Table 1.
Clinical parameters of HIV-1+ patients.
Fig 1.
IFNAR2 expression following HIV-1 infection of LPMCs.
LPMCs from healthy donors were infected with R5-tropic HIV-1NL4.3 and cultivated for 4 days. (A) Surface expression of IFNAR2 (black line) on mock-treated and infected cells was determined via flow cytometry on CD4+ T cells, CD8+ T cells, B cells and NK cells using fluorescence minus one controls (tinted area). Histograms and dot plots of one representative LPMC sample are shown. (B) Individual frequencies of IFNAR2-expressing cells and mean values (+SEM) are shown as dots and bars (n = 11). Differences between the groups were analyzed by paired student’s t-test. (C) P24 expression of mock-treated and HIV-infected samples was analyzed by ELISA, p24 expression and mean values (+SEM) are shown by bars (n = 8). Differences between the groups were analyzed by paired student’s t-test, **** indicates p<0.0001.
Fig 2.
Soluble IFNAR2a expression in HIV-1-infected LPMCs and plasma samples.
Supernatant of mock-treated and R5-tropic HIV-1NL4.3-infected LPMCs from days 1, 4 and 7 post infection (A, n = 6) and plasma samples (B) from healthy donors (n = 13), cART-naïve (n = 22) and cART-experienced HIV-1+ patients (n = 22) were analyzed for sIFNAR2a expression via ELISA. The dotted line indicates the detection limit of 0.16 ng/ml. Individual frequencies of sIFNAR2a-expressing samples and mean values (+SEM) are shown as dots and bars. Statistically significant differences between the groups were analyzed by paired student‘s t-test (LPMCs) and ordinary one way ANOVA analysis with Bonferroni’s multiple comparison (plasma samples).
Fig 3.
IFNAR1 and IFNAR2 mRNA expression in HIV-1-infected LPMCs.
LPMCs were infected with R5-tropic HIV-1NL4.3 and cultivated for 4 days. Following CD4+ T cell isolation, mRNA expression levels of IFNAR1, IFNAR2b and IFNAR2c were analyzed via qRT-PCR. Quantification of the target mRNA was normalized according to the delta-delta Ct (ΔΔCt) method and the expression fold change (2-ΔΔCt) using GAPDH as reference. The mean gene fold expression (+SEM) of each target is shown in bars (n = 4). Differences between the groups were analyzed by paired student’s t-test.
Fig 4.
Titration of individual IFNα subtype activity.
The recombinant human reporter cell line ISRE-Luc was stimulated with series dilutions of each IFNα subtype. Maximal and minimal effective concentrations were determined and the EC50 was calculated. The upper and lower dotted lines indicate maximal and minimal effective concentration of the respective IFNα subtype, the middle dotted line indicates the EC50.
Fig 5.
IFNAR2 expression following stimulation with IFNα subtypes.
LPMCs were stimulated with the EC50 of IFNα1, IFNα2, IFNα8 and IFNα14 for 15 min, 30 min, 2 h and 24 h. Surface expression of IFNAR2 on unstimulated and stimulated cells was determined via flow cytometry on CD4+ T cells, CD8+ T cells, B cells and NK cells. Individual frequencies of IFNAR2-expressing cells and mean values (+SEM) are shown as dots and bars (n = 4). Differences between the groups were analyzed by ordinary one way ANOVA analysis and Bonferroni’s multiple comparisons.
Fig 6.
IFNAR1 and IFNAR2 mRNA expression in IFNα subtype-stimulated cells.
After isolation of CD4+ T cells from LPMCs, cells were stimulated with the EC50 of IFNα1, IFNα2, IFNα8 and IFNα14 for 30 min (A) and 24 h (B). Following mRNA isolation and reverse transcription, transcription levels of IFNAR1, IFNAR2b and IFNAR2c were analyzed via qRT-PCR. Quantification of the target mRNA was normalized according to the delta-delta Ct (ΔΔCt) method and the expression fold change (2-ΔΔCt) using GAPDH as reference. The mean gene fold expression (+ SEM) of each target is shown in bars (n = 3). Differences between the groups were analyzed by ordinary one way ANOVA analysis and Bonferroni’s multiple comparisons.