Association of genetic variation in co-stimulatory molecule genes with outcome of liver transplant in Iranian patients

CD28, cytotoxic T-lymphocyte associated antigen 4 (CTLA4), inducible costimulator (ICOS) and programmed cell death 1 are closely-linked genes located on chromosome 2q and encode co-stimulatory molecules, which are T-cell activity regulators. The principal assignment of T-cell mediated immune response in allograft rejection is an interesting topic of multiple studies. Although the variation in these genes may influence the graft survival and the amount of immunosuppression needed, the studies so far have been restricted solely to the CTLA4 gene. In 145 patients who underwent liver allograft transplantation, 10 single nucleotide polymorphisms of CD28, CTLA4, ICOS, and PD.1 genes were defined. To distinguish the polymorphisms of all 10 SNPs, PCR-RFLP method was used and according to the standard criteria, acute rejection episodes were determined. CTLA4-1661, AA genotype was significantly more frequent in the patients with acute rejection and AG genotype was significantly more frequent in the patients without rejection. Frequencies of CTLA4+49 AG A allele and CTLA4-1661AG A allele were significantly higher than those of CTLA4+49 AG and CTLA4-1661AG, G allele in the patients with acute rejection. ICOS+693, GG genotype and G allele were significantly less frequent in the patients with acute rejection and CD28 CT genotype was significantly more in patients with acute rejection. The present results demonstrate that potentially functional genetic variation in T-cell co-stimulatory molecules including ICOS, CTLA4 and CD28 can influence liver transplant outcome.     

Genetic polymorphism of the human ICOS gene

Inducible costimulator (ICOS) is a novel receptor belonging to the same family as CD28 and CTLA4, which regulate T-lymphocyte activation in the immune response. The genes for these molecules are located adjacent to each other on Chromosome 2q33. Many autoimmune diseases have been found to be genetically linked to or associated with genetic markers near the CTLA4 gene. However, as all three genes are closely linked and have related functions, it is possible that the findings could be explained by variation in CD28 or ICOS. Few data on genetic variation in the ICOS gene are available. We sequenced the ICOS gene in 13 healthy unrelated individuals and found eight single nucleotide polymorphisms. One was located in the first intron, and the others in the untranslated region of the last exon. The allele frequencies and linkage disequilibrium were determined from a population sample of 63 Finnish individuals. The results show that the ICOS gene is polymorphic, but no variation in the coding sequence was detected, implying that the genetic linkage of this gene region to autoimmune diseases may not result from structural variation in the ICOS molecule. These polymorphisms, however, should be useful in genetic studies of this candidate gene.     

Cutting edge: the related molecules CD28 and inducible costimulator deliver both unique and complementary signals required for optimal T cell activation

Optimal T cell activation requires engagement of CD28 with its counterligands B7-1 and B7-2. Inducible costimulator (ICOS) is the third member of the CD28/CTLA4 family that binds a B7-like protein, B7RP-1. Administration of ICOS-Ig attenuates T cell expansion following superantigen (SAg) administration, but fails to regulate either peripheral deletion or anergy induction. ICOS-Ig, but not CTLA4-Ig, uniquely regulates SAg-induced TNF-alpha production, whereas IL-2 secretion is modulated by CTLA4-Ig, but not ICOS-Ig. In contrast, both ICOS and CD28 are required for complete attenuation of IL-4 production. Our data suggest that ICOS and CD28 regulate T cell expansion and that ligation of either CD28 or ICOS can either uniquely regulate cytokine production (IL-2/TNF-alpha) or synergize for optimal cytokine production (IL-4) after SAg administration.     

Polymorphisms in the CD28/CTLA4/ICOS genes: role in malignant melanoma susceptibility and prognosis?

