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.
Dendritic cells (DCs) are professional APCs that play a key role in bridging the innate and adaptive immune responses. Upon Ag exposure, DCs undergo phenotypic and functional maturation and migrate to secondary lymphoid tissues where they initiate adaptive T cell responses (1–3). Distinct subsets of circulating DCs can be identified in blood, including myeloid dendritic cells (mDCs; Lin−HLA-DR+CD11c+) and plasmacytoid DC (pDCs; Lin−HLA-DR+CD123+), which show different phenotypic and functional properties (4, 5). Functionally impaired DCs have been proposed to be responsible for the failure of T cell immunity and antiviral immune responses (6). During SIV or HIV infection, both mDCs and pDCs have been shown to decrease in frequency and/or function (7). HIV-infected DCs also facilitate transfer of virus to CD4+ T cells in vitro, which may play a role in HIV-1 transmission and CD4+ T cell loss (8). Thus, DCs play a key role in regulation of immune responses and disease progression in HIV-1–infected individuals.
B7-H1 (also called programmed death [PD]-L1) is a member of the B7 family of costimulatory molecules, which are emerging as important mediators of various host immune responses. B7-H1 is differentially expressed on various cell subsets and to different extents on human and murine cells. Human B7-H1 is constitutively expressed at low levels on DCs, activated T cells (compared with high expression on activated murine T cells), and is highly expressed on monocytes and tumor cells (9). Both B7-H1 and B7-DC (PD-L2) are ligands for the PD-1 receptor, which is a negative regulatory receptor on activated T and B cells. For example, B7-H1 expression has been correlated with increased IL-10 production (a Th2 or T cell “dampening” cytokine), suppression of CTL responses, induction of apoptosis of tumor-specific T cells, FoxP3+ regulatory T cell production, as well as various states of unresponsiveness including T cell anergy and exhaustion (10–14). Blocking PD-L1, but not PD-L2, significantly restored Ag-specific T cell responses and promoted CD8 T cell differentiation into functional CTLs (15, 16), also suggesting a role for B7-H1 in the inhibition of T cell responses.
Several groups have shown that PD-1 expression is elevated on HIV-specific (17–19), hepatitis B virus-specific (20), and hepatitis C virus-specific T cells (21). B7-H1 is also reported to be upregulated on peripheral lymphocytes and associated with HIV-1 disease progression (22). In this study, we show that upregulation of B7-H1 on both mDCs and pDCs in peripheral and mucosal tissues from SIV-infected macaques correlates with PD-1 upregulation on T cell subsets and with declining peripheral CD4+ T cell counts. Furthermore, blockade of B7-H1 on monocyte-derived DCs improved SIV-specific T cell function and proliferation. These findings suggest that upregulation of B7-H1 on DCs may contribute to suppression of SIV-specific immune responses and disease progression. These studies were designed to address the significance and impact of this phenomenon in regulation of SIV infection and disease progression.
Materials and Methods
Animals and virus
A total of 21 adult rhesus macaques (Macaca mulatta) of Indian origin, which were initially negative for HIV-2, SIV, type D retrovirus, and simian T-lymphotropic virus type 1 infection, were examined in this study. All animals were housed at the Tulane National Primate Research Center (Covington, LA) in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care International standards. All studies were reviewed and approved by the Tulane University Institutional Animal Care and Use Committee. Of these animals, five were uninfected controls. The 16 animals left were infected with SIVmac251 and were either euthanized in acute infection (8–10 d; n = 4); euthanized in chronic asymptomatic stage, in which no overt signs of disease were detected (n = 4); euthanized in symptomatic AIDS stage (n = 4); or were animals previously infected with high viremia but subsequently controlled viral replication to undetectable levels (<125 copies per milliliter) in plasma (elite controllers; n = 4). Macaques with AIDS all had either opportunistic infections including Pneumocystis carinii pneumonia, disseminated Mycobacterium avium infection, or SIV encephalitis. All tissue samples were collected at necropsy except for those from elite controllers, which were collected by biopsy, as these animals remain clinically healthy and are considered important for further study.
