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. 2022 Jul 7;18(7):e1010611.
doi: 10.1371/journal.ppat.1010611. eCollection 2022 Jul.

Multiple modes of antigen exposure induce clonotypically diverse epitope-specific CD8+ T cells across multiple tissues in nonhuman primates

Jennifer Simpson  1 , Carly E Starke  1 , Alexandra M Ortiz  1 , Amy Ransier  2 , Sam Darko  2 , Daniel C Douek  2 , Jason M Brenchley  1
Affiliations

Multiple modes of antigen exposure induce clonotypically diverse epitope-specific CD8+ T cells across multiple tissues in nonhuman primates

Jennifer Simpson et al. PLoS Pathog. .

Abstract

Antigen-specific CD8+ T cells play a key role in the host's antiviral response. T cells recognize viral epitopes via the T cell receptor (TCR), which contains the complementarity-determining region-3 (CDR3), comprising the variable, diversity and joining regions of the TCRβ gene. During chronic simian immunodeficiency virus (SIV) infection of Asian macaque nonhuman primates, tissue-specific clonotypes are identifiable among SIV-specific CD8+ T cells. Here, we sought to determine level of antigen exposure responsible for the tissue-specific clonotypic structure. We examined whether the priming event and/or chronic antigen exposure is response for tissue-specific TCR repertoires. We evaluated the TCR repertoire of SIV-specific CD8+ T cells after acute antigen exposure following inoculation with a SIV DNA vaccine, longitudinally during the acute and chronic phases of SIV, and after administration of antiretrovirals (ARVs). Finally, we assessed the TCR repertoire of cytomegalovirus (CMV)-specific CD8+ T cells to establish if TCR tissue-specificity is shared among viruses that chronically replicate. TCR sequences unique to anatomical sites were identified after acute antigen exposure via vaccination and upon acute SIV infection. Tissue-specific clones also persisted into chronic infection and the clonotypic structure continued to evolve after ARV administration. Finally, tissue-specific clones were also observed in CMV-specific CD8+ T cells. Together, these data suggest that acute antigen priming is sufficient to induce tissue-specific clones and that this clonal hierarchy can persist when antigen loads are naturally or therapeutically reduced, providing mechanistic insight into tissue-residency.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. SIV-specific CD8+ T cell repertoire fluctuates throughout infection and during ARV treatment.
PBMCs, LN biopsies and BAL were sampled from 7 different SIVmac239X or SIVmac239-infected rhesus macaques during acute infection, chronic infection and after 2–7 months of ARV treatment. (A-G). Clonotypes consisting of more than 1% of the TCR repertoire were represented as percentage of the TCR repertoire during acute and chronic infection and with ARV treatment. “Public” clonotypes are the same clonotype (same V and J segments and same CDR3 amino acid sequence) found in multiple animals in this study, and matching clonotypes previously identified in the VDJdb database. “Shared” clonotypes were those found only in one animal but observed in multiple tissues. “Private” clonotypes were identified in a single anatomical site in a single animal. n = 7 animals. Total cell and TCR sequences numbers are listed above each column.
Fig 2
Fig 2. SIV-specific CD8+ T cells exhibit similar TCR diversity and similarity throughout SIV infection.
PBMCs, LN biopsies and BAL were sampled from SIVmac239X or SIVmac239-infected rhesus macaques during acute infection, chronic infection and after 2–6 months of ARV treatment. (A) The number of unique clonotypes in multiple anatomical sites at all timepoints. (B) The normalized Shannon-Weiner diversity index for the TCR repertoires of all tissues throughout SIV infection. (C) The d50 diversity index for the TCR repertoires of all tissues throughout SIV infection. (D) The Jaccard similarity index for comparisons between each tissue’s TCR repertoire. In (A-D), data is represented by mean and SD, with each individual animal represented by a unique symbol. (E) MDS plot of the TCR repertoires of each animal, tissue and timepoint throughout infection. Two-way ANOVA was used to determine statistical significance for panels A to D. n = 7 animals.
Fig 3
Fig 3. SIV-gag DNA vaccine induced a diverse TCR repertoire and tissue-specific clonotypes.
