Cytotoxic T-lymphocyte-associated Protein 4 Expression Research Articles

Abstract PO3-25-01: CTLA4 expression profiles and their association with clinical outcomes of breast cancer

Abstract The cytotoxic T-lymphocyte-associated protein 4 (CTLA4) is involved in the progression of various cancers, but its biological roles in breast cancer (BRCA) remain unclear. Therefore, we performed a systematic multiomic analysis to expound on the prognostic value and underlying mechanism of CTLA4 in BRCA. First, the expression of CTLA4 in pan-cancer was evaluated using the platforms of Oncomine. Afterward, the web-based bioinformatics platforms of GEPIA and UCLAN were used to compare the expression of CTLA4 in breast cancer and normal tissues. Next, we assessed the relationship between CTLA4 mRNA expression and prognosis in breast cancer patients using the PrognoScan Database, Kaplan-Meier Plotter, and R2: Kaplan-Meier Scanner. Additionally, the correlation between CTLA4 expression and immune cell infiltration in breast cancer and functional enrichment analysis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways was performed to elucidate the potential biological functions of CTLA4. Finally, we used the STRINGdb and cytoHubba plugin Cytoscape to create a PPI network and identify known and predicted structural essentials for CTLA4 functions and the top five hub genes. CTLA4 was highly expressed in BRCA tumor tissue compared to normal tissue (p< 0.01). The CTLA4 mRNA levels in BRCA based on breast cancer subtypes of luminal, Human epidermal growth factor receptor 2 (HER-2), and Triple-negative breast cancer (TNBC) were considerably higher than in normal tissues (p< 0.001). However, the overexpression of CTLA4 was associated with a better prognosis in BRCA (P< 0.001), and was correlated with clinicopathological characteristics including age, T-stage, estrogen receptors (ER), progesterone receptors (PR), and PAM50 (P< 0.01). The infiltration of multiple immune cells was associated with increased CTLA4 expression in BRCA (P< 0.001). CTLA4 was highly enriched in antigen binding, immunoglobulin complexes, lymphocyte-mediated immunity, and cytokine-cytokine receptor interaction. This study provides suggestive evidence of the prognostic role of CTLA4 in breast cancer, which may be a therapeutic target for breast cancer. Furthermore, CTLA4 may influence breast cancer prognosis through antigen binding, immunoglobulin complexes, lymphocyte-mediated immunity, and cytokine-cytokine receptor interaction. These findings help us understand how CTLA4 plays a role in breast cancer and set the stage for more research. Citation Format: Woo Chul Noh, Young Bum Yoo, Kyoung Sik Park, Sang Eun Nam, Seung Hwan Lim. CTLA4 expression profiles and their association with clinical outcomes of breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-25-01.

"Expression and prognostic relevance of PD-1, PD-L1 and CTLA-4 immune checkpoints in adrenocortical carcinoma".

Adrenocortical carcinoma (ACC) is a rare endocrine malignancy with poor prognosis in advanced stages. While therapies targeting the checkpoint molecules programmed cell death 1 (PD-1), its ligand PD-L1 and the cytotoxic T lymphocyte-associated protein 4 (CTLA-4) have revolutionized treatment in many cancers, the results in ACC were heterogeneous. Their expression in ACC has not been systematically studied and might explain the variable response to checkpoint inhibitors. The expression of PD-1, PD-L1 and CTLA-4 was examined in 162 tumor samples from 122 ACC patients by immunohistochemistry (threshold of >1%) and correlated with tumoral T lymphocyte infiltration and clinical endpoints. Finally, uni- and multivariate analyses of progression-free and overall survival were performed. PD-1 and PD-L1 were expressed in 26.5% and 24.7% of samples, respectively, with low expression in most tumor samples (median positive cells: 2.1% and 21.7%. In contrast, CTLA-4 expression was observed in 52.5% of ACC with a median of 38.4% positive cells. Positive PD-1 expression was associated with longer progression-free survival (HR: 0.50, 95% CI 0.25-0.98, p=0.04) even after considering prognostic factors. In contrast, PD-L1 and CTLA-4 did not correlate with clinical outcome. Additionally, PD-1 and PD-L1 expression correlated significantly with the amount of CD3+, CD4+, FoxP3+ and CD8+ T cells. The heterogeneous expression of PD1, PD-L1, and CTLA-4 in this large series of well-annotated ACC samples might explain the heterogeneous results of the immunotherapies in advanced ACC. In addition, PD-1 expression is a strong prognostic biomarker that can easily be applied in routine clinical care and histopathological assessment.

CTLA4 expression profiles and their association with clinical outcomes of breast cancer: a systemic review.

