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* Dermatology Branch and
Experimental Retrovirology Branch, Center for Cancer Research, National Cancer Institute; and
Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892
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Abstract |
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Introduction |
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The use of monocyte-derived DC, first described in 1994 (22, 23), has greatly facilitated studies into numerous aspects of DC biology. These cells are now the most extensively studied subtype of DC. The popularity of using monocyte-derived DC is largely due to the ability to easily generate and purify large numbers of these cells. Because of this, many studies performed using monocyte-derived DC could never have been conducted using Langerhans cells or other tissue-derived DC, where isolation of sufficient numbers of purified cells is extremely difficult. One important caveat, however, is that research findings involving monocyte-derived DC have not always correlated with data obtained using tissue-derived DC or with studies done on DC in vivo.
In this study, the aim was to re-examine the question of whether HIV infection impairs the APC function of DC. Initially, HIV infection of monocyte-derived DC was noted to cause marked down-regulation of cell surface CD4. This observation allowed for the enrichment of HIV-infected DC in heterogeneous cultures containing uninfected and infected DC (by depleting populations of HIV-uninfected CD4+ DC). Extensive functional analyses using highly enriched populations of HIV-infected DC were then performed, directly comparing these cells with unenriched HIV-infected DC populations and uninfected DC. Interestingly, HIV infection of monocyte-derived DC impaired their ability to stimulate CD4+ T cell proliferation and IL-2 production. This effect was mediated by soluble HIV gp120 produced in these cultures. Taken together, these findings suggest that DC function, altered by HIV infection and production of soluble HIV gp120, directly affects CD4+ T cell-mediated immune responses.
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Materials and Methods |
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All of the murine mAbs were purchased from BD PharMingen (San Diego, CA). Immunex (Seattle, WA) kindly provided soluble trimeric recombinant human CD40 ligand (CD40L). Zidovudine and didanosine (ddI) were purchased from Sigma-Aldrich (St. Louis, MO) and JE2147 was obtained from Japan Energy (Tokyo, Japan). The latter is a relatively new potent protease inhibitor that has been used in previous laboratory studies (24, 25). Dr. R. Offord at the University of Geneva (Geneva, Switzerland) kindly provided aminooxypentane (AOP)-RANTES, Dr. N. Fujii at Kyoto University (Kyoto, Japan) kindly provided AMD3100, and Genentech (Vacavill, CA) kindly provided soluble (s) CD4. Recombinant HIV gp120IIIB was supplied by the National Institutes of Health AIDS Research and Reference Reagent Program (Rockville, MD).
Preparation of cells
As previously described (15, 17, 26), plastic-adherent PBMC from healthy adults were cultured for 1 wk in RPMI-based complete medium containing 10% heat-inactivated FBS (Biofluids, Rockville, MD), 1000 U/ml recombinant human GM-CSF (Immunex), and 1000 U/ml recombinant human IL-4 (R&D Systems, Minneapolis, MN). At day 7, DC were harvested, washed, and resuspended in a mixture containing the following mouse anti-human mAbs (each at 5 µg/ml): anti-CD3, anti-CD14, anti-CD16, and anti-CD19. Cells were incubated with the mixture for 30 min at 4°C, with gentle agitation every 10 min. During this incubation, magnetic beads (5 beads/cell) coated with sheep anti-mouse IgG Abs (Dynal, Great Neck, NY) were washed in HBSS/10% FCS (wash medium) using an MPC-1 magnetic particle concentrator (Dynal) and resuspended in 1 ml of wash medium. After incubation with the mAb mixture, DC were washed, mixed with the magnetic bead suspension, and incubated for an additional 30 min at 4°C. Contaminating lineage-specific PBMC were then separated from DC through a series of washes using the magnetic particle concentrator. As assessed by flow cytometry, DC were always >99% pure using this method. Cryopreserved allogeneic PBMC were enriched for CD4+ T cells by negative selection using a commercially prepared mAb mixture/complement reagent (Lympho-Kwik; One Lambda, Los Angeles, CA;>90% pure as assessed by flow cytometry).
