Fig 2: HIV tetramer-positive CD8 T cells express more GITR compared to total CD8 T cells in PWH on ART ex vivo(A–F) Expression of GITR in tetramer-positive (green) and tetramer-negative (black) CD8 T cells from an HIV aviremic donor is shown as a contour plot and histogram (A). The percentage of cells expressing GITR (left) and PD-1 (right) among tetramer-negative (tet-, black) and tetramer-positive (tet+, green) CD8 T cells from seven HIV aviremic donors. Columns indicate mean (B). Contour plots showing tetramer-negative (black) and tetramer-positive (green) CD8 T cells in PBMCs stimulated with cognate HIV peptide over 10 days from one representative aviremic donor. Gate shows frequency of tetramer-positive cells (C). Percentage of cells expressing GITR (D), PD-1 (E), and CD25 (F) within tetramer-positive (green) and tetramer-negative (black) CD8 T cells over time course following HIV peptide stimulation of PBMC isolated from aviremic donors (n = 7) Horizontal line indicates mean. Data were examined for normal distribution and skewed data were log transformed to potentially achieve normal distribution. Paired t test or non-parametric test (Mann-Whitney-U test) were then applied. p value <0.0001∗∗∗∗, <0.001∗∗∗, <0.1∗∗, <0.05∗, ≥0.05 ns.

Fig 3: GITR stimulation decreases cytotoxicity of tetramer-positive CD8 T cells in PWH on ART ex vivo(A–H) Graphical depiction of PBMC culture conditions including media (gray), GITR-L (blue), HIV peptide (black) or GITR-L plus HIV peptide (bright green) (A). The proportion of cell trace violet (CTV) and tetramer-positive CD8 T cells after stimulation with anti-CD3 and anti-CD28 for 4 days in different culture conditions. Contour plot (left) and histograms (right) from a representative aviremic donor in different culture conditions, i.e., media (M, gray), GITR-L (G, blue), peptide (P, black), or GITR-L plus peptide (G/P, bright green). Bar graph shows percentage of tetramer-positive CD8 T cells in the different culture conditions. Height of the bar indicates mean (B). The percentage of IFNγ+ (C) and mean fluorescence intensity (MFI) of Granzyme B (GzmB) (D) of tetramer-positive CD8 T cells after 4 days in the different culture conditions. Height of the bar indicates mean (C and D). Graphical depiction of HIV-specific CD8 T cell killing assay. PBMC from PWH on ART (aviremic) were expanded with cognate HIV peptides in presence or absence of GITR-L for 13 days. On day 13, purified autologous CD4 T cells were CTV labeled with a high and low concentration of CTV. CTV-low cells were pulsed with same peptide used to recall CD8 T cell for 13 days and then mixed with CTV-high, unloaded CD4 T cells at a 1:1 ratio. The 1:1 pool of CD4 “target” T cells was cocultured with HIV-peptide-expanded and -purified CD8 “effector” T cells at various effector:target ratios for 4 h at 37°C. A representative pseudocolor plot shows the “target” only readout (left) and the “effector:target” readout (right) demonstrating decreased frequency of the CTV-low CD4 population (red number) consistent with target specific lysis (E). Dot plots show tetramer-positive CD8 T cells expanded with peptide alone (black) or peptide+ GITR-L (green) on day 13 of culture (F). The frequency (left) and absolute cell numbers (right) of tetramer-positive CD8 T cells in those cultures is shown. Height of the bar indicates mean (G). The percentage specific lysis of HIV specific CD8 T cells cultured with CTV-stained target CD4 T cells with peptide alone (black) versus peptide plus GITR-L (bright green) at various effector:target ratios. Height of the bar indicates mean (H). Data were examined for normal distribution and skewed data were log transformed to potentially achieve normal distribution. Paired t test or non-parametric test (Mann-Whitney-U test) was then applied. p value <0.0001∗∗∗∗, <0.001∗∗∗, <0.1∗∗, <0.05∗, ≥0.05 ns.

