continuing doxycycline induction. By using the O-propargyl-puromycin to track the newly synthesized proteins, we found that the RUNX1 mut-on and RUNX1 mut-off mice showed comparable protein synthesis rate at full-blown leukemia stage, indicating that the RUNX1 mutation does not play a restrictive role to protein synthesis in transformed leukemia cells. The c-Kit+ AML cells cultured from the RUNX1 mut-off mice showed reduced viability and significantly reduced CFU-C activities, which are accompanied by a lineage switch in a decrease in expression of Mac1 and an increase in expression of B220. In vivo, the RUNX1 mut-off mice showed a slightly prolonged survival compared with RUNX1 mut-on group (182 vs. 126 days, p=0.0036). An analysis of the lineage distribution in their peripheral blood and bone marrow revealed that the RUNX1 mut-off AML mice underwent a dramatic lineage switch from myelocytes to lymphocytes (Fig. 1): while the Mac1+Gr1+population remained high in the RUNX1 mut-on AML mice, turning off RUNX1 mut expression caused an increased B cell population in expense of the Mac1+Gr1+population. Pathological analysis also showed a drastic decrease of the myeloid marker MPO and increased lymphoid marker B220 in the RUNX1mu-off mice. Consistently, a RT-PCR test of the BM c-Kit+ cells further showed that upon turning off the RUNX1mut, critical B cell regulatory genes including TCF3, EBF1, PAX5, CD79A were significantly up regulated whereas myeloid regulatory genes such as PU.1 were downregulated. In addition, RUNX1 S291fsX300 expression in the AML cells confers a resistance to chemotherapy (Ara-C) treatment compared with that of WT RUNX1 expression, indicating that the RUNX1 mutation is responsible for the AML resistance to therapy.Conclusion:Although RUNX1 mutations found in AML patient may not be essential for the hyperproliferative and growth phenotype of the AML, they play a causal role in maintaining myeloid lineage restriction, expansion and chemo-resistance. Correction or removal of the RUNX1 mutation in AML cells leads to a disease switch from AML to B-ALL, effectively changing the difficult-to-treat drug-resistant AML to another disease with more favorable CR (B-ALL). [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Notch Activation Rescues Exhaustion in CISH-Deleted Human iPSC-Derived Natural Killer Cells to Promote In Vivo Persistence and Enhance Anti-Tumor Activity

