The Characteristic of TCR Signaling Pathway in T Cell from Patients with Aplastic Anemia

A number of abnormalities of T cell immune function leading to immune dysregulation have been reported in aplastic anemia (AA). Our previous study showed not only the abnotmal distribution and clonal expansion of TCR repertoire,but also significantly higher expression of CD3ζ in T cells from AA patients. Alternative CD3ζ gene expression level may directly represent a characteristic of abnormal T cell activation. Thus, we further investigated the expression level of CD28 and CTLA-4 which involved in mediating T cell activation; CD3ζ-regulating factors, the expression levels of Cbl-b and distribution of the CD3ζ 3’ untranslated region (3’-UTR) splice variant. The splice variant  of CD3ζ 3’-UTR were identified in peripheral blood mononuclear cells (PBMCs) from 55 healthy individuals and 68 patients with AA ( 36 cases with SAA and 32 cases with AA) respecitively by RT-PCR. The expression levels of the Cbl-b, CD28, CTLA4 and CD3ζ genes were analyzed by real-time quantitative PCR. The results showed that increased expression level of CD28 (0.91±0.67) and CD3ζ (1.72±2.02) were found in AA group compared with healthy group (CD28: 0.71±0.58, P = 0.08; CD3ζ:1.07±0.76, P<0.05), While significantly decreased expression level of CTLA-4 (0.16±0.17) and Cbl-b (0.34±0.31) were found in AA group compared with healthy group (CTLA-4: 0.22±0.14, P<0.05; Cbl-b: 0.62±0.35, P<0.01). There are not significantly differences of the expression levels from all detected genes between SAA and AA group. Two types of splicing variants forms, wild type CD3ζ 3’-UTR (WT) and alternatively spliced CD3ζ 3’-UTR (AS) which has significantly lower stability and translation of CD3ζ, were detected in all of the healthy individuals. However, 64.7% AA patients contained WT⁺AS⁺CD3ζ 3′-UTR, 22.1% AA patients contained WT⁺AS^-CD3ζ 3′-UTR and 13.2% AA patients WT^-AS⁺CD3ζ 3′-UTR. The frequencies of three types of CD3ζ 3′-UTR also have no significantly differences between SAA and AA patients. Based on the CD3ζ 3'-UTR isoform expression characteristic, we divided the AA into three subgroups: WT⁺AS⁺, WT⁺AS^- and WT^-AS⁺ group. Significantly higher expression level of CD3ζ gene was found in WT^-AS⁺ group than that in  WT⁺AS⁺ groups. In conclusion, to our knowledge, this is the first study on mechanism of increased CD3ζ gene in AA. The increased expression level of CD3ζ gene may be associated with decreased expression level of Cbl-b gene in AA. Additionally, the expression feature of CD3ζ 3'-UTR isoform may be affect the expression of CD3ζ gene in some AA patient. Increased expression level of CD3ζ in AA patients contain WT^-AS⁺ CD3ζ 3'-UTR may be feedback regulation. Meanwhile, aberrant of T cell activation in AA may be related to changed expression of CD28 and CTLA-4.