Renal cell carcinoma (RCC) is an immunogenic and proangiogenic cancer. Although antivascular endothelial growth factor (VEGF) therapies achieve impressive responses in some patients, many tumors eventually develop resistance to such therapy. The B7 family molecules such as CTLA-4, PD-1, and PD-L1 are pivotal players in immune checkpoints that positively or negatively regulate various immune responses. Recently, immunotherapy based on blocking immune checkpoints with anti-CTLA4, anti-PD-1, or anti-PD-L1 antibodies has been proposed as a potential new approach to the treatment of metastatic RCC. Higher expression of PD-L1 and B7-H4 in the tumors is associated with a poor prognosis in RCCs, however, the clinical impact of serum levels of B7 family molecules has not been elucidated in patients with metastatic RCCs receiving VEGF-targeted agents. We assessed the preoperative serum levels of B7 family molecules, including CD80, CD86, PD-1, PD-L1, B7-H3, B7-H4, and CTLA-4, and CD28 in RCC patients, and determined their relations with various clinicopathological characteristics. Elevated preoperative serum levels of PD-L1 and B7-H4 were correlated with less differentiated tumors, higher invasive and metastatic potential, a worse response to anti-VEGF therapy, and shorter overall survival. These findings suggested that investigating preoperative serum levels of PD-L1 and B7-H4 might not only be useful to assess the biological aggressiveness of RCCs, but also to predict the efficacy of anti-VEGF therapy and the eventual prognosis, indicating the future design of clinical trials of therapies targeting immune checkpoint in advanced RCCs.

The purpose of this study was to assess the effect of neoadjuvant chemotherapy (NACT) on immune activation in stage IIIC/IV tubo-ovarian high-grade serous carcinoma (HGSC), and its relationship to treatment response.

We obtained pre- and posttreatment omental biopsies and blood samples from a total of 54 patients undergoing platinum-based NACT and 6 patients undergoing primary debulking surgery. We measured T-cell density and phenotype, immune activation, and markers of cancer-related inflammation using IHC, flow cytometry, electrochemiluminescence assays, and RNA sequencing and related our findings to the histopathologic treatment response.

There was evidence of T-cell activation in omental biopsies after NACT: CD4(+) T cells showed enhanced IFNγ production and antitumor Th1 gene signatures were increased. T-cell activation was more pronounced with good response to NACT. The CD8(+) T-cell and CD45RO(+) memory cell density in the tumor microenvironment was unchanged after NACT but biopsies showing a good therapeutic response had significantly fewer FoxP3(+) T regulatory (Treg) cells. This finding was supported by a reduction in a Treg cell gene signature in post- versus pre-NACT samples that was more pronounced in good responders. Plasma levels of proinflammatory cytokines decreased in all patients after NACT. However, a high proportion of T cells in biopsies expressed immune checkpoint molecules PD-1 and CTLA4, and PD-L1 levels were significantly increased after NACT.

NACT may enhance host immune response but this effect is tempered by high/increased levels of PD-1, CTLA4, and PD-L1. Sequential chemoimmunotherapy may improve disease control in advanced HGSC. Clin Cancer Res; 22(12); 3025-36. ©2016 AACR.

OBJECTIVE

Define an immunologic response using the tetramer and enzyme-linked immunospot (ELISPOT) assays.

METHODS

Ten healthy subjects and 21 patients with melanoma (all HLA-A*0201) donated a total of 121 blood samples to determine the lower limit of detection (LLD), analytic coefficient of variation (aCV), and physiologic CV (pCV) of the tetramer and ELISPOT assays. The mean, SD, and reference change value (RCV) were calculated to define changes beyond the assay imprecision, and its application was tested in the monitoring of T-cell expansion after CTLA4 blockade with ticilimumab (CP-675,206).

RESULTS

The LLD for the tetramer assay was 0.038% CD8+ cells and seven spots per 10(5) peripheral blood mononuclear cells for the ELISPOT assay. The aCV of the tetramer assay was <10% and was higher for the ELISPOT (24.69-36.32%). There was marked between-subject variability on baseline homeostatic values, which was correlated to prior antigen exposure. An immunologic response was defined as an increase beyond the mean + 3 SD in antigen-specific cells for subjects with baseline levels below the LLD, or beyond the assay RCV for baseline levels above the LLD. In four patients receiving ticilimumab, expansions of antigen-specific T cells beyond the assay variability were noted for EBV and MART1 antigens.

