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. 2016 Dec 15;214(12):1808-1816.
doi: 10.1093/infdis/jiw463. Epub 2016 Oct 4.

Inflammatory Function of CX3CR1+ CD8+ T Cells in Treated HIV Infection Is Modulated by Platelet Interactions

Joseph C Mudd  1   2 , Soumya Panigrahi  1 , Benjamin Kyi  1 , So Hee Moon  1 , Maura M Manion  1   2 , Souheil-Antoine Younes  1 , Scott F Sieg  1 , Nicholas T Funderburg  3 , David A Zidar  4 , Michael M Lederman  1 , Michael L Freeman  1
Affiliations

Inflammatory Function of CX3CR1+ CD8+ T Cells in Treated HIV Infection Is Modulated by Platelet Interactions

Joseph C Mudd et al. J Infect Dis. .

Abstract

Increases in inflammation, coagulation, and CD8+ T-cell numbers are associated with an elevated cardiovascular disease (CVD) risk in human immunodeficiency virus (HIV)-infected antiretroviral therapy (ART) recipients. Circulating memory CD8+ T cells that express the vascular endothelium-homing receptor CX3CR1 (fractalkine receptor) are enriched in HIV-infected ART recipients. Thrombin-activated receptor (PAR-1) expression is increased in HIV-infected ART recipients and is particularly elevated on CX3CR1+ CD8+ T cells, suggesting that these cells could interact with coagulation elements. Indeed, thrombin directly enhanced T-cell receptor-mediated interferon γ production by purified CD8+ T cells but was attenuated by thrombin-induced release of transforming growth factor β by platelets. We have therefore identified a population of circulating memory CD8+ T cells in HIV infection that may home to endothelium, can be activated by clot-forming elements, and are susceptible to platelet-mediated regulation. Complex interactions between inflammatory elements and coagulation at endothelial surfaces may play an important role in CVD risk in HIV-infected ART recipients.

Keywords: CD8+ T-cell expansion; atherosclerosis; non–AIDS-related morbidities; platelets.

