Published June 28, 2019, doi:10.4049/jimmunol.1801511 The Journal of Immunology

NK Response Correlates with HIV Decrease in Pegylated IFN-a2a–Treated ART-Suppressed Subjects

Emmanouil Papasavvas,* Livio Azzoni,* Andrew V. Kossenkov,* Noor Dawany,† Knashawn H. Morales,‡ Matthew Fair,* Brian N. Ross,* Kenneth Lynn,x Agnieszka Mackiewicz,* Karam Mounzer,{ Pablo Tebas,‡ Jeffrey M. Jacobson,‖ Jay R. Kostman,# Louise Showe,* and Luis J. Montaner*

We previously reported that pegylated IFN-a2a (Peg–IFN-a2a) 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-a2a administration (ART, baseline), after 5 wk of ART+Peg–IFN-a2a, and after 12 wk of Peg–IFN-a2a monotherapy (primary endpoint). After 5 wk of ART+Peg–IFN-a2a, 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-a2a monotherapy was associated with 1) higher levels of NK cell activity and IFN-g–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-a–induced NK cytotoxicity after 5 wk of ART+Peg–IFN-a2a. 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-a2a–mediated HIV control. The Journal of Immunology, 2019, 203: 000–000.

nterferon-a is a type I IFN produced by leukocytes as part of lysis of autologous HIV-infected CD4+ T cell targets ex vivo the host’s TLR-mediated antiviral response (1). Type I IFNs (11, 12), suggesting that IFN-a immunotherapy may activate I modulate cellular antiviral immune responses in vivo either similar mechanisms in vivo to control HIV infection. directly by activation of antiviral host restriction factors (2) or Several human clinical trials in which IFN-a immunotherapy indirectly via stimulation of innate NK cell–mediated responses was administered without antiretroviral therapy (ART) in HIV-

by guest on October 1, 2021. Copyright 2019 Pageant Media Ltd. (3–6). Clinical trials with IFN-a support modulation of cell- infected viremic individuals support a predominantly anti-HIV mediated responses as an outcome of immunotherapy, leading to effect without advancement of disease progression (13–23). In increased perforin expression in NK and CD8+ T cells (7, 8), the absence of ART, temporal reductions of HIV plasma viral load increases in CD16+CD56+ NK cell numbers (9), and activation of following administration of pegylated IFN-a2a (Peg–IFN-a2a) CD56+ NK cells (10). Activation of innate host restriction factors were also associated with activation of baseline levels of host and NK responses have been associated with control of HIV and restriction factors, indicating a role for IFN-induced gene induction

*The Wistar Institute, Philadelphia, PA 19104; †The Children’s Hospital of Philadelphia, The microarray data presented in this article have been submitted to the Gene ‡ http://classic.jimmunol.org Philadelphia, PA 19104; Perelman School of Medicine, University of Pennsylvania, Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under accession number Philadelphia, PA 19104; xPresbyterian Hospital–University of Pennsylvania Hos- GSE107549. pital, Philadelphia, PA 19104; {Jonathan Lax Immune Disorders Treatment Center, Address correspondence and reprint requests to Dr. Louise Showe and Dr. Luis J. Philadelphia Field Initiating Group for HIV-1 Trials, Philadelphia, PA 19107; ‖ Montaner, The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104. E-mail Lewis Katz School of Medicine, Temple University, Philadelphia, PA 19140; addresses: [email protected] (L.S.) and [email protected] (L.J.M.) and #John Bell Health Center, Philadelphia Field Initiating Group for HIV-1 Trials, Philadelphia, PA 19107 The online version of this article contains supplemental material. ORCIDs: 0000-0002-1536-0418 (A.V.K.); 0000-0003-2983-3893 (N.D.); 0000-0001- Abbreviations used in this article: ADCC, Ab-dependent cytotoxicity; ART,

Downloaded from 5345-7942 (P.T.); 0000-0002-7818-4220 (L.S.). antiretroviral therapy; AUC, area under the curve; BD, Becton Dickinson; BDCA1, blood DC Ag 1; C3AR1, CC3a receptor 1; CCR7, CCR type 7; Ct, cycle threshold; Received for publication November 14, 2018. Accepted for publication June 3, 2019. DC, dendritic cell; FDR, false discovery rate; GSEA, gene set enrichment analysis; This work was supported by National Institutes of Health Grants U01AI065279 and HCV, hepatitis C virus; HIC, HIV-1 controller; IFI44L, IFN-induced protein 44 like; UM1 AI126620 (to L.J.M.). Additional support was provided by The Philadelphia IPA, Ingenuity Pathway Analysis; ISG, IFN-stimulated gene; KIR2DL1, killer cell Foundation (Robert I. Jacobs Fund), a Kean Family Professorship, Ken Nimblett and Ig-like receptor, two Ig domains and long cytoplasmic tail 1; KIR2DL2/DL3, the Summerhill Trust, AIDS funds from the Commonwealth of Pennsylvania, and KIR2DL, two domains, long cytoplasmic tail 2/3; MDC, myeloid DC; MFI, mean from the Commonwealth Universal Research Enhancement Program, Pennsylvania fluorescence intensity; miRNA, microRNA; N, primary endpoint nonresponder; NES, Department of Health, the Penn Center for AIDS Research (Grant P30 AI 045008), normalized enrichment score; NN, ISG nonresponder/primary endpoint nonre- and Wistar Cancer Center Grant P30 CA10815. The funders had no role in study sponder; PDC, plasmacytoid DC; Peg–IFN-a2a, pegylated IFN-a2a; R, primary design, data collection and analysis, decision to publish, or preparation of the endpoint responder; RN, ISG responder/primary endpoint nonresponder; RNU, nu- manuscript. cleolar RNA; RR, ISG responder/primary endpoint nonresponder; RT, room temperature. M.F., B.N.R., and A.M. performed experimental work. K.M., J.R.K., P.T., and J.M.J. selected patients. K.L. performed patient recruitment. A.V.K. and N.D. performed Ó analysis of the microarray data. K.H.M. performed the statistical analysis of immune Copyright 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 and clinical data. E.P., L.A., L.S., and L.J.M. designed the study, evaluated the results, and wrote the manuscript. All authors have read and approved the manuscript.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1801511 2 INNATE CORRELATES OF IFN-a MEDIATED SUPPRESSION IN HIV

