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High-throughput sequencing and analyses of the TCR repertoire

ABSTRACT: We co-cultured T cells with Mo-DCs exposed to ConA for 24 h and used multiplex PCR and next-generation sequencing to detect the TCRα repertoire.The length of the amino acids of CDR3 in each group was 4–59 amino acids, and all presented a normal distribution. Moreover, the length of the amino acids of CDR3 was not different between ConA and control groups. The number of clonotypes and unique clonotypes in the ConA group was increased significantly compared with that in control groups. However, the difference of high-frequency CDR3 amino-acid clonotypes between ConA and control groups was not significant (cutoff = 0.2%). Our results for the V and J gene segments of TCR diversity showed a marked increase in the number of clonotypes in the ConA group. The Shannon–Wiener Diversity Index of ConA was lower than that in the control group, and the top-20 V and J gene segments of ConA showed a higher usage frequency than that in the control group, but the difference between ConA and the control group was not significant. Collectively, these data indicated that ConA could reduce CDR3 diversity and augment usage of high-frequency CDR3.

ORGANISM(S): Homo sapiens

PROVIDER: GSE163933 | GEO | 2022/05/15

REPOSITORIES: GEO

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Project description:We investigated the immune responses in the fetuses and trafficked maternal cells following in-utero transplantation (IUT) of maternal (mIUT) and paternal (pIUT) bone marrow donor mononuclear cells (MNC). Fetal cells and trafficked maternal cells were isolated from the 1 week old neonates by magnetic and FACS sorting and subjected to bulk RNA-sequencing. The gene expression profile in maternal and recipient cells of mIUT and pIUT show enriched clusters for RNA and protein metabolism, hemopoiesis and immune system development. In recipients of DT+ pIUT recipient clusters additionally represented T-cell regulation. Upregulated pIUT and DT+pIUT maternal clusters represented mitogen-activated protein kinases (MAPK) cascades, T-cell activation, and immune system development. All groups shared common genes represented for cytokine stimulus response, immune system regulation, B-cell mediated immunity and adaptive immune response. Analysis of receptor clonotypes reveals, the retrieval frequency of maternal-derived clonotypes increased and clonotypes diversity was reduced following mIUT and pIUT compared to uninjected controls. In DT+pIUT, decreased maternal clonotype retrieval and increased diversity towards baseline. In contrast, mIUT and pIUT recipient-derived clonotypes showed increased diversity while decreased frequency in DT+pIUT recipients. Maternal-derived top 5 clonotypes in each group were expanded with IUT and diminished with DT+, while recipient-derived top 5 clonotypes were most abundant in uninjected pups. We observed a large number of public maternal-derived clonotypes between DT+pIUT and controls, and a substantial number of public recipient-derived clonotypes between mIUT, pIUT and DT+pIUT. Complementarity-determining region 3 (CDR3) of naïve and antigen-experienced clonotypes are longer and shorter respectively, reflecting antigen driven selection. We also observed uniqueness in V/J-segment usage between uninjected control, mIUT and pIUT clonotypes. Our data indicate that both maternal and recipient immune cells respond to donor cells and that only maternal TCR and BCR diversity is affected by maternal DC depletion.

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High-throughput sequencing and analyses of the TCR repertoire

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