HIV infection of DC
Purified, pelleted, and titered HIV-1Ba-L (an R5 virus; stock at 50% tissue culture-infective dose of 107.17/ml) and HIV-1IIIB (an X4 virus; stock at 50% tissue culture-infective dose of 108/ml) were purchased from Advanced Biotechnologies (Columbia, MD). HIV (1/100 dilution of each stock) was added to cultures containing 2 x 106 DC suspended in 1 ml of complete medium with GM-CSF/IL-4 (1.8 x 108 HIV-1Ba-L particles/2 x 106 DC, 6.7 x 108 HIV-1IIIB particles/2 x 106 DC). After overnight incubation with HIV, DC were washed, resuspended in complete medium supplemented with GM-CSF/IL-4, and cultured for 10–12 additional days. DC were cultured for this length of time before functional analyses to allow for sufficient spreading of HIV infection in a larger percentage of cells. Half of the total volume of medium was replaced with fresh complete medium and GM-CSF/IL-4 every other day.
Phenotypic characterization of HIV-infected DC
DC were collected 10–12 days after HIV exposure and incubated with 10 µg/ml PE-conjugated mAbs against surface molecules. Cells were then incubated with Dead Red (Molecular Probes, Eugene, OR), fixed/permeabilized with Cytofix/Cytoperm reagents (BD PharMingen), incubated with 10 µg/ml unconjugated rat anti-HIV-1 p24 mAbs (BioSource International, Camarillo, CA), and finally incubated with FITC-conjugated goat anti-rat IgG F(ab')2 (BioSource International). Live cells were examined using a FACScan (BD Biosciences, Mountain View, CA) equipped with Lysis II software (BD Biosciences). For each Ag, specific staining was confirmed in at least three separate experiments. Of note, as previously shown, the number of single HIV-infected DC determined by intracellular HIV p24 staining and flow cytometry correlates well with levels of HIV p24 protein released into supernatants containing HIV-infected DC (26).
Enrichment for HIV-infected DC
Ten to 12 days after HIV exposure, DC were repurified by immunomagnetic bead separation as described above. Because down-regulation of surface CD4 on HIV 24+ DC was a consistent and reproducible finding (Fig. 1A and Table I), enriched populations of HIV-infected DC could be generated for further functional studies. For these experiments, HIV-exposed DC were enriched for HIV+ cells by immunomagnetic bead negative selection with additional mAbs directed against CD4. In initial experiments to confirm and quantify enrichment, CD4-depleted and CD4-nondepleted DC were fixed/permeabilized, stained with anti-HIV p24 mAbs, and examined by flow cytometry (Fig. 1B). For later functional experiments using enriched DC, cells were not fixed and permeabilized (Figs. 2–6)
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Each functional experiment was performed with five groups of DC (cultured from a single individual): uninfected DC, HIVBa-L-infected unenriched DC, HIVBa-L-infected enriched DC, HIVIIIB-infected unenriched DC, and HIVIIIB-infected enriched DC. All experiments were performed with particular emphasis on paired data obtained from uninfected DC vs HIVBa-L-infected enriched DC, as well as paired data from uninfected DC vs HIVIIIB-infected enriched DC. DC were obtained from many different individual donors. Because of MHC mismatch differences, T cell stimulation values done on different days with DC obtained from different donors were not directly compared. Instead, comparisons were always between the five groups of cells cultured from a single individual.
Briefly, 2 x 105 allogeneic CD4+ T cells were resuspended in complete medium (as above, except that 10% FBS was replaced with 2.5% human AB serum) and cocultured with varying numbers of DC for 5 days. [3H]Thymidine (1 µCi) was added to cultures 16–18 h before harvesting and thymidine incorporation was measured. Stimulation ratio was defined as the cpm of cultures containing 5 x 103 HIV-infected DC and 2 x 105 CD4+T cells divided by the cpm of cultures containing 5 x 103 uninfected DC and 2 x 105 CD4+ T cells. IL-2 production ratio was defined as the IL-2 detected in day 2 supernatants in cultures containing 5 x 103 HIV-infected DC and 2 x 105 CD4+ T cells divided by the IL-2 detected in day 2 supernatants in cultures containing 5 x 103 uninfected DC and 2 x 105 CD4+ T cells as measured by ELISA (R&D Systems).
In some experiments, DC were fixed with 1% paraformaldehyde (PFA) before coculture with T cells. In other experiments, DC were preincubated with various concentrations of ddI (1–10 µM), JE2147 (10–100 nM), sCD4 (0.3–10 nM), AOP-RANTES (1–100 nM), AMD3100 (2–200 nM), or combinations of these drugs for 16 h, then incubated with CD4+ T cells in the presence of these same drugs at the preincubation concentrations. In experiments using uninfected allogeneic DC and CD4+ T cells, recombinant HIV gp120 was added in doses ranging from 0.001 to 1 nM.
