Levels Of CTLA-4 Research Articles

Clinical Relevance of Soluble Forms of Immune Checkpoint Molecules sPD-1, sPD-L1, and sCTLA-4 in the Diagnosis and Prognosis of Ovarian Cancer

It is crucial to find new diagnostic and prognostic biomarkers. A total of 80 patients were enrolled in the study. The study group consisted of 37 patients with epithelial ovarian cancer, and the control group consisted of 43 patients with benign ovarian cystic lesions. Three proteins involved in the immune response were studied: PD-1, PD-L1, and CTLA-4. The study material was serum and peritoneal fluid. The ROC curve was plotted, and the area under the curve was calculated to characterize the sensitivity and specificity of the studied parameters. Univariate and multivariate analyses were performed simultaneously using the Cox regression model. The cut-off level of CTLA-4 was 0.595 pg/mL, with the sensitivity and specificity of 70.3% and 90.7% (p = 0.000004). Unfavorable prognostic factors determined in serum were: PD-L1 (for PFS: HR 1.18, 95% CI 1.11–1.21, p = 0.016; for OS: HR 1.17, 95% CI 1.14–1.19, p = 0.048) and PD-1 (for PFS: HR 1.01, 95% CI 0.91–1.06, p = 0.035). Unfavorable prognostic factors determined in peritoneal fluid were: PD-L1 (for PFS: HR 1.08, 95% CI 1.01–1.11, p = 0.049; for OS: HR 1.14, 95% CI 1.10–1.17, p = 0.045) and PD-1 (for PFS: HR 1.21, 95% CI 1.19–1.26, p = 0.044). We conclude that CTLA-4 should be considered as a potential biomarker in the diagnosis of ovarian cancer. PD-L1 and PD-1 concentrations are unfavorable prognostic factors for ovarian cancer.

Single-cell landscape reveals active cell subtypes and their interaction in the tumor microenvironment of gastric cancer

Background: Gastric cancer remains the third most common cause of cancer-related death worldwide. The development of novel therapeutic strategies for gastric cancer requires a deep understanding of the tumor cells and microenvironment of gastric cancer.Methods: We performed the single-cell RNA sequencing (scRNA-seq) on nine untreated non-metastatic gastric cancer patients. The transcriptomic atlas and ligand-receptor-based intercellular communication networks of the single cells were characterized.Results: Here, we profiled the transcriptomes of 47,304 cells from nine patients with gastric cancer. Tregs cells were significantly enriched in the gastric tumor tissues with increased expression of immune suppression related genes, which suggest a more immunosuppressive microenvironment. We also observed the absence of separate exhausted CD8+ T cell cluster, and the low expression level of exhaustion markers PDCD1, CTLA4, HAVCR2, LAG-3, and TIGIT in those specific cells. These may serve as molecular-level evidence for the limited benefit of immunotherapy among gastric cancer patients. In addition, we found ACKR1 specifically expressed in tumor endothelial cells, associated with poor prognosis in the cohort data and potentially provided a novel target of gastric cancer treatment. Furthermore, the tight interaction between endothelial cells and fibroblast implied the important roles of fibroblast in tumor angiogenesis and the maintenance of tumor vasculature.Conclusions: In conclusion, this single-cell atlas provide understanding the cellular heterogeneity from molecular level in gastric cancer and will serve as a valuable resource for developing innovative early and companion diagnostics, as well as discovering novel targeted therapies for gastric cancer.

The association between parameters of oral mucosal immunity and 25-hydroxyvitamin D in patients with rampant caries

Aim. To determine the saliva level of immunoregulatory proteins in patients with rampant caries and 25-hydroxyvitamin D (25(OH)D) deficiency and evaluate the association of their concentration with 25(OH)D plasma level.Materials and methods. The study was performed in two groups. The experimental group included 15 patients aged 20–22 years with rampant caries and the 25(OH)D plasma level of < 20 ng / ml. The control group encompassed 15 healthy age-matched volunteers with the 25(OH)D plasma level of 20–100 ng / ml. The concentrations of B7.2 (CD86), free active TGF-β1, CTLA-4, PD-1, Tim-3, LAG-3, IGFBP-4, and ICAM-1 were assessed using flow cytometry. The levels of LL-37 and secretory immunoglobulin A (sIgA) were measured using ELISA. The Spearman’s rank correlation coefficient was used to reveal a correlation between the indicated proteins and the 25(OH)D plasma level.Results. A decrease in B7.2 (CD86), PD-1, Tim-3, sIgA, and LL-37 and elevation of IGFBP-4 and ICAM-1 saliva levels were detected in patients with rampant caries and 25-hydroxyvitamin D deficiency. A positive Spearman’s rank correlation coefficient was revealed between plasma 25(OH)D and saliva levels of free active TGF-β1, CTLA4, B7.2 (CD86), LL-37, and sIgA. A negative correlation was revealed between 25(OH)Dand ICAM-1.Conclusion. 25(OH)D deficiency in patients with rampant caries is associated with decreased levels of B7.2 (CD86), PD-1, Tim-3, sIgA, and LL-37 and elevated levels of IGFBP-4 and ICAM-1 in the saliva.

