Co-stimulatory signal B7(CD80/CD86):CD28 is needed in order to activate T cells in immune response. Cytotoxic T lymphocyte-associated antigen-4-immunoglobulin (CTLA4-Ig) binding to the B7 molecules on antigen-presenting cells downregulates this activation and represents a recent biological treatment in rheumatoid arthritis (RA). Objectives of the study were to investigate the presence of the B7.2 (CD86) molecule and its masking by CTLA4-Ig on cultures of both RA synovial macrophages (RA SM), and of macrophages differentiated from THP-1 cells (M). In addition, the anti-inflammatory effects of CTLA4-Ig on co-cultures of RA SM and M with activated T cells were tested.
All macrophages were co-cultured for 24 hours with activated T cells, without or with CTLA4-Ig (10, 100, 500 μg/ml for 1 hour, 3 hours and overnight, respectively). Immunofluorescence (IF) staining for B7.2, and an analysis of inflammatory cytokine expression (interleukin (IL) -6, tumor necrosis factor (TNF) α, IL-1β, transforming growth factor (TGF) β) by immunocytochemistry (ICC), western blot (WB) and reverse transcriptase-polymerase chain reaction (RT-PCR) were performed.
Macrophages showed intense B7.2 expression. CTLA4-Ig/B7.2 masking was evident for all macrophages, even after only 1 hour of cell culture (range from 10 to 100 μg/ml). ICC of co-cultures showed a dose-dependent decrease in inflammatory cytokines (P < 0.001 for IL-6, TNFα, IL-1β and TGFβ). Data were confirmed by WB and RT-PCR analysis.
Optimal concentrations of CTLA4-Ig for the CTLA4-Ig/B7.2 masking on activated macrophages were identified and were found to induce significant downregulation in the cell production of IL-6, TNFα, IL1-β and TGFβ. In conclusion, macrophages would appear to be a sensitive target for CTLA4-Ig treatment in RA.
Juvenile idiopathic arthritis (JIA) is an autoimmune disorder mediated by Th1-immune responses. Cytotoxic T-lymphocyte Antigen 4 (CTLA4), expressed on the T-cell surface, plays a negative role in regulating T-cell activation. Single nucleotide polymorphisms (SNPs) in CTLA4 have been implicated in susceptibility to several autoimmune disorders, including JIA. Our objective was to test three functional CTLA4 variants for association with JIA.
Families of 531 children with JIA were genotyped for SNPs located in the promoter region (C-318T), exon-1 (A49G), and the 3’ untranslated region (CT60) of CTLA4 by PCR amplification and digestion. Family-based association test (FBAT) was used to test CTLA4 SNPs and haplotypes for association with JIA. A second independent cohort of more than 300 children with JIA and 500 controls were genotyped for case-control analyses. Case-control analyses of the combined cohorts, as well as meta-analyses of published association studies between CTLA4 and JIA, were performed.
There were no deviations of transmission of any of the CTLA4 variants to children with JIA, or JIA subtypes, by FBAT. There were also no significant associations between CTLA4 C-318T or A49G SNPs in 650 JIA cases and 350 controls. Similarly, there were also no significant associations between CT60 variants with over 800 JIA cases and 500 controls. The meta-analysis also failed to confirm an association between JIA and CTLA4 variants.
These results suggest that C-318T, A49G or CT60 or haplotypes tagged by these CTLA4 SNPs are not associated with JIA or major JIA subtypes.
CTLA4; JRA; genetics; autoimmune; association; juvenile idiopathic arthritis
Cytotoxic T lymphocyte-associated antigen (CTLA-4), also known as CD152, is a co-inhibitory molecule that functions to regulate T cell activation. Antibodies that block the interaction of CTLA-4 with its ligands B7.1 and B7.2 can enhance immune responses, including anti-tumor immunity. Two CTLA-4 blocking antibodies are presently under clinical investigation: ipilimumab and tremelimumab. CTLA-4 blockade has shown promise in treatment of patients with metastatic melanoma, with a recently completed randomized, double-blind Phase III trial demonstrating a benefit in overall survival (OS) in the treated population. However, this approach appears to benefit only a subset of patients. Understanding the mechanism(s) of action of CTLA-4 blockade and identifying prognostic immunologic correlates of clinical endpoints to monitor are presently areas of intense investigation. Several immunologic endpoints have been proposed to correlate with clinical activity. This review will focus on the endpoints of immune monitoring described in studies to date and discuss future areas of additional work needed.
