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Subjects In Vitro by Modulating MonocytesRecall Responses from HIV-1-Infected IL-13 Acutely Augments HIV-Specific and

Karam Mounzer, Jay R. Kostman and Luis J. MontanerMoore, Brian Thiel, Rob Roy MacGregor, Adrian Minty, Emmanouil Papasavvas, Junwei Sun, Qi Luo, Elizabeth C.

http://www.jimmunol.org/content/175/8/5532doi: 10.4049/jimmunol.175.8.5532

2005; 175:5532-5540; ;J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2005 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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IL-13 Acutely Augments HIV-Specific and Recall Responsesfrom HIV-1-Infected Subjects In Vitro byModulating Monocytes1

Emmanouil Papasavvas,* Junwei Sun,* Qi Luo,* Elizabeth C. Moore,* Brian Thiel,*Rob Roy MacGregor,† Adrian Minty,‡ Karam Mounzer,§ Jay R. Kostman,†§ andLuis J. Montaner2*

We show in this study that acute exposure of PBMCs derived from HIV-infected subjects to IL-13 results in increased recall Tcell lymphoproliferative responses against HIV-1 p24 (n � 30, p < 0.0001) and other recall Ags (influenza, n � 43, p < 0.0001;purified protein derivative tuberculin, n � 6, p � 0.0299). This effect is due to a mechanism that acutely targets APC function inthe adherent monocyte subset, as shown by the expansion of CD4� T cell responses following coculture of IL-13-treated enrichedCD14� monocytes with donor-matched enriched CD4� T cells and Ag. Exposure to IL-13 over 18–72 h resulted in a significantenhancement of monocyte endocytosis (n � 11, p � 0.0005), CD86 expression (n � 12, p � 0.001), and a significant decrease inspontaneous apoptosis (n � 8, p � 0.008). Moreover, IL-13 exposure induced a significant decrease of significantly elevatedconstitutive levels of PBMC-secreted TNF-� (n � 14, p < 0.001) and IL-10 (n � 29, p < 0.001) within 18 h of exposure ex vivo,also reflected by decreased gene expression in the adherent cell population. Our data show that IL-13 is able to acutely enhancethe function of the CD14� cell subset toward supporting Ag-specific cell-mediated responses in chronic HIV-1 infection. TheJournal of Immunology, 2005, 175: 5532–5540.

I mmune dysfunction in HIV-1 infection is associated with aloss of CD4� T cell lymphoproliferative responses (1) inassociation with dysregulation of Ag presentation function to

include impaired costimulatory molecule expression (CD80/CD86) (2) and cytokine production (3–5). Decreased levels offunctional cell-mediated responses against HIV-1 and other recallAgs have been associated with delayed disease progression andremain a target of antiretroviral adjunct immunotherapy (6). How-ever, direct modulation of Ag presentation with or without anti-retroviral therapy has not been largely pursued by current immune-based therapy approaches, despite its central role during animmune response and its well-characterized impairment in HIV-1infection (7).

IL-13 is secreted by activated T cells, basophils, mast cells, andNK cells (8). IL-13, unlike IL-4 or IL-10, has no direct regulationon T cell function (9) and modulates human monocytes/macro-phages and B cells (10, 11). Regarding modulation of humanmonocytes/macrophages, exposure to IL-13 results in an increaseof MHC class II expression (12), priming for increased IL-12 se-cretion upon stimulation (13), and direct inhibition of inflamma-

tory cytokines such as TNF-� and IL-1� (11, 14). In HIV-1 in-fection, secretion of IL-13 by CD4� and CD8� T cell subsetsfollowing anti-CD3/anti-CD28 stimulation ex vivo is decreased inparallel with IFN-� secretion and the loss of CD4� T cell count�500 (15). In vitro studies have also shown that IL-13 inhibitsHIV-1 in monocyte-derived macrophages (16, 17) by blocking thecompletion of reverse transcription, decreasing virus production,and reducing the infectivity of progeny virus (17). Based on itsanti-inflammatory activity and Ag presentation function modula-tion properties, we addressed the potential that a single short-termexposure of PBMC from HIV-infected persons to IL-13 wouldenhance the Ag presentation function of adherent monocytes andresult in enhanced recall lymphoproliferative responses. We showthat IL-13 significantly increases HIV-specific and recall CD4� Tcell-mediated responses in HIV-1 infection independently of IL-12and in association with modulation of monocyte Ag presentationfunction, as reflected by effects on cell surface molecule expres-sion, Ag uptake, apoptosis, and cytokine secretion.

