Polyclonal CD8⁺ T Cell Effector Capacity by ERK1/2 Signaling Ability.

†<p>IQR = Interquartile Range.</p>††<p>GMF = Geometric Mean Fluorescence Intensity.</p

Study Participant Characteristics.

†<p>IQR = Interquartile Range.</p>††<p>ART = Antiretroviral Treatment.</p

p-ERK1/2-Refractory CD8⁺ T cells exhibit low per cell effector function in response to polyclonal stimulation.

<p>(A–E) Response of total CD8+ T cells to140 minutes PMA+I. (<b>A</b>) Gating for CD8⁺ p-ERK1/2-refractory versus responsive T cell subsets. (B) Frequency of p-ERK1/2-refractory cells. (C) Gating for IFN-γ (dashed gates) and perforin (solid gates) expression within p-ERK1/2 subsets. (D) IFN-γ expression by ERK1/2 signaling response. Left graph displays frequency of IFN-γ⁺ cells contained within the parent population. Right graph, the IFN-γ geometric mean fluorescence intensity (GMF) of IFN-γ⁺ cells (E) Perforin expression by ERK1/2 signaling response. Left graph, frequency of perforin⁺ cells. Right graph, perforin GMF of perforin⁺ cells. (F–G) Response of highly activated (CD38+HLA-DR+) CD8+ T cells. (F) Gating for p-ERK1/2-refractory versus responsive subsets. (G) Frequency of p-ERK1/2-refractory cells within the CD38+HLA-DR+ compartment. (H) Gating for IFN-γ and perforin expression within activated p-ERK1/2 subsets. (I) IFN-γ expression by ERK1/2 signaling response: Left graph, frequency of IFN-γ⁺ cells. Right graph, IFN-γ GMF of IFN-γ⁺ cells. (J) Perforin expression by ERK1/2 signaling response. Left graph, frequency of perforin⁺ cells. Right graph, perforin GMF in perforin⁺ cells. Significance Not Significant (NS) p>0.01, Marginal (M) p<0.01, *p<0.05, **p<0.005, ***p<0.0005. (A,C,F,H, n = 1; D,E,I,J, n = 19).</p

p-ERK1/2-refractory CD8⁺ T cells are distinct from classical exhaustion, remain stable over time and predict HIV-1 viral load.

<p>(A–D) CD8⁺ T cells following 20 minutes PMA+I<b>.</b> (A) Panels from left to right: ERK1/2 phosphorylation in total CD8⁺ T cells. Gating for PD1 expression. Gating for p-ERK1/2-refractory versus responsive subsets within the PD1⁺ compartment. (B) Frequency of p-ERK1/2-refractory cells within the PD1⁺ compartment. (C) Panels from left to right: The ERK1/2 phosphorylation response in total CD8⁺ T cells, Gating for Tim-3 expression in total CD8⁺ T cells. Gating for Tim-3 expression in total CD8⁺ T cells. (D) Frequency of p-ERK1/2-refractory cells contained within the Tim-3⁺ compartment. (E–F) Smoothed moving average plots displaying the frequency of p-ERK1/2-refractory (E) and CD38⁺HLADR⁺ (F) CD8⁺ T cells from HIV-1-infected treatment-naïve adults followed longitudinally over the first 2.5 years of infection. (G) Lowess plots displaying average viral load over time in patients stratified by high or low p-ERK1/2-refractory measurement at study entry. Open squares with black line represents individuals with a first clinical visit % p-ERK1/2-refractory CD8⁺ T cell measurement above the median frequency. Closed triangles with grey line represents individuals below the median frequency. Individuals with a high p-ER1/2-refractory measurement during early infection maintain significantly higher viral loads over time. (A,C n = 1; B n = 11; D, n = 20; E–F, n = 27 with 2–4 time points per individual, G, n = 74).</p

p-ERK1/2-refractory CD8⁺ T cells exhibit low per cell effector function in response to HIV-1 Gag stimulation.