ACSL4 Expression Is Associated With CD8+ T Cell Infiltration and Immune Response in Bladder Cancer.

ObjectiveThis study aimed to explore the role of ACSL4 in CD8+ T cell tumor infiltration and outcomes of bladder cancer (BLCA) patients after immunotherapy.MethodsThe correlation between ACSL4 expression and tumor infiltration of immune cells was analyzed using the Tumor Immune Estimation Resource database. The prognostic significance of ACSL4 in BLCA was analyzed using Kaplan–Meier curves. Immunohistochemistry was used to detect CD8+ T cell infiltration in tumors with high and low ACSL4 expression obtained from patients at the Fudan University Shanghai Cancer Center. The relationships between immune checkpoint genes and immune response were analyzed using The Cancer Genome Atlas and IMvigor 210 cohorts. The molecular functions, cellular components, and biological processes involving ACSL4 were explored using Kyoto Encyclopedia of Genes and Genomes and Gene Ontology enrichment pathway analyses.ResultsThe expression level of ACSL4 was significantly correlated with the infiltration of CD8+ T cells in BLCA tumors (r = 0.192, P = 2.22e-04). Elevated ACSL4 was associated with suppressed tumor progression and better outcomes for BLCA patients. The higher expression level of ACSL4 predicted better immunotherapeutic responses and was associated with higher expression levels of core immune checkpoint genes, including CD274, CTLA4, PDCD1, and LAG3, compared with the low ACSL4 expression group.ConclusionThis study demonstrated for the first time that elevated ACSL4 correlated significantly with CD8+ T cell infiltration and contributed to better immunotherapeutic responses in BLCA patients. Furthermore, ACSL4 serves as a novel biomarker for predicting patient outcomes after immunotherapeutic treatments, which may improve the development of individualized immunotherapy for BLCA.

MTOR-dependent translation drives tumor infiltrating CD8+ effector and CD4+ Treg cells expansion

We performed a systematic analysis of the translation rate of tumor-infiltrating lymphocytes (TILs) and the microenvironment inputs affecting it, both in humans and in mice. Measurement of puromycin incorporation, a proxy of protein synthesis, revealed an increase of translating CD4+ and CD8+ cells in tumors, compared to normal tissues. High translation levels are associated with phospho-S6 labeling downstream of mTORC1 activation, whereas low levels correlate with hypoxic areas, in agreement with data showing that T cell receptor stimulation and hypoxia act as translation stimulators and inhibitors, respectively. Additional analyses revealed the specific phenotype of translating TILs. CD8+ translating cells have enriched expression of IFN-γ and CD-39, and reduced SLAMF6, pointing to a cytotoxic phenotype. CD4+ translating cells are mostly regulatory T cells (Tregs) with enriched levels of CTLA-4 and Ki67, suggesting an expanding immunosuppressive phenotype. In conclusion, the majority of translationally active TILs is represented by cytotoxic CD8+ and suppressive CD4+ Tregs, implying that other subsets may be largely composed by inactive bystanders.

Tumor-Infiltrating Immune-Related Long Non-Coding RNAs Indicate Prognoses and Response to PD-1 Blockade in Head and Neck Squamous Cell Carcinoma.

Long non-coding RNAs (lncRNAs) in immune cells play critical roles in tumor cell–immune cell interactions. This study aimed to characterize the landscape of tumor-infiltrating immune-related lncRNAs (Ti-lncRNAs) and reveal their correlations with prognoses and immunotherapy response in head and neck squamous cell carcinoma (HNSCC). We developed a computational model to identify Ti-lncRNAs in HNSCC and analyzed their associations with clinicopathological features, molecular alterations, and immunotherapy response. A signature of nine Ti-lncRNAs demonstrated an independent prognostic factor for both overall survival and disease-free survival among the cohorts from Fudan University Shanghai Cancer Center, The Cancer Genome Atlas, GSE41613, and GSE42743. The Ti-lncRNA signature scores in immune cells showed significant associations with TP53 mutation, CDKN2A mutation, and hypoxia. Inferior signature scores were enriched in patients with high levels of PDCD1 and CTLA4 and high expanded immune gene signature (IGS) scores, who displayed good response to PD-1 blockade in HNSCC. Consistently, superior clinical response emerged in melanoma patients with low signature scores undergoing anti-PD-1 therapy. Moreover, the Ti-lncRNA signature was a prognostic factor independent of PDCD1, CTLA4, and the expanded IGS score. In conclusion, tumor-infiltrating immune profiling identified a prognostic Ti-lncRNA signature indicative of clinical response to PD-1 blockade in HNSCC.