Materials and MethodsParticipants

A total number of 68 HIV� and 175 HIV� age-matched subjects partici-pated in the study. As defined in Results, not all subjects were used amongall assays performed. Regarding the HIV� subjects in total, 73% were maleand 19% were female; median age was 46 years (25th-75th interquartile(IQR):3 42–53), while median year of HIV infection diagnosis was 1993(25th-75th IQR: 1990–1996). Ethnic distribution for HIV� subjects was64.5% African American, 21% Caucasian, 6% Hispanic, and 0.5% NativeAmerican. Sixty-eight percent of patients were confirmed on antiretroviraltherapy. Apart from six HIV� subjects with confirmed tuberculosis, asdescribed in Results, no additional information for opportunistic infection

*The Wistar Institute, Philadelphia, PA 19104; †Infectious Diseases Division, Uni-versity of Pennsylvania, Philadelphia, PA 19104; ‡Molecular and Functional Genom-ics Department, Sanofi-Synthelabo Recherche, Labege, France; and §PhiladelphiaField Initiating Group for HIV-1 Trials, Philadelphia, PA 19107

Received for publication April 8, 2005. Accepted for publication August 2, 2005.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This study was supported by National Institutes of Health AI40379, the PhiladelphiaFoundation (Jacobs Fund), M. Stengel Miller’s support of the HIV-1 PartnershipProgram for Basic Research, and funds from the Commonwealth Universal ResearchEnhancement Program, Pennsylvania Department of Health.2 Address correspondence and reprint requests to Dr. Luis J. Montaner, The WistarInstitute, 3601 Spruce Street, Philadelphia, PA 19104. E-mail address:montaner@wistar.org

3 Abbreviations used in this paper: IQR, interquantile; Flu, influenza A virus PR8H1/M1; IL-1r�, IL-1 receptor antagonist; LPA, lymphoproliferative assay; MFI,mean fluorescent intensity; PPD, purified protein derivative; RPA, RNase protectionassay; RT, room temperature; SI, stimulation index.

The Journal of Immunology

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was available on subjects. Median CD4 count for the cohort was 392cells/�l (25th-75th IQR: 232–604), while median plasma HIV-1 RNA was1500 copies/ml (25th-75th IQR: 400–14,973). HIV-1-uninfected controlswere derived from the Wistar Institute phlebotomy center that recruits/monitors HIV-1 and hepatitis C seronegative subjects without any knowncomplicating health conditions. Demographic data for the HIV� subjectsused were as following: 54% were male, 69% African American, 23%Caucasian, 8% Hispanic, and 0% Native American.

Human experimentation guidelines of the US Department of Health andHuman Services and the authors’ institutions were followed. All subjectsprovided signed informed consent. Venous peripheral blood of HIV� andHIV� subjects was collected in a cross-sectional pattern. Plasma HIV-1RNA and CD4� T cell count were determined by Quest Diagnostic at thetime of blood collection.

Cell preparation

PBMC were isolated by standard Ficoll-Hypaque (Pharmacia), as previ-ously described (18). Monocytes were prepared from PBMC by adherenceto plastic and extensive washings to remove nonadherent cells, as previ-ously described (19, 20). Unless indicated otherwise, PBL were removedby extensive washings following a 1 h culture of PBMC over tissue cul-ture-treated plastic at 37°C. Adherent cells were subsequently harvestedwith cold 1�PBS and 45 mM EDTA and phenotyped for CD14 puritylevels before each experiment. Routinely, we observed CD14 levels �87%in adherent fraction with �3% CD3� T cells.