Various phenotype and functional T-cell abnormalities are observed in multiple myeloma (MM) patients. The aim of this study was to investigate the association between polymorphisms in the gene encoding cytotoxic T-lymphocyte antigen-4 (CTLA-4), a negative regulator of the T-lymphocyte immune response and susceptibility to multiple myeloma in a Polish population. Two hundred MM patients and 380 healthy subjects were genotyped for the following polymorphisms: CTLA-4c.49A>G, CTLA-4g.319C>T, CTLA-4g.*642AT(8_33), CT60 (CTLA-4g.*6230G>A), Jo31 (CTLA-4g.*10223G>T). Our study is the largest and most comprehensive evaluation to date of the association between genetic polymorphisms in the CTLA-4 molecule and multiple myeloma. It was found that CTLA-4c.49A>G[G], CT60[G], and Jo31[G] alleles were more frequently observed in MM patients than in controls (0.50 vs. 0.44, p = 0.03, 0.65 vs. 0.58, p = 0.04, and 0.63 vs. 0.57, p = 0.03, respectively). Moreover, the haplotype CTLA-4c.49A>G[G], CTLA-4g.319C>T[C], CTLA-4g.*642AT(8_33) , CT60[G], Jo31[G] including all susceptibility alleles increases the risk of MM about fourfold (OR: 3.79, 95%CI: 2.08–6.89, p = 0.00001). These findings indicate that genetic variations in the CTLA-4 gene play role in susceptibility to multiple myeloma and warrant further investigation through replication studies.
CTLA-4; Gene polymorphisms; MM
The CTLA-4 pathway is a key regulator of T cell activation and a critical failsafe against autoimmunity. While early models postulated that CTLA-4 transduced a negative signal, in vivo evidence suggests that CTLA-4 functions in a cell-extrinsic manner. That multiple cell-intrinsic mechanisms have been attributed to CTLA-4, yet its function in vivo appears to be cell-extrinisic, has been an ongoing paradox in the field. Whilst CTLA-4 expressed on conventional T cells (Tconv) can mediate inhibitory function, it is unclear why this fails to manifest as an intrinsic effect. Here we show that Tconv-expressed CTLA-4 can function in a cell-extrinsic manner in vivo. CTLA-4+/+ T cells, from DO11/rag−/− mice that lack Treg, were able to regulate the response of CTLA4−/− T cells in co-transfer experiments. This observation provides a potential resolution to the above paradox, and suggests CTLA-4 function on both Tconv and Treg can be achieved through cell-extrinsic mechanisms.
Cytotoxic T lymphocyte–associated antigen 4 (CTLA-4) is an essential negative regulator of T cell activation. Recent evidence suggests that CTLA-4 association with the immunological synapse during contact with antigen-presenting cells is important for its inhibitory function. In the present study, we observed a direct interaction of CTLA-4 with the phosphorylated form of T cell receptor (TCR)ζ within the glycolipid-enriched microdomains associated with the T cell signaling complex. In this setting, CTLA-4 regulated the accumulation/retention of TCRζ in the signaling complex, as the lipid raft fractions from CTLA-4KO T cells contained significantly higher amounts of the TCR components when compared with wild-type littermates. In contrast, coligation of CTLA-4 with the TCR during T cell activation selectively decreased the amount of TCRζ that accumulated in the rafts. These results suggest that CTLA-4 functions to regulate T cell signaling by controlling TCR accumulation and/or retention within this a critical component of the immunological synapse.
costimulation; T cells; GEM; immunological synapse; negative signal
Cytotoxic T lymphocyte antigen 4 (CTLA4) appears to negatively regulate T cell activation.
One mechanism by which CTLA4 might antagonize T cell function is through inhibition of
CD28 signaling by competing for their shared ligands B7-1 and B7-2. In addition, CTLA4 ligation could initiate a signaling cascade that inhibits T cell activation. To address whether
CTLA4 could inhibit immune responses in the absence of CD28, rejection of heart allografts
was studied in CD28-deficient mice. H-2q hearts were transplanted into allogeneic wild-type
or CD28-deficient mice (H-2b). Graft rejection was delayed in CD28-deficient compared with
wild-type mice. Treatment of wild-type recipients with CTLA4-immunoglobulin (Ig), or with
anti–B7-1 plus anti–B7-2 mAbs significantly prolonged allograft survival. In contrast, treatment
of CD28-deficient mice with CTLA4-Ig, anti–B7-1 plus anti–B7-2 mAbs, or a blocking anti-CTLA4 mAb induced acceleration of allograft rejection. This increased rate of graft rejection
was associated with more severe mononuclear cell infiltration and enhanced levels of IFN-γ
and IL-6 transcripts in donor hearts of untreated wild-type and CTLA4-Ig– or anti-CTLA4
mAb–treated CD28-deficient mice. Thus, the negative regulatory role of CTLA4 extends beyond its potential ability to prevent CD28 activation through ligand competition. Even in the
absence of CD28, CTLA4 plays an inhibitory role in the regulation of allograft rejection.
