Characterization of CD28, CTLA4, and ICOS polymorphisms in three Brazilian ethnic groups

Costimulatory molecules CD28, CTLA-4 (cytotoxic T-lymphocyte-associated antigen-4), and ICOS (inducible costimulator) genes lie within the 300-kb chromosome region 2q33. CD28, CTLA-4, and ICOS have been described to be important regulators of T-cell activation. With the objective to study ethnic variations in allelic frequencies and linkage disequilibrium (LD) patterns, we genotyped CD28 intron 3 (+17 T>C), CTLA4 promoter (-319 C>T), CTLA4 exon 1 (+49 A>G), and ICOS 3' UTR (1564 T>C) polymorphisms in white (n = 103), mulatto (n = 97), and black (n = 79) Brazilian healthy individuals. No significant deviations from Hardy-Weinberg equilibrium were found in any of the population samples. A higher frequency of CD28 +17 C allele was detected in white (27%) in comparison with mulatto (15%) and black (13%) (p = 0.005) populations. LD between CD28 +17 C and CTLA4 -319 T alleles was observed in whites (p < 0.0001), mulattos (p = 0.0001), and blacks (p = 0.0002).     

CD28 and inducible costimulator (ICOS) signalling can sustain CD154 expression on activated T cells

The biological outcome of receptor-mediated signalling often depends on the duration of engagement. Because CD40 signalling is controlled by the regulated expression of its ligand, CD154, the mechanisms that regulate CD154 expression probably determine the strength and duration of CD40 signalling. Here, we demonstrate that CD154 expression on the surface of mouse CD4 T cells can be separated into an early phase, occurring between 0 and 24 hr after T-cell activation, and a later extended phase, occurring after 24 hr. The early phase of CD154 expression did not require costimulation and was probably influenced by the strength of T-cell receptor (TCR) signalling alone. However, later CD154 expression was highly dependent on costimulation through either CD28 or inducible costimulator (ICOS). Although CD28 signalling interleukin (IL)-2 secretion, ICOS not, suggesting that costimulation enhance CD154 expression independently of IL-2 production. In fact, anti-CD28 treatment could still induce late-phase CD154 on anti-CD3-stimulated CD4 T cells expressing a mutated form of CD28 that not lead to the induction of IL-2. However, this CD154 induction was somewhat weaker than that of wild-type CD28-expressing cells, suggesting that direct signalling and IL-2-mediated signalling co-operatively responsible for the levels of CD154 induced by CD28. Finally, we show that the second phase of CD154 expression negatively regulated B-cell terminal differentiation and antibody secretion. These results demonstrate that TCR signalling and costimulation each regulate different phases of CD154 expression and control the biological outcome of CD40 signalling on B cells.     

Role of the CD28 receptor in T-cell activation

Antigen-specific T-cell activation is initiated through the T-cell receptor. Recent evidence has shown that a number of additional T-cell surface receptors serve to regulate the responses of antigen-activated T cells. One such molecule, CD28, is a member of a heterophilic cell adhesion complex, and is the receptor for the B-cell-restricted B7/BB-1 antigen. As Carl June, Jeffrey Ledbetter, Peter Linsley and Craig Thompson review here, CD28 serves as the surface component of a novel signal transduction pathway that modulates T-cell lymphokine production and increases the resistance of T-cell responses to various immunosuppressive agents.     