Identification of critical ferroptosis regulators in lung adenocarcinoma that RRM2 facilitates tumor immune infiltration by inhibiting ferroptotic death

Ferroptosis is a novel form of cell death characterized by heavy iron accumulation and lipid peroxidation that plays a critical role in the tumor microenvironment. However, promising biomarkers associated with tumor immune cell infiltration and the immunotherapy response to ferroptosis regulators remain to be elucidated in lung adenocarcinoma (LUAD) patients. In this study, we defined ferroptosis regulators in LUAD through database analysis and experimental validation to determine the implementation of genes associated with clinical relevance, immunotherapy response and tumor microenvironment in LUAD patients. Multiomics data analysis was performed to explore the CNV features, molecular mechanisms and immunogenic characteristics of ferroptosis regulators in LUAD patients. Then, univariate and multivariate Cox regression analyses were used to identify three genes (DDIT4, RRM2, and SLC2A1) that were closely associated with the prognosis of LUAD patients. The prognostic model based on the determination of these three genes was an independent prognostic factor (p < 0.05, HR = 2.838), and patients with superior predictive performance and higher prognostic risk were more likely to have poor survival rates than those with lower prognostic risk in the training group (p < 0.001, HR = 3.19) and the test group (p < 0.001, HR = 2.94; p < 0.001, HR = 3.44). Activated immune cells, including T helper cells and activated CD8 T cells, were lower in the high-risk group, while type 2 T cells were higher (p < 0.05). Patients with higher prognostic risk were less likely to benefit from immunotherapy, partly due to low CTLA4 levels and an immunosuppressive microenvironment (p < 0.05). Combined with LUAD tissue samples and mouse trials, RRM2 was found to influence lung cancer progression and affect tumor immune cell infiltration. RRM2 inhibition effectively promoted M1 macrophage polarization and suppressed M2 macrophage polarization in vitro and in vivo. And ferroptosis inhibitor ferrostatin-1 treatment effectively re-blanced macrophage polarization mediated by RRM2 inhibition. Taken together, the results of the multiomics data analysis and experimental validation identified ferroptosis regulators as promising biomarkers and therapeutic targets associated with tumor immune infiltration in LUAD patients.

Characterization of Exosome-Related Gene Risk Model to Evaluate the Tumor Immune Microenvironment and Predict Prognosis in Triple-Negative Breast Cancer.

BackgroundAs a kind of small membrane vesicles, exosomes are secreted by most cell types from multivesicular endosomes, including tumor cells. The relationship between exosomes and immune response plays a vital role in the occurrence and development of tumors. Nevertheless, the interaction between exosomes and the microenvironment of tumors remains unclear. Therefore, we set out to study the influence of exosomes on the triple-negative breast cancer (TNBC) microenvironment.MethodOne hundred twenty-one exosome-related genes were downloaded from ExoBCD database, and IVL, CXCL13, and AP2S1 were final selected because of the association with TNBC prognosis. Based on the sum of the expression levels of these three genes, provided by The Cancer Genome Atlas (TCGA), and the regression coefficients, an exosome risk score model was established. With the median risk score value, the patients in the two databases were divided into high- and low-risk groups. R clusterProfiler package was employed to compare the different enrichment ways between the two groups. The ESTIMATE and CIBERSORT methods were employed to analyze ESTIMATE Score and immune cell infiltration. Finally, the correlation between the immune checkpoint-related gene expression levels and exosome-related risk was analyzed. The relationship between selected gene expression and drug sensitivity was also detected.ResultsDifferent risk groups exhibited distinct result of TNBC prognosis, with a higher survival rate in the low-risk group than in the high-risk group. The two groups were enriched by immune response and biological process pathways. A better overall survival (OS) was demonstrated in patients with high scores of immune and ESTIMATE rather than ones with low scores. Subsequently, we found that CD4+-activated memory T cells and M1 macrophages were both upregulated in the low-risk group, whereas M2 macrophages and activated mast cell were downregulated in the low-risk group in patients from the TCGA and GEO databases, respectively. Eventually, four genes previously proposed to be targets of immune checkpoint inhibitors were evaluated, resulting in the expression levels of CD274, CTLA4, LAG3, and TIM3 being higher in the low-risk group than high-risk group.ConclusionThe results of our study suggest that exosome-related risk model was related to the prognosis and ratio of immune cell infiltration in patients with TNBC. This discovery may make contributions to improve immunotherapy for TNBC.

Glucose Metabolism Reprogramming of Regulatory T Cells in Concanavalin A-Induced Hepatitis.

Autoimmune hepatitis (AIH) is an inflammatory liver disease caused by a dysregulated immune response. Although the pathogenesis of AIH remains unclear, impaired regulatory T cells (Tregs) have been considered a driver of AIH development. Unlike autoreactive T cells, Tregs mainly utilize oxidative phosphorylation (OXPHOS) as their energy supply. Elevated glycolysis has been reported to limit the suppressive functions of Tregs. However, whether glucose metabolism reprogramming in Tregs is involved in AIH etiology remains unknown. The aim of this study was to examine alternations in Treg numbers and functions in AIH patients and concanavalin A (Con A)-induced hepatitis, while exploring associations between impaired Tregs and glucose metabolism. The frequency of Tregs was decreased in the peripheral blood but increased in liver biopsies of AIH patients. Moreover, immunosuppressive therapy rescued circulating Tregs in AIH. In Con A-induced immune hepatitis, enhanced intrahepatic Treg accumulation was observed over time, accompanied by reduced splenic Treg numbers. To investigate whether functional impairment of Tregs occurs in AIH, Tregs were isolated from experimental AIH (EAH) model mice and normal controls and the former displayed downregulated mRNA levels of FOXP3, CTLA4, CD103, TIGIT, CD39, and CD73. EAH model-derived Tregs also produced fewer anti-inflammatory mediators (TGF-β and IL-35) than control Tregs. Moreover, enhanced glycolysis and reduced OXPHOS were found in Tregs from EAH model mice, as reflected by elevated levels of key glycolytic enzymes (HK2, PK-M2, and LDH-A) and a decreased ATP concentration. This study revealed a decreased peripheral Treg frequency and abnormal intrahepatic Treg infiltration in AIH. It is first reported that glucose metabolism reprogramming is associated with decreases and functional impairments in the Treg population, promoting AIH development. Targeting glucose metabolism may provide novel insights for the treatment of AIH.

