Background
Aplastic anemia (AA) is a serious hematological disorder characterized by pancytopenia [
1‐
3]. AA is an immune-mediated destruction of hematopoietic cells caused by abnormally activated T cells for most cases [
4,
5]. 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]. An abnormal CD3ζ gene expression level may directly represent a characteristic of abnormal T cell activation.
T cell recognition of antigenic peptide/major histocompatibility complexes plays a pivotal role in the initiation and regulation of the adaptive immune response [
7‐
9]. TCR activation plays a crucial role in T cell function. The TCR itself does not possess signaling domains. Instead, the TCR is non-covalently coupled to a conserved multisubunit signaling apparatus, i.e., the CD3 complex, which comprises the CD3εγ, CD3εδ, and CD3ζζ dimmers [
10]. However, the TCR/CD3 signaling complex alone is insufficient for antigen-specific T cell responses and a second pathway, co-stimulatory signaling, is required for T cell immune responses. The co-stimulatory signaling molecule CD28 that is found on T cells must bind B7-1 and B7-2, which are expressed on antigen presenting cells (APCs), to trigger T cell activation [
11,
12]. 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].
Optimal T cell activation requires signaling through the TCR and the CD28 co-stimulatory receptor. CD28 co-stimulation is believed to set the threshold for T cell activation. Casitas B-lineage lymphoma proto-oncogene-b (Cbl-b), a RING finger E3 ubiquitin-protein ligase, is involved in CD28-dependent T cell activation [
19]. Results from T cell activation assays in vitro have shown that CD28 co-stimulation promotes TCR-induced Cbl-b degradation, whereas CTLA-4-B7 interaction potentiates TCR-induced Cbl-b re-expression [
20].
Expression of the CD3ζ gene is regulated at the transcriptional, posttranscriptional, and posttranslational levels [
21]. As previously described, there are two isoforms of the CD3ζ 3′-UTR: a wild type (WT) isoform (906 bp) and an alternatively spliced (AS) isoform (344 bp). Abnormal CD3ζ expression was found in T cell from SLE patients, and this may be associated with decreased stability and translation of CD3ζ mRNAs that contain AS CD3ζ 3′-UTRs [
22]. However, the mechanism of upregulating the CD3ζ mRNA expression level in AA patients is unclear.
Thus, we investigated the expression level of the CD28, CTLA-4, Cbl-b, and CD3ζ genes, the SNP rs231775 in CTLA-4 gene, CD3ζ-regulating factors, and the distribution of the CD3ζ 3′-UTR splice variant. We concluded that analysis of these factors may facilitate the comprehensive understanding of the abnormal T cell immune characteristics of AA.
Discussion
AA is an immune-mediated disease in which T cell target hematopoietic cells [
23,
24]. The precise mechanism of T cell activation in AA pathogenesis remains unknown. TCR signaling plays an important role in T cell activation [
25,
26]. In this study, we analyzed the expression of TCR signaling molecules and related factors that are involved in mediating TCR signaling in AA patients.
T cell activation requires the delivery of at least two signals. The first signal is antigen-specific and MHC-TCR ligation, and the second is a co-stimulatory signal [
13,
27,
28]. Co-stimulatory signals occur via the binding of CD28, which is located on T cell, with B7 family members on APCs. Initial activation of T cell after antigen exposure is mediated by the CD28/B7 interaction and leads to the proliferation and differentiation of effector T cell [
29]. Co-stimulation cannot proceed unchecked; otherwise, an overwhelming immune response will ultimately ruin the host. The co-stimulatory system consists of peculiar mechanisms that can dampen T cell activation signals, resulting in immune homeostasis. CTLA-4, an inhibitory signaling molecule that prevents T cell activation, becomes expressed on activated T cell when T cell activation has reached its peak [
30,
31]. CD28 and CTLA-4 control the threshold for T cell activation by regulating the level of Cbl-b expression [
19,
20].
In this study, we analyzed the expression characteristics of the CD3ζ, CD28, CTLA-4, and Cbl-b genes, and our data indicate significantly decreased CTLA-4 and Cbl-b and increased CD3ζ and CD28 expression in AA patients. These results suggest that there may be aberrant T cell activation, which may be related to the first and second signals of the T cell activation signaling molecules in AA. However, do the other signaling molecules of TCR signaling such as CD3γ, CD3δ, CD3ε, and ZAP-70 [
32,
33] also have similar change trend in AA, it is needed further investigation, and it is suggested to characterize the alteration of gene expression profile. Downregulated Cbl-b gene expression in AA suggests that the threshold for T cell activation is different from that in healthy individuals. There is variation in the CD3 gene expression level in SAA and NSAA patients, which indicates that an aberrant T cell activation is more obvious in NSAA patients and may help distinguish between SAA and NSAA patients for immunosuppressive treatments in the clinic.