Phenotyping blood and tissue mononuclear cells
Peripheral blood, lymph node, and intestinal tissues were isolated and processed using previously described techniques (23). Briefly, tissues were collected from the jejunum and mesenteric lymph nodes within minutes of euthanasia and processed immediately to prepare cell suspensions. Multicolor flow cytometry was used to enumerate percentages of various cell types and to characterize phenotypically macaque pDCs and mDCs in EDTA-treated whole blood or single-cell tissue suspensions of mesenteric lymph node and jejunum using previously described criteria (24). Briefly, CD123+ pDCs or CD11c+ mDCs were identified within HLA-DR+ (clone L243) and lineage-negative (Lin−
Phenotyping DCs in tissues by multilabel confocal microscopy
Three-color immunofluorescent staining for CD4, CD3 (T cells), and PD-1 and/or B7-H1 was performed on mesenteric lymph nodes of SIV-infected animals to visualize the distribution of PD-1+ and B7-H1+ subsets by confocal microscopy. Tissues were stained using unconjugated primary Abs and then with secondary Abs conjugated to Alexa 488 (green), Alexa 568 (red), or Alexa 633 (blue) (Molecular Probes, Eugene, OR). Confocal microscopy was performed using a Leica TCS SP2 confocal microscope equipped with three lasers (Leica Microsystems, Exton, PA). Individual optical slices representing 0.2 μm and 32 to 62 optical slices were collected at 512 × 512 pixel resolution. Image version 1.62 (National Institutes of Health, Bethesda, MD) and Adobe Photoshop version 7.0 (Adobe, San Jose, CA) were used to assign colors to the channels collected: HNPP/Fast Red (2-hydroxy-3-naphtoic acid-2′-phenylanilide phosphate), which fluoresces when exposed to a 568-nm wavelength laser, appears red; Alexa 488 (Molecular Probes) appears green; Alexa 633 (Molecular Probes) appears blue; and the differential interference contrast image is grayscale. The four channels were collected simultaneously. In some tissues and to differentiate between individual cells, To-pro3 (nuclear marker; Molecular Probes) was used at 1 μg/ml, incubated for 5 min, and tissues were then washed in PBS. Colocalization of Ags is demonstrated by the addition of colors as indicated in the legends to figures of this article.
Preparation of monocyte-derived DCs from blood for in vitro assays
Monocyte-derived dendritic cells (MoDCs) from chronically SIV-infected rhesus macaques (n = 3) were generated from heparinized peripheral blood using previously established protocols with minor modifications (25). In brief, CD14+ monocytes were positively selected using magnetic beads (Miltenyi Biotec, Auburn, CA), as assessed by a minimum 95% purity for CD14 expression by flow cytometry, and cultured at 1.5 × 106 to 2 × 106 cells/3 ml in RPMI 1640 medium, supplemented with 2 mM l2 (Sigma-Aldrich, St. Louis, MO) for another 24 h at 5 × 105
Cytospin preparation of SIV Ag-loaded DCs
For examining Ag ingestions and presentation of DC, viable SIV Ag-loaded and unloaded DCs (5 × 103 to 10 × 103 cells/slide) were spun onto glass slides, dried overnight, and examined for p28 Ag by immunofluorescence. Images were acquired at ×100 magnification by confocal microscopy.
Enrichment of T cells
To enrich for rhesus macaque resting T cells, 1 × 108/ml PBMCs were labeled with PE-conjugated anti-human HLA-DR mAb (BD Biosciences), followed by anti–PE-mAb–conjugated magnetic beads (Miltenyi Biotec). HLA-DR− cells were then used as responder cells in T cell response assays described later. Purity of T cells as monitored by staining with FITC-conjugated mAb to CD3 was ≥90% in all experiments.