The cytotoxic T-lymphocyte-associated protein 4 (CTLA4) is involved in the progression of various cancers, but its biological roles in breast cancer (BRCA) remain unclear. Therefore, we performed a systematic multiomic analysis to expound on the prognostic value and underlying mechanism of CTLA4 in BRCA. We assessed the effect of CTLA4 expression on BRCA using a variety of bioinformatics platforms, including Oncomine, GEPIA, UALCAN, PrognoScan database, Kaplan-Meier plotter, and R2: Kaplan-Meier scanner. CTLA4 was highly expressed in BRCA tumor tissue compared to normal tissue (P < 0.01). The CTLA4 messenger RNA levels in BRCA based on BRCA subtypes of Luminal, human epidermal growth factor receptor 2, and triple-negative BRCA were considerably higher than in normal tissues (P < 0.001). However, the overexpression of CTLA4 was associated with a better prognosis in BRCA (P < 0.001) and was correlated with clinicopathological characteristics including age, T stage, estrogen receptors, progesterone receptors, and prediction analysis of microarray 50 (P < 0.01). The infiltration of multiple immune cells was associated with increased CTLA4 expression in BRCA (P < 0.001). CTLA4 was highly enriched in antigen binding, immunoglobulin complexes, lymphocyte-mediated immunity, and cytokine-cytokine receptor interaction. This study provides suggestive evidence of the prognostic role of CTLA4 in BRCA, which may be a therapeutic target for BRCA. Furthermore, CTLA4 may influence BRCA prognosis through antigen binding, immunoglobulin complexes, lymphocyte-mediated immunity, and cytokine-cytokine receptor interaction. These findings help us understand how CTLA4 plays a role in BRCA and set the stage for more research.

Vactosertib, a Novel TGFb Type I Receptor Kinase Inhibitor, Improves T-Cell Fitness:a Single-Arm, Phase 1b Trial in Relapsed/Refractory Multiple Myeloma

The prognosis for Multiple Myeloma (MM) patients (pts) has improved substantially over the past two decades with the success of immunomodulatory drugs (IMiDs), monoclonal antibodies, chimeric antigen receptor (CAR) T-cells and bispecific T-cell engagers (BiTEs) that broke the circulus vitiosus of tumor-induced immunosuppression to reengage the immune system. Functional blockade of the transforming growth factor-beta (TGF) signaling pathway improves the efficacy of cytotoxic and immune therapies, therefore we investigator the impact of TGF-inhibition in an steroid free regimn. Methods: We conducted a phase 1b study to determine the safety, efficacy, and maximal tolerated dose (200 mg po bid) of the potent, orally-available TGF type I receptor kinase inhibitor vactosertib in relapsed and/or refractory multiple myeloma (RRMM) pts who had received -2 lines of chemoimmunotherapy in combination with standard dosage of Pomalidomide without any corticosteroids. Results: Twenty one pts were enrolled in the phase 1b dose escalation cohort and received vactosertib plus pomalidomide as indicated (Fig. 1A). Pts' characteristics are described in Table 1The summary of grade 1-4 adverse events is shown (Table 2). A single grade 4 elevated bilirubin (5%) and a single grade 4 elevated lipase (5%) were observed as well as four grade 4 events of decreased neutrophils (19%). Four pts demonstrated a PR, two had a MR, nine had SD and four had disease progression (Fig. 1B). The median time to response for all pts and the responders eight weeks and median duration of response was ~12 weeks. The best overall response for individual pts to treatment is shown (Fig. 1C). Clinical activity was observed across all four dose levels (Table 3). To probe the tumor intrinsic anti-myeloma activity of vactosertib, we first determined the relative effect of vactosertib compared to the IMiDs pomalidomide and lenalidomide (Fig. 2A, B). The effect of vactosertib was greater than that observed with either lenalidomide or pomalidomide. MMCLs and patient CD138 + cells were then treated with vactosertib in combination with pomalidomide, and importantly, vactosertib with pomalidomide synergistically reduced myeloma viability (Fig. 3A). We next probed the systemic, tumor extrinsic effects of vactosertib by analyzing cytokines immune and tumor cells present within patient BM samples. (Fig. 4A). (Fig. 4B). Therefore, we explored the immunophenotype of CD8 + T-cells isolated from patient BM prior to and at the EOT. The relative percentage of T-cells that expressed the immunosuppressive markers programmed cell death-1 (PD-1), T-cell immunoglobulin and mucin domain-containing protein 3 (TIM3), B and T lymphocyte attenuator (BTLA), and cytotoxic T-lymphocyte associated protein 4 (CTLA-4) was reduced following treatment relative to their expression prior to treatment (Fig. 4C). Results indicated that vactosertib and pomalidomide treatment in vivo reduced PD-1, TIM3, BTLA and CTLA-4 expression on patient CD8 + T-cells. Next, CD8 + T-cells were isolated from patient peripheral blood and cultured in the presence of TGF-1, vactosertib, pomalidomide, or vactosertib plus pomalidomide (Fig. 5B). Treatment of patient CD8 + T-cells with vactosertib decreased PD-1 expression up to 40% and decreased TIM3 expression up to 65%. Pomalidomide alone did not significantly reduce PD-1, TIM3, BTLA, or LAG-3 on patient CD8 + T-cells. Vactosertib and pomalidomide also reduced TIM3 levels on CD8 + T-cells from most pts. (Fig. 5C, D). (Fig. 5 G, H). Results indicated that vactosertib or pomalidomide did not increase CD138 + cell death (Fig. 5I). Conclusion: Vactosertib combined with pomalidomide was well-tolerated at all doses, had a manageable adverse event profile and induced durable responses with 80% progression-free survival (PFS-6) at 6 months, Vactosertib reduced TGF- in patient bone marrow and suppressed PD-1 expression on CD8 + T-cells and lead to reduction of PD-L1/PD-L2 expression on CD138 + cells and enhanced autologous T-cell cytotoxicity. Taken together, our results support the safety and efficacy of vactosertib to treat RRMM and revealed that vactosertib modulates the T-cell immunophenotype and reinvigorates T-cell fitness.