Radiochemotherapy upregulates expression of checkpoint receptors on circulating T cells

Purpose This study assesses changes of circulating leukocyte subpopulations and the expression of checkpoint receptors in T cells in patients undergoing radiochemotherapy. Materials and methods Fifty-seven patients with either esophageal cancer or cervical cancer who received radiochemotherapy were recruited into this study. Serial blood collection was carried out before and during treatments. Leukocyte subpopulations and the level of PD-1 and CTLA-4 in T cells were determined by flow cytometry. The plasma concentrations of 34 human cytokines, chemokines, and growth factors were quantified. Results Significant decreases of lymphocyte count and percentage of T cells and B cells were observed during radiochemotherapy. Percentages of PD-1hi and CTLA-4hi populations in T cells increased after treatments. Proportion of activated T cells showed no significant difference. No significant changes in the plasma concentrations of the 34 humoral mediators except mild decreases of six cytokines. Conclusion Elevated expression of PD-1 and CTLA-4 in T cells in patients receiving radiochemotherapy, which suggests that exhaustion-like T-cell dysfunction develops during cancer cytotoxic treatments.

Chronically Activated T-cells Retain Their Inflammatory Properties in Common Variable Immunodeficiency

PurposeImmune dysregulation complications cause significant morbidity and mortality in common variable immunodeficiency (CVID), but the underlying pathophysiology is poorly understood. While CVID is primarily considered a B-cell defect, resulting in the characteristic hypogammaglobulinemia, T-cells may also contribute to immune dysregulation complications. Here, we aim to further characterize T-cell activation and regulation in CVID with immune dysregulation (CVIDid).MethodsFlow cytometry was performed to investigate T-cell differentiation, activation and intracellular cytokine production, negative regulators of immune activation, regulatory T-cells (Treg), and homing markers in 12 healthy controls, 12 CVID patients with infections only (CVIDio), and 20 CVIDid patients.ResultsBoth CD4 + and CD8 + T-cells in CVIDid showed an increased activation profile (HLA-DR + , Ki67 + , IFNγ +) when compared to CVIDio, with concomitant upregulation of negative regulators of immune activation PD1, LAG3, CTLA4, and TIGIT. PD1 + and LAG3 + subpopulations contained equal or increased frequencies of cells with the capacity to produce IFNγ, Ki67, and/or GzmB. The expression of PD1 correlated with serum levels of CXCL9, 10, and 11. Treg frequencies were normal to high in CVIDid, but CVIDid Tregs had reduced CTLA-4 expression, especially on CD27 + effector Tregs. Increased migratory capacity to inflamed and mucosal tissue was also observed in CVIDid T-cells.ConclusionCVIDid was characterized by chronic activation of peripheral T-cells with preserved inflammatory potential rather than functional exhaustion, and increased tissue migratory capacity. While Treg numbers were normal in CVIDid Tregs, low levels of CTLA-4 indicate possible Treg dysfunction. Combined studies of T-cell dysfunction and circulating inflammatory proteins may direct future treatment strategies.

Immune Checkpoint Blockade Enhances Mutated Calreticulin-Induced T Cell Immunity in Myeloproliferative Neoplasms

