INK4 cyclin-dependent kinase inhibitors as potential prognostic biomarkers and therapeutic targets in hepatocellular carcinoma

The INK4 family is an important family of cyclin-dependent kinase inhibitors (CDKIs) and consists of CDKN2A, CDKN2B, CDKN2, and CDKN2D. Abnormal expression of CDKN2A has been reported in hepatocellular carcinoma (HCC) and is associated with the prognosis of patients and infiltration of immune cells. However, there is a lack of systematic research on the roles of the other INK4 family members in the diagnosis, prognosis, and immune regulation of HCC. Using online public databases and clinical samples, we comprehensively analyzed the INK4 family in HCC. All four INK4 proteins were overexpressed in HCC and correlated with advanced cancer stage and poor prognosis. INK4 expression accurately distinguished tumor from normal tissue, particularly CDKN2A and CDKN2C. The INK4 family participated in cell-cycle regulation and the DNA damage repair pathway, which inhibited genotoxic-induced apoptosis in tumorigenesis. INK4 proteins were positively correlated with the infiltration of immune cells (B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells) and immune checkpoints (CTLA-4, PD1, and PD-L1). CDKN2D had the highest correlation (correlation coefficient >0.3) with all the above-mentioned infiltrating immune cells and immune checkpoints, indicating that it may be useful as an immunotherapy target. The INK4 family was valuable for diagnosis and predicting the prognosis of HCC and participated in the occurrence, progression, and immune regulation of HCC, demonstrating its potential as a diagnostic and prognostic biomarker and therapeutic target in HCC.     

A dual immune signature of CD8+ T cells and MMP9 improves the survival of patients with hepatocellular carcinoma

The 5-year survival of hepatocellular carcinoma (HCC) is difficult due to the high recurrence rate and metastasis. Tumor infiltrating immune cells (TICs) and immune-related genes (IRGs) bring hope to improve survival and treatment of HCC patients. However, there are problems in predicting immune signatures and identifying novel therapeutic targets. In the study, the CIBERSORT algorithm was used to evaluate 22 immune cell infiltration patterns in gene expression omnibus (GEO) and the cancer genome atlas (TCGA) data. Eight immune cells were found to have significant infiltration differences between the tumor and normal groups. The CD8+ T cells immune signature was constructed by least absolute shrinkage and selection operator (LASSO) algorithm. The high infiltration level of CD8+ T cells could significantly improve survival of patients. The weighted gene co-expression network analysis (WGCNA) algorithm identified MMP9 was closely related to the overall survival of HCC patients. K-M survival and tROC analysis confirmed that MMP9 had an excellent prognostic prediction. Cox regression showed that a dual immune signature of CD8+ T cells and MMP9 was independent survival factor in HCC. Therefore, a dual prognostic immune signature could improve the survival of patient and may provide a new strategy for the immunotherapy of HCC.     

Correlation of zinc finger protein 2, a prognostic biomarker,with immune infiltrates in liver cancer

Purpose: The expression and clinical value of zinc finger protein 2 gene (ZIC2) in hepatocellular carcinoma (HCC) were analyzed by mining gene information from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases.Methods: Gene chip data sets were retrieved from GEO and TCGA and screened for differentially expressed genes in HCC. Gene expression profile interaction analysis (GEPIA) and Kaplan–Meier curves were used to analyze the relationship between differentially expressed genes (DEGs) and survival and prognosis in patients with HCC. Moreover, the Genecards database was used to extract ZIC2-related proteins and to analyze the physiological process of protein enrichment. Furthermore, the relationships between ZIC2 gene and tumor cell immune invasion and that between immune cell infiltration and the 5-year survival rate were studied using the tumor immune evaluation resource (TIMER) database.Results: Datasets from GEO and TCGA revealed that ZIC2 was differentially expressed in HCC tissues and normal tissues (P<0.05). High ZIC2 expression was associated with overall survival (OS) and progress-free survival in HCC patients. Overall, 25 ZIC2 related proteins, including Gli3, PRKDC, and rnf180 were identified and protein enrichment analysis indicated these were associated with four types of cell components, six types of cell functions, and eight types of biological processes. ZIC2 was positively correlated with immune infiltration cells in patients with HCC, and higher expression of ZIC2 mRNA CD4+T cells is associated with a better 5-year survival.Conclusion: ZIC2 gene may be used as an immune response marker in liver cancer to predict the prognosis of HCC.     

