Immune Checkpoint-Related Gene Polymorphisms Are Associated With Primary Immune Thrombocytopenia.

Cancer immunotherapy by immune checkpoint blockade has been effective in the treatment of certain tumors. However, the association between immune checkpoints and autoimmune diseases remains elusive and requires urgent investigation. Primary immune thrombocytopenia (ITP), characterized by reduced platelet count and a consequent increased risk of bleeding, is an autoimmune disorder with a hyper-activated T cell response. Here, we investigated the contribution of immune checkpoint-related single-nucleotide polymorphisms (SNPs), including CD28, ICOS, PD1, TNFSF4, DNAM1, TIM3, CTLA4, and LAG3 to the susceptibility and therapeutic effects of ITP. In this case-control study, 307 ITP patients and 295 age-matched healthy participants were recruited. We used the MassARRAY system for genotyping immune checkpoint-related SNPs. Our results revealed that rs1980422 in CD28 was associated with an increased risk of ITP after false discovery rate correction (codominant, CT vs. TT, OR = 1.788, 95% CI = 1.178-2.713, p = 0.006). In addition, CD28 expression at both the mRNA and protein levels was significantly higher in patients with CT than in those with the TT genotype (p = 0.028 and p = 0.001, respectively). Furthermore, the T allele of PD1 rs36084323 was a risk factor for ITP severity and the T allele of DNAM1 rs763361 for corticosteroid-resistance. In contrast, the T allele of LAG3 rs870849 was a protective factor for ITP severity, and the T allele of ICOS rs6726035 was protective against corticosteroid-resistance. The TT/CT genotypes of PD1 rs36084323 also showed an 8.889-fold increase in the risk of developing refractory ITP. This study indicates that immune checkpoint-related SNPs, especially CD28 rs1980422, may be genetic factors associated with the development and treatment of ITP patients. Our results shed new light on prognosis prediction, disease severity, and discovering new therapeutic targets.

Clinical Implications of Peripheral CD3+CD69+ T-Cell And CD8+CD28+ T-Cell Proportions in Patients Prior to Radical Prostatectomy.

PURPOSE: To investigate the clinical implications of CD3+CD69+ T-cells and CD8+CD28+ T-cells in the peripheral blood of patients prior to radical prostatectomy. MATERIALS AND METHODS: A total of 91 prostate cancer (PCa) patients and 50 benign prostatic hyperplasia (BPH) patients were enrolled from January 2016 to December 2017. The proportions of CD3+CD69+ T-cells and CD8+CD28+ T-cells in the peripheral blood of PCa and BPH patients were detected by flow cytometry, and the association of these T-cell populations with pathological Grade Group and pathological TNM classification was evaluated. Data analysis was performed with SAS version 9.4 software. RESULTS: The proportions of CD3+CD69+ and CD8+CD28+ T-cells in peripheral blood were higher in PCa patients than those in BPH patients. Multivariate analysis identified a higher CD3+CD69+ T-cell proportion as a risk factor for PCa (odds ratio (OR) = 4.783, P = 0.0013), but the diagnostic efficacy of the CD3+CD69+ T-cell proportion (area under the curve (AUC)=0.6833, P = 0.0003) for PCa was still inferior to that of the tPSA level (AUC=0.7531, P < 0.0001). The AUCs for CD3+CD69+ T-cell and CD8+CD28+ T-cell proportions for PCa were 0.6959 (P = 0.0372) and 0.6935 (P = 0.0395), respectively, among men with tPSA levels of 4.0-10.0 ng/mL. A lower CD3+CD69+ T-cell proportion was associated with higher pathological Grade Group (P=0.0074). CONCLUSION: The proportions of CD3+CD69+ T-cells and CD8+CD28+ T-cells in peripheral blood are potential diagnostic indicators for PCa. The preoperative proportion of CD3+CD69+ T-cells in peripheral blood may have prognostic value in terms of the pathological Grade Group in PCa.

Six Cases of CD20-Positive Adult T-Cell Leukemia.