▪Somatic mutations in the calreticulin (CALR) gene are key drivers of cellular transformation in myeloproliferative neoplasms (MPN) and are the second most common driver mutations in essential thrombocythemia (ET) and myelofibrosis (MF) patients. Each CALR mutation leads to a shift in the reading frame resulting in the formation of an altered protein with an identical 36-amino acid sequence in the C-terminus. We hypothesized that the recurrence and uniformity of the novel sequence would provide the mutated CALR an attractive candidate as a MPN-specific tumor antigen that might elicit anti-tumor immune responses. Therefore, we investigated the immunogenicity of the altered CALR by employing in silico peptide binding prediction algorithms to interrogate the binding affinities of mutated CALR peptides to human leukocyte antigens (HLA). These analyses indicated that the mutated epitopes can bind to class I and II HLA alleles with high affinity supporting the hypothesis that mutant CALR could be immunogenic in vivo . We then tested the immunogenicity of the overlapping peptides spanning the mutated region in vitro by utilizing intracellular staining (ICS) and Enzyme-Linked ImmunoSPOT (ELISPOT) assays. Naïve T cells derived from the peripheral blood mononuclear cells (PBMNCs) isolated from healthy donors displayed effector functions after priming with the mutated peptides but not with the corresponding wild type peptides . Incubation withthe mutated CALR peptides induced T cells proliferation, upregulation of CD137 (4-1BB) and production of interferon (IFN)-γ and tumor necrosis factor (TNF)-α. Moreover, as suggested by in silico peptide-HLA binding predictions, both CD8+ and CD4+ T cell precursors displayed increased IFN-γ production in response to stimulation with mutated CALR peptides. There was an overall 4±3-fold increase in IFN-γ production by CD4+ T cells and a 2.5±0.5-fold increase by CD8+ T cells after stimulation with mutated peptides as compared to control self-antigens (n = 14 healthy donors out of which 11 showed CD4+ and 5 showed CD8+ T cell responses). These results demonstrate that mutated CALR elicits T cell responses in vitro supporting its potential immunogenic functions in vivo .We next investigated the presence of mutant CALR-specific T cell immunity in MPN patients with CALR mutations. PBMNCs from a total of 18 CALR-mutated MPN patients with ET (7), MF (5) or MF arising from ET (6) were evaluated. MPN T cells were then stimulated with the pooled mutant CALR peptides and T cell responses were assessed by ELISPOT. Out of 18 patients, increased IFN-γ production upon stimulation was observed in only 2 patients (one with ET and one with ET-MF). The less than robust MPN T cell responses suggested that immune suppressive mechanisms might be operating. To this end, we evaluated the expression of cell surface inhibitory receptors, programmed cell death (PD)-1 and the cytotoxic T-lymphocyte-associated antigen (CTLA)-4, by T cells from MPN patients. Both CD8+ and CD4+ T cells from MPN patients exhibited increased levels of PD-1 and CTLA-4 when compared to the healthy controls (HC). For instance, 9.3±8.2% of MPN patients CD4+ T cells expressed PD-1 (n = 12) as compared to 2.5±1.7% of HC CD4+ T cells (n=10) (p value = 0.0482). Similarly, there were twice as many PD-1 expressing CD8+ T cells in MPN patients as compared to HC. We next assessed MPN T cell responses against mutated peptides in the presence of neutralizing PD-1 or CTLA-4 antibodies. PD-1 inhibition restored T cell immunity in cultures derived from 3 MPN patients and CTLA-4 inhibition restored response in one patient (n=16). Of note, following the PD-1 or CTLA-4 inhibition, the number of IFN-γ producing cells and the amount of IFN-γ produced on a per-cell-basis increased upon stimulation with mutant-CALR peptides as compared to control peptides. These results indicate that the limited MPN T cell responses to CALR mutated peptides were due to T cell exhaustion caused, at least in part, by activation of the PD-1/CTLA-4 pathway.In summary, our studies establish mutated CALR as a MPN-specific tumor antigen thereby providing a rationale for the development of immunotherapies targeting mutated CALR in patients. Moreover, our data indicate that immune checkpoint signaling might be responsible for T cell exhaustion in some MPN patients thereby supporting the use of checkpoint blockade inhibitors for treatment of these patients. DisclosuresMascarenhas:Incyte: Other: Clinical Trial Steering Committee , Research Funding; CTI Biopharma: Research Funding; Janssen: Research Funding; Merck: Research Funding; Novartis: Other: DSMB member , Research Funding; Promedior: Research Funding. Bhardwaj:Merck & Co., Inc.: Membership on an entity's Board of Directors or advisory committees; Dendreon Corporation: Membership on an entity's Board of Directors or advisory committees; Dynavax Technologies Corporation: Consultancy; Crucell: Patents & Royalties; Neostem, Inc.: Patents & Royalties.

Immune Checkpoint (PD-1, PD-L1, PD-L2, and CTLA-4) Expression in Plasma Cell Myeloma