The statistical significance of both the stimulation ratios and the IL-2 production ratios (compared with uninfected) for each of the four HIV-infected DC groups (BaL, E-BaL, IIIB, and E-IIIB) was determined by a t test using 1.0 as the null hypothesis for the ratio, after confirmation that the data to be analyzed were normally distributed. The difference between the stimulation ratios and IL-2 production ratios for BaL vs E-BaL, and IIIB vs E-IIIB, were also determined using a paired t test after confirmation that these paired differences were normally distributed. The statistical significance of the results was determined after an adjustment for multiple comparisons using the Hochberg method (27). Thus, all reported p values are adjusted and are two tailed.
Characterization of cytokine production by HIV-infected DC
Each cytokine production experiment was performed with five groups of DC (as described above). Again, all experiments were performed with particular emphasis on paired data obtained from uninfected DC vs HIVBa-L-infected enriched DC as well as paired data from uninfected DC vs HIVIIIB-infected enriched DC. The five groups of DC (5 x 105/ml) were incubated with or without CD40L (1 µg/ml) for 48 h and IL-12p70 (R&D Systems) and IL-10 (BD PharMingen) were measured by ELISA.
Comparisons between the paired data for BaL vs E-BaL, as well as between IIIB and E-IIIB, were made using the Wilcoxon signed rank test because of the non-normal appearance and limited amount of data. An analysis of the "trend" from HIV-uninfected to BaL to E-BaL and from HIV-uninfected to IIIB to E-IIIB was done using Fisher’s method for combining p values as applied to the individual segments of the graphs, i.e., from HIV-uninfected to BaL and from BaL to E-BaL and similarly from HIV-uninfected to IIIB and from IIIB to E-IIIB.
HIV p24 and HIV gp120 detection by ELISA and Western blot analyses
Supernatants from cocultures of HIV-infected DC and CD4+ T cells were collected on days 2, 4, and 5 and HIV p24 (Coulter, Miami, FL) and HIV gp120 (Advanced Biotechnologies) protein content was measured by ELISA. In addition, 1.5 ml of supernatant fluid collected on day 5 of cocultures were passed through 0.45-µm filters and ultracentrifuged to pellet virions. Virion-derived protein was subjected to electrophoresis under reducing conditions followed by electroblotting. HIV Gag and Env proteins were visualized using anti-p24 and anti-gp120 antisera, respectively (28).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because of the need to generate sufficient numbers of infected cells for functional studies, relatively high amounts of virus were used to infect monocyte-derived DC. DC were double stained with mAbs against surface Ags of interest and intracellular HIV p24 10 days following HIVBa-L or HIVIIIB infection. Expression levels of MHC class II and costimulatory molecules on HIV p24+ DC were similar to levels on HIV p24- DC and on uninfected DC (representative phenotypic data in Fig. 1A and summary of all phenotypic data in Table I). However, expression levels of MHC class I, CD1a, and CD4 on HIV p24+ DC were down-regulated by 
30, 60, and 80%, respectively (Fig. 1A and Table I), consistent with previous results observed in HIVBa-L-infected epidermal Langerhans cells (29). Of note, expression levels of MHC class II and costimulatory molecules on HIV p24+ DC that were matured in the presence of CD40L for 2 days were also similar to levels on mature HIV p24- DC and on mature uninfected DC (data not shown), indicating that HIV infection of immature DC did not interfere with their ability to respond appropriately to a maturation stimulus.
For functional assays, HIV p24+ DC were enriched by depletion of CD4+HIV p24- DC using immunomagnetic bead separation. The percentage of HIV p24+ DC after CD4+ cell depletion was substantially increased in HIVBa-L-infected DC (mean, 83.2%; range, 79–91%, n = 5) and HIVIIIB-infected DC (mean, 77.8%; range, 54–95%, n = 4), compared with nonenriched HIVBa-L-infected DC (mean, 25.8%; range, 16–38%, n = 5) and nonenriched HIVIIIB-infected DC (mean, 14.0%; range, 6–21%, n = 4; representative enrichment data in Fig. 1B).
