Composition and diversity analysis of the B-cell receptor immunoglobulin heavy chain complementarity-determining region 3 repertoire in patients with acute rejection after kidney transplantation using high-throughput sequencing

摘要

The aim of the present study was to assess the genetic diversity of the B-cell receptor (BCR) in kidney transplant recipients with acute rejection. A total of three patients with acute rejection after kidney transplantation were examined by performing a composition and diversity analysis of the BCR immunoglobulin heavy chain (IGH) complementarity-determining region 3 (H-CDR3) repertoire. The peripheral blood mononuclear cells of patients were collected at 1 day prior to (Pre1), as well as 1 day (Post1) and 7 days (Post7) after the transplantation, and DNA was extracted. High-throughput sequencing technology was applied to determine the BCR repertoire. Raw sequences in FASTQ format were analyzed with the Basic Local Alignment Search Tool. The diversity of the BCR repertoire was assessed by calculating Shannon entropy, Simpson's diversity index, the Gini coefficient and highly expanded clone distributions. The diversity of the BCR repertoire at Pre1 was greater than that at Post1 or Post7. The diversity of the BCR repertoire was the lowest at Post1 and increased at Post7 but failed to reach the pre-transplantation levels. Patients exhibited the loss of seven IGH variable (IGHV)3 family genes, while five new genes were expressed at a low frequency. Furthermore, five IGHV-IGH joining (IGHJ) gene pairings, including IGHJ6-IGHV3-11, were detected in the patients. Up- and downregulated genes were assessed by calculating the expression frequencies of the IGH diversity and IGHV gene families at Post1 and Post7. The results of the H-CDR3 length distribution and H-CDR3 amino acid (AA) usage analyses indicated that in Case 1 and 2, the AA length was similar at mostly 14–18 AA, while that in Case 3 was relatively stable at 12–16 AA. In conclusion, the present results illustrate the diversity of H-CDR3 in patients with acute rejection after kidney transplantation may provide novel ideas, methods and means of monitoring and analyzing the immune status of patients under physiological and pathological conditions.