As indicated in Results, parallel enrichment for CD3�CD4� T cells andCD14� cells was performed for 11 separate donors by using the rosetteSephuman CD4� T cell and human monocyte enrichment mixtures (StemCellTechnologies), according to the manufacturer’s instructions. The purity ofthe enriched populations was further confirmed by flow cytometry, as de-scribed below, showing preparations of CD3�CD4� T cells enriched up to92%, while same donor-matched CD14 enrichment was achieved to�82%.

Proliferation assays

Ag-specific proliferation was measured by lymphoproliferative assays(LPA) or by usage of CFSE.

Regarding LPA, PBMC (250,000 cells/well) were isolated and culturedon the day of collection in a six-replicate format, as previously described(18), in the presence or absence of IL-13 (20 ng/ml, as described by Mon-taner et al. (21)), added once and at the time of PBMC isolation along withno stimulation; positive control; Ag stimulation: 1) UV-inactivated influ-enza A virus PR8 H1/M1 (Flu) at 100 HAU/ml (Fisher UVXL-1000 UVCrosslinker, 1200 �W/cm2, 15 min, 4°C), 2) purified protein derivative(PPD) tuberculin (5 �g/ml; Aventis Pasteur), 3) HIV-1 p24 (5 �g/ml;Protein Sciences) (18), 4) keyhole limpet hemocyanin (5 �g/ml; Sigma-Aldrich), or 5) PHA (5 �g/ml, positive control; Sigma-Aldrich). All Agswere added following 18 h PBMC isolation/IL-13 exposure. The number ofAgs tested was decreased if PBMC yields were restricted, while PPD Agwas used only if a previous tuberculosis diagnosis was confirmed. Whereindicated, neutralizing mouse anti-human IL-12 (10 �g/ml, in house pro-duction clone C8.6.2) was used in LPA. Analysis was done by stimulationindex (SI) defined as: SI � Ag-stimulated mean cpm (with or withoutcytokine)/unstimulated mean cpm (with or without cytokine). An SI �3was interpreted as positive.

For CD14� and CD3�/CD4� T cell enrichment experiments, LPA wasperformed in target cell populations pre-exposed or not to IL-13 (18 h, 20ng/ml) and subsequently stimulated or not with Flu Ag, as indicated above.In addition, all IL-13-exposed cell populations were tested by either wash-out of cytokine at 18 h (time of addition of Ag) or its continuous presence.Briefly, the following conditions were tested: 1) CD3�/CD4� T-enrichedcells with or without IL-13 as negative controls, 2) CD14�-enriched cellswith or without IL-13 as negative controls, 3) coculture of CD3�/CD4�

T-enriched cells without IL-13 and donor-matched CD14�-enriched cellswithout IL-13, and 4) coculture of CD3�/CD4� T-enriched cells withoutIL-13 and donor-matched IL-13-exposed CD14�-enriched cells. All con-ditions were tested in the presence or absence of Flu (experimental con-ditions) using a similar method as that described for PBMC above. Cultureswere initiated at a CD14�:CD3�/CD4� T cell ratio as determined byCD14� and CD3�/CD4� cell subset ratio in donors’ PBMC at isolation.All conditions were tested in triplicate with a total cell content of 300,000cells/well.

Regarding CFSE assays, measurement of proliferation was based on theVybrant CFSE cell tracer kit (Molecular Probes). Briefly, cells were en-riched for CD14� and CD3�/CD4� cell subsets; culture conditions wereprepared, as described above; and cells were incubated with CFSE (1.5�M) for 5 min at room temperature (RT) previous to Ag stimulation. At the

end of the incubation with CFSE, 100 �l of FBS (Cansera) was added andcells were washed once with complete medium (RPMI 1640 (Mediatech)supplemented with 10% FBS, 100 U/ml penicillin/100 �g/ml streptomycin(Invitrogen Life Technologies), and 2 mM glutamine (Invitrogen LifeTechnologies)). Cells were resuspended at a final concentration of 106