CONCLUSIONS

A combined approach of change from negative (below the LLD) to positive (above the LLD) and a percentage change beyond the assay variability using the RCV score can be computed to define which change in circulating antigen-specific T cells represents a response to immunotherapy.

Activation of T lymphocytes requires the recognition of peptide-major histocompatibility complexes (MHCs) and costimulatory signals provided by antigen-presenting cells (APCs). It has been shown that T-cell activation without costimulation can lead to anergy. In this study, we developed a novel strategy to inhibit expression of B7 molecules (CD80/86) by transfecting APCs with a gene construct encoding a modified cytotoxic T lymphocyte antigen 4 (CTLA4) molecule (CTLA4-KDEL) that is targeted to the endoplasmic reticulum (ER). APCs expressing this construct failed to express CD80/86 on their surface, were unable to stimulate allogeneic and peptide-specific T-cell responses, and induced antigen-specific anergy of the responding T cells. Cells expressing CTLA4-KDEL do not up-regulate the indoleamine 2, 3-dioxygenase enzyme, unlike cells treated with soluble CTLA4-immunoglobin (Ig). This gene-based strategy to knock out surface receptors is an attractive alternative to using immature dendritic cells for preventing transplant rejection and treating of autoimmune diseases.

Allograft rejection is dependent on T cell activation, which requires both the engagement of the T cell receptor by antigen in the context of the MHC molecules and costimulatory signals delivered by cell surface molecules such as B7-CD28/CTLA4 pathway. CTLA4-Ig is a fusion protein that blocks this pathway and has previously been shown to prolong both allograft and xenograft survival. The current study demonstrates markedly prolonged murine cardiac allograft survival and specific prolongation of secondary skin grafts using a combination of CTLA4-Ig plus donor bone marrow. A role for hematopoietic chimerism in the establishment of CTLA4-Ig-induced transplantation tolerance was investigated using reverse transcriptase polymerase chain reaction analysis of recipient tissues. Expression of donor-specific MHC class II transcripts in both peripheral and lymphoid tissues was demonstrated at greater than 200 days after transplant. To investigate the functional significance of this observation, heart donors, and donor bone marrow were irradiated before transplantation in CTLA4-Ig-treated recipients. A reduction in allograft survival was associated with irradiation of both the donor heart and the bone marrow. These results suggest that there may be a donor-derived radiosensitive element that enhances allograft survival in this model. Reverse transcriptase polymerase chain reaction analysis of allografts of tolerant and control animals at days 5, 8, and 12 after transplantation failed to demonstrate a dramatic difference in the expression of interleukin (IL)-2, IL-4, IL-10, and interferon-gamma message. Cytotoxicity effector transcripts were largely intact in CTLA4-Ig + bone marrow-treated recipients as they showed no decrease in intragraft granzyme, perforin, Fas, or Fas ligand transcripts during thr first 8 days after transplant. These results imply that complex mechanisms may be important for the induction and maintenance of transplantation tolerance in the CTLA4-Ig plus bone marrow murine cardiac allograft model.

The Type I Diabetes Genetics Consortium genotyped 24 single-nucleotide polymorphisms (SNPs) in the CTLA4 locus in 2298 type I diabetes (T1D) nuclear families (11 159 individuals, 5003 affected) to evaluate the recognized T1D association. The 24 CTLA4 SNPs span approximately 43 kb from the 5' flanking to 3' flanking region of the gene in the middle of an extended region of linkage disequilibrium of more than 100 kb. The genotyping was performed using two technologies (Illumina GoldenGate and Sequenom iPlex) on the same samples. The genotype calls by both the methods were highly consistent (the majority >99%). Previously reported T1D association from both the +49G>A and the CT60 SNPs was replicated. The reported association of the -319C>T SNP was not replicated. Although associated with T1D risk, it is likely that neither SNP is causative, as the peak of T1D association was from the SNP rs231727 at 3' flanking of the CTLA4 gene. Comprehensive resequencing and fine mapping of the CTLA4 region are still needed to clarify the causal variants.

