Influence of Plasma Viremia on Defects in Number and Immunophenotype of Blood Dendritic Cell Subsets in Human Immunodeficiency Virus 1Infected Individuals

Divisions of ¹Infectious Diseases and ²Clinical Immunology, Department of Medicine, and ³Department of Preventive Medicine and Biometrics, University of Colorado Health Sciences Center, Denver

Received 17 April 2002; revised 10 September 2002; electronically published 13 December 2002.

     Presented in part: 8th Conference on Retroviruses and Opportunistic Infections, Chicago, 48 February 2001 (abstract B15e); 9th Conference on Retroviruses and Opportunistic Infections, Seattle, 2428 February 2002 (abstract B16e).
     The study was approved by the Colorado Multiple Institutional Review Board at the University of Colorado Health Sciences Center (UCHSC; Denver), and informed consent was obtained from all study participants. Human experimentation guidelines of the US Department of Health and Human Services and those of the UCHSC were followed in the conduct of clinical research.
     Financial support: National Institutes of Health (grants KO8 AI-01459 and PO1 AI-43664 to C.C.W. and AI-07447 5T32 and AmFAR 2-5-20188 to M.A.B.)
     Reprints or correspondence: Dr. Cara C. Wilson, University of Colorado Health Sciences Center, Div. of Clinical Immunology, Dept. of Medicine, 4200 E. Ninth Ave., Box B164, Denver, CO 80262 .

     Dendritic cells (DCs) are potent antigen (Ag)presenting cells (APCs) that are capable of activating and expanding naive, Ag-specific CD4⁺ and CD8⁺ T lymphocytes [1]. There are several lines of evidence suggesting that DCs may be involved in the transmission and immunopathogenesis of HIV-1 [2]. In addition to the impact that DCs may have on HIV-1 disease as a result of their ability to transmit HIV-1 to T cells [3, 4], it has been suggested that DCs themselves might be adversely effected by replicating HIV-1. Several investigators have reported decreased numbers of epidermal or blood DCs in HIV-1infected persons with advanced disease [4 9]. In addition, there is evidence that DCs freshly isolated from HIV-1infected individuals or DCs infected with HIV-1 in vitro are dysfunctional in their ability to induce a primary immune response. This dysfunction may result from direct effects of viral infection, such as down-regulation of major histocompatibility complex molecules [7], or indirect effects, such as exposure to a cytokine environment that is less conducive to the generation of cellular immune responses.

     Blood DCs have been traditionally characterized by the absence of leukocyte lineage (Lin)specific Ags and by surface expression of HLA-DR and CD4 [10, 11]. They have been subcategorized into myeloid DCs (mDCs), denoted as Lin^-HLA-DR⁺CD11c⁺, and plasmacytoid DCs (pDCs), denoted as Lin^-HLA-DR⁺CD11c^-IL-3R^Hi [12, 13]. In addition to phenotypic differences, different functional attributes, including the ability to stimulate specific T helper subsets, were thought to be associated with the individual subsets on the basis of their lineage [12, 14]. However, it has recently been suggested that the DC lineage per se may not actually be the basis of the Th1/Th2 polarization observed. The activation state of the DC, the type of activation signal it encounters, and the cytokine environment all have been postulated to contribute to T helper polarization by DCs [15 18].

     The role of both blood DC subtypes in HIV-1 immunopathogenesis remains an area of intense research. A decrease in the proportion or absolute number of blood DCs expressing CD11c⁺ (mDCs) has been observed in a number of cohorts of HIV-1infected donors [5, 6, 9, 19]. However, a recent article by Chehimi et al. [20] that examined levels of blood DC subsets in both treated and untreated HIV-1infected subjects reported a median frequency of mDCs in HIV-1infected subjects similar to that of uninfected control subjects. In that study, a decrease in mDC number relative to that of control subjects was only noted in HIV-1infected subjects with active viral replication.

     It was recently reported that pDCs might be the natural interferon (IFN)producing cells (IPCs) of the immune system [21, 22] and may serve as a link between innate and adaptive immune systems. Therefore, the role of this blood DC subset in HIV-1 disease is of great interest. In a study that examined IPCs in HIV-1infected patients, IPC numbers were decreased in patients with AIDS, and a negative correlation was found between IPC number and plasma HIV-1 RNA levels [23]. Others have shown a similar decrease in the number of pDCs [5, 6, 20] and decreased IFN- production with viral stimulation [6, 20] in patients with HIV-1 infection.