Impaired Function of CD4+ T Follicular Helper (Tfh) Cells Associated with Hepatocellular Carcinoma Progression

MethodsA total of 85 HCC patients with hepatitis B virus (HBV) infection, 25 HBV-relative liver cirrhosis (LC) patients, and 20 healthy controls (HC) were randomly enrolled. Flow cytometric analysis, immunohistochemical staining, and relative function (i.e., cytokine secretion, B cell maturation) assays were used to analyze the properties of CXCR5+CD4+ T cells. In addition, the relationship between the frequency of CXCR5+CD4+ T cells and overall survival rates or disease-free survival rates was also analyzed by the Kaplan-Meier method.ResultsThe frequency of circulating CXCR5+CD4+ T cells was significantly decreased in HCC patients compared with HBV-relative liver cirrhosis (LC) patients and healthy controls, and the decrease in circulating CXCR5+CD4+ T cells correlated with disease progression. The proportion of infiltrated CXCR5+CD4+ T cells was significantly decreased in tumor regions compared with nontumor regions. Furthermore, compared with healthy controls, the function of circulating CXCR5+CD4+ T cells in HCC was impaired, with reduced IL-21 secretion and dysfunction in promoting B cell maturation. Importantly, follow-up data indicated that a decreased frequency of circulating CXCR5+CD4+ T cells was also associated with reduced disease-free survival time in HCC patients.ConclusionsImpairment of CD4+ T follicular helper cells may influence the development of HBV-associated HCC. Decreased CD4+ T follicular helper cells may represent a potential prognostic marker and serve as a novel therapeutic target for HCC patients.     

Spontaneous and vaccine induced AFP-specific T cell phenotypes in subjects with AFP-positive hepatocellular cancer

We are investigating the use of Alpha Fetoprotein (AFP) as a tumor rejection antigen for hepatocellular carcinoma (HCC). We recently completed vaccination of 10 AFP+/HLA-A2.1+ HCC subjects with AFP peptide-pulsed autologous dendritic cells (DC). There were increased frequencies of circulating AFP-specific T cells and of IFNγ-producing AFP-specific T cells after vaccination. In order to better understand the lack of association between immune response and clinical response, we have examined additional aspects of the AFP immune response in patients. Here, we have characterized the cell surface phenotype of circulating AFP tetramer-positive CD8 T cells and assessed AFP-specific CD4 function. Before vaccination, HCC subjects had increased frequencies of circulating AFP-specific CD8 T cells with a range of naïve, effector, central and effector memory phenotypes. Several patients had up-regulated activation markers. A subset of patients was assessed for phenotypic changes pre- and post-vaccination, and evidence for complete differentiation to effector or memory phenotype was lacking. CD8 phenotypic and cytokine responses did not correlate with level of patient serum AFP antigen (between 74 and 463,040 ng/ml). Assessment of CD4+ T cell responses by ELISPOT and multi-cytokine assay did not identify any spontaneous CD4 T cell responses to this secreted protein. These data indicate that there is an expanded pool of partially differentiated AFP-specific CD8 T cells in many of these HCC subjects, but that these cells are largely non-functional, and that a detectable CD4 T cell response to this secreted oncofetal antigen is lacking.     

MicroRNA-26b-5p enhances T cell responses by targeting PIM-2 in hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is a common tumor malignancy threatening a significant number of people worldwide. Although microRNAs (miRNAs) have been shown to play essential role in tumorigenesis, little is known about their role in T cells functions during HCC progression.MethodsThe abundances of miR-26b-5p were detected in HCC tissues or cells, T cells and H22 cells by quantitative real-time polymerase chain reaction (qRT-PCR). Regulation effect of miR-26b-5p on proviral integrations of moloney virus 2 (PIM2) was investigated by qRT-PCR, western blot (WB) and immunohistochemical analysis. The effect of miR-26b-5p and PIM-2 on cytokines secretion in CD4+ and CD8+ cells was evaluated by commercial enzyme linked immunosorbent assays (ELISA) kit. The interaction between miR-26b-5p and PIM-2 was probed by luciferase activity and RNA immunoprecipitation (RIP). H22 subcutaneous model was established to investigate the interaction of miR-26b-5p with HCC and immune competence.ResultsThe abundance of miR-26b-5p was decreased in HCC and associated with poor survival. Addition of miR-26b-5p contributed to secretion of tumor necrosis factor α (TNF-α), interferon-γ (IFN-γ), interleukin-6 (IL-6) and IL-2 in CD4+ and CD8+ cells. Interestingly, PIM-2 was negatively regulated by miR-26b-5p and PIM-2 knockdown reversed anti-miR-26b-5p-mediated immunosuppression. Moreover, inhibitory effect of miR-26b-5p on HCC tumorigenesis was dependent on immune competence.ConclusionsmiR-26b-5p enhanced T cells responses by targeting PIM-2 in HCC, uncovering a promising therapeutic opportunity of HCC through reactivating immune system.     