Molecular and serological biomarkers to predict trastuzumab responsiveness in HER-2 positive breast cancer.

HER-2-positive breast cancer is characterized by its aggressive nature, poor prognosis, and reduced overall survival. The emergence of trastuzumab resistance is currently considered a global problem. The immune system plays a pivotal role in tumor progression and development. Cytotoxic T lymphocyte-associated protein-4 (CTLA-4) and other immune checkpoint proteins may be potential prognostic factors and therapeutic targets for breast cancer. This study aimed to determine the correlation between CTLA-4 expression in peripheral blood and insulin-like growth factor-1 (IGF-1) serum levels and their impact on trastuzumab responsiveness in HER-2-positive patients with breast cancer. CTLA-4 expression was analyzed in peripheral blood cells using quantitative PCR, while IGF-1 serum levels were assessed through electrochemiluminescence assays. There was a significant increase in CTLA-4 expression at cycle 9, which continued to increase until it reached 4.6 at cycle 17. High IGF-1 levels were observed in newly diagnosed HER-2 positive patients before trastuzumab therapy, significantly decreasing post-therapy (p=0.001). Co-targeting HER-2 and IGF-1 receptors may reduce the risk of recurrence and improve outcomes. In addition, targeted CTLA-4 molecules may improve patient survival and prevent recurrence.

Digital spatial profiling of CD4+ T cells in classic Hodgkin lymphoma.

Classic Hodgkin lymphoma (CHL) harbors a small number of Hodgkin-Reed-Sternberg (HRS) cells scattered among numerous lymphocytes. HRS cells are surrounded by distinct CD4+ T cells in a rosette-like manner. These CD4+ T cell rosettes play an important role in the tumor microenvironment (TME) of CHL. To elucidate the interaction between HRS cells and CD4+ T cell rosettes, we completed digital spatial profiling to compare the gene expression profiles of CD4+ T cell rosettes and other CD4+ T cells separated from the HRS cells. Immune checkpoint molecules including OX40, programed cell death-1 (PD-1), and cytotoxic T lymphocyte associated protein 4 (CTLA-4) expression was higher in CD4+ T cell rosettes compared to other CD4+ T cells. Immunohistochemistry confirmed variable PD-1, CTLA-4, and OX40 expression in the CD4+ T cell rosettes. This study introduced a new pathological approach to study the CHL TME, and provided deeper insight into CD4+ T cells in CHL.

Comprehensive analysis of alternative polyadenylation regulators of CTLA4 and immune infiltration in clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is the most common renal cancer. Alternative polyadenylation (APA) plays an important role in the progression and immunity of multiple tumors. Although immunotherapy has emerged as an important treatment option for metastatic renal cell carcinoma, whether APA affects the tumor immune microenvironment (TIME) in ccRCC remains unclear. Patients with ccRCC were classified into two groups by performing a consensus clustering analysis of APA factor expression profiles. The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) databases were used to assess the association between APA regulators and ccRCC prognosis. Through the use of the R package, GSVA, the correlation between SNRNP70 expression and tumor immune features were analyzed. The TCGA data revealed that APA regulators were associated with Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA4) expression. Cluster 1 exhibited a higher grade and histological tumor stage, as well as a worse prognosis compared to Cluster 2. A ssGSEA analysis demonstrated that Cluster 2 possessed an extensively higher level of immune infiltration. Moreover, high SNRNP70 expression was found to be positively correlated with CTLA4 expression and a poor prognosis in ccRCC. Thus, SNRNP70 might represent a novel immune-related prognostic biomarker in ccRCC. A pan-cancer analysis suggested that SNRNP70 may also play a role in other types of cancer by affecting the TIME. The data from this study indicate that APA regulators play a key role in immune infiltration in ccRCC. SNRNP70 is a promising prognostic biomarker and a potential target for ccRCC's immunotherapy.

