Cytotoxic T Lymphocyte-associated Molecule-4 Research Articles

Immune Checkpoint Inhibitors: Recent Clinical Advances and Future Prospects.

Immune checkpoints are vital molecules and pathways of the immune system with defined roles of controlling immune responses from being destructive to the healthy cells in the body. They include inhibitory receptors and ligands, which check the recognition of most cancers by the immune system. This happens when proteins on the surface of T cells called immune checkpoint proteins identify partner proteins on the cancer cells and bind to them, sending brake signals to the T cells to evade immune attack. However, drugs called immune checkpoint inhibitors block checkpoint proteins from binding to their partner proteins, thereby inhibiting the brake signals from being sent to T cells. This eventually allows the T cells to destroy cancer cells and arbitrate robust tumor regression. Many such inhibitors have already been approved and are in various developmental stages. The well-illustrated inhibitory checkpoints include the cytotoxic T lymphocyte-associated molecule-4 (CTLA-4), programmed cell death receptor-1 (PD-1), and programmed cell death ligand-1 (PD-L1). Though many molecules blocking these checkpoints have shown promise in treating many malignancies, such treatment options have limited success in terms of the immune response in most patients. Against this backdrop, exploring new pathways and next-generation inhibitors becomes imperative for developing more responsive and effective immune checkpoint therapy. Owing to the complex biology and unexplored ambiguities in the mechanistic aspects of immune checkpoint pathways, analysis of the activity profile of new drugs is the subject of strenuous investigation. We herein report the recent progress in developing new inhibitory pathways and potential therapeutics and delineate the developments based on their merit. Further, the ensuing challenges towards developing efficacious checkpoint therapies and the impending opportunities are also discussed.

Targeting immune checkpoints in anti-neutrophil cytoplasmic antibodies associated vasculitis: the potential therapeutic targets in the future.

Anti-neutrophil cytoplasmic autoantibodies (ANCA) associated vasculitis (AAV) is a necrotizing vasculitis mainly involving small blood vessels. It is demonstrated that T cells are important in the pathogenesis of AAV, including regulatory T cells (Treg) and helper T cells (Th), especially Th2, Th17, and follicular Th cells (Tfh). In addition, the exhaustion of T cells predicted the favorable prognosis of AAV. The immune checkpoints (ICs) consist of a group of co-stimulatory and co-inhibitory molecules expressed on the surface of T cells, which maintains a balance between the activation and exhaustion of T cells. CD28, inducible T-cell co-stimulator (ICOS), OX40, CD40L, glucocorticoid induced tumor necrosis factor receptor (GITR), and CD137 are the common co-stimulatory molecules, while the programmed cell death 1 (PD-1), cytotoxic T lymphocyte-associated molecule 4 (CTLA-4), T cell immunoglobulin (Ig) and mucin domain-containing protein 3 (TIM-3), B and T lymphocyte attenuator (BTLA), V-domain Ig suppressor of T cell activation (VISTA), T-cell Ig and ITIM domain (TIGIT), CD200, and lymphocyte activation gene 3 (LAG-3) belong to co-inhibitory molecules. If this balance was disrupted and the activation of T cells was increased, autoimmune diseases (AIDs) might be induced. Even in the treatment of malignant tumors, activation of T cells by immune checkpoint inhibitors (ICIs) may result in AIDs known as rheumatic immune-related adverse events (Rh-irAEs), suggesting the importance of ICs in AIDs. In this review, we summarized the features of AAV induced by immunotherapy using ICIs in patients with malignant tumors, and then reviewed the biological characteristics of different ICs. Our aim was to explore potential targets in ICs for future treatment of AAV.

Differential Expression of the TLR4 Gene in Pan-Cancer and Its Related Mechanism.

