CTLA-4 gene polymorphism at position 49 in exon 1 reduces the inhibitory function of CTLA-4 and contributes to the pathogenesis of Graves' disease

Activation of T cells requires at least two signals transduced by the Ag-specific TCR and a costimulatory ligand such as CD28. CTLA-4, expressed on activated T cells, binds to B7 present on APCs and functions as a negative regulator of T cell activation. Our laboratory previously reported the association of Graves' disease (GD) with a specific CTLA-4 gene polymorphism. In theory, reduced expression or function of CTLA-4 might augment autoimmunity. In the present study, we categorized autoimmune thyroid disease patients and normal controls (NC) by genotyping a CTLA-4 exon 1 polymorphism and investigated the function of CTLA-4 in all subjects. PBMCs and DNA were prepared from GD (n = 45), Hashimoto's thyroiditis (HT) (n = 18), and NC (n = 43). There were more GD patients with the G/G or A/G alleles (82.2% vs 65.1% in NC), and significantly fewer patients with the A/A allele (17.8% vs 34.9% in NC). In the presence of soluble blocking anti-human CTLA-4 mAb, T cell proliferation following incubation with allogeneic EBV-transformed B cells was augmented in a dose-dependent manner. Augmentation induced by CTLA-4 mAb was similar in GD and NC (GD, HT, NC = 156%, 164%, 175%, respectively). We related CTLA-4 polymorphism to mAb augmentation of T cell proliferation in each subgroup (GD, HT, NC). Although PBMC from individuals with the G/G alleles showed 132% augmentation, those with the A/A alleles showed 193% augmentation (p = 0.019). CTLA-4 polymorphism affects the inhibitory function of CTLA-4. The G allele is associated with reduced control of T cell proliferation and thus contributes to the pathogenesis of GD and presumably of other autoimmune diseases.     

Targeted CTLA-4 engagement induces CD4+CD25+CTLA-4high T regulatory cells with target (allo)antigen specificity

CTLA-4 (CD152) is actively involved in down-regulating T cell activation and maintaining lymphocyte homeostasis. Our earlier studies showed that targeted engagement of CTLA-4 can down-modulate T cell response and suppress allo- and autoimmune responses. In this study, we report that targeted CTLA-4 engagement can induce immune tolerance to a specific target through selective induction of an Ag-specific CD4(+)CD25(+)CTLA-4(high) regulatory T cell (Treg cell) population. Allogeneic cells coated with anti-CTLA-4 Ab induced immune hyporesponsiveness through suppression of proinflammatory cytokines IFN-gamma and IL-2, and up-regulation of the regulatory cytokines IL-10, TGF-beta1, and IL-4, presumably through the engagement of CTLA-4 on activated T cells. Although rechallenge with alloantigen failed to break the unresponsiveness, a transient recovery from tolerance was observed in the presence of high concentrations of exogenous IL-2, saturating concentrations of neutralizing anti-TGF-beta1 and anti-IL-10 Abs, and blocking anti-CTLA-4 Ab, and upon depletion of CD4(+)CD25(+) Treg cells. The CD4(+)CD25(+)CTLA-4(high) Treg cells from tolerant mice suppressed the effector function of CD25(-) T cells from Ag-primed mice. Adoptive transfer of these Treg cells into Ag-primed mice resulted in a significantly reduced alloantigen-specific response. Further characterization demonstrated that the Treg cells with memory phenotype (CD62L(-)) were more potent in suppressing the alloantigen-specific T cell response. These results strongly support that the targeted engagement of CTLA-4 has therapeutic potential for the prevention of transplant rejection.     