CTLA-4, CD28, and ICOS gene polymorphism associations with non-small-cell lung cancer

Polymorphisms in genes encoding CD28, ICOS, and CTLA-4 were demonstrated to be associated with susceptibility to malignancies. To the best of our knowledge, no study on this association has been performed in a Caucasian population for non-small-cell lung cancer (NSCLC). In the present work, we investigated the polymorphisms CTLA-4c.49A>G (rs231775), CTLA-4g.319C>T (rs5742909), CTLA-4g.*642AT(8_33), CTLA-4g.*6230G>A (CT60) (rs3087243), CTLA-4g.*10223G>T (Jo31) (rs11571302), CD28c.17+3T>C (rs3116496), and ICOSc.1554+4GT(8_15) in 208 NSCLC patients and 326 controls. The distributions of the allele and genotype were similar in both groups for CTLA-4, CD28, and ICOS gene polymorphisms. However, we noted a tendency toward overrepresentation of individuals possessing the CTLA-4c.49A>G[A] allele in NSCLC patients compared with controls (0.84 vs 0.79, p = 0.09). The association became significant compared with controls in women for the CTLA-4c.49A>G[A] allele and CTLA-4c.49A>G[AA] genotype (0.67 vs 0.54, p = 0.01, and 0.47 vs 0.30, p = 0.02; respectively). Moreover, the constellation of alleles CTLA-4c.49A>G[A]/CT60[G]/CD28c.17+3T>C[T]/ICOSc.1554+4GT(8_15)[>10] increased the risk of NSCLC about 2-fold (p = 0.002). The same constellation of alleles combined with smoking, CTLA-4g.319C>T[T], and ICOSc.1554+4GT(8_15)[>10] was associated with a decreased overall survival rate. In conclusion, the constellation of specific alleles in CTLA-4, CD28, and ICOS genes contributes to the susceptibility and clinical course of NSCLC.     

The T cell activation molecule H4 and the CD28-like molecule ICOS are identical

The recently cloned CD28-like molecule ICOS displays striking similarities with H4, characterized some years ago in the mouse and recently in humans. Both molecules are selectively expressed by activated and germinal center T cells, display similar structure, and display co-stimulatory activities. H4 displays lateral association with the CD3/TCR and is expressed by mature thymocytes. In the mouse, H4 is also expressed at high levels by thymic NKT cells that are resistant to negative selection. The aim of this work was to evaluate whether H4 and ICOS are the same molecule using the C398.4A (binding human and mouse H4) and F44 (binding human ICOS) monoclonal antibody (mAb) in parallel experiments on human T cells. ICOS and H4 displayed the same expression pattern in a panel of T cell lines and the same expression kinetics in phytohemagglutinin-activated T cells. C398.4A completely blocked cell staining by F44, whereas F44 partially blocked C398.4A. H4 and ICOS immunoprecipitates displayed identical SDS-PAGE patterns and H4 immunoprecipitation completely removed ICOS from cell lysates. Finally, the C398.4A mAb specifically stained cells transfected with the human or mouse ICOS. These data prove that H4 and ICOS are the same molecule and that F44 and C398.4A bind partially different epitopes.     

Intrinsic antiviral immunity

Intrinsic antiviral immunity refers to a form of innate immunity that directly restricts viral replication and assembly, thereby rendering a cell nonpermissive to a specific class or species of viruses. Intrinsic immunity is conferred by restriction factors that are mostly preexistent in certain cell types, although these factors can be further induced by viral infection. Intrinsic virus-restriction factors recognize specific viral components, but unlike other pattern-recognition receptors that inhibit viral infection indirectly by inducing interferons and other antiviral molecules, intrinsic antiviral factors block viral replication immediately and directly. This review focuses on recent advances in understanding of the roles of intrinsic antiviral factors that restrict infection by human immunodeficiency virus and influenza virus.     

Proliferative arrest and cell cycle regulation in CD8(+)CD28(-) versus CD8(+)CD28(+) T cells

