Single-Cell–Based High-Throughput Ig and TCR Repertoire Sequencing Analysis in Rhesus Macaques

Walsh, Evan; Tollison, Tammy S; Brochu, Hayden; Shaw, Brian I.; Diveley, Kayleigh; Chou, Hsuan; Law, G. Lynn; Kirk, Allan D.; Gale, Michael; Peng, Xinxia

doi:10.4049/jimmunol.2100824

Cited by 4 publications

(2 citation statements)

References 44 publications

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“…γδ T cells are attractive candidates for novel vaccines against TB because they are not MHC I restricted, and thus, can be broadly stimulated in the target population ( 15 ). Early studies identified simple phosphoantigens such as isopentenyl pyrophosphate (IPP) as potent polyclonal stimulators of virtually all γ 9 δ 2 T cells ( 16 ). However, our previous studies have shown that only a subset of the IPP or other phosphoantigen-responsive γδ T cells expressing a more limited diversity of γδ TCR, are capable of recognizing Mtb-infected human monocytes and mediating inhibitory effects on intracellular mycobacteria ( 17 ).…”

Section: Discussionmentioning

confidence: 99%

Impact of BCG vaccination on the repertoire of human γδ T cell receptors

et al. 2023

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IntroductionTuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) infection is a serious threat to human health. Vaccination with BCG prevents the development of the most severe forms of TB disease in infants and was recently shown to prevent Mtb infection in previously uninfected adolescents. γδ T cells play a major role in host defense at mucosal sites and are known to respond robustly to mycobacterial infection. However, our understanding of the effects of BCG vaccination on γδ T cell responses is incomplete.MethodsIn this study we performed γδ T cell receptor (TCR) repertoire sequencing of samples provided pre- and post-BCG vaccination from 10 individuals to identify specific receptors and TCR clones that are induced by BCG.ResultsOverall, there was no change in the diversity of γTCR or δTCR clonotypes in post- vs pre-BCG samples. Furthermore, the frequencies of TCR variable and joining region genes were minimally modulated by BCG vaccination at either the γTCR or δTCR loci. However, the γTCR and δTCR repertoires of individuals were highly dynamic; a median of ~1% of γTCR and ~6% of δTCR in the repertoire were found to significantly expand or contract in post- vs pre-BCG comparisons (FDR-q < 0.05). While many of the clonotypes whose frequency changed after BCG vaccination were not shared among multiple individuals in the cohort, several shared (i.e., “public”) clonotypes were identified with a consistent increase or decrease in frequency across more than one individual; the degree of sharing of these clonotypes was significantly greater than the minimal sharing that would be expected among γTCR and δTCR repertoires. An in vitro analysis of Mtb antigen-reactive γδ T cells identified clonotypes that were similar or identical to the single-chain γTCRs and δTCRs that changed consistently after BCG vaccination; pairings of γTCRs and δTCRs that increased after BCG vaccination were significantly over-represented among the Mtb-reactive γδ T cells (p = 1.2e-6).DiscussionThese findings generate hypotheses about specific γδTCR clonotypes that may expand in response to BCG vaccination and may recognize Mtb antigens. Future studies are required to validate and characterize these clonotypes, with an aim to better understand the role of γδ T cells in Mtb immunity.

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Section: Discussionmentioning

confidence: 99%

Impact of BCG vaccination on the repertoire of human γδ T cell receptors

et al. 2023

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“…Our analysis of these primers shows them to be suitable for enrichment of TRA and TRB genes from rhesus macaques, and therefore this set of primers can be used for both species of macaques [ 25 ]. Similarly, nested primers for rhesus TCR enrichment using the 10X Genomics platform can also be used for cynomolgus macaques as the regions to which they anneal are 100% conserved [ 35 , 36 ].…”