<p>(A–E) Response of total CD8⁺ T cells to 12 hours HIV-1 Gag peptides and 20 minutes PMA+I. (<b>A</b>) Gating for CD8⁺ p-ERK1/2-refractory versus responsive T cell subsets. (B) Frequency of p-ERK1/2-refractory cells. (C) Gating for IFN-γ (dashed gate) and perforin expression (solid gate) within p-ERK1/2 subsets. (D) IFN-γ expression by ERK1/2 signaling response. Left graph displays frequency of IFN-γ⁺ cells contained within the parent population. Right graph, the IFN-γ geometric mean fluorescence intensity (GMF) of IFN-γ⁺ cells (E) Perforin expression by ERK1/2 signaling response. Left graph, frequency of perforin⁺ cells. Right graph, perforin GMF of perforin⁺ cells. (F–J) Response of highly activated (CD38⁺HLA-DR⁺) CD8⁺ T cells. (F) Gating for p-ERK1/2-refractory versus responsive subsets. (G) Frequency of p-ERK1/2-refractory cells within the CD38⁺HLA-DR⁺ compartment. (H) Gating for IFN-γ and perforin expression within activated p-ERK1/2 subsets. (I) IFN-γ expression by ERK1/2 signaling response: Left graph, frequency of IFN-γ⁺ cells. Right graph, IFN-γ GMF of IFN-γ⁺ cells. (J) Perforin expression by ERK1/2 signaling response. Left graph, frequency of perforin⁺ cells. Right graph, perforin GMF in perforin⁺ cells. (K–L) CD8⁺ T cells, (K) Gating for IFN-γ⁺CD107α⁺ expression and frequency of IFN-γ⁺CD107α⁺ cells within p-ERK1/2 subsets. (M) CD107α expression within IFN-γ⁺ cells by ERK1/2 signaling response: Left graph, frequency of CD107α⁺ cells. Right graph, CD107α GMF of CD107α⁺ cells. Significance Not Significant (NS) p>0.01, Marginal (M) p<0.01, *p<0.05, **p<0.005, ***p<0.0005. (A,C,F,H,K, n = 1; D,E,I,J, n = 30; L,M, n = 14).</p

Activation levels on total and antigen specific T cells by differentiation stage.

<p>In the first column the proportion of total T cells (CD8+ and CD4+) expressing CD38 and HLA-DR activation markers by T cell maturation category are displayed and defined as early memory (CD27+CD28+) EM, intermediate memory (CD27+CD28−) IM, and late memory (CD27−CD28−) LM. In columns 2 the CD38 MFI level is shown by maturation stage for CD8+ T cell IFN-γ responses to both HIV-1 Gag (red) and CMV pp65 (green). And in column 3 the CD38 MFI is shown for each maturation stage of CD4+ T cell IFN-γ responses to HIV-1 Gag (red) and CMV pp65 (green). Row 1 displays measurements for visit 1, prior to antiretroviral therapy. Row 2 displays measurements for visit 2, during a virologically suppressive anti-retroviral regimen, and row 3 displays measurements for visit 3, after patients had halted an anti-retroviral regimen and viremia had rebounded. Black bars indicate differences between activation levels by response categories (Wilcoxon 2 Sample Test). Red dots notes a significant change from baseline (visit 1) values (Sign Rank Test).</p

T cell activation declines during anti-retroviral therapy but maturation profile of Gag specific CD8+ T cells does not change

<p>HIV-1 RNA levels, total CD8+ T cell activation levels, HIV-1 specific CD8+ T cell activation levels and the proportion of HIV-1 Gag specific IM and LM CD8+ T cells at visit 1 (Pre-ART), visit 2 (On ART) and visit 3 (Post-ART). P-values for changes in viral load, Total and HIV-1 specific CD8+ T cell activation are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004408#pone-0004408-t001" target="_blank">Table 1</a>. HIV-1 RNA, total CD8+ T cell activation levels and HIV-1 gag specific CD8+ T cell activation levels all declined after initiation of ART. Black bars display p-values for a test of change in HIV-1 Gag specific CD8+ T cell IM and LM fractions from visit 1 to visit 2, and visit 3. Neither the Gag specific IFN-γ+ CD8+ IM or LM pool changed significantly from pre-therapy levels after therapy was initiated (On ART), or later halted (Post-ART).</p

A More Differentiated Gag Specific CD8+ T Cell Response is Protective in Early HIV-1 Infection.

<p>Spearman Correlation results shown. Correlation performed on data from pre-treatment, Visit 1. Patients with higher CD4+ T cell counts during early infection and prior to anti-retroviral therapy tended to have higher HIV-1 Gag specific IFN-γ late memory (CD27−CD28−) CD8+ T cells</p

Expression of Maturation and Activation markers on Total CD4+ and CD8+ T cells.

<p>(A): before initiation of ART (i); after treatment (ii); and post-ART (iii). Activation is also shown on CD27 and CD28 subsets from CD8+ T cells post-ART (iv). Expression of Maturation and Activation markers on HIV Gag- and CMV pp65-specific T cells (B). CD27 and CD28 expression and CD38 mean fluorescent intensity (MFI) is shown on IFN-γ+ CD8+T cells. (GM = Geometric Mean).</p

Relationship of Total CD8+ T Cell Activation to T Cell Maturation Profiles.

<p>Relationship between total CD8+ T cell activation levels at visit 1 (pre-treatment) on the x-axis and the maturation profile of CD8+ and CD4+ T cell populations, total or antigen specific. In the first panel total CD8+ T cell activation is higher when a greater fraction of total CD8+ T cells fall in the intermediate memory (CD27+CD28−) phenotype. In the second panel total CD8+ T cell activation levels are higher when a greater fraction of HIV-1 Gag specific CD8+ T cells fall in the intermediate memory (CD27+CD28−) phenotype. In the third panel total CD8+ T cell activation levels are lowest when a lower fraction of HIV-1 Specific CD4+ T cells fall into the early memory (CD27+CD28+) phenotype.</p