     Distilling the findings of all these recent reports into a unifying hypothesis about the fate and function of blood DCs in the setting of HIV-1 infection has been difficult because of the diversity of subject cohorts studied, including subjects who differ in both stage of disease and treatment status. In addition, the role that blood DC defects play in mediating the observed cellular immune dysfunction characteristic of progressive HIV-1 infection, and the degree of reversal in DC defects after initiating highly active antiretroviral therapy (HAART), has yet to be fully elucidated. Blood DCs and their progeny are likely to play a critical role in priming T cell responses to HIV-1 and opportunistic pathogens in vivo. An understanding of the impact of HIV-1 replication on the numbers of circulating blood DCs, their maturation state, and their ability to present antigens to naive and memory T cells is likely to be important in the development of vaccines and immune-based therapies for HIV-1 infection. In the present study, we sought to further characterize the defects in blood DC subsets observed during HIV-1 infection and to assess the effect of HIV-1 plasma viremia on blood DC number and phenotype. In addition, we sought to determine whether a relationship existed between circulating blood DCs and attributes of T cell function in the setting of treated or untreated HIV-1 infection.

MATERIALS AND METHODS

     Study population.     Forty-two HIV-1infected subjects who received their care through the University of Colorado Health Sciences Center (UCHSC) Infectious Diseases Group Practice were studied. For comparison, 20 HIV-1seronegative control subjects were also studied.

     Complete blood counts.     Complete blood count data were obtained from heparinized blood using an ADVIA 120 (Bayer Diagnostics) or from EDTA lavender top vacutainer tubes (Becton-Dickinson [BD]) using a CELL-DYN 4000 (Abbot Diagnostics).

     Flow cytometry.     Peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood using standard Ficoll-Paque (Pharmacia Biotech AB) density-gradient centrifugation. Three-color flow cytometric analysis was performed on freshly-isolated PBMC stained with the following panel of monoclonal antibodies (MAbs): fluorescein isothiocyanate (FITC)labeled anti-Lineage (Lin^-) panel (CD3/CD14/CD16/CD19/ CD20/CD56) MAb (BD), FITC-labeled anti-CD34 MAb (BD), peridinin chlorophyll proteinlabeled antiHLA-DR (BD), and either phycoerythrin (PE)labeled anti-CD11c (PharMingen [Ph]), -CD123 (IL3R) (BD), -CD40 (Ancell), -CD4 (Ph), -CD86 (Ancell), -CXCR4 (Ph), or -CCR5 (Ph) MAbs. Four-color analysis was performed on fresh PBMC using FITC-labeled anti-Lin anti-CD34 MAbs, tricolor-labeled antiHLA-DR MAbs (Caltag), allophycocyanin conjugatelabeled anti-CD11c (BD), or biotinylated anti-CD123 (IL-3R) (Ph) that was then labeled with streptavidin-conjugated APC (Ph) per the manufacturer's instructions, and either PE-labeled anti-CD40, -CD4, -CD86, -CXCR4, or -CCR5 MAbs. Cells were also labeled with appropriate isotype control antibodies in each experiment. After staining for 30 min with MAbs, cells were washed, resuspended in fixation buffer (PBS plus 1% paraformaldehyde), and stored at 4°C in the dark before analysis. Three- and 4-color FACS analysis was performed using a FACScan cytofluorometer (BD Immunocytometry Systems). For each sample, 50,000200,000 events were acquired and gated on Lin^-CD34^-/HLA-DR⁺ expression, and a scatter gate was designed to include only viable leukocytes. The values for percentage of positive cells or the mean fluorescence intensity (MFI) of all gated cells reported reflect subtraction of the isotype control values. DC subsets were defined as follows: mDCs were defined as Lin^-CD34^-HLA-DR⁺CD11c⁺, and pDCs were defined as Lin^-CD34^-HLA-DR⁺CD11c^-IL-3R^Hi. Absolute DC count was derived by using the percentage of cells in relation to the mononuclear fraction determined by the automatic differential blood count and is expressed as number of cells per milliliter of blood.

     Lymphoproliferative assay (LPA).     Fresh PBMC were isolated by Ficoll density centrifugation, resuspended at 1 × 10⁶ cells/mL in RPMI 1640 medium with 10% human AB serum (Sigma), and 100 L of cells was added to 96-well plates that contained 100 L of HIV-1 p24 and p66 baculovirus-expressed recombinant proteins (NY5 and IIIB strains, respectively; Protein Sciences; final concentration, 1 g/mL). Phytohemagglutinin (5 g/mL; Sigma) and whole candida protein (10 mg/mL; Greer) were used as positive controls in each assay. Cells were incubated at 37°C in a humidified 5% CO² atmosphere for 6 days. Plates were pulsed with 1 Ci/well of tritiated thymidine for 6 h, cells were harvested, and radioactivity was counted on a -counter (Packard).