Previous studies have revealed the relationship between toll-like receptor 4 (TLR4) polymorphisms and cancer susceptibility. However, the relationship between TLR4 and prognosis and immune cell infiltration in pan-cancer patients is still unclear. Through the Genotype-Tissue Expression (GTEx) and The Cancer Genome Atlas (TCGA) databases, the distinct expression of the TLR4 gene in 24 tumors and normal tissues was analyzed. Univariate Cox proportional hazards regression analysis was used to identify the cancer types whose TLR4 gene expression was related to prognosis. The relationship between TLR4 and tumor cell immune invasion was studied. Spearman’s rank correlation coefficient was used to analyze the relationship among TLR4 and immune neoantigens, tumor mutation burden (TMB), microsatellite instability (MSI), DNA repair genes, and DNA methylation. Gene Set Enrichment Analysis (GSEA) was used to identify the tumor-related pathways that the TLR4 gene was highly expressed in; the expression of the TLR4 gene was verified with the Human Protein Atlas (HPA) database. Low expression of TLR4 was associated with an inferior prognosis in kidney renal clear cell carcinoma (KIRC), skin cutaneous melanoma (SKCM), and uterine corpus endometrial carcinoma (UCEC), while high expression was related to a poor prognosis in head and neck squamous cell carcinoma (HNSC), prostate adenocarcinoma (PRAD), stomach adenocarcinoma (STAD), and testicular germ cell tumor (TGCT). The expression of TLR4 was negatively correlated with the expression of B cells in STAD. The expression of TLR4 was positively correlated with the infiltration of B cells, CD4 and CD8 T cells, neutrophils, macrophages, and dendritic cells in STAD, KIRC, UCEC, TGCT, and SKCM. The expression of the TLR4 gene in KIRC, SKCM, STAD, TGCT, and UCEC was highly correlated with inducible T-cell costimulator (ICOS), cytotoxic T lymphocyte-associated molecule 4 (CTLA4), and CD28 immune checkpoints. Spearman’s rank correlation coefficient showed that the expression of TLR4 gene was significantly correlated with TMB in STAD and UCEC and was prominently correlated with MSI in TGCT, STAD, and SKCM. The expression of the TLR4 gene was highly correlated with MLH1, MSH2, and MSH6 in KIRC, SKCM, and STAD. The expression of the TLR4 gene was remarkably correlated with the methyltransferases DNA methyltransferase 2 (DNMT2) and DNA methyltransferase 3-beta (DNMT3B) in SKCM and STAD. Enrichment analysis showed that TLR4 was highly expressed in the chemokine signaling pathway and the cell adhesion molecule and cytokine receptor interaction pathway. In summary, the expression of TLR4 is linked to the prognosis of KIRC, SKCM, STAD, TGCT, and UCEC patients and the level of immune infiltration of CD4, CD8 T cells, macrophages, neutrophils, and dendritic cells.

Co-stimulatory agonists: An insight into the immunotherapy of cancer.

Immune checkpoint pathways consist of stimulatory pathways, which can function like a strong impulse to promote T helper cells or killer CD8+ cells activation and proliferation. On the other hand, inhibitory pathways keep self-tolerance of the immune response. Increasing immunological activity by stimulating and blocking these signaling pathways are recognized as immune checkpoint therapies. Providing the best responses of CD8+ T cell needs the activation of T cell receptor along with the co-stimulation that is generated via stimulatory checkpoint pathways ligation including Inducible Co-Stimulator (ICOS), CD40, 4-1BB, GITR, and OX40. In cancer, programmed cell death receptor-1 (PD-1), Programmed cell death ligand-1(PD-L1) and Cytotoxic T Lymphocyte-Associated molecule-4 (CTLA-4) are the most known inhibitory checkpoint pathways, which can hinder the immune responses which have specifically anti-tumor characteristics and attenuate T cell activation and also cytokine production. The use of antagonistic monoclonal antibodies (mAbs) that block CTLA-4 or PD-1 activation is used in a variety of malignancies. It has been reported that they can lead to an increase in T cells and thereby strengthen anti-tumor immunity. Agonists of stimulatory checkpoint pathways can induce strong immunologic responses in metastatic patients; however, for achieving long-lasting benefits for the wide range of patients, efficient combinatorial therapies are required. In the present review, we focus on the preclinical and basic research on the molecular and cellular mechanisms by which immune checkpoint inhibitor blockade or other approaches with co-stimulatory agonists work together to improve T-cell antitumor immunity.