Ag presentation assay
Purified T cells (5 × 105 T cells) were cocultured with autologous SIV Ag-loaded and unloaded MoDCs at DC/T cell ratios of 1:20, 1:60, and 1:180 in the presence of 10 μg/ml anti–B7-H1 or control Ig. All samples were incubated for 1 h at 37°C, followed by the addition of brefeldin A (5 μg/ml; BD Biosciences) and incubated for an additional 5 h. T cells (5 × 105) in media with and without staphylococcal enterotoxin B (1 μg/ml) were used as positive and negative controls, respectively. Cells were then stained with Pacific blue–anti-CD3, allophycocyanin–anti-CD4, and PE-TxR–anti-CD8, followed by fixation and permeabilization for subsequent intracellular staining with PE-Cy7–anti–IFN-γ. Samples were acquired on a FACSAria (Becton Dickinson) and data analyzed using FlowJo software.
T cell proliferation with Ag-loaded DC cocultures
Proliferation assays were based on CFSE dilution assays. Responder T cells were prepared as above and labeled with 5 μM CFSE (Molecular Probes, Invitrogen) and adjusted to 1 × 106/ml. SIV lysate Ag-loaded and unloaded MoDCs were added at DC to T cell ratios of 1:20, 1:60, 1:180 to 105 autologous CFSE-T cells in the presence of 10 μg/ml anti–B7-H1 or control Ig. Controls consisted of 105 T cells with and without staphylococcal enterotoxin B as described earlier. On day 6, the cells were stained using allophycocyanin-conjugated CD3 Ab. T cell proliferation was evaluated via dilution of CFSE on a FACSCalibur flow cytometer. Percentages of cells with diluted CFSE (CFSE+low) were determined in gated populations of total CD3+ T cells. Cytokines in supernatants were detected using a BD FACSAarray Bioanalyzer (BD Biosciences) according to the manufacturer’s instructions.
Graphical presentation and statistical analysis of the data were performed using GraphPad Prism 4.0 (GraphPad Software, San Diego, CA). Comparisons among groups were analyzed by a one-way ANOVA and Mann–Whitney t test. Those p values <0.05 were considered statistically significant. Correlations between samples were calculated and expressed using the Spearman's coefficient of correlation.
Distribution, phenotype, and B7-H1 expression on mDCs and pDCs in SIV-infected rhesus macaques
To evaluate a potential link between expression of B7-H1 on DCs and the immunopathology of SIV-infected rhesus macaques, we examined the two major DC subsets previously described (24, 26) as lineage-negative mDCs (HLA-DR+CD11c+) or pDCs (HLA-DR+CD123+) in PBMCs from blood, or mononuclear cells isolated from mesenteric lymph node and jejunum lamina propria, and B7-H1 expression on mDCs or pDCs was examined (Fig. 1A). The percentage of mDCs and pDCs composed between 5–60% and 1–5% of the Lin−, HLA-DR+ fraction in normal (uninfected) blood, respectively (data not shown). B7-H1 was constitutively expressed at low levels on both mDCs and pDCs in normal animals but significantly upregulated on DCs from blood, mesenteric lymph node, and jejunum lamina propria after SIV infection (Fig. 1B, 1C). B7-H1+ DCs were comparable between SIV-infected and uninfected animals. For mDCs, the percentage of B7-H1+ mDCs in blood from acute and chronic stage were significantly higher than that in healthy controls (p < 0.05), and expression levels of B7-H1 on mDCs were also markedly upregulated in mesenteric lymph node and jejunum lamina propria in the acute-, chronic-, or AIDS-stage animals (p < 0.05). In addition, significant upregulation of B7-H1 on mDCs was observed in the mesenteric lymph node (p < 0.001) after SIV infection compared with that of controls (Fig. 1B). Similar to mDCs, upregulation of B7-H1 on pDCs was also observed, including in blood-, mesenteric lymph node-, and jejunum lamina propria-derived pDCs. Percentages of B7-H1+ pDCs from jejunum lamina propria were significantly higher in SIV-infected animals than that in healthy controls (p < 0.001) (Fig. 1C). Notably, there was no significant difference in frequency of B7-H1+ circulating mDCs or pDCs between controllers and normal controls. These data suggests that upregulation of B7-H1 on both mDCs and pDCs correlates with SIV disease progression.