It is known that CD3ζ is the key regulator of the TCR/CD3 complex in T cell activation [
34,
35]. The increased CD3ζ expression in AA samples indicated abnormal T cell activation at least in some subsets of T cell in the patients, while a differently significant CD3ζ gene expression pattern was found for SAA and NSAA in this study. However, the underlying mechanism remains unclear because a previous study showed that different distributions of the CD3ζ 3′-UTR isoforms may affect CD3ζ and its related gene expression. Therefore, we compared the CD3ζ expression characteristics in subgroups containing different combinations of the CD3ζ 3′-UTR isoforms.
In general, the WT and AS 3′-UTR play different roles in CD3ζ transcript stability and generation of the CD3ζ protein [
36]. In this study, we first characterized the distribution of the WT and AS 3′-UTRs in AA patients. Interestingly, we found that only 64 % of the AA samples expressed WT
+AS
+CD3ζ 3′-UTR, which was thought to be the normal genotype. We also found two different subgroups, WT
+AS
−CD3ζ 3′-UTR (22 %) and WT
−AS
+CD3ζ 3′-UTR (14 %), in the AA patients, which may be related to the change in the CD3ζ level in the patients. Because we could not find a difference in the CD3ζ expression level in the total AA samples, we further compared the SAA and NSAA groups. Notably, an increased CD3ζ expression level was found in the WT
−AS
+ CD3ζ 3′-UTR SAA group compared with both the WT
+AS
+ CD3ζ 3′-UTR and WT
+AS
− CD3ζ 3′-UTR SAA groups. Interestingly, there was no significant difference in the CD3ζ expression level between the WT
+AS
+, WT
−AS
+, and WT
+AS
− subgroups for the NSAA patients. Therefore, there may be a difference in the regulation of the CD3ζ expression level and T cell activation between SAA and NSAA. At a minimum, it is thought that the CD3ζ 3′-UTR isoforms may influence the CD3ζ expression level in T cells from SAA, which may be one reason for the abnormal T cell activation.
It has been reported that aberrant CD3ζ expression may be associated with the decreased stability and translation of CD3ζ mRNAs with an AS CD3ζ 3′-UTR in SLE [
22,
36]. In this study, the CD3ζ gene expression level in some SAA patients was thought to be affected by different splicing variants of the CD3ζ 3′-UTR. As our results demonstrated, there was an increased CD3ζ expression level in SAA patients with the WT
-AS
+ CD3ζ 3′-UTR genotype, and whether this is feedback regulation remains an open question. The T cell activation function in SAA patients will be examined in the future to evaluate the influence of the CD3ζ 3′-UTR isoforms. To characterize the positive and negative regulatory factors of the TCR signal pathway, it is necessary to analyze the characteristics of the negative factors e.g., CTLA-4 and Cbl-b. It has been reported that SNP rs231775 in CTLA-4 gene is associated with an increased frequency of autoimmune diseases such as Graves’ disease, autoimmune hypothyroidism, type I diabetes, and multiple sclerosis [
37‐
39]. ]s To determine whether this SNP also plays a role in AA, we analyzed its distribution in AA samples. To our knowledge, this is the first study to analyze this SNP in Chinese AA patients. Interestingly, we found that the homozygous GG mutant was significantly higher in AA patients, indicating that this CTLA-4 variant may have an association with susceptibility to developing AA. However, this result is in contrast with a report by Svahn J et al. who demonstrated that there were no significant differences in the genotype or allele frequency of SNP rs231775 in CTLA-4 gene between AA patients and healthy individuals in the Caucasian population [
40]. However, the CTLA-4 mRNA expression level had no significant association with the genotype of SNP rs231775 in CTLA-4 gene in this study because SNP rs231775 is associated with the protein level [
17,
18]. Further study is needed to analyze the CTLA-4 protein level in T cell from AA patients to confirm this result.
CTLA-4 regulates TCR signals via Cbl-b, and it plays an important role in regulating it at the transcriptional level [
20]. However, little is known about the mRNA expression pattern of CTLA-4 and Cbl-b in AA patients. In this study, we found a significant positive correlation between CTLA-4 and Cbl-b in healthy individuals with the AA and AG genotypes of SNP rs231775 in CTLA-4 gene but not in those with the GG genotype of SNP rs231775 in CTLA-4 gene. However, there was no significant correlation between CTLA-4 and Cbl-b in any of the patients. These results indicate that CTLA-4 loses its proper regulatory role of Cbl-b expression at the transcriptional level in AA patients.
Acknowledgements
This study was sponsored by grants from the National Natural Science Foundation of China (#81370605 and #81460026), a China Post-doctoral Science Foundation funded project (#20070410840), the Natural Science Foundation of Guangdong Province (#S2012010008794 and #S2013020012863), the Technology Program of Guangdong Province (#2014A020212209), the Foundation for High-level Talents in Higher Education of Guangdong, China (#[2013]246-54), a Science and Information Technology of Guangzhou funded basic research for application project (#2011 J4100028), and Fundamental Research Funds for the Central Universities (#21612425).
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
BL and YQL contributed to concept development and study design. BL, LXG, YKX, MJW, and LLZ performed the laboratory studies. YPZ, XL, SHC, and LJY were responsible for collection of clinical data. BL and YQL coordinated the study and helped draft the manuscript. All authors read and approved the final manuscript.