Inherited polymorphisms in immuno-modulatory genes may contribute to variations in immune function and genetic susceptibility for complex diseases, including cancer. We report results from a comprehensive study to discover novel single nucleotide polymorphisms (SNPs) and to estimate allelic frequency for both novel and known coding and regulatory region SNPs in genes encoding proteins that have been implicated in the immune response to tumors. We identified 12 novel nucleotide substitution variants and one deletion variant in 17 genes analyzed (TGFBETA;R, BETA;2M, IFNGAMMA;, TNFALPHA;, TNFALPHA;R, LTALPHA;, IL-6, IL-12, IL-2, IL-1ALPHA;, IL-1BETA;, IL-1RN, IL-10, CTLA4, CD40L, FAS and FASL). We determined the frequency of these novel polymorphisms, as well as 17 previously identified polymorphisms, in a control sample of 158 individuals, approximately half of which were Caucasian (n = 74) and half of which were African American (n = 84). Significant differences in allele frequencies were observed between the two racial groups for 13/17 genes tested. These allelic variations maybe associated with alterations in immune function and thus susceptibility to a number of complex disease states such as cancer.

We recently have developed a method to generate primary P815-specific CTL in vitro from normal syngeneic splenocytes by employing transfection of B7-1 combined with exogenous IL-12 and IL-6. Surprisingly, when the homologous costimulator molecule B7-2 was substituted for B7-1 in this system, no specific CTL activity was obtained. Similarly, B7-1- but not B7-2-transfected P815 cells generated alloantigen-specific CTL activity from C57BL/6, accessory cell- and CD4(+)-depleted splenocytes, and costimulated proliferation of CD8+ lymphocytes in the presence of low doses of anti-CD3 mAb. In all systems, combined expression of both B7-1 and B7-2 costimulated as effectively as B7-1 alone, arguing against delivery of a dominant negative signal by B7-2. Proliferation of allogeneic, CD4(+)-depleted splenocytes in response to P815 cells, which relies on costimulation by normal accessory cells, was inhibited by anti-B7-1 but not anti-B7-2 mAbs. Finally, indirect evidence suggested a higher avidity of B7-1+ cells than B7-2+ cells for CTLA4. Thus, at least in the context of primary stimulation by irradiated P815 transfectants, B7-1 appears to be superior to B7-2 at costimulation of CD8+ T lymphocytes.

Recent success in pancreatic islet allotransplantation has raised expectations but has equally highlighted the acute shortage of donor tissue. The use of xenogeneic tissue would help to address this shortage; however, strong cellular immunity limits the application of this approach. T-cell responses to xenogeneic tissues involve recognition of intact species-mismatched major histocompatibility complex (MHC) molecules, the direct pathway, and xenogeneic proteins presented as peptides by responder-type MHC molecules, the indirect pathway. In this study, we exploited the species difference to selectively and sequentially inhibit direct and indirect xenoresponses after transplantation of porcine islets into mice. Selective inhibition of the direct response was achieved using porcine CTLA4-Ig, which binds preferentially to pig versus mouse B7 molecules. Selective inhibition of the indirect response was achieved using murine CTLA4-Ig, which binds preferentially to mouse B7 molecules. Administration of porcine CTLA4-Ig alone caused modest prolongation of islet survival. Injection of murine CTLA4-Ig alone had a minimal effect. However, the injection of the porcine fusion protein early and the murine homolog late after grafting led to permanent survival of the porcine islets, in the absence of any other immunosuppression. These results suggest that a similar approach could have clinical utility in porcine islet xenotransplantation.

Talk of numerous genetic risk factors for rheumatoid arthritis (RA) and psoriasis has been percolating for years, but with the exception of the human leukocyte antigen (HLA) region, none have been definitively identified. Recently the results of multiple, well powered, genetic case-control studies have begun to appear providing convincing statistical evidence for at least ten non-HLA related risk genes or loci (C5/TRAF1, CD40, CTLA4, KIF5A/PIP4K2C, MMEL1/TNFRSF14, PADI4, PRKCQ, PTPN22, STAT4, and TNFAIP3/OLIG3) for RA and six (IL12B, IL13, IL23R, STAT2/IL23A, TNFAIP3, and TNIP1) for psoriasis. These initial, novel findings are beginning to shed light on the molecular pathways pertinent to the individual diseases and highlight the pleiotropic effects of several risk factors as well as the allelic heterogeneity underlying susceptibility to these and other autoimmune diseases.