     Statistical analysis.     All statistical analyses assumed a 2-sided significance level of .05. Because of small sample sizes, nonparametric statistics were used. The Mann-Whitney U test was used for primary comparisons between HIV-1infected and uninfected control subjects, Wilcoxon signed-rank tests were used for within group comparisons, and Spearman's  was used to describe correlations;  > 0.3 or  < -0.3 was considered to be clinically significant. Overall P values for secondary comparisons between the control group and HIV-1infected subgroups used a Kruskal-Wallis test (a nonparametric analysis of variance), and, given an overall P < .05, pairwise comparisons to the control group were conducted using Dunn's multiple comparison adjustment. LPA data were analyzed using univariate logistic regression. Data analyses were performed with GraphPad Prism (version 3.00), Splus (Insightful), and SAS (SAS Institute) software.

RESULTS

     Blood DC subtypes are decreased in the blood of HIV-1infected individuals and serve as markers of immune status.     Using 3-color flow cytometry, Lin^-HLA-DR⁺ cells from 34 HIV-1infected and 20 uninfected subjects were analyzed for surface expression of CD11c (mDCs), CD123 (pDCs), costimulatory molecules (CD40 and CD86), and the HIV-1 coreceptor CD4. The HIV-1infected patients evaluated had a wide range of peripheral CD4⁺ T cell counts and plasma HIV-1 RNA levels and included those receiving HAART, as well as untreated subjects. HIV-1infected subjects as a group displayed decreases in blood mDC and pDC subsets relative to uninfected control subjects, both when DCs were measured as a percentage of total PBMC (median mDCs: control subjects, 0.41%; HIV-1infected subjects, 0.23%; P = .002, Mann-Whitney U test; median pDCs: control subjects, 0.30%; HIV-1infected subjects, 0.15%; P = .001, Mann-Whitney U test), and when measured as absolute numbers in the blood (median mDC count: control subjects, 8070 cells/mL; HIV-1infected subjects, 5505 cells/mL; P = .001, Mann-Whitney U test; and median pDC count: control subjects, 5970 cells/mL; HIV-1infected subjects, 3180 cells/mL; P = .009, Mann-Whitney U test). When the relationship between blood DC numbers and peripheral CD4⁺ T cell count was evaluated in HIV-1infected subjects, absolute numbers of both mDCs and pDCs were found to correlate positively with peripheral CD4⁺ T cell counts ( and ).

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Figure 1.        Blood dendritic cell (DC) subsets serve as markers of immune status in human immunodeficiency virus (HIV) type 1infected individuals. Both myeloid DCs (mDCs; A) and plasmacytoid DCs (pDCs; B) nos. correlated positively with peripheral CD4⁺ T cell counts in HIV-1infected subjects. Nos. of CD11c⁺ mDCs and IL-3R^Hi+ pDCs in highly active antiretroviral therapy (HAART)suppressed (HS), HAART failure (HF), and HAART-naive (HN) subjects were decreased (C and D), compared with uninfected control subjects (HIV^-). Bars represent median values (results summarized in ).

     To better define the effect of plasma viremia on DC phenotype and function, subsequent analyses were performed with the 34 HIV-1infected subjects separated into 3 groups on the basis of their treatment status and plasma HIV-1 RNA levels. HAART-suppressed (HS) subjects were defined as those receiving combination antiretroviral therapy, with plasma HIV-1 RNA level <400 copies/mL for 6 consecutive months. HAART-failure (HF) subjects were receiving combination antiretroviral therapy without suppression of viral replication (HIV-1 RNA level >2000 copies/mL) for 6 consecutive months. HAART-naive (HN) subjects had never received antiretroviral therapy prior to enrolling in the study and had active plasma viremia (minimum plasma HIV-1 RNA level, 2000 copies/mL). The clinical, immunologic, and virological characteristics of the HIV-1infected subjects are shown in . The median duration of HAART therapy was 26 months in the HS group versus 31 months in the HF group. White blood cell counts of the HIV-1infected subjects (median, 4.63 × 10⁹ cells/L) and control subjects (median, 5.36 × 10⁹ cells/L) were not statistically different (P = .09, Mann-Whitney U test).

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Table 1.          Human immunodeficiency virus (HIV) type 1infected donor characteristics.

     The ratio of mDCs to pDCs in the blood was not significantly different between the uninfected control group and the HIV-1infected groups or among the HS, HF, and HN groups (data not shown), with an average mDC : pDC ratio of 1.39 found across groups. All 3 HIV-1infected groups had absolute mDC and pDC numbers lower than those of control subjects, but only those HIV-1infected subjects with active viral replication (HF and HN groups) had median DC numbers that were statistically significantly lower than those of control subjects ( and ). Of note, both HIV-1infected groups with active viral replication also had lower median counts of both mDC and pDC subtypes than did the HS group . When relationships between absolute numbers of DC subsets in the blood and plasma HIV-1 RNA levels were evaluated in HIV-1infected subjects, statistically significant direct correlations between mDCs or pDCs and plasma HIV-1 RNA levels were not found (mDC: Spearman's , -0.314; P = .071; pDC: Spearman's , -0.267; P = .127; data not shown).