Expression of PD-1 on T cells of SIV-infected macaques in situ
Recent studies have shown that the PD-1–B7-H1 pathway plays a major role in regulating T cell exhaustion and unresponsiveness (27). Further, HIV-specific CD4 and CD8 T cells express high levels of PD-1 in blood of patients with high viremia (17–19). In this study, immunohistochemistry and confocal microscopy were performed to compare PD-1 expression on T cells in lymph nodes between normal and infected macaques. As expected, routine histopathology demonstrated expanded germinal centers and margination of the T cell zones typical of lymph nodes in SIV infection compared with that of normal controls (Fig. 2a, 2b, respectively). However, while PD-1 expression was constitutively expressed in the small follicular regions of uninfected animals (Fig. 2c), PD-1+ cells were markedly expanded throughout the expanded germinal centers and even in T cell zones of lymph nodes in SIV-infected macaques (Fig. 2d). Three-color confocal microscopy confirmed expanded B cell germinal centers and macrophages interspersed with large numbers of PD-1+ cells that were of lymphocyte morphology and expressed neither B cell nor macrophage markers (Fig. 2e, 2f) Note PD-1 expression (green) was significantly increased on lymphocytes within these germinal centers in infected macaques (Fig. 2f) compared with that in controls (Fig. 2e).
Expression of B7-H1 on DCs correlates with PD-1 on T cells in SIV infection
To identify whether B7-H1+ cells interact with PD-1+ T cells in lymphoid tissues from SIV-infected animals, lymph node sections were further analyzed by multifluorescent immunohistochemistry to determine their identity and distribution in situ. Colabeling with CD4 confirmed most of the PD-1+ cells in lymphocytes were CD4+ T cells (Fig. 3a). Further, both B7-H1+ and PD-1+ cells were predominately distributed within the germinal centers of follicles, yet these were markedly expanded in SIV-infected animals (Figs. 2 and 3). As shown in Fig. 3, most PD-1+ cells have a lymphocyte morphology and coexpress CD3 and CD4. In contrast, B7-H1+ cells displayed obvious dendritic morphology consistent with DC and were in close proximity to, and even occasionally surrounding, PD-1+ T cells (Fig. 3b). The expanded number and distribution of PD-1+ CD4+ T cells in association with B7-H1+ DCs may facilitate greater interaction of these cells, and may regulate immune responses within these crucial secondary lymphoid tissues.
We next compared coexpression of PD-1 on total CD3+ T cells, CD4+, and CD8+ T cells in blood, mesenteric lymph nodes, and jejunum lamina propria (major target tissues for HIV) in acute, chronic, and AIDS stages of SIV infection and in blood from controllers by flow cytometry (Fig. 4). Prior to infection, ~21% of total CD3+ T cells in blood expressed PD-1, but this frequency was significantly elevated in acute and chronic stages of SIV infection (p < 0.01). In mesenteric lymph nodes, PD-1+CD3+ T cells were significantly increased compared with that in normal controls (p < 0.01) (Fig. 4A). Further, levels of PD-1 expression were elevated on CD4+ T cells in blood in acute, mesenteric lymph nodes in chronic, and jejunum in AIDS stages compared with those of normal controls (p < 0.05) (Fig. 4B). In fact, increases in PD-1 expression appeared to be limited to CD4+ T cells in the gut in SIV infection, as there was not a significant increase on CD8+ or total (CD3+) T cells (Fig. 4A–C). During the acute and chronic stages of infection, the frequency of PD-1+ CD8+ T cells in blood and mesenteric lymph nodes was higher than that in normal macaques (p < 0.01), and this frequency was also higher in mesenteric lymph nodes in macaques with AIDS. The jejunum maintained high frequencies of PD-1+ CD8+ T cells yet did not show significant differences (Fig. 4C). In addition, SIV-specific CD8+ T cells (gag CM-9 and tat SL-8 responsive) had high levels of surface PD-1 expression (Fig. 4D). Notably, the frequency of PD-1+ T cells in blood was similar between controllers and normal controls. Combined, these results clearly demonstrate upregulation of PD-1 on both CD4+ and CD8+ T cell subsets in both mucosal and systemic lymphoid tissues of SIV-infected macaques, yet the dynamic responses of each appear to differ between tissues.