cytotoxic T lymphocyte antigen 4; CD28-deficient; cytotoxic T lymphocyte
antigen 4–immunoglobulin; transplantation; T lymphocyte
The critical role of cytotoxic lymphocyte antigen 4 (CTLA-4) in inhibiting antigen-driven T cell responses upon engagement with its ligands, B7-1 and B7-2, and its importance for peripheral T cell tolerance and T cell homeostasis has been studied intensively. The CTLA-4 splice variant, li-CTLA-4 is expressed in naïve and activated T cells and can actively alter T cell signaling despite its lack of a B7 binding domain. To study the effect of li-CTLA-4 in regulating T cell responses in the context of autoimmunity, we engineered a B6.CTLA-4 (floxed-Exon2)-BAC-transgene, resulting in selective expression of li-CTLA-4 upon Cre-mediated deletion of Exon 2. Introducing the B6.BAC into the NOD background, which is genetically deficient for li-CTLA-4, restores mRNA levels of li-CTLA-4 to those observed in B6 mice. Further, re-expressing this ligand non-binding isoform in NOD mice reduced IFN-γ-production in T effector cells accompanied by a significant decrease in insulitis and T1D frequency. However, selective expression of li-CTLA-4 could not fully rescue the CTLA-4KO disease phenotype when bred onto NOD.BDC2.5.CTLA-4KO background due to the requirement of the full-length, B7-binding CTLA-4 molecule on T effector cells. Thus, the li-CTLA-4 form, when expressed at physiologic levels in the CTLA-4 sufficient NOD background can suppress autoimmunity, however, the functionality of the li-CTLA-4 isoform depends on the presence of the full-length molecule to alter effector T cell signaling.
It is now clear that functionally specialized regulatory T (Treg) cells exist as part of the normal immune repertoire, preventing the development of pathogenic responses to both self- and intestinal antigens. Here, we report that the Treg cells that control intestinal inflammation express the same phenotype (CD25+CD45RBlowCD4+) as those that control autoimmunity. Previous studies have failed to identify how CD25+ Treg cells function in vivo. Our studies reveal that the immune-suppressive function of these cells in vivo is dependent on signaling via the negative regulator of T cell activation cytotoxic T lymphocyte–associated antigen 4 (CTLA-4), as well as secretion of the immune-suppressive cytokine transforming growth factor β. Strikingly, constitutive expression of CTLA-4 among CD4+ cells was restricted primarily to Treg cells, suggesting that CTLA-4 expression by these cells is involved in their immune-suppressive function. These findings raise the possibility that Treg cell function contributes to the immune suppression characteristic of CTLA-4 signaling. Identification of costimulatory molecules involved in the function of Treg cells may facilitate further characterization of these cells and development of new therapeutic strategies for the treatment of inflammatory diseases.
inflammatory bowel disease; CD4+ T lymphocyte; T lymphocyte suppressor; interleukin 2 receptor; autoimmunity
Immunosuppressive cytotoxic T lymphocyte associated antigen-4 immunoglobulin fusion proteins (CTLA4-Ig) block the CD28:CD80/86 costimulatory pathway. On a cellular level, CTLA4-Ig is understood to dampen T cell responses. As a mechanism, CTLA4-Ig has been reported to affect dendritic cell (DC) function via inducing the immunosuppressive indoleamine 2,3 dioxygenase (IDO) pathway and promoting a DC regulatory phenotype. We here probed cellular mechanisms of CTLA4-Ig immunoregulation in an allogeneic setting using C57BL/6 splenic or bone marrow derived DCs (BMDCs) as stimulators of allogeneic Balb/c derived T cells. To address whether CTLA4-Ig immunosuppression affected DCs, we pre-exposed C57BL/6 splenic or BMDCs to CTLA4-Ig and removed unbound CTLA4-Ig before co-culture with allogeneic T cells. CTLA4-Ig disappeared rapidly (within 4 h) from the cell membrane by combined internalization and dissociation. These CTLA4-Ig pre-exposed DCs were fully capable of stimulating allogeneic T cell proliferation, suggesting that CTLA4-Ig does not impair the DC stimulatory capacity. Only the presence of CTLA4-Ig during DC/T cell co-culture resulted in the expected inhibition of proliferation. C57BL/6 splenic or BMDCs exposed to CTLA4-Ig did not display IDO activity. We conclude that CTLA4-Ig immunosuppressive activity does not depend on a DC regulatory phenotype but on its presence during DC/T cell interaction.
► CTLA4-Ig binding to CD80/86 on murine DC surface is short lived. ► CTLA4-Ig does not confer a DC regulatory phenotype ► CTLA4-Ig does not induce IDO ► CTLA4-Ig mediates immune inhibition by affecting DC/T cell interaction
CTLA4-Ig; Abatacept; IFN-γ; IDO; Reverse signaling
Background: The cytotoxic T lymphocyteassociated
antigen 4 gene (CTLA-4) encode the
T cell receptor involved in the control of T cell
proliferation and mediates T cell apoptosis.