Blockade of the CD28/CTLA4/B7 costimulatory pathway using CTLA4-Ig has great therapeutic potential, and has been shown to prolong allograft survival in a variety of animal models. To gain further insight into the mechanism by which costimulatory blockade prevents allograft rejection, we studied cardiac allograft survival in the complete absence of B7 costimulation using mice lacking B7-1 and B7-2 (B7-1/B7-2-/- mice). To determine the role of B7 on donor vs recipient cells, we used B7-1/B7-2-/- mice as either donors or recipients of allografts. Wild-type (WT) recipients acutely reject fully allogeneic hearts from both WT and B7-1/B7-2-/- mice. In contrast, B7-1/B7-2-/- recipients allow long-term survival of grafts from both WT and B7-1/B7-2-/- mice, with minimal histologic evidence of either acute or chronic rejection in grafts harvested after 90 days. The B7-1/B7-2-/- mice acutely reject B7-1/B7-2-/- allografts if CD28 stimulation is restored by the administration of Ab to CD28 and can mount an alloresponse in mixed lymphocyte reactions. Therefore, B7-1/B7-2-/- mice are capable of generating alloresponses both in vivo and in vitro. Our results demonstrate that in the alloresponse to mouse heterotopic cardiac transplantation, B7 molecules on recipient cells rather than donor cells provide the critical costimulatory signals. The indefinite survival of allografts into B7-1/B7-2-/- recipients further shows that the absence of B7 costimulation alone is sufficient to prevent rejection.

CD4+CD25+ T cells regulate a variety of autoimmune and alloimmune responses including the development of autoimmune diabetes in non-obese diabetic (NOD) mice. We have examined the role of CD28/CTLA4/B7 interactions in the expansion and survival of CD4+CD25+ regulatory T cells (T(reg)) in this setting. CD28/ B7 interactions are essential in the development of T(reg) in the thymus and for their survival in the periphery. The CD28-mediated homeostasis of these cells is independent of Il2, OX40, CD40L, and survival factor Bcl-XI. In addition, analysis of T(reg) from CTLA4-deficient mice suggests that CTLA4 expression is not required for their development or function. However, non-activating anti-CTLA4 antibodies blocked the suppressor activity of regulatory cells in vitro. Thus, clinical application of co-stimulatory blockade using agents such as CTLA4Ig in the treatment of autoimmune disease may result in complicated outcomes.

Genetic variation at a linkage disequilibrium block encompassing the cytotoxic T-lymphocyte antigen-4 (CTLA4) gene influences susceptibility to autoimmunity, but identifying the polymorphism(s) responsible for this effect has been challenging. Recently, a single-nucleotide polymorphism (SNP) located 3' to the known polyadenylation site of CTLA4 (+6230G>A) and strongly associated with autoimmune disease was reported to regulate levels of soluble CTLA4 isoform (sCTLA4) but not the full-length isoform. The purpose of the present study is to define the mechanistic effect of the 3'SNP on the isoforms of CTLA4 (alternative splicing vs polyadenylation vs effects on RNA stability). However, using allele-specific single-nucleotide primer extension, we found no difference between mRNA transcripts derived from either +6230G>A allele in 11 heterozygous individuals, in either of the two known CTLA4 isoforms. We also found no effect of this polymorphism on ICOS (inducible costimulator), a putative downstream target. In addition, repeated attempts at 3' RACE (3'rapid amplification of cDNA ends) were unsuccessful in amplifying any contiguous sequence past the known CTLA4 polyadenylation site and no such sequence was found in the EST databases. We conclude that the mechanism of the observed association of the +6230 SNP with autoimmune disease remains to be determined, but does not involve modulation of steady-state mRNA of any known CTLA4 isoform.

BACKGROUND

Maternal atopic background and stimulation of the adaptive immune system with allergen interact in the development of allergic disease. Stimulation of the innate immune system through microbial exposure, such as activation of the innate Toll-like-receptor 2 (TLR2), may reduce the development of allergy in childhood. However, little is known about the immunological effects of microbial stimulation on early immune responses and in association with maternal atopy.

METHODS

We analyzed immune responses of cord blood mononuclear cells (CBMC) from 50 healthy neonates (31 non-atopic and 19 atopic mothers). Cells were stimulated with the TLR2 agonist peptidoglycan (Ppg) or the allergen house dust mite Dermatophagoides farinae (Derf1), and results compared to unstimulated cells. We analyzed lymphocyte proliferation and cytokine secretion of CBMC. In addition, we assessed gene expression associated with T regulatory cells including the transcription factor Foxp3, the glucocorticoid-induced TNF receptor (GITR), and the cytotoxic lymphocyte antigen 4 (CTLA4). Lymphocyte proliferation was measured by 3H-Thymidine uptake, cytokine concentrations determined by ELISA, mRNA expression of T cell markers by real-time RT-PCR.