Background: Malignant cells evade host immunity and change the milieu in the tumor microenvironment by activation of immune checkpoints, such as PD-1, PD-L1, PD-L2, and CTLA-4. Plasma cell myeloma (PCM) is one of the hematologic malignancies caused by immune dysregulation described as loss of effector T-cell populations and bone marrow (BM) microenvironment promoting immune escape. Though some investigators recently reported immune checkpoints in PCM, their results were controversial, and the application of immune checkpoints by means of treatment has not been clarified. In this study, we evaluated the expression of PD-1, PD-L1, PD-L2, and CTLA-4 in PCM patients according to disease course, genetic abnormalities and stages of Revised International Staging System (R-ISS).Methods: We obtained 192 BM samples from 166 PCM patients (3 with R-ISS stage I, 37 with stage II and 17 with stage III) at Asan medical center in Korea between May 2016 and May 2017, including 85 at diagnosis and 107 after treatment, and normal BM samples were collected from 25 individuals with no evidence of hematologic malignancies as the controls. Expression percentages of PD-1 and CTLA-4 on T cells, and those of PD-L1 and PD-L2 on plasma cells and monocytes were measured using FACSCanto II (Becton-Dickinson, Sunnyvale, CA, USA) by flow cytometry.Results: At diagnosis, the median percentage of PD-1 expressed on CD4+ T cells was 24.8%, which shows no difference with that of the controls (Fig. 1). After stem cell transplantation (SCT) regardless residual disease, the expression levels of PD-1 on T cells were higher than those at the diagnosis (P < 0.001). The expression levels of PD-1 on CD4+ T cells in refractory disease after chemotherapy were significantly higher than in both of diagnosis (P= 0.001) and complete remission after chemotherapy (P= 0.044). CTLA-4 expression levels on CD4+ T cells positively correlated with the ratio of involved/uninvolved free light chain (P= 0.004, rho = 0.341). CTLA-4 expression on CD4+ T cell showed higher percentages in PCM patients with R-ISS stage III than those with R-ISS stage II disease (P= 0.03) (Fig. 2). Expression of PD-1 and CTLA-4 of CD4+ and/or CD8+ T cells showed higher values depending on the genetic abnormalities such as monosomy 13, 1q gain, complex chromosome and hypodiploidy. There was no meaningful tendency in the expression percentages of immune checkpoints on CD8+ T cells, on neoplastic plasma cells and on monocytes among PCM patients with different states.Conclusion: We found that high expression levels of PD1 on CD4+ T cells associated with refractoriness against chemotherapy. We also found that PD-1 expression was upregulated by SCT and associated with several genetic abnormalities. The positive correlation between the expression levels of CTLA-4 on CD4+ T cells and R-ISS suggests that overexpression of CTLA-4 might depress anti-tumor host immune responses. We expect immune checkpoint blockades have therapeutic potential in refractory PCM patients after chemotherapy or SCT. [Display omitted] DisclosuresLee:Boehringer Ingelheim: Research Funding.

Isolation and expansion of thymus-derived regulatory T cells for use in pediatric heart transplant patients.

Regulatory T-cells (Tregs) are a subset of T cells generated in the thymus with intrinsic immunosuppressive properties. Phase I clinical trials have shown safety and feasibility of Treg infusion to promote immune tolerance and new studies are ongoing to evaluate their efficacy. During heart transplantation, thymic tissue is routinely discarded providing an attractive source of Tregs. In this study, we developed a GMP-compatible protocol for expanding sorted thymus-derived CD3+ CD4+ CD25+ CD127- (Tregs) as well as CD3+ CD4+ CD25+ CD127- CD45RA+ (RA+ Tregs) cells. We aimed to understand whether thymic RA+ Tregs can be isolated and expanded offering an advantage in terms of stability as it has been previously shown for circulating adult CD45RA+ Tregs. We show that both Tregs and RA+ Tregs could be expanded in large numbers and the presence of rapamycin is essential to inhibit the growth of IFN-γ producing cells. High levels of FOXP3, CTLA4, and CD25 expression, demethylation of the FOXP3 promoter, and high suppressive ability were found with no differences between Tregs and RA+ Tregs. After freezing and thawing, all Treg preparations maintained their suppressive ability, stability, as well as CD25 and FOXP3 expression. The number of thymic Tregs that could be isolated with our protocol, their fold expansion, and functional characteristics allow the clinical application of this cell population to promote tolerance in pediatric heart transplant patients.

Nivolumab/ipilimumab primed immunotransplant in post-CAR-T and post-ASCT DLBCL.

2593 Background: Patients with refractory diffuse large b cell lymphoma (DLBCL) have poor outcomes with &lt; 30% surviving for 12 months and especially poor outcomes for those who progress after chimeric antigen receptor T cells (CAR-T) or autologous stem cell transplant (ASCT). These therapies utilize lymphodepleting chemotherapy which induces homeostatic T cell proliferation. We have demonstrated these expanding T cells express high levels of PD1 and CTLA4. In pre-clinical models, addition of dual checkpoint blockade (DCB) with anti-PD1/anti-CTLA-4 to adoptive T cell transfer after lymphodepletion achieved a synergistic anti-tumor effect. Based on these studies, we developed a phase Ib/II study of Nivolumab/Ipilimumab primed “immunotransplant” for relapsed/refractory (R/R) DLBCL (NCT03305445). Methods: Phase Ib of the trial enrolled 6 patients with progressive disease following at least one line of standard therapy. Patients received two cycles of DCB with ipilimumab (1mg/kg) and nivolumab (3mg/kg) given at three-week intervals followed by immunotransplant (i.e. peripheral blood T cell harvest, lymphodepletion with fludarabine/cyclophosphamide, T cell reinfusion), followed by two further cycles of DCB and nivolumab maintenance. Results: Five patients received at least two cycles of DCB and the autologous T cell transfer, while one patient had progressive disease during initial DCB and required salvage chemotherapy. Treatment emergent AE (TEAE) occurred in 100% of patients. As expected with lymphodepleting chemotherapy, the most common TEAE were neutropenia (66.7% grade 1, 66.7% grade ≥ 3), fatigue (83.3%, 16.7%), fever (66.7%, 0%), and dyspnea (66.7%, 0.0%). One patient (16.7%) died during the intervention period (grade 5 TEAE), though relation to study drug is unclear. Three patients (50.0%) experienced clinical benefit with immunotransplant. One patient (a 58yo M with progression after seven lines of therapy including ASCT and CAR-T) is experiencing partial metabolic response after 4 months on protocol. One 77yo F with multiple prior lines of therapy including ASCT has experienced an extended complete metabolic response, currently 31 months post immunotransplant. A third (a 50yo F) experienced mixed radiographic response, and has not received subsequent therapy 30 months following immunotransplant. Conclusions: Nivolumab/Ipilimumab primed immunotransplant is well tolerated in patients with R/R DLBCL for whom there are few treatment options. Preliminary results demonstrate remissions in heavily pre-treated patients, including prior ASCT and CAR-T. Pre-clinical models in melanoma, non-small cell lung cancer, and T cell lymphoma all demonstrate synergy when DCB is administered with lymphodepletion and autologous T cell transfer. These data support further investigation of DCB-primed immunotransplant. Clinical trial information: NCT03305445.

