PMC

Inflated DYS⁺ CD4⁺ T cells have nearly monoclonal productive CDR3s. Productive and unique TCRβ CDR3 clones of DYS⁺ and DYS⁻ T_EM and/or T_EMRA subsets from Subjects (A) 10027, (B) 10040, (C) 10069, (D) 10032 and (E) 20. Data represent single experiments.

Inflated DYS⁺ CD4⁺ T cells have a wider but not significantly higher response magnitude of CD38⁺HLA-DR⁺ co-expression. Ex vivo comparisons of (A) CD38⁺HLA-DR⁺ (B) CD38 and (C) HLA-DR magnitudes on DYS⁺ CD4⁺ T_EM of HIV⁺ HCMV⁺ subjects to DYS⁻ CD4⁺ T_EM of HIV⁻ HCMV⁻ controls. HIV⁺ HCMV⁺: n=7, HIV⁻ HCMV⁺: n=10 and HIV⁻ HCMV⁻: n=10. Graphs represent single experiments for each subject and mean±95% CI for all subjects.

Inflated DYS⁺ CD4⁺ T cells have a CD127⁻ TIGIT⁻ Granzyme B⁺ phenotype. Ex vivo comparisons of (A) CD127, (B) PD-1, (C) TIGIT, (D) Granzyme B expressions or (E) Bcl-2 median fluorescence intensity (MFI) of DYS⁺ CD4⁺ T_EM of HIV⁺ HCMV⁺ subjects to DYS⁻ CD4⁺ T_EM of HIV⁻ HCMV⁻ controls. PBMCs were stained with tetramer and mAbs for either surface PD-1, CD127 and TIGIT or intracellular granzyme B and Bcl-2 proteins. HIV⁺ HCMV⁺: n=7, HIV⁻ HCMV⁺: n=10 and HIV⁻ HCMV⁻: n=10. Graphs represent single experiments for each subject, with mean±95% CI for all subjects.

Productive TCRβ V and J gene pairs of bulk-sorted, inflated DYS⁺ CD4⁺ T cells are highly restricted. TCRβV and TCRβJ gene family pairings of T_EM and/or T_EMRA subsets of productive DYS⁺ and DYS⁻ CD4 TCRs from Subjects (A) 10027, (B) 10040, (C) 10069, (D) 10032, and (E) 20. Data shown represent single experiments. V and J gene pairs are connected by stems between their arcs. Arc lengths reflect gene family proportions within the sample’s repertoire. High magnitude V and J gene families are emphasized.

Inflated DYS⁺ CD4 T cells are CX₃CR1^high, not replicatively senescent and do not cross-react with HIV gag proteins. A–B: Ex vivo comparisons of (A) CX₃CR1 MFI and (B) Ki-67 response magnitude of DYS⁺ CD4⁺ T_EM of HIV⁺ HCMV⁺ subjects to DYS⁻ CD4⁺ T_EM of HIV⁻ HCMV⁻ controls. HIV⁺ HCMV⁺: n=7, HIV⁻ HCMV⁺: n=10 and HIV⁻ HCMV⁻: n=10. (C) CD57 and CD28 expressions of Subject 10027 DYS⁺ and DYS⁻ CD4⁺ T cells. (D) Luciferase luminescence response of 19 DR7-presented HIV gag epitopes stimulation of Subject 10027’s DYS⁺ TCR expressed in the Jurkat cell line. (E) DYS⁺ CD4 magnitude change from tp1 (HIV viremia) to tp2 (HIV aviremia). A–B, E: graphs show mean±95% CI for all subjects and represent single experiments with no replicates except (E) showing technical replicates. D: data shows mean±SD of technical duplicates.

HLA-DR7-restricted HCMV glycoprotein B DYS-epitope specific CD4⁺ T cells undergo memory inflation in HIV⁺ HLA-DR7⁺ subjects. A–B: CD4-enriched or untouched peripheral blood mononuclear cells (PBMCs) from HIV⁺ DR7⁺ subjects were stained with DR7:CLIP or DR7:DYS tetramer for their (A) tp1 (no ART) (n=8), and (B) tp2 (on ART) samples (n=4). (C) DR7:DYS tetramer staining of HIV⁻ DR7⁺ subjects’ PBMCs (n=10). (D) Response magnitude comparisons of DYS⁺ and EPD⁺ CD4⁺ T cells from HIV⁺ HCMV⁺ DR7⁺ (n=8; tp1 of Subject 10013, and tp2 of Subjects 10004, 10027 and 10032), HIV⁻ HCMV⁺ DR7⁺ (n=10) and HIV⁺ HCMV⁺ DR7⁻ (n=5–7) subjects determined simultaneously from the same samples per subject. E–F: Longitudinal (E) response magnitudes and (F) absolute counts of DYS⁺ and EPD⁺ CD4⁺ T cells from Subjects 10027 and 10069. Values in (D) represent ≥3 biological replicates with means, except for the HIV⁺ HCMV⁺ DR7⁻ cohort with no replicates.

Expressed clonal TCR of inflated DYS⁺ CD4⁺ T cells recognize DR7-restricted DYS epitope. (A) Subject 10027’s DYS⁺ single cell α:β TCR CDR3 sequences, and NFAT-mediated luciferase luminescence response of DR7⁺ LCL-presented, serially diluted DYS epitope stimulation of DYS⁺ TCR. Subject 10027’s DYS⁺ α:β TCR gene sequences were determined from single cell sorting and expressed using plasmids in an NFAT-luciferase reporter Jurkat cell line for stimulation. Graph shows mean±SD: conditions with two data-points represent technical replicates, while those with four represent two biological replicates of two technical replicates. (B) Clonality comparison of productive CDR3s of DYS⁺ to DYS⁻ CD4⁺ T_EM and T_EMRA subsets from Subjects 10027, 10040, 10069, 10032 and 20. Clonality fractions were bioinformatically determined after productive entropy normalization. Values near 1: more clonal. (C) Fractional comparison of productive V(D)J rearrangements of DYS⁺ to DYS⁻ CD4⁺ T_EM and T_EMRA subsets from all five subjects. Values near 1: fewer out-of-frame sequences or stop codons. B–C: Data represent single experiments for each subject with mean±95% CI.

CD4⁺ T cells specific for DYS epitope consist of T_EM and T_EMRA subsets, and secrete cytokines upon DYS stimulation. (A) Response magnitude comparisons of DYS⁺ and DYS⁻ CD4⁺ T cell subsets from HIV⁺ HCMV⁺ DR7⁺ subjects (n=7). PBMCs were stained with the tetramer and memory markers to identify the subsets. Plots show grand means and represent at least two biological replicates. (B) Normalized magnitudes of CD4 subsets within DYS⁺ or DYS⁻ CD4⁺ T cells from HIV⁻ HCMV⁺ DR7⁺ subjects with sufficient tetramer⁺ response for analyses. (C) CD45RO and CD45RA staining of Subject 10027’s PBMCs. D–E: Background-corrected IFN-γ ELISpot responses of PBMCs from (D) HIV⁺ HCMV⁺ DR7⁺ (n=4; tp1) and (E) HIV⁻ HCMV⁺ DR7⁺ (n=3) subjects upon stimulation with 0.001 μg/ml of DYS, EPD, FRD, PRS, LIN epitopes, and of the following controls: HCMV pp65 overlapping 15-mer peptide pool, HIV’s gag overlapping 15-mer peptide pool and anti-human CD3. Data represent technical triplicates except in conditions without mean±SD. (F) Intracellular cytokine staining of Subject 10027’s PBMCs after DYS or SEB stimulation.