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Figures

Figure 1.
Figure 1.
Circulating CX3CR1+ CD8+ T cells are increased in human immunodeficiency virus (HIV)–infected antiretroviral therapy recipients. A, Surface CX3CR1 expression on CD8+ or CD4+ T cells from a representative HIV-uninfected donor (n = 17). B, CD8+ T-cell expression of CX3CR1 and CCR7, CD27, PD-1, or KLRG-1 in a representative HIV-uninfected donor (top). Percentage of CX3CR1− (open circles) or CX3CR1+ (closed circles) CD8+ T cells that express CCR7, CD27, PD-1, and KLRG-1 in HIV-uninfected donors (n = 7–16; bottom). P values were determined by the Mann–Whitney test. C, Percentage of naive (CD45RO−CCR7+), central memory (CM; CD45RO+CCR7+; **P = .0093), effector memory (EM; CD45RO+CCR7−; ***P = .001), and terminal effector memory RA (TEMRA; CD45RO−CCR7−; ****P < .001) CD8+ T cells that express surface CX3CR1. P values were calculated by the Kruskal–Wallis test with the Dunn multiple comparisons posttest.
Figure 2.
Figure 2.
A, Percentage of CD8+ T cells that are CX3CR1+CCR7− in human immunodeficiency virus (HIV)–uninfected donors (n = 12) and HIV-infected antiretroviral therapy (ART)–recipient donors (n = 9). The P value was determined by the Mann–Whitney test. B, Expression of CX3CR1 on CD8+ T cells from the lymph nodes or peripheral blood of HIV-uninfected donors (open circles; n = 2) or HIV-infected ART-recipient donors (filled circles; n = 3). The P value was determined by the paired t test. C, Correlation of CX3CR1 expression on CD8+ T cells and the ratio of CD4+ to CD8+ T cells in HIV-infected ART recipients (n = 35). P and r values were determined by Spearman correlation analysis.
Figure 3.
Figure 3.
CX3CR1+ CD8+ T cells express the thrombin receptor protease-activated receptor 1 (PAR-1). A, Percentage of CCR7+ or CX3CR1+ CD8+ T cells that exhibit surface PAR-1 expression (n = 8). P values were determined by the Mann–Whitney test. B, Percentage of CD8+ T cells from human immunodeficiency virus (HIV)–infected antiretroviral therapy (ART) recipients that coexpress CX3CR1 and PAR-1, by cytomegalovirus (CMV) seronegativity (n = 8) or seropositivity (n = 7). The P value was determined by the Mann–Whitney test. C, CD8+ expression and tetramer/peptide complex binding on CD8+ T cells (top). CMV pp65–specific cells (red) or influenza virus M1–specific (blue) cells are indicated. CX3CR1 and PAR-1 expression on total (gray), CMV pp65–specific (red), and influenza virus M1–specific (blue) CD8+ T cells (bottom). Numbers indicate the percentage of virus-specific CD8+ T cells in each quadrant (n = 4–7). D, Percentage of CD8+ T cells that coexpress CX3CR1 and PAR-1 from HIV-infected ART-recipient donors that are CMV pp65 specific (n = 7) or influenza virus M1 specific (n = 4). The P value was determined by the Mann–Whitney test.
Figure 4.
Figure 4.
Thrombin activation induces protease-activated receptor 1 (PAR-1) internalization. Peripheral blood mononuclear cells (PBMCs) were incubated with indicated concentrations of thrombin for 5 minutes and assayed by flow cytometry. Surface PAR-1 expression on CCR7neg CD8+ T cells (experiment representative of 2; left). Surface PAR-1 was determined following incubation with 5 µM vorapaxar for 20 minutes prior to thrombin activation (experiment representative of 2; right).
Figure 5.
Figure 5.
Thrombin activation mediates CD8+ T-cell function. A, CD8+ and interferon γ (IFN-γ) expression on gated CCR7neg CD8+ T cells from 2 human immunodeficiency virus (HIV)–uninfected donors after stimulation of purified T cells for 6 hours in the presence of thrombin (0.5 U/mL), TFLLR (5 µM), or vorapaxar (5 µM), and anti-CD3/anti-CD28 (left). Percentage of CCR7neg CD8+ T cells expressing IFN-γ in purified T-cell cultures after 6 hours of stimulation with anti-CD3/anti-CD28 in the absence (0 U/mL) or presence (0.5 U/mL) of thrombin (n = 8; right). The P value was determined by the Wilcoxon matched-pairs signed rank test. B, Left IFN-γ expression among CCR7neg CD8+ T cells from an HIV-uninfected donor after 6 hours of stimulation of peripheral blood mononuclear cells (PBMCs) treated as described in panel A (left). Percentage of CCR7neg CD8+ T cells expressing IFN-γ in PBMC cultures after stimulation with anti-CD3/anti-CD28 for 6 hours in the absence (0 U/mL) or presence (0.5 U/mL) of thrombin (n = 9; right). The P value was determined by the Wilcoxon matched-pairs signed rank test. C, Change in IFN-γ expression (ΔIFN-γ) after anti-CD3/anti-CD28 (αCD3αCD28) stimulation in the absence or presence (0.5 U/mL) of thrombin, normalized to total IFN-γ expression following stimulation without thrombin, as described in “Methods” section. The P value was calculated by the Mann–Whitney test).
Figure 6.
Figure 6.
Platelets bind to CX3CR1+ CD8+ T cells. A, CX3CR1 and PSGL-1 expression on CD8+ T cells (left). Percentage of CCR7+ or CX3CR1+ CD8+ T cells that are PSGL-1hi (n = 7; right). The P value was calculated by the Mann–Whitney test. B, Acquisition of CD62P (representative of platelet binding) to CX3CR1+ CD8+ T cells in a purified T-cell/purified platelet coculture after thrombin stimulation (n = 6–8). C, Interferon γ (IFN-γ) expression and CD62P adhesion on CCR7neg CD8+ T cells after anti-CD3/anti-CD28 stimulation in the presence or absence of gel-purified platelets (n = 8). Top numbers indicate the percentage of cells that are IFN-γ+/CD62Pneg, and bottom numbers indicate the percentage of cells that are IFN-γneg/CD62P+.
Figure 7.
Figure 7.
Activated platelets modulate CD8+ T-cell function via transforming growth factor β (TGF-β) release. A, Expression of CD62P (left) or TGF-β (right) on the surface of gel-purified platelets after thrombin activation (n = 3–13). ***P < .001, by the Kruskal–Wallis test with the Dunn multiple comparisons posttest. B, Quantification of TGF-β protein by enzyme-linked immunosorbent assay in supernatants of thrombin-activated platelets (n = 5). *P = .0474 and **P = .0047, by the Kruskal–Wallis test with the Dunn multiple comparisons posttest. C, CD8+ and interferon γ (IFN-γ) expression by CCR7neg CD8+ T cells in peripheral blood mononuclear cell cultures treated with 0.5 U/mL thrombin and mouse immunoglobulin G1 (isotype), anti–TGF-β, LY2157299, or vorapaxar. D, Change in IFN-γ expression (ΔIFN-γ) after anti-CD3/anti-CD28 (αCD3αCD28) stimulation (0.5 U/mL vs 0 U/mL thrombin), normalized to total IFN-γ expression following stimulation without thrombin, as described in “Methods” section. *P = .0481, by the Kruskal–Wallis test with the Dunn multiple comparisons posttest.
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