Adult T-cell leukaemia/lymphoma (ATLL) is a neoplasm originating in mature CD4+ peripheral T cells. However, rare cases of CD20+ ATLL have been reported. Here, we describe six cases of CD20+ ATLL diagnosed in our department. The median age was 79 years (range, 54-90 years); two patients were men, and four were women. Elevated lactate dehydrogenase was observed in four cases. All cases were lymphoma type and positive for human T-lymphotropic virus-1 (HTLV-1). HTLV-1 proviral DNA was detected in four cases. The Ann Arbor stage was I, II, or IV in one patient each and III in three patients. The clinical course was poor in almost all cases. Tumour cells were large in all cases, and flow cytometry revealed CD20+ lymphoma cells in five of six cases. Immunohistochemistry revealed lymphoma cells positive for CD20, CD3, CD4, and CCR4 and negative for CD8, CD79a, and PAX5 in all cases. CD20 expression was lower than that in normal B cells. One case was initially misdiagnosed as diffuse large B-cell lymphoma. Thus, combined use of an antibody panel and molecular genetic studies is important to avoid misdiagnosing ATLL as B-cell lymphoma.

BAFF upregulates CD28/B7 and CD40/CD154 expression and promotes mouse T and B cell interaction in vitro via BAFF receptor.

AIM: B cell-activating factor belonging to the TNF family (BAFF) is a member of TNF family and required for peripheral B cell survival and homeostasis. BAFF has been shown to promote the proliferation of T and B cells. In this study we examined whether and how BAFF mediated the interaction between mouse T and B cells in vitro. METHODS: BAFF-stimulated B or T cells were co-cultured with T or B cells. The interactions between T and B cells were analyzed by measuring the expression of co-stimulatory molecules (CD28/CD80 or CD40/CD154), the proliferation and secretion of T and B cells and other factors. Two siRNAs against the transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) and BAFF receptor (BAFF-R) were used to identify the receptors responsible for the actions of BAFF. RESULTS: BAFF-stimulated B cells significantly promoted the proliferation and activity of co-cultured T cells, and increased the percentages of CD4(+)CD28(+) and CD4(+)CD154(+) T cells. Similarly, BAFF-stimulated T cells significantly promoted the proliferation and activity of co-cultured B cells, and increased CD19(+)CD80(+) and CD19(+)CD40(+)B cell subpopulations. BAFF-R siRNA-silenced B cells showed significantly lower expression of CD40 and CD80 than the control B cells. When the BAFF-R siRNA-silenced B cells were stimulated with BAFF, then co-cultured with T cells, the expression of CD28 and CD154 on T cells was not increased. TACI siRNA-silenced B cells exhibited higher expression of CD40 and CD80 than the control B cells. When the TACI siRNA-silenced B cells were stimulated with BAFF, then co-cultured with T cells, the expression of CD28 and CD154 on T cells was significantly increased. CONCLUSION: BAFF upregulates CD28/B7 and CD40/CD154 expression, and promotes the interactions between T and B cells in a BAFF-R-dependent manner.

CD4+ T-cell gene expression of healthy donors, HIV-1 and elite controllers: Immunological chaos.

OBJECTIVES: T-cell repertoire dysfunction characterizes human immunodeficiency virus type 1 (HIV-1) infection, but the pathogenic mechanisms remain unclear. Disease progression is probably due to a profound dysregulation of Th1, Th2, Th17 and Treg patterns. The aim of this study was to analyze the features of CD4+ T cells in HIV-positive patients with different viroimmunological profile. METHODS: we used a gene expression dataset of CD4+ T cells from healthy donors, HIV+ naive patients and Elite Controllers (EC), obtained from the NCBI Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/, accession number GSE18233). RESULTS: Principal Component Analysis (PCA) showed an almost complete overlap between the HIV-infected and EC patients, which cannot easily explain the different responses to HIV infection of these two group of patients. We have found that HIV patients and the EC showed an upregulation of the Th1 pro-inflammatory cytokines and chemokines, compared to the controls. Also, we have surprisingly identified IL28B, which resulted downregulated in HIV and EC compared to healthy controls. We focused attention also on genes involved in the constitution of the immunological synapse and we showed that HLA class I and II genes resulted significantly upregulated in HIV and in EC compared to the control. In addition to it, we have found the upregulation of others syncytial molecules, including LAG3, CTLA4, CD28 and CD3, assisting the formation of syncytia with APC cells. CONCLUSIONS: Understanding the mechanisms of HIV-associated immunological chaos is critical to strategically plan focused interventions.