as part of the anti-HIV mechanism of action (13). Short-term CD16-allophycocyanin-Cy7; 3) CD158a (killer cell Ig-like receptor, two Ig treatment with Peg–IFN-a2a added to ART in acute infection domains and long cytoplasmic tail 1 [KIR2DL1]–FITC, CD158b (KIR2DL, also led to a decrease in HIV reservoirs (24). two domains, long cytoplasmic tail 2/3 [KIR2DL2/DL3]–PE, CD3-PerCP- Cy5.5, CD94-allophycocyanin, and CD56-allophycocyanin-Cy7; 4) CD1c We and others have tested the potential for Peg–IFN-a2a im- (blood DC Ag 1 [BDCA1]–FITC, CD303 (BDCA2)–PE, CD304 (BDCA4)– munotherapy to reduce the size of the HIV reservoir (measured PE, CD11c-PerCP-Cy5.5, CD197 (CCR type 7 [CCR7])–allophycocyanin, and as the integrated HIV DNA levels) in chronic ART-suppressed CD19-allophycocyanin-Cy7; 5) CD95-FITC, CD25-PE, CD3-PerCP-Cy5.5, HIV or hepatitis C virus (HCV)/HIV–infected subjects (25–27). CD38-allophycocyanin, and CD4-allophycocyanin-Cy7; and 6) CD3- PerCP-Cy5.5, HLA-DR-allophycocyanin, and CD4-allophycocyanin-Cy7. Together, these studies suggest that IFN-a can suppress plasma Isotypes used included IgG1k-FITC, IgG1k-PE, IgG1k-PerCP-Cy5.5, + viral load and act to decrease CD4 T cell integrated HIV DNA IgG1k-allophycocyanin, and IgG1k-allophycocyanin-Cy7. All Abs were levels, as reported in our NCT00594880 clinical study (25). from Becton Dickinson (BD) Biosciences (San Diego, CA) except BDCA1- However, the mechanisms underlying the in vivo responses de- FITC and BDCA2-PE, which were from Miltenyi Biotec (San Diego, CA). scribed for our NCT00594880 clinical study remain undefined. Stainings 1–3 allowed for the assessment of markers of activation/inhibition (HLA-DR, CD94, CD25, and KIRs) on NK cell subsets (30, 31), identified as We now describe innate activity, NK cytotoxicity, and gene the following: 1) CD32CD56bright and CD32CD56dim with or without CD16, expression as correlates of retained plasma viral load suppres- and 2) CD32CD562CD16+,CD32CD56+CD16+,andCD32CD56+CD162. sion and CD4+ T cell integrated HIV DNA reduction after ART Staining 4 allowed for the assessment of maturation markers (CCR7) on DC + + interruption and Peg–IFN-a2a monotherapy. subsets (32), identified as BDCA2 BDCA4 (plasmacytoid DC [PDC]) and CD192BDCA1+CD11c+ (myeloid DC [MDC]). Finally, stainings 5 and 6 allowed for the assessment of markers of activation (i.e., CD38, CD95, CD25, Materials and Methods and HLA-DR) on T cells (CD3+CD4+ and CD3+CD42). Cells were analyzed Participants on an LSRII cytometer (BD Biosciences) by collecting .200,000 events, and data were analyzed using FlowJo software (Version 8.8.4; Tree Star, Fresh whole blood samples were obtained from 20 HIV-infected sub- Ashland, OR). Gating was originally done on singlets and then on jects on suppressive ART (with .6moat$400 CD4+ T cells/mm3 [nadir 3 “live lymphocyte” (for NK and T cells) or “all live cell” (for DC) gates $ 200 cells/mm ] and undetectable HIV viral load measurement defined by size and granularity in forward scatter and side scatter. , of 50 copies/ml at screening) participating in an open-label longitudi- Thresholds were set by isotype-matched negative controls and unstained nal study (NCT00594880) investigating the antiviral activity of 90 or cells. Results were expressed as mean fluorescence intensity (MFI), 180 mg/wk Peg–IFN-a2a when administered with ART for 5 wk, followed percentages, and cells/mm3. by ART interruption and Peg–IFN-a2a monotherapy for 12 wk as primary endpoint. The details and clinical findings of this study have been pub- Assessment of the in vitro role of IFN-a on STAT-1 lished elsewhere (25). Briefly, and as described in our prior publication (25), 9 of 20 subjects phosphorylation within PBMC subsets , maintained plasma HIV viral load 400 copies/ml by the 12-wk study Freshly isolated PBMC (2 3 106/ml) were stained for 1) CD3-FITC, primary endpoint (primary endpoint responders [R]). Of the remaining . CD14-FITC, CD19-allophycocyanin, CD20-allophycocyanin, CD16– 11/20 subjects, seven exhibited virologic failure (viral load 400 copies/ml) Pacific Blue, and CD56-PECy7; 2) CD14-FITC, BDCA2-allophycocyanin, prior to the 12-wk study primary endpoint, and four discontinued the study BDCA4-allophycocyanin, and CD3–Pacific Blue; or 3) corresponding because of moderate depression scores (n = 3) and grade 3 neutropenia (n =1); isotypes (IgG1k-FITC, IgG2ak-FITC, IgG1-allophycocyanin, IgG1k– according to the study design, all of these subjects were considered to have Pacific Blue, and IgG1k-PECy7) for 30 min at 4˚C, washed with 13 PBS achieved primary endpoint failure (primary endpoint nonresponders [N]). In at 1500 rpm for 5 min, and resuspended in warm 13 PBS. PBMC were addition, and as described in our prior publication (25), responders at primary then treated for 10 min at 37˚C with medium, IFN-a (5000 U/ml; PBL, by guest on October 1, 2021. Copyright 2019 Pageant Media Ltd. endpoint had a significant drop in the levels of integrated HIV DNA in PBMC, Piscataway, NJ), or IFN-g (10 ng/ml; R&D Systems, Minneapolis, MN). as detected by Alu-Gag PCR between baseline (ART) and primary Cells were then fixed with paraformaldehyde (5% final concentration) for endpoint (Peg–IFN-a2a). Levels of integrated viral DNA did not change 10 min at 37˚C, washed, and permeabilized with PhosFlow buffer (BD in nonresponders. Biosciences) for 30 min at room temperature (RT). PBMC were then In the current study, results are described for the 20 subjects participating washed in FACS washing buffer at 2200 rpm for 10 min, stained with an in NCT00594880 over three time points. All available peripheral blood Ab against p–STAT-1 (p–STAT-1–PE) or corresponding isotype IgG2ak- samples were used as obtained prior to Peg–IFN-a2a initiation (baseline, PE for 1 h at RT, washed with FACS washing buffer, and analyzed in the ART only), after 5 wk of ART+Peg–IFN-a2a, and after 12 wk of Peg–IFN- CyAn cytometer as described above. Staining 1 allowed for the assessment a2a monotherapy (primary endpoint). of NK cell subsets (identified as Lin32CD56+CD16+,Lin32CD56+CD162,or 2 2 + Ethics statement Lin3 CD56 CD16 , with Lin3 consisting of CD3, CD14, CD19, and CD20), whereas staining 2 allowed for the identification of monocytes (identified http://classic.jimmunol.org 2 2 2 Written informed consent was obtained from patients according to the as CD3 CD14+) and PDC (identified as CD3 CD14 BDCA2+BDCA4+). directives of the institutional review boards at the Wistar Institute, STAT-1 phosphorylation was expressed as MFI of p–STAT-1 (constitutive or University of Pennsylvania, Philadelphia FIGHT, and Drexel University. in vitro IFN-a–orIFN-g–induced defined as in vitro IFN-a–orIFN-g– All study participants were adults. induced MFI of p–STAT-1/constitutive MFI of p–STAT-1) for all the above described cell subsets. All Abs were from BD Biosciences except BDCA2- Sample usage allophycocyanin, BDCA4-allophycocyanin, and IgG1-allophycocyanin, which were purchased from Miltenyi Biotec. Fresh blood samples were used for clinical assessment, immune subset Downloaded from characterization by flow cytometry, and for plasma and PBMC iso- lation. PBMC were isolated as previously described using a stan- Assessment of constitutive CD107a expression on NK cells dard Ficoll-hypaque density gradient centrifugation (28). Plasma was Fresh PBMC (1 3 106) were incubated with CD107a-PE or corre- cryopreserved and used for cytokine assessment. All available PBMC sponding isotype (IgG1k-PE) for 4 h at 37˚C, blocked with 10% serum were either used fresh for ex vivo assessment of constitutive and/or for 10 min at RT, and stained for 30 min on ice with CD3-FITC, CD14- IFN-a–induced innate functionality (flow cytometry–based assays for 51 FITC, CD19-FITC, CD20-FITC, CD56-PE-Cy7, and CD16–Pacific STAT-1 phosphorylation and CD107a expression and a standard Cr Blue, or corresponding isotypes IgG1k-FITC, IgG2ak-FITC, IgG1k- release assay) or after cryopreservation for host gene expression studies PE-Cy7, and IgG1k–Pacific Blue. All Abs were from BD Biosci- in PBMC. ences. PBMC were washed with 2 ml of FACS washing buffer at 1500 Phenotypic characterization of innate and adaptive cell subsets rpm at 4˚C for 5 min, lysed with 1 ml of BD FACS Lyse for 10 min at 37˚C, washed again with 2 ml of FACS washing buffer, and resus- Immunophenotypic characterization of NK cells, dendritic cells (DC), and pended in 100 ml of FACS washing buffer. Cells were analyzed on a T cell subsets was performed by same-day whole blood five-color staining as CyAn cytometer as described above. This staining allowed for the as- previously described (28, 29) using the following combinations of directly sessment of markers of CD107a (marker of degranulation) and NKp46 fluorochrome-conjugated anti-human cell-surface mAbs: 1) CD56–FITC, (activating receptor) on NK cell subsets (identified as Lin32CD56bright and CD3-PerCP-Cy5.5, CD94–allophycocyanin, and CD16-allophycocyanin-Cy7; Lin32CD56dim with or without CD16, with Lin3 consisting of CD3, CD14, 2) CD56-FITC, CD25-PE, CD3-PerCP-Cy5.5, HLA-DR-allophycocyanin, and CD19, and CD20). The Journal of Immunology 3