The receptor protein is a specific T lymphocyte
surface antigen that is detected on cells only
after antigen presentation. Thus, CTLA-4 is
directly involved in both immune and autoimmune
responses and may be involved in the
pathogenesis of multiple T cell-mediated
autoimmune disorders. There is polymorphism
at position 49 in exon 1 of the CTLA-4 gene,
providing an A-G exchange. Moreover, we
assessed the CTLA-4 49 (Thr/Ala) polymorphism
in diabetic patients and first-degree relatives
as compared to control subjects.
Research design and methods: Three loci
(HLA-DQB1, DQA1 and CTLA-4) were analysed in 62 type 1 diabetic patients, 72 firstdegree
relatives and 84 nondiabetic control
subjects by means of PCR-RFLP.
Results: A significant enrichment in DQB1
alleles encoding for an amino acid different
from Asp in position 57 (NA) and DQA1 alleles
encoding for Arg in position 52 was
observed in diabetic subjects and first-degree
relatives as compared to controls. The genotype
and allele frequencies of these polymorphisms
in type 1 diabetic patients and firstdegree
relatives differed significantly from
those of controls (p< 0.001 and 0.05 respectively).
CTLA-49 Ala alleles frequencies were
75.8% in type 1 diabetic patients and 68.1% in
first-degree relatives in comparison to 35.7% in
control subjects. The Ala/Ala genotype conferred
a relative risk of 18.8 (p < 0.001).
Conclusion: The CTLA-4 49 Ala allele confers
an increased risk of type 1 diabetes, independent
of age and HLA-DQ genetic markers.
The mechanisms that regulate the strength and duration of CD8+ cytotoxic T cell activity determine the effectiveness of an antitumor immune response. To better understand the antitumor effects of anti-cytotoxic T lymphocyte–associated antigen 4 (CTLA-4) antibody treatment, we analyzed the effect of CTLA-4 signaling on CD8+ T cells in vitro and in vivo. In vitro, cross-linking of CTLA-4 on purified CD8+ T cells caused decreased proliferative responses to anti-CD3 stimulation and rapid loss of activation marker expression. In vivo, blockade of CTLA-4 by neutralizing anti–CTLA-4 mAb greatly enhanced the accumulation, activation, and cytotoxic activity of CD8+ T cells induced by immunization with Ag on dendritic cells (DC). This enhanced response did not require the expression of MHC class II molecules on DC or the presence of CD4+ T cells. These results demonstrate that CTLA-4 blockade is able to directly enhance the proliferation and activation of specific CD8+ T cells, indicating its potential for tumor immunotherapy even in situations in which CD4+ T cell help is limited or absent.
CTLA-4 (CD152); CD8+ T cells; dendritic cells; CD4+ T cells; cytotoxicity
Cytotoxic T-lymphocyte associated protein 4 (CTLA4) is a negative regulator of T-cell proliferation. Polymorphisms in CTLA4 have been inconsistently associated with susceptibility to rheumatoid arthritis (RA) in populations of European ancestry but have not been examined in African Americans. The prevalence of RA in most populations of European and Asian ancestry is ∼1.0%; RA is purportedly less common in black Africans, with little known about its prevalence in African Americans. We sought to determine if CTLA4 polymorphisms are associated with RA in African Americans. We performed a 2-stage analysis of 12 haplotype tagging single nucleotide polymorphisms (SNPs) across CTLA4 in a total of 505 African American RA patients and 712 African American controls using Illumina and TaqMan platforms. The minor allele (G) of the rs231778 SNP was 0.054 in RA patients, compared to 0.209 in controls (4.462×10−26, Fisher's exact). The presence of the G allele was associated with a substantially reduced odds ratio (OR) of having RA (AG+GG genotypes vs. AA genotype, OR 0.19, 95% CI: 0.13–0.26, p = 2.4×10−28, Fisher's exact), suggesting a protective effect. This SNP is polymorphic in the African population (minor allele frequency [MAF] 0.09 in the Yoruba population), but is very rare in other groups (MAF = 0.002 in 530 Caucasians genotyped for this study). Markers associated with RA in populations of European ancestry (rs3087243 [+60C/T] and rs231775 [+49A/G]) were not replicated in African Americans. We found no confounding of association for rs231778 after stratifying for the HLA-DRB1 shared epitope, presence of anti-cyclic citrullinated peptide antibody, or degree of admixture from the European population. An African ancestry-specific genetic variant of CTLA4 appears to be associated with protection from RA in African Americans. This finding may explain, in part, the relatively low prevalence of RA in black African populations.