Immune modulation of effector CD4+ and regulatory T cell function by sorafenib in patients with hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a difficult to treat cancer characterized by poor tumor immunity with only one approved systemic drug, sorafenib. If novel combination treatments are to be developed with immunological agents, the effects of sorafenib on tumor immunity are important to understand. In this study, we investigate the impact of sorafenib on the CD4+CD25? effector T cells (Teff) and CD4+CD25+ regulatory T cells (Tregs) from patients with HCC. We isolated Teff and Treg from peripheral mononuclear cells of HCC patients to determine immune reactivity by thymidine incorporation, ELISA and flow cytometry. Teff cultured alone or with Treg were supplemented with different concentrations of sorafenib. The effects of sorafenib on Teff responses were dose-dependent. Pharmacologic doses of sorafenib decreased Teff activation by down regulating CD25 surface expression. In contrast, sub-pharmacologic concentrations of sorafenib resulted in Teff activation. These low doses of sorafenib in the Teff cultures led to a significant increase in Teff proliferation, IL2 secretion and up-regulation of CD25 expression on the cell surface. In addition, low doses of sorafenib in the suppression Teff/Treg cocultures restored Teff responses by eliminating Treg suppression. The loss of Treg suppressive function correlated with an increase in IL2 and IL6 secretion. Our findings show that sub-pharmacologic doses of sorafenib impact subsets of T cells differently, selectively increasing Teff activation while blocking Treg function. In conclusion, this study describes novel immune activating properties of low doses of sorafenib by promoting immune responsiveness in patients with HCC.     

The underlying pathophysiology association between the Type 2-diabetic and hepatocellular carcinoma

Type 2-diabetic (T2D) disease has been reported to increase the incidence of liver cancer, however, the underlying pathophysiology is still not fully understood. Here, we aimed to reveal the underlying pathophysiology association between the T2D and hepatocellular carcinoma (HCC) and, therefore, to find the possible therapeutic targets in the occurrence and development of HCC. The methylation microarray data of T2D and HCC were extracted from the Gene Expression Omnibus and The Cancer Genome Atlas. A total of 504 differentially methylated genes (DMGs) between T2D samples and the controls were identified, whereas 6269 DMGs were identified between HCC samples and the control groups. There were 336 DMGs coexisting in diabetes and HCC, among which 86 genes were comethylated genes. These genes were mostly enriched in pathways as glycosaminoglycan biosynthesis, fatty acid, and metabolic pathway as glycosaminoglycan degradation and thiamine, fructose and mannose. There were 250 DMGs that had differential methylation direction between T2D DMGs and HCC DMGs, and these genes were enriched in the Sphingolipid metabolism pathway and immune pathways through natural killer cell-mediated cytotoxicity and ak-STAT signaling pathway. Eight genes were found related to the occurrence and development of diabetes and HCC. Moreover, the result of protein-protein interaction network showed that CDKN1A gene was related to the prognosis of HCC. In summary, eight genes were found to be associated with the development of HCC and CDKN1A may serve as the potential prognostic gene for HCC.     