Blockade of T cell activation using a surface-linked single-chain antibody to CTLA-4 (CD152)

CTLA-4 (CD152) engagement can down-regulate T cell activation and promote the induction of immune tolerance. However, the strategy of attenuating T cell activation by engaging CTLA-4 has been limited by sharing of its natural ligands with the costimulatory protein CD28. In the present study, a CTLA-4-specific single-chain Ab (scFv) was developed and expressed on the cell surface to promote selective engagement of this regulatory molecule. Transfectants expressing anti-CTLA-4 scFv at their surface bound soluble CTLA-4 but not soluble CD28. Coexpression of anti-CTLA-4 scFv with anti-CD3epsilon and anti-CD28 scFvs on artificial APCs reduced the proliferation and IL-2 production by resting and preactivated bulk T cells as well as CD4+ and CD8+ T cell subsets. Importantly, expression of anti-CTLA-4 scFv on the same cell surface as the TCR ligand was essential for the inhibitory effects of CTLA-4-specific ligation. CTLA-4-mediated inhibition of tyrosine phosphorylation of components of the proximal TCR signaling apparatus was similarly dependent on coexpression of TCR and CTLA-4 ligands on the same surface. These findings support a predominant role for CTLA-4 function in the modification of the proximal TCR signal. Using T cells from DO11.10 and 2C TCR transgenic mice, negative regulatory effects of selective CTLA-4 ligation were also demonstrated during the stimulation of Ag-specific CD4+ and CD8+ T cells by MHC/peptide complexes. Together these studies demonstrate that selective ligation of CTLA-4 using a membrane-bound scFv results in attenuated T cell responses only when coengaged with the TCR during T cell/APC interaction and define an approach to harnessing the immunomodulatory potential of CTLA-4-specific ligation.     

CTLA-4+49A>G基因多态性与结直肠癌的相关性研究

目的:探讨细胞毒性T淋巴细胞相关抗原-4(CTLA-4)+49 AG位点多态性与结直肠癌发生的相关性,为早期预测结直肠癌的发生提供临床参考依据。方法:选取结直肠癌病例231例未实验组和正常健康体检者325例为对照组,取其空腹外周静脉血提取DNA后,采用聚合酶链式反应(PCR)技术对CTLA-4基因第1外显子区+49位点DNA进行扩增,产物用限制性片段长度多态性(RFLP)方法检测CTLA-4第1外显子区+49AG位点的多态性,比较两组杂合子AG和纯合子AA、GG基因型发生的频率、A等位基因与G等位基因的分布,分析CTLA-4+49 AG位点多态性与结直肠癌发生的相关性。结果:两组间CTLA-4基因外显子1区+49AG位点杂合子AG和纯合子AA、GG基因型发生的频率以及A等位基因与G等位基因的分布比较差异均无统计学意义(P0.05)。结论:CTLA-4基因外显子1区+49 AG基因多态性与我省汉族人群结直肠癌的发病无显著相关性。     

ICOS costimulation requires IL-2 and can be prevented by CTLA-4 engagement

We investigated the relationship between ICOS, CD28, CTLA-4, and IL-2 to gain a better understanding of this family of costimulatory receptors in the immune response. Using magnetic beads coated with anti-CD3 and varying amounts of anti-ICOS and anti-CTLA-4 Abs, we show that CTLA-4 ligation blocks ICOS costimulation. In addition to inhibiting cellular proliferation, CTLA-4 engagement prevented ICOS-costimulated T cells from producing IL-4, IL-10, and IL-13. Both an indirect and direct mechanism of CTLA-4's actions were examined. First, CTLA-4 engagement on resting cells was found to indirectly block ICOS costimulation by interferring with the signals needed to induce ICOS cell surface expression. Second, on preactivated cells that had high levels of ICOS expression, CTLA-4 ligation blocked the ICOS-mediated induction of IL-4, IL-10, and IL-13, suggesting an interference with downstream signaling pathways. The addition of IL-2 not only overcame both mechanisms, but also greatly augmented the level of cellular activation suggesting synergy between ICOS and IL-2 signaling. This cooperation between ICOS and IL-2 signaling was explored further by showing that the minimum level of IL-2 produced by ICOS costimulation was required for T cell proliferation. Finally, exogenous IL-2 was required for sustained growth of ICOS-costimulated T cells. These results indicate that stringent control of ICOS costimulation is maintained initially by CTLA-4 engagement and later by a requirement for exogenous IL-2.     

