NK Response Correlates with HIV Decrease in Pegylated IFN-α2a-Treated Antiretroviral Therapy-Suppressed Subjects

Abstract

We previously reported that pegylated IFN-α2a (Peg-IFN-α2a) added to antiretroviral therapy (ART)-suppressed, HIV-infected subjects resulted in plasma HIV control and integrated HIV DNA decrease. We now evaluated whether innate NK cell activity or PBMC transcriptional profiles were associated with decreases in HIV measures. Human peripheral blood was analyzed prior to Peg-IFN-α2a administration (ART, baseline), after 5 wk of ART+Peg-IFN-α2a, and after 12 wk of Peg-IFN-α2a monotherapy (primary endpoint). After 5 wk of ART+Peg-IFN-α2a, immune subset frequencies were preserved, and induction of IFN-stimulated genes was noted in all subjects except for a subset in which the lack of IFN-stimulated gene induction was associated with increased expression of microRNAs. Viral control during Peg-IFN-α2a monotherapy was associated with 1) higher levels of NK cell activity and IFN-γ-induced protein 10 (IP-10) on ART (preimmunotherapy) and 2) downmodulation of NK cell KIR2DL1 and KIR2DL2/DL3 expression, transcriptional enrichment of expression of genes associated with NK cells in HIV controller subjects, and higher ex vivo IFN-α-induced NK cytotoxicity after 5 wk of ART+Peg-IFN-α2a. Integrated HIV DNA decline after immunotherapy was also associated with gene expression patterns indicative of cell-mediated activation and NK cytotoxicity. Overall, an increase in innate activity and NK cell cytotoxicity were identified as correlates of Peg-IFN-α2a-mediated HIV control.

Trial registration: ClinicalTrials.gov NCT02227277.

Figure 1.

Study time points used for sample analysis. (A) Time points of the open label longitudinal study () used for sample analysis are indicated by red arrows. ART is represented by yellow boxes, Peg-IFN-α2a by green boxes and no ART by white boxes. Primary endpoint responders (R) and primary endpoint non-responders (N) were defined according to study protocol. (B) Response groups definitions used in the current study.

Figure 2.

Changes in clinical and immune variables after 5 weeks of combined administration of ART+Peg-IFN-α2a. Whole blood measures are shown at baseline (ART) and 5 weeks after Peg-IFN-α2a and ART. Cell count (cells/mm³) is shown for (A) Total whole blood cells, (B) neutrophils, (C) CD4⁺ T cells. Lymphocyte frequencies are shown for: (D) CD3⁺CD4⁺, (E) CD3⁺CD4⁺CD38⁺, and (F) CD3⁺CD4⁻CD38⁺. Frequencies of innate cell subsets are shown for (G) CD3⁻CD56^dimCD16⁺, (H) CD19⁻BDCA1⁺CD11c⁺, and (I) CD19⁻BDCA2⁺BDCA4⁺. Plasma levels are shown for (J) IL-8, and (K) MCP-1. Available data are shown for study subjects together with the mean of distribution and significant (<0.05) p values.

Figure 3.

Gene profiles after Peg-IFN-α2a immunotherapy between primary endpoint responders (R) and primary endpoint non-responders (N). (A) Hierarchical clustering of all subjects before and after 5 weeks of Peg-IFN-α2a and ART using the 1436 probes that were significantly differentially expressed between these time points at FDR<5%. Star (*) and red fonts indicate subjects with gene expression profiles that are similar on ART and after 5 weeks of ART+ Peg-IFN-α2a, suggesting that the overall IFN response is muted in this subset of subjects. Green boxes illustrate responders as defined by primary endpoint outcome (R), while orange boxes illustrate non-responders as defined by primary endpoint outcome (N). (B) Gene expression fold changes for the top 30 known ISGs after 5 weeks of ART+Peg-IFN-α2a (i.e. gene expression on ART+Peg-IFN-α2a / gene expression on ART) are shown for RRs, RNs and NNs. (C) Heatmap of expression changes for the 77 mRNA probes that were significantly more upregulated (FDR<5%) in RR vs NN patients. 12 miRNA significantly more upregulated in NN vs RR patients (p<0.05) are shown along with their predicted or experimentally confirmed target genes (purple boxes). All genes targeted by at least one miRNA are highlighted. Codes used: ISG responders/primary endpoint responders (RR) shown with black shapes; ISG responders/primary endpoint non-responders (RN) shown with grey shapes; ISG non-responders/primary endpoint non-responders (NN) shown with white shapes. Genes annotated by number are found together with miRNA lists in the same order in Supplemental Table 2. (D) GSEA analysis investigating whether other relevant gene sets are differentially induced between the RR and NN groups, using a gene set that was recently reported to characterize a state of activation of NK cells from HIV-1 controllers (HIC).