cells/ml and were either re-exposed or not to IL-13 (20 ng/ml), as describedabove, along with no stimulation or Ag stimulation (UV-inactivated Flu(100 HAU/ml)) for 4 days. Samples were analyzed by flow cytometry forcell-specific proliferative changes in the CD3�/CD4� T cell subset viastaining with Abs against CD14, CD4, and CD3, as described below. Livegates were set manually, and detection thresholds were set according toisotype-matched negative controls. Acquisition of data and analysis ofCFSE dilution were performed on a DakoCytomation Cyan flow cytometer(DakoCytomation) using the FlowJo 4.5.9 (Tree Star) software package, asfurther described below.

Flow cytometry analysis

Flow cytometry was used for: 1) characterization of the cell subsets inproliferation assays described above, to assess CD3�/CD4� T cell andCD14� cell distribution in PBMC, as well as to assess purity of isolatedadherent cells, PBL, CD3�/CD4� T cell, and CD14�-enriched cell prep-arations; 2) measurement of cell surface expression of molecules associ-ated with Ag presentation in adherent cell fractions derived from PBMCfollowing culture for 18, 48, or 72 h with or without IL-13 (20 ng/ml addedonce and at the time of PBMC isolation); 3) cell-based quantification ofendocytosis (further described below); and 4) cell-based measurements ofapoptosis (further described below). Briefly, cells were washed with 1�PBS, blocked with blocking buffer (1� PBS, 0.1% gelatin, 0.1% NaN3, 5%human AB serum, 5% mouse serum (Sigma-Aldrich)) for 15 min at RT,and then stained for 30 min at 4°C with the fluorochrome-conjugated mAbs(when needed, incubation with secondary Ab was done for an additional 30min at RT and cells were washed three times in FACS washing buffer (1�PBS, 0.1% BSA, 0.02% NaN3)). Following staining, cells were washedthree times in FACS washing buffer, fixed in 1� PBS, 4%paraformaldeyde for 20 min at RT or in FACS Lyse (BD Biosciences) for10 min at RT, washed once in FACS washing buffer, and resuspended in100 �l of FACS washing buffer. Samples were analyzed as indicated on aBD Biosciences FACSCalibur flow cytometer using the CellQuest soft-ware package for acquisition and analysis, or on a DakoCytomation Cyanflow cytometer using the FlowJo 4.5.9 software package for acquisitionand analysis. Either instrument was used consistently for data collection inrespective applications of flow cytometry methods to avoid variability.Live cell gates were set manually, and detection thresholds were set ac-cording to isotype-matched negative controls. Results were expressed asmean fluorescent intensity (MFI) and percent positive.

The following anti-human mAbs from the following sources were usedlisted by source: 1) BD Biosciences: IgG1 CD8-allophycocyanin, IgG1CD3-PE-Cy7, IgG1 CD4 allophycocyanin-Cy7, IgG2a CD14-PE, IgG2bHLA-DR-allophycocyanin, isotypes (mouse): IgG1-allophycocyanin,IgG1-PE-Cy7, IgG1-allophycocyanin-Cy7, and IgG2a-PE; 2) BD Pharm-ingen: IgG1 CD3-PE, IgG1 CD86-PE, IgG1 CD86-FITC, IgG1 CD40-TriColor, IgG2b HLA-DR-FITC, isotypes (mouse): IgG1-PE, IgG1-FITC,IgG2b-FITC, IgG1-allophycocyanin, and IgG1-TriColor; 3) Caltag Labo-ratories: IgG1 CD3-FITC, IgG1 CD14-FITC, IgG1 CD16-FITC, IgG1CD19-FITC, IgG1-CD20-FITC, IgG1 HLA-DR-allophycocyanin, isotype(mouse) IgG1-FITC, and IgG2b-allophycocyanin; and 4) Beckman-Coulter: IgG1 HLA-DR-EnergyCoupledDye, IgG1 CD80, and isotype(mouse): IgG1-EnergyCoupledDye. Goat anti-mouse IgG-FITC (Sigma-Aldrich) was used as secondary Ab when needed.