The appearance of vitiligo and spontaneous regression of the primary lesion in melanoma patients illustrate a relationship between tumor immunity and autoimmunity. T lymphocytes play a major role both in tumor immunity and autoimmunity. CD28, Cytotoxic T lymphocyte antigen 4 (CTLA4) and inducible costimulator (ICOS) molecules are important secondary signal molecules in the T lymphocyte activation. Single nucleotide polymorphisms (SNPs) in the CD28/CTLA4/ICOS gene region were reported to be associated with several autoimmune diseases including, type-1 diabetes, SLE, autoimmune thyroid diseases and celiac disease. In this study, we investigated the association of SNPs in the CD28, CTLA4 and ICOS genes with the risk of melanoma. We also assessed the prognostic effect of the different polymorphisms in melanoma patients. Twenty-four tagging SNPs across the three genes and four additional SNPs were genotyped in a cohort of 763 German melanoma patients and 734 healthy German controls. Influence on prognosis was determined in 587 melanoma cases belonging to stage I or II of the disease. In general, no differences in genotype or allele frequencies were detected between melanoma patients and controls. However, the variant alleles for two polymorphisms in the CD28 gene were differentially distributed in cases and controls. Similarly no association of any polymorphism with prognosis, except for the rs3181098 polymorphism in the CD28 gene, was observed. In addition, individuals with AA genotype for rs11571323 polymorphism in the ICOS gene showed reduced overall survival. However, keeping in view the correction for multiple hypothesis testing our results suggest that the polymorphisms in the CD28, CTLA4 and ICOS genes at least do not modulate risk of melanoma and nor do those influence the disease prognosis in the investigated population.     

CD28 and inducible costimulatory protein Src homology 2 binding domains show distinct regulation of phosphatidylinositol 3-kinase,Bcl-xL,and IL-2 expression in primary human CD4 T lymphocytes

Ligation of either CD28 or inducible costimulatory protein (ICOS) produces a second signal required for optimal T cell activation and proliferation. One prominent difference between ICOS- and CD28-costimulated T cells is the quantity of IL-2 produced. To understand why CD28 but not ICOS elicits major increases in IL-2 expression, we compared the abilities of these molecules to activate the signal transduction cascades implicated in the regulation of IL-2. Major differences were found in the regulation of phosphatidylinositol 3-kinase activity (PI3K) and c-jun N-terminal kinase. ICOS costimulation led to greatly augmented levels of PI3K activity compared with CD28 costimulation, whereas only CD28 costimulation activated c-jun N-terminal kinase. To examine how these differences in signal transduction affected IL-2 production, we transduced primary human CD4 T cells with a lentiviral vector that expressed the murine CD28 extracellular domain with a variety of human CD28 and ICOS cytoplasmic domain swap constructs. These domains were able to operate as discrete signaling units, suggesting that they can function independently. Our results show that even though the ICOS Src homology (SH) 2 binding domain strongly activated PI3K, it was unable to substitute for the CD28 SH2 binding domain to induce high levels of IL-2 and Bcl-x(L). Moreover, the CD28 SH2 binding domain alone was sufficient to mediate optimal levels of Bcl-x(L) induction, whereas the entire CD28 cytoplasmic tail was required for high levels of IL-2 expression. Thus, differences within their respective SH2 binding domains explain, at least in part, the distinct regulation of IL-2 and Bcl-x(L) expression following ICOS- or CD28-mediated costimulation.     

Association studies of CTLA-4, CD28, and ICOS gene polymorphisms with type 1 diabetes in the Japanese population

Co-stimulatory molecules of CD28, cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), and the newly identified inducible co-stimulator (ICOS) are expressed on cell surfaces and provide regulatory signals for T-cell activation. Their genes are candidate susceptibility genes for type 1 diabetes because they co-localize to Chromosome 2q33 with the IDDM12 locus. After determining the genomic structure and screening for polymorphisms of the ICOS gene, we performed association studies between newly identified polymorphisms of the ICOS gene, together with known polymorphisms of CD28 and CTLA-4 genes, and type 1 diabetes. The 49A/G dimorphism in exon 1 and the (AT)n in the 3' untranslated region of the CTLA-4 gene were significantly associated with type 1 diabetes. Evaluation of the CTLA-4 49A-3'(AT)n 86-bp haplotype frequency in patients and controls confirmed the results from the analysis of each polymorphic site. Dimorphism in intron 3 of the CD28 gene was associated with type 1 diabetes only in the early-onset group. In contrast, there was no association with the microsatellite polymorphisms in the ICOS gene or dimorphisms in the promotor region of CTLA-4. Of the three genes encoding co-stimulatory molecules, the CTLA-4 gene appears to confer risks for the development of type 1 diabetes.     