Higher frequency of the CTLA-4+ LAG-3+ T-cell subset in patients with newly diagnosed acute myeloid leukemia.

Immune suppression based on alternative regulation of immune checkpoint proteins, for example, programmed cell death receptor-1 (PD-1) and cytotoxic T lymphocyte-associated molecule-4 (CTLA-4), which results in T-cell exhaustion, contributes to cancer development and progression. In this study, we sought to characterize the distribution of CTLA-4 and T-cell lymphocyte activation gene-3 (LAG-3) expression on exhausted T cells in different T-cell subsets from patients with acute myeloid leukemia (AML). The coexpression of CTLA-4 and LAG-3 on exhausted CD244+ and CD57+ T cells from the CD3+ , CD4+ , and CD8+ T-cell subsets in peripheral blood from 12 patients with newly diagnosed AML was analyzed by multicolor flow cytometry assay. A significantly higher percentage of CTLA-4+ CD3+ , CD4+ and CD8+ T cells was found in patients with AML. In addition, higher numbers of both CTLA-4+ CD244+ and CTLA-4+ CD57+ CD3+ T cells were detected. Interestingly, the increased CTLA-4+ CD244+ T cells were predominantly CD4+ T cells. In contrast, the increased CTLA-4+ CD57+ T cells primarily consisted of the CD8+ T-cell subset. A high proportion of LAG-3+ T cells was found in only a few cases with AML; however, a significantly higher proportion of coexpression of CTLA-4 and LAG-3 in the CD3+ and CD8+ T-cell subsets was detected. We for the first time observed higher CTLA-4+ CD244+ CD4+ , CTLA-4+ CD57+ CD8+ , CTLA-4+ LAG-3+ CD3+ and CTLA-4+ LAG-3+ CD8+ T cells in patients with AML, whereas the upregulated expression of LAG-3 on T cells was only found in a subset of the cases. These data may provide further information by complementing the heterogeneity of immune checkpoints expression in AML.

Serum sCTLA-4 levels and clinical manifestations in ankylosing spondylitis patients.

T cell abnormal activation is thought to have a main role in the etiology of ankylosing spondylitis (AS). While cytotoxic T lymphocyte-associated molecule-4 (CTLA-4) is suppressing the immune system, in previous studies serum soluble CTLA-4 (sCTLA-4) was detected at high amounts in autoimmune disorders. We sought to evaluate the association between soluble CTLA-4 in serum and disease activity in AS patients. Thirty-eight patients with AS, 28 rheumatoid arthritis (RA) patients, and 27 disease-free controls were enrolled to the study. The levels of sCTLA-4 were determined for each participant using an enzyme-linked immunosorbent assay. The erythrocyte sedimentation rate (ESR), C-reactive peptide, and demographic characteristics were documented. The data were analyzed by using relevant statistical methods. In comparison with RA patients and controls, patients with AS showed high sCTLA-4 levels (p<0.001). The sCTLA-4 levels did not correlate with the severity of the disease in AS patients (p=0.370). The ESR levels and Bath Ankylosing Spondylitis Disease Activity Index were correlated in AS patients (p=0.012). We evaluated the association between the disease severity of AS and sCTLA-4. Although, the correlation was not shown, sCTLA-4 was highest in the AS group. Further studies with larger samples should be completed to attain a better understanding of the AS etiology.

