The pathogenesis of type 2 diabetes is insulin resistance and impaired islet β-cells function of the pancreas, accounting for approximately 90% of diabetic patients [14]. T2D patients have a greatly increased risk of cardiovascular, renal, brain complications and so on, among which cardiovascular complications caused the highest death in non-communicable diseases each year, with about 17.6 million people [15].
T1D is a typical organ-specific autoimmune disease in which the predominant immune response is mediated by T cells targeting the islet insulin-producing beta cells [16]. Previous investigators had analyzed the profiling of TCR repertoire in the peripheral blood of T1D patients by high-throughput sequencing, which could identify high abundance unique CDR3 amino acid sequence, providing support for specific TCR sequences as biomarkers and potential therapeutic targets [17].However, T2D is not an autoimmune disease, but recent researches showed that T cells could remove to the visceral adipose tissue (VAT) with a TCR repertoire changed, which demonstrated the expansion of antigen-special T cells were occurred in the VAT [18]. Another study showed that all Vβ families of resident T cells in the adipose tissue of T2D patients were significantly altered, suggesting that adipose tissue-resident T cells have specific restricted TCR repertoires [19]. TCR repertoire bias of tissue-resident T cells occurred frequently in organ-special autoimmune diseases [20], but was little reported in T2D patients.
An important feature of T2DM is chronic low-grade inflammation. Abnormal lipid metabolism can trigger chronic inflammatory response. This chronic inflammation may activate T cells, leading to abnormal immune responses.
In the present study, we analyzed the characteristics of the TCR repertoires in the peripheral blood of T2D patients with and without complications by using the techniques of high-throughput sequencing. We found that although there was no statistical difference in the quantity of V-J combinations between with and without complications, there was a decreasing trend in the patients with complications. PCA analysis had better distinguished the V-J combination frequency profile between T2D patients with and without complications. Both D50 Diversity and Gini indexes indicated that the diversity of TCR profiles decreased in complication group compared with control group. The above data indicate that the TCR repertoires of the complication group were significantly changed compared with the control group, which may be caused by the specific amplification of TCRs against antigen. Moreover, we screened out the specifically amplified fragments (TRBV27, TRBV28, TRBV3-2, and TRBJ1-6), and also 25 differentially expressed CDR3 sequences (8 up-regulated and 17 down-regulated) in the complication group, which had the potential to be a marker to predict complications of T2D patients.
To distinguish the profiles of TCR repertoires in T2D patients with different complications, we also analyzed and compared patients with LVD and Ne complications using high-throughput sequencing. We found that though there was no statistically significant, the quantity of V-J combinations in patients with LVD was more. And PCA on the V-J combination frequency profile could distinguish between LVD and Ne groups. Though there was no statistically significant, the diversity of TCR composition showed by D50 Diversity and Gini indexes in the group with LVD tended to increase. Moreover, the motif diagram of top 50 abundant CDR3 sequences showed that selecting suitable amino acid sequence can effectively distinguish patients with LVD or Ne complications.
TCR is a special marker that can determine the antigen specificity of specific T cells. Due to the development of sequencing methods and computational analysis techniques, TCR repertoires have become important drug candidates for a new generation of cell-free T lymphocyte markers. TCR biomarkers have the following advantages: 1) can be used for assays that do not require live T cells; 2) reduce intra- and inter-assay variability due to cell condition and operator performance; 3) newly developed high Throughput sequencing technology can detect rare and poorly responsive T cells.
Recently, high-throughput sequencing for TCR repertoires has been used to display T cells function in different conditions, such as immune diseases or cancers, suggesting that TCR repertoire may have the potential to reflect the immune status or serve as the prognostic marker [21]. Previous research on T-cell immunity and T1D evaluation was mainly carried out by methods such as Western blot, T cell proliferation, ELISPot and flow cytometry. These methods can distinguish T1D patients from normal controls, but their sensitivity and specificity are lower than using their own antibodies [22]. However, the immunoblot and T-cell proliferation assays usually required freshly isolated cells, which limited their application in the detection of a large number of clinical samples [23]. ELISpot and MHC polymer detection technologies could accurately identify target antigen epitopes and T lymphocyte phenotypes, but due to the limitations of sample conditions and HLA, multi-center studies were greatly hindered. However, through high-throughput sequencing for the profiling of TCR repertoires, researchers now can identify T cell clones, phenotypes, and the specific epitopes of T cells independent of HLA haplotypes [24].
There are still some limitations in this study that should be addressed. First, the current results are limited by the retrospective nature of our study. The relationship between TCR diversity and T2D development must be verified in prospective studies. Second, the relative small sample size might compromise the reliability of conclusions, which could be further confirmed by large-scale clinical cohorts. In addition, the causality and the predominant effects of TCR repertoire on T2D development remain unclear, and further study on the underlying immunological mechanisms is required.
There were several limitations of this study. First, all of the samples analyzed here were collected from one center, and the sample size was modest. Second, this analysis was based on bulk tissue samples, and these could not be used to identify immunological cells extracted from tissues. Finally, the specific function of each immune clonotype could not be identified. Therefore, large-scale investigations in the future are warranted.
In summary, this study revealed the divergence in TCR repertoire in T2D patients with (LVD or Ne) or without complications. Our findings provided novel insights into the role of T cell immunity in T2D incidence and progression, which may support the development of early diagnostic methods and targeting immunotherapies.
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