Polarization of naive CD4+ T cells toward the Th1 subset by CTLA-4 costimulation

In this study, we examined in vitro the role of CTLA-4 costimulation in the polarization of naive CD4+ T cells toward the Th1 subset. When CTLA-4 costimulation was blocked by the inclusion of anti-CTLA-4 Fab in cultures during priming of naive CD4+ T cells with anti-CD3 in the presence of splenic adherent cells, they were polarized toward the Th2 subset. Conversely, the engagement of CTLA-4 with immobilized anti-CTLA-4 or with CD80-P815 cells polarized naive CD4+ T cells costimulated with anti-CD3 and anti-CD28 toward the Th1 subset. The CTLA-4 costimulation during priming augmented TGF-beta1 mRNA accumulation in naive CD4+ T cells, and the inclusion of anti-TGF-beta in cultures for priming suppressed the effect of CTLA-4 costimulation on the Th1 polarization. The addition of low doses of TGF-beta1 in cultures for priming of naive CD4+ T cells enhanced the production of Th1 cytokines upon secondary stimulation, although Th2 cytokine production was not affected by the doses of TGF-beta1. The CTLA-4 costimulation was also shown to suppress IL-4 production of naive CD4+ T cells upon priming. These results indicate that the costimulation against CTLA-4 drives polarization of naive CD4+ T cells toward the Th1 subset independent of IL-12 through, at least in part, the enhancement of TGF-beta1 production, and it also hampers Th2 subset differentiation by affecting IL-4 production of naive CD4+ T cells.     

Importance of IL-10 for CTLA-4-mediated inhibition of tumor-eradicating immunity

In this study, we show that engagement of CTLA-4 on tumor-infiltrating lymphocytes from low-dose melphalan (L-phenylalanine mustard (L-PAM))-treated MOPC-315 tumor bearers led to IL-10 secretion. In addition, the inhibitory activity of CTLA-4 ligation for IFN-gamma secretion following stimulation with anti-CD3 plus anti-CD28 mAb depended on IL-10 production. Consistent with the importance of IL-10 for CTLA-4-mediated inhibition, administration of neutralizing anti-IL-10 mAb to low-dose L-PAM-treated MOPC-315 tumor bearers (administration of blocking anti-CTLA-4 mAb) resulted in enhanced tumor-infiltrating lymphocyte-mediated anti-MOPC-315 cytotoxicity and led to complete tumor eradication in a higher percentage of mice than that observed with low-dose L-PAM alone. The percentage of MOPC-315 tumor-bearing mice cured following administration of neutralizing anti-IL-10 mAb to low-dose L-PAM-treated MOPC-315 tumor bearers was comparable to that observed following administration of blocking anti-CTLA-4 mAb. Moreover, IL-10 neutralization together with CTLA-4 blockade did not provide added therapeutic benefits to low-dose L-PAM-treated MOPC-315 tumor bearers. Taken together, these results indicate that CTLA-4 blockade improves the therapeutic outcome of low-dose L-PAM for MOPC-315 tumor bearers by inhibiting IL-10 secretion as a consequence of blocking CTLA-4 ligation.     

Cutting edge: targeted ligation of CTLA-4 in vivo by membrane-bound anti-CTLA-4 antibody prevents rejection of allogeneic cells

Natural engagement of CTLA-4 on host B7 limits T cell activation. We hypothesized that therapeutic cross-linking of CTLA-4 in vivo may further inhibit T cell function and prevent allograft rejection. However, none of the currently available CTLA-4-binding reagents have ligating properties when injected in vivo. The observation that surface-immobilized anti-CTLA-4 mAb inhibits T cell activation in vitro prompted us to develop a membrane-bound single-chain anti-CTLA-4 Ab (7M). To model whether tissue expression of 7M could suppress allograft rejection, we examined the ability of H-2L(d)-specific TCR-transgenic T cells to reject 7M-expressing allogeneic tumor cells injected s.c. Expression of 7M significantly inhibited allogeneic rejection in mice that received CTLA-4(+/+) but not CTLA-4(-/-) T cells. Furthermore, CTLA-4(+/+) T cells that had encountered 7M-expressing tumors in vivo acquired defects in cytokine production and cytotoxicity. Thus, deliberate ligation of CTLA-4 in vivo potently inhibits allogeneic T cell responses.     