Rheumatoid arthritis (RA) is a systemic autoimmune condition affecting the synovial membranes of diarthrodial joints. The etiology of RA is unclear but is thought to result from an environmental trigger in the context of genetic predisposition. We report that a single nucleotide polymorphism (SNP) (rs231778) in CTLA4, which encodes a negative regulator of T cell activation, is associated (p = 2.4×10−28) with protection from developing RA among African Americans. rs231778 is only polymorphic in populations of African ancestry. Protective alleles such as this one may contribute to the purported lower prevalence of RA in African Americans. Our finding appears to be independent from confounding by linkage with the HLA-DRB1 shared epitope or by genetic admixture. Furthermore, we did not replicate associations of CTLA4 SNPs with RA or other autoimmune diseases previously reported in Asians and Caucasians, such as rs3087243 (+60C/T) and rs231775 (+49A/G). The associations of different SNPs with RA susceptibility specific to different populations highlight the importance of CTLA4 in the pathogenesis of RA and demonstrate the ethnic-specific genetic background that contributes to its susceptibility.
CTLA-4 encodes cytotoxic T lymphocyte-associated antigen-4, a cell-surface molecule providing a negative signal for T-cell activation. CTLA-4 gene polymorphisms have been widely studied in connection with genetic susceptibility to various autoimmune diseases, but studies have led to contradictory results in different populations. This case-control study sought to investigate whether CTLA-4 CT60 and/or +49A/G polymorphisms were involved in the genetic predisposition to primary Sjögren syndrome (pSS). We analysed CTLA-4 CT60 and +49A/G polymorphisms in a first cohort of 142 patients with pSS (cohort 1) and 241 controls, all of Caucasian origin. A replication study was performed on a second cohort of 139 patients with pSS (cohort 2). In cohort 1, the CTLA-4 +49A/G*A allele was found on 73% of chromosomes in patients with pSS, compared with 66% in controls (p = 0.036; odds ratio (OR) 1.41, 95% confidence interval (CI) 1.02 to 1.95). No difference in CTLA-4 CT60 allelic or genotypic distribution was observed between patients (n = 142) and controls (n = 241). In the replication cohort, the CTLA-4 +49A/G*A allele was found on 62% of chromosomes in patients with pSS, compared with 66% in controls (p = 0.30; OR 0.85, 95% CI 0.63 to 1.16). Thus, the CTLA-4 +49A/G*A allele excess among patients from cohort 1 was counterbalanced by its under-representation in cohort 2. When the results from the patients in both cohorts were pooled (n = 281), there was no difference in CTLA-4 +49A/G allelic or genotypic distribution in comparison with controls. Our results demonstrate a lack of association between CTLA-4 CT60 or +49A/G polymorphisms and pSS. Premature conclusions might have been made if a replication study had not been performed. These results illustrate the importance of case-control studies performed on a large number of patients. In fact, sampling bias may account for some contradictory results previously reported for CTLA-4 association studies in autoimmune diseases.
T cells play a central role in cell-mediated immunity, atopic disease, and asthma. The balance of CD28/cytotoxic T-lymphocyte antigen 4 (CTLA4)-derived signal transduction plays an important role in the activation of T cells and an increased immunoglobulin E (IgE) response. The aim of the current study was to investigate the association between polymorphisms in the genes encoding both CTLA4 and the high-affinity IgE receptor 1B (FCER1B) and serum IgE levels in Korean children with asthma.
We enrolled 238 controls and 742 children with asthma. The CTLA4 +49A/G and FCER1B -654C/T polymorphisms were genotyped by PCR-restriction fragment length polymorphism analysis.
We observed no difference in the distribution of CTLA4 +49A/G among controls, children with asthma, and those with atopic asthma. In contrast, the GA genotype of CTLA4 +49A/G in children with atopic asthma was significantly higher compared to that in those with non-atopic asthma. Moreover, significantly higher log Dp/Df-specific IgE levels were found in children with asthma and those with atopic asthma carrying one or two copies of the CTLA4 +49A versus those homozygous for +49G. Gene-gene interactions between CTLA4 and FCER1B with the heterozygote and homozygote of variant genotypes were associated with the log Dp/Df-specific IgE levels, but not asthma development. In addition, children with Dp/Df (+) asthma carried an elevated combined genotype of risk allele compared to those with Dp/Df (-) asthma.
The CTLA4 +49A/G polymorphism may contribute to the production of IgE in Korean children with asthma, especially in Dp/Df-specific IgE levels, but not in the direct development of asthma. In addition, Dp/Df-specific IgE levels with a FCER1B -654C/T polymorphism may involve additive effects.
Asthma; CTLA4; FCER1B; polymorphism; Dermatophagoides pteronyssinus; Dermatophagoides farinae; immunoglobulin E
The cytotoxic T lymphocyte antigen 4 (CTLA‐4) gene encodes for a membrane bound (mCTLA‐4) and a soluble (sCTLA‐4) isoform, which are both involved in regulation of T cell function. The CTLA‐4 +49A/G single nucleotide polymorphism (SNP) influences expression of mCTLA‐4; +6230G/A SNP affects the production of sCTLA‐4.