Novel Immunologic Approaches to Melanoma Treatment

Approaches to treating melanoma have changed radically since the introduction of immunotherapy, and survival figures are now higher than possible with earlier therapies. The immunomodulators currently available mainly block CTLA-4 (cytotoxicT lymphocyte-associated molecule-4) and PD-1 (programed cell death protein 1) translocated to the cell surface, where they inhibit the antitumor immune response. Treatments blocking these molecules are being more widely used. Research now seeks new molecular targets, the best combinations of available drugs, and biomarkers that can identify ideal candidates for each one

Immunogenomics: using genomics to personalize cancer immunotherapy.

While the use of genomic data has the potential to revolutionize patient care, there is still much work to be done with regard to the transformation of host-tumor interactions into favorable clinical outcomes for our patients. High-throughput technologies, such as next-generation sequencing (NGS), have rapidly advanced our understanding of oncology, and we are learning that most tumors do not simply possess consistently mutated genes that are responsible for tumorigenesis, facilitating the need for personalized cancer therapy. A T cell-dependent mechanism of cancer progression was discovered in 2012, providing a potential link to cancer immunotherapy. Since then, an antibody against cytotoxic T lymphocyte-associated molecule-4 (CTLA-4), ipilimumab, and three programmed death-1 (PD-1)/programmed death ligand-1 (PD-L1) inhibitors, pembrolizumab (Keytruda), nivolumab (Opdivo), and atezolizumab (Tecentriq), were approved by the Food and Drug Administration (FDA) in the USA. In this review article, based on evidence that has been emerging in the literature over the last decade, we will discuss the basis for including genomic data in immunotherapy regimens, the current progress in identifying biomarkers targetable by immune checkpoint blockade, and the application of these therapies in modern oncology programs. Going forward, the clinical application of NGS in personalized oncology programs could include dose monitoring and adjustment or the development of individualized vaccines or other personalized therapies based onthe mutational landscape. The continued identification of new neoantigens and the efficient mobilization of tumor-reactive lymphocytes in patients with cancer will promote the advancement of immunotherapy using personalized NGS-guided technologies.

Novedades en inmunología del melanoma

Approaches to treating melanoma have changed radically since the introduction of immunotherapy, and survival figures are now higher than possible with earlier therapies. The immunomodulators currently available mainly block CTLA-4 (cytotoxicT lymphocyte-associated molecule-4) and PD-1 (programed cell death protein 1) translocated to the cell surface, where they inhibit the antitumor immune response. Treatments blocking these molecules are being more widely used. Research now seeks new molecular targets, the best combinations of available drugs, and biomarkers that can identify ideal candidates for each one.

Expression and prognostic significance of programmed death protein 1 and programmed death ligand-1, and cytotoxic T lymphocyte-associated molecule-4 in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common malignancies and causes of death worldwide. In this study, we assessed the correlation between clinicopathologic factors with programmed cell death protein 1 (PD-1) and programmed cell death ligand-1 (PD-L1), and cytotoxic T lymphocyte-associated molecule-4 (CTLA-4) expressions. Furthermore, we analyzed the prognostic significance of these proteins in a subgroup of patients. We retrospectively evaluated the PD-1, PD-L1, and CTLA-4 expressions in 294 HCC tissue microarray samples using immunohistochemistry. PD-1 and PD-L1 expressions were significant related to high CD8+ tumor-infiltrating lymphocytes (TILs) (r = 0.664, p < 0.001 and r = 0.149, p = 0.012). Only high Edmondson-Steiner grade was statistically related to high PD-1 expression. High PD-L1 expression was demonstrated as an independent poor prognostic factor for disease-free survival in addition to previous known factors, size >5 cm and serum albumin ≤3.5 g/dL in high CD8+ TILs group. We have demonstrated that the combined high expression of PD-L1 and CD8+ TIL is an important prognostic factor related to the immune checkpoint pathway in HCC and furthermore, there is a possibility that it could be used as a predictor of therapeutic response. Also, this result would be helpful in evaluating the applicable group of PD-1/PD-L1 blocking agent for HCC patients.

