Activation associated ERK1/2 signaling impairments in CD8+ T cells co-localize with blunted polyclonal and HIV-1 specific effector functions in early untreated HIV-1 infection

Timothy Q Crawford, Fredrick M Hecht, Christopher D Pilcher, Lishomwa C Ndhlovu, Jason D Barbour, Timothy Q Crawford, Fredrick M Hecht, Christopher D Pilcher, Lishomwa C Ndhlovu, Jason D Barbour

Abstract

We recently observed that a large proportion of activated (CD38(+)HLA-DR(+)) CD8(+) T cells from recently HIV-1-infected adults are refractory to phosphorylation of ERK1/2 kinases (p-ERK1/2-refractory). Given that the ERK1/2 pathway mediates intracellular signaling critical for multiple T cell functions, including key effector functions, the loss of ERK1/2 responsiveness may have broad consequences for CD8(+) T cell function. In the current study, we hypothesized that the p-ERK1/2-refractory population, localized largely within the activated CD38(+)HLA-DR(+) CD8(+) T cell population, would display impairments in CD8(+) T cell effector functions, such as cytokine production and degranulation, compared to CD8(+) p-ERK1/2-responsive cells. We further hypothesized that the p-ERK1/2-refractory phenotype is persistent over time during untreated infection, and would correlate with poorer virologic control, in a manner independent of CD8(+) T cell activation level. We performed single-cell resolution, flow cytometric assays of phospho-kinase responses paired to intracellular cytokine staining in one assay to examine IFN-γ, perforin and CD107α responses in CD8(+) T cells by ERK1/2 signaling profile. On a per cell basis, p-ERK1/2-refractory cells, which fall predominantly within the activated CD8(+) T cell compartment, produced less IFN-γ in response to polyclonal or HIV-1 antigen-specific stimulation, and expressed lower levels of perforin and CD107α. The p-ERK1/2 refractory cell population displayed minimal overlap with the PD-1 and Tim-3 inhibitory exhaustion markers and predicted high viral load independent of activation, suggesting that ERK1/2 may be a unique marker and point of intervention for improving CD8(+) T cell function. Blunted effector functions, secondary to ERK1/2 signaling deficits concentrated within activated CD8(+) T cells, may contribute to immunodeficiency and underlie the predictive capacity of CD8(+) T cell activation on HIV-1 disease progression. (270/300).

Conflict of interest statement

Competing Interests: Jason Barbour serves as an academic editor for PLOS ONE. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1. p-ERK1/2-Refractory CD8 + T cells…
Figure 1. p-ERK1/2-Refractory CD8+ T cells exhibit low per cell effector function in response to polyclonal stimulation.
(A–E) Response of total CD8+ T cells to140 minutes PMA+I. (A) 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).
Figure 2. p-ERK1/2-refractory CD8 + T cells…
Figure 2. p-ERK1/2-refractory CD8+ T cells exhibit low per cell effector function in response to HIV-1 Gag stimulation.
(A–E) Response of total CD8+ T cells to 12 hours HIV-1 Gag peptides and 20 minutes PMA+I. (A) 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).
Figure 3. p-ERK1/2-refractory CD8 + T cells…
Figure 3. p-ERK1/2-refractory CD8+ T cells are distinct from classical exhaustion, remain stable over time and predict HIV-1 viral load.
(A–D) CD8+ T cells following 20 minutes PMA+I. (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).

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