RESULTS

Ppg stimulation induced primarily IL-10 cytokine production, in addition to IFN-gamma, IL-13 and TNF-alpha secretion. GITR was increased following Ppg stimulation (p = 0.07). Ppg-induced IL-10 production and induction of Foxp3 were higher in CBMC without, than with maternal atopy (p = 0.04, p = 0.049). IL-10 production was highly correlated with increased expression of Foxp3 (r = 0.53, p = 0.001), GITR (r = 0.47, p = 0.004) and CTLA4 (r = 0.49, p = 0.003), independent of maternal atopy.

CONCLUSIONS

TLR2 stimulation with Ppg induces IL-10 and genes associated with T regulatory cells, influenced by maternal atopy. Increased IL-10 and Foxp3 induction in CBMC of non-atopic compared to atopic mothers, may indicate an increased capacity to respond to microbial stimuli.

T cell activation is the result of antigen-specific interactions with the TCR/CD3 complex and costimulation via other T cell surface receptors. Prevention of costimulation can result in clonal anergy. CTLA4Ig is a fusion protein that binds with high-affinity to the B7/BB1 ligand and blocks the interaction of this ligand with CD28 and CTLA4. We explored the immunosuppressive effects of CTLA4Ig in a murine nonvascularized heterotopic cardiac transplant model and in a model of cell mediated immunity. CTLA4Ig administered in vivo for two days at the time of transplantation resulted in significant prolongation of allograft survival (55 +/- 2.0 vs. 12.2 +/- 0.5 days for control, P < 0.03). Administration at later times or to previously primed animals produced no prolongation of graft survival. CTLA4Ig administered during in vivo immunization to the hapten TNP suppressed the contact sensitivity response and inhibited the subsequent in vitro generation of secondary TNP-specific CTL. CTLA4Ig administered in vivo had no effect on subsequent primary alloantigen-specific CTL or MLR responses--however, when added to culture the fusion protein inhibited the MLR response by 80%, but not the alloantigen-specific CTL response. CTLA4Ig inhibited CD4+ and CD8+ proliferative and cytokine responses to alloantigen. Flow cytometry showed no changes in distribution of subpopulations of T cells. These results confirm the immunosuppressive activity of CTLA4Ig in vivo in an allograft model and show that both CD4+ and CD8+ T cells are suppressed by CTLA4Ig. The most efficacious time of administration is during priming of the immune response at the time of antigen presentation.

Tumor necrosis factor α (TNF-α) is a key regulator of inflammation and rheumatoid arthritis (RA). TNF-α blocker therapies can be very effective for a substantial number of patients, but fail to work in one third of patients who show no or minimal response. It is therefore necessary to discover new molecular intervention points involved in TNF-α blocker treatment of rheumatoid arthritis patients. We describe a data analysis strategy for predicting gene expression measures that are critical for rheumatoid arthritis using a combination of comprehensive genotyping, whole blood gene expression profiles and the component clinical measures of the arthritis Disease Activity Score 28 (DAS28) score. Two separate network ensembles, each comprised of 1024 networks, were built from molecular measures from subjects before and 14 weeks after treatment with TNF-α blocker. The network ensemble built from pre-treated data captures TNF-α dependent mechanistic information, while the ensemble built from data collected under TNF-α blocker treatment captures TNF-α independent mechanisms. In silico simulations of targeted, personalized perturbations of gene expression measures from both network ensembles identify transcripts in three broad categories. Firstly, 22 transcripts are identified to have new roles in modulating the DAS28 score; secondly, there are 6 transcripts that could be alternative targets to TNF-α blocker therapies, including CD86--a component of the signaling axis targeted by Abatacept (CTLA4-Ig), and finally, 59 transcripts that are predicted to modulate the count of tender or swollen joints but not sufficiently enough to have a significant impact on DAS28.

Myasthenia gravis (MG) in thymoma patients depends critically on intratumorous generation and export of mature autoreactive CD4+ T cells. Why non-MG thymomas fail to produce CD4+ T cells is unknown. We studied three single-nucleotide polymorphisms of the cytotoxic T-lymphocyte-associated antigen 4(CTLA4) gene in thymoma patients, nonthymoma early-onset MG patients, and control subjects. Surprisingly, the CTLA4high genotype +49A/A, which is protective against several autoimmune diseases, exerted a prominent predisposing effect to paraneoplastic MG in thymoma patients. The unusual disease association with a CTLA4high genotype implies a unique pathogenesis of paraneoplastic MG, with high CTLA4 levels possibly supporting the nontolerogenic selection of CD4+ T cells in MG-associated thymomas.