CD8(+)CD28(-) T cells have been characterized by oligoclonal expansions, impaired proliferative responses, but preserved cytotoxicity and reduced telomeres. To examine this subset further and define the underlying mechanisms of proliferation arrest, we investigated several features of this cell type compared with CD8(+)CD28(+) controls. We analyzed expression of various activation markers, thymidine incorporation upon activation, T-cell receptor (TCR) zeta-chain phosphorylation, cell cycle characteristics, and cell cycle related gene expression. Flow cytometry revealed higher expression of CD11b, CD29, CD57, and CD94, and lower expression of CD25 in CD8(+)CD28(-) compared with CD8(+)CD28(+) T cells. Sorted CD8(+)CD16(-)CD28(-) cells exhibited decreased phosphorylation of the TCR zeta-chain in three of four probands. Proliferation of these T cells was impaired, even when activated with mitogens that bypass TCR signaling. Cell cycle profiles demonstrated a lower percentage of cycling cells and significantly higher levels of cyclin dependent kinase inhibitor p16(INK4a) in the CD28(-) subset compared with the CD28(+) control. These observations suggest that expanded CD8(+)CD28(-) T cells in normal elderly individuals have reduced proliferation concomitant with increased p16(INK4a) expression. Defects in TCR signaling were associated with altered TCR zeta-chain phosphorylation.     

Genetic diversity of CD14, CD28, CTLA-4 and ICOS gene promoter polymorphism in African and American sickle cell disease

Variable immune response to external stimuli remains a major concern in sickle cell disease (SCD), with such responses predicted to be contributors to disease pathogenesis. Elucidating the diversity of host genes contributing to immune response would assist to clarify differing outcomes among and between disease groups. We hypothesize that there is a significant interethnic diversity in the CD14 (rs2569190), CD28 (rs35593994), CTLA-4 (rs5742909) and ICOS (rs4404254) gene polymorphisms among and between SCD groups. We genotyped single nucleotide polymorphisms of the 4 loci among African and African American SCD and control groups and between SCD groups. In all, 375 individuals from Mali (145 SCD and 230 controls) and 700 DNA samples from the United States (321 SCD and 379 controls) were subjected to a PCR-RFLP assay. We found no intraethnic difference in genotypic and allelic frequencies of the 4 loci among Africans and African Americans, potentially significant in disease association studies, including a similar observation for interethnic frequencies of CD28, CTLA-4 and ICOS genes, but not CD14. The CD14 (rs2569190) gene promoter demonstrated a significant difference (p < 0.02) between African and African American SCD groups, with the mutant variant (−159 T/T) more frequent (p < 0.0002) in African American SCD (38.9% versus 26.2%). The higher frequency of CD14 mutants among African Americans without an accompanying defect in CD28, CTLA-4 and ICOS diversity possibly indicates a defective innate response, driven by CD14, is untethered to downstream T cell differentiation or effector function. Additionally, we show that CD28 (rs35593994) mutant variants have no impact on T cell differentiation, as the ICOS gene provides an alternative pathway to override this impairment. We conclude that in spite of the defect in CD14, T cell selection and differentiation is unimpeded and a robust adaptive immune response initiated.     

Genetic analysis of the CD28/CTLA4/ICOS (CELIAC3) region in coeliac disease

Abstract:  In order to extend our previous findings of genetic linkage to the CD28/CTLA4/ICOS region on chromosome 2q33 ( CELIAC3 ) in coeliac disease (CD), we have investigated 22 genetic markers in 325 Norwegian/Swedish multiplex and simplex CD families. We found both linkage and association with several markers, primarily in the multiplex material. We observed strong linkage disequilibrium (LD) between SNPs (Single Nucleotide Polymorphisms) within an LD block delimited by MH30 and D2S72. A haplotype of this region marked by the alleles −1147*T: + 49*A:CT60*G:CT61*A was significantly associated with CD, suggesting that one or more polymorphisms of this haplotype, possibly −1147*T, are involved in CD susceptibility. The CT60 SNP, a polymorphism found to be most strongly associated with some other immune-mediated diseases, was not associated with CD, as this SNP was part of both associated and non-associated haplotypes. Moreover, our results suggest that CELIAC3 harbours several independent loci contributing to CD susceptibility.     