Cytokine-inducible SH2-containing protein (CIS) is a critical negative regulator of IL-15 signaling in natural killer (NK) cells and is encoded by CISH gene in human. Recent studies in a murine model demonstrate that CIS is a potent inhibitory checkpoint in NK cell-mediated tumor immunity. However, it is unclear if CIS similarly regulates human NK cell-mediated anti-tumor activity. Unlike CTLA-4 and PD1 that are expressed at the cell surface and can be blocked by antibody-mediated therapy, CIS is expressed intracellularly. Therefore, we deleted CISH gene in human induced pluripotent stem cells (iPSCS) using CRISPR/Cas9 technology and characterized the CISH-knockout iPSC lines. Our study demonstrates that CISH does not regulate maintenance of undifferentiated human pluripotent stem cells. We then derived NK cells from these gene-modified iPSCs using a two-stage in vitro hematopoietic-lymphoid differentiation protocol developed by our group. Initial hematopoietic differentiation of the iPSCs was unaffected by CISH deletion. However, deletion of CISH in iPSCs markedly delayed the second stage of in vitro NK cell differentiation. Specifically, whereas NK cell differentiation was typically fully complete with >90% NK cells after 4 weeks using WT iPSCs, the CISH-/- iPSC- cells only produced ~10% CD45+CD56+ NK cells at 4 weeks, though by 5 weeks these cultures were >80% NK cells. The CISH-/- iPSC-derived NK cells demonstrated typical NK surface maker expression, including CD94, CD16, NKG2D, NKp44, NKp46, FasL, and KIRs. Initial studies demonstrated that CISH-/- human iPSC-derived NK cells had significantly reduced ability to expand in vitro with evidence of NK cell exhaustion, including increased TIM-3 expression, decreased IFN-γ production and decreased cytotoxicity. RNA-seq analysis also confirmed that expression of exhaustion-related genes, including TIM3, CTLA4, and 2B4, were all significantly increased in CISH-/- NK cells. Interestingly, mass cytometric (CyTOF) analysis of CISH-/- NK cells with a panel of 36 phenotypic and functional NK cell markers identified an exhausted sub-population (increased expression of exhaustion marker TIM3 and inhibitory receptors such as ILT2 and Siglec7, as well as decreased proliferation marker Ki67). Importantly, this exhaustion could be rescued by co-culturing CISH-/- NK cells with Notch ligand-expressing OP9-DL4 stromal cells, leading to production of fully functionally mature NK cells. The CISH-/- NK cells stimulated by Notch maintained better expansion and improved cytotoxic function with low concentrations of either IL15 or IL2 compared to WT-iPSC-derived NK cells. CISH-/- NK cells demonstrate increased cytotoxic activity against leukemia cell lines K562 and MOLM-13 cells in vitro. Moreover, single-cell cytokine response analysis of CISH-/- NK cells after Notch stimulation showed >10 fold enrichment of polyfunctional cell subsets with effector cytokine production (Granzyme B, IFN-γ, MIP-1α, Perforin, TNF-α) compared with WT NK cells or NK cells from peripheral blood. In a MOLM-13 xenograft model, CISH-/- NK cells displayed significantly increased persistence in peripheral blood in comparison with WT NK cells (CISH-/- NK cells 6.81 ± 0.85 % vs WT NK cells 2.05 ± 0.25 N=5). More importantly, CISH-/- NK cells show significantly better control of tumor progression in the MOLM-13 xenograft model compared with WT NK cells. Together, these studies demonstrate CISH plays a key role to regulate NK cell activation-induced exhaustion and that Notch activation prevents this exhaustion to enable production of functionally hyperactive NK cells. DisclosuresGuan:Vivace: Equity Ownership. Malmberg:Fate Therapeutics Inc.: Consultancy, Research Funding. Kaufman:Fate Therapeutics: Consultancy, Research Funding.

The relationship between homeobox B7 expression and the clinical characteristics of patient with prostate cancer.

The prognosis of patients with prostate cancer (PCa) remains poor. GSE16560, GSE32448, GSE79957, GSE17951, and TCGA-PRAD were reanalyzed to evaluate the expression of homeobox B7 (HOXB7) between PCa tissues and normal prostate tissues and to characterize the correlation between the expression of HOXB7 and the clinicopathological features ofpatients with PCa. Gene set enrichment analysis was conducted to investigate the mechanisms. HOXB7 was upregulated in PCa tissues (P = 0.0005). Both the univariate and multivariate analyses demonstrated that the expression of HOXB7 was correlated with the Gleason score and TNM staging of patients with PCa. The Gleason score and TNM staging were higher in the HOXB7 high expression group. The overall survival (hazard ratio [HR] = 0.632; 95% confidence interval [CI]: 0.4773-0.8369; P = 0.0014) and progression-free survival (HR = 0.544; 95% CI: 0.3157-0.9373; P = 0.0283) favored patients with PCa in HOXB7 low expression group over those in HOXB7 high expression group. PCa samples in HOXB7 low expression group were enriched in gene sets associated with the epithelial mesenchymal transition, apical junction, angiogenesis, ultraviolet response, and hypoxia. HOXB7 might be an independent prognostic factor of patients with PCa.

IL-33 drives the antitumor effects of dendritic cells via the induction of Tc9 cells.