Prognostic significance of PD-1, CTLA-4, CD4, and CD8 expression in olfactory neuroblastoma.

There are limited data regarding immune surveillance mechanisms in olfactory neuroblastoma. We investigated the expression of programmed cell death protein 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), CD4, and CD8 in olfactory neuroblastoma to identify potential therapeutic targets. Immunohistochemistry was used to detect PD-1 and CTLA-4 and measure the numbers of CD4+ and CD8+ T cells in 56 patients with olfactory neuroblastoma. The relationships between these molecules in tumor microenvironment, clinicopathological features, and survival were analyzed. The prevalence of PD-1 in Kadish C stage was 24.14%, significantly greater than in Kadish A and B stage. CD4+ T-cell and CD8+ T-cell levels correlated with higher Hyams histological grade and Kadish stage. In addition, PD-1 was related positively with CTLA-4, CD4+ T cells, and CD8+ T cells in olfactory neuroblastoma. Univariate survival analysis showed that higher PD-1 positivity, CD8+ T cells, and Hyams grade correlated with worse clinical outcome. Multivariate analysis showed that the expression of PD-1 was an independent parameter for poor prognosis. In conclusion, olfactory neuroblastoma with PD-1 expression had more aggressive clinicopathological features and worse prognosis. PD-1 may potentially predict the outcome of olfactory neuroblastoma patients.

Unequal distribution of genetically-intact HIV-1 proviruses in cells expressing the immune checkpoint markers PD-1 and/or CTLA-4.

HIV-1 persists in resting CD4+ T-cells despite antiretroviral therapy (ART). Determining the cell surface markers that enrich for genetically-intact HIV-1 genomes is vital in developing targeted curative strategies. Previous studies have found that HIV-1 proviral DNA is enriched in CD4+ T-cells expressing the immune checkpoint markers programmed cell death protein-1 (PD-1) or cytotoxic T-lymphocyte associated protein-4 (CTLA-4). There has also been some success in blocking these markers in an effort to reverse HIV-1 latency. However, it remains unclear whether cells expressing PD-1 and/or CTLA-4 are enriched for genetically-intact, and potentially replication-competent, HIV-1 genomes. We obtained peripheral blood from 16 HIV-1-infected participants, and paired lymph node from four of these participants, during effective ART. Memory CD4+ T-cells from either site were sorted into four populations: PD-1-CTLA-4- (double negative, DN), PD-1+CTLA-4- (PD-1+), PD-1-CTLA-4+ (CTLA-4+) and PD-1+CTLA-4+ (double positive, DP). We performed an exploratory study using the full-length individual proviral sequencing (FLIPS) assay to identify genetically-intact and defective genomes from each subset, as well as HIV-1 genomes with specific intact open reading frames (ORFs). In peripheral blood, we observed that proviruses found within PD-1+ cells are more likely to have intact ORFs for genes such as tat, rev and nef compared to DN, CTLA-4+ and DP cells, all of which may contribute to HIV-1 persistence. Conversely, we observed that CTLA-4 expression is a marker for cells harbouring HIV-1 provirus that is more likely to be defective, containing low levels of these intact ORFs. In the lymph node, we found evidence that CTLA-4+ cells contain lower levels of HIV-1 provirus compared to the other cell subsets. Importantly, however, we observed significant participant variation in the enrichment of HIV-1 proviruses with intact genomes or specific intact ORFs across these memory CD4+ T-cell subsets, and therefore consideration of additional cellular markers will likely be needed to consistently identify cells harbouring latent, and potentially replication-competent, HIV-1.

CTLA-4 on thymic epithelial cells complements Aire for T cell central tolerance

Medullary thymic epithelial cells (mTECs) are essential for the establishment of T cell central tolerance. The transcription factor Aire plays a key role in this process, but other factors remain understudied. We found that a small population of mTECs expressed the coinhibitory receptor cytotoxic T lymphocyte-associated protein 4 (CTLA-4). These CTLA-4+ cells were detectable in perinates, peaked around young adulthood and expanded sixfold in the absence of Aire. Single-cell transcriptomics revealed CTLA-4+ mTECs to express a distinct gene signature encoding molecules associated with antigen presentation and interferon-gamma signaling. Mice conditionally lacking CTLA-4 in thymic epithelial cells had no major immunological deficiencies but displayed a mildly increased inflammatory tone and a partial defect in the generation of Foxp3+CD4+ regulatory T cells. Consequently, these mice developed modest levels of autoantibodies and lymphocytic infiltration of peripheral tissues. Thus, CTLA-4 expression in mTECs complements Aire to establish T cell central tolerance.