The inducible costimulator plays the major costimulatory role in humoral immune responses in the absence of CD28

CD28 plays crucial costimulatory roles in T cell proliferation, cytokine production, and germinal center response. Mice that are deficient in the inducible costimulator (ICOS) also have defects in cytokine production and germinal center response. Because the full induction of ICOS in activated T cells depends on CD28 signal, the T cell costimulatory capacity of ICOS in the absence of CD28 has remained unclear. We have clarified this issue by comparing humoral immune responses in wild-type, CD28 knockout (CD28 KO), and CD28-ICOS double-knockout (DKO) mice. DKO mice had profound defects in Ab responses against environmental Ags, T-dependent protein Ags, and vesicular stomatitis virus that extended far beyond those observed in CD28 KO mice. However, DKO mice mounted normal Ab responses against a T-independent Ag, indicating that B cell function itself was normal. Restimulated CD4(+) DKO T cells that had been primed in vivo showed decreased proliferation and reduced IL-4 and IL-10 production compared with restimulated CD4(+) T cells from CD28 KO mice. Thus, in the absence of CD28, ICOS assumes the major T cell costimulatory role for humoral immune responses. Importantly, CD28-mediated ICOS up-regulation is not essential for ICOS function in vivo.     

Characterization of human inducible costimulator ligand expression and function

The inducible costimulator (ICOS) is the newest member of the CD28/CD152 receptor family involved in regulating T cell activation. We constructed a soluble-Ig fusion protein of the extracellular domain of human ICOS and used it as a probe to characterize expression patterns of the ICOS ligand (ICOSL). ICOSIg did not bind to CD80- or CD86-transfected Chinese hamster ovary cell lines, demonstrating that ICOSL is distinct from those ligands identified for CD28/CD152. ICOSIg showed selective binding to monocytic and B cell lines, whereas binding was undetectable on unstimulated monocytes and peripheral blood T and B cells. Expression of ICOSL was induced on monocytes after integrin-dependent plastic adhesion. Pretreatment of monocytes with mAb to the beta2-integrin subunit CD18 decreased adhesion and abolished ICOSL up-regulation but had no effect on CD80/86 (CD152 ligand (CD152L)) expression. Both ICOSL and CD152L were up-regulated on monocytes by IFN-gamma but by distinct signaling pathways. Unlike CD152L expression, ICOSL expression did not change when monocytes were differentiated into dendritic cells (DCs) or after DCs were induced to mature by LPS, TNF-alpha, or CD40 ligation. Addition of ICOSIg to allogeneic MLRs between DCs and T cells reduced T cell proliferative responses but did so less efficiently than CTLA4Ig (CD152Ig) did. Similarly, ICOSIg also blocked Ag-specific T cell proliferation to tetanus toxoid. Thus, ICOSL, like CD80/86, is expressed on activated monocytes and dendritic cells but is regulated differently and delivers distinct signals to T cells that can be specifically inhibited by ICOSIg.     

PI3-kinase and MAP-kinase signaling cascades in AILIM/ICOS- and CD28-costimulated T-cells have distinct functions between cell proliferation and IL-10 production

Both AILIM/ICOS and CD28 provide positive costimulatory signals for T-cell activation, resulting in proliferation and cytokine production. In this study, we attempted to clarify the key signaling molecules in T-cell proliferation, and also IL-2 and IL-10 production, during T-cell activation by CD3 induced by costimulation with either AILIM/ICOS or CD28. We examined the role of both the PI3-kinase/Akt pathway and MAP kinase family members such as ERK1/2, JNK, and p38 kinase in this process. PI3-kinase and Erk1/2 were shown to potentially regulate primary T-cell activation and subsequent proliferation via both AILIM/ICOS- or CD28-mediated costimulation and the Erk signaling cascade was essential for this proliferation induction and also for IL-2 production. The JAK inhibitor, AG490, inhibited this induction. Our studies indicate that IL-2 is necessary for induction of T-cell proliferation and that the quantities of IL-2 produced by AILIM/ICOS ligation are also sufficient for T-cells to proliferate. In contrast, inhibition of Akt and p38, that are phosphorylated by both AILIM/ICOS and CD28-ligation, could downregulate IL-10 production but not T-cell proliferation. These data raise the interesting possibility that the signaling cascades between T-cell proliferation and IL-10 production are regulated by different molecules in AILIM/ICOS- and CD28-costimulated T-cells.     