Blockade of CTLA-4 decreases the generation of multifunctional memory CD4+ T cells in vivo

CTLA-4 is known as a central inhibitor of T cell responses. It terminates T cell activation and proliferation and induces resistance against activation induced cell death. However, its impact on memory formation of adaptive immune responses is still unknown. In this study, we demonstrate that although anti-CTLA-4 mAb treatment during primary immunization of mice initially enhances the number of IFN-γ-producing CD4(+) T cells, it does not affect the size of the memory pool. Interestingly, we find that the CTLA-4 blockade modulates the quality of the memory pool: it decreases the amount of specialized "multifunctional" memory CD4(+) T cells coproducing IFN-γ, TNF-α, and IL-2 in response to Ag. The reduction of these cells causes an immense decrease of IFN-γ-producing T cells after in vivo antigenic rechallenge. Chimeric mice expressing CTLA-4-competent and -deficient cells unmask, which these CTLA-4-driven mechanisms are mediated CD4(+) T cell nonautonomously. In addition, the depletion of CD25(+) T cells prior to the generation of Ag-specific memory cells reveals that the constitutively CTLA-4-expressing natural regulatory T cells determine the quality of memory CD4(+) T cells. Taken together, these results indicate that although the inhibitory molecule CTLA-4 damps the primary immune response, its engagement positively regulates the formation of a high-quality memory pool equipped with multifunctional CD4(+) T cells capable of mounting a robust response to Ag rechallenge.     

CTLA-4 blockade by a human MAb enhances the capacity of AML-derived DC to induce T-cell responses against AML cells in an autologous culture system

BACKGROUND: Cells from AML patients can differentiate into the phenotype of DC when cultured with GM-CSF and IL-4. Such cytokine-treated AML-derived DC (AML-DC) can stimulate autologous T cells. In this study we examined whether an anti-CTLA-4 MAb (MDX-010) could enhance the generation of autologous anti-AML T cells. METHODS: MAb MDX-010 was added to AML PBMC cultures in the presence of GM-CSF and IL-4, a previously reported AML-DC culture method of generating anti-AML T cells. T-cell activation and proliferation were examined thereafter. RESULTS: Addition of MDX-010 to GM-CSF/IL-4-conditioned AML-DC cultures induced a mean seven-fold increase in the numbers of autologous T cells compared with cultures without MDX-010 (P < 0.007). T cells stimulated by AML-DC with CTLA-4 blockade were significantly more cytotoxic towards autologous AML cells than those without MDX-010 (42 +/- 23% vs. 26 +/- 15%, E:T ratio of 20). T cells stimulated by AML-DC with CTLA-4 blockade had significantly greater proportions of T cells producing IFN-gamma in response to autologous AML cells than those cultured with AML-DC alone (10.7 +/- 4.7% vs. 4.5 +/- 2.4% for CD4+ IFN-gamma+ CD69+ and 9.8 +/- 4.1% vs. 4 +/- 2.1% for CD8+ IFN-gamma+ CD69+ with or without MDX-010; n = 7; P < 0.007, P < 0.003, respectively). DISCUSSION: CTLA-4 blockade enhances the activity and numbers of AML-reactive T cells that can be stimulated by autologous AML-DC and may enhance the efficacy of adoptive immunotherapy of AML.     

Association between the CTLA-4 +49 A/G polymorphism and susceptibility to rheumatoid arthritis: a meta-analysis

The aim of this study was to explore whether the cytotoxic T lymphocyte antigen-4 (CTLA-4) +49 A/G polymorphism confers susceptibility to rheumatoid arthritis (RA). A meta-analysis was conducted on the associations between CTLA-4 +49 A/G polymorphism and RA using; 1) allele contrast, 2) the recessive model, 3) the dominant model, and 4) an additive model. A total of 19 studies, 5,752 RA patients and 5,508 controls, encompassing 9 Caucasian, 8 Asian, 1 Mexican, and 1 Tunisian population were included in this meta-analysis. Ethnicity-specific meta-analysis was performed on Caucasian and Asian populations. Meta-analysis of the CTLA-4 +49 A/G polymorphism revealed an association between RA and the CTLA-4 +49 G allele in all 11,260 study subjects (odds ratio (OR) 1.118, 95% confidence interval (CI) 1.033–1.210, P = 0.005). Stratification by ethnicity showed an association between the CTLA-4 +49 G allele and RA in Asians (OR 1.164, 95% CI 1.056–1.283, P = 0.002), but no evidence of an association in Caucasians (OR 1.085, 95% CI 0.973–1.209, P = 0.431). Furthermore, associations were found between RA and the CTLA-4 +49 A/G polymorphism in Asians using the dominant and additive models, but not using the recessive model. On the other hand, no association was found between RA and the CTLA-4 +49 A/G polymorphism using the recessive, dominant, or additive models in Caucasians. This meta-analysis demonstrates that the CTLA-4 +49 A/G polymorphism confers susceptibility to RA in Asians, but not in Caucasians.     