Restoring anti-tumor functions of T cells via nanoparticle-mediated immune checkpoint modulation

The core purpose of cancer immunotherapy is the sustained activation and expansion of the tumor specific T cells, especially tumor-infiltrating cytotoxic T lymphocytes (CTLs). Currently, one of the main foci of immunotherapy involving nano-sized carriers is on cancer vaccines and the role of professional antigen presenting cells, such as dendritic cells (DCs) and other phagocytic immune cells. Besides the idea that cancer vaccines promote T cell immune responses, targeting immune inhibitory pathways with nanoparticle delivered regulatory agents such as small interfering RNA (siRNA) to the difficultly-transfected tumor-infiltrating T cells may provide more information on the utility of nanoparticle-mediated cancer immunotherapy. In this study, we constructed nanoparticles to deliver cytotoxic T lymphocyte-associated molecule-4 (CTLA-4)-siRNA (NPsiCTLA-4) and showed the ability of this siRNA delivery system to enter T cells both in vitro and in vivo. Furthermore, T cell activation and proliferation were enhanced after NPsiCTLA-4 treatment in vitro. The ability of direct regulation of T cells of this CTLA-4 delivery system was assessed in a mouse model bearing B16 melanoma. Our results demonstrated that this nanoparticle delivery system was able to deliver CTLA-4-siRNA into both CD4+ and CD8+ T cell subsets at tumor sites and significantly increased the percentage of anti-tumor CD8+ T cells, while it decreased the ratio of inhibitory T regulatory cells (Tregs) among tumor infiltrating lymphocytes (TILs), resulting in augmented activation and anti-tumor immune responses of the tumor-infiltrating T cells. These data support the use of potent nanoparticle-based cancer immunotherapy for melanoma.

Cancer Immunotherapy Researchers Focus on Refining Checkpoint Blockade Therapies

The origins of cancer immunotherapy date back more than 2 decades to when researchers explored ways to amplify the immune system to attack tumor cells. At the time, scientists hypothesized that tumors were able to evade immune survei l lance because tumor cells lacked costimulatory molecules required to signal T cells to the presence of the tumor. However, in the mid-1990s, James P. Allison, PhD, and his colleagues at the University of California, Berkeley, Cancer Research Laboratory discovered that rather than lack costimulatory molecules, tumor cel ls and other immune cel ls expressed inhibitory molecules that disabled T cells from recognizing and eliminating tumors. These early experiments compared tumor growth in mice that had received injections of cytotoxic T lymphocyte– associated molecule 4 (CTLA-4) antibodies with tumor growth in mice in a control group (Leach DR et al. Science. 1996;271: 1734-1736). Allison and his colleagues hypothesized that the antibodies would turn off CTLA-4, a receptor that dampens T-cell activity, and thus unleash an attack on the tumors. The experiment results were striking. “The idea was that the brakes come on at a certain point. If we just disabled the brakes, [the immune system] can keep going until it has time to kill the tumor mass. In the first experiment, in the control group, all the mice had to be euthanized because they grew big tumors. In the [treated] mice, the tumors had gone away,” recalled Allison, who at the time was director of the Cancer Research Laboratory. But when the data were published in Science in 1996, the response from the research community was more like a collective shrug than a buzz of excitement, recalled Allison, now the executive director of the Immunology Platform and chair of the department of immunology at the MD Anderson Cancer Center in Houston. “Cancer immunotherapy had a checkeredhistory,”he said, noting thatdisillusionment had set in among researchers after interleukin-2 and cancer vaccines failed to meet high expectations for their success. Despite the initial muted response to his work, and nearly 2 decades after the publication in Science, Allison received both the 2015 Lasker-DeBakey Clinical Medical Research Award and the 2015 American Society of Clinical Oncology’s Science of Oncology Award for his work that contributed to the development of cancer immunotherapy. Now, dozens of clinical trials are under way to study how these drugs work on several different types of cancers, and oncology researchers are trying to determine why these drugs are lifesavers for some patients but fail to help others.