Section: Discussionmentioning

confidence: 99%

Identification and characterization of the T cell receptor (TCR) repertoire of the cynomolgus macaque (Macaca Fascicularis)

et al. 2022

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Background Cynomolgus macaque (Macaca fascicularis) is an attractive animal model for the study of human disease and is extensively used in biomedical research. Cynomolgus macaques share behavioral, physiological, and genomic traits with humans and recapitulate human disease manifestations not observed in other animal species. To improve the use of the cynomolgus macaque model to investigate immune responses, we defined and characterized the T cell receptor (TCR) repertoire. Result We identified and analyzed the alpha (TRA), beta (TRB), gamma (TRG), and delta (TRD) TCR loci of the cynomolgus macaque. The expressed repertoire was determined using 22 unique lung samples from Mycobacterium tuberculosis infected cynomolgus macaques by single cell RNA sequencing. Expressed TCR alpha (TRAV) and beta (TRBV) variable region genes were enriched and identified using gene specific primers, which allowed their functional status to be determined. Analysis of the primers used for cynomolgus macaque TCR variable region gene enrichment showed they could also be used to amplify rhesus macaque (M. mulatta) variable region genes. Conclusion The genomic organization of the cynomolgus macaque has great similarity with the rhesus macaque and they shared > 90% sequence similarity with the human TCR repertoire. The identification of the TCR repertoire facilitates analysis of T cell immunity in cynomolgus macaques.

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The reverse TRBV30 gene of mammals: a defect or superiority in evolution?

Wu,

Yao

et al. 2024

BMC Genomics

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At the 3’ end of the C2 gene in the mammalian TRB locus, a distinct reverse TRBV30 gene (named TRBV31 in mice) has been conserved throughout evolution. In the fully annotated TRB locus of 14 mammals (including six orders), we observed noteworthy variations in the localization and quality of the reverse V30 genes and Recombination Signal Sequences (RSSs) in the gene trees of 13 mammals. Conversely, the forward V29 genes and RSSs were generally consistent with the species tree of their corresponding species. This finding suggested that the evolution of the reverse V30 gene was not synchronous and likely played a crucial role in regulating adaptive immune responses. To further investigate this possibility, we utilized single-cell TCR sequencing (scTCR-seq) and high-throughput sequencing (HTS) to analyze TCRβ CDR3 repertoires from both central and peripheral tissues of Primates (Homo sapiens and Macaca mulatta), Rodentia (Mus musculus: BALB/c, C57BL/6, and Kunming mice), Artiodactyla (Bos taurus and Bubalus bubalis), and Chiroptera (Rhinolophus affinis and Hipposideros armige). Our investigation revealed several novel observations: (1) The reverse V30 gene exhibits classical rearrangement patterns adhering to the ‘12/23 rule’ and the ‘D-J rearrangement preceding the V-(D-J) rearrangement’. This results in the formation of rearranged V30-D2J2, V30-D1J1, and V30-D1J2. However, we also identified ‘special rearrangement patterns’ wherein V30-D rearrangement preceding D-J rearrangement, giving rise to rearranged V30-D2-J1 and forward Vx-D2-J. (2) Compared to the ‘deletional rearrangement’ (looping out) of forward V1-V29 genes, the reverse V30 gene exhibits preferential utilization with ‘inversional rearrangement’. This may be attributed to the shorter distance between the V30 gene and D gene and the ‘inversional rearrangement’ modes. In summary, in the mammalian TRB locus, the reverse V30 gene has been uniquely preserved throughout evolution and preferentially utilized in V(D)J recombination, potentially serving a significant role in adaptive immunity. These results will pave the way for novel and specialized research into the mechanisms, efficiency, and function of V(D)J recombination in mammals.

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Single-Cell–Based High-Throughput Ig and TCR Repertoire Sequencing Analysis in Rhesus Macaques

Cited by 4 publications

References 44 publications

Impact of BCG vaccination on the repertoire of human γδ T cell receptors

Impact of BCG vaccination on the repertoire of human γδ T cell receptors

Identification and characterization of the T cell receptor (TCR) repertoire of the cynomolgus macaque (Macaca Fascicularis)

The reverse TRBV30 gene of mammals: a defect or superiority in evolution?

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