DOCK8 Eksikliği Olan Hastalarda T Hücre Reseptör Repertuvarının Yeni Nesil Dizileme İle Belirlenmesi

Abstract

Identification of immüne repertoire is crucial to characterize adaptive immüne responses in terms of disease approach. V(D)J recombination is one of the most crucial factors in the development of the immune repertoire. The repertoire diversity that results from V (variable), D (diversity), and J (joining) genes and the structure of CDR3 (complementarity determining region 3) can be investigated by using next-generation sequencing (NGS) technologies and bioinformatics softwares. In this study, the T cell receptor (TCR) repertoire was evaluated in patients with DOCK8 deficiency. First, lymphocyte isolation was performed from the patients. By using magnetic negative separation technique, CD4+ and CD8+ T cells were separated from lymphocytes. Total RNA was extracted from the collected CD4+ and CD8+ T cells, and total RNA concentration were determined. TCR β chain amplification was performed with SMARTer technology. The TCR region was sequenced by Illumina Miseq. Bioinformatics and statistical analyzes of the results were carried out by using Cogent, Immunarch and IMGT softwares. The results showed that there was no significant difference between the CD4+ T cell repertoire diversity and unique clonotype numbers of patients and controls. It was observed that CD4+ T cell V-J gene usage of the patients was different than the controls. The patients' CD8+ T cell repertoire richness and number of distinct clonotypes were considerably lower than those of the control group. It was observed that the frequency of the usage of diverse V-J genes in CD8+ T cells of the patients was significantly lower than in the control. It was revealed that the distribution of CDR3 lengths of CD8+ T cells was lower than those of CD4+ T cells. The results showed that patients with DOCK8 deficiency have drastically altered CD8+ T cell repertoires, which suggests that DOCK8 mutations may affect how CD4+ and CD8+ cell repertoires are generated. DOCK8 protein may be thought to play a key role in the differentiation. This study indicates that the skewed CD8+ T cell repertoire may influence the susceptibility to infections by intracellular pathogens and cutaneous infections observed in DOCK8 deficiency.