The association of autoimmunity with antitumor immunity challenges a paradigm of selective surveillance against tumors. Aided with well-characterized models of robust autoimmunity, we show that self-antigen-specific effector T (Teff) cell clones could eradicate tumor cells. However, a tumor microenvironment reinforced by Treg cells and myeloid-derived suppressor cells (MDSCs) presented a barrier to the autoimmune effectors, more so in tumors than in healthy tissues. This barrier required optimal CTLA4 expression in Teff cells. In a spontaneous model of breast cancer, subtle reductions in CTLA4 expression impeded tumor onset and progression, providing the first direct evidence that CTLA4 inhibits spontaneous tumor development. In an adoptive therapy model of lymphoma, self-antigen-specific Teff cells were potentiated by even a modest reduction of CTLA4. A subtle reduction of CTLA4 did not curtail Treg-cell suppression. Thus, Teff cells had an exquisite sensitivity to physiological levels of CTLA4 variations. However, both Treg and Teff cells were impacted by anti-CTLA4 antibody blockade. Therefore, whether CTLA4 impacts through Treg cells or Teff cells depends on its expression level. Overall, the results suggest that the tumor microenvironment represents an "immunoprivileged self" that could be overcome practically and at least partially by RNAi silencing of CTLA4 in Teff cells.

Genetic predisposition is recognized as an important factor for the development of autoimmune hepatitis (AIH). To assess the potential contribution of MHC and non-MHC genes, type 2 AIH was reproduced in three mice strains, taking advantage of their different genetic makeup with regard to MHC and non-MHC genes. Mice (C57BL/6, 129/Sv and BALB/c) were DNA vaccinated with a pCMV-CTLA4-CYP2D6-FTCD plasmid coding for the extracellular region of CTLA-4 and for the antigenic region of the CYP2D6 and FTCD, and with pCMV-IL12. ALT and total IgG levels, liver histology, FACS analysis and liver T-cell cytotoxicity assays were monitored up to 8 months post-injection. C57BL/6 mice showed elevated serum ALT levels, autoantibodies, antigen-specific cytotoxic T-cells and lobular and periportal inflammatory infiltrate. The 129/Sv mice showed slightly elevated ALT levels, sparse liver lobular infiltrate and cytotoxic T-cells. The BALB/c mice showed no liver inflammation. All mice had elevated total serum IgG levels. This murine model of type 2 AIH shows that MHC and non-MHC genes contribute to the susceptibility to autoimmune hepatitis. The understanding of the genetic determinants implicated in AIH development will be a major advance in the study of its pathogenesis and could lead to a better diagnostic approach and preventive strategies.

In this study, we show that costimulation required for mucosal IgA responses is strikingly different from that needed for systemic responses, including serum IgA. Following oral immunization with cholera toxin (CT) adjuvant we found that whereas CTLA4-H1 transgenic mice largely failed to respond, CD28-/- mice developed near normal gut mucosal IgA responses but poor serum Ab responses. The local IgA response was functional in that strong antitoxic protection developed in CT-immunized CD28-/- mice. This was in spite of the fact that no germinal centers (GC) were observed in the Peyer's patches, spleen, or other peripheral lymph nodes. Moreover, significant somatic hypermutation was found in isolated IgA plasma cells from gut lamina propria of CD28-/- mice. Thus, differentiation to functional gut mucosal IgA responses against T cell-dependent Ags does not require signaling through CD28 and can be independent of GC formations and isotype-switching in Peyer's patches. By contrast, serum IgA responses, similar to IgG-responses, are dependent on GC and CD28. However, both local and systemic responses are impaired in CTLA4-Hgamma1 transgenic mice, indicating that mucosal IgA responses are dependent on the B7-family ligands, but require signaling via CTLA4 or more likely a third related receptor. Therefore, T-B cell interactions leading to mucosal as opposed to serum IgA responses are uniquely regulated and appear to represent separate events. Although CT is known to strongly up-regulate B7-molecules, we have demonstrated that it acts as a potent mucosal adjuvant in the absence of CD28, suggesting that alternative costimulatory pathways are involved.