Expression profiling of murine double-negative regulatory T cells suggest mechanisms for prolonged cardiac allograft survival

Recent studies have demonstrated that both mouse and human alpha beta TCR(+)CD3(+)NK1.1(-)CD4(-)CD8- double-negative regulatory T (DN Treg) cells can suppress Ag-specific immune responses mediated by CD8+ and CD4+ T cells. To identify molecules involved in DN Treg cell function, we generated a panel of murine DN Treg clones, which specifically kill activated syngeneic CD8+ T cells. Through serial cultivation of DN Treg clones, mutant clones arose that lost regulatory capacity in vitro and in vivo. Although all allogeneic cardiac grafts in animals preinfused with tolerant CD4/CD8 negative 12 DN Treg clones survived over 100 days, allograft survival is unchanged following infusion of mutant clones (19.5 +/- 11.1 days) compared with untreated controls (22.8 +/- 10.5 days; p < 0.001). Global gene expression differences between functional DN Treg cells and nonfunctional mutants were compared. We found 1099 differentially expressed genes (q < 0.025%), suggesting increased cell proliferation and survival, immune regulation, and chemotaxis, together with decreased expression of genes for Ag presentation, apoptosis, and protein phosphatases involved in signal transduction. Expression of 33 overexpressed and 24 underexpressed genes were confirmed using quantitative real-time PCR. Protein expression of several genes, including Fc epsilon RI gamma subunit and CXCR5, which are >50-fold higher, was also confirmed using FACS. These findings shed light on the mechanisms by which DN Treg cells down-regulate immune responses and prolong cardiac allograft survival.     

Decreased expression of interleukin-36α correlates with poor prognosis in hepatocellular carcinoma

Interleukin-36α (IL-36α) has been found to have a prominent role in the pathogenesis of inflammatory disorders; however, little is known about the role of IL-36α in cancer. In this study, we investigated the expression, prognostic value, and the underlying antitumor mechanism of IL-36α in hepatocellular carcinoma (HCC). From immunohistochemistry analysis, IL-36α expression was lower in poorly differentiated HCC cells. In clinicopathological analysis, low IL-36α expression significantly correlated with tumor size, histological differentiation, tumor stage, and vascular invasion, and low intratumoral IL-36α expression had significantly worse overall survival rates and shorter disease-free survival rates. Moreover, intratumoral IL-36α expression was an independent risk factor for overall survival. Consecutive sections were used to detect CD3⁺, CD8⁺, and CD4⁺ tumor-infiltrating lymphocytes (TILs), and we found that high-IL-36α-expressing tumor tissues exhibited a significantly higher proportion of intratumoral CD3⁺ and CD8⁺ TILs, but not CD4⁺ TILs. Our in vitro model confirmed that supernatant from IL-36α-overexpressing human HCC cells had an increased capacity to recruit CD3⁺ and CD8⁺ T cells. Consistently, mouse HCC cells engineered to overexpress IL-36α demonstrated markedly delayed growth in vivo, as well as higher levels of intratumoral CD3⁺ and CD8⁺ TILs, compared with control mice. In vitro chemotaxis analysis also showed that mouse HCC cells overexpressing IL-36α could recruit more number of CD3⁺ and CD8⁺ T cells. These results show that IL-36α expression may play a pivotal role in determining the prognosis of patients with HCC, which we attribute to the activation of adaptive T cell immunity, especially CD8⁺ T cell immune response.     

Lung T-cell subset composition at the time of surgical resection is a prognostic indicator in non-small cell lung cancer

NSCLC arises in the complex environment of chronic inflammation. Depending on lung immune polarization, infiltrating immune cells may either promote or suppress tumor growth. Despite the importance of the immune microenvironment, current staging techniques for NSCLC do not take into consideration the immune milieu in which the neoplasms arise. T-cell subset content was compared between paired tumor-bearing and contralateral lungs, patient and control peripheral blood. The relationship between T-cell subset distribution and survival were evaluated. CD4 and CD8+ T cells were subsetted by CD45RA/CD27 and analyzed for expression of activation, adhesion, and homing markers. Strikingly, T-cell content was indistinguishable between lungs. Compared with peripheral blood, naïve CD4 and CD8 T cells were rare in BAL. CD4+ BAL T cells showed increased CD95 (higher apoptotic potential) and CD103 expression (epithelial adhesion), but decreased CD38 (activation) and CCR7 expression (lymph node homing). CD8+ BAL T cells showed increased CD103 expression and decreased CD28 expression (co-stimulation). Differences in CD28, CD95, and CCR7 expression were more pronounced within memory cells, while differences in CD4+ CD103 expression were more prominent in effector/memory cells. Of these populations, the absence of lung CD4 T cells with an effector-like phenotype (CD45RA+/CD27?) emerged as a predictor of favorable outcome. Patients with a low proportion (≤0.44%) had 90% 5-year survival (n = 10, median survival 2,343 days), compared with 0% (n = 9, median survival 516 days) of patients with a higher proportion. Further study is required to confirm this association prospectively and define the function of this subpopulation.     