To examine whether these functional SNPs influence the rate of rejection after liver transplantation.
Patients and methods
Liver graft recipients (n = 483) were genotyped for both SNPs, and haplotypes were reconstructed. Association with rejection was tested by the log rank test using the Kaplan‐Meier method with time to the first acute rejection episode as outcome. Multiple analysis of SNPs together with demographic factors was performed by Cox regression.
Three haplotypes were observed in the cohort: +49A/+6230A, +49A/+6230G, and +49G/+6230G. The +49A/+6230G haplotype was significantly and dose dependently associated with acute rejection (p = 0.01). Of the demographic factors tested, only underlying liver disease was significantly associated with rejection. Adjusted for underlying liver disease, each additional +49A/+6230G haplotype allele resulted in a significantly higher risk of acute rejection (risk ratio 1.34 (95% confidence interval 1.04–1.72); p = 0.02). Patients who lacked this haplotype had the lowest, carriers an intermediate, and homozygotes the highest risk of acute rejection.
The CTLA‐4 +49A/+6230G haplotype, which encodes for normal mCTLA‐4 expression but reduced sCTLA‐4 production, is a co‐dominant risk allele for acute rejection after clinical liver transplantation. This implies that even under immunosuppression, CTLA‐4 is critically involved in the regulation of the human immune response to allogeneic grafts.
cytotoxic T lymphocyte antigen 4; liver transplantation; rejection; haplotype; single nucleotide polymorphism
Blockade of the Cytotoxic T Lymphocyte-associated Antigen 4 (CTLA-4), a down-regulator of T cell activation, can cause cancer regression in patients with metastatic melanoma. However, not all patients respond well to the therapy and some develop severe autoimmune reactions. We hypothesized that common genetic variation in the CTLA4 gene could contribute to response to CTLA-4 blockade and the occurrence of autoimmune reactions. We investigated seven common single-nucleotide polymorphisms, SNPs, (rs733618, rs4553808, rs11571317, rs5742909, rs231775, rs3087243 and rs7565213) in 152 Caucasian melanoma patients who received CTLA-4 blockade. Three SNPs were associated with response to therapy: proximal promoter SNPs, rs4553808 (p=0.002, OR 3.39, 95%CI:1.62–7.10) and rs11571327 (p=0.02, OR 2.89, 95%CI: 1.23–6.83) and the non-synonymous SNP rs231775 (Thr17Ala, p=0.009, OR 0.39; 95% CI 0.18–0.82). A haplotype analysis including the 7 SNPs suggested that the common haplotype, TACCGGG could be associated with no response (p=0.02) whereas the haplotype TGCCAGG (p=0.06, OR: 4.13, 95%CI: 1.17–14.5) could be associated with response to the treatment. No significant association was observed for occurrence of severe autoimmune reactions (grade III/IV) either by single SNP or haplotype analyses. Our results suggest that genetic variation in CTLA4 could influence response to CTLA-4 blockade therapy in metastatic melanoma patients, but further studies are necessary to confirm the observed associations.
CTLA4; Single Nucleotide Polymorphism; CTLA-4 blockade; melanoma
Ligation of cytotoxic T lymphocyte antigen 4 (CTLA4) appears to inhibit T cell responses.
Four mechanisms have been proposed to explain the inhibitory activity of CTLA4: competition for B7-1 and B7-2 binding by CD28; sequestration of signaling molecules away from
CD28 via endocytosis; delivery of a signal that antagonizes a CD28 signal; and delivery of a signal that antagonizes a T cell receptor (TCR) signal. As three of these potential mechanisms involve functional antagonism of CD28, an experimental model was designed to determine
whether CTLA4 could inhibit T cell function in the absence of CD28. TCR transgenic/recombinase activating gene 2–deficient/CD28–wild-type or CD28-deficient mice were generated and immunized with an antigen-expressing tumor. Primed T cells from both types of mice
produced cytokines and proliferated in response to stimulator cells lacking B7 expression.
However, whereas the response of CD28+/+ T cells was augmented by costimulation with B7-1,
the response of the CD28−/− T cells was strongly inhibited. This inhibition was reversed by
monoclonal antibody against B7-1 or CTLA4. Thus, CTLA4 can potently inhibit T cell activation in the absence of CD28, indicating that antagonism of a TCR-mediated signal is sufficient to explain the inhibitory effect of CTLA4.