Tumor CTLA-4 overexpression predicts poor survival in patients with nasopharyngeal carcinoma.

The expression levels of CTLA-4 and CD28 were analyzed in 191 nasopharyngeal carcinoma (NPC) patients diagnosed and treated at our hospital between January 2010 and November 2011. The 3-year overall survival (OS) rate (91.4% vs. 81.2%,p = 0.043), failure-free survival (FFS) rate (82.8% vs. 68.0%, p = 0.009) and distant failure-free survival (D-FFS) rate (85.8% vs. 72.3%, p = 0.006) in the low tumor CTLA-4 expression group was higher than in the high tumor CTLA-4 group. There were no differences between the locoregional failure-free survival (LR-FFS) rates in the high and low tumor CTLA-4 expression groups. Moreover, no differences in the OS, FFS, D-FFS, or LR-FFS were observed between the groups with high and low lymphocyte CTLA-4 levels, high and low tumor CD28 levels, or high and low lymphocyte CD28 levels. Cox regression analysis confirmed the prognostic value of tumor CTLA-4 expression, particularly for D-FFS, in NPC patients (p = 0.044). NPC patients with high tumor CTLA-4 expression had a poorer prognosis than those with low expression.

Structural Design of Engineered Costimulation Determines Tumor Rejection Kinetics and Persistence of CAR T Cells.

T cell engineering is a powerful means to rapidly generate anti-tumor T cells. The costimulatory properties of second-generation chimeric antigen receptors (CARs) determine the overall potency of adoptively transferred T cells. Using an in vivo "stress test" to challenge CD19-targeted T cells, we studied the functionality and persistence imparted by seven different CAR structures providing CD28 and/or 4-1BB costimulation. One configuration, which uses two signaling domains (CD28 and CD3ζ) and the 4-1BB ligand, provided the highest therapeutic efficacy, showing balanced tumoricidal function and increased T cell persistence accompanied by an elevated CD8/CD4 ratio and decreased exhaustion. Remarkably, induction of the IRF7/IFNß pathway was required for optimal anti-tumor activity. Thus, 1928z-41BBL T cells possess strikingly potent intrinsic and immunomodulatory qualities.

Gene expression changes reflect clinical response in a placebo-controlled randomized trial of abatacept in patients with diffuse cutaneous systemic sclerosis.