CTLA-4 Facilitates DNA Damage-Induced Apoptosis by Interacting With PP2A.

Cytotoxic T-lymphocyte–associated protein 4 (CTLA-4) plays a pivotal role in regulating immune responses. It accumulates in intracellular compartments, translocates to the cell surface, and is rapidly internalized. However, the cytoplasmic function of CTLA-4 remains largely unknown. Here, we describe the role of CTLA-4 as an immunomodulator in the DNA damage response to genotoxic stress. Using isogenic models of murine T cells with either sufficient or deficient CTLA-4 expression and performing a variety of assays, including cell apoptosis, cell cycle, comet, western blotting, co-immunoprecipitation, and immunofluorescence staining analyses, we show that CTLA-4 activates ataxia–telangiectasia mutated (ATM) by binding to the ATM inhibitor protein phosphatase 2A into the cytoplasm of T cells following transient treatment with zeocin, exacerbating the DNA damage response and inducing apoptosis. These findings provide new insights into how T cells maintain their immune function under high-stress conditions, which is clinically important for patients with tumors undergoing immunotherapy combined with chemoradiotherapy.

Nicotinic Acetylcholine Receptor Subunit Alpha-7 Mediates PD-L1 and CTLA-4 Expression in HepG2 Cells

Background: The liver is the largest solid organ in the body and has several unique immunological roles. The alpha7 subtype of nicotinic acetylcholine receptor (α7nAChR) is expressed in the liver and has different immunoregulatory effects. Programmed death ligand 1 (PD-L1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) are two major checkpoints that act as a negative regulator of the immune system. In this study, we examined the effects of activation (with nicotine) and inhibition (with specific siRNA) of α7nAChR on the PD-L1 and CTLA-4 genes expression. Methods: Human hepatocellular carcinoma (HepG2) cell line was used to investigate the effects of treatment with low dose (1 μM) and high dose (10 μM) of nicotine on the PD-L1 and CTLA-4 expression. In addition to this, the effects of treatment with 100 nM concentration of specific siRNA targeting α7nAChR (α7-siRNA) were examined using quantitative real-time PCR. Results: Compared to untreated cells, the HepG2 cells treated with nicotine exhibited significant dose-dependent decreases in PD-L1 expression. Furthermore, nicotine has a biphasic effect on the CTLA-4 expression and decreased its expression in 10 μM concentration. The qRT-PCR revealed that α7-siRNA significantly reduced the mRNA levels of α7nAChR (P &lt; 0.01). Also, compared to control cells, α7-siRNA treated cells exhibited significant increases in both PD-L1 and CTLA-4 expression. Conclusion: These experiments determined that nicotine treatment leads to decreases in PD-L1 and CTLA-4 expression. These effects are reversed by treatment with α7-siRNA, which indicates the involvement of α7nAChR related mechanisms in these processes.

Lupus susceptibility region containing CTLA4 rs17268364 functionally reduces CTLA4 expression by binding EWSR1 and correlates IFN-\u03b1 signature

BackgroundDysregulation of T cells mediated immune responses is a hallmark in the development of systemic lupus erythematosus (SLE). Recent genome wide association study (GWAS) revealed the genetic contribution of variants located in the cytotoxic T lymphocyte-associated protein-4 (CTLA4)-inducible T cell co-stimulator (ICOS) intergenic region to SLE susceptibility. Our aim is to find a functional variant in this region.MethodsThe genetic association results in the CTLA4-ICOS region from previous GWAS were adopted to select the potential variant which was further replicated in two independent cohorts (Henan cohort 2053 SLE patients and 1845 healthy controls, Beijing cohort 2303 SLE patients and 19,262 healthy). In order to explore the functional significance in SLE, bioinformatics with validation experiments (including electrophoretic mobility shift assay and luciferase reporter assay) and mRNA expression analysis were also performed.ResultsA variant located in the CTLA4-ICOS intergenic region, rs17268364, was associated with susceptibility to SLE patients in Chinese populations (risk allele, pmeta = 7.02×10−11, OR 1.19, 95%CI 1.13–1.26). The bioinformatics suggested that rs17268364 might affect the expression of CTLA4, not ICOS. The rs17268364 risk G allele containing sequence reduced the expression of the reporter gene by binding transcriptional repressor Ewing sarcoma breakpoint region 1 (EWSR1). Following genotype-mRNA expression, the analysis also showed the risk allele of rs17268364 was associated with low CTLA4 expression in lupus nephritis (LN) patients. Healthy individuals carrying rs17268364 risk G allele was significantly correlated with higher levels of IFN-α signature including increased lymphocyte antigen 6E (LY6E) (p=0.031), interferon-stimulated gene 15 (ISG15) (p=0.038), interferon regulatory factor 9 (IRF9) (p=0.028), and interferon regulatory factor 5 (IRF5) (p=0.040) mRNA expression.ConclusionsThe present study confirmed the functional role of rs17268364 in the CTLA4-ICOS intergenic region that increased SLE susceptibility in the Chinese population.