Correlation of B7-H1 on circulating DCs with PD-1 on T cells, CD4+ T cells, and viremia in SIV infection
Because both B7-H1 expression on DCs and its receptor PD-1 on T cells were significantly upregulated after SIV infection, we sought a correlation between B7-H1 expression on DCs and PD-1 on T cells in normal and SIV-infected animals. In blood, percentages of B7-H1+ mDCs significantly correlated with percentages of PD-1+ CD3+ T cells (R2 = 0.5278, p = 0.0022), CD4+ T cells (R2 = 0.284, p = 0.048), and CD8+ T cells (R2 = 0.4786, p = 0.0043) (Fig. 5). Percentages of B7-H1+ pDCs also positively correlated with PD-1+ CD3+ T cells (R2 = 0.632, p = 0.0004), CD4+ T cells (R2 = 0.271, p = 0.0387), and CD8+ T cells (R2 = 0.7728, p < 0.001) (Fig. 5). Combined, these data indicate B7-H1 expression on mDCs and pDCs correlates with PD-1 expression on T cells in blood and major lymphoid targets of SIV infection.
Because progression of HIV infection is associated with reductions in CD4+ T cells, increased viral loads, and immune activation, we sought correlations between B7-H1 expression on mDCs and/or pDCs, CD4+ T cells, and viral loads in plasma. There was a highly significant inverse correlation between percentages of CD4 cells and both B7-H1+ mDCs (R2 = 0.5559, p < 0.0001) and pDCs (R2 = 0.4346, p < 0.0016) (Fig. 6A). Similarly, there was a positive correlation between SIV viremia and both B7-H1+ mDCs (R2 = 0.5003, p < 0.01) and pDCs (R2 = 0.861, p < 0.001) (Fig. 6B). These data indicate B7-H1 expression on DCs is markedly increased in animals with declining levels of CD4+ T cells in SIV infection and suggest B7-H1 expression on DCs may be involved in progression to AIDS.
Blockade of B7-H1 increases DC-mediated SIV-specific T cell responses
It is well known that DCs are potent APCs, and DC maturation is associated with increased T cell stimulatory capacity. To determine if B7-H1 upregulation on DCs is involved in suppression of T cell responses, monocytes were obtained from PBMCs, and MoDCs were prepared as described in Materials and Methods including 6 d of inoculation with IL-4 and GM-CSF. The immature DCs were incubated with SIV lysate at day 6 and finally stimulated by maturation mixture for 48 h. FACS analysis was performed to analyze maturation markers on immature DCs and Ag-loaded mature DCs, respectively. As shown in Fig. 7A, mature DCs including the SIV Ag-loaded mature DCs exhibited uniform upregulation of CD80, CD83, and CD86 compared with that of immature DCs. Meanwhile, B7-H1high expression on mature MoDCs was also induced, consistent with previously reports (14, 28).
To examine the effect of B7-H1 expression on activation of SIV-specific T cell responses by mature DCs, immature DCs (mDCs) were loaded with SIV lysate and further matured in maturation mixture. Matured (pDCs) DCs from infected macaques captured significant amounts of SIV Ag as detected by immunostaining for SIV p28 protein. Note that intense staining (green) for SIV p28 was detected in cells (Fig. 7B). These SIV-loaded mature DCs were cocultured with autologous SIV-primed T cells at different ratios and monitored for T cell activation by measuring IFN-γ release and T cell proliferation.
For measuring T cell activation, intracellular IFN-γ–producing CD4+ or CD8+ T cells were examined after coculture of T cells with Ag-loaded mature DCs. SIV Ag-loaded DCs consistently increased IFN-γ production from both CD4+ and CD8+ T cells upon addition of anti–B7-H1 Ab compared with irrelevant control Ab at different ratios (CD4+ T-cell, p = 0.2 for 1:20, p = 0.4 for both 1:60 and 1:180; CD8+ T-cell, p = 0.0571 for 1:20, p = 0.4 for both 1:60 and 1:180) (Fig. 8A). We also purified CD8+ T cells from PBMCs in macaques and examined the effects of B7-H1 blockade on cytokine expression in purified CD8+ T cells alone. Adding anti–B7-H1 did not result in any effect on CD8+ T cell cytokine production (data not shown). This confirms the activation effect was due to blocking B7-H1 on MoDCs rather than on CD8+ T cells. Thus, B7-H1 on MoDCs could inhibit T cell function through B7-H1 and PD-1 interactions, resulting in a decrease of IFN-γ production by T cells, but this inhibitory signal could be rescued by treatment with anti–B7-H1 of MoDCs.