Cutting edge: identification of GL50, a novel B7-like protein that functionally binds to ICOS receptor

By the genetic selection of mouse cDNAs encoding secreted proteins, a B7-like cDNA clone termed mouse GL50 (mGL50) was isolated encoding a 322-aa polypeptide identical with B7h. Isolation of the human ortholog of this cDNA (hGL50) revealed a coding sequence of 309 aa residues with 42% sequence identity with mGL50. Northern analysis indicated GL50 to be present in many tissues including lymphoid, embryonic yolk sac, and fetal liver samples. Of the CD28, CTLA4, and ICOS fusion constructs tested, flow cytometric analysis demonstrated only mouse ICOS-IgG binding to mGL50 cell transfectants. Subsequent phenotyping demonstrated high levels of ICOS ligand staining on splenic CD19+ B cells and low levels on CD3+ T cells. These results indicate that GL50 is a specific ligand for the ICOS receptor and suggest that the GL50-ICOS interaction functions in lymphocyte costimulation.     

Opposing effects of ICOS on graft-versus-host disease mediated by CD4 and CD8 T cells

ICOS, a CD28 family member expressed on activated CD4(+) and CD8(+) T cells, plays important roles in T cell activation and effector function. Here we studied the role of ICOS in graft-vs-host disease (GVHD) mediated by CD4(+) or CD8(+) T cells in allogeneic bone marrow transplantation. In comparison of wild-type and ICOS-deficient T cells, we found that recipients of ICOS(-/-) CD4(+) T cells exhibited significantly less GVHD morbidity and delayed mortality. ICOS(-/-) CD4(+) T cells had no defect in expansion, but expressed significantly less Fas ligand and produced significantly lower levels of IFN-gamma and TNF-alpha. Thus, ICOS(-/-) CD4(+) T cells were impaired in effector functions that lead to GVHD. In contrast, recipients of ICOS(-/-) CD8(+) T cells exhibited significantly enhanced GVHD morbidity and accelerated mortality. In the absence of ICOS signaling, either using ICOS-deficient donors or ICOS ligand-deficient recipients, the levels of expansion and Tc1 cytokine production of CD8(+) T cells were significantly increased. The level of expansion was inversely correlated with the level of apoptosis, suggesting that increased ability of ICOS(-/-) CD8(+) T cells to induce GVHD resulted from the enhanced survival and expansion of those cells. Our findings indicate that ICOS has paradoxical effects on the regulation of alloreactive CD4(+) and CD8(+) T cells in GVHD.     

Association of the costimulatory molecule gene polymorphisms and active cytomegalovirus infection in hematopoietic stem cell transplant patients