INTRODUCTION: Systemic sclerosis is an autoimmune disease characterized by inflammation and fibrosis of the skin and internal organs. We sought to assess the clinical and molecular effects associated with response to intravenous abatacept in patients with diffuse cutaneous systemic. METHODS: Adult diffuse cutaneous systemic sclerosis patients were randomized in a 2:1 double-blinded fashion to receive abatacept or placebo over 24 weeks. Primary outcomes were safety and the change in modified Rodnan Skin Score (mRSS) at week 24 compared with baseline. Improvers were defined as patients with a decrease in mRSS of ≥30% post-treatment compared to baseline. Skin biopsies were obtained for differential gene expression and pathway enrichment analyses and intrinsic gene expression subset assignment. RESULTS: Ten subjects were randomized to abatacept (n = 7) or placebo (n = 3). Disease duration from first non-Raynaud's symptom was significantly longer (8.8 ± 3.8 years vs. 2.4 ± 1.6 years, p = 0.004) and median mRSS was higher (30 vs. 22, p = 0.05) in the placebo compared to abatacept group. Adverse events were similar in the two groups. Five out of seven patients (71%) randomized to abatacept and one out of three patients (33%) randomized to placebo experienced ≥30% improvement in skin score. Subjects receiving abatacept showed a trend toward improvement in mRSS at week 24 (-8.6 ± 7.5, p = 0.0625) while those in the placebo group did not (-2.3 ± 15, p = 0.75). After adjusting for disease duration, mRSS significantly improved in the abatacept compared with the placebo group (abatacept vs. placebo mRSS decrease estimate -9.8, 95% confidence interval -16.7 to -3.0, p = 0.0114). In the abatacept group, the patients in the inflammatory intrinsic subset showed a trend toward greater improvement in skin score at 24 weeks compared with the patients in the normal-like intrinsic subset (-13.5 ± 3.1 vs. -4.5 ± 6.4, p = 0.067). Abatacept resulted in decreased CD28 co-stimulatory gene expression in improvers consistent with its mechanism of action. Improvers mapped to the inflammatory intrinsic subset and showed decreased gene expression in inflammatory pathways, while non-improver and placebos showed stable or reverse gene expression over 24 weeks. CONCLUSIONS: Clinical improvement following abatacept therapy was associated with modulation of inflammatory pathways in skin. TRIAL REGISTRATION: ClinicalTrials.gov NCT00442611. Registered 1 March 2007.

A novel multicolor immunostaining method using ethynyl deoxyuridine for analysis of in situ immunoproliferative response.

Immune responses are generally accompanied by antigen presentation and proliferation and differentiation of antigen-specific lymphocytes (immunoproliferation), but analysis of these events in situ on tissue sections is very difficult. We have developed a new method of simultaneous multicolor immunofluorescence staining for immunohistology and flow cytometry using a thymidine analogue, 5-ethynyl-2'-deoxyuridine (EdU). Because of the small size of azide dye using click chemistry and elimination of DNA denaturation steps, EdU staining allowed for immunofluorescence staining of at least four colors including two different markers on a single-cell surface, which is impossible with the standard 5-bromo-2'-deoxyuridine method. By using two rat models, successfully detected parameters were the cluster of differentiation antigens including phenotypic and functional markers of various immune cells, histocompatibility complex antigens, and even some nuclear transcription factors. Proliferating cells could be further sorted and used for RT-PCR analysis. This method thus enables functional in situ time-kinetic analysis of immunoproliferative responses in a distinct domain of the lymphoid organs, which are quantitatively confirmed by flow cytometry.

Chemokine CX3CL1 and its receptor CX3CR1 are associated with human atherosclerotic lesion volnerability.

BACKGROUND: CX3CL1 and its receptor CX3CR1 have been emphasized in atherosclerosis recently. In this study we investigated the role of the chemokines CX3CL1 and their receptor CX3CR1 in atherogenesis and identified whether the genetic variations in CX3CL1 and CX3CR1 impacted the atherosclerosis process in coronary artery disease (CAD) or not. METHODS: CX3CL1/CX3CR1 expression in coronary and carotid artery specimens were analysed by immunohistochemistry. CX3CR1 expression on CD4(+) CD28(-) T cells was analysed by flow cytometry. We also screened for CX3CL1/CX3CR1 sequence variations selected from the hapmap database and examined the association between CX3CL1/CX3CR1 and CAD in the Chinese Han population. RESULTS: Immunohistochemical staining of tissue from CAD patients showed increased CX3CL1/CX3CR1 expression in atherosclerotic coronary and carotid artery plaques compared with normal arteries. CX3CL1/CX3CR1 expression was correlated with the severity of the atherosclerosis lesion. Patients with CAD also showed an increased number of CX3CR1(+) CD4(+) CD28(-) T cells. Compared with their corresponding wild-type genotypes, CX3CL1 rs170364 and CX3CR1 rs17793056 were associated with increased susceptibility to CAD. CONCLUSIONS: CX3CL1 and CX3CR1 may contribute to the formation of coronary atherosclerotic plaque in CAD.CX3CL1 rs170364 and CX3CR1 rs17793056 polymorphisms may be independent genetic risk factors for CAD.

