(A–D) GrB activity or ICE with D#0 (A and B) or D#6 (C and D) cells plotted against true E:T ratios based on measurements of IFN-γ-secreting cells (Figure 4B) and p24-expressing targets (Figure 4D, top panel). GrB activity is shown after subtraction of background. Analysis of covariance was used for quantifying the difference in GrB activity and ICE in LTNPs and progressors over the range of E:T ratios.

(A and B) Representative marker expression after 6 day Gag peptide stimulation (A) or 6 day stimulation followed by 6 hr Gag peptide restimulation (B) is shown for an HLA B*57⁺ LTNP (top row) and progressor (bottom row). Values indicate the percentage of gated tetramer⁺ CD8⁺ T cells.
(C) Summary pie charts showing median percentage expression of GrB, perforin, and CD107 in LTNPs (n = 8, top row) and progressors (n = 8, bottom row).

(A) HIV-1 RNA amounts quantified to 1 copy/ml were compared between LTNPs (n = 27) and treated progressors who maintain <50 copies/ml (Rx < 50, n = 50).
(B) Total IFN-γ-producing HIV-specific CD8⁺ T cell frequencies for individuals in (A).
(C) Comparison of HIV-1 RNA amounts between LTNPs (n = 19) and Rx < 50 (n = 28) with detectable viremia (≥1 copy/ml).
(D) Total IFN-γ-producing HIV-specific CD8⁺ T cell frequencies for individuals in (C). Horizontal lines indicate median values. Comparisons were made with the Wilcoxon two-sample test. Only significant p values are shown.

(A and B) Summary data of the total cytotoxic response with GrB activity (A) or ICE (B) in LTNPs (n = 18), progressors (n = 18 and 19 in [A] and [B], respectively), and Rx < 50 (n = 16). Data are representative of three experiments. Comparisons were made with the Wilcoxon two-sample or signed-rank tests. Horizontal lines indicate median values. Only significant p values are shown.
(C) With D#6 CD8⁺ T cells, GrB target cell activity correlates directly with ICE (n = 52).
(D and E) With D#6 CD8⁺ T cells, the perforin content of HIV-specific CD8⁺ T cells directly correlates with both GrB target cell activity (D) and ICE (E) in a subset of LTNPs, viremic progressors, and Rx < 50 (n = 17). Statistical analyses were performed with the Spearman correlation.

(A) Gating scheme for identifying three cell populations (right plot) is shown: CD8⁺ T cell effectors (target LIVE DEAD label negative), CD4⁺ T-lymphoblast targets (LIVE DEAD label positive), and cells that have died prior to the incubation of CD8⁺ T cells and CD4⁺ T cell targets (LIVE DEAD label bright, off scale).
(B) D#0 and D#6 HIV-specific CD8⁺ T cells are measured as percentages of CD3⁺ CD8⁺ lymphocytes expressing IFN-γ.
(C) GrB activity in gated targets after adding no cells, D#0 cells, or D#6 CD8⁺ cells in a representative LTNP. Red values indicate percentages of targets with increased GrB activity after subtracting background values (data not shown), which were derived from measurements of GrB activity in uninfected targets mixed with D#0 (for middle panel) or D#6 cells (for bottom panel), respectively.
(D) Cells from (C) after fixation, permeabilization, and staining for CD4 and intracellular p24 expression. Quadrant values indicate percentages of gated targets. Infected cell elimination (ICE) was calculated with p24⁺ targets (sum of upper quadrants) as described in Experimental Procedures.

(A–D) After anti-CD3+anti-CD28 (A and C) or PMA+Io (B and D) treatment, PBMCs were incubated for 18 days and then stimulated with Gag peptides and IL-2 (2 IU/ml) for 6 more days. Some cells, which had been CFSE labeled on day 18, were stained with three B57- or 2 B27-HIV Gag tetramers and assessed for proliferation on day 24 (A and B). Quadrant values indicate percentages of gated CD8⁺ T cells. GrB activity in peptide-pulsed targets was measured with day 24 cells (C and D). Values indicate percentages of targets after subtracting background.
(E) Summary data of GrB activity plotted against E:T ratios of D#6 cells incubated with Gag peptides or 24 days after treatment with CD3+CD28 antibodies or PMA+Io in three B*27⁺ and seven B*57⁺ progressors. Linear mixed and generalized estimating equations approaches were used for inference.
(F) Representative plots of Image Stream analysis of CD8⁺ T cells incubated in medium alone, HIV KF11 peptide, and PMA+Io control conditions. Representative images corresponding to quadrants are shown in lower panels.
(G) Summary data of NFAT nuclear translocation in HIV tetramer⁺ cells for LTNPs (n = 17), viremic progressors (n = 22), and Rx < 50 (n = 13). Horizontal lines indicate median values. Comparisons were made with the Wilcoxon two-sample test. Only significant p values are shown.

(A) GrB activity in PBMC targets pulsed with three B57-restricted Gag epitopes after adding day 0 (D#0, center column) or day 6 (D#6, right column) cells in a B*57⁺ LTNP (top row) and progressor (bottom row). Values indicate percentages of targets with increased fluorescence due to GrB substrate cleavage. Red values indicate GrB activity after subtracting background (left column for D#0 cells and not shown for D#6 cells).
(B) Light-scatter characteristics of gated targets from (A).
(C) Measurement of peptide-specific CD8⁺ T cells with three B57 HIV tetramers complexed to the same peptides used in (A) and (B). Values indicate the percentage of gated CD8⁺ T cells.
(D and E) Summary data of the total cytotoxic response (sum of the individual responses when more than one epitope was recognized) with D#0 cells of LTNPs (red circles, n = 8) and progressors (blue circles, n = 15, [D]) or D#6 cells of LTNPs (n = 16) and progressors (n = 24, [E]). Horizontal lines indicate median values. Comparisons were made with the Wilcoxon two-sample test. Only significant p values are shown.
(F and G) Data in (D) and (E) plotted against E:T ratios on the basis of HIV-tetramer frequencies (C). Curves represent trends for LTNPs (red) and progressors (blue). Analysis of covariance was used for quantifying the logit of GrB activity over the range of logged E:T ratios.