Abstract
BackgroundA previous study has demonstrated a significantly increased CD3ζ gene expression level in aplastic anemia (AA). However, the mechanism underlying the upregulated CD3ζ mRNA expression level and that of T cell activation signaling molecules in AA patients remains unclear. Thus, we investigated the expression levels of the CD3ζ, CD28, CTLA-4, and Cbl-b genes, the SNP rs231775 in the CTLA-4 gene, and the distribution of the CD3ζ 3′-UTR splice variant in AA patients.MethodsCD3ζ 3′-UTR splice variants were identified in peripheral blood mononuclear cells (PBMCs) from 48 healthy individuals and 67 patients with AA [37 cases of severe aplastic anemia (SAA) and 30 cases of non-sever aplastic anemia (NSAA)] by RT-PCR. CD3ζ, CD28, CTLA-4, and Cbl-b gene expression was analyzed by real-time quantitative PCR. The SNP rs231775 in CTLA-4 gene was analyzed by PCR-RFLP.ResultsCD3ζ and CD28 expression was significantly higher, while CTLA-4 and Cbl-b expression was significantly lower in AA patients compared with healthy individuals. Significantly higher CD3ζ expression was found in the NSAA subgroup compared with the SAA subgroup. 64 % of the AA samples had the same genotype (WT+AS+CD3ζ 3′-UTR); 22 % of the AA patients had a WT+AS−CD3ζ 3′-UTR genotype, and 14 % of the AA patients had a WT−AS+CD3ζ 3′-UTR genotype. The CD3ζ expression level of WT−AS+ subgroup was the highest in the SAA patients. A significantly higher frequency of the GG genotype (mutant type, homozygous) of SNP rs231775 in CTLA-4 gene was found in the AA patients. Positive correlation between the CTLA-4 and Cbl-b gene expression levels was found in healthy individuals with the AA and AG genotypes, but not in the AA patients.ConclusionsThis is the first study analyzing the expression characteristics of the CD28, CTLA-4, and Cbl-b genes in AA. Our results suggest that aberrant T cell activation may be related to the first and second signals of T cell activation in AA. The GG genotype of SNP rs231775 in CTLA-4 gene might be associated with AA risk in the Chinese population. The characteristics of CD3ζ 3′-UTR alternative splicing may be an index for evaluating the T cell activation status in AA patients, particularly in SAA patients.
Highlights
A previous study has demonstrated a significantly increased CD3ζ gene expression level in aplastic anemia (AA)
The expression levels of CD3ζ, CD28, cytotoxic T-lymphocyte antigen-4 (CTLA-4), and Casitas B-lineage lymphoma proto-oncogene-b (Cbl-b) in AA The expression levels of the CD3ζ, CD28, CTLA-4, and Cbl-b genes in cDNA from peripheral blood mononuclear cells (PBMCs) from 67 AA patients before treatment and 48 healthy individuals were quantitatively assessed by real-time polymerase chain reaction (PCR) using the SYBR Green I technique
The results demonstrated an increased expression level for CD28 and CD3ζ in AA patients compared with healthy individuals (CD28 0.49, P < 0.01; CD3ζ 0.95, P < 0.05; Fig. 1), while significantly decreased CTLA-4 and Cbl-b expression was found (CTLA-4 0.18, P < 0.01; Cbl-b 0.55, P < 0.01; Fig. 1)
Summary
A previous study has demonstrated a significantly increased CD3ζ gene expression level in aplastic anemia (AA). Our previous study has shown that, in addition to abnormal distribution and clonal expansion of the T cell receptor (TCR) repertoire, there is significantly higher CD3ζ expression in T cells in AA patients [6]. Upon T cell activation, cytotoxic T-lymphocyte antigen-4 (CTLA-4) is induced and outcompetes CD28 for B7-1 and B7-2 ligands, thereby preventing excessive T cell expansion [13, 14]. This mechanism provides a key checkpoint in the regulation of T cell immunity [15, 16]. The SNP rs231775, A > G transition mutation which is located at exon 1 in the CTLA-4 gene, has been reported to potentially influence the inhibitory function of CTLA-4 by reducing its cell surface expression [17, 18]
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