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a b Supplementary Figure 1 Supplementary Figure 1. mRNA levels of GrzB, Perforin, T-bet, and Eomes in blood and rectal mucosal CD8+ T-cells. (a) Gene expression levels of GrzB, perforin, T-bet, and Eomes in unstimulated ex vivo blood and rectal CD8+ T-cells. (b) Gene expression levels of GrzB, perforin, T-bet, and Eomes in ex vivo blood and rectal CD8+ T-cells stimulated with SEB or DMSO (vehicle control) for 2.5 hours. Gene expression levels were calculated using the delta Ct method with 18S rRNA as the reference gene. Wide horizontal bars represent medians; narrow whiskers indicate interquartile ranges.

Supplementary Figure 2 Supplementary Figure 2. Distribution of CD8+ T-cell memory/effector subsets in the blood and rectal mucosa in HIV+ and seronegative (SN) participant groups. No significant difference was detected in the frequency of CD8+ T-cell memory/effector subsets between HIV+ and seronegative participant groups in either blood or rectal mucosa. Wide horizontal bars represent medians; narrow whiskers indicate interquartile ranges.

a b Supplementary Figure 3 HIV+ SN b HIV+ SN Supplementary Figure 3. Co-expression analysis of perforin, granzyme B, T-bet, and eomesodermin in unstimulated blood (a) and rectal (b) CD8+ T-cells from HIV+ and seronegative (SN) participant groups. SPICE software was used to generate co-expression data. Wide horizontal bars represent medians; narrow whiskers indicate interquartile ranges; asterisks show level of significance as follows: * P <0.05.

a b Supplementary Figure 4 Supplementary Figure 4. Correlations between expression of perforin and GrzB and transcription factors T-bet and Eomes in blood and rectal mucosal CD8+ T-cells. (a) Correlation between the frequency of unstimulated ex vivo perforin+ CD8+ T-cells and the frequency of either T-betHigh or EomesHigh CD8+ T-cells in blood and rectal mucosa. (b) Correlation between the frequency of unstimulated ex vivo GrzB+ CD8+ T-cells and the frequency of either T-betHigh or EomesHigh CD8+ T-cells in blood and rectal mucosa. Correlations were determined using Spearman correlation and linear regression analysis.

a b c Supplementary Figure 5 Supplementary Figure 5. Neutralization of TGF-β for 24 hours increases perforin expression in rectal CD8+ T cells ex vivo. Representative flow cytometry plot showing intracellular perforin expression as measured by (a) the frequency of perforin+ CD8+ T-cells (gated as viable cells, CD3+, CD8+, and displayed versus the SSC parameter for clarity) or (b) perforin fluorescence intensity of rectal CD8+ T-cells incubated for 24h with PBS, IgG isotype control, or pan anti-TGF-β antibody (colors as indicated on right). (c) Increase in perforin expression after 24h incubation, expressed as the percentage of perforin+ CD8+ T-cells (left) or as median fluorescence intensity (MFI) of rectal CD8+ T-cells in the PE (perforin) channel (right) following incubation with pan anti-TGF-β antibody, PBS or IgG isotype control. Wide horizontal bars represent medians; narrow whiskers indicate interquartile ranges; asterisks indicate level of significance as follows: ***P<0.001.