Study Participant Characteristics.

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

HIV-1 Gag CD8⁺ T Cell Effector Capacity by ERK1/2 Signaling Ability.

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

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

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

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

Comparison of measured urine biomarkers of HIV-infected participants on ART with and without albuminuria⁽^a⁾.

<p>Comparison of measured urine biomarkers of HIV-infected participants on ART with and without albuminuria⁽<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153758#t004fn001" target="_blank">^a</a>⁾.</p

Comparison of immunological parameters of HIV-infected participants on ART with and without albuminuria⁽^a⁾.

<p>Comparison of immunological parameters of HIV-infected participants on ART with and without albuminuria⁽<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153758#t002fn001" target="_blank">^a</a>⁾.</p

Multivariable linear regression analysis^{(^a)} of non-classical (CD14^+/lowCD16⁺⁺) monocyte counts as a predictor of albuminuria in HIV-infected participants on ART while adjusting for risk factors (n = 96).

<p>Multivariable linear regression analysis^{(^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153758#t003fn001" target="_blank">a</a>})} of non-classical (CD14^+/lowCD16⁺⁺) monocyte counts as a predictor of albuminuria in HIV-infected participants on ART while adjusting for risk factors (n = 96).</p

Comparison of demographic and clinical parameters of HIV-infected participants on ART with and without albuminuria⁽^a⁾.

<p>Comparison of demographic and clinical parameters of HIV-infected participants on ART with and without albuminuria⁽<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153758#t001fn001" target="_blank">^a</a>⁾.</p