A pan-cancer study of PD-1 and CTLA-4 as therapeutic targets.

BackgroundImmunotherapy is a new and powerful weapon against tumors, represented by inhibitors of programmed death-1 (PD-1) and cytotoxic T lymphocyte-associated protein-4 (CTLA-4). This study aimed to determine the similarities and differences between PD-1 and CTLA-4 in 33 cancers in The Cancer Genome Atlas (TCGA) and the impact of subtypes of the immune environment on tumor production and treatment.MethodsFrom the Xena browser, we downloaded TNM stage, immune subtypes, and tumor microenvironment scores for 33 tumors from TCGA. Expression of CTLA-4 and PD-1 in normal and tumor samples were compared for various tumors with normal tissue sample sizes greater than five. The relationship between expression and overall survival was investigated using one-way Cox analysis. The immune scores of 33 tumors were assessed using ESTIMATE prediction software to predict the degree of immune cell infiltration across tumors and calculate the correlation between PD-1 and CTLA-4 expression with the tumor microenvironment and tumor stem cells. We also examined the correlation between genes and drug sensitivity.ResultsPD-1 and CTLA-4 were highly expressed in breast invasive carcinoma (BRCA), cholangiocarcinoma (CHOL), esophageal carcinoma (ESCA), and kidney renal clear cell carcinoma (KIRC) (P<0.05), highly correlated with immune subtypes C2 (IFN-γ-dominant) and C6 (TGF-β-dominant), and positively correlated with tumor microenvironmental immune scores (P<0.05). In renal clear cell carcinoma, PD-1 and CTLA-4 expression was positively correlated with clinical stage and microenvironmental score (r>0.7, P<0.05).ConclusionsThe finding that PD1 and CTLA-4 are associated with the prognosis of most tumour patients and are closely related to the tumour microenvironment is of great value and provides a research direction for the screening of populations benefiting from immunotherapy.

Cytotoxic T-lymphocyte-associated protein 4 in gastric cancer: Prognosis and association with PD-L1 expression.

Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is one of the most studied immune checkpoint in gastric cancer (GC). However, the prognostic role of CTLA-4 expression in GC is poorly described. This study aimed to evaluate CTLA-4 expression in GC and its impact on survival, including patients treated with standard platinum-based chemotherapy (CMT), and association with PD-L1 expression. All GC patients who underwent D2-gastrectomy were investigated retrospectively. Tumor samples were examined for CTLA-4 and PD-L1 by immunohistochemistry. Tumor-infiltrating inflammatory cells, including CD4 + and CD8 + , were also examined. Among the 284 GC patients included, 159 (56%) were CTLA-4 positive and the remaining 125 (44%) were classified as negative. CTLA-4 positive GC was associated with increased inflammatory cell infiltration (p < 0.001), high CD8 + T cells (p = 0.016) and PD-L1 expression (p = 0.026). Considering GC referred for treatment, CTLA-4 negative patients who received CMT had a significant improvement in disease-free survival compared to untreated CLTA-4 negative (p = 0.028). In multivariate analysis, GC positive for both CTLA-4 and PD-L1 had a prognostic impact on survival. CTLA-4 positive was associated with PD-L1 expression and a high tumor-infiltrating CD8 + T cells. Accordingly, positivity for both CTLA-4 and PD-L1 was an independent factor associated to better survival in GC patients.

Infliximab modifies regulatory T cells and co-inhibitory receptor expression on circulating T cells in psoriasis