Figure 4.

Functional gene expression categories in association with change in integrated HIV DNA on CD4⁺ T cells. (A) Heatmap of the genes with changes at 5 weeks of ART+Peg-IFN-α2a (at which time ART was interrupted; top), and after 12 weeks of Peg-IFN-α2a immunotherapy (bottom) that were significantly associated with HIV DNA changes per circulating CD4⁺ T cells. Together with these genes, functional categories significantly enriched among those genes are also shown. Enriched category color indicates predicted category activation score (z-score by IPA) with orange for functions predicted to be more active in primary endpoint responders (R) or purple for functions more active in primary endpoint non-responders (N). (B) Expression of genes from each functional category that were significantly associated with HIV DNA changes per circulating CD4⁺ T cells at 5 weeks of ART+Peg-IFN-α2a, and after 12 weeks of Peg-IFN-α2a immunotherapy were averaged and plotted for every patient. Methods used for this analysis are described in “Subject, material and methods” section. Briefly, for changes after 5 weeks of ART+Peg-IFN-α2a or at primary endpoint (Peg-IFN-α2a) versus ART, in HIV DNA per circulating CD4⁺ T cells: Genes were selected to have significant Pearson correlation (nominal p<0.05) with log2 ART+Peg-IFN-α2a / ART [or primary endpoint (Peg-IFN-α2a) / ART] ratio of the HIV DNA measurement. 1260 probes (992 unique genes) for visit ART+Peg-IFN-α2a and 880 probes (703 unique genes) for primary endpoint (Peg-IFN-α2a) were analyzed for functional enrichments using IPA and significantly enriched functions (p<10⁻³) were then manually categorized into major classes. Expression of the genes involved in the functional categories then shown on a heatmap with shared genes that belonged to multiple categories shown within the category with the fewest genes. Only enriched categories with predicted activation (z-score>1 calculated by IPA based on direction of correlation of member genes) were considered.

Figure 5.

Immune correlates of clinical response after Peg-IFN-α2a immunotherapy on ART. Shown are whole blood cell frequency and PBMC functional measures distinguishing primary endpoint responders (R) from primary endpoint non-responders (N). Panels A-D show in N and R groups the following variables on ART: (A) Frequency of CD3⁺CD4⁻CD38⁺, (B) MFI of HLA-DR on CD3⁺CD4⁻, (C) CD3⁻C56^brightKIR2DL2/DL3⁺, and (D) plasma IP-10 (pg/ml). Panels E-H show Δchange [(ART+Peg-IFN-α2a) – (ART)] between N and R groups for: (E) CD3⁻CD56^dimCD16⁻CD25⁺HLA-DR⁺, (F) CD3⁻CD56^dimKIR2DL1⁺, (G) CD3⁻CD56^brightKIR2DL2/DL3⁺, and (H) CD19⁻BDCA1⁺CD11c⁺CCR7⁺. Panels A-D show available data for N an R, together with the mean of distribution, and significant (<0.05) p values. Data in panels E-H are shown as mean of the distribution for each group (left) and per patient for each group (right), together with significant (<0.05) p values.

Figure 6.

NK cytotoxic responses as a correlate to HIV control after Peg-IFN-α2a immunotherapy on ART. (A) In vitro constitutive NK cytotoxic responses against K562 are shown as AUC over 50:1 to 6:1 at baseline (ART) and 5 weeks of ART+Peg-IFN-α2a, (B) In vitro IFN-α stimulated NK cytotoxic responses against K562 (shown as IFN-α stimulated - constitutive cytotoxicity) are shown as AUC over 50:1 to 6:1 at baseline (ART) and 5 weeks of ART+Peg-IFN-α2a, (C) In vitro IFN-α stimulated NK cytotoxic responses against K562 (shown as IFN-α stimulated - constitutive cytotoxicity) are shown as AUC over 50:1 to 6:1 at 5 weeks of ART+Peg-IFN-α2a in N and R groups, (D) AUC responses are shown at 5 weeks of ART+Peg-IFN-α2a (blue bars) and at 12 weeks of Peg-IFN-α2a monotherapy (red bars) in N and R subjects (study numbers are shown for each group), (E) Frequency of Lin3⁻CD56^dimCD16⁻CD107a⁺ at 5 weeks of ART+Peg-IFN-α2a in the absence of in vitro stimulation with IFN-α in primary endpoint responders (R) and primary endpoint non-responders (N). Panels A-C and E show available data for study subjects, together with the mean of distribution, and significant (<0.05) p values. Panel D shows data in N and R per patient.