Impaired allogeneic CD4+ T cell proliferation and IL-2 production following stimulation by HIV-infected DC
To test APC capacity of HIV-infected DC, DC were cocultured with allogeneic CD4+ T cells (at various ratios) and T cell proliferation and IL-2 production were measured. Interestingly, when stimulated by enriched HIV-infected DC, CD4+ T cell proliferation was impaired when compared with that of T cells stimulated by nonenriched HIV-infected DC or uninfected DC (Fig. 2, A and B). The p values were as follows: 0.0008 (BaL vs uninfected); 0.0003 (E-BaL vs uninfected); 0.0123 (BaL vs E-BaL); 0.0001 (IIIB vs uninfected); 0.0001 (E-IIIB vs uninfected); and 0.0002 (IIIB vs E-IIIB). Similar alterations were observed in IL-2 production by CD4+ T cells (summary of data shown in Fig. 2C). The p values were as follows: 0.0043 (BaL vs uninfected); 0.0042 (E-BaL vs uninfected); 0.0345 (BaL vs E-BaL); 0.0046 (IIIB vs uninfected); 0.0012 (E-IIIB vs uninfected); and 0.0036 (IIIB vs E-IIIB). Thus, in general, CD4+ T cell proliferation and IL-2 production were inversely proportional to the number of HIV p24+ DC in the cocultures. Of note, DC pretreated with anti-CD4 mAbs exhibited a similar APC capacity to DC pretreated with control IgG (data not shown), suggesting that impaired APC function of HIV-infected DC was not due to loss of CD4 on the surface of DC.
Next, the mechanism(s) responsible for impaired allogeneic T cell stimulation by HIV-infected DC was studied. DC were fixed with 1% PFA before coculture with allogeneic CD4+ T cells. Fixed HIV-infected DC and fixed uninfected DC exhibited comparable APC capacity (Fig. 2D), suggesting that cell surface costimulatory molecules on HIV-infected DC were functionally intact. This finding is consistent with the phenotypic data shown above (Fig. 1A and Table I). These data also suggest that HIV-infected DC were secreting an immunosuppressive soluble factor.
Increased IL-12p70 and decreased IL-10 production by HIV-infected DC
To test whether secreted IL-12p70 and/or IL-10 produced by HIV-infected DC contributed to T cell dysfunction, DC were matured in the presence of CD40L for 48 h and these cytokines were measured in DC culture supernatants. Unstimulated HIV-infected DC and unstimulated uninfected DC produced low levels of IL-12p70 (<5 pg/ml) and IL-10 (<20 pg/ml). Interestingly, enriched HIVBa-L- or HIVIIIB-infected DC stimulated by CD40L produced higher amounts of IL-12p70 and lower amounts of IL-10 compared with both nonenriched HIV-infected DC and uninfected DC (Fig. 3, A and B). Consequently, the ratio of IL-12p70 production to IL-10 production by enriched HIV-infected DC was substantially more than that of uninfected DC (Fig. 3C). The p values were as follows: 0.0625 (BaL vs E-BaL) and 0.0625 (IIIB vs E-IIIB). Using Fisher’s method to assess "trends" in the IL-12p70 and IL-10 production data, the p values were as follows: 0.0078 (from uninfected DC to BaL to E-BaL) and 0.0078 (from uninfected DC to IIIB to E-IIIB). Thus, these data clearly showed that the IL-12p70:IL-10 production ratio was proportional to the number of HIV p24+ DC in the cell populations studied. Surprisingly, these results are in contrast to the altered cytokine production profile reported for HIV-infected monocytes/macrophages (30, 31, 32).
Impaired APC capacity of HIV-infected DC is reversed by sCD4, but not by antiretroviral drugs
To determine whether CD4+ T cell dysfunction was due to DC-mediated transfer of HIV infection to these cells, combinations of antiretroviral drugs (the reverse transcriptase inhibitor ddI and the relatively new protease inhibitor JE2147) were added to mixtures of DC and CD4+ T cells. As expected, this antiretroviral drug combination blocked HIV p24 production in cocultures of HIV-infected DC and CD4+ T cells (Fig. 4A). Furthermore, JE2147 alone (and not ddI alone) blocked HIV p24 release in DC-T cell cocultures as efficiently as the combination of ddI and JE2147 (Fig. 4A). As controls for these experiments, the addition of either AOP-RANTES or AMD3100, which bind to the HIV coreceptors CCR5 and CXCR4, respectively, failed to alter HIV p24 production in cocultures (data not shown). Since ddI, AOP-RANTES, and AMD3100 block de novo productive infection of cells, these data suggest that HIV p24 produced in the presence of these compounds was derived from DC infected before the time of coculture. Although JE2147 effectively blocked release of HIV p24 in DC-CD4+ T cell cocultures, it failed to restore T cell proliferation (Fig. 4A) or IL-2 production (data not shown). Similarly, culturing HIV-infected DC and CD4+ T cells in the presence of ddI, AOP-RANTES, or AMD3100 did not affect CD4+ T cell function (Fig. 4A and data not shown).