51Cr release assay for NK cytotoxicity stimulated at least 10-fold by IFN-a were considered as IFN-stimulated genes (ISGs). The top 30 of those ISGs were then used to illustrate dif- NK function was assessed measuring constitutive and IFN-a (5000 U/ml)– 51 ferences in expression among ISG responders/primary endpoint responders induced NK cell–mediated cytotoxicity by standard Cr release assay, (RR), ISG responders/primary endpoint nonresponders (RN), and ISG as previously described, using fresh PBMC preparations as effector cells nonresponders/primary endpoint nonresponders (NN). against the tumor-derived erythroblastoid cell line K562 (29, 33). Briefly, Unpaired t tests were used for the assessment of a gene signature on viable fresh PBMC preparations from whole blood (effector cells) were ART that can distinguish R from N. Principal component analysis was then treated for 18 h at 37˚C with medium alone or IFN-a (5000 U/ml; PBL). performed using expression of genes that passed a p , 0.001 threshold. Erythroblastoid MHC-null K562 cells, which served as targets, were Association of gene expression changes after 5 wk of ART+Peg–IFN- 51 ∼ labeled with Na2 CrO4 ( 50 mCi) for 1.30 h at 37˚C, washed, and a2a (n = 11) and by primary endpoint (Peg–IFN-a2a, n = 6) with inte- 3 5 resuspended at a concentration of 1 10 cells/ml in culture medium. grated HIV DNA changes was tested using Pearson correlation, and genes Effectors and labeled K562 targets were cultured in triplicate to yield the with p , 0.05 were considered for enrichment analysis. Enrichment desired E:T ratios in 0.2-ml volume (usually 50:1, 25:1, 12.5:1, and 6.25:1) analysis was done using IPA, and significantly enriched biological func- in round-bottom 96-well plates and incubated for 4 h. Percent lysis was tions that passed a p , 0.001 threshold were considered. Functions with 2 determined by the following formula: [(experimental counts spontaneous predicted activation (z-score . 1 calculated by IPA based on the direction 2 3 released counts)/(total counts spontaneous released counts)] 100. of correlation of member genes) were then manually categorized into Results were expressed as area under the curve (AUC) for E:T ratios major classes with maximum z-scoreassignedtoeachoftheclasses. of 50:1, 25:1, 12.5:1, and 6.25:1 for both constitutive and in vitro Expression of the genes involved in the functional categories was shown IFN-a–induced NK function. on an expression heatmap, with shared genes that belong to multiple Cytokine assessment in plasma categories illustrated within a category with the fewest genes. In addition, gene set enrichment analysis (GSEA) was also performed on Cryopreserved plasma was used for cytokine profile definition by using the the genes ranked by the significance and direction of change between RR Human Cytokine 30-Plex Panel (Invitrogen Multiplex Bead Immunoassay and NN and baseline expression of R and N, as well as correlation of week Kit) with the Luminex 100 or 200 dual-laser detection system. This panel 5 and week 8 changes with HIV DNA changes. Results that passed allowed for the quantitative determination of epidermal growth factor FDR , 15% were considered significant. (EGF), eotaxin, basic fibroblast growth factor, G-CSF, GM-CSF, hepatocyte Analysis of the microarray data was done using MATLAB 7.10.0. The growth factor (HGF), IFN-a, IFN-g, IL-1Ra, IL-1b, IL-2, IL-2R, IL-4, data were submitted to the Gene Expression Omnibus (https://www.ncbi. IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 (p40/p70), IL-13, IL-15, IL-17, nlm.nih.gov/geo/) under accession number GSE107549. IP-10, MCP-1, monokine induced by IFN-g (MIG), macrophage inflam- matory protein 1a (MIP-1a), MIP-1b, RANTES, TNF-a, and vascular Statistical analysis endothelial growth factor (VEGF). Phenotypic and functional data are reported as means with SD. The ef- Microarray fect of 5 wk of ART+Peg–IFN-a2a on study variables was assessed by paired t tests, and two-sided p values ,0.05 were considered statistically Isolation of total RNA and DNA from cryopreserved PBMC was performed significant. Comparisons between groups were performed by nonpaired using Trireagent (Sigma-Aldrich, St. Louis, MO) according to the manu- t tests with a cutoff of p ,0.05. Analysis was done by using R.2.5.1. facturer’s instructions. For the gene expression microarrays, amplified cRNA was generated from 100 ng of RNA using the TargetAmp Nano-g Biotin- aRNA Labeling Kit (Epicentre, Madison WI) and then hybridized to the Results HumanHT-12 v4 Expression BeadChips (Illumina, San Diego, CA). An Study schema and patient groups additional 200 ng of RNA was used for microRNA (miRNA) assays using TaqMan OpenArray Human miRNA Panels (Life Technologies, Grand Study schema, time points used for analysis, and response group

by guest on October 1, 2021. Copyright 2019 Pageant Media Ltd. Island, NY) with Megaplex Reverse Transcription Primers and PreAmp definitions used in the current study are shown in Fig. 1. The Human Pools Set v3.0 (Life Technologies); 100 ng was used for each of the number of patients used in the current study for analysis of the two primers, A and B. Raw gene expression microarray data were quantile-normalized and different sets of data (immune variables, integrated DNA, and log2-scaled. Noninformative probes, either expressed at background level gene expression) was dependent on sample availability and is or showing ,1.2-fold change between all samples pairs, were removed shown along with the different response groups used in the prior to further analysis. For miRNA data preprocessing, cycle threshold current study in Supplemental Table I. (Ct) values were converted to expression levels. The small nucleolar RNAs (RNU) RNU44 and RNU48 were used as endogenous controls (house- Innate cell subsets and inflammatory protein changes after 5 keeping genes) to normalize the expression levels of the samples and wk of dual treatment with Peg–IFN-a2a and ART compute relative amounts for each miRNA (DCt). First, the average Ct of http://classic.jimmunol.org the RNUs (RNUavg) was calculated. Ct values were then restricted Consistent with the expected leukopenic effects of Peg–IFN-a2a to ,24, as suggested by the manufacturer, and the maximum DCt value immunotherapy, 5 wk of ART+Peg–IFN-a2a resulted in reduc- 2 was designated as DCt24 (where DCt24 = 24 RNUavg). The DCt values tions in whole blood cell count, neutrophil count, and CD4+ T cell were then converted to absolute expression levels by calculating 2DCt24 – DCt. DCt values exceeding DCt24 were considered unreliable and were floored to count (p = 0.0012, p = 0.0074, and p = 0.0009, respectively; + the DCt24 value for the comparative analyses. miRNAs with DCt values at Fig. 2A–C). The reduction in CD4 T cell count did not reflect a the DCt24 level across all samples were filtered out. Expression levels were selective loss of CD4+ T cell percentage within PBMC (Fig. 2D). log2-scaled for further analysis. + Downloaded from As with CD4 T cell frequencies, no change was detected in T cell Microarray data analysis activation or in the major NK or DC subsets (Fig. 2E–I). However, a clear reduction in STAT-1 phosphorylation was noted in all Expression level comparisons between ART and 5 wk of ART+Peg–IFN- a2a were done using paired t tests with correction for multiple testing done leukocytes (NK, DC, and monocytes) after immunotherapy when according to Storey et al. (34). Genes that passed a false discovery rate challenged ex vivo with exogenous IFN-a (Supplemental Fig. 1A, (FDR) , 5% were called significant and used for hierarchical clustering 1B), in contrast to a retention of IFN-g–induced STAT-1 phos- of samples with standardized Euclidean distance and average linkage. phorylation observed in monocytes and PDC (Supplemental The significance of the difference of changes due to 5 wk of combined Fig. 1B). With regard to soluble plasma cytokine changes, we administration of ART+Peg–IFN-a2a between R and N was calculated using unpaired t tests, and genes that passed FDR , 5% and miRNAs with detected an increase in plasma levels of IL-8 (p = 0.0093) and p , 0.05 were considered. Ingenuity Pathway Analysis (IPA, www.qiagen. MCP-1 (p = 0.0005) after 5 wk of ART+Peg–IFN-a2a (Fig. 2J, 2K). com/ingenuity; QIAGEN, Redwood City, CA) was used for information None of the significant changes detected in clinical parameters, linking miRNAs and their targets where 1) information was experimentally leukocyte cell subset distribution, or ex vivo IFN-a–induced STAT-1 confirmed and 2) information was predicted by a TargetScan algorithm with high/moderate confidence. phosphorylation after 5 wk of ART+Peg–IFN-a2a were associated Genes that were significantly detected above the microarray background with HIV plasma viral load control or changes in integrated proviral were annotated using the Interferome database (35), and those known to be DNA, as measured between R and N (data not shown). 4 INNATE CORRELATES OF IFN-a MEDIATED SUPPRESSION IN HIV