We also investigated the effect of MoDC B7-H1 blockade on SIV-specific CD3+ T cell proliferation by coculture of CFSE-labeling CD3+ T cells and SIV Ag-loaded MoDCs at different ratios. As shown in Fig. 8B and 8C, the frequency of CFSE− CD3+ T cells increased significantly when treated with neutralizing B7-H1 Ab at all ratios tested compared with control Ab (p < 0.05 for 1:20 and 1:60). This suggests B7-H1 upregulation on DCs is responsible for the decreased CD3+ T cell expansion observed in SIV-infected macaques. Further, B7-H1 expression may be associated with suppression of virus-specific T cell proliferation in vivo, and blockade of B7-H1 may restore the proliferative capacity of T cells.
Finally, to determine SIV-specific cytokine release from T cell–DC cocultures after B7-H1 blockade, we activated autologous SIV-primed T cells with SIV Ag-loaded MoDCs at 60:1 ratio for 6 d, and cytokine levels in supernatants were compared. The data showed blockade of B7-H1 increased IL-2 production (p = 0.079) and significantly enhanced IFN-γ (p < 0.05) and decreased IL-10 release (p < 0.05) (Fig. 8D). These results strongly suggest upregulation of B7-H1 expression was associated with suppression of SIV-specific T cell immune responses after SIV infection.
To evade host immunity, HIV-1 uses numerous strategies to impair normal DC function including preventing activation of potentially protective antiviral immune responses by increasing IL-10 and decreasing IL-12 and IFN-γ production, as well as downregulating expression of surface costimulatory molecules (25, 29–31). Using the unique model of pathogenic SIV infection in nonhuman primates, we simultaneously compared expression of B7-H1 on DCs, and its receptor PD-1 on T cells, in the peripheral blood, lymph node, and GALT in various stages of SIV infection and in blood of elite controllers. These findings demonstrate that upregulation of B7-H1 on circulating mDCs and pDCs positively correlates with viremia and disease progression in SIV-infected rhesus macaques and that DCs may be involved in mediating T cell suppression and dysfunction through the B7-H1–PD-1 pathway in mucosal and secondary lymphoid tissues of infected hosts.
PD-1 expression on virus-specific CD8+ T cells is linked with impairment of immune function (18, 19). Further, PD-1 is upregulated upon activation, and functional high-level expression is maintained even by seemingly “exhausted” CD8 T cells (17, 27, 31). PD-1 and its ligands have important roles in regulating immune defenses against tumors or infectious agents. Recent studies indicate that the interaction between B7-H1 and its receptor PD-1 mediate inhibition of T cell responses and negatively regulate cytokine production and proliferation of T cells (33, 34). This inhibitory effect has been shown for CD4+ as well as for CD8+ T cells (35, 36). In this study, immunohistochemistry analysis showed that PD-1 expression increased on T cells in general, and especially on CD4+ T cells within germinal centers of lymph nodes. Notably, however, the percentages of PD-1+ CD8+ T cells were similar to those of CD4+ T cells by flow cytometry. However, when the mean fluorescence intensity (MFI) was compared, we found that the percentages did not correlate with the MFI values between CD4+ and CD8+ T cells in lymph nodes of chronically SIV-infected macaques. The mean percentage of PD-1+ CD4+ T cells was 27.17 ± 1.09 but 51.48 ± 9.525 for CD8+ T cells, whereas the MFI of PD-1 expression was 200 ± 76.97 on CD4+ cells and 118.9 ± 21.33 on CD8+ T cells. Thus, CD4+ T cells in germinal center clearly expressed higher levels of PD-1 than those of CD8+ cells, which is likely associated with more frequent DC–T cell interaction in this region as evidenced by colocalization of PD-1 on T cells and B7-H1 on DCs (Fig. 3). The PD-1–PD-L pathway thus appears to be a key determinant of the outcome of infection, regulating the delicate balance between effective antimicrobial immune defenses and immune-mediated tissue damage (37, 38). Thus, these data support that upregulation of B7-H1 on DCs and PD-1 on T cells contributes to suppression of T cell function during SIV infection. B7-H1 upregulation could also contribute to functional impairment of DCs as a mechanism of immune evasion by SIV. Several studies in vivo have found a greater effect of anti–B7-H1 blockade compared with that of anti–PD-1 or anti–PD-L2 blockade, and these suggest targeting B7-H1 could be a better strategy for treating chronic viral infections (32).