Association of HLA-DR, -DQ genotype and CTLA-4 gene polymorphism with Graves' disease in Japanese children

OBJECTIVE: Childhood onset Graves' disease (GD) has been documented to be clinically distinct from adult onset GD, and an association with the genes encoding HLA and CTLA-4 (cytotoxic T lymphocyte antigen-4) has been reported in both Caucasian and Japanese adult GD patients. The aim of this study was to determine whether HLA-DR, -DQ and CTLA-4 are associated with childhood onset GD in Japanese individuals. METHODS: We investigated the genotype of HLA class II (DRB1, DQB1) and the A/G transition polymorphism of CTLA-4 exon 1 position 49 in 43 GD patients and in healthy controls for comparison. The CTLA-4 alleles were identified by the polymerase chain reaction (PCR) of genomic DNA and restriction fragment-length polymorphism analysis (PCR-RFLP) with Ita1. RESULTS: The frequency of both HLA-DRB1*0405 and DQB1*0401 was increased in the patient group (DRB1*0405: 26.7%, p < 0.001; DQB1*0401: 25.6%, p < 0.005) compared with the controls. Patients with GD had a significantly lower frequency of the AA genotype of CTLA-4 than the controls, but there was no difference in allele frequency between the G and A allele. CONCLUSIONS: the association of HLA-DRB1 and DQB1 genotype with susceptibility to childhood onset GD differs from that in adult onset GD, whereas the association between CTLA-4 gene polymorphism and childhood onset GD is similar to that in adult onset GD in Japanese individuals, but the association is weak.     

Role of CTLA-4 in the activation of single- and double-positive thymocytes

CTLA-4, a homologue of CD28, is a negative regulator of T cell activation in the periphery and is transiently expressed on the cell surface after T cell activation. However, the role of CTLA-4 in T cell activation in the thymus is not clear. This investigation was initiated to determine the role of CTLA-4 in the activation of CD4(+)CD8(+) double-positive (DP) and CD4(+)CD8(-) and CD4(-)CD8(+) single-positive (SP) thymocytes using fetal thymic organ cultures (FTOC) of MHC class II-restricted, OVA(323-339)-restricted TCR transgenic mice (DO11.10). We found that treatment of the FTOC with anti-CTLA-4-blocking Ab during activation with OVA(323-339) increased the proportion and number of DP thymocytes, but decreased the proportion and number of SP thymocytes compared with OVA(323-339)-stimulated FTOC without anti-CTLA-4 Ab treatment. In addition, anti-CTLA-4 Ab treatment inhibited OVA(323-339)-induced expression of the early activation marker, CD69, in DP thymocytes, but increased CD69 in SP thymocytes. Similarly, CTLA-4 blockage decreased phosphorylation of ERK in DP thymocytes by Ag-specific TCR engagement, but increased phosphorylation of ERK in SP thymocytes. CTLA-4 blockage inhibited deletion of DP thymocytes treated with a high dose of OVA(323-339), whereas CTLA-4 blockage did not inhibit deletion of DP thymocytes treated with a low dose of OVA(323-339). We conclude that CTLA-4 positively regulates the activation of DP thymocytes, resulting in their deletion, whereas blocking CTLA-4 suppresses the activation of DP thymocytes, leading to inhibition of DP thymocyte deletion. In contrast, CTLA-4 negatively regulates the activation of SP thymocytes.     