DNA甲基化在急性冠脉综合征患者CD4+CD28-T细胞CD28表达缺失中的作用

目的 通过研究急性冠脉综合征(acute coronary syndrome,ACS)患者外周血CD4+T细胞CD28 mRNA水平和CD28基因启动子调节序列的甲基化状态,旨在探讨DNA甲基化在ACS患者CD4+CD28-T细胞CD28表达缺失中的作用.方法 免疫磁珠分离CD4+T细胞经逆转录后,实时定量PCR(real time-PCR)技术检测CD4+T细胞CD28mRNA的表达水平,亚硫酸氢钠测序检测CD28基因启动子调节序列的甲基化状态.结果 与正常对照组相比,ACS患者CD4+T细胞CD28mRNA表达水平显著减低,差异具有统计学意义[正常对照组比ACS组:(1.066±0.162)比(0.401±0.069),P<0.05].CD28基因启动子区域的甲基化水平显著增高,差异有统计学意义[正常对照组比ACS组:(24.47±3.17)%比(43.33±1.52)%,P<0.05].CD28基因启动子区域DNA甲基化水平与CD28mRNA表达水平呈显著负相关(P=0.01,r=-0.579).结论 ACS患者CD4+T细胞CD28基因启动子区域高甲基化调控了CD28基因转录抑制.DNA甲基化参与了CD4+CD28-T细胞的形成.     

Costimulation in antiviral immunity: differential requirements for CD4(+) and CD8(+) T cell responses

A key step toward improving vaccines is understanding the molecular interactions responsible for inducing antiviral T cell responses. An emerging theme from recent studies is that CD4(+) and CD8(+) T cell responses require distinct costimulatory pathways for activation. In addition, these costimulatory interactions can play a crucial role during the death phase of the T cell response and determine the number of effector T cells that survive to become memory T cells.     

Analysis of the CTLA-4, CD28, and inducible costimulator (ICOS) genes in autoimmune thyroid disease

The cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) gene on 2q33 is associated with autoimmune thyroid diseases (AITDs). Our earlier study in 56 families showed linkage of 2q33 to the presence of thyroid antibodies (TAbs). The goals of this study were to confirm the linkage of the 2q33 region to TAbs, to fine map this region, and study the ICOS gene. We performed a linkage study in an expanded data set of 99 multiplex AITD-TAb families (529 individuals). The highest two-point LOD score of 2.9 was obtained for marker D2S325 on 2q33. To fine map this locus, we genotyped 238 Caucasian AITD patients and 137 controls for five additional markers in the linked locus, which contained the CTLA-4, CD28, and ICOS genes. The A/G single-nucleotide polymorphism at position 49 of CTLA-4 was associated with AITD (P=0.01, OR=1.5), while markers inside CD28 and ICOS were not. Functional studies have shown that the G allele was associated with reduced inhibition of T-cell proliferation by CTLA-4. We concluded that: (1) the AITD gene in the 2q33 locus is the CTLA-4 gene and not the CD28 or ICOS genes; and (2) the G allele is associated with decreased function of CTLA-4.     

Autophagy and antiviral immunity

Autophagy is an ancient pathway designed to maintain cellular homeostasis by degrading long-lived proteins and organelles in the cytosol. Recent studies demonstrate that autophagy is utilized by the cells of the innate and adaptive immune systems to combat viral infections. Autophagy plays a key role in recognizing signatures of viral infection, and represents a critical effector mechanism to restrict viral replication. On the other hand, autophagosomes have been exploited by certain viruses as a niche for viral replication. Furthermore, autophagy can be used to deliver endogenous viral antigens to the MHC class II loading compartment, allowing activation of CD4 T cells. In this review, we describe recent advances in the field of autophagy as it relates to innate and adaptive antiviral immune responses.     