Comprehensive analysis reveals a prognostic and therapeutic biomarker CD3D in the breast carcinoma microenvironment

Breast carcinoma (BRCA) is the most common carcinoma among women worldwide. Despite the great progress achieved in early detection and treatment, morbidity and mortality rates remain high. In the present study, we make a systematic analysis of BRCA using TCGA database by applying CIBERSORT and ESTIMATE computational methods, uncovered CD3D as a prognostic biomarker by intersection analysis of univariate COX and protein–protein interaction (PPI). It revealed that high CD3D expression was strongly associated with poor survival of BRCA, based on The Cancer Genome Atlas (TCGA) database and online websites. Gene Set Enrichment Analysis (GSEA) revealed that the high CD3D expression group was mainly enriched for the immune-related pathways and the low CD3D expression group was mainly enriched for metabolic-related activities. Based on CIBERSORT analysis, the difference test and correlation test suggested that CD3D had a strong correlation with T cells, particularly CD8 + T cells, which indicated that CD3D up-regulation may increase T cell immune infiltration in the TME and induce antitumor immunity by activating T lymphocytes. Furthermore, the correlation analysis showed that CD3D expression had a strongly positive correlation with immune checkpoints, which indicating that the underlying mechanism involves CD3D mediated regulation of T cell functions in BRCA, and single cell RNA-seq analysis revealed that CD3D correlate with CD8 + T cells and it is itself highly expressed in CD8 + T cells. In summary, we identified a prognostic biomarker CD3D in BRCA, which was associated with lymphocyte infiltration, immune checkpoints and could be developed for innovative therapeutics of BRCA.     

Athymic nude CD4+8- T cells produce IL-2 but fail to proliferate in response to mitogenic stimuli

A comparative study of immune functions of CD4+8- T cells isolated from normal and athymic nude mice by electronic cell sorting was performed. Athymic nude CD4+8- T cells expressed the TCR-associated CD3 molecule but the level of expression was significantly lower than that of normal CD4+8- T cells. Proliferative responses were studied upon stimulation by 1) the T cell mitogen Con A; 2) anti-CD3 mediated cross-linking of the CD3:TCR complex, and 3) the combined action of PMA + ionomycin. All three mitogenic stimuli caused readily detectable cell division in normal (euthymic) CD4+8- T cells. In marked contrast, none of the mitogenic stimuli induced significant proliferation in athymic nude CD4+8- T cells. The failure of athymic nude CD4+8- T cells to proliferate occurred over a wide range of mitogen concentrations and over a 4-day observation period. Neither exogenously supplied rIL-2 or mixed lymphocyte culture supernatant had any effect on the impaired proliferative response by athymic nude CD4+8- T cells. Although IL-2 was produced by athymic nude CD4+8- T cells at a reduced level when compared to normal CD4+8- T cells, it was nevertheless readily detected upon stimulation with either Con A or anti-CD3. Furthermore, stimulation of athymic nude CD4+8- T cells by anti-CD3 induced the expression of the p55 chain of IL-2R on the cell surface. Therefore, despite production of IL-2 and induced expression of IL-2R, athymic nude CD4+8- T cells failed to undergo cell division.     

Effects of IL-7 and dexamethasone: induction of CD25, the high affinity IL-2 receptor, on human CD4+ cells

Since we have previously shown that dexamethasone (Dex) enhances the proportion of murine Treg cells, we tested the effect of IL-7, a promoter of T cell survival, together with Dex on human CD4+CD25+ Treg cells in an in vitro setting. The results showed that IL-7 in concert with Dex markedly augmented the generation of CD4+CD25+ T cells. To discern the origin of the induced CD4+CD25+ T cells, MACS-purified CD4+CD25-, and CD4+CD25+ cells were cultured in the presence of Dex and/or IL-7 for 4 days. Although two thirds of CD4+CD25- T cells became CD4+CD25+ T cells, they had no suppressive activity. In contrast, the original CD4+CD25+ T cells maintained suppressive activity after Dex/IL-7 treatment, however, there was not a significant expansion in their cell number. Dex and IL-7 did not induce additional Treg cells, but additively induced the expression of the activation marker CD25 by CD4+CD25- T cells. This combination may provide a novel means of priming CD4 T cells to respond to IL-2 and may prove useful in up-regulation of normal immune responses in immune deficient diseases.     