Immune checkpoints programmed death 1 ligand 1 and cytotoxic T lymphocyte associated molecule 4 in gastric adenocarcinoma

ABSTRACTRemarkable efficacy of immune checkpoint inhibition has been reported for several types of solid tumors and early studies in gastric adenocarcinoma are promising. A detailed knowledge about the natural biology of immune checkpoints in gastric adenocarcinoma is essential for clinical and translational evaluation of these drugs. This study is a comprehensive analysis of cytotoxic T lymphocyte associated molecule 4 (CTLA-4) and programmed death 1 ligand 1 (PD-L1) expression in gastric adenocarcinoma. PD-L1 and CTLA-4 were stained on tumor sections of 127 Caucasian patients with gastric adenocarcinoma by immunohistochemistry (IHC) and somatic mutation profiling was performed using targeted next-generation sequencing. Expression of PD-L1 and CTLA-4 on lymphocytes in tumor sections, tumor-draining lymph nodes (TDLN) and peripheral blood were studied by flow-cytometry and immune-fluorescence microscopy in an additional cohort. PD-L1 and CTLA-4 were expressed in 44.9% (57/127) and 86.6% (110/127) of the analyzed gastric adenocarcinoma samples, respectively. Positive tumor cell staining for PD-L1 or CTLA-4 was associated with inferior overall survival. Somatic mutational analysis did not reveal a correlation to expression of PD-L1 or CTLA-4 on tumor cells. Expression of PD-1 (52.2%), PD-L1 (42.2%) and CTLA-4 (1.6%) on tumor infiltrating T cells was significantly elevated compared to peripheral blood. Of note, PD-1 and PD-L1 were expressed far higher by tumor-infiltrating lymphocytes than CTLA-4. In conclusion, specific immune checkpoint-inhibitors should be evaluated in this disease and the combination with molecular targeted therapies might be of benefit. An extensive immune monitoring should accompany these studies to better understand their mode of action in the tumor microenvironment.

FRI0145 The Role of Sctla-4 in Pathogenesis of Ankylosing Spondylitis and Evaluation of Its Relation with Clinical Manifestations

BackgroundAnkylosing Spondylitis (AS) is characterized by abnormal immune responses including T cell activation. Although Cytotoxic T lymphocyte associated molecule-4 (CTLA-4) is involved in down-regulating immune responses, a soluble form of...

The associations between the polymorphisms in the CTLA-4gene and the risk of Graves’ disease in the Chinese population

BackgroundThe associations between the polymorphisms in Cytotoxic T lymphocyte-associated molecule-4 (CTLA-4) gene and Graves’ disease (GD) have been extensively investigated in Chinese population. However, the results were inconsistent. The objective of this study is to investigate the associations between the polymorphisms in CTLA-4 gene and the risk of GD by meta-analysis.MethodsWe searched Pubmed database, Medline (Ovid) database, CNKI database and Wanfang database, covering all studies until August 11, 2012. Statistical analysis was performed by using the Revman4.2 software and the Stata10.0 software.ResultsA total of 28 case–control studies concerning the most widely studied three polymorphisms [+49A/G(rs231775), -318C/T(rs5742909) and CT60(rs3087243)] for Chinese population in 21 publications were included. The results suggested that the G allele carriers (GG+GA) might have an increased risk of GD when compared with the AA homozygote carriers for the +49A/G polymorphism (GG+GA vs. AA: OR = 2.57, 95%CI = 1.87-3.52). However, as to the -318C/T polymorphism and CT60 polymorphism, the results indicated that the variant allele carriers might have decreased risks of GD when compared with the homozygote carriers (−318C/T: TT+TC vs. CC: OR = 0.78, 95%CI = 0.62-0.97; CT60: AA+AG vs. GG: OR = 0.64, 95%CI = 0.52-0.78).ConclusionsThe current meta-analysis indicated that the polymorphisms in the CLTA-4 gene might be risk factors for GD in the Chinese population. In future, more large-scale case–control studies are needed to validate these results.