Defective mismatch repair associated mutational signatures, a prognostic and predictive biomarker in endometrial cancer.

5528 Background: Mismatch repair (MMR) deficiency is the distinguishing molecular feature of a significant portion of endometrial cancers (UCEC), and tumors with MMR deficiency have been identified as candidates for immune checkpoint blockade therapy. We studied MMR deficiency in UCEC using defective mismatch repair associated mutational signatures (MMRd-ams). Methods: WES-derived somatic mutation data of 531 UCEC samples from TCGA Pan-Cancer Atlas were analyzed. COSMIC mutational signatures for each sample were calculated using the R package deconstructSigs. MMRd-ams were correlated with clinical and molecular features for 507 TCGA samples (cBioPortal). Samples were divided into High MMRd-ams ( n = 192) and Low MMRd-ams ( n = 315) groups by the average of the representative MMRd-ams (0.2396). Fractions of tumor immune infiltrates were derived from CIBERSORT. Results: A significantly higher percentage of patients (47/192, 24.5%) in the High MMRd group had somatic putative driver mutations in at least 1 of the MMR genes ( MLH1, MLH3, MSH2, MSH3, MSH6, PMS1, PMS2 and EPCAM), compared with patients in the Low MMRd group (37/315, 11.8%, p = 0.0003). 15% (54/359) of tumors in the non-MSI subtypes expressed significant MMRd-ams and were categorized in the High MMRd-ams group. Patients in the High MMRd-ams group had longer progression-free survival (PFS) ( p = 0.0457, log-rank). Analysis of the inferred composition of tumor immune infiltrates revealed that the High MMRd-ams group had significantly higher fraction of CD8+ T cells ( p &lt; 0.0001), higher fraction of T follicular helper cells ( p &lt; 0.0001) and lower fraction of M2 macrophages ( p &lt; 0.001). Tumors in the High MMRd-ams group also displayed higher mRNA expression levels of immune checkpoint genes: PDCD1 ( p = 0.0013), and CTLA4 ( p = 0.0016). Conclusions: MMRd-ams may be a prognostic and predictive biomarker with significant clinical impact. High MMRd-ams patients prognostically demonstrated longer PFS. Predictively, high MMRd-ams was associated with increased tumor immune infiltrates and elevated expression levels of CTLA4 and PDCD1 , known immune checkpoint genes exploitable by immune checkpoint therapies. MMRd-ams importantly characterized a subset of patients that were non-MSI but fit the MMR deficient phenotype by mutational signature. Together these findings open an avenue for recognizing and treating a previously unidentifiable group of patients.

Role of Inhibitory Check Point (CTLA-4) in Iraqi Women with Breast Cancer

Breast cancer is the most important and frequently diagnosed cancer among women worldwide. The CTLA4 is a member of Immunoglobulin super family and binds to the CD80 and CD86 co-stimulatory molecules.(binds to CD80/86 on APCs with a higher affinity than CD28, thereby inhibiting co-stimulatory signals.CTLA-4 (Gene ID:1493, MIM number: 123890) is a new member of the immunoglobulin super family known as insulin-dependent diabetes mellitus 12 (IDDM 12) ,and cluster of differentiation 152 (CD152).The CTLA-4 protein consists of a 37 amino acid leader peptide, an extracellular immunoglobulin (Ig) V like domain or the ligand-binding domain (116 amino acid), a hydrophobic trans membrane region (37 amino acid), and a cytoplasmic domain. The aim of the present study of the association of CTLA-4 serum level and polymorphism with breast cancer in Iraqi women suffers from breast cancer. Materials and Methods: peripheral Blood samples were collected from 45 Iraqi patients women diagnosed with breast cancer patient, and 45 healthy women were matched with patients as a control. ELISA technique has been used to determine the serum level of sCTLA-4.The polymerase chain reaction (PCR) performed on 45 patients and 45 control, to determine the genetic variation in the +49 exon 1 region of CTLA-4 gene. Results: the serum level of understudying groups recorded an non-significant difference increasing in the serum level of CTLA-4 under (p>0.05). in mean ± SE (3.17 ± 0.82 ng/ml) patients as compared to control (2.72 ± 0.29 ng/ml) under (p>0.05). Genetic polymorphism of CTLA.4 gene (rs231775) which illustrated the distribution of genotypes of CTLA.4 in patients and control. The heterozygous genotype AG recorded high frequency in patients (35.56%) than control (0.00%) with a highly significant difference under (P<0.05). Homozygote genotype AA recorded high frequency in control (95.56%) than patients (64.44) and has a highly significant difference, and the homozygous genotype GG frequency (0.00%) was non- significant in patients compared to control (4.44%). The allele frequency for allele A was (0.82%) in patients compared with control (0.96%) while for the frequency of allele G was (0.18%) in patients compared with control (0.04%) with a significant difference. Conclusion: elevation of serum concentration of sCTLA-4 could consider as a clinical biomarker for prognosis breast cancer. There are some difference noticed in CTLA-4 gene with SNP (rs231775A/G) which showed a significant differences between patients and control, A allele of SNP (rs231775A/G) may have role to prevent the risk of breast cancer in Iraqi females, while allele G has an etiological fraction.