CD28 family of costimulatory receptors is comprised of molecules with a single V-type extracellular Ig domain, a transmembrane and an intracytoplasmic region with signaling motifs. CD28 and cytotoxic T lymphocyte antigen-4 (CTLA4) homologs have been recently identified in rainbow trout. Other sequences similar to mammalian CD28 family members have now been identified using teleost, Xenopus and chicken databases. CD28- and CTLA4 homologs were found in all vertebrate classes whereas inducible costimulatory signal (ICOS) was restricted to tetrapods, and programmed cell death-1 (PD1) was limited to mammals and chicken. Multiple B and T Lymphocyte Attenuator (BTLA) sequences were found in teleosts, but not in Xenopus or in avian genomes. The intron/exon structure of btlas was different from that of cd28 and other members of the family. The Ig domain encoded in all the btla genes has features of the C-type structure, which suggests that BTLA does not belong to the CD28 family. The genomic localization of these genes in vertebrate genomes supports the split between the BTLA and CD28 families.

Immune checkpoint molecules have been identified as crucial regulators of the immune response, which motivated the emergence of immune checkpoint-targeting therapeutic strategies. However, the prognostic significance of the immune checkpoint molecules PD-1, CTLA4, TIM-3 and LAG-3 remains controversial. The aim of our study was to conduct a systematic assessment of the expression of these immune checkpoint molecules across different cancers in relation to treatment response, tumor-infiltrating immune cells and survival. Oncomine and PrognoScan database analyses were used to investigate the expression levels and prognostic values of these immune checkpoint molecule genes across various cancers. Then, we used Kaplan-Meier plotter to validate the associations between the checkpoint molecules and cancer survival identified in the PrognoScan analysis. TIMER analysis was used to evaluate immune cell infiltration data from the TCGA. Finally, we used GEPIA to investigate the prognostic value of these four checkpoint molecules and assess the correlations between these four checkpoint molecules and genetic markers. These immune checkpoint molecules may potentially serve as prognostic factors and therapeutic targets in breast cancer, ovarian cancer and lung cancer. The prognostic roles of these checkpoint molecules varied greatly across cancers, which implied a noteworthy amount of heterogeneity among tumors, even within the same molecular subtype. In addition, the expression patterns of these checkpoint molecules were closely associated with treatment response and provided some useful direction when choosing chemotherapeutic drugs. These findings enhance our understanding of these checkpoints in cancer treatment and identify strategies to promote synergistic activities in the context of other immunotherapies. This article is protected by copyright. All rights reserved.

OBJECTIVE

Recent studies have shown that Graves' disease (GD) is linked to and associated with alleles of the cytotoxic T lymphocyte antigen-4 (CTLA4) locus. However, the true pathogenic polymorphism(s) at this locus remains uncertain. Moreover, the association studies of the promoter CTLA4(-318)C/T polymorphism in white GD populations have produced conflicting results. Therefore, we have analysed three CTLA4 single nucleotide polymorphisms, including promoter CTLA4(-318)C/T, exon 1 CTLA4(49)A/G and intron 1 CTLA4(1822)C/T in our GD cohort from the UK.

METHODS

We studied 301 white patients with GD and 349 healthy ethnically matched local controls. Amongst GD probands, 129 had significant thyroid-associated orbitopathy (TAO; NOSPECS class III or worse). The CTLA4(-318)C/T, CTLA4(49)A/G and CTLA4(1822)C/T polymorphisms were genotyped by using the restriction enzymes MseI, Bst71I and HaeIII, respectively.

RESULTS

We found no association between GD and alleles of CTLA4(-318)C/T. GD was found to be associated with the G allele of CTLA4(49)A/G[P = 5.9 x 10(-6), odds ratio (OR) 1.65] and the T allele of CTLA4(1822)C/T (P = 7.7 x 10(-6), OR 1.64). The frequencies of these alleles were significantly higher in GD probands with significant TAO than in those without TAO (G allele: P = 0.001, OR 1.68; T allele: P = 0.001, OR 1.70).

CONCLUSIONS

The promoter CTLA4(-318)C/T polymorphism is not in linkage disequilibrium with the pathogenic polymorphism(s) at the CTLA4 locus. The alleles of both the exon 1 CTLA4(49)A/G and the intron 1 CTLA4(1822)C/T polymorphisms are associated with GD, which is stronger in patients with TAO.