MicroRNA Profile of Circulating CD4-positive Regulatory T Cells in Human Adults and Impact of Differentially Expressed MicroRNAs on Expression of Two Genes Essential to Their Function

Regulatory T cells (Tregs) are characterized by a high expression of IL-2 receptor α chain (CD25) and of forkhead box P3 (FOXP3), the latter being essential for their development and function. Another major player in the regulatory function is the cytotoxic T-lymphocyte associated molecule-4 (CTLA-4) that inhibits cytotoxic responses. However, the regulation of CTLA-4 expression remains less well explored. We therefore studied the microRNA signature of circulating CD4(+) Tregs isolated from adult healthy donors and identified a signature composed of 15 differentially expressed microRNAs. Among those, miR-24, miR-145, and miR-210 were down-regulated in Tregs compared with controls and were found to have potential target sites in the 3'-UTR of FOXP3 and CTLA-4; miR-24 and miR-210 negatively regulated FOXP3 expression by directly binding to their two target sites in its 3'-UTR. On the other hand, miR-95, which is highly expressed in adult peripheral blood Tregs, positively regulated FOXP3 expression via an indirect mechanism yet to be identified. Finally, we showed that miR-145 negatively regulated CTLA-4 expression in human CD4(+) adult peripheral blood Tregs by binding to its target site in CTLA-4 transcript 3'-UTR. To our knowledge, this is the first identification of a human adult peripheral blood CD4(+) Treg microRNA signature. Moreover, unveiling one mechanism regulating CTLA-4 expression is novel and may lead to a better understanding of the regulation of this crucial gene.

Effect of cytotoxic T lymphocyte‐associated molecule 4 1661 gene polymorphism on its expression and transcription in ulcerative colitis

Our aim was to investigate the expression of cytotoxic T lymphocyte-associated molecule 4 (CTLA-4) in ulcerative colitis (UC) and to evaluate the effect of CTLA-4 gene -1661A/G polymorphism on CTLA-4 expression and transcription. A total of 20 UC patients and 22 healthy controls matched by age and sex were enrolled at Zhongnan Hospital of Wuhan University in central China. The CTLA-4 -1661A/G polymorphism was genotyped by the polymerase chain reaction-restriction fragment length polymorphism method. A Western blot analysis was performed to determine the full length CTLA-4 (flCTLA-4) protein expression in the peripheral blood of the UC patients. Serum-soluble CTLA-4 (sCTLA-4) levels were measured by enzyme-linked immunosorbent assay. CTLA-4-1661G mutant promoter transcription function was analyzed by site-directed PCR-based mutagenesis. CTLA-4 protein expression on CD4(+) T cells in UC patients was lower than that in the healthy controls (P < 0.001) while serum sCTLA-4 in the UC patients was significantly higher than that in the healthy controls (P < 0.001). No correlation was found between flCTLA-4 and sCTLA-4 expression levels and the -1661 A/G polymorphism of the CTLA-4 gene. Meanwhile, CTLA-4 -1661 allele A had no significant impact on the promoter activity compared with allele G (P > 0.05). CTLA-4 expressions were aberrant in UC patients compared with the healthy controls. CTLA-4 -1661A/G polymorphism had no significant impact on CTLA-4 expression and transcription in the peripheral CD4 T cells of UC patients.

