Single-Cell Approach to Influenza-Specific CD8+ T Cell Receptor Repertoires Across Different Age Groups, Tissues, and Following Influenza Virus Infection

Abstract

CD8+ T cells recognizing antigenic peptides derived from conserved internal viral proteins confer broad protection against distinct influenza viruses. As memory CD8+ T cells change throughout the human lifetime and across tissue compartments, we investigated how T cell receptor (TCR) composition and diversity relate to memory CD8+ T cells across anatomical sites and immunological phases of human life. We used ex vivo peptide-HLA tetramer magnetic enrichment, single-cell multiplex RT-PCR for both the TCR-alpha (TCRα) and TCR-beta (TCRβ) chains, and new TCRdist and grouping of lymphocyte interactions by paratope hotspots (GLIPH) algorithms to compare TCRs directed against the most prominent human influenza epitope, HLA-A*02:01-M158–66 (A2+M158). We dissected memory TCR repertoires directed toward A2+M158 CD8+ T cells within human tissues and compared them to human peripheral blood of young and elderly adults. Furthermore, we compared these memory CD8+ T cell repertoires to A2+M158 CD8+ TCRs during acute influenza disease in patients hospitalized with avian A/H7N9 virus. Our study provides the first ex vivo comparative analysis of paired antigen-specific TCR-α/β clonotypes across different tissues and peripheral blood across different age groups. We show that human A2+M158 CD8+ T cells can be readily detected in human lungs, spleens, and lymph nodes, and that tissue A2+M158 TCRαβ repertoires reflect A2+M158 TCRαβ clonotypes derived from peripheral blood in healthy adults and influenza-infected patients. A2+M158 TCRαβ repertoires displayed distinct features only in elderly adults, with large private TCRαβ clonotypes replacing the prominent and public TRBV19/TRAV27 TCRs. Our study provides novel findings on influenza-specific TCRαβ repertoires within human tissues, raises the question of how we can prevent the loss of optimal TCRαβ signatures with aging, and provides important insights into the rational design of T cell-mediated vaccines and immunotherapies.

Divergent SATB1 expression across human life span and tissue compartments

“…To further understand whether the observed downregulation of SATB1 expression in T cells occurs following antigenic encounters, we analyzed antigen‐specific CD8 + T cells present in human adult blood for two immunodominant HLA‐A*02:01‐restricted epitopes, influenza‐derived M1 58–66 (GILGFVFTL; referred to as A2/M1 58 ) and EBV‐derived BMLF1 280–288 (GLCTLVAML; referred to as A2/GLC). IAV‐ and EBV‐specific CD8 + T cells were enriched from four HLA‐A*02:01‐positive donors using A2/M1 58 ‐ and A2/GLC‐tetramers using the well‐established tetramer‐associated magnetic enrichment approach . Enriched tetramer‐positive CD8 + T cells were then analyzed for T‐cell differentiation phenotype and co‐expression of SATB1 and the immune checkpoint protein PD‐1, shown to be pivotal in T‐cell exhaustion following chronic infections or cancer and regulated by SATB1 …”

“…HLA‐A*02:01‐GILGFVFTL (A2/M1 58 ) and HLA‐A*02:01/GLCTLVAML (A2/GLC) monomer (ImmunoID, University of Melbourne, Melbourne, VIC, Australia) was tetramerized with streptavidin‐phycoerythrin (PE) (BD Biosciences, San Jose, CA, USA). PBMCs from HLA‐A*02:01‐expressing donors were enriched for A2/M1 58 ‐ and A2/GLC‐specific CD8 + T cells using the A2/M1 58 ‐ and A2/GLC‐PE conjugated‐tetramers, respectively, followed by magnetic enrichment as previously described using anti‐PE microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Enriched, unenriched and flow‐through samples were then stained for flow cytometry.…”

“…IAV-and EBV-specific CD8 + T cells were enriched from four HLA-A*02:01-positive donors using A2/M1 58 -and A2/GLC-tetramers using the well-established tetramer-associated magnetic enrichment approach. [23][24][25] Enriched tetramer-positive CD8 + T cells were then analyzed for T-cell differentiation phenotype and co-expression of SATB1 and the immune checkpoint protein PD-1, shown to be pivotal in T-cell exhaustion following chronic infections or cancer and regulated by SATB1. 22 Data from four donors showed tetramer-positive CD8 + T populations with different mean precursor frequencies, specific for either IAV-M1 and/or EBV-GLC epitopes following tetramer-associated magnetic enrichment (Figure 4a).…”

Characterization of Human Mucosal‐associated Invariant T (MAIT) Cells

“…This protocol provides a nested multiplex PCR method for deriving the sequence of the V(D)J junction of the rearranged TCRα and TCRβ chains of MAIT cells at the singlecell level, which allows determination of TCR V(D)J usage and CDR3 loop sequences (Nguyen et al, 2017;Sant et al, 2018;Valkenburg et al, 2016;Wang et al, 2012). Single MAIT cells are isolated based on phenotypic identification as in Basic Protocol 1, but using fluorescence-activated cell sorting (FACS) as per Support Protocol 3 instead of flow cytometric analysis.…”

“…Human MAIT cells in tissues can be characterized as in Basic Protocol 1 following the generation of a single-cell suspension. As an example, the following protocol describes a method for generating a single-cell suspension from lymph nodes (LNs) using enzymatic digestion as (Koutsakos et al, , 2019Sant et al, 2018).…”