FIGURE 1. Study time points used for sample analysis. (A) Time points of the open-label longitu- dinal study (NCT00594880) used for sample analy- sis are indicated by red arrows. ART is represented by yellow boxes, Peg–IFN-a2a by green boxes, and no ART by white boxes. R and N were defined according to the study protocol. (B) Response group definitions used in the current study. by guest on October 1, 2021. Copyright 2019 Pageant Media Ltd.

Gene expression analysis identifies gene signatures that inform subjects who showed a response (as defined by modulation of clinical outcomes ISG) to 5 wk of ART+Peg–IFN-a-2a treatment but did not show a Gene expression (mRNA and miRNAs) was analyzed in PBMC response at primary endpoint (RN), and 3) subjects who did not samples obtained on ART and after 5 wk of ART+Peg–IFN-a2a. A show a response (as defined by modulation of ISGs) to 5 wk of total of 1436 probes were found to be significantly differentially ART+Peg–IFN-a-2a treatment and did not show a response at http://classic.jimmunol.org expressed between these two time points (FDR , 5%). Hierar- primary endpoint (NN). The results clearly showed that induction chical clustering of the samples based on these 1436 probes sep- of ISG expression alone was not able to segregate persons that arated them into two main arms based on the presence or absence controlled viral load under monotherapy at primary endpoint, yet of immunotherapy (i.e., ART versus 5 wk of ART+Peg–IFN-a2a; the lack of ISG induction was only observed within persons failing Fig. 3A). Overall, there was no separation between R and N with to control plasma viral load at primary endpoint (Fig. 3B). regard to overall gene expression within ART or after 5 wk of Apart from the 30-gene ISG induction criteria, a comparison of

Downloaded from ART+Peg–IFN-a2a, although in four subjects (008, 009, 013, and the changes in the expression levels for genes and miRNAs after 029, indicated with asterisks in Fig. 3A), the samples after 5 wk of 5 wk of ART+Peg–IFN-a2a treatment between the RR and RN or ART+Peg–IFN-a2a clustered with their corresponding samples on between the RR and NN groups was performed. No significant ART, suggesting that these four subjects had no detectable gene differences in the response were found between the RR and RN modulation after ART+Peg–IFN-a2a treatment. groups (FDR . 95% for all genes, only five genes with nominal To further investigate the lack of response observed in these p , 0.001). In contrast, comparison between the RR and NN four subjects, the average expression fold change after 5 wk groups identified 111 gene probes with significant differences of ART+Peg–IFN-a2a treatment (i.e., ART+Peg–IFN-a2a ISG (FDR , 5%; Supplemental Table IIA) with the majority expression/ART ISG expression) was calculated for the top 30 (85 probes corresponding to 77 unique genes) being upregulated known IFN-a–stimulated genes [shown in other studies to be after 5 wk of ART+Peg–IFN-a2a treatment in the RR group stimulated at least 10-fold, as shown in the Interferome database (Fig. 3C). Of the 77 genes found to be significantly upregulated (35)].The following groups were defined based on ISG expression after 5 wk of ART+Peg–IFN-a2a treatment in the RR group when change and primary endpoint outcome: 1) subjects who showed a compared with the NN group, 46 genes (60%) were previously response (as defined by modulation of ISG) to 5 wk of ART+Peg– described to be stimulated at least 10-fold by IFN-a, based on IFN-a2a treatment and a response at primary endpoint (RR), 2) information from the Interferome database (35), representing an The Journal of Immunology 5 by guest on October 1, 2021. Copyright 2019 Pageant Media Ltd. http://classic.jimmunol.org

FIGURE 2. Changes in clinical and immune variables after 5 wk of combined administration of ART+Peg–IFN-a2a. Whole blood measures are shown at baseline (ART) and 5 wk after Peg–IFN-a2a and ART. Cell count (cells per cubic millimeter) is shown for (A) total whole blood cells, (B) neutrophils, and Downloaded from (C) CD4+ T cells. Lymphocyte frequencies are shown for (D) CD3+CD4+,(E) CD3+CD4+CD38+, and (F) CD3+CD42CD38+. Frequencies of innate cell subsets are shown for (G) CD32CD56dimCD16+,(H) CD192BDCA1+CD11c+, and (I) CD192BDCA2+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.

enrichment of 34-fold over the total prevalence of such genes putative regulation by the corresponding miRNA, high-confidence (1.7%) among all the genes detected by microarrays. matches were noted for IFN-induced protein 44 like (IFI44L) (36), In addition, 12 significantly differentially changed miRNAs Ral guanine nucleotide dissociation stimulator like 1 (RGL1) (37), (nominal p , 0.05) were found to be more upregulated in the NN plant homeodomain finger protein 11 (PHF11) (38), mitochondrial group, with nine of them having prior evidence of targeting at ribosomal protein L17 (MRPL17) (39), and miR-19b (40, 41), as least one gene from the upregulated list in the RR group (noted well as for membrane associated ring-CH-type finger 1 (MARCH1) below). A combined heatmap for the 77 genes and the 12 miRNAs (42), miR-155 (43, 44), IFN-a and b receptor subunit 1 (IFNAR1) is shown in Fig. 3C, with all predicted or experimentally con- (45), and miR-370 (43, 46). In addition, experimentally confirmed firmed target genes for the miRNAs highlighted. Among the matches were found for TLR7 (1) and miR-17 (47, 48), IFN- identified matches between the expression of a target gene and the induced protein with tetratricopeptide repeats 5 (IFIT5) (49, 50) 6 INNATE CORRELATES OF IFN-a MEDIATED SUPPRESSION IN HIV by guest on October 1, 2021. Copyright 2019 Pageant Media Ltd. http://classic.jimmunol.org Downloaded from

