Molecular alterations in the TCR signaling pathway in patients with aplastic anemia

Abstract

Background: A 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.

Methods: CD3ζ 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.

Results: CD3ζ 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.

Conclusions: This 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.

Keywords: AA; CD28; CD3ζ; CD3ζ 3′-UTR splice variant; CTLA-4; Cbl-b; Gene expression level; SNP.

Fig. 1

The relative gene expression levels of the CD3ζ, CD28, CTLA-4, and Cbl-b in AA and HIs. a The relative gene expression levels of CD3ζ in AA and HIs; b the relative gene expression levels of CD28 in AA and HIs; c the relative gene expression levels of CTLA-4 in AA and HIs; d the relative gene expression levels of Cl-b in AA and HIs

Fig. 2

The relative gene expression levels of CD3ζ, CD28, CTLA-4, and Cbl-b in SAA and NSAA. a The relative gene expression levels of CD3ζ in SAA and NSAA; b the relative gene expression levels of CD28 in SAA and NSAA; c the relative gene expression levels of CTLA-4 in SAA and NSAA; d the relative gene expression levels of Cbl-b in SAA and NSAA

Fig. 3

CD3ζ 3′-UTR splicing variant isoforms in AA and HIs. a The WT and AS isoforms could be detected in all of the healthy individuals (lane 1), 64 % of the AA patients contained WT⁺AS⁺CD3ζ 3′-UTR (lane 2), 22 % of the AA patients had only the WT⁺AS⁻CD3ζ 3′-UTR (lanes 4 and 5), and 14 % of the AA patients had only the WT^-AS⁺CD3ζ 3′-UTR (lanes 6 and 7). Lane 3 is a 100 bp DNA ladder; b the expression level of the CD3ζ gene with different CD3ζ 3′-UTR splicing variant isoforms in SAA

Fig. 4

The genotype characteristic of SNP rs231775 in CTLA-4 gene in AA and HIs. a Distribution of the genotype of SNP rs231775 in AA patients (n = 67); b distribution of the genotype of SNP rs231775 in HIs (n = 84); c agarose gel electrophoresis results for SNP rs231775 in CTLA-4 gene, lane 1 is the AG genotype, lane 2 is the GG genotype, lane 3 is a 50 bp DNA ladder, lane 4 is the AA genotype, and lane 5 is a PCR product without TseI digestion; d sequencing results of AA genotype; e sequencing results of AG genotype; f sequencing results of GG genotype; the red arrow indicates the single-nucleotide polymorphism sites

Fig. 5

Correlation analysis of the CTLA-4 and Cbl-b expression levels for different genotypes of SNP rs231775 in CTLA-4 gene in AA and HIs. a CTLA-4 and Cbl-b in AA with the AA genotype, b CTLA-4 and Cbl-b in AA with the AG genotype, c CTLA-4 and Cbl-b in AA with the GG genotype, d CTLA-4 and Cbl-b in HIs with the AA genotype, e CTLA-4 and Cbl-b in HIs with the AG genotype, and f CTLA-4 and Cbl-b in HIs with the GG genotype