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Table 2.          Summary of 3-color flow cytometric analysis comparing uninfected control subjects with human immunodeficiency virus (HIV) type 1infected subgroups.

     Effect of HIV-1 plasma viremia on blood DC expression of costimulatory molecules and CD4.     As DCs are activated to mature, their surface expression of the costimulatory molecules CD86 and CD40 tends to increase [1]. Expression of these surface markers on Lin^-HLA-DR⁺ blood DCs was found to differ among the subject groups tested ( and  ). The median surface expression of CD86 was higher on DCs from HIV-1infected subjects with plasma viremia (HF and HN groups), relative to that of DCs from the HS subjects and uninfected control subjects. DC expression of CD40 was also higher in viremic subjects than in uninfected control subjects or HS subjects, but this difference only reached statistical significance for the HF group . In addition, a strong positive correlation was found between CD86 expression on DCs and plasma HIV-1 RNA copies/mL , and a negative correlation was found between peripheral CD4⁺ T cell counts and CD86 expression on DCs (Spearman's , -0.496; P = .004; data not shown). Significant correlations between blood DC expression of CD40 and either plasma HIV-1 RNA levels (Spearman's , 0.242; P = .168) or peripheral CD4⁺ T cell counts (Spearman's , -0.129; P = .467) were not observed (data not shown).

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Figure 2.        Blood dendritic cell (DC) expression of costimulatory molecules and CD4. CD86 (A) and CD40 (B) expression on Lin^-HLA-DR⁺ blood DCs in uninfected control subjects (human immunodeficiency virus (HIV)negative [HIV^-]), highly active antiretroviral therapy (HAART)suppressed (HS), HAART failure (HF), and HAART-naive (HN) subjects. C, Correlation between plasma HIV-1 RNA levels and DC expression of CD86. D, Percentage of Lin^-HLA-DR⁺ blood DCs expressing CD4 for each subject cohort. E, Relationship between DC expression of CD4 and HIV-1 RNA levels. Bars in each graph represent median values. Results are summarized in .

     Blood DCs have been shown to express the surface marker CD4, a molecule that also serves as a coreceptor for HIV-1. The surface expression of CD4 was measured on Lin^-HLA-DR⁺ blood DCs in the different HIV-1infected cohorts. The percentage of blood DCs expressing CD4 was significantly decreased in all the HIV-1infected groups, compared with uninfected control subjects . In addition, the percentage of Lin^-HLA-DR⁺CD4⁺ DCs in total PBMC was also significantly decreased in all HIV-1infected subgroups . A significant inverse correlation was found between plasma HIV-1 RNA levels and percentage of blood DCs expressing CD4 .

     Blood DC subtypes have differential expression of surface costimulatory molecules and HIV-1 coreceptors.     When examining Lin^-HLA-DR⁺ populations using 3-color flow cytometry, it was noted that 20% of the cells in this population in uninfected control subjects were neither CD11c⁺ nor IL3-R^Hi+, which suggests that they did not fall into either mDC or pDC subsets . The percentage of non-mDC/non-pDC cells in the Lin^-HLA-DR⁺ populations of HIV-1infected subjects was generally greater than that of uninfected control subjects . To assess the respective expression of HIV-1 coreceptors and DC costimulatory molecules directly on mDCs or pDCs within the Lin^-HLA-DR⁺ population, 4-color flow cytometric analysis was performed on blood obtained from 8 additional HIV-1infected subjects with HIV-1 plasma viremia and 8 uninfected control subjects. The 8 HIV-1infected subjects studied had a median peripheral CD4⁺ T cell count of 183.5 cells/L (range, 26690 cells/L) and a median HIV-1 RNA level of 47,870 copies/mL (range, 17,400750,000 copies/mL) and were selected on the basis of plasma HIV-1 RNA levels 2000 copies/mL for 6 consecutive months without regard to their HAART treatment status.

     We first compared the expression of CD40, CD86, and CD4 and the additional HIV-1 coreceptors CXCR4 and CCR5 on the 2 DC subtypes in the blood of individual seronegative control subjects . In these donors, pDCs expressed higher levels of HIV-1 coreceptors (CD4, CXCR4, and CCR5) than did mDCs, whereas mDCs expressed higher levels of DC costimulatory or maturation markers (CD86 and CD40)  and 4). In general, these trends in surface phenotype of mDC and pDCs were recapitulated in HIV-1infected subjects, and these findings confirm those of Donaghy et al. [5].

