Fig 2: Dose-dependent HIV-inhibitory activity of IFN-a subtypes as examined in primary cells infected ex vivo. (A and B) CD4+ T cells, PBMCs, or MDMs were pretreated with the indicated IFN-a subtype for 2 h and then inoculated with HIV YU-2 overnight. The next day, the cultures were washed and the IFN-as added back. At days 0, 4, 8, and 12, supernatant was harvested to monitor HIV replication by quantifying p24 Ag. The area under the curve of the p24 Ag over time was calculated and normalized to that of the control (i.e., no IFN-a). (A) Data exploring anti-HIV activities of IFN-a1, -a2, -a6, and -a14 in subsets of six donors’ CD4+ T cells and three donors’ PBMCs at an extended dose range. (B) Data for IFN-a1, -a2, and -a6 in cells from larger numbers of donors (12, 9, and 9 donors, respectively), focusing on higher dosages of the IFN-a subtypes. Responses were compared to those measured in the IFN-a2-treated groups by significance testing with RM two-way ANOVAs, comparing all groups to the IFN-a2 group. Symbols for comparisons with IFN-a2 are as follows: *, IFN-a1; #, IFN-a6. (C) Data for fold changes in anti-HIV activities compared to that of IFN-a2 for IFN-a subtypes and donor cells as described in the legend to panel B. (D) Percentages of inhibition in donor-matched cell subsets for IFN-a1, -a2, and -a6 at 10 pg/ml. Significance testing was done for each IFN-a subtype using RM one-way ANOVAs, comparing the responses in each cell type to one another.

Fig 3: Signaling activities of IFN-α subtypes converge at high treatment doses, which translates into comparable ISG induction profiles between subtypes. (A) Dose-dependent induction of gene expression by all 12 IFN-α subtypes in cells carrying an ISRE-luciferase construct. Significance testing was done for each IFN-α dose using one-way ANOVAs on fold activity relative to the activity of IFN-α2. Symbols for comparisons with IFN-α2 are as follows: *, IFN-α1; #, IFN-α6. Presentation of data is described in “Statistical analysis” in Materials and Methods for all figures. (B) Heat map showing genes induced in primary CD4+ T cells from three independent donors stimulated with 1,000 pg/ml of IFN-α1, -α2, -α6, or -α14 compared to their induction in unstimulated controls (i.e., fold upregulation normalized to the expression in the unstimulated control; the values −3 to 115 represent the extremes of down- and upregulation as normalized to the result for the unstimulated control), and hierarchical clustering of the induced-gene-response profiles. For this assay, the interferons and receptors RT2 Profile PCR array (Qiagen) was used, but only results for genes of interest (ISGs) are shown; see Fig. S2 in the supplemental material for results for the full array of genes included in the PCR array. (C) Detailed view of genes quantitatively differentially induced by IFN-α1, -α2, -α6, or -α14. Lines connect responses from each donor, and the horizontal dashed line represents 2-fold change in gene expression compared to expression in unstimulated controls. These 10 genes represent the set of genes that showed significantly different expression levels between at least one pair of IFN-α subtypes in repeated measures (RM) two-way ANOVAs of the data set shown in panel B (ANOVA results are summarized in Table S2). Note that all 10 genes were induced >2-fold.

Fig 4: IFN subtypes converge in their anti-HIV activities at 1,000 pg/ml, with the exception of IFN-a1. PBMCs were pretreated with the IFN-a subtypes and infected with HIV YU-2 overnight. The next day, the cultures were washed and IFN-as added back. Vres was assessed for all subtypes (n = 3 donors, in duplicates). Significance testing was done using RM one-way ANOVAs, comparing all IFN-a treatment groups to IFN-a2.

Fig 5: Auto-Abs against other subtypes of IFN-I. The presence of auto-Abs against other subtypes of IFN-I was assessed by in-house ELISA in all sera with anti-IFN-a2 auto-Abs detected with the Thermo Fisher Kit (n = 21, from left to right, increasing order of concentration of anti-IFN-a2 using the Thermo Fisher Kit). APS-1 patient's serum was used as positive control, and sera from two healthy controls were used as negative controls.