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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.

T cells plays important role in controlling the infection. Previous research findings have suggested that many immunodominant antigens are not similarly immunogenic. We attempt to discover novel antigen that can be developed as a potential vaccine candidate. Low dose Mycobacterium tuberculosis (Mtb) infection in Cynomolgus macaque results in human like granuloma formation. It is comprised of T cells, B cells, NK cells, and several other cells. Upon FDG-PET scan it has been shown that some of these granulomas are permissive or progressive in nature and some are restrictive or controlling. Our aim is to discover Mtb antigens that can be recognized by the T cells present in the granuloma. To investigate our hypothesis, we have infected Cynomolgus macaque with low dose of Mtb. T cells were isolated from the low and high burden granuloma. They were subjected to scRNA sequencing. Using bioinformatic tools and algorithms we have identified high value TCRs from controlling granulomas. The TCRs were reconstructed and transfected in TCR deficient Jurkat cells with NFAT promoter and luciferase reporter gene. The Mtb ORFeome library was cloned and expressed in E. coli. For antigen preparation, IPTG induced recombinant E. coli were killed by Peracetic acid or heat-killed and presented to the high value TCRs expressed in the reporter Jurkat cells by autologous BLCLs. The recognition was measured using luciferase activity. The selected unique TCRs show remarkable luciferase activity in response to PMA and Ionomycin stimulation. We believe that the antigen recognized by these TCRs will be equally immunogenic and can be developed as the new target for the vaccine against tuberculosis. Supported by BAA-NIAID-NIHAI201700104

Background: Non-human primates (NHP) are desirable as animal models of human disease because they share behavioral, physiological, and genomic traits with people. Hence, NHP recapitulates manifestations of disease not observed in other animal species. The Macaca fascicularis (i.e., Cynomolgus macaque) is an NHP species extensively used for biomedical research, but the TCR repertoire has not been characterized yet. Result: We used the genomic sequences to design primers to identify the expressed TCR repertoire by single-cell RNAseq. The data analysis from 22 unique samples was used to assign a functional status to each TCR gene. We identified and analyzed the TRA/D, TRB, and TRG loci of the Cynomolgus macaque. Conclusion: The genomic organization of the Cynomolgus macaque has great similarity with Macaca mulatta (i.e., Rhesus macaque) and they shared >90% sequence similarity with the human TCR repertoire. These data will facilitate the analysis of T cell immunity in Cynomolgus macaques.