Association between CTLA-4 exon-1 +49A/G polymorphism and systemic lupus erythematosus: an updated analysis

The results of studies on association between CTLA-4 exon-1 +49A/G (rs231775) polymorphism and susceptibility to systemic lupus erythematosus are controversial. To derive a more precise estimation of the relationship between the CTLA-4 exon-1 +49A/G polymorphism and SLE, a meta-analysis of 18 published case-control studies was performed. 18 studies meeting our inclusion criteria comprising 1806 SLE cases and 2,490 controls were included. The effect summary odds ratio (OR) and 95 % confidence intervals were obtained. Publication bias was tested by funnel plot, Egger's test and heterogeneity was assessed. The combined results showed that there were significant differences in genotype distribution between SLE cases and control on the basis of all studies, GG versus AA (OR = 1.53, 95 % CI: 1.12-2.10), GG versus GA/AA (OR = 1.30, 95 % CI: 1.04-1.64), GG versus GA (OR = 1.27, 95 % CI: 1.03-1.55). When stratifying for the race, the phenomenon was found that SLE cases had a significantly higher frequency of GG/GA versus AA (OR = 1.58, 95 % CI: 1.23-2.03), GG versus AA (OR = 1.89, 95 % CI: 1.23-2.91), GG versus GA/AA(OR = 1.39, 95 % CI: 1.03-1.89), GA versus AA(OR = 1.38, 95 % CI: 1.06-1.80) and G versus A(OR = 1.34, 95 % CI: 1.07-1.67) than control in Asians. Our meta-analysis results suggest that CTLA-4 exon-1 +49A/G polymorphism might be a risk factor for SLE susceptibility, at least in Asians. The large sample and well-designed study based on different ethnic groups should be considered in future associated studies to clarify the association of CTLA-4 exon-1 +49A/G polymorphism with SLE susceptibility.     

Evidence for functional relevance of CTLA-4 in ultraviolet-radiation-induced tolerance

Hapten sensitization through UV-exposed skin induces hapten-specific tolerance that can be adoptively transferred by injecting T lymphocytes into naive recipients. The exact phenotype of T cells responsible for inhibiting the immune response and their mode of action remain unclear. Evidence exists that CTLA-4 negatively regulates T cell activation. We addressed whether CTLA-4 is involved in the transfer of UV-induced tolerance. Injection of lymph node cells from mice that were sensitized with dinitrofluorobenzene (DNFB) through UV-irradiated skin inhibited induction of contact hypersensitivity against DNFB in the recipient animals. When CTLA-4+ cells were depleted, transfer of suppression was lost. Likewise, significantly fewer lymphocytes enriched for CTLA-4+ cells were necessary to transfer suppression than unfractionated cells. Expression of CTLA-4 appears to be functionally relevant, since in vivo injection of a blocking anti-CTLA-4 Ab was able to break UV-induced tolerance and inhibited transfer of suppression. Upon stimulation with dendritic cells in the presence of the water-soluble DNFB analogue, DNBS, CTLA-4+ T cells from DNFB-tolerized mice secreted high levels of IL-10, TGF-beta, and IFN-gamma; low levels of IL-2; and no IL-4, resembling the cytokine pattern of T regulatory 1 cells. Ab blocking of CTLA-4 resulted in inhibition of IL-10 release. Accordingly, transfer of tolerance was not observed when recipients were treated with an anti-IL-10 Ab. Hence we propose that T cells, possibly of the T regulatory 1 type, transfer UV-mediated suppression through the release of IL-10. Activation of CTLA-4 appears to be important in this process.     

The study of CTLA-4 and vitamin D receptor polymorphisms in the Romanian type 1 diabetes population

Several studies suggested that part of the genetic susceptibility for Type 1 diabetes (TIDM) is encoded by some polymorphisms of CTLA-4 gene (2q33) and of Vitamin D Receptor gene (VDR; 12q12-14). Our aim was to assess their contribution to TIDM genetic susceptibility in the Romanian population. We typed CTLA-4 49 A/G and VDR Fok I (F/f), Apa I (A/a) and Taq I (T/t) polymorphisms by Sequence Specific Primer PCR (SSP-PCR) in 204 Romanian diabetic families (756 individuals: 212 TIDM probands and 544 unaffected parents and siblings). We studied alleles transmission using the Transmission Disequilibrium Test (TDT). We found an increased transmission of CTLA-4 49G allele to diabetics (54.8%, p=0.11). The transmission of F (56.1%, p=0.063), a (55.7%, p=0.061) and T (51.8%, p=0.37) alleles of VDR gene to diabetics was increased but did not reach statistical significance. In conclusion we found the same increased transmission of CTLA-4 49 G allele to diabetics as previously reported. VDR Foq I F allele seems to be predisposing while Taq I T allele seems to be protective.     