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Figure 3.        Example of blood dendritic cell (DC) subset analysis by 4-color flow cytometry. First, a gate was drawn around Lin^-HLA-DR⁺ cells (A); then the gates were set on Lin^-HLA-DR⁺CD11c⁺ cells (myeloid DCs , R3) (B), with subsequent expression of CD86 (C), CD40 (D), CD4 (E), CXCR4 (F), and CCR5 (G) analyzed on the mDCs in the R3 gate. HM, A similar analysis was performed on Lin^-HLA-DR⁺IL-3R^Hi+ cells (plasmacytoid DCs , R4). The shaded histogram represents the staining with specific monoclonal antibody, and the outlined histogram represents the respective isotype antigen-presenting cell (APC) or phycoerythrin (PE) control subjects.

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Figure 4.        Four-color flow cytometric analysis of dendritic cell (DC) subtypes. Expression of human immunodeficiency virus (HIV) type 1 coreceptors and costimulatory molecules was examined on myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) using 4-color flow cytometry in uninfected subjects and HIV-1infected subjects with HIV-1 RNA levels >2000 copies/mL. A significant difference between CD4 (A) and CXCR4 (B) expression was not found (see text). However, mDC expression of CCR5 (C) was significantly increased in the viremic patients (P = .028, Mann-Whitney U test). CD40 mean fluorescence intensity (MFI) was significantly higher on DCs from HIV-1infected individuals (mDC, P = .029; pDC, P = .0721, Mann-Whitney U test) (D). DC expression of CD86 was also higher in HIV-positive donors, but these differences were not statistically significant (E). *P < .05.

     We next compared the respective mDC and pDC expression of CD4, CXCR4, CCR5, CD40, and CD86 between uninfected control subjects and HIV-1infected, viremic subjects . CD4 has been reported to be highly expressed on blood DC subsets and to decrease on mDCs with culture [10, 24]. Although the percentage of CD4⁺ mDCs and CD4⁺ pDCs was slightly lower in HIV-1infected, viremic subjects relative to uninfected control subjects, these differences did not reach statistical significance ( percentage of CD4⁺ mDCs, P = .279; percentage of CD4⁺ pDCs, P = .279, Mann-Whitney U test for both comparisons). These results suggest that the marked decrease in Lin^-HLA-DR⁺ cells expressing CD4 in HIV-1infected subjects using 3-color flow cytometry likely reflects decreases in absolute numbers of pDCs and mDCs in the blood of those subjects rather than a true decrease in the surface expression of CD4 on those cells.

     DC expression of CXCR4 on mDCs and pDCs was slightly higher in HIV-1infected subjects than in control subjects, but, again, these differences were not statistically significant ( percentage of CXCR4⁺ mDCs, P = .383; percentage of CXCR4⁺ pDCs, P = .645, Mann-Whitney U test for both comparisons). Expression of CCR5 on mDCs, expressed at very low levels in uninfected subjects, was significantly increased in HIV-1infected donors . The expression of CCR5 on pDCs did not significantly differ between the donor groups. Higher expression of CD40 was observed on both mDCs and pDCs in HIV-1infected subjects with plasma viremia, relative to uninfected control subjects . Although the median CD86 expression on both DC subsets was slightly higher in viremic subjects than in control subjects, these differences were not statistically significant, perhaps because of the small sample size . These results are consistent with the findings of increased CD40 and CD86 expression on the Lin^-HLA-DR⁺ DC population in HIV-1infected viremic subjects using 3-color flow cytometry, so they likely represent true immunophenotypic changes related to HIV-1 replication.

     Longitudinal analysis of blood DCs in antiretroviral-naive HIV-1infected subjects after starting HAART.     To evaluate the effect of in vivo virus suppression on blood DC number and phenotype, blood DCs were evaluated by flow cytometry in 5 antiretroviral therapynaive, HIV-1infected subjects from the initial analysis over a period of 6 months after institution of HAART. Of the 5 subjects studied, 2 had baseline peripheral CD4⁺ T cells counts >200 cells/L (range, 251368 cells/L), and 3 had CD4⁺ T cells counts <200 cells/L (range, 57110 cells/L). All 5 subjects achieved good viral suppression during HAART, with undetectable plasma HIV-1 RNA levels (<200 copies/mL) measured in all subjects after 6 months on therapy ( and ). All the subjects had an increase in their peripheral CD4⁺ T cells counts after the initiation of therapy.

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Figure 5.        Longitudinal analysis of dendritic cell (DC) subsets and immunophenotype in human immunodeficiency virus (HIV) type 1infected subjects after institution of highly active antiretroviral therapy (HAART). CD4⁺ T cell counts, HIV-1 RNA levels, blood myeloid DC (mDC) and plasmacytoid DC (pDC) nos., and Lin^-HLA-DR⁺ DC expression of CD86 and CD40 were measured before and 6 months (mo) after starting HAART. For technical reasons, analysis of CD86 expression on DCs was performed on only 4 of 5 original subjects. MFI, mean fluorescence intensity.