Cytokine assays

Same day isolated PBMC (250,000 cells/well) were cultured with or with-out IL-13 (20 ng/ml, 18 h) before collection of cell-free supernatants.TNF-� and IL-10 were measured by RIA in cell-free supernatants, as pre-viously described, using the mAb pairs B154.9.2/B154.7.1 and 9D7/12G8,respectively (22, 23). Thresholds of TNF-� and IL-10 detection were 1.69and 0.8 pg/ml, respectively.

RNA isolation

PBMC from 11 HIV� subjects under no antiretroviral therapy were iso-lated and PBL were removed following 1 h incubation at 37°C. For eachof three experiments, adherent cells (with or without IL-13, 20 ng/ml, 5 h,18 h) from three to four subjects were detached with cold 1� PBS and 45mM EDTA and pooled together before isolation of nuclear RNA. NuclearRNA was isolated using the TRI-REAGENT (Molecular Research Center),according to the manufacturer’s instructions.

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RNase protection assay (RPA)

The Ambion’s RPA II kit (Ambion) with the hck2 and hck3 MultiProbeTemplate Set (BD Pharmingen) was used following the manufacturer’sinstructions. Radioactivity was detected using the PhosphorImager 445 SI(Amersham Pharmacia Biotech). Data were analyzed using the IMAGE-QUANT software version 5.0.

RT-PCR

cDNA was generated using Ambion’s RETROscript First-Strand SynthesisKit for RT-PCR following the manufacturer’s instructions. Primers forhuman IL-10, the classic 18S primer set, and competitor primers were alsoobtained from Ambion. [32P]dCTP was also included. Amplification wasperformed using a Peltier Model PTC 200 Thermal Cycler (MJ Research)and 0.2-ml thin-wall reaction tubes, as follows: 1) heat: 5 min, 95°C; 2) 23cycles: 20 s, 94°C; 30 s, 56°C; and 40 s, 72°C; 3) hold: 5 min, 72°C. Theamplified product was run on a 6% denaturing polyacrylamide gel, andradioactivity was detected using the PhosphorImager 445 SI. Data wereanalyzed using the IMAGEQUANT software version 5.0.

Cell population-based quantification of endocytosis

PBMC were isolated and PBL were removed following 1 h incubation at37°C, as described. Adherent cells were cultured for 18, 48, or 72 h in thepresence or absence of IL-13 (20 ng/ml, added once and at the initiation ofthe adherent cell culture). Endocytic uptake was measured with dextranAlexa 647 (Molecular Probes) or HRP (Sigma-Aldrich).

Measurement of cell-specific endocytosis by dextran Alexa 647 uptakewas performed by incubating cells with dextran Alexa 647 (10,000 m.w.,500 �g/ml, 60 min) or dextran amino (Molecular Probes; 10,000 m.w.,negative control); subsequently washed with 1� PBS and stained withsurface Abs for CD3, CD4, CD14, CD19, and HLA-DR; and analyzed byflow cytometry, as described above. Monocytes were defined as CD3�/CD4�/CD14�/HLA-DR�. Results were expressed as MFI and percentpositive.

HRP endocytosis as a monocyte population-based assay was measuredas follows: adherent cells were incubated with HRP (1 mg/ml, 60 min),washed six times with 1� PBS and 1% FBS, and lysed in 0.5% TritonX-100 (Boehringer Mannheim). Total protein in cell lysates was deter-mined by Dc protein assay, according to the manufaturer’s instructions(Bio-Rad). The amount of HRP in the lysate was quantified by mixing eachlysate with HRP substrate and analyzing in a kinetic absorbance reader at460 nm (Rainbow Reader; STL), as previously described (21). HRP con-tent (nanograms per milliliter) was determined using an HRP standardcurve, correcting for any cell loss by expressing uptake values per micro-gram of adherent cells protein in lysates. Results were expressed as ng ofHRP uptake/�g adherent cell protein in lysates.