FIGURE 3. Gene profiles after Peg–IFN-a2a immunotherapy between R and N. (A) Hierarchical clustering of all subjects before and after 5 wk of Peg–IFN-a2a and ART using the 1436 probes that were significantly differentially expressed between these time points at FDR , 5%. Asterisks (*) and red font indicate subjects with gene expression profiles that are similar on ART and after 5 wk of ART+Peg–IFN-a2a, suggesting that the overall IFN response is muted in this subset of subjects. Green boxes illustrate responders as defined by primary endpoint outcome (R), whereas orange boxes illustrate nonre- sponders as defined by primary endpoint outcome (N). (B) Gene expression fold changes for the top 30 known ISGs after 5 wk of ART+Peg–IFN-a2a (i.e., gene expression on ART+Peg–IFN-a2a/gene expression on ART) are shown for RR, RN, and NN. (C) Heatmap of expression changes for the 77 mRNA probes that were significantly more upregulated (FDR , 5%) in RR versus NN patients. Twelve miRNAs significantly more upregulated in NN versus 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: RR shown with black shapes; RN shown with gray shapes; NN shown with white shapes. Genes annotated by number are found together with miRNA lists in the same order in Supplemental Table IIA. (D) GSEA 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 HIC. The Journal of Immunology 7 by guest on October 1, 2021. Copyright 2019 Pageant Media Ltd. http://classic.jimmunol.org Downloaded from

FIGURE 4. Functional gene expression categories in association with changes in integrated HIV DNA on CD4+ T cells. (A) Heatmap of the genes with changes at 5 wk of ART+Peg–IFN-a2a (at which time ART was interrupted; top) and after 12 wk of Peg–IFN-a2a 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 R or purple for functions more active in N. (B) Expression of genes from each functional category that were significantly associated with HIV DNA changes per circulating CD4+ T cells at 5 wk of ART+Peg–IFN-a2a and after 12 wk of Peg–IFN-a2a immunotherapy was averaged and plotted for every patient. Methods used for this analysis are described in the Materials and Methods section. Briefly, for changes after (Figure legend continues) 8 INNATE CORRELATES OF IFN-a MEDIATED SUPPRESSION IN HIV

and let7e (43, 51), and ankyrin repeat and FYVE domain con- integrated HIV DNA in PBMC between baseline (ART) and taining 1 (ANKFY1) (52, 53) and miR-155 (43). primary endpoint (Peg–IFN-a2a). We evaluated whether these We also tested by GSEA whether other relevant gene sets are changes were associated with specific gene expression patterns. A differentially induced between the RR and NN groups, including total of 1260 probes (992 unique genes) identified after 5 wk of a gene set that was recently reported to characterize a state of ART+Peg–IFN-a2a and 880 probes (703 unique genes) identified activation of NK cells from HIV-1 controllers (HIC) (54). We at primary endpoint (Peg–IFN-a2a) were analyzed for func- found that the genes that were upregulated in the RR as compared tional enrichments using IPA. Significantly enriched functions with the NN group were significantly associated with the NK (p , 0.001) were then manually categorized into major classes. genes reported to be upregulated in HIC patients when compared Only enriched categories with predicted activation state (z-score with progressor patients (normalized enrichment score [NES] = . 1 calculated by IPA based on direction of correlation of 1.6, p = 0.0062, FDR = 0.89%; Fig. 3D). Briefly, the 21 genes member genes) were considered. As demonstrated in Fig. 4A and detected to be enriched in RR when compared with NN were Supplemental Table IIB and IIC this analysis showed that subjects CC3a receptor 1 (C3AR1); MX dynamin like GTPase 2 who experienced reduction in integrated HIV DNA exhibited an (MX2); placenta specific 8 (PLAC8); hematopoietic SH2 enrichment in genes associated with leukocyte proliferation and domain containing (HSH2D); tripartite motif containing 22 survival, leukocyte chemotaxis and recruitment, leukocyte acti- (TRIM22); IFI44L; nucleic acid binding protein 1 (NABP1); vation, cytotoxicity/cell-mediated response, NK cytotoxicity, and GTPase, IMAP family member 8 (GIMAP8); sphingosine-1- Ab-dependent cytotoxicity (ADCC). In contrast, subjects without phosphate receptor 1 (S1PR1); zinc finger protein 143 (ZNF143); decreases in integrated HIV DNA showed enrichment for genes cytokine inducible SH2 containing protein (CISH); DExD/H-box associated with leukocyte cell death and cancer/neoplasia. No helicase 58 (DDX58); tetratricopeptide repeat and ankyrin repeat enrichment for ISG groupings was detected in association with containing 1 (TRANK1); GIMAP4; C-X3-C motif chemokine re- changes in integrated HIV levels. Importantly, a similar pattern in ceptor 1 (CX3CR1); GIMAP6; GIMAP7; tubulin d 1(TUBD1); gene enrichment was observed in data from two independent time grancalcin (GCA); tRNA nucleotidyl transferase 1 (TRNT1); and points between subjects analyzed either after 5 wk of ART+Peg– ZNF518A. Of these genes, four were significantly upregulated IFN-a2a or during Peg–IFN-a2a monotherapy (Fig. 4 and in the RR group when compared with the NN group (IFI44L Supplemental Table IIB, IIC showing the same genes across both [p =0.02],MX2[p =0.02],PLAC8[p =0.04],andC3AR1 time points). Interestingly, GSEA after 5 wk of ART+Peg–IFN-a2a [p = 0.01]). or during Peg–IFN-a2a monotherapy further supported an associ- In addition, analysis of the baseline preimmunotherapy gene ation between immune responses and reduction in HIV. Briefly, expression between groups controlling or not controlling plasma subjects who experienced reduction in integrated HIV DNA had an viral load upon primary endpoint identified a 30-gene signature enrichment in genes associated with immune functions such as with significant differential expression (p , 0.001; Supplemental NK cell–mediated immunity (NES = 21.68, p =0.01,FDR=6%), Fig. 2A). Principal component analysis using the expression of regulation of leukocyte mediated cytotoxicity (NES = 21.46, p =0.04, these genes confirmed a separation of the RR group using pre- FDR = 18%), and T cell proliferation (NES = 21.93, p = 0.001, immunotherapy gene expression alone (Supplemental Fig. 2B). FDR = 0.4%). In addition, GSEA REACTOME pathways anal-

by guest on October 1, 2021. Copyright 2019 Pageant Media Ltd. Baseline gene expression differentiating R from N was also ex- ysis showed an enrichment in these subjects of genes associated plored by GSEA. Results revealed one gene set (GSE18791) (55) with IFN signaling (NES = 22.21, p , 0.001, FDR = 0.01%), to be significant in association with genes that were more upreg- HIV life cycle (NES = 21.85, p , 0.001, FDR = 2%), antiviral ulated in R when compared with N (NES = 2.1, p , 0.001, FDR = mechanism of ISGs (NES = 21.56, p = 0.01, FDR = 11%), and 4.7%). This gene set was identified as a regulatory network un- apoptosis (NES = 22.1, p , 0.001, FDR = 0.07%). derlying the antiviral state transition during the first 18 h following Overall, these data indicate that reductions in cell-associated the in vitro infection of DC with Newcastle disease virus (55). integrated HIV DNA following Peg–IFN-a2a administration are Interestingly, we also identified an association of genes that associated with activation of NK and cell-mediated gene expres- were upregulated in R compared with N with the gene set sion programs. http://classic.jimmunol.org of HALLMARK_INTERFERON_ALPHA_RESPONSE (NES = Increase in innate activity and cytotoxic responses before and 1.87, p , 0.001), although with FDR = 43.8%. after Peg–IFN-a2a immunotherapy in subjects controlling In summary, microarray data analysis allowed for the identifi- HIV during Peg–IFN-a2a monotherapy cation of gene signatures at baseline (on ART) and after 5 wk of ART+Peg–IFN-a2a with regard to R and N outcomes. We analyzed whether innate variables measured on ART and after 5 wk of ART+Peg–IFN-a2a were related to plasma viral control Enrichment in expression of genes that are associated with

