Increased B7-H1 expression on dendritic cells correlates with programmed death 1 expression on T cells in simian immunodeficiency virus-infected macaques and may contribute to T cell dysfunction and disease progression

Abstract

Suppression of dendritic cell (DC) function in HIV-1 infection is thought to contribute to inhibition of immune responses and disease progression, but the mechanism of this suppression remains undetermined. Using the rhesus macaque model, we show B7-H1 (programmed death [PD]-L1) is expressed on lymphoid and mucosal DCs (both myeloid DCs and plasmacytoid DCs), and its expression significantly increases after SIV infection. Meanwhile, its receptor, PD-1, is upregulated on T cells in both peripheral and mucosal tissues and maintained at high levels on SIV-specific CD8(+) T cell clones in chronic infection. However, both B7-H1 and PD-1 expression in SIV controllers was similar to that of controls. Expression of B7-H1 on both peripheral myeloid DCs and plasmacytoid DCs positively correlated with levels of PD-1 on circulating CD4(+) and CD8(+) T cells, viremia, and declining peripheral CD4(+) T cell levels in SIV-infected macaques. Importantly, blocking DC B7-H1 interaction with PD-1(+) T cells could restore SIV-specific CD4(+) and CD8(+) T cell function as evidenced by increased cytokine secretion and proliferative capacity. Combined, the results indicate that interaction of B7-H1-PD-1 between APCs and T cells correlates with impairment of CD4(+) Th cells and CTL responses in vivo, and all are associated with disease progression in SIV infection. Blockade of this pathway may have therapeutic implications for HIV-infected patients.

Figure 1

(a) Rhesus macaque myeloid DC and plasmacytoid DC were identified in blood as HLA-DR+, lineage negative (Lin-) cells as depicted. PBMCs were first selected based on side scatter and HLA-DR+ (R1 gate) and further defined as HLA-DR+ Lin- cells (R2 gate). CD11c+HLA-DR+ (mDCs) and CD123+ HLA-DR+ (pDCs) events within R2 were defined in R3 and R4, respectively. B7-H1 expression on macaque mDCs or pDCs in blood is shown in representative histograms. (b and c) Expression of B7-H1 on myeloid DC and plasmacytoid DC from blood and mucosal tissues in SIV-infected macaques. Note marked and significant increases in B7-H1 expression in blood, mes LN, and jejunum pDCs and mDCs in SIV infected animals in acute and chronic infection. Mean values, SE, and statistically significant differences are shown.

Figure 2

Microarchitecture of the lymph node and expression of PD-1 in tissues post SIV infection. (a,b) Representative Hematoxylin and Eosin (H&E) staining of lymph node sections from a normal (top left, a) and SIV-infected macaque (top right, b; 3 months p.i). Scale bar=100μm. (c and d) Representative PD-1 expression in lymph node of a normal (c) and SIV-infected macaque (d) demonstrating markedly expanded populations of PD-1+ cells in follicular regions after SIV infection (brown). Scale bar = 50 μm. (e) Lymph node from a representative normal macaque showing distribution of B cells (CD20+; red), macrophages (CD68+; blue) in relation to normal PD-1 expression within B cell zones and germinal centers (inset shown in Figure to the right as indicated). (f) Lymph node from a chronically infected macaque (3 mos) showing markedly expanded germinal centers and increased PD-1 expressing cells specifically within germinal centers. Note that PD-1 is expressed only on cells with a lymphocyte morphology and not on B cells or macrophages/DC. Colors indicate B cells (CD20+ red); macrophages (CD68+, blue) and PD-1 (green).

Figure 3

(a) Distribution and co-expression of PD-1 on CD4+ T (CD3+) cells in lymph nodes of a representative SIV-infected macaque by confocal microscopy. Immunohistochemistry demonstrates PD-1 (green) expression is primarily co-expressed on CD4+ T cells in germinal centers in chronic SIV infection. (scale bar = 20μm). (b) Co-localization of B7-H1 (red) and PD-1 (green) expression in lymph node of a chronically infected macaque 3 months after infection. Note B7-H1+ cells (red) have a dendritic morphology and co-localize with PD-1+ cells (green) which have a lymphocyte morphology and shown to be CD4+ T cells in (a). B7-H1+ cells (red) are mostly found in follicular areas, and usually in close proximity and often direct contact with PD-1+CD4+ T cells. (scale bar = 20μm).

Figure 4

PD-1 expression on T cells in blood, mesenteric LN and lamina propria in SIV-infected macaques. (a, b and c) Percentage of PD-1 expression on CD3+ (a), CD4+ (b) and CD8+ T cells (c) in normal, acute, chronic, and AIDS stage infection, and symptomatic AIDS. Note marked expansion of PD-1+ T cells occurs and persists in most tissues throughout SIV infection. (d) Surface PD-1 is predominantly expressed on SIV-specific CD8+ T cells clone (CM9 and SL-8) from chronic SIV-infected macaques. Mean values, SE, and statistically significant difference are shown.

Figure 5

Positive correlations between B7-H1 levels on mDC (left panels) and pDC (right panels) with PD-1+ on T cell subsets including total T Cells (CD3+; top panels); CD4+ T cells (middle) and CD8+ T cells (bottom).

Figure 6

Correlations of B7-H1 levels on mDCs (left) and pDCs (right) with percentages of circulating CD4+ T cells and viral load in plasma of SIV-infected rhesus macaques. (a) Inverse correlation of percentage of B7-H1 on circulating DCs and CD4+ T cells in SIV-infected rhesus macaques. (b) Note a positive correlation of percentage of B7-H1 on circulating mDCs (filled hexagon), or pDCs (open hexgon) and SIV viral load in plasma in SIV-infected rhesus macaques.

Figure 7

Identification of mature DCs and SIV antigen-loaded DCs by flow cytometry (top) and fluorescent microscopy (bottom). (a) Histogram of mature monocyte-derived macaque DCs. Monocyte-derived immature DCs (iDC) were cultured with maturation cocktail and phenotyped by FACS analysis of FITC-anti-CD80 or -CD86, PE-anti-CD83 or -B7-H1, and PerCP-anti-HLA-DR. (b) Immunofluorescent staining of SIV lysate pulsed DCs. Immature DCs were pulsed with SIV lysate for 24 hours, and then incubated for 2 days in DC maturation cocktail. SIV lysate-unloaded and -loaded DCs were cytospun onto slides, fixed and stained with a control antibody (left), or anti-p28 monoclonal antibody (green). Nuclei are stained in blue. Representative fields (scale bar = 20μm) SIV p28 antigen staining of loaded and unloaded DCs are shown.

Figure 8

Effects of B7-H1 blockade on SIV-specific antigen function. (a) Loaded and unloaded DCs were added to 5×10⁵ autologous T cells at shown ratios with control or anti-B7-H1 Ab added at dilutions indicated. Intracellular IFN-γ staining was performed 6 hours after incubation. Means ±SEM of triplicate data are shown. (b and c) Effects of B7-H1 blockade on SIV-specific T cell proliferation. Ranging doses of the loaded DCs were added to 10⁵ autologous T cells, with control or anti-B7-H1 Ab added as indicated and T cell proliferation was assessed by CFSE dilution 6 days later. b, histogram of CFSE dilution. Means (±SEM) of triplicate data are provided. DCs and T cells alone (±SIV lysates) served as negative controls for both assays, which showed background levels less than the unloaded DC-T cells controls. (d) IL-2, IFN-γ and IL-10 cytokine levels in supernatants of T/DC co-culture at 60:1 after 6 days incubation. Bars reflect means of three independent data points.