Determinative associations may exist between costimulatory molecule gene polymorphisms with a variety of post hematopoietic stem cell transplantation (HSCT) viral related clinical outcomes especially acute graft versus host disease (aGVHD). Therefore in this study the associations between costimulatory molecule gene polymorphisms including: cytotoxic T-lymphocyte antigen-4 (CTLA4), programmed cell death-1 (PD-1), inducible T cell costimulator (ICOS), and cluster differentiation 28 (CD28) with active cytomegalovirus (CMV) infection were evaluated in HSCT patients. The 72 allogeneic HSCT patients with and without aGVHD were enrolled in this cross sectional study between years: 2004–2011. The single nucleotide polymorphisms in loci of the costimulatory molecules including: CTLA4 gene (?318 C/T, 1722 T/C, 1661 A/G, +49 A/G), PD-1 gene (PD-1.3 A/G, PD-1.9 C/T), ICOS gene (1720 C/T), and CD28 gene (+17 C/T) were analyzed in studied HSCT patients by PCR-RFLP methods. The active CMV infection was evaluated in fresh EDTA-treated blood samples of each allogeneic HSCT patients by CMV antigenemia kit according to manufacturer’s instruction. Active CMV infection was found in 11 of 72 (15.27 %) of allogeneic HSCT patients. The T allele and TT genotype of the CD28 +17 C/T were significantly higher frequency in active CMV infected allogeneic HSCT patients experienced aGVHD. The G allele and GG genotype of the CTLA4 ?1661 A/G were significantly higher frequent in active CMV infected allogeneic HSCT patients experienced low grade of aGVHD. Finally, finding of significant associations between CD28 +17 C/T and CTLA4 ?1661 A/G genotypes with CMV active infection in allogeneic HSCT patients experienced aGVHD emphasize on the importance of the genetic pattern of costimulatory genes in outcomes of active CMV infection in HSCT patients needs completed studies.     

LICOS, a primordial costimulatory ligand?

In mammals, the classical B7 molecules expressed on antigen-presenting cells, B7-1 (CD80) and B7-2 (CD86), bind the structurally related glycoproteins CD28 and CTLA-4 (CD152), generating costimulatory signals that regulate the activation state of T cells. A recently identified human CD28-like protein, ICOS, also induces costimulatory signals in T cells when crosslinked with antibodies, but it is unclear whether ICOS is part of a B7-mediated regulatory pathway of previously unsuspected complexity, or whether it functions independently and in parallel. Here, we report that, rather than binding B7-1 or B7-2, ICOS binds a new B7-related molecule of previously unknown function that we call LICOS (for ligand of ICOS). At 37 degrees C, LICOS binds only to ICOS but, at lower, non-physiological temperatures, it also binds weakly to CD28 and CTLA-4. Sequence comparisons suggest that LICOS is the homologue of a molecule expressed by avian macrophages and of a murine protein whose expression is induced in non-lymphoid organs by tumour necrosis factor alpha (TNFalpha). Our results define the components of a distinct and novel costimulatory pathway and raise the possibility that LICOS, rather than B7-1 or B7-2, is the contemporary homologue of a primordial vertebrate costimulatory ligand.     

Tetraspanins in cellular immunity

Tetraspanins are a superfamily of integral membrane proteins involved in the organization of microdomains that consist of both cell membrane proteins and cytoplasmic signalling molecules. These microdomains are important in regulating molecular recognition at the cell surface and subsequent signal transduction processes central to the generation of an efficient immune response. Tetraspanins, both immune-cell-specific, such as CD37, and ubiquitously expressed, such as CD81, have been shown to be imp-ortant in both innate and adaptive cellular immunity. This is via their molecular interaction with important immune cell-surface molecules such as antigen-presenting MHC proteins, T-cell co-receptors CD4 and CD8, as well as cytoplasmic molecules such as Lck and PKC (protein kinase C). Moreover, the generation of tetraspanin-deficient mice has enabled the study of these proteins in immunity. A variety of tetraspanins have a role in the regulation of pattern recognition, antigen presentation and T-cell proliferation. Recent studies have also begun to elucidate roles for tetraspanins in macrophages, NK cells (natural killer cells) and granulocytes.     

Cooperation between 4-1BB and ICOS in the immune response to influenza virus revealed by studies of CD28/ICOS-deficient mice