Downloaded from after 12 wk of Peg–IFN-a2a monotherapy. activation of NK and cell-mediated responses in subjects Consistent with baseline values as indicative of viral control with decreased levels of integrated HIV DNA following outcomes postimmunotherapy, R had a higher baseline frequency a Peg–IFN- 2a immunotherapy of CD3+CD42 T cells expressing CD38 (p = 0.0459) and HLA- Results from our NCT00594880 study (25) indicated that R, when DR (p = 0.0079), higher baseline frequencies of NK cells bearing compared with N, exhibited a significant drop in the levels of inhibitory receptors (i.e., CD32CD56brightKIR2DL2/DL3+, p = 0.0397),

5 wk of ART+Peg–IFN-a2a or at primary endpoint (Peg–IFN-a2a) 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-a2a/ART) or log2 (Peg–IFN-a2a/ART) ratio of the HIV DNA measurement. A total of 1260 probes (992 unique genes) for 5 wk ART+Peg–IFN-a2a and 880 probes (703 unique genes) for primary endpoint (Peg–IFN-a2a) were analyzed for functional enrichments using IPA, and significantly enriched functions (p , 1 3 1023) were then manually categorized into major classes. Expression of the genes involved in the functional categories was 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 cor- relation of member genes) were considered. The Journal of Immunology 9 by guest on October 1, 2021. Copyright 2019 Pageant Media Ltd.

http://classic.jimmunol.org FIGURE 5. Immune correlates of clinical response after Peg–IFN-a2a immunotherapy on ART. Shown are whole blood cell frequencies and PBMC functional measures distinguishing R from N. (A)–(D) show in N and R groups the following variables on ART: (A) frequency of CD3+CD42CD38+,(B)MFIof HLA-DR on CD3+CD42,(C)CD32C56brightKIR2DL2/DL3+,and(D) plasma IP-10 (picograms per milliliter). (E)–(H)showDchange [(ART+Peg–IFN-a2a) 2 (ART)] between N and R groups for (E)CD32CD56dimCD162CD25+HLA-DR+,(F)CD32CD56dimKIR2DL1+,(G)CD32CD56brightKIR2DL2/DL3+,and(H) CD192BDCA1+CD11c+CCR7+.(A)–(D) show available data for N and R, together with the mean of distribution and significant (,0.05) p values. Data in (E)–(H) are shown as the mean of the distribution for each group (left) and per patient for each group (right), together with significant (,0.05) p values. Downloaded from and higher baseline plasma levels of IP-10 (p = 0.0395; monotherapy (R) had a higher ex vivo IFN-a–induced NK Fig. 5A–D) when compared with N. cytotoxic response (Fig. 6C, 6D). Supporting higher NK After 5 wk of ART+Peg–IFN-a2a, R also showed changes not degranulation in vivo, we also detected a trend for higher observed in N, such as a decrease in the frequencies of CD25+HLA- constitutive expression of CD107a in CD56dim NK cells 2 2 DR+ NK cells (i.e., CD32CD56dimCD162CD25+HLA-DR+, (i.e., Lin3 CD56dimCD16 CD107a+, p = 0.099; Fig. 6E) in R p = 0.017; Fig. 5E) and of NK cells bearing inhibitory markers when compared with N. (i.e., CD32CD56dimKIR2DL1+ [p = 0.0235], CD32CD56brightKIR2DL2/ Overall, we identify lower KIR expression and higher NK cy- DL3+ [p = 0.0184]; Fig. 5F, 5G). In addition, R also totoxicity as correlates of HIV plasma viral load control upon showed an increase in the frequencies of mature MDC Peg–IFN-a2a monotherapy. (i.e., CD192BDCA1+CD11c+CCR7+, p = 0.0318; Fig. 5H). NK cytotoxicity was tested ex vivo at baseline and after 5 wk of Discussion ART+Peg–IFN-a2a, showing no detectable effect of treatment on In this study, we show that increases in innate activity, NK cyto- constitutive or in vitro IFN-a–induced cytotoxicity (Fig. 6A, 6B). toxicity, and gene expression are correlates of integrated HIV DNA However, subjects controlling viral rebound during Peg–IFN-a2a decrease and plasma viral load control after ART interruption and 10 INNATE CORRELATES OF IFN-a MEDIATED SUPPRESSION IN HIV

FIGURE 6. NK cytotoxic responses as a correlate to HIV control after Peg–IFN-a2a 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 wk of ART+Peg–IFN-a2a. (B) In vitro IFN-a–stimulated NK cytotoxic responses against K562 (shown as IFN-a–stimulated constitutive cytotoxicity) are shown as AUC over 50:1 to 6:1 at baseline (ART) and 5 wk of ART+Peg–IFN-a2a. (C) In vitro IFN-a–stimulated NK cytotoxic responses against K562 (shown as IFN-a–stimulated constitutive cytotoxicity) are shown as AUC over 50:1 to 6:1 at 5 wk of ART+Peg–IFN-a2a in N and R groups. (D) AUC responses are shown at 5 wk of ART+Peg–IFN-a2a (blue bars) and at 12 wk of Peg–IFN-a2a monotherapy (red bars) in N and R subjects (study numbers are shown for each group). (E) Frequency of Lin32CD56dimCD162CD107a+ at 5 wk of ART+Peg–IFN-a2a in the absence of in vitro stimulation with IFN-a in R and N. (A)–(C) and (E)show available data for study subjects, together with the mean of distribution and significant (,0.05) p values. (D) shows data in N and R per patient. by guest on October 1, 2021. Copyright 2019 Pageant Media Ltd.

continued Peg–IFN-a2a immunotherapy. Specifically, we identify correlates of IFN modulation on or off ART, as induction of ISGs modulations in KIR expression, NK cytotoxicity, and mature from preimmunotherapy levels was noted as a correlate of temporal MDC as innate cell changes that are associated with HIV control viral control of Peg–IFN-a2a administered to viremic subjects (13). after ART interruption and Peg–IFN-a2a monotherapy. We also By contrast, our study indicates that when Peg–IFN-a2a is ad- introduce a baseline gene signature able to identify subjects more ministered in suppressed subjects on ART, the activation of innate likely to control viremia after Peg–IFN-a2a monotherapy, as well cellular activity may be a greater correlate to control of HIV than as a gene signature able to identify subjects that fail to modulate ISG induction. Importantly, two independent NK cell changes http://classic.jimmunol.org ISGs after adding Peg–IFN-a2a to ART or to control viremia after (lower inhibitory KIR expression in NK cells and higher NK Peg–IFN-a2a monotherapy. Interestingly, we describe that sub- cytotoxicity) were correlated with control of plasma viral load, jects failing to modulate ISG gene expression may include active whereas two independent assessments by IPA gene expression mRNA expression patterns actively countering gene expression supported NK cell activity (among other cell-mediated activity) after Peg–IFN-a2a. Although future independent validation of as being significantly correlated with a decrease in integrated described gene signatures is needed, our data following Peg–IFN- HIV DNA by primary endpoint.