Strikingly, when sCD4 was added to DC-CD4+ T cell cocultures, the APC capacity of HIV-infected DC was completely restored (Fig. 4A). This ability of sCD4 to restore CD4+ T cell proliferation when cocultured with HIV-infected DC was dose dependent (Fig. 4B). By contrast, the addition of sCD4 did not alter accumulation of HIV p24 in coculture supernatants (Fig. 4). Thus, the amount of HIV replication, as measured by HIV p24 levels in coculture supernatants, did not correlate in any way with proliferation and IL-2 production by T cells.
HIV gp120 is produced and secreted by DC in the presence of sCD4 or antiretroviral drugs
HIV gp120 protein levels in the supernatants of DC-T cell cocultures were measured. Interestingly, although the protease inhibitor JE2147 blocked p55 cleavage and HIV p24 production, it did not inhibit HIV gp160 cleavage and subsequent HIV gp120 production by DC (Fig. 5). These findings are consistent with the known mode of action of JE2147, which has been shown to block release of mature HIV virions from infected cells, but does not inhibit release of HIV gp120 secreted in the form of immature defective virions (24). The latter process is mediated by a cellular protease and thus is not affected by drugs that impair function of the HIV protease.
Recombinant HIV gp120 impairs proliferation of CD4+ T cells stimulated by uninfected allogeneic DC in a dose-dependent manner: reversal of defects by sCD4
To confirm that HIV gp120 suppressed T cell proliferation in MLR, uninfected DC and uninfected allogeneic CD4+ T cells were cocultured in the presence of exogenous recombinant HIV gp120. CD4+ T cell proliferation in this context was significantly inhibited in the presence of HIV gp120 (Fig. 6). This suppression of CD4+ T cell proliferation by HIV gp120 was dose dependent (Fig. 6A) and was observed at varying DC:T cell ratios (Fig. 6B). DC pretreated with HIV gp120, washed, and then cocultured with T cells exhibited normal APC capacity (data not shown). Interestingly, sCD4 reversed HIV gp120-mediated suppression of CD4+ T cell proliferation in a dose-dependent manner (Fig. 6C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Using HIV-enriched vs nonenriched populations of DC, HIV-induced functional defects in cellular immunity were documented (Fig. 2). Enriched HIV-infected DC exhibited a poor capacity to stimulate primary immune responses to alloantigen. Several potential mechanisms for this defect were explored and found not to be responsible for impaired allostimulatory capability of HIV-infected DC. Specifically, HIV-infected DC 1) did not decrease expression of surface MHC class II and costimulatory molecules (Fig. 1A and Table I); 2) exhibited normal APC capacity when fixed with PFA (Fig. 2D); 3) produced increased amounts of IL-12p70 and decreased amounts of IL-10 (Fig. 3); and 4) failed to retain allostimulatory capacity in the presence of a combination of antiretroviral drugs that blocked HIV replication in DC-CD4+ T cell cocultures (Fig. 4). Since in vitro infection of monocyte/macrophages with HIV leads to decreased MHC class II expression, decreased IL-12p70 production, and increased IL-10 production (30, 31, 32), these current DC data highlight the differential effects of HIV infection in DC vs monocytes/macrophages. A similar discrepancy between these two cell types has been reported in Leishmania major and Mycobacterium tuberculosis infection; infection of monocytes/macrophages with either of these organisms inhibits IL-12p70 production (34, 35, 36), whereas infection, in the presence of IFN-
, induces DC to release IL-12p70 (35, 36). Our current results are different from those where we showed that immune function of monocyte-derived DC isolated from HIV-infected patients was normal (17). DC used in that previous study were quite different from those used in the current study. Specifically, in the previous work, monocytes were isolated from HIV-infected individuals and differentiated into DC during a 1-wk culture period with high doses of GM-CSF and IL-4, culture conditions that likely enhanced APC capacity, overcame any immunologic defect in DC present in vivo, and selected for HIV uninfected cells. By contrast, in the present study, fully differentiated DC were exposed to HIV, enriched for HIV infection, and then assessed for APC capacity.