T lymphocytes; costimulation; cytotoxic T lymphocyte antigen 4; B7; transgenic/knockout
Co-stimulation blockade can be used to modulate the immune response for induction of organ transplantation tolerance, treatment of autoimmune disease as well as cancer treatment. Cytotoxic T-Lymphocyte Antigen-4 (CTLA-4), also known as CD152, is an important co-stimulatory molecule which serves as a negative regulator for T cell proliferation and differentiation. CTLA-4/CD28-CD80/CD86 pathway is a critical co-stimulatory pathway for adaptive immune response. T cell activation through the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for CD80 and CD86. MGH MHC-defined miniature swine provide a unique large animal model useful for preclinical studies of transplantation tolerance and immune regulation. In this study, we have expressed the codon-optimized soluble porcine CTLA-4 in the yeast Pichia pastoris system. The secreted porcine CTLA-4 was captured using Ni-Sepharose 6 fast flow resin and further purified using strong anion exchange resin Poros 50HQ. Glycosylation analysis using PNGase F demonstrated the N-linked glycosylation on Pichia pastoris expressed soluble porcine CTLA-4. To improve the expression level and facilitate the downstream purification we mutated the two potential N-linked glycosylation sites with non-polarized alanines by site-directed mutagenesis. Removal of the two N-glycosylation sites significantly improved the production level from ~2 mg/L to ~8 mg/L. Biotinylated glycosylated and non-N-glycosylated soluble porcine CTLA-4 both bind to a porcine CD80-expressing B-cell lymphoma cell line (KD = 13 nM) and competitively inhibit the binding of an anti-CD80 monoclonal antibody. The availability of soluble porcine CTLA-4, especially the non-N-glycosylated CTLA-4, will provide a very valuable tool for assessing co-stimulatory blockade treatment for translational studies in the clinically relevant porcine model.
Porcine CTLA-4; Pichia pastoris expression; purification; porcine CD80; glycosylation
Cytotoxic T lymphocyte antigen 4 (CTLA-4) is an inhibitory regulator of the T-cell immune response against tumor cells. Ipilimumab is a monoclonal antibody directed against CTLA-4.
This review describes the basic mechanism of ipilimumab and discusses data available to date with regards to its safety and efficacy profile.
Data from clinical trials including abstracts was reviewed using the PubMed Database, as well as the American Society of Clinical Oncology Abstract Database.
CTLA-4 inhibition with a monoclonal antibody is usually well tolerated and has efficacy as a therapeutic agent in a variety of cancers. The classical response interpretation has changed because of the delayed mechanism of action. The toxicities are autoimmune events and guidelines for treatment of these effects are discussed. Therapy with ipilimumab leads to durable responses. The first two Phase III randomized studies showed an improvement of survival at 1, 2, and 3 years. Other studies are currently underway to better understand the optimal treatment administration of ipilimumab in melanoma.
melanoma; CTLA-4; ipilimumab; immune-related adverse events; melanoma; modified immune response criteria
Cytotoxic T lymphocyte–associated antigen (CTLA)-4 can inhibit T-cell responses and is involved in tolerance against self antigens. We previously reported autoimmune manifestations and objective cancer regressions in patients with metastatic melanoma treated with CTLA-4 blockade. The possibility of activating tumor-reactive T cells while removing inhibitory activity with CTLA-4 blockade has stimulated interest in using anti–CTLA-4 antibodies in combination with other cancer immunotherapies to improve clinical outcomes. In this study, we assessed the antitumor activity and autoimmune toxicity of CTLA-4 blockade in combination with an immune-activating stimulus, interleukin (IL)-2, in patients with metastatic melanoma.
Thirty-six patients received anti–CTLA-4 antibody every 3 weeks. Three patients per cohort received doses of .1, .3, 1.0, and 2.0 mg/kg. Twenty-four patients received 3.0 mg/kg. All patients received IL-2 therapy (720,000 IU/kg every 8 hours to a maximum of 15 doses).
Eight patients (22%) experienced objective tumor responses (three complete and five partial), including metastases in the lungs, lymph nodes, mediastinum, and subcutaneous tissues. Six of the eight patients have ongoing objective responses at 11 to 19 months. Five patients (14%) developed grade III/IV autoimmune toxicities secondary to anti–CTLA-4 administration, including four patients with enterocolitis and one with arthritis and uveitis.
There is not evidence to support a synergistic effect of CTLA-4 blockade plus IL-2 administration, because the 22% objective response rate is that expected from the sum of these two agents administered alone. Durable cancer regressions were seen in patients treated with this combination.
Cytotoxic T lymphocyte-associated antigen 4; Interleukin 2; Melanoma; Autoimmunity
Rheumatoid arthritis is a chronic inflammatory disease with a strong MHC class II component and where many patients develop characteristic autoantibodies towards the noncoding amino acid citrulline. Such anti-citrullinated protein antibodies (ACPA) have recently been put forward as an independent predictive factor for treatment response by co-stimulation blockade by CTLA4-Ig (abatacept). We have performed a mechanism of action study to dissect T cell functionality in RA patients with long-standing disease undergoing abatacept treatment and the influence of ACPA status.