Fig 4: Constitutive and inducible expression of GITR following TCR stimulation of T cells isolated from blood from PWH on and off ART(A–C) PBMC from HIV uninfected (U, black), PWH off ART (V, viremic; red), and PWH on ART (A, aviremic; green) were stimulated with plate-bound anti-CD3 and CD28 overnight (O/N) and expression of GITR was quantified by flow cytometry. A representative example of GITR positive CD4 T cells at baseline (unstimulated) and following stimulation with anti-CD3 and anti-CD28 (stimulated) is shown (A). The percentage of GITR positive cells (median +/− IQR) in total CD4 and CD8 T cells and T cell subsets (TEMRA and naive (TN) = naive; TCM = central memory; TEM = effector memory; TTD = terminally differentiated; TTM = transitional memory; Treg = regulatory T cells) at baseline (no stim) or after stimulation with anti-CD3 and anti-CD28 overnight (stim) and the fold change expression of GITR in stimulated compared to unstimulated cells is shown in the three participant populations (B). The percentage of PD-1+CD8 T cells is shown as a contour plot and the percentage of GITR positive cells of the PD-1+CD8 T cells (median +/− IQR) at baseline (no stim), after anti-CD3 and anti-CD28 (stim) and fold change of expression of GITR in stimulated compared to unstimulated cells is shown for the three participant populations (C). Data were examined for normal distribution using and visual inspection of frequency histograms and quantile-quantile plots. Skewed data were log transformed to potentially achieve normal distribution. Parametric (t-test) or non-parametric test (Mann-Whitney-U test) was then applied as appropriate with statistical comparisons between experimental conditions performed by paired t test or signed-rank test. p value < 0.0001∗∗∗∗, <0.001∗∗∗, <0.1∗∗, <0.05∗, ≥0.05 ns.

Fig 5: Stimulation with GITR-L of PBMC from PWH on ART ex vivo increases proliferation of CD4 Tconv and abrogates Treg suppression(A–H) Representative histogram overlay shows proliferation peaks of CTV labeled CD4 Tconv cells from PWH on ART (top histogram, gray) either cocultured with autologous, non-CTV labeled CD4 Tconv (middle histogram, purple) or autologous CD4 Tregs (bottom histogram, orange) (A). The percentage of proliferating CD4 T cells in co-cultures with different culture conditions, i.e., media (gray), GITR-L (blue), anti-CD3 antibody (black) or anti-CD3 antibody plus GITR-L (bright green). Height of the bar indicates mean (B). The fold change of proliferating CD4 Tconv cells co-cultured with either autologous CD4 Tconv or Tregs in the presence of anti-CD3 antibody (black) or anti-CD3 antibody plus GITR-L (bright green), normalized to the same culture conditions containing CD4 Tconv only. Height of the bar indicates mean (C). The percentage of proliferating CD4 T cells in co-cultures that have been stimulated with anti-CD3 antibody plus GITR-L and contain various Treg:Tconv ratios. Height of the bar indicates mean (D) Contour plot shows CD4 T cells from Treg:Tconv co-cultures, gate indicates CD4+Foxp3+Tregs. Histogram overlay shows CD25 MFI (middle) of Tregs in Tconv co-cultures stimulated with anti-CD3 antibody (black) or anti-CD3 plus GITR-L (bright green). Contour plot overlay (left) shows PD-1 and GITR expression on Tregs from those co-cultures. Bar graphs show percentage of GITR expressing Tregs and MFI of PD-1 and CD25 (E). Bar graph shows frequency of CD25+Foxp3+ Tregs from “suppressive” Tconv co-cultures (F). Histograms show proliferation peaks of CTV labeled, purified CD4 Tconv cells that have been cultured in media (gray), with GITR-L (blue) anti-CD3 antibody (black) and anti-CD3 antibody plus GITR-L (bright green). Bar graph shows percentage of proliferating CD4 Tconv from those cultures (G). Bar graph depicts memory subsets (TCM, TN, TEM, and TTD) of proliferating CD4 Tconv cells (H). Data were examined for normal distribution and skewed data were log transformed to potentially achieve normal distribution. Paired t test or non-parametric test (Mann-Whitney-U test) was then applied. p value <0.0001∗∗∗∗, <0.001∗∗∗, <0.1∗∗, <0.05∗, ≥0.05 ns.