Regulatory T cells attenuate experimental periodontitis progression in mice

The aim of this study was to identify the presence and characterize the function of regulatory T cells (Tregs) in experimental periodontitis in mice. C57Bl/6 mice infected with Actinobacillus actinomycetemcomitans, treated or not with anti-glucocorticoid-inducible tumour necrosis factor receptor (anti-GITR) to inhibit Tregs function, were analysed regarding inflammatory cell and Tregs influx, alveolar bone loss and cytokine expression/production (analysed by real-time polymerase chain reaction and ELISA) throughout experimental periodontitis. A. actinomycetemcomitans inoculation in mice resulted in periodontal disease characterized by marked alveolar bone loss and an influx of inflammatory cells. Flow cytometry evaluation of inflammatory cells demonstrated an increased number of CD4(+)CD25(+) and CD4(+)FOXp3(+) cells, characterizing the presence of Tregs in the periodontal environment in a late stage after infection. Tregs-associated cytokines interleukin-10 (IL-10), cytotoxic T lymphocyte-associated molecule 4 (CTLA-4) and transforming growth factor-beta (TGF-beta) were found to be expressed/produced in a kinetics that resembles Tregs migration. Treatment with anti-GITR, which inhibits Tregs function, showed increased alveolar bone loss and inflammatory cell migration. A reduction in IL-10, CTLA-4 and TGF-beta levels was also observed, while interferon-gamma, tumour necrosis factor-alpha and receptor activator for nuclear factor kappaB ligand levels were increased. However, bacterial load and C-reactive protein serum did not show any differences. Taken together, our results showed that the presence of Treg cells attenuates the severity of experimental periodontitis without impairment in the control of infection.

Increased production of soluble CTLA-4 in patients with spondylarthropathies correlates with disease activity

IntroductionSpondylarthropathies (SpA) are characterized by abnormal immune responses including T cell activation. Cytotoxic T lymphocyte associated molecule-4 (CTLA-4) is involved in down-regulating immune responses. A soluble form of CTLA-4 (sCTLA-4), resulting from an alternative splicing, has been identified and was found increased in several autoimmune diseases. Here, we evaluated circulating levels of sCTLA-4 as a marker of immune dysregulation in SpA. Intracellular CTLA-4 and levels of CTLA-4 transcript expression in peripheral blood lymphocytes (PBL) were also studied.MethodsSera from 165 patients with SpA were evaluated for sCTLA-4 measurements. Results were compared with those from 71 patients with rheumatoid arthritis (RA) and 88 healthy subjects. In 32 patients with SpA, 22 patients with RA and 15 healthy controls, we analyzed the intracellular CTLA-4 expression in CD4+ T cells, CD8+ T cells, activated (HLA-DR+Foxp3-) CD4+ T cells, CD4+ regulatory (CD25+Foxp3+) T cells and in CD3 negative cells by flow cytometry. Expression of the full length (coding for membrane CTLA-4) and spliced form (coding for sCTLA-4) of CTLA-4 transcripts in PBL were analyzed by quantitative real-time polymerase chain reaction (QRT-PCR).ResultsHigh levels of sCTLA-4 were found in the SpA group compared to the RA group and healthy controls (P < 0.0001). Soluble CTLA-4 serum levels strongly correlated with clinical index of disease activity BASDAI (r = 0.42, P < 0.0001) and C-reactive protein (CRP) levels (r = 0.17, P = 0.037). In contrast to RA patients, SpA patients did not exhibit changes in intracellular CTLA-4 expression in the different PBL subsets tested. Finally, the SpA group showed a preferential expression of the spliced CTLA-4 mRNA (P = 0.0014) in PBL.ConclusionsSpA patients exhibit high levels of circulating sCTLA-4 that may result from an alternative splicing of CTLA-4 transcripts. This may influence immune activation and regulation in SpA.

Cytotoxic T lymphocyte-associated molecule-4 and systemic lupus erythematosus

Cytotoxic T lymphocyte-associated molecule-4 (CTLA-4), a member ofB7/CD28 family, is expressed mainly by immune cells. It has multiple immunologic effects and plays a central role in down-regulating immune responses. Animal experiments and clinical practices have confirmed the efficacy of CTLA-4 immunoglobulin and anti-CTLA-4 monoclonal antibody in the prevention and treatment of autoimmune diseases, transplantation rejection and tumors. Systemic lupus erythematosus (SLE), a common autoimmune disease involving multiple organs, is immunologically characterized by dysfunction of T cells and dysregulation of B-cell activity, which leads to an increase in the activity of and autoantibody production by B cells. Key words: Lupus erythematosus,systemic; T-lymphocytes,cytotoxic; Immunity