PBMCs, BAL, LN, and jejunum (jej) biopsies were sampled from rhesus macaques who had been administered with SIV-gag DNA vaccine. (A) CM9-specific CD8+ T cells in all tissues pre- and post- vaccine administration as a percentage of total CD8+ cells. (B) Clonotypes consisting of more than 1% of the TCR repertoire are represented as percentage of the TCR repertoire. “Public” clonotypes are the same clonotype (same V and J segments and same CDR3 amino acid sequence) found in multiple animals in this study, and matching clonotypes previously identified in the VDJdb database. “Shared” clonotypes were those found only in one animal but observed in multiple tissues. “Private” clonotypes were identified in a single anatomical site in a single animal. Total cell and TCR sequences numbers are listed above each column. (C) The normalized Shannon-Weiner diversity index for the TCR repertoires of all tissues. (D) The d50 diversity index for the TCR repertoires of all tissues. (E) The Jaccard similarity index for comparisons between each tissue’s TCR repertoire. In (C) to (E), data are presented as mean, with individual data points shown. Two-way ANOVA (A) and one-way ANOVA (C-E) were used to determine statistical significance. n = 5 animals.
Fig 4
Fig 4. CMV-specific CD8+ T cells exhibit public, shared and tissue-specific clonotypes upon natural infection.
PBMCs, LN, liver biopsies, and BAL were sampled from rhesus macaques who had been naturally infected with CMV. (A) The number of AN10-specific CD8+ T cells in multiple anatomical sites as a percentage of total CD8+ cells. (B) The number of VY9-specific CD8+ T cells in multiple anatomical sites as a percentage of total CD8+ cells. (C) Clonotypes consisting of more than 1% of the TCR repertoire of AN10-specific CD8+ T cells are represented as a percentage of the total TCR repertoire. “Public” clonotypes are the same clonotype (same V and J segments and same CDR3 amino acid sequence) found in multiple animals in this study, and matching clonotypes previously identified in the VDJdb database. “Shared” clonotypes were those found only in one animal but observed in multiple tissues. “Private” clonotypes were identified in a single anatomical site in a single animal. Total cell and TCR sequences numbers are listed above each column. (D) Clonotypes consisting of more than 1% of the TCR repertoire of VY9-specific CD8+ T cells are represented as a percentage of the total TCR repertoire. (E) The normalized Shannon-Weiner diversity index for the TCR repertoires of VY9-specific CD8+ T cells. (F) The d50 diversity index for the TCR repertoires of VY9-specific CD8+ T cells. (G) The Jaccard similarity index for comparisons between each tissue’s VY9-specific CD8+ T cell repertoire. In (A), (B) and (E)-(G), data are presented as mean, with individual data points. One-way ANOVA or mixed effects analysis was used to determine statistical significance. n = 2 to 5 animals.
Fig 5
Fig 5. The TCR repertoires of SIV- and CMV- specific CD8+ T cells exhibit similar diversity and tissue similarity.
PBMCs, LN and BAL were sampled from rhesus macaques who were chronically infected with SIVmac239X (without ARV treatment) or naturally infected with CMV. (A) The normalized Shannon-Weiner diversity index of the TCR repertoires. (B) The d50 diversity index of the TCR repertoires. (C) The Jaccard similarity index for comparisons between each tissue’s TCR repertoire. Data are presented as mean, with individual data points. One way ANOVA was used to determine statistical significance. n = 3 to 6 animals.
Fig 6
Fig 6. Expression patterns of CD69 and CD103 among virus-specific CD8+ T cells.
The expression of CD69 and CD103 on antigen specific CD8+ T cells were assessed by flow cytometry across all experimental groups. (A-E) Average percentage of CD69-CD103-, CD69+CD103-, CD69-CD103+ and CD69+CD103+ antigen-specific (CM9) CD8+ T cells after exposure to SIV-gag DNA vaccine (A); during acute (B), chronic (C), and chronic SIVmac239X or SIVmac239 infection with ARV treatment (D); or during CMV infection (E). (F) The number of CD69+CD103+ antigen-specific CD8+ T cells during all experimental conditions. In (F) data are presented as mean with SD. Two-way ANOVA was used to determine statistical significance in (F). n = 3 to 6 animals.
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Grants and funding

Funding for this study was provided by the divisions of intramural research of The National Institute of Allergy and Infectious Diseases. JMB received a funding award from The National Institute of Allergy and Infectious Diseases, NIH (NUMBER: 1ZIAAI001029). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.