Although a combination of antiretroviral drugs blocked production of mature HIV virions in DC-CD4+ T cell cocultures, these drugs had no effect on HIV gp120 levels in these cultures (Fig. 5). This is consistent with the fact that HIV gp120 release from infected cells is under the control of a cellular protease, as opposed to Gag release, which is regulated by the HIV protease enzyme. In contrast to antiretroviral drugs, sCD4 completely reversed T cell dysfunction caused by HIV gp120 released from HIV-infected DC (Fig. 4) and exogenous recombinant HIV gp120 (Fig. 6), presumably by blocking HIV gp120 from binding CD4 on the surfaces of T cells. These findings are consistent with many previous reports that describe HIV gp120-mediated inhibition of T cell signaling and proliferation (37, 38, 39, 40, 41). Unlike other work, however, HIV gp120 did not induce apoptosis in DC-T cell cocultures (data not shown). In fact, the addition of sCD4 led to complete restoration of CD4+ T cell proliferation and IL-2 production.
It is unclear whether HIV gp120 contributes to impaired cellular immunity in HIV-infected individuals, although our results complement some recent findings that demonstrate how HIV replication in vivo affects cellular immune responses. It has been known for many years that HIV infection causes diminished responses to recall Ags well in advance of CD4+ T cell depletion. In fact, proliferative responses to HIV Ags are not observed in the majority of untreated HIV-infected individuals. However, this is not due to the absence of HIV-specific CD4+ T cells; HIV-specific CD4+ T cells are detected by intracellular IFN- staining in the majority of infected patients (42, 43, 44, 45, 46). In one recent study, HIV viremia during structured treatment interruption caused suppression of HIV-specific and non-HIV-specific proliferative responses measured ex vivo, although Ag-specific cells were clearly detectable in peripheral blood (45). Thus, these recent results indicate that poor proliferative responses to Ags in HIV disease are not due to depletion of Ag-specific cells, but are instead a direct result of viremia. Although the frequency of HIV-infected DC in vivo is probably low, these cells may directly mediate a suppressive effect on CD4+ T cell proliferative responses to HIV or non-HIV Ags.
Our findings, taken in the context of previous clinical studies, suggest there may be some therapeutic role for sCD4 when used in combination with highly active antiretoviral therapy (HAART). sCD4 completely restored CD4+ T cell function in vitro, yet did not affect viral replication (Figs. 4 and 5). Conversely, a combination of antiretroviral drugs suppressed viral replication, but did not affect CD4+ T cell function (Figs. 4 and 5). These results highlight the disparity between suppression of viral load and restoration of cell-mediated immunity. Although previous clinical trials using sCD4 for HIV-infected patients have been unsuccessful (47, 48, 49, 50, 51), these studies were performed before the era of HAART (when most patients had poor control of plasma viremia). Despite poor control of viremia, it is very interesting to note that clear evidence of enhanced cellular immunity was documented in most patients who received sCD4 (47). Thus, we hypothesize that HAART plus sCD4 may be an effective combination for suppressing HIV replication while stimulating CD4+ T cell-mediated immune responses.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: DC, dendritic cell; HAART, highly active antiretroviral therapy; sCD4, soluble CD4; PFA, paraformaldehyde; AOP, aminooxypentane; CD40L, CD40 ligand; ddI, didanosine.
Received for publication August 16, 2002. Accepted for publication February 6, 2003.
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References |
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production in HIV-infected patients correlates with numerical and functional deficiencies in circulating type 2 dendritic cell precursors. Clin. Immunol. 101:201.[Medline]
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1 and increased viral replication with CD40 ligand-induced maturation. Eur. J. Immunol. 31:360.[Medline]
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, uninfected DC; 
, enriched HIVBa-L-infected DC; 
, enriched HIVIIIB-infected DC) were preincubated with the indicated compounds, washed, and then cocultured with allogeneic CD4+ T cells at a CD4+ T cell:DC ratio of 40:1 in the presence of the indicated compounds (JE2147 at 100 nM, ddI at 10 µM, and sCD4 at 10 nM). Thymidine incorporation was measured on day 5 and HIV p24 protein levels in supernatants were measured by ELISA on day 4. B, Similar to A, except that varying doses of sCD4 were used. Data shown are from single experiments performed in triplicate and are representative of at least five independent experiments.
) of a fixed concentration (1 nM) of recombinant HIVIIIB gp120. C, Similar to A, except that varying concentrations of sCD4 were added in the presence (•) or absence (