Downloaded from a2a treatment are consistent with prior observations with Our data do not exclude a role for ISGs in HIV control after panobinostat suggesting that induction of ISGs together with Peg–IFN-a2a on ART, as all R did increase ISGs. However, the changes in NK activation correlate with decreases in integrated observation that four of the nine N had gene expression profiles HIV DNA after treatment with panobinostat in well controlled indicative of a refractory response indicates the possibility of HIV-infected subjects on ART (56). Furthermore, we do find a identifying subjects that will not control HIV upon Peg–IFN-a2a significant association between genes that were upregulated in monotherapy. Importantly, the lack of response in these subjects PBMC from subjects controlling viral load under Peg–IFN-a2a includes evidence for increases in miRNAs together with the monotherapy and genes described to be upregulated in isolated downregulation of their corresponding gene targets. Among these NK cells from HIC (54). miRNA gene targets were genes associated with cancer [e.g., Combined administration of ART+Peg–IFN-a2a for 5 wk re- IFI44L (36), RGL1 (37), MARCH1 (42), and MRPL17 (39)] and sulted in a clear induction of IFN-mediated genes in the majority antiviral activity [e.g., IFIT5 (57) and TLR7 (1)]. Taken together of subjects; however, the levels of antiviral IFN-induced genes with the upregulation of miRNAs (e.g., miR-155, let-7e, miR-370, alone were not enough to segregate between R and N. The fact that miR-192, and miR-1275) that target IFN-inducible antiviral not all subjects that induced upregulation of these genes were R effector molecules (43), it is interpreted that these subjects may highlights a potential difference between the dominant antiviral exemplify a response profile to Peg–IFN-a2a that would The Journal of Immunology 11

predict a return of plasma viral load, as was observed upon be excluded that this loss of KIR expression (which is skewed to ART interruption. more differentiated NK cells) could also imply a reduction in Previous data by Hua et al. (58) in HIV-1/HCV-coinfected more differentiated NK cells in favor of less-differentiated NK patients showed an increase in NK subsets after treatment with cell subsets or an expansion of KIR-negative NK cells, we inter- Peg–IFN-a2a as well as an association between reduction in viral pret that lower inhibitory receptor activity may contribute to an reservoir and NK cells coexpressing activation markers NKG2D increase in NK-mediated killing. Previous studies have shown that and NKp30. In our study, 5 wk of ART+Peg–IFN-a2a did not treatment with IFN-a can increase NK cells’ cytokine secretion, result in changes in the major NK subsets (besides an increase in viral suppressive capacity, and cytotoxic function (12, 64–67). CD56bright NK cells) or in activated NK, suggesting the lack of Gene analysis further supported this interpretation via an enrich- a direct effect of treatment in these subsets. Furthermore, we ment of genes associated with leukocyte activation, proliferation, observed in subjects able to control plasma viral load a decrease in survival, chemotaxis, and recruitment, as well as cytotoxicity/cell- CD25+HLA-DR+CD56dimCD162 NK cells, supporting an asso- mediated response and NK ADCC in persons with reductions in ciation between levels of NK activation and HIV burden. Impor- integrated HIV DNA. Although our data support higher NK cy- tantly, the observed differences between our study and the study totoxic function, they do not address induction of viral suppressive by Hua et al. likely reflect differences in study design (e.g., patient activity. Our study also does not address an additional role for NK population studied: HIV-1/HCV in the study by Hua et al. versus cells in viral control, as indicated in nonpathogenic SIV infection, HIV in our study; and treatment duration: average of 11 mo in the in which NK cells migrate into lymph node follicles in association study by Hua et al. versus 5 wk in our study). Of interest, the most with reductions in viral levels within lymph nodes (68). uniform change noted across all subjects receiving Peg–IFN-a2a Finally, unpaired t tests allowed for the identification of a sig- was a reduction in the ex vivo IFN-a–induced p–STAT-1, sug- nature that consisted of the top 30 most significantly differentially gesting a lower cellular capacity to signal through the type I IFN expressed genes at baseline (Supplemental Fig. 2) and was able receptor. Interestingly, constitutive levels of p–STAT-1 in circu- to segregate R from N. This signature consisted mainly of genes lating leukocytes were not higher after Peg–IFN-a2a immuno- that have been reported to be associated with gene suppression therapy, indicating that drug levels in circulation can maintain [e.g., DICER1 (69, 70) and Wolf–Hirschhorn Syndrome candidate gene expression changes without sustained higher levels in con- 1 (WHSC1L1) (71)], negative regulation of innate immune re- stitutive p–STAT-1. We also document that the reduction in type I sponses [e.g., G-patch domain containing 3 (GPATCH3) (72)], or IFN receptor response did not affect type II IFN receptor re- cancer [e.g., annexin A2 pseudogene 1 (ANXA2P1) (73), Rho sponses within the same cells, indicating a receptor-level mech- associated coiled-coil containing protein kinase 1 (ROCK1) (74), anism of unresponsiveness. The decrease in type I IFN receptor and hemogen (HEMGN) (75)]. The presence in both R and N of activity, together with the ex vivo data showing higher IFN- high levels of expression of genes coding for proteins that par- induced NK cytotoxicity as a correlate of HIV control, sug- ticipate in transcriptional repression (i.e., WHSC1L1 (71) and gests that despite noted decreases in IFN-induced p–STAT-1, DICER1 (69, 70), respectively) underlies uncharacterized associ- the retained induction of NK responses was retained within ations between gene expression and IFN-a–mediated viral control. subjects controlling plasma viral load. Furthermore, no clear segregation was observed between subjects

by guest on October 1, 2021. Copyright 2019 Pageant Media Ltd. Hansen et al. (59) have also shown that IFN-a can increase T and that were able to upregulate ISGs and those that did not, as shown NK cells’ cytotoxicity by boosting the IL-15–induced signaling and by an upregulation in the RN and NN groups of the same cytotoxic activity of these cells. In our study, no increased levels of genes. Future studies will be needed to validate and reconfirm IL-15 were observed in plasma after 5 wk of ART+Peg–IFN-a2a. this baseline gene signature on an independent validation set. This could be explained because soluble IL-15 can generally not be As no T cell responses were measured in our study, our data do detected in physiological fluids, as it binds with a very high affinity not address the effects of Peg–IFN-a2a on T cell–mediated viral to IL-15Ra at the surface of APCs. IL-15Ra gene expression was control. Paradoxically, recent data from humanized murine models also not detected at a significant level over the background in infected with HIV and treated temporally with ART suggest a microarrays in our study. However, GSEA exploring whether detrimental role for type I IFNs by acting against CD8+ T cell– http://classic.jimmunol.org IL-15–responsive genes are differentially induced between RR mediated anti-HIV responses (76, 77). However, the lack of NK and NN groups using the datasets GSE70214 (60), GSE120904 effector responses in humanized mice (78) and the absence of (database reported by Cezari et al. at the National Center for qualification of the effects of IFN-b versus IFN-a on the impair- Biotechnology Information on October 16, 2018), GSE22886 ment of CD8+ T cell responses (79, 80) limit the inference from (61), GSE22919 (62), and GSE7764 (63), which have been this animal model data to human responses using Peg–IFN-a2a as described to be modulated by IL-15, revealed three of these described in this study.

Downloaded from gene sets to be significant (p , 0.05 and FDR , 5%) in as- Our study has limitations. First, as a pilot study, the sample size sociation with both upregulated and downregulated genes is limited, and the uncovered associations and gene signatures (GSE70214, GSE120904, and GSE22886; data not shown). described in this study need to be further validated in larger, in- These results suggest that IFN-a may have increased NK cells’ dependent studies. Second, our data do not address an ART inter- cytotoxicity by boosting IL-15 effects. Future studies will ruption without Peg–IFN-a2a monotherapy because, as described in need to further investigate the connection between Peg–IFN- our prior publication (25), the U.S. Food and Drug Administration a2a and IL-15. mandated the removal of that control group. Third, the gene sig- In this study, we show an increase in cytotoxic NK response natures described are obtained in PBMC as opposed to indepen- against K562 targets at 5 wk of ART+Peg–IFN-a2a among sub- dent cell subsets, and the relationship between gene expression jects able to control plasma viral load. These results support the and protein levels remains to be determined. Fourth, as no infor- interpretation that NK cells contribute to a reduction in viral mation on treatment history is available on study subjects, the role control via direct cell-mediated lysis of infected cells. Con- of time to ART initiation in the observed baseline differences sistent with higher cytotoxicity, we also detected a decrease in between groups remains to be determined. Finally, all measures CD32CD56dimKIR2DL1+ and CD32CD56brightKIR2DL2/DL3+ described were performed in peripheral blood, whereas no mea- NK cells in subjects able to control viral load. Although it cannot sures in tissue were performed. 12 INNATE CORRELATES OF IFN-a MEDIATED SUPPRESSION IN HIV