A novel immune-related gene-based prognostic signature to predict biochemical recurrence in patients with prostate cancer after radical prostatectomy.

Accumulating evidences indicates that the immune landscape signature dramatically correlates with tumorigenesis and prognosis of prostate cancer (PCa). Here, we identified a novel immune-related gene-based prognostic signature (IRGPS) to predict biochemical recurrence (BCR) after radical prostatectomy. We also explored the correlation between IRGPS and tumor microenvironment. We identified an IRGPS consisting of seven immune-related genes (PPARGC1A, AKR1C2, COMP, EEF1A2, IRF5, NTM, and TPX2) that were related to the BCR-free survival of PCa patients. The high-risk patients exhibited a higher fraction of regulatory T cells and M2 macrophages than the low-risk BCR patients (P < 0.05) as well as a lower fraction of resting memory CD4 T cells and resting mast cells. These high-risk patients also had higher expression levels of CTLA4, TIGIT, PDCD1, LAG3, and TIM3. Finally, a strong correlation was detected between IRGPS and specific clinicopathological features, including Gleason scores and tumor stage. In conclusion, our study reveals the clinical significance and potential functions of the IRGPS, provides more data for predicting outcomes, and suggests more effective immunotherapeutic target strategies for PCa.

WEE1 inhibition reverses trastuzumab resistance in HER2-positive cancers.

To date, many efforts have been made to understand the resistance mechanism of trastuzumab in human epidermal growth factor receptor 2 (HER2)-positive breast and gastric cancer. However, there is still a huge unmet medical need for patients with trastuzumab resistance. In our study, we generated four trastuzumab-resistant (HR) cancer cell lines from ERBB2-amplified gastric and biliary tract cancer cell lines (SNU-216, NCI-N87, SNU-2670, and SNU-2773). Here, we found higher PD-L1 expression in trastuzumab-resistant (HR) HER2-positive cancer cells than in parental cells, and blocking PD-L1 reversed the resistance to trastuzumab in HR cells. Trastuzumab upregulated PD-L1 expression via NF-κB activation in both parental and HR cells, however, led to DNA damage only in parental cells. The WEE1 inhibitor adavosertib, which downregulates PD-L1 expression, enhanced trastuzumab efficacy by blocking BRCA1-CMTM6-PD-L1 signals and the HER2-CDCP-1-SRC axis. Additionally, the levels of galectin-9, CD163, FoxP3, and CTLA-4 were diminished by blocking WEE1 in the presence of human PBMCs in vitro. Taken together, the strategy of co-targeting HER2 and WEE1 could overcome resistance to trastuzumab in HER2-positive cancers, supporting further clinical development in HER2-positive cancer patients.

In vitro immunoregulatory activity and anti-inflammatory effect of Echinococcus granulosus laminated layer

The Laminated layer of Echinococcus granulosus (LL) is the outer layer of the hydatic cyst. It plays a pivotal role in protecting the metacestode from host immunity. In our current study, we investigated the immunomodulatory effect of the LL on mouse spleen cells in presence of Lipopolysaccharide (LPS). Mouse spleen cells were cultured with or without LL in presence of LPS. After 24 h, the nitrites level representative of Nitric oxide (NO) production was measured in the culture supernatant by Griess-modified method. In addition, the mRNA expression levels of cytokines (IFN-γ, IL-1β, TGF-β, IL-10), Foxp3, and CTLA-4 were measured by quantitative Real-Time Polymerase chain reaction (qRT-PCR). Interestingly, our results showed a significant decrease (p< 0.01) in NO production and IFN-γ mRNA level (p< 0.001) from LPS- induced spleen cells in response to LL after 24h of culture. Moreover, LPS induced high level of IL-1β that was significantly (p<0.05) down regulated by LL. Importantly, mRNA levels of TGF-β (p< 0.01), Foxp3 and IL-10 (p< 0.05) were significantly upregulated by LL. In conclusion, our data indicated the in vitro immuno-regulatory and anti-inflammatory effects of the hydatic Laminated Layer on mouse spleen cells. These effects are related to an innate response implicating up-regulation of Foxp3, IL-10 and TGF-β expression and down-regulation of IFN-γ and IL-1β expression. LL could constitute a potential candidate for controlling inflammation during inflammatory disease.