Dendritic cell (DC) tumor vaccines exert their antitumor effects through the induction of effector T cells. We recently identified Tc9 cells as a new potent antitumor effector T cell subset. However, approaches to direct DCs to preferably prime antitumor Tc9 cells should be further exploited. Here, we demonstrate that the addition of interleukin (IL)-33 potently promotes the induction of Tc9 cells by DCs in vitro and in vivo. IL-33 treatment also drives the cytotoxic activities of DC-induced Tc9 cells. Notably, IL-33 treatment enhances cell survival and proliferation of DC-primed CD8+ T cells. More importantly, the addition of IL-33 during in vitro priming of tumor-specific Tc9 cells by DCs increases the antitumor capability of Tc9 cells. Mechanistic studies demonstrated that IL-33 treatment inhibits exhaustive CD8+ T cell differentiation by inhibiting PD-1 and 2B4 expression and increasing IL-2 and CD127 (IL-7 receptor-α, IL-7Rα) expression in CD8+ T cells. Finally, the addition of IL-33 further promotes the therapeutic efficacy of DC-based tumor vaccines in the OT-I mouse model. Our study demonstrates the important role of IL-33 in DC-induced Tc9 cell differentiation and antitumor immunity and may have important clinical implications.

2B4 Mediates Inhibition of CD8+ T Cell Responses via Attenuation of Glycolysis and Cell Division.

We recently showed that 2B4 expression on memory T cells in human renal transplant recipients was associated with reduced rates of rejection. To investigate whether 2B4 functionally underlies graft acceptance during transplantation, we established an experimental model in which 2B4 was retrogenically expressed on donor-reactive murine CD8+ T cells (2B4rg), which were then transferred into naive recipients prior to skin transplantation. We found that constitutive 2B4 expression resulted in significantly reduced accumulation of donor-reactive CD8+ T cells following transplantation and significantly prolonged graft survival following transplantation. This marked reduction in alloreactivity was due to reduced proliferation of CD8+ Thy1.1+ 2B4rg cells as compared with control cells, underpinned by extracellular flux analyses demonstrating that 2B4-deficient (2B4KO) CD8+ cells activated in vitro exhibited increased glycolytic capacity and upregulation of gene expression profiles consistent with enhanced glycolytic machinery as compared with wild type controls. Furthermore, 2B4KO CD8+ T cells primed in vivo exhibited significantly enhanced ex vivo uptake of a fluorescent glucose analogue. Finally, the proliferative advantage associated with 2B4 deficiency was only observed in the setting of glucose sufficiency; in glucose-poor conditions, 2B4KO CD8+ T cells lost their proliferative advantage. Together, these data indicate that 2B4 signals function to alter T cell glucose metabolism, thereby limiting the proliferation and accumulation of CD8+ T cells. Targeting 2B4 may therefore represent a novel therapeutic strategy to attenuate unwanted CD8+ T cell responses.

Immunomodulation Mediated by Anti-angiogenic Therapy Improves CD8 T Cell Immunity Against Experimental Glioma.

Glioblastoma (GBM) is a lethal cancer of the central nervous system with a median survival rate of 15 months with treatment. Thus, there is a critical need to develop novel therapies for GBM. Immunotherapy is emerging as a promising therapeutic strategy. However, current therapies for GBM, in particular anti-angiogenic therapies that block vascular endothelial growth factor (VEGF), may have undefined consequences on the efficacy of immunotherapy. While this treatment is primarily prescribed to reduce tumor vascularization, multiple immune cell types also express VEGF receptors, including the most potent antigen-presenting cell, the dendritic cell (DC). Therefore, we assessed the role of anti-VEGF therapy in modifying DC function. We found that VEGF blockade results in a more mature DC phenotype in the brain, as demonstrated by an increase in the expression of the co-stimulatory molecules B7-1, B7-2, and MHC II. Furthermore, we observed reduced levels of the exhaustion markers PD-1 and Tim-3 on brain-infiltrating CD8 T cells, indicating improved functionality. Thus, anti-angiogenic therapy has the potential to be used in conjunction with and enhance immunotherapy for GBM.