Regulation of the small GTPase Rap1 and extracellular signal-regulated kinases by the costimulatory molecule CTLA-4

The mitogen-activated protein kinase extracellular signal-regulated kinase (ERK) is activated following engagement of the T-cell receptor and is required for interleukin 2 (IL-2) production and T-cell proliferation. This activation is enhanced by stimulation of the coreceptor CD28 and inhibited by the coreceptor CTLA-4. We show that the small G protein Rap1 is regulated in the opposite manner; it is inhibited by CD28 and activated by CTLA-4. Together, CD3 and CTLA-4 activate Rap1 in a sustained manner. To delineate T-cell function in the absence of Rap1 activity, we generated transgenic mice expressing Rap1GAP1, a Rap1-specific GTPase-activating protein. Transgenic mice showed lymphadenopathy, and transgenic T cells displayed increased ERK activation, proliferation, and IL-2 production. More significantly, the inhibitory effect of CTLA-4 on T-cell function in Rap1GAP1-transgenic T cells was reduced. We demonstrate that CTLA-4 activates Rap1, and we propose that intracellular signals from CTLA-4 antagonize CD28, at least in part, at the level of Rap1.     

Blockade of CTLA-4 signals inhibits Th2-mediated murine chronic graft-versus-host disease by an enhanced expansion of regulatory CD8+ T cells

CTLA-4 (CD152) is thought to be a negative regulator of T cell activation. Little is known about the function of CTLA-4 in Th2-type immune responses. We have investigated the effect of initial treatment with anti-CTLA-4 mAb on murine chronic graft-vs-host disease. Transfer of parental BALB/c splenocytes into C57BL/6 x BALB/c F1 mice induced serum IgE production, IL-4 expression by donor CD4+ T cells, and host allo-Ag-specific IgG1 production at 6-9 wk after transfer. Treatment with anti-CTLA-4 mAb for the initial 2 wk significantly reduced IgE and IgG1 production and IL-4 expression. Analysis of the splenic phenotype revealed the enhancement of donor T cell expansion, especially within the CD8 subset, and the elimination of host cells early after anti-CTLA-4 mAb treatment. This treatment did not affect early IFN-gamma expression by CD4+ and CD8+ T cells and anti-host cytolytic activity. Thus, blockade of CTLA-4 greatly enhanced CD8+ T cell expansion, and this may result in the regulation of consequent Th2-mediated humoral immune responses. These findings suggest a new approach for regulating IgE-mediated allergic immune responses by blockade of CTLA-4 during a critical period of Ag sensitization.     

Murine malaria is exacerbated by CTLA-4 blockade

Cytolytic T lymphocyte-associated Ag-4 (CD152) is a negatively regulating molecule, which is primarily expressed on T cells following their activation. In this study, we have examined the role of CTLA-4 expression in experimental blood-stage malaria. Similar to human malaria, CTLA-4 is expressed on CD4(+) T cells of C57BL/6 mice after infection with Plasmodium berghei. A kinetic analysis revealed that CTLA-4 expression was increased on day 5 postinfection and reached a peak on day 9 postinfection, when almost 10% of splenic CD4(+) T cells expressed CTLA-4. Blockade of CTLA-4 in vivo by a specific mAb and subsequent challenge with P. berghei caused neurological signs reminiscent of murine cerebral malaria and earlier death. Histologic examination of brain sections from anti-CTLA-4-treated mice revealed pathologic changes such as hemorrhages and edema, which were absent in control mice. Furthermore, treatment with anti-CTLA-4 also reversed the extensive loss of CD4(+) T cells and the suppressed T cell response occurring during blood-stage malaria. Our data suggest that CTLA-4 expression prevents immune pathology by restricting T cell activation during malaria. They also indicate that the development of cerebral malaria is mediated by a failure to down-regulate T cell activation.