Induced expression of FcγRIIIa (CD16a) on CD4+ T cells triggers generation of IFN-γhigh subset.

Whether or not CD4(+) T-cells express low affinity receptor FcγRIIIa (CD16a) in disease pathology has not been examined in great detail. In this study, we show that a subset of activated CD4(+) T-cells in humans express FcγRIIIa. The ligation of FcγRIIIa by immune complexes (ICs) in human CD4(+) T-cells produced co-stimulatory signal like CD28 that triggered IFN-γ production. The induced expression of FcγRIIIa on CD4(+) helper T-cells is an important finding since these receptors via ITAM contribute to intracellular signaling. The induced expression of FcγRIIIa on CD4(+) T helper cells and their ability to co-stimulate T-cell activation are important and novel findings that may reveal new pathways to regulate adaptive immune responses during inflammation and in autoimmunity.

The suppressive function of human CD8(+) iTregs is inhibited by IL-1ß and TNFα.

CD8(+) Tregs display an immunoregulatory activity and may play an essential role in the immunopathology of several diseases. Therefore, their therapeutic potential is exquisite and further studies on their differentiation and function are essential. The aim of this study was to evaluate the role of the innate immune system in CD8(+) iTreg differentiation and function. Naive human CD8(+) CD25(-) CD45RA(+) T cells were cultured in Treg-inducing conditions with or without IL-1ß, TNFα or monocyte-derived dendritic cells (DCs). The differentiation of CD8(+) CD127(-) CD25(hi) FoxP3(hi) -induced Tregs (CD8(+) iTregs) is dependent on TGF-ß1 and IL-2, which had synergistic effect upon their differentiation. CD8(+) iTregs were also induced in a coculture with allogeneic mature DCs (mDCs). The CD8(+) iTregs suppressive function was confirmed, which was diminished in the presence of IL-1ß and TNFα. The IL-1ß-prevented suppressive function was associated with reduced secretion of IL-10 and IFNγ, whereas the presence of TNFα did not affect their secretion. Furthermore, the presence of TNFα reduced IL-10 and TGF-ß1 secretion by CD8(+) iTregs, whereas only IL-10 secretion was decreased by IL-1ß. Together, these results suggest that IL-1ß and TNFα prevent IL-2- and TGF-ß1-driven CD8(+) iTregs suppressive function in human T cells. Such pro-inflammatory innate immune response possibly mediates its negative tolerogenic effect through reduced IFNγ-, IL-10- and TGF-ß1-driven mechanism.

CD45RA-Foxp3(low) non-regulatory T cells in the CCR7-CD45RA-CD27+CD28+ effector memory subset are increased in synovial fluid from patients with rheumatoid arthritis.

Increased numbers of regulatory T (Treg) cells are found in synovial fluid from patients with rheumatoid arthritis (RASF) compared with peripheral blood. However, Treg cells in RASF have been shown to have a decreased capacity to suppress T cells. Here we phenotypically classified CD4+ T cells in RASF into six subsets based on the expression of CD45RA, CCR7, CD27 and CD28, and demonstrated that the CCR7-CD45RA-CD27+CD28+ TEM subset was significantly increased in synovial fluid compared with peripheral blood. In addition, the proportion of Foxp3+ Treg cells in the CCR7-CD45RA-CD27+CD28+ TEM subset was significantly increased in RASF. Furthermore, most of the Foxp3+ Treg cells in RASF were non-suppressive CD45RA-Foxp3(low) non-Treg cells, and the frequency of the non-Treg cells in the CCR7-CD45RA-CD27+CD28+ TEM subset was significantly increased in RASF. Our findings suggest that the pro-inflammatory environment in RA joints may induce the increase of CD45RA-Foxp3(low) non-Treg cells in synovial fluid.

The influence of intrinsic and extrinsic factors on immune system aging.

