A: Representative flow cytometry plots from an uninfected rhesus macaque, showing the gating strategy used to determine PD-1 expression on memory T cell subsets. Histograms depict PD-1 expression levels on CD8⁺ T cell subsets in the peripheral blood as indicated. PD-1 fluorescent signals in the respective CD8⁺ T cell subsets (black open histogram) are shown in an overlay with a FMO control in the same subset (grey open histogram).
B and C: Summary of data showing the frequency (%) of PD-1⁺ CD8⁺ T cells (B) and CD4⁺ T cells (C) in the indicated T cell subsets in the peripheral blood of 15 SIV-seronegative, healthy rhesus macaques. T_EM = effector memory T cells, T_CM = central memory T cells. Horizontal bars represent mean values. p-values were calculated using the Mann-Whitney nonparametric test.

A: Summary of data showing the frequency of PD-1⁺ cells (%) in naïve, T_CM, T_EM CD8+ T cell subsets, in total CD8⁺ T cells, and within CD8⁺ T cells specific for the Mamu-A*01-restricted epitopes Gag_181-189CM9 and Tat_28-35SL8, and the Mamu-A*02-restricted epitope Nef_159-167YY9 in Mamu-A*01⁺ (n=10) and Mamu-A*02⁺ (n=7) animals chronically infected with either SIVmac239 or SIVmac251. Horizontal bars represent mean values. *** p ≤ 0.0005, *** p ≤ 0.005, * p ≤ 0.05. p-values were calculated using the Kruskal-Wallis test with Dunn’s post test.
B: Representative flow cytometry plots from one scSIV-vaccinated, Mamu-A*01⁺ rhesus macaque at the indicated times post-challenge with SIVmac239, showing PD-1 expression levels on Tat_28-35SL8-specific CD8⁺ T cells (upper panel, black open histograms) and Gag_181-189CM9-specific CD8⁺ T cells (lower panel, black open histograms) in an overlay with a FMO control (grey open histograms).
C: Summary of data showing the frequency of PD-1 expression (%) on CD8⁺ T cells specific for the SIV epitopes Gag_181-189CM9, Tat_28-35SL8, and Nef_159-167YY9 at 4 weeks (left panel) and 8 weeks (right panel) post-infection with SIVmac239 in scSIV-vaccinated (colored circles) and unvaccinated (open and black circles) Mamu-A*01⁺ (n=5) and Mamu-A*02⁺ (n=4) rhesus macaques. Horizontal bars represent mean values. p-values were calculated using the Kruskal-Wallis test with Dunn’s post-test.
D: Frequency of CD8⁺ T cells specific for the SIV epitopes Gag_181-189CM9, Nef_159-167YY9, and at_28-35SL8 in the peripheral blood of Mamu-A*01⁺ (n=5) and Mamu-A*02⁺ (n=4) rhesus macaques after infection with SIVmac239. Symbols represent means, vertical bars standard deviation.
E: Table summarizing amino acid sequence variation in the Gag_181-189CM9, Tat_28-35SL8, and Nef_159-167YY9 epitopes in viral isolates from scSIV-vaccinated and unvaccinated, Mamu-A*01⁺ and Mamu-A*02⁺ rhesus macaques 8 weeks post-infection with SIVmac239. Bold letters in the epitope amino acid sequences indicate published CTL escape mutations. “wt” indicates that the sequence is identical to the SIVmac239 epitope sequence of the inoculum, listed on top of the column. Numbers in parentheses in columns marked with “#” indicate the frequency with which the associated sequence has been detected per clones sequenced. * indicates bulk PCR sequencing was performed. N/A = not applicable.
F: Summary of data showing the expression of CD28 on CD8⁺ T cells specific for the SIV epitopes Gag_181-189CM9, Tat_28-35SL8, and Nef_159-167YY9 in the Mamu-A*01⁺ and Mamu-A*02⁺ animals described in Fig. 3A. Horizontal bars represent mean values. p-values were calculated using the Mann-Whitney nonparametric test.
G: Summary of data showing PD-1 expression as the frequency of Gag_181-189CM9-specific CD8⁺ T cells (%, left panel) and PD-1 MFI on Gag_181-189CM9-specific CD8⁺ T cells (right panel) within the T_CM and T_EM CD8⁺ T cell subsets in the Mamu-A*01⁺ and Mamu-A*02⁺ animals described in Fig. 3A. Horizontal bars represent mean values. p-values were calculated using the Mann-Whitney nonparametric test.