     Blood DC subsets, mDCs and pDCs, were measured before therapy and after 6 months of receiving HAART ( and ). The median baseline (pre-HAART) number of mDCs in the HIV-1infected subjects was 5390 cells/mL (range, 85017,940 cells/mL), and the baseline number of pDCs was 2770 cells/mL (range, 85010,260 cells/mL). Some degree of recovery of both mDCs and pDCs was observed after 6 months of virus suppression, with a more marked and consistent increase with therapy noted in the pDC subset than in the mDC subset (mean change in mDC number, 530 cells/mL; mean change in pDC number, 4510 cells/mL), although these changes relative to pre-HAART values did not reach statistical significance (mDC, P = .625; pDC, P = .125, Wilcoxon signed-rank test). Despite the increase in DC numbers from baseline after starting therapy, posttreatment pDC and mDC numbers remained lower than the median numbers measured in control subjects.

     Surface expression of CD40 and CD86 on Lin^-HLA-DR⁺ blood DCs was also evaluated before and after initiation of HAART in this cohort of subjects ( and ). Four subjects were evaluated with respect to CD86 expression of DCs, and all 4 displayed some decrease in CD86 expression on Lin^-HLA-DR⁺ blood DCs after virus suppression . CD86 expression on Lin^-HLA-DR⁺ blood DCs decreased from a median MFI of 35.85 before therapy to 20.96 at 6 months of HAART, resulting in values after virus suppression similar to those observed in uninfected control subjects (median CD86 MFI, 19.45; ). A decrease in DC expression of CD40 was also observed in 3 of 5 subjects tested after 6 months of therapy, with the median MFI decreasing from 89.53 to 61.40 . In uninfected control subjects, the median CD40 MFI was 52.96 .

     Relationship of blood DC numbers to T cell lymphoproliferation.     Because DCs are potent APCs important in both priming and maintaining Ag-specific T lymphocyte responses in vivo, we reasoned that a relationship might exist between the number of circulating DCs and measured T cell lymphoproliferative function. To address this hypothesis, standard LPAs were performed using PBMC from 30 of the initial 34 HIV-1infected study subjects. To determine whether numbers of mDC or pDC subsets or other surrogate markers of HIV-1 disease, such as peripheral CD4⁺ T cell counts and plasma HIV-1 RNA levels, were predictive of the probability of a positive proliferative response to recall or HIV-1 antigens in the LPA (defined here as a stimulation index 5), univariate logistic-regression analyses were carried out . Peripheral CD4⁺ T cell counts and plasma HIV-1 RNA levels were statistically predictive of the probability of a positive LPA response to HIV-1 p24, HIV-1 p66, and cytomegalovirus (CMV) antigens. The absolute numbers of blood mDCs or pDCs were not predictive of a response to either HIV-1 p24 or p66 Ags. However, mDC numbers were predictive of a CMV-specific LPA response, and the predictive value of pDC numbers for a CMV-specific response trended toward significance. None of the parameters measured was predictive of an LPA response to Candida Ag. When a direct relationship between blood DC numbers and magnitude of lymphoproliferative responses was assessed, significant positive correlations were observed between blood mDC numbers and CMV LPA responses (Spearman's , 0.449; P = .010) and between blood pDC numbers and LPA responses directed against HIV-1 p24 (Spearman's , 0.407; P = .021; data not shown.).

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Table 3.          Summary of logistic univariate regression analysis of lymphoproliferative responses.

DISCUSSION

     To date, the role of blood DC subtypes in the immunopathogenesis of HIV-1 infection has not been well defined. In our analysis, we found that absolute mDC and pDC numbers in the peripheral blood, as well as the percentage of each DC subset in total PBMCs, were significantly decreased in HIV-1infected individuals, compared with control subjects. This deficit was more notable in subjects with detectable plasma HIV-1 RNA levels. However, a direct correlation between DC numbers or the percentage of CD11c⁺ or CD11c^-IL-3R^HI+ DCs and plasma HIV-1 RNA levels was not found in our study, either when analyzing all HIV-1infected subjects or only those with detectable plasma viremia.