Sex and age-matched wild-type and TCR transgenic mice were infected with cytomegalovirus (CMV) at 6 months of age and followed for 12 additional months to examine aging of the immune system. It was found that viral infection of C57Bl/6 mice resulted in accelerated aging of the immune system as shown by a loss of CD8(+)28(+) cells and an accumulation of KLRG1(+) T cells. CMV infection of OT-1 transgenic mice had no influence on immune aging of these mice which nonetheless demonstrated an accumulation of CD8(+)28(-) and KLRG1(+) T cells with time. CD4(+) T cells were unaffected in either strain of mice. Thus, immunological aging was found to be due to both cell-intrinsic and cell-extrinsic factors. Persistent viral infections may accelerate immunological aging but consideration must be given to individual variation in the aging process.

Infectious versus non-infectious loosening of implants: activation of T lymphocytes differentiates between the two entities.

PURPOSE: Loosening of implants occurs mainly for two reasons: bacterial infection of the implant or "aseptic loosening" presumably due to wear particles derived from the implant. To gain further insight into the pathomechanism, we analysed activation of the T cell response in these patients. METHODS: Activation of peripheral T lymphocytes was determined by cytofluorometry as down-regulation of CD28 and up-regulation of CD11b. In addition, tissue samples obtained during surgery were analysed by quantitative RT-PCR for gene expression of CD3, CD14 and cathepsin K, as markers for T cells, monocytes/macrophages or osteoclasts, respectively. RESULTS: Activated T lymphocytes were detected in patients with infection but not in patients with aseptic loosening. Gene expression of CD3 was significantly enhanced in tissues of patients with infection compared to those with aseptic loosening. Expression of CD14 and of cathepsin K did not differ between the two groups. CONCLUSION: Implant-associated infection and aseptic loosening are associated with a local inflammatory response, which eventually results in osteoclastogenesis and bone resorption. Systemic T cell activation, in contrast, occurs only in patients with implant-associated infection, and hence analysis of T cell activation markers could serve as a diagnostic tool to differentiate between the two entities.

Ubiquitination regulates expression of the serine/arginine-rich splicing factor 1 (SRSF1) in normal and systemic lupus erythematosus (SLE) T cells.

T cells from patients with systemic lupus erythematosus (SLE) exhibit reduced expression of the critical T cell receptor (TCR)-associated CD3ζ signaling chain and are poor producers of the vital cytokine IL-2. By oligonucleotide pulldown and mass spectrometry discovery approaches, we identified the splicing regulator serine/arginine-rich splicing factor (SRSF) 1 or splicing factor 2/alternative splicing factor (SF2/ASF) to be important in the expression of CD3ζ chain. Importantly, increases in the expression of SRSF1 rescued IL-2 production in T cells from patients with SLE. In this study, we investigated the regulation of SRSF1 expression in resting and activated human T cells. We found that T cell stimulation induced a rapid and significant increase in mRNA expression of SRSF1; however, protein expression levels did not correlate with this increase. Co-engagement of CD28 induced a similar mRNA induction and reduction in protein levels. Proteasomal but not lysosomal degradation was involved in this down-regulation as evidenced by blocking with specific inhibitors MG132 and bafilomycin, respectively. Immunoprecipitation studies showed increased ubiquitination of SRSF1 in activated T cells. Interestingly, T cells from patients with SLE showed increased ubiquitination of SRSF1 when compared with those from healthy individuals. Our results demonstrate a novel mechanism of regulation of the splicing factor SRSF1 in human T cells and a potential molecular mechanism that controls its expression in SLE.

Costimulatory molecule CD28 participates in the process of embryo implantation in mice.

Embryo implantation is a complex process requiring reciprocal interactions between implantation-competent blastocysts and receptive uteri. Accumulating literatures have indicated that T cells are involved in this process. The first signal mediated by T-cell receptor/CD3 complex and the second signal delivered by costimulatory molecules are essential for the differentiation of T cell into an effector cell. Expression and function of CD28, an important costimulatory molecule, during early pregnancy in mice is still unclear. In the present study, we investigated the expression pattern of CD28 in mouse uterus during early pregnancy and pseudopregnancy by real-time quantitative polymerase chain reaction, Western blotting, in situ hybridization, and immunohistochemistry (IHC). We found that injection of the uterine horn with CD28 antisense oligodeoxynucleotides leads to a decreased number of implantation sites. The expression pattern of CD3 protein examined by IHC is similar to that of CD28. These findings suggest that CD28 participates in the process of embryo implantation in mice, which might play its role through delivering the second costimulatory signal.