Overall, this study provides, to our knowledge, the first proof-of- trial of interferon alpha therapy for HIV type 1 infection. AIDS Res. Hum. Retroviruses 16: 183–190. concept that in ART-suppressed subjects, immunotherapy with 19. Lane, H. C., J. A. Kovacs, J. Feinberg, B. Herpin, V. Davey, R. Walker, type I IFNs resulting in a reduction of HIV measures is associated L. Deyton, J. A. Metcalf, M. Baseler, N. Salzman, et al. 1988. Anti-retroviral with activation of NK responses. A randomized trial to formally effects of interferon-alpha in AIDS-associated Kaposi’s sarcoma. Lancet 2: 1218–1222. test whether reductions in HIV measures on ART are the result of 20. Rozenbaum, W., S. Gharakhanian, M. S. Navarette, R. De Sahb, B. Cardon, and IFN-a immunotherapy is now underway (NCT02227277). C. Rouzioux. 1990. Long-term follow-up of 120 patients with AIDS-related Kaposi’s sarcoma treated with interferon alpha-2a. J. Invest. Dermatol. 95(6 Suppl)161S–165S. Acknowledgments 21. Skillman, D. 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Downloaded from Supporting information (Supplemental Figures, Figures legends, Supplemental

Table 1)

Supplemental Figure 1 A B MFI p-STAT-1: Constitutive MFI p-STAT-1: In vitro IFN-α/constitutive MFI: 3.2 In vitro IFN-α-induced (IFN-α/Constitutive)

+ Constitutive MFI: 4.58 p=0.0002 100 In vitro IFN-α MFI: 14.7 10 6 MFI p-STAT-1: + + CD16

+ 80 5 In vitro IFN-γ-induced 60 7.5 (IFN-γ/Constitutive) CD16 CD16 + + 4

CD56 40 - 20 5 3 + CD56 CD56 Lin3 - 0 - 1 2 3 2 0 10 10 10 2.5 2.2 Lin3 Lin3 1 BDCA4 MFI p-STAT-1 + 0 0 1.8 (ART) ART ART ART ART + + BDCA2 Peg-IFN-α-2a Peg-IFN-α-2a - 1.4 In vitro IFN-α/constitutive MFI: 1.57 CD14 - + + p=0.0004 1 1

Constitutive MFI: 8.11 - 20 CD3

+ ART ART 100 15 + BDCA4 BDCA4 + In vitro IFN-α MFI: 12.8 +

STAT 15 CD16 80 Peg-IFN-α-2a - + 60 10 40 10 BDCA2 CD56 MFI p BDCA2 - - 20 - 40 0 5 Lin3 5 + CD14 CD14 - 1 2 3 - 0 10 10 10 30 CD3 CD3

0 0 CD14 MFI p-STAT-1 ART ART ART ART - 20 (ART +Peg-IFN-α-2a)

+ + CD3 Peg-IFN-α-2a Peg-IFN-α-2a 10

p<0.0001 0 25 ART ART 20 + + + 20 Peg-IFN-α-2a 15 15 CD14 CD14 - - 10 10 CD3 CD3

5 5

0 0 ART ART ART ART + + Peg-IFN-α-2a Peg-IFN-α-2a

Supplemental Figure 1. Preservation of constitutive STAT-1 phosphorylation and

decreased responsiveness to ex vivo IFN-α stimulation after 5 weeks of ART+Peg-IFN-

α2a. (A) Representative constitutive (red line) and in vitro IFN-α stimulated (blue line) phosphorylated STAT-1 (p-STAT-1) on Lin3-CD56+CD16+ are shown as MFI values on

ART (top) and after 5 weeks of ART+Peg-IFN-α2a (bottom) for subject 025. (B) Left:

constitutive p-STAT-1 on Lin3-CD56+CD16+ (NK cells, top), CD3-CD14-BDCA2+BDCA4+

(PDC, middle), and CD3-CD14+ (monocytes, bottom) cells on ART and after 5 weeks of

ART+Peg-IFN-α2a; middle: ratio of in vitro IFN-α stimulated/constitutive p-STAT-1 for the

same cell subsets on ART and after 5 weeks of ART+Peg-IFN-α2a; right: ratio of in vitro

IFN-γ stimulated/constitutive p-STAT-1 for PDC (top), and monocytes (bottom) on ART and after 5 weeks of ART+Peg-IFN-α2a. B shows available data for study subjects together with the mean of distribution and significant (<0.05) p values. Supplemental Figure 2 A B 002 005 011 012 023 025 031 032 006 007 020 022 027 008 009 013 029

Gene ANXA2P1 10 SMA4 ROCK1

DICER1 5 LIG4 009 PPP2CB 032 013 031 008 UQCRH 011 029 005 0 023 022 IP6K1 012 025 020 002 VPS26 027 007 RALBP1 006 DUS2L -5 TMED1 Principal componentPrincipal 2 (11%) RPL37A RCRR RN NN UBR5 -10 FAM177A1 -10 -5 0 5 10 ZC3H7A Principal component 1 (56%) FAM90A3 NUDT16L1 RFPL3S TH1L EPB41 CUL1 C7orf59 WHSC1L1 AQR DIDO1 GPATCH3 POLR1D PSMD3 HEMGN

Supplemental Figure 2. Baseline gene expression on ART distinguishes primary endpoint responders (R) from primary endpoint non-responders (N). (A) Expression heatmap for 30 coding genes showing significant differences at baseline (p<0.001) between responders (RR) and one of non-responder classes (RN or NN). (B) Principal component analysis based on the expression of the 30 coding genes. 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. Supplemental Table 1. Study patients and response groups

Groups by HIV viral load at primary Groups by primary endpoint Groups by ISG modulation Groups by ISG modulation, primary Groups by primary endpoint Groups by HIV viral load at Patients endpoint used in immune variables outcome and integrated HIV and primary endpoint endpoint outcome and integrated HIV outcome primary endpoint analysis DNA outcome DNA

PEG-002 R R R R RR RR

PEG-005 R R R R RR RR

PEG-006 N N N N RN RN

PEG-007 N N N N RN RN

PEG-008 N NN

PEG-009 N NN

PEG-010 N N

PEG-011 R R R R RR RR

PEG-012 R R R R RR RR

PEG-013 N N N N NN NN

PEG-014 N

PEG-019 R R R R

PEG-020 N N N N RN RN

PEG-022 N N N N RN RN

PEG-023 R R R RR

PEG-025 R R R RR

PEG-027 N N N RN

PEG-029 N N N NN

PEG-031 R R R R RR RR

PEG-032 R R R R RR RR

R, subjects who successfully reached the primary endpoint with viral load<400 copies/ml; N, subjects with viral load that re-bounced back (>400 copies/ml) before reaching primary endpoint or who did not reach primary endpoint because they discontinued the study; RR, subjects who showed a response (as defined by modulation of ISG) to 5 weeks of ART+Peg-IFN-α2a treatment and a response at primary endpoint (ISG responders/primary endpoint responders); RN, subjects who showed a response (as defined by modulation of ISG) to 5 weeks of ART+Peg-IFN-α2a treatment but did not show a response at primary endpoint (ISG responders/primary endpoint non-responders); NN, subjects who did not show a response (as defined by modulation of ISGs) to 5 weeks of

ART+Peg-IFN-α2a treatment and did not show a response at primary endpoint (ISG non-responders/primary endpoint non-responders). Any absent group designation in the Table is due to missing data.