Immature and mature CD8alpha+ dendritic cells prolong the survival of vascularized heart allografts.

CD8alpha+ and CD8alpha- dendritic cells (DCs) arise from committed bone marrow progenitors and can induce or regulate immune reactivity. Previously, the maturational status of CD8alpha-(myeloid) DCs has been shown to influence allogeneic T cell responses and allograft survival. Although CD8alpha+ DCs have been implicated in central tolerance and found to modulate peripheral T cell function, their influence on the outcome of organ transplantation has not been examined. Consistent with their equivalent high surface expression of MHC and costimulatory molecules, sorted mature C57BL/10J (B10; H2(b)) DCs of either subset primed naive, allogeneic C3H/HeJ (C3H; H2(k)) recipients for Th1 responses. Paradoxically and in contrast to their CD8alpha- counterparts, mature CD8alpha+ B10 DCs given systemically 7 days before transplant markedly prolonged B10 heart graft survival in C3H recipients. This effect was associated with specific impairment of ex vivo antidonor T cell proliferative responses, which was not reversed by exogenous IL-2. Further analyses of possible underlying mechanisms indicated that neither immune deviation nor induction of regulatory cells was a significant contributory factor. In contrast to the differential capacity of the mature DC subsets to affect graft outcome, immature CD8alpha+ and CD8alpha- DCs administered under the same experimental conditions significantly prolonged transplant survival. These observations demonstrate for the first time the innate capacity of CD8alpha+ DCs to regulate alloimmune reactivity and transplant survival, independent of their maturation status. Mobilization of such a donor DC subset with capacity to modulate antidonor immunity may have significant implications for the therapy of allograft rejection.     

基于SNP共表达网络肝癌分子分型及预后分析

基于SNP突变数据与mRNA表达谱关联分析,构建一种肝癌分子分型方法并对比不同分型预后的差异,并对不同分型肝癌的发生发展机制进一步研究。首先通过TCGA数据库收集359例肝细胞癌患者的SNP突变数据和mRNA表达数据,采用Wilcoxon秩和检验,筛选突变后差异表达基因,并通过生物信息学工具String和Cytoscape构建差异表达基因的蛋白互作网络,筛选连接度最高的10个Hub基因。利用Consensus Cluster Plus软件包,基于Hub基因mRNA表达水平构建NMF分子分型模型,再结合生存数据评估各分型患者的预后。最后利用加权基因共表达网络分析(WGCNA),识别与肝癌分子分型相关的模块,并针对关键模块的基因进行通路富集,从而对不同分型肝癌的基因表达谱进行比较。结果：NMF模型将肝癌分为高危、低危2个分型,其中CDKN2A和FOXO1基因对分型贡献度高。生存分析显示低危组患者的生存情况显著优于高危组,高危组富集多个与肿瘤细胞侵蚀、转移、复发过程相关的信号通路,低危组则与细胞周期和胰液分泌相关。本研究在无先验性信息的前提下,基于突变后显著差异表达的Hub基因表达水平构建的...     

CD28 regulates glucocorticoid-induced TNF receptor family-related gene expression on CD4+ T cells via IL-2-dependent mechanisms

The glucocorticoid-induced TNF-related gene receptor (GITR) is the newest member of the costimulatory molecule family and is expressed on both resting CD4+CD25+ regulatory T (T(R)) cells and activated CD4+ T cells. We investigated the endogenous mechanisms that regulate GITR expression on both T(R) and CD4+ T cells, as well as the functional interaction between GITR and other costimulatory molecules. CD28 stimulation increased GITR expression on both T(R) and CD4+ T cells via IL-2-dependent mechanisms. In addition, ligation of GITR and/or CD28 increased the level of CD4+ T cell proliferation and effector function under both APC-dependent and -independent conditions, suggesting that these costimulatory molecules cooperate to regulate CD4+ T cell activation and function by directly signaling to the CD4+ T cell. Thus, GITR may serve opposing functional roles on CD4+ T(R) and effector cells and alterations in GITR expression and/or function may tip the balance between immune tolerance and effector function.