Anti-HIV designer T cells progressively eradicate a latently infected cell line by sequentially inducing HIV reactivation then killing the newly gp120-positive cells.

The current antiretroviral therapy (ART) can effectively reduce plasma HIV loads to undetectable levels, but cannot eliminate latently infected resting memory CD4 T cells that persist for the lifetime of infected patients. Therefore, designing new therapeutic approaches to eliminate these latently infected cells or the cells that produce HIV upon reactivation from latency is a priority in the ART era in order to progress to a cure of HIV. Here, we show that "designer" T cells expressing chimeric antigen receptor (CAR), CD4-CD28-CD3ζ, can target and kill HIV Env-expressing cells. Further, they secrete effector cytokines upon contact with HIV Env+ target cells that can reactivate latent HIV in a cell line model, thereby exposing those cells to recognition and killing by anti-HIV CAR+ T cells. Taken to the limit, this process could form the basis for an eventual functional or sterilizing cure for HIV in patients.

[CD28 expression on CD4(+);T cells and its relationship with IFN-γ/IL-10 ratio in patients with primary immune thrombocytopenia].

OBJECTIVE: To investigate CD28 expression on CD4(+);T cells and its relationship with IFN-γ/IL-10 ration in patients with primary immune thrombocytopenia (ITP) before and after treatment. METHODS: The expression of CD4(+);CD28(+); was detected by flow cytometry, the levels of IFN-γ and IL-10 were determined by double-antibody sandwich ELISA and platelet count was tested by blood cells automatical counter in peripheral blood of 30 patients with ITP before and after glucocorticoid treatment and 26 cases of normal controls. Then the correlations between the outcomes were analyzed. RESULTS: The expression of CD4(+);CD28(+); of ITP patients before treatment was higher than that of the normal control group (P<0.05), however, after treatment the expression between the two groups had no statistical difference (P>0.05). Compared with the control group, the ITP patients before treatment showed the significantly higher level of IFN-γ and the significantly decreased level of IL-10, and the ratio of IFN-γ/IL-10 was significantly raised (P<0.01); while ITP patients after treatment had no statistically significant difference in the above indexes from the normal controls (P>0.05). Moreover, CD4(+);CD28(+); was positively correlated with IFN-γ/IL-10 ratio (P<0.05), and was negatively correlated with platelet count in ITP patients before treatment (P<0.05). CONCLUSION: Co-stimulatory molecule CD4(+);CD28(+); was closely related with immune disorder of ITP. It maybe played a role in the pathogenesis of ITP through involving Th1 advantage state formation.

Ndfip1 enforces a requirement for CD28 costimulation by limiting IL-2 production.

Although the pathways that permit IL-2 production and the full activation of T cells upon Ag encounter are fairly well defined, the negative regulatory circuits that limit these pathways are poorly understood. In this study, we show that the E3 ubiquitin ligase adaptor Ndfip1 directs one such negative regulatory circuit. T cells lacking Ndfip1 produce IL-2, upregulate IL-2Rα, and proliferate, in the absence of CD28 costimulation. Furthermore, T cells in mice lacking both Ndfip1 and CD28 become activated, produce IL-4, and drive inflammation at barrier surfaces. Ndfip1 constrains T cell activation by limiting the duration of IL-2 mRNA expression after TCR stimulation. Ndfip1 and IL-2 have a similar expression pattern, and, following TCR stimulation, expression of both Ndfip1 and IL-2 requires the activity of NFAT and Erk. Taken together, these data support a negative regulatory circuit in which factors that induce IL-2 expression downstream of TCR engagement also induce the expression of Ndfip1 to limit the extent of IL-2 production and, thus, dampen T cell activation.