DCs are a heterogeneous population of APCs important for bridging the initiation and regulation of innate and adaptive immune responses. mDCs are phenotypically and functionally similar to MoDCs, inducing Th1 cell responses (39). pDCs are potent producers of α IFNs in response to enveloped viruses and appear to direct T cell responses (40). Blockade of B7-H1 on MoDCs may thus restore SIV-specific T cell function and is supported in this study by the finding that B7-H1 expression on DCs is involved in functional T cell suppression in SIV infection.
It has been reported that B7-H1 mRNA in lymphoid tissue and PBMCs increases in HIV-infected patients, that B7-H1 is inducible by IL-10, and that increased B7-H1 expression correlates with HIV-1 disease progression (22). B7-H1 expression on DCs is also involved in the induction and maintenance of T cell anergy (41). In this study, we show that B7-H1 expression is normally expressed at low levels on mDCs and pDCs in healthy macaques but is significantly elevated in frequency on both B7-H1+ mDCs and pDCs in blood and mucosal tissues and that levels inversely correlate with peripheral CD4+ T cell counts after SIV infection. However, B7-H1 expression on DCs was low and similar to uninfected controls in animals controlling infection. Further, immunohistochemistry demonstrated B7-H1+ cells and PD-1+ T cells colocalized in the hyperplastic/expanded follicles of lymph nodes of SIV-infected macaques. Although there is no single specific marker suitable for identifying DCs in tissues by immunohistochemistry, B7-H1+ cells clearly displayed DC-like morphology in B7-H1–PD-1 double-positive regions of the lymph node (Fig. 4). Combined, these data indicate interactions between B7-H1+ DCs and PD-1+ T cells facilitate regulation of immune responses and, in particular, suppress T cell function during SIV infection, at least in organized lymphoid tissues. B7-H1 upregulation on DCs could also contribute to functional impairment of DCs as a mechanism of immune evasion by SIV. Regardless, B7-H1–PD-1 interactions appear to be a key correlate of the outcome of SIV infection and may be involved in regulating the delicate balance between effective immune defense and immune dysfunction.
In summary, this work represents, to our knowledge, the first description that B7-H1 upregulation on mDCs and pDCs parallels upregulation of PD-1 expression on T cells in mucosal and systemic lymphoid tissues and inversely correlates with CD4+ T cell loss in SIV-infected rhesus macaques. Further, T cell effector function could be rescued by blocking B7-H1–PD-1 interaction in cells from SIV-infected macaques. The suppressive interaction between DCs and T cells involving the B7-H1–PD-1 pathway in HIV infection suggests that targeted therapies exploiting this pathway may represent a promising approach for enhancing T cell immunity in infected patients.
We thank Julie Bruhn, Calvin Lanclos, and Desiree Waguespachek for flow cytometry support and Janell LeBlanc, Kelsi Rasmussen, Maryjane Dodd, and Maury Duplantis for technical support.
Disclosures The authors have no financial conflicts of interest.
This work was supported by National Institutes of Health Grants AI49080, AI084793, and RR000164. The James B. Pendleton Charitable Trust Foundation provided an instrumentation grant.
Abbreviations used in this paper:
- dendritic cell
- lymph nodes
- myeloid dendritic cell
- monocyte-derived dendritic cell
- programmed death
- plasmacytoid dendritic cell.
- Received May 25, 2010.
- Accepted October 3, 2010.