     The HIV-1infected subjects in the present study were grouped by clinical treatment status into HS, HF, or HN cohorts for the purpose of analysis. We observed that the number of mDCs and pDCs was markedly decreased in the 2 cohorts with active plasma viremia (HF and HN subjects), compared with uninfected control subjects, whereas the number of mDCs and pDCs in subjects with effective virus suppression while receiving HAART (HS) were lower, but not significantly lower, than those in control subjects. It remains unclear whether the decrease in DC numbers we observed in the blood of subjects with active HIV-1 replication is a result of DC death and deletion, migration to lymph nodes where active viral replication is occurring, or a down-regulation of traditional DC surface markers. Regardless of the mechanism, our longitudinal data from HAART-treated subjects suggest that these defects may, in part, be reversible.

     It has been proposed that pDCs play a role in controlling plasma viremia through their production of antiviral type I interferons [23]. As such, pDCs are likely important APCs that bridge the gap between innate and adaptive immunity and also exert an antiviral effect. Their deletion in the blood of HIV-1infected subjects has been associated with disease progression and development of opportunistic infections [23]. Chehimi et al. [20] found a sustained decrease in pDC frequency in untreated subjects that was not normalized in HAART-treated, virus-suppressed subjects. However, the results of our longitudinal analysis suggest that pDC numbers do recover to some degree after virus suppression with HAART and that they do so more quickly and consistently than do mDCs. This degree of recovery is encouraging. The difference in the rate and degree of reconstitution of the different DC subtypes during therapy suggests that the mechanisms of their depletion from the blood during active viremia may also differ. Understanding the mechanism of the observed defects in blood DC numbers will require further investigation.

     In addition to assessing the effect of HIV-1 replication on blood DC numbers, we evaluated the relationship between peripheral CD4⁺ T cell counts and numbers of both mDCs and pDCs in the blood of our HIV-1infected subjects, finding a strong positive correlation between these 2 parameters and confirming the findings of others [5, 9]. A similar relationship between peripheral CD4⁺ T cell and DC numbers was observed in the setting of antiretroviral therapy, as evidenced in our longitudinal analysis of DCs before and after initiation of HAART. This positive correlation between blood DC and peripheral CD4⁺ T cell count suggests that the level of blood DCs may serve as another measure of immune competence during HIV-1 infection. In addition, the positive correlation may speak to an intricate relationship between blood DCs and CD4⁺ T cells. In fact, several murine studies have shown that DCs may influence the survival of CD4⁺ T cells, naive CD4⁺ T cells in particular, in the periphery [25, 26].

     The finding that both mDC and pDC numbers were decreased in the setting of HIV-1 plasma viremia also led us to investigate whether immunophenotypic abnormalities might be present on DCs in this setting. CD4, CXCR4, and CCR5 are surface receptors expressed on cells, including CD4⁺ T lymphocytes, monocytes, and some DCs, that are susceptible to infection with HIV-1 [27 30]. We sought to examine the effect of HIV-1 infection and active HIV-1 plasma viremia on DC surface expression of these HIV-1 coreceptors. Using 3-color flow cytometry, expression of CD4 on the total Lin^-HLA-DR⁺ blood DC population was first evaluated. We observed that the percentage of Lin^-HLA-DR⁺ blood DCs expressing CD4 was significantly decreased in all HIV-1infected subjects, regardless of treatment status, and that the percentage of CD4⁺ DCs correlated inversely with plasma HIV-1 RNA levels. This initially raised the question as to whether the DCs that expressed CD4 had become actively infected with HIV-1 and, as a consequence, were deleted from the blood or whether surface expression of CD4 on blood DCs had been down-regulated. When the respective DC subsets were examined using 4-color flow cytometry, the percentage of each DC subset expressing CD4 did not significantly differ between the control group and the viremic subjects. These results suggest that, in all likelihood, the lower numbers of CD4⁺ Lin-HLA-DR⁺ DCs observed initially in the blood of HIV-1infected subjects reflects an absolute decrease in numbers of CD4-expressing DCs in both DC subsets rather than an actual down-regulation of CD4 expression on DCs. Whether CD4⁺ blood DCs have been deleted from the peripheral DC pool or have migrated out of the peripheral blood remains unclear.

     CCR5 and CXCR4 serve both as chemokine receptors and as coreceptors for HIV-1 infection, and surface expression of these receptors on DCs has been reported to change as a result of DC maturation [31 33]. As such, their expression on each DC subset might be involved in mediating susceptibility to HIV-1 infection [24, 32], be involved in trafficking of blood DCs to tissues and lymph nodes [31], or may simply reflect an altered DC activation state. Our results, as well as those of some previous studies, showed that the HIV-1 coreceptors CD4, CCR5, and CXCR4 were more highly expressed on the surface of pDCs than on mDCs in control subjects, suggesting a possible mechanism for increased susceptibility of pDCs to HIV-1 infection and their subsequent deletion in the blood [5, 32]. This hypothesis also finds support in several in vitro studies that reported an increased susceptibility of pDCs, compared with mDCs, to infection with certain strains of HIV-1 [24, 32].

