Study Participant Characteristics.

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

Per cell expression levels of IL-1β and TNF by each monocyte subset before and after stimulation.

<div><p>Geometric mean fluorescence (GMF) intensities are shown by monocyte subset for IL-1β (left column) and TNF (right column) in the basal (top row) state, and after oxLDL (middle row) or LPS (bottom row) stimulation. In HIV-infected subjects, IL-1β was expressed at highest levels by the Mono1 (CD14++CD16-) and Mono2 (CD14++CD16+) populations. TNF expression was observed in all subsets after stimuli, with the brightest monocyte subsets being Mono2 (CD14++CD16+) and Mono3 (CD14+ CD16+). Statistical significance was adjusted for multiple comparisons (p = 0.05/4 comparisons per stimulation condition: p = 0.0125).</p> <p>**** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.0125.</p></div

Monocyte production of pro-inflammatory cytokines in the basal state and upon stimulation.

<div><p>HIV-infected subjects are shown in red circles and HIV-uninfected subjects are shown in blue triangles. In the no stimulation condition (basal state), HIV-infected subjects showed higher levels of IL-1β (upper left panel) and IL-8 (lower left panel). Upon stimulation with either oxLDL or LPS, HIV-infected subjects exhibited higher levels of IL-1β, IL-8 and IL-6 (upper right panel) compared to the HIV-uninfected subjects. While HIV-1 infected subjects did tend to have higher TNF (lower right panel) responses upon stimulation, these differences were not significant.</p> <p>**** p < 0.0001, *** p < 0.001, ** p < 0.01.</p></div

Gating strategy for identification of total monocytes, monocyte subsets and detection of cytokine expression.

<p>Gating Strategy. Panel A. Identification of total monocytes from peripheral blood mononuclear cells by exclusion of doublets, dead cells, CD3, CD56, CD19, CD20 and low HLA-DR expressing cells. Double-negative CD14-CD16- cells were also excluded. Forward scatter (FSC) vs. side scatter (SSC) plots are shown comparing total PBMC (doublet and dead cells excluded) and total monocytes. Panel B. Monocyte subsets were identified based on the expression of CD14 and CD16 (left column). The diagram at the bottom of the left column is a visual guide for the terminology of monocyte subsets employed in this report. Intracellular cytokines (IL-1β, IL-8/CXCL8, IL-6 and TNF) produced in total monocytes were detected in response to no stimulus, oxidized low density lipoprotein (oxLDL) or lipopolysaccharide (LPS). Fluorescence minus one control condition, in which the antibody conjugate in question is omitted to guide creation of the gate that defines positive expression of that target, is shown on the bottom row. The subject presented is HIV-infected and displays high but representative responses to stimuli.</p

HIV-1 infected subjects with well-controlled viremia have a greater proportion of classical (Mono1, CD14++CD16-) monocytes and a lower frequency of intermediate (Mono2, CD14++CD16+) monocytes, which correlates with production of IL-1β.

<p>HIV-infected subjects are shown in red circles, and HIV-uninfected subjects are shown in blue triangles. Panel A. HIV-infected subjects had a greater fraction of monocytes that fall into the Mono1 (CD14++CD16- classical) subset, and a lower fraction that fall into the Mono2 (CD14++CD16+ intermediate) subsets. Upper right diagram represents monocyte gating scheme (Mono 1-4). Panel B. A lower proportion of monocytes in the Mono2 subset was associated with higher basal IL-1β production. **** p < 0.0001, *** p < 0.001,.</p