BackgroundPsoriasis is a T cell–mediated autoimmune skin disease. Accumulating evidence has demonstrated that co-inhibitory receptors (CIRs) play a vital role in regulating T cell–mediated immune response, especially in neoplasm and autoimmunity. However, the immuno-function of CIRs in the development of psoriasis remains unclear. ObjectiveWe investigated the expression of CIRs on the circulating T lymphocytes of psoriasis patients before and after anti–tumor necrosis factor-α (TNF-α) therapy. MethodsWe enrolled 17 patients with moderate-to-severe plaque psoriasis, 17 patients with mild plaque psoriasis, and 18 healthy controls in this study. Fourteen of the moderate-to-severe psoriasis patients were treated with infliximab, a monoclonal antibody against TNF-α. Peripheral blood was collected, and peripheral blood mononuclear cells were extracted. The proportion of T cell subsets along with their expression of CIRs, namely T cell immunoreceptor with Ig and ITIM domains (TIGIT), lymphocyte activating gene 3 (LAG-3), cytotoxic T–lymphocyte associated protein 4 (CTLA-4), B and T lymphocyte–associated protein (BTLA), endothelial protein C receptor (PROCR), podoplanin (PDPN), programmed cell death 1 (PD-1), and T cell immunoglobulin mucin family containing molecule 3 (TIM-3), were determined by flow cytometric assay. ResultsThe moderate-to-severe plaque psoriasis patients had less circulating Tregs, which increased after infliximab treatment. They also had decreased TIGIT, LAG-3 but increased PDPN expression on peripheral CD4+ T cells. Infliximab enhanced TIGIT, LAG-3, CTLA-4 but reduced PROCR expression on circulating CD4+ T cells. Remarkably, both the frequency of circulating Tregs and the expression level of TIGIT on CD4+ T cells at baseline (pre-treatment) negatively correlated with the extent of PASI score reduction benefited from infliximab therapy. ConclusionAnti–TNF-α therapy increased the frequency of Tregs and TIGIT, LAG-3, CTLA-4 expression but reduced PROCR expression on circulating CD4+ T cells in psoriasis patients. The baseline proportion of Tregs and the expression level of TIGIT on circulating CD4+ T cells might serve as predictive markers for the degree of disease remission benefited from infliximab treatment.

Cytotoxic T-Lymphocyte-Associated Protein-4 and Lymphocyte Activation Gene-3 Expression in Orbitally-Invasive Versus Nodular Basal Cell Carcinoma.

To compare the expression of cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) and lymphocyte activation gene-3 (LAG-3) in nodular and orbitally-invasive forms of basal cell carcinoma (BCC). Immunohistochemical staining for CTLA-4 and LAG-3 was performed on the pathology specimens of BCC from orbital exenteration and nodular forms. The numbers of positively-staining cells/×40 field were counted across 5 consecutive fields of each specimen and statistical analysis was performed to calculate the difference in expression between the 2 groups. Nine cases of orbitally-invasive BCC and 6 cases of nodular BCC were studied. The mean numbers of CTLA-4-positively staining cells were 11.51 cells/×40 field (median = 6.60 cells/×40 field, range = 0.4-31.8 cells/×40 field) in invasive BCC and 0.90 cells/×40 field (median = 0.60 cells/×40 field, range = 0.0-2.8 cells/×40 field) in nodular specimens. The difference between the 2 groups was statistically significant (p = 0.0030). The mean number of LAG-3-positively staining cells was 0.58 cells/×40 field (median = 0.0, range = 0.0-2.8 cells/×40 field) in invasive BCC and 3.13 cells/×40 field (median = 0.0, range = 0.0-18.18 cells/×40 field). There was no significant difference in LAG-3 positivity between tumor groups (p = 0.5564). CTLA-4 expression was enriched in orbitally invasive BCC compared with nodular forms of BCC, whereas LAG-3 expression did not differ between these entities. CTLA-4 mediated immune suppression may facilitate the development of orbitally invasive BCC. Treatment strategies that use existing medications to target CTLA-4 may decrease the requirement for orbital exenteration and provide enhanced survival outcomes.

Diagnostic Dilemma on Novel Pathogenic Variant of Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) in a Family with Chronic ITP and Immune Dysregulation

Inborn errors of immunity are increasingly linked to autoimmune cytopenias, especially in families with immunodeficiency and immune dysregulation. We present a large family study with several members presenting with chronic immune thrombocytopenia (ITP) and variable combinations of immune dysregulation and infections. The proband and kindred were diagnosed with a novel variant of unknown significance (VUS) in CTLA-4. Diagnostic approach to confirm the association between clinical phenotype and the pathogenicity of this novel variant is described. Retrospective chart review yielded demographic information, clinical history, immunological phenotype, genetic panel testing and treatment response. Functional testing included CTLA-4 expression and transendocytosis assay. Proband a 50-year-old female, presented with recurrent pneumonia, chronic diarrhea, hypothyroidism, granulomatous interstitial lung disease, and ITP. Immune phenotyping confirmed hypogammaglobulinemia requiring immunoglobulin replacement therapy. Proband, along with three other family members, had a novel CTLA4-VUS (c.173G>C p.Cys58Ser). Proband and her adult cousin had chronic ITP and hypogammaglobulinemia; however, two children with this VUS only had history of milk-induced colitis and asthma, respectively. Internalization of CD80/86 was impaired in CTLA-4 trans-endocytosis assay performed on T cells from proband’s cousin, revealing a functional impairment of CTLA-4. For the proband, abatacept (CTLA4-Ig) was initiated and resulted in marked improvement in respiratory and gastrointestinal symptoms along with decreased frequency of infections. The segregation of clinical phenotype within the family, functional testing, and response to abatacept support this CTLA-4 VUS as pathogenic variant. Our study highlights the importance of genetic testing and functional studies in patients with immune dysregulation and immunodeficiency.