A: Representative flow cytometry plots showing the identification of total CD8⁺ and HIV-1-specific CD8⁺ T cells using MHC class I tetramers (left panels, gated on CD3⁺ lymphocytes), and the subsequent determination of PD-1 expression on both subsets in an overlay (right panels; HIV-1-specific cells depicted by solid blue line, total CD8⁺ T cells by red dashed line). Data are from an untreated individual during chronic HIV-1 infection, following staining of cryopreserved PBMC with tetramers specific for the HLA-A*0301-restricted QK10 epitope.
B: Summary of data from elite controllers (elite, n=12) and individuals during chronic, untreated HIV-1 infection (chronic untreated, n=9), showing PD-1 expression (frequency, left panel and MFI, right panel) on total CD8⁺ T cells and cells specific for 10 immunodominant HIV-1 epitopes as represented by the colors in the legend. For the total CD8⁺ T cell population, mean values were calculated for subjects for whom PD-1 expression was determined for more than one tetramer, and each symbol represents one individual. For the HIV-specific CD8⁺ T cell population, each symbol represents one epitope-specific response. Horizontal bars represent mean values. p-values were calculated using the Mann-Whitney nonparametric test.

A and B: Summary of data showing the frequency of PD-1⁺ CD8⁺ T cells (A) or CD4⁺ T cells (B) as a percentage of the respective T cell subset in the peripheral blood of SIV-seronegative macaques (uninfected, n=14), macaques infected with SIVmac239 or SIVmac251 (wt SIV, n=24), and macaques infected with the LASIV strains SIVmac239Δnef or SIVmac239Δ3 (LASIV, n=8). T_CM: central memory T cells; T_EM: effector memory T cells. Horizontal bars represent means. *** p ≤ 0.0005, ** p ≤ 0.005, * p ≤ 0.05, ns = not significant. p-values were calculated using the Kruskal-Wallis test with Dunn’s post test. Data from uninfected animals presented in Figure 1 are shown for comparison.
C: Correlation between the frequency of peripheral PD-1⁺ CD8⁺ T_CM (open circles) and PD-1⁺ CD8⁺ T_EM (black circles) with plasma viral load in 24 rhesus macaques infected with SIVmac239 or SIVmac251. p-values were calculated using the Spearman-Rank nonparametric test.
D and E: Correlation between the frequency of peripheral PD-1⁺ total CD4⁺ T cells and viral load (D) or blood CD4⁺ T cell counts (number of CD4⁺CD3⁺ lymphocytes/μl blood, E) in 24 rhesus macaques infected with SIVmac239 or SIVmac251. p-values were calculated using the Spearman-Rank nonparametric test.

A: Representative flow cytometry plots showing PD-1 expression on CD8⁺ T cells from a Mamu-A*01⁺ rhesus macaque 21 weeks after infection with SIVmac251 (upper panel), and a Mamu-A*01⁺ rhesus macaque 58 weeks after infection with SIVmac239Δnef (lower panel). Gag_181-189CM9-specific cells were identified using MHC class I tetramers. The depicted population was gated on CD8⁺CD3⁺ lymphocytes.
B: Summary of data showing PD-1 expression as the frequency of Gag_181-189CM9-specific CD8⁺ T cells (%, left panel) and density of expression per Gag_181-189CM9-specific CD8⁺ T cell (MFI, right panel) in Mamu-A*01⁺ rhesus macaques during acute (4–7 weeks p.i., n=8) and chronic (20–70 weeks p.i., n=9) infection with SIVmac239 or SIVmac251, and Mamu-A*01⁺ rhesus macaques infected with SIVmac239Δnef (18–58 weeks p.i., n=10). Horizontal bars represent mean values. p-values were calculated using the Kruskal-Wallis test with Dunn’s post test.
C: Comparison of the frequency of PD-1⁺ Gag_181-189CM9-specific CD8⁺ T cells (% of total CD8⁺ T cells, upper panels) and the PD-1 expression levels (geometric MFI, lower panels) in animals infected with SIVmac251 (left panels, n=5), SIVmac239Δnef (middle panel, n=7 for %PD-1 and n=3 for MFI), or VSV-G trans-complemented single-cycle SIV (scSIV, right panel, n=5). Early and late time points after inoculation were as follows: SIVmac251, acute—4–7 weeks p.i., chronic—21–29 weeks p.i.; SIVmac239Δnef, acute—2 or 4 weeks p.i., chronic—12 or 15 weeks p.i.. p-values were calculated using the Wilcoxon signed rank test.