Peripheral blood samples were collected from RA patients as they started CTLA4-Ig treatment and 3 and 6 months later. A general decrease of regulatory T cell subsets was observed in the cohort. Additionally within the ACPA-positive group significant down-regulation of all key T cell effector subsets including Th1, Th2, and Th17 was observed by analyzing cytokines by intracellular flow cytometry and in cell culture supernatants.
RA synovial fluid samples were cultured in vitro in the presence or absence of CTLA4-Ig (abatacept). T cell cytokine production was diminished, but without increasing the functional capacity of CD4+CD25hi regulatory T cells as previously demonstrated in the context of TNF-blockade and anti-IL6R therapy.
Our immunological study of T cell functionality in RA patients, both ACPA-positive and ACPA-negative, starting biological therapy with the co-stimulation blockade abatacept (CTLA4-Ig) supports the recently published registry study implicating ACPA seropositivity as an independent predictive factor to treatment response as we observed the most striking effect on T cell subset modulation in ACPA-positive patients. These data further support the notion of RA as a disease with several sub-entities, where the ACPA-positive fraction represents a classical HLA-associated autoimmune disorder while ACPA-negative patients may have other driving forces apart from classical adaptive immune responses.
Rheumatoid arthritis; Autoimmunity; T lymphocyte; Cytokines; Regulatory T cells; Abatacept; ACPA
T-cell costimulatory molecules deliver positive or negative signals to govern the ultimate fate of immune responses. These ligands and receptors that negatively costimulate T cells (including cytotoxic T-lymphocyte antigen [CTLA]-4, B7-H1, programmed death [PD]-1, B7-H3 and B7x) have received significant interest recently owing to their proposed ability to form a molecular shield for tumor cells. CTLA-4 represents the most extensively studied receptor in the costimulatory pathway and functions as a potent inhibitor of T-cell-mediated immunity. Clinical trials with anti-CTLA-4 are ongoing, although numerous objective responses have been observed in heavily pretreated patients, albeit with autoimmune side effects. In renal cell carcinoma, B7-H1, PD-1 and B7x have been observed to be expressed on tumor cells or infiltrating lymphocytes and are individually associated with adverse pathologic features and poor clinical outcome. In prostate cancer, B7-H3 and B7x immunostaining intensity correlate with disease spread, clinical cancer recurrence and cancer-specific death. External validation and prospective studies are now needed to confirm these findings, while further development of humanized monoclonal antibodies, similar to the experience with anti-CTLA-4, are underway. Herein, we review the B7–CD28 family as it applies to urologic malignancies.
lymphocyte activation; prostatic neoplasm; regulatory T lymphocyte; renal cell carcinoma; urinary bladder neoplasm
Cytotoxic T lymphocyte antigen-4 (CTLA-4) plays a critical role in negatively regulating T cell responses and has also been implicated in the development and function of natural FOXP3+ regulatory T cells. CTLA-4–deficient mice develop fatal, early onset lymphoproliferative disease. However, chimeric mice containing both CTLA-4–deficient and –sufficient bone marrow (BM)–derived cells do not develop disease, indicating that CTLA-4 can act in trans to maintain T cell self-tolerance. Using genetically mixed blastocyst and BM chimaeras as well as in vivo T cell transfer systems, we demonstrate that in vivo regulation of Ctla4−/− T cells in trans by CTLA-4–sufficient T cells is a reversible process that requires the persistent presence of FOXP3+ regulatory T cells with a diverse TCR repertoire. Based on gene expression studies, the regulatory T cells do not appear to act directly on T cells, suggesting they may instead modulate the stimulatory activities of antigen-presenting cells. These results demonstrate that CTLA-4 is absolutely required for FOXP3+ regulatory T cell function in vivo.
Cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) is a potent immunoregulatory molecule that downregulates T-cell activation and thus influences the antitumor immune response. CTLA-4 polymorphisms are associated with various cancers, and CTLA-4 mRNA/protein increased expression is found in several tumor types. However, most of the studies are based on peripheral blood mononuclear cells, and much less is known about the relationship between CTLA-4 expression, especially gene expression, and its polymorphic variants in cancer tissue. In our study we assessed the distribution of CTLA-4 two polymorphisms (+49A/G and −318C/T), using TaqMan probes (rs231775 and rs5742909, resp.), and CTLA-4 gene expression in real-time PCR assay in non-small-cell lung cancer (NSCLC) tissue samples. The increased CTLA-4 expression was observed in the majority of NSCLC patients, and it was significantly correlated with TT genotype (−318C/T) and with tumor size (T2 versus T3 + T4). The presence of G allele and GG genotype in cancer tissue (+49A/G) was significantly associated with the increased NSCLC risk. Additionally, we compared genotype distributions in the corresponding tumor and blood samples and found statistically significant differences. The shift from one genotype in the blood to another in the tumor may confirm the complexity of gene functionality in cancer tissue.