Cytomegalovirus impairs antiviral CD8(+) T cell immunity by recruiting inflammatory monocytes

Inflammatory monocytes are key early responders to?infection that contribute to pathogen-host interactions in diverse ways. Here, we report that the murine cytomegalovirus-encoded CC chemokine, MCK2, enhanced CCR2-dependent recruitment of these cells to modulate antiviral immunity, impairing virus-specific CD8(+) T?cell expansion and differentiation into effector cytotoxic T lymphocytes, thus reducing the capacity to eliminate viral antigen-bearing cells and slowing viral clearance. Adoptive transfer of inflammatory monocytes into Ccr2(-/-)Ccl2(-/-) mice impaired virus antigen-specific clearance. Cytomegalovirus therefore enhances?a natural CCR2-dependent immune regulatory network to modulate adaptive immunity via nitric oxide production, reminiscent of the monocytic subtype of myeloid-derived suppressor cells primarily implicated in cancer immunomodulation.     

Role of IgM antibodies versus B cells in influenza virus-specific immunity

We have compared the role of IgM antibodies with the role of B cells in control of primary influenza virus infection. Mice deficient in IgM (IgM(-/-)), but capable of producing other Igisotypes, exhibited increased pulmonary virus titers compared to wild-type mice. However, IgM(-/-) mice were less susceptible compared to B cell-deficient micro MT) mice. CD4(+) T cells from spleen and lung draining lymph nodes of infected micro MT mice showed reduced proliferation upon virus re-stimulation in vitro. Furthermore, numbers of IFN-gamma-producing CD4(+) effector T cells were reduced in the alveolar lavage (BAL) of micro MT mice but not IgM(-/-) mice. In contrast, total number of virus-specific CTL was almost comparable in BAL of micro MT and wild-type mice. Pulmonary recruitment of inflammatory macrophages and neutrophils occurred normally in both micro MT and IgM(-/-) mice. Interestingly, virus-specific IgG2a and IgG2b antibody responses were affected locally in the BAL and in the serum of IgM(-/-) mice, while IgG1 responses remained largely normal. Taken together, our data suggest a role for B cells to promote effector T cell responses and a role of both IgM and IgG antibodies in the defense against acute influenza virus infection.     

ICOS and CD28 reversely regulate IL-10 on re-activation of human effector T cells with mature dendritic cells

With newly generated ICOS-ligand (ICOS-L)-specific monoclonal antibodies we determined that human Langerhans cells in situ express similar levels of ICOS-L, CD80, and CD86, compared to immature dendritic cells (DC) derived from monocytes in vitro. Maturation of DC strongly up-regulated CD80 and CD86 but did not significantly change ICOS-L levels. On coculture of "naive"CD4(+) T cells with mature DC in the presence of superantigen, ICOS was highly up-regulated on T cells, but played only a secondary role in the CD28-dominated release of TNF-alpha and IFN-gamma, and did not participate in the induction of IL-2. Cocultures of "effector" CD4(+) T cells with mature DC revealed CD28 as the driving force for the secretion of IL-2, IFN-gamma, IL-6, and IL-13, with no apparent contribution of ICOS. In contrast, the release of IL-10 was differentially regulated. Interaction of ICOS with ICOS-L strongly promoted IL-10 secretion, whereas the CD28/B7 pathway acted as a potent attenuator of IL-10 release. Our data thus indicate a selective regulation of IL-10 secretion by ICOS on re-activation of effector T cells with professional antigen-presenting cells (bearing CD80 and CD86) in lymphoid tissue.     

Epigenetic regulation in antiviral innate immunity

Emerging life-threatening viruses have posed great challenges to public health. It is now increasingly clear that epigenetics plays a role in shaping host-virus interactions and there is a great need for a more thorough understanding of these intricate interactions through the epigenetic lens, which may represent potential therapeutic opportunities in the clinic. In this review, we highlight the current understanding of the roles of key epigenetic regulators — chromatin remodeling and histone modification — in modulating chromatin openness during host defense against virus. We also discuss how the RNA modification m6A (N6-methyladenosine) affects fundamental aspects of host-virus interactions. We conclude with future directions for uncovering more detailed functions that epigenetic regulation exerts on both host cells and viruses during infection.