Categorisation of patients based on immune profiles: a new approach to identifying candidates for response to checkpoint inhibitors.

ObjectivesInhibitors to the checkpoint proteins cytotoxic T‐lymphocyte‐associated protein 4 (CTLA‐4) and programmed cell death protein 1 (PD‐1) are becoming widely used in cancer treatment. However, a lack of understanding of the patient response to treatment limits accurate identification of potential responders to immunotherapy.MethodsIn this study, we assessed the expression of PD‐1 and CTLA‐4 on 19 leucocyte populations in the peripheral blood of 74 cancer patients. A reference data set for PD‐1 and CTLA‐4 was established for 40 healthy volunteers to determine the normal expression patterns for these checkpoint proteins.ResultsUnsupervised hierarchical clustering found four immune profiles shared across the solid tumor types, while chronic lymphocytic leukaemia patients had an immune profile largely unique to them. Furthermore, we measured these leucocyte populations on an additional cohort of 16 cancer patients receiving the PD‐1 inhibitor pembrolizumab in order to identify differences between responders and non‐responders, as well as compared to healthy volunteers (n = 20). We observed that cancer patients had pre‐treatment PD‐1 and CTLA‐4 expression on their leucocyte populations at different levels compared to healthy volunteers and identified two leucocyte populations positive for CTLA‐4 that had not been previously described. We found higher levels of PD‐1+ CD3+ CD4− CD8− cells in patients with progressive disease and have identified it as a potential biomarker of response, as well as identifying other significant differences in phenotypes between responders and non‐responders.ConclusionThese results are suggestive that categorisation of patients based on immune profiles may differentiate responders from non‐responders to immunotherapy for solid tumors.

Accumulation of TNFR2-expressing regulatory T cells in malignant pleural effusion of lung cancer patients is associated with poor prognosis.

BackgroundRegulatory T cells (Tregs) may represent a major cellular mechanism in immune suppression by dampening the anti-tumor response in malignant pleural effusion (MPE). Tumor necrosis factor receptor type II (TNFR2) has emerged as a novel identification for the maximally suppressive subset of Tregs in the tumor environment. At present, the significance of TNFR2 expression on Tregs in MPE remains unclear.MethodsThe distribution of TNFR2+cells in Tregs and effector T cells (Teffs) in MPE, peripheral blood (PB), and tuberculosis pleural effusion (TPE) were determined. The associations between TNFR2+Tregs frequencies present in MPE and the clinical and laboratorial characteristics of patients with lung cancer were investigated. The immunosuppressive phenotype of TNFR2+Tregs in MPE was analyzed. The effects of the TNF-TNFR2 interaction on the immunosuppressive function of Tregs was explored. The efficacy of targeting TNFR2 for MPE therapy was examined. The source of TNF in MPE was identified.ResultsWe observed that markedly higher levels of TNFR2 were expressed in MPE Tregs compared with the levels expressed in MPE Teffs, PB Tregs, or in TPE Tregs. The frequencies of TNFR2+Tregs were positively correlated with the number of tumor cells in MPE, as well as the volume of MPE. High frequencies of TNFR2+Tregs in MPE indicated short survival time and poor performance status for MPE patients. Compared to TNFR2-Tregs, TNFR2+Tregs expressed higher levels of immunosuppressive molecules cytotoxic T lymphocyte-associated protein 4 (CTLA-4), programmed cell death-ligand 1 (PD-L1), and replicating marker Ki-67. Consequently, the proportions of interferon gamma (IFN-γ)-producing cytotoxic T lymphocytes (CTLs) were significantly increased after TNFR2 blockade. Furthermore, tumor necrosis factor (TNF), through interaction with TNFR2, enhanced the suppressive capacity of Tregs by up-regulating CTLA-4 and PD-L1 expression. Interestingly, T helper 1 (Th1) and T helper 17 (Th17) cells are the major source of TNF in MPE, suggesting that MPE Teffs may paradoxically promote tumor growth by boosting MPE Treg activity via the TNF-TNFR2 pathway.ConclusionsOur data expanded the immunosuppressive mechanism present in MPE induced by Tregs, and provides novel insight for the diagnosis, disease evaluation, and treatment of MPE patients.