Cell population-based measurement of spontaneous apoptosis

PBMC (107 cells/well) were cultured for 48 h in complete medium with orwithout IL-13 (20 ng/ml, added once and at the time of PBMC isolation).PBL were subsequently removed, as described above, and adherent cellswere detached with cold 1� PBS and 45 mM EDTA. PBL and donormatched-adherent cell apoptosis was qualified using the In Situ Cell DeathDetection kit, Fluorescein (Boehringer Mannheim), according to the man-ufacturer’s instructions, and analyzed by flow cytometry. FluoresceindUTP incorporated in the DNA strand breaks of the cells was measured ona BD Biosciences FACSCalibur flow cytometer using the CellQuest soft-ware package for acquisition and analysis. Results were expressed as MFIand percent positive.

Single cell imaging of endocytosis and apoptosis

Single cell imaging of endocytosis was performed by dextran uptake assaysand confocal microscopy. Adherent cells in 5-cm2 glass Gold Seal cover-slips (BD Biosciences) were exposed to IL-13 (20 ng/ml, 48 h), pulsed with70 Kd dextran-Texas Red (1 mg/ml, 60 min; Molecular Probes), washed sixtimes with warm 1� PBS, and fixed with 2% paraformaldehyde (30 min,4°C). Apoptosis was detected by in situ specific end labeling of the DNAfragments using the ApoptTag Fluorescein In Situ Apoptosis Detection kit(Serologicals), following the manufacturer’s instructions. Images were ob-tained at The Wistar Institute Microscopy Facility using rhodamine andFITC filters on a Leica Microsystems confocal microscope with FocusImagecorder Plus software.

Statistical analysis

All descriptive analysis and statistical tests were performed using JMP 4.0(SAS Institute), as previously described (24). All tests were two tailed,unless specified in text. All tests applied an � of 0.05.

ResultsSingle addition of IL-13 augments recall and HIV-1-specificlymphoproliferative responses

Exposure of PBMC from HIV� subjects (n � 14) or HIV�

subjects (n � 43) to IL-13 and subsequent stimulation with Ag(Flu) resulted in significant increase in LPA response ( p � 0.03and p � 0.0001, respectively; Fig. 1A). In support of thesefindings, a significant increase of T cell LPA responses againstHIV-1 p24 (n � 30, p � 0.0001) and PPD tuberculin (n � 6HIV-1-infected patients with confirmed diagnosis of past tuber-culosis, p � 0.0299) was also observed (Fig. 1B), indicatingthat IL-13 had a general effect in increasing LPA recall re-sponses. IL-13-mediated increases in HIV-specific LPA werenot directly associated with CD4� T cell count or viral load( p � 0.05) at the time of analysis, even though a greater amountof LPA responses in the absence of IL-13 was observed athigher CD4� T cell count, as previously noted by others (6)(data not shown). Although we did not find an association withviral load, we also addressed whether recall responses and theeffects of IL-13 were different in subjects with or without an-tiretroviral therapy. The results of this analysis showed no dif-ference in the ability of IL-13 to increase LPA responses (Flu:patients on therapy, n � 35, p � 0.0001, and patients withouttherapy, n � 6, p � 0.03; p24: patients on therapy, n � 19, p �0.0001, and patients without therapy, n � 10, p � 0.01). Me-dian CD4 count for patients without therapy was 510 cells/�l(25th-75th IQR: 379 –709), while median plasma HIV-1 RNAwas 1025 copies/ml (25th-75th IQR: 400-9519). The specificityof IL-13 in increasing recall and anti-HIV-1 responses was alsosupported by the lack of enhancement of non-Ag-specificPBMC responses to mitogen (PHA, n � 13, p � 0.094; Fig. 1B)or neoantigen stimulation (keyhole limpet hemocyanin, n � 8,p � 0.383; data not shown).

FIGURE 1. IL-13 significantly enhances recall CD4� T cell responsesagainst HIV-1 and recall Ags in HIV�- and HIV�-infected PBMCs. A,LPA responses (mean SI � SE) against Flu � IL