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

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.

Regulatory γδ T cells induced by G-CSF participate in acute graft-versus-host disease regulation in G-CSF-mobilized allogeneic peripheral blood stem cell transplantation

“…After the initial denaturation at 95 °C for 2 min, 45 cycles consisting of 95 °C 15 s, 60 °C 60 s and 82 °C 1 s for plate reading were performed using MJ Research DNA Engine Opticon 2 PCR cycler (BIO-RAD, USA). The relative mRNA expression levels of relative gene in each sample were calculated according to the comparative cycle time method [ 17 , 20 ].…”

Aplastic anemia is related to alterations in T cell receptor signaling

“…Results have shown that CD28 promotes Cbl-b degradation, whereas CTLA-4 promotes Cbl-b re-expression (28). An increased CD28 and decreased CTLA-4 and Cbl-b mRNA expression level were also found in patients newly diagnosed with AA (20). These results suggest that aberrant T cell activation in AA not only associates with the initial signals, but it is also related to the secondary signals in the TCR signaling pathway.…”

“…In most reports, a significant increase in the CD3γ, CD3δ, CD3ε, and CD3ζ expression levels was detected in peripheral blood samples from newly diagnosed AA patients, which may result in enhancing T cell activation (19,20). Interestingly, the CD3ζ expression level was higher in NSAA compared with SAA patients.…”

“…It has been reported that aberrant CD3ζ expression is associated with different CD3ζ 3'-UTR isoforms. There are two CD3ζ 3′-UTR isoforms: wild type (WT) and an alternatively spliced (AS) isoform (23,24 (20). Therefore, there may be a difference in the regulation of the CD3ζ expression level and T cell activation between SAA and NSAA patients.…”

Yin and Yang of mesenchymal stem cells and aplastic anemia

“…In addition, CD8+ T-cells are expanded in AA patients and they show restricted T-cell receptor (TCR) expression[ 13 ]. Some studies suggest that the TCRs themselves show increased expression of CD3-zeta and co-stimulatory molecule CD28 promoting T-cell activation[ 14 ], whereas other studies suggest a restricted TCR with decreased CD3-zeta expression but with aberrant activity[ 15 ].…”