     However, as noted above, increased expression of CCR5 was observed on mDCs in viremic, HIV-1infected subjects, potentially rendering these DCs more susceptible than usual to infection with R5-tropic strains of HIV-1, as well as making them more likely to migrate in response to inflammatory stimuli [33]. DC subsets from viremic subjects also expressed slightly higher levels of CXCR4 than did DCs from seronegative donors. DC surface expression of CXCR4 has been reported to increase [31] or to remain unchanged [32, 33] with maturation in some studies, whereas surface expression of CCR5 was observed to decrease with maturation in one study [33]. Whether or not the changes in blood DC chemokine receptor expression that we observed in viremic subjects are implicated in causing the defect in DC numbers, or whether these changes simply reflect differences in DC activation or maturation, has yet to be determined.

     The DC costimulatory molecules CD40 and CD86 increase as DCs mature and are important in T cell activation through interactions with CD40L and CD28, respectively [1]. We observed that DC expression of both CD40 and CD86 was higher in HIV-1infected subjects than in control subjects. Among the HIV-1infected subjects, those with plasma viremia had higher median expression of these costimulatory molecules, compared with those who were HAART suppressed, and a significant positive correlation was observed between DC expression of CD86 and plasma HIV-1 RNA levels. These changes in costimulatory molecule expression on Lin^-HLA-DR⁺ blood DCs were also observed on pDC and mDC subsets in subjects with viremia. Of interest, HN subjects treated with effective antiretroviral therapy had decreases in the expression of CD40 and CD86 on DCs over time with virus suppression, which suggests that these abnormalities may, in part, be reversible with treatment. Increased DC surface expression of CD40 and CD86, taken together with the correlation observed between CD86 and plasma viremia, suggests that HIV-1 may serve as an activation or maturation stimulus to DCs, either directly or indirectly via production of proinflammatory cytokines such as tumor necrosis factor (TNF) [34]. Proinflammatory cytokines, such as TNF and interleukin-1, are known to be potent DC maturation factors [35 37], and elevated levels of these cytokines have been measured in the blood of HIV-1infected individuals [38]. It has also been shown that maturation of blood DCs may be associated with an up-regulation of CCR7 expression, theoretically resulting in migration of blood DCs to lymph nodes and other tissues [39 41]. Thus, the decrease in blood DC numbers observed in HIV-1infected subjects may not simply reflect deletion of these subsets but may represent an adaptive mechanism by which DC precursors deal with a large pathogen load by migrating from the blood to T cellrich areas in lymphoid organs, where they present Ag and stimulate T cells. Finally, one potential adverse consequence of increased DC costimulatory molecule expression might be a subsequent increase in nonspecific activation of T cells by DCs, thereby implicating DCs in the cycle of chronic immune activation associated with unchecked HIV-1 infection [42].

     To better define the relationship between blood DC numbers and T cell function in our HIV-1infected cohort, we examined the relationship between DC numbers and CD4⁺ lymphoproliferative ability. CD4⁺ T cell counts and plasma HIV-1 RNA levels were statistically predictive of the probability of an LPA response to most Ags, as might be expected. Although, in general, blood DC numbers did not predict a significant lymphoproliferative response to HIV-1 Ags, they did appear to predict T cell responses to CMV Ag, responses that are generally larger in magnitude than those directed against HIV-1 Ags. The inability of DC numbers to predict an HIV-1specific LPA response does not negate the likely importance of DCs in initiating or maintaining these Ag-specific T cell responses in vivo. The relationship between DCs and T cell function in the setting of HIV-1 infection may not be based simply on numbers of DCs but perhaps on more complex aspects of DC function. As mentioned, blood DCs may play a role in inducing primary immune responses to evolving viral Ags in the setting of active viremia or may be involved in sustaining naive T cells in the periphery during immune restoration on HAART. Sorting out the complex relationship between blood DCs and T cells will require further investigation.

     Defining the defects in blood DC number and function in the setting of HIV-1 infection should aid in the design of therapies aimed at fully restoring immune function in chronically HIV-1infected individuals. The results presented here further delineate the impact of active HIV-1 replication on the numbers and immunophenotype of blood mDCs and pDCs and suggest a possible role for blood DC subsets in the immunopathogenesis of HIV-1 disease.

Acknowledgments

     We thank the physicians, staff, and patients in the Infectious Diseases Group Practice at the University of Colorado Health Sciences Center, for their assistance and participation in our study; Karen Helm and Mike Ashton, for help with the flow cytometry; Ian McNiece and Steven Rosinski, for technical advice; Robert Schooley, for critical review of the manuscript; and the National Institutes of Health Division of AIDS, Vaccine and Prevention Research Program, for providing reagents used in the study.

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