CD28, ICOS, and 4-1BB each play distinct roles in the CD8 T cell response to influenza virus. CD28-/- mice are severely impaired in primary CD8 T cell expansion and fail to mount a secondary response to influenza. Influenza-specific CD8 T cells expand normally in ICOS-/- mice, with only a small and transient defect late in the primary response and an unimpaired secondary response. Conversely, 4-1BB/4-1BBL interaction is dispensable for the primary CD8 T cell response to influenza, but maintains CD8 T cell survival and controls the size of the secondary response. Previous results showed that a single dose of agonistic anti-4-1BB Ab at priming allowed partial restoration of primary CD8 T cell expansion and full recovery of the secondary CD8 T cell responses to influenza in CD28-/- mice. In this study we show that anti-4-1BB fails to correct the CD8 T cell defect in CD28-/-ICOS-/- mice, suggesting that ICOS partially compensates for CD28 in this model. In support of this hypothesis, we found that anti-4-1BB enhances ICOS expression on both T cell subsets and that anti-4-1BB and anti-ICOS can synergistically activate CD4 and CD8 T cells. Furthermore, ICOS and 4-1BB can cooperate to directly stimulate isolated CD28-/- CD8 T cells. These results reveal a novel interaction between the ICOS and 4-1BB costimulatory pathways as well as unexpected redundancy between CD28 and ICOS in primary CD8 T cell expansion. These findings have implications for costimulation of human T cell responses in diseases such as AIDS or rheumatoid arthritis, in which CD28- T cells accumulate.     

Mouse inducible costimulatory molecule (ICOS) expression is enhanced by CD28 costimulation and regulates differentiation of CD4+ T cells

The inducible costimulatory (ICOS) molecule is expressed by activated T cells and has homology to CD28 and CD152. ICOS binds B7h, a molecule expressed by APC with homology to CD80 and CD86. To investigate regulation of ICOS expression and its role in Th responses we developed anti-mouse ICOS mAbs and ICOS-Ig fusion protein. Little ICOS is expressed by freshly isolated mouse T cells, but ICOS is rapidly up-regulated on most CD4(+) and CD8(+) T cells following stimulation of the TCR. Strikingly, ICOS up-regulation is significantly reduced in the absence of CD80 and CD86 and can be restored by CD28 stimulation, suggesting that CD28-CD80/CD86 interactions may optimize ICOS expression. Interestingly, TCR-transgenic T cells differentiated into Th2 expressed significantly more ICOS than cells differentiated into Th1. We used two methods to investigate the role of ICOS in activation of CD4(+) T cells. First, CD4(+) cells were stimulated with beads coated with anti-CD3 and either B7h-Ig fusion protein or control Ig fusion protein. ICOS stimulation enhanced proliferation of CD4(+) cells and production of IFN-gamma, IL-4, and IL-10, but not IL-2. Second, TCR-transgenic CD4(+) T cells were stimulated with peptide and APC in the presence of ICOS-Ig or control Ig. When the ICOS:B7h interaction was blocked by ICOS-Ig, CD4(+) T cells produced more IFN-gamma and less IL-4 and IL-10 than CD4(+) cells differentiated with control Ig. These results demonstrate that ICOS stimulation is important in T cell activation and that ICOS may have a particularly important role in development of Th2 cells.     

Antiviral immune responses in CTLA4 transgenic mice.

The role of B7 binding CD28 in the regulation of T- and B-cell responses against viral antigens was assessed in transgenic mice expressing soluble CTLA4-Hgamma1 (CTLA4-Ig tg mice) that blocks B7-CD28 interactions. The results indicate that transgenic soluble CTLA4 does not significantly alter cytotoxic T-cell responses against replicating lymphocytic choriomeningitis virus (LCMV) or vaccinia virus but drastically impairs the induction of cytotoxic T-cell responses against abortively replicating vesicular stomatitis virus (VSV). While the T-independent neutralizing immunoglobulin M (IgM) responses were within normal ranges, the switch to IgG was reduced 4- to 16-fold after immunization with abortively replicating VSV and more than 30-fold after immunization with an inert VSV glycoprotein antigen in transgenic mice. IgG antibody responses to LCMV, as detected by enzyme-linked immunosorbent assay and by neutralizing action, were reduced about 3- to 20-fold and more than 50-fold, respectively. These results suggest that responses in CTLA4-Ig tg mice are mounted according to their independence of T help. While immune responses to nonreplicating or poorly replicating antigens are in general most dependent on T help and B7-CD28 interactions, they are most impaired in CTLA4-Ig tg mice. The results of the present experiments also indicate that highly replicating viruses, because of greater quantities of available antigens and by inducing as-yet-undefined factors and/or cell surface changes, are capable of compensating for the decrease in T help caused by the blocking effects of soluble CTLA4.