Expression of PD-L1 and PD-L2 on human macrophages is up-regulated by HIV-1 and differentially modulated by IL-10

Abstract

PD-1 plays an important role in T cell exhaustion during HIV infection. PD-1 has two ligands: PD-L1, expressed on hematopoietic and nonhematopoietic cells, and PD-L2, limited to DCs and macrophages. Little is known about PD-L1 expression and regulation in human macrophages. Previous reports have found few immediate effects of macrophage exposure to HIV, suggesting that macrophages lack PRRs for this virus. Using quantitative confocal microscopy and a multiplexed cytokine bead array, we measured induction of PD-L1, PD-L2, and innate response cytokines in human MDMs in response to chemically inactivated HIV virions. Consistent with previous reports, no cytokines were induced by HIV virion exposure. Whereas PD-L1 and PD-L2 had low baseline expression, TLR ligands (LPS and CL097) up-regulated PD-L1 but not PD-L2. Unlike what we found for cytokine expression, PD-L1 and PD-L2 were up-regulated in response to exposure with inactivated HIV virions or with replication-competent HIV. Expression of PD-L1 was differentially modulated by IL-10, which induced up-regulation of PD-L1 but not of PD-L2, and IL-10 blockade enhanced only PD-L2 expression. We discuss implications for innate recognition of HIV by macrophages and potential, different roles for PD-L1 and PD-L2 in immunity and pathogenesis.

Figure 1.. Expression of PD-L1 and PD-L2 by human MDM.

MDMs were differentiated on coverslips for 5 days and stimulated with or without LPS [nonstimulated (NS)] for 48 h. PD-L1 and PD-L2 expression was analyzed by laser-scanning confocal microscopy and quantified with Metamorph software. (A) Confocal microscopy images corresponding to PD-L1 (upper panels) and PD-L2 (lower panels) staining with respective isotype controls. MDMs strongly up-regulated PD-L1 (upper right) but not PD-L2 (lower right) expression after LPS treatment. Blue represents DAPI-stained cell nuclei, and red shows PD-L1 or PD-L2 as indicated. Original magnification, 20×. (B) Quantitative analysis of PD-L1 and PD-L2 fluorescence intensity. Bars represent the mean value ± sem from the analysis of four different confocal fields for each condition. ***P < 0.0001. Representative result of four independent experiments with different donors. (C) Percent of PD-L1 (black bars)- or PD-L2 (gray bars)-positive cells in the presence or absence of LPS. LPS stimulation did not alter the percent-positive or intensity of staining with isotype control (not shown). Bars represent mean value ± sem from four independent experiments with different donors.

Figure 2.. Up-regulation of PD-L1 after exposure to HIV.

(A) Expression of PD-L1 (upper panels) and PD-L2 (lower panels) in MDMs by confocal microscopy after stimulation with control MV (second column), AT-2 HIV (third column), or CL097 (right column). MV are an AT-2-treated preparation lacking HIV virions. Blue represents DAPI-stained nuclei, and red represents PD-L1 or PD-L2 as indicated. (B) Quantitative analysis of the confocal microscopy fields with Metamorph software. Bars represent the mean value ± sem from the analysis of four different microscopy fields for each condition. Representative result of six independent experiments with six different donors. ***P < 0.0001; **P < 0.001; *P < 0.05. (C) Magnified view of HIV- or LPS-stimulated MDMs to illustrate morphological differences observed under different conditions.

Figure 3.. Cytokine secretion by MDM.

Supernatants from MDM cultures stimulated under different conditions were analyzed by Luminex for cytokine secretion. LPS and CL097 treatments significantly induced cytokine secretion, however no differences were found between control conditions and AT-2 HIV-exposed cells. Bars represent the mean ± sem from four different donors. ***P < 0.0001; **P < 0.001; *P < 0.05. For all conditions, including untreated controls, levels of IL-8 were above the maximum standard, and levels of IL-12p70 were below the level of detection (data not shown).

Figure 4.. Modulation of PD-L1 in MDMs by IL-10.

(A) Laser-scanning confocal microscopy images of nonstimulated MDMs or AT-2-HIV-stimulated macrophages (third column) plus IL-10 or plus IL-10 blockade (right column). Blue represents DAPI-stained nuclei, and red shows PD-L1 or PD-L2 as indicated. (B) Quantitative analysis of fluorescent cell intensity of nonstimulated macrophages (white bars) and AT-2 HIV-stimulated macrophages (black bars) in the presence of IL-10 stimulation or IL-10 blockade. Bars represent the mean value ± sem from the analysis of four different microscopy fields for each condition. Representative result of three independent experiments for AT-2-stimulated macrophages and two independent experiments for nonstimulated macrophages. Consistent with data shown in Fig. 2, PD-L2 expression in HIV-treated MDMs was significantly higher than untreated cells in a direct comparison, but significance of enhancement was lost after correction for multiple comparisons shown here. ***P < 0.001; **P < 0.01. (C) Secretion of TNF-α by MDMs treated with AT-2 HIV in the absence (gray bars) and presence (black bars) of anti-IL-10-blocking antibodies. Right panel shows fold increased in TNF-α secretion in anti-IL-10-blocking antibody-treated MDMs (black bar) relative to untreated MDMs (gray bar); bars represent mean ± sem from three different donors.

Figure 5.. Up-regulation of PD-L1 in MDMs after HIV-infection.

(A) Laser-scanning confocal microscopy images of uninfected MDMs (NI; left panels) or HIV-BaL-infected macrophages (BaL; right panel) after 7 days of infection. Blue represents DAPI-stained nuclei, and red shows PD-L1 or PD-L2 as indicated. (B) Quantitative analysis of fluorescent cell intensity of uninfected macrophages (white bars) and HIV-BaL-infected macrophages (BaL; black bars). Bars represent the mean value ± sem from the analysis of four different microscopy fields for each condition. ***P < 0.001. (C) Flow cytometry analysis of HIV-infected MDMs, which were stained for PD-L1, PD-L2, and intracellular p24 after 7 days of infection. Macrophage population was separated into p24-negative and p24-positive cells. Overlay histrograms represent the uninfected control (filled gray histograms), the HIV-BaL- or HIV-NL4.3 p24-negative population (dashed lines), and p24-positive population (solid lines). SSH, side scatter height. (D) Flow cytometry analysis of AT-2-HIV-treated MDMs after 2 days of stimulation. Histograms represent the unstimulated cells (gray filled histogram) and the AT-2 HIV-stimulated cells (solid lines). (E) Mean fluorescence intensity fold increase for PD-L1 and PD-L2 in AT-2-HIV-treated cells (gray bars), in the whole population of HIV-BaL (BaL; black bars)-infected cells, and in the p24-positive cells (BaL p24+; black bars). Bars represent the mean ± sem from three independent experiments.