Figure 1.
Levels of soluble and cell-associated CD40 ligand in HIV-1 infected patients (pat.) and controls (ctrl.).
(a) Baseline counts of natural killer (NK) cells, monocytes, CD4+ and CD8+ T cells, B cells, plasmacytoid (PDC) and myeloid dendritic cells (MDC) in HIV-1 infected individuals and uninfected controls, given as percentages (%) of peripheral blood mononuclear cells (PBMC). (b) Plasma levels of interleukin (IL-)3 and soluble CD40 ligand (sCD40L), the latter also analyzed in patients on antiretroviral therapy (HAART). (c) Expression of cell-associated CD40L (cCD40L) and (d) CD40 at the cell surface, evaluated by flow cytometry as percentage of positive cells and delta mean fluorescence intensity after subtraction of isotype controls. Data are presented as median, interquartile ranges (boxes), and 10% and 90% values (whiskers). *p<0.05, **p<0.01, ***p<0.001 (Mann-Whitney test).
Figure 2.
Deficits in interferon (IFN) alpha production in HIV-1 infected patients (pat.) versus control donors (ctrl.).
Peripheral blood mononuclear cells (PBMC) were exposed to (a) the CpG-A oligonucleotide 6016 (0.75 µM) and (b) the CpG-P oligodeoxynucleotide 21798 (0.25 µM) for 20 hours (h). The IFN-alpha production is given before and after normalization for the percentage of plasmacytoid dendritic cells (PDC). The data of HIV-1 infected patients were also analyzed for samples with CD4+ T cell counts below 250/µl (<250), between 250–500/µl (250–500), and above 500/µl (>500). Data are presented as median, interquartile ranges (boxes), and 10% and 90% values (whiskers). *p<0.05, **p<0.01, ***p<0.001 (Mann-Whitney test). Part of the data were presented in another study [31]. Similar data were obtained using S-27609 (data not shown).
Figure 3.
Suppression of interferon (IFN) alpha production by CD40 ligand.
(a) Preincubation of peripheral blood mononuclear cells (PBMC) of controls without (w/o) or with (w/t) a cell-culture grade commercial soluble CD40 ligand (sCD40L; 3 µg/ml) for 48 hours (h), followed by stimulation with CpG-P 21798. Data represent 11 and 5 separate experiments for IL-3 levels of 0 and 5–5,000 pg/ml, respectively. The arrow indicates physiological IL-3 levels. (b) Neutralization of sCD40L with a cell-culture grade anti-CD40L antibody with subsequent CpG-P stimulation in two donors. Similar data were obtained using CpG-A (data not shown). (c) Direct effect of sCD40L (3 µg/ml) on the CpG-P induced IFN-alpha production by plasmacytoid dendritic cells (PDC) of control donors. (d) Expression of cell-associated CD40L (cCD40L) on baby hamster kidney (BHK) cells. (e) Shedding of sCD40L into the cell culture supernatants after 24 h and 48 h of culture. (f) Western Blot of concentrated supernatants of CD40L-expressing BHK cells and a commercial sCD40L preparation (R&D Systems) using reducing conditions. Data represent three separate experiments. No bands were detected in the supernatants of BHK wild type cells (data not shown). (g) Inhibition of CpG-P induced IFN-alpha production using sCD40L from R&D Systems and BHK cells. Statistics were calculated between PBMC of HIV-1 infected patients (pat.) (n = 5) and controls (ctrl.) (n = 10) exposed to sCD40L (R&D Systems). (h) cCD40L expression on CD4+ and CD8+ T cells of control donors after stimulation with mock (grey filled curve) or PMA/ionomycin (black dotted curve). (i) Inhibition of CpG-P induced IFN-alpha production by increasing counts of untreated (unstim.) or PMA/ionomycin-stimulated (stim.) CD4+ T cells, which were left unfixed or fixed used 4% paraformaldehyde, and coincubated with 1×106 PBMC of five control donors. Similar results were obtained with CpG-A (data not shown). (j) Inhibition of CpG-P induced IFN-alpha production by paraformaldehyde-fixed cCD40L-expressing and wild type (WT) BHK cells using PBMC of eight control donors. Data are presented as mean and standard error. *p<0.05, **p<0.01, ***p<0.001 (Student's t-test).
Figure 4.
Effect of CD40L neutralization on the suppression of CpG-P induced interferon (IFN)-alpha production.
Peripheral blood mononuclear cells (PBMC) of three control donors were preincubated with 50,000 unstimulated (unstim.) or PMA/ionomycin-stimulated (stim.) CD4+ T cells of the respective donors for 48 hours, in the absence or presence of a cell-culture grade anti-CD40L antibody (30 µg/ml), and then stimulated with CpG-P 21798 (0.25 µM) for additional 24 hours. Data are presented as mean and standard error. *p<0.05, **p<0.01 (Tukey HSD test).
Figure 5.
Correlation of CpG-induced interferon (IFN)-alpha production with clinical parameters.
Spearman rank order correlation of CpG-A and CpG-P induced IFN-alpha production with (a) the percentage of PDC, (b) the CD4+ T cell count, and (c) the viral load in untreated HIV-1 infected individuals (n = 40).
Table 1.
Correlation of the CpG-induced interferon (IFN) alpha production with sCD40L plasma levels and the expression of CD40 on the PDC in HIV-1 infected subjects.
Figure 6.
Effect of soluble CD40 ligand (sCD40L) on the cytokine profile.
Peripheral blood mononuclear cells (PBMC) of HIV-infected patients (pat.) and control (ctrl.) donors were exposed to low-dose sCD40L (50 pg/ml) for 48 hours and subsequently stimulated with the CpG-P oligodeoxynucleotide 21798 for 20 hours. The levels of interleukin (IL-)2, interferon gamma (IFN-g), tumor necrosis factor alpha (TNF-a), IL-6, IL-8, and IL-10 were measured using a fluorescent bead assay. IL-4, IL-5, IL-12, TNF-beta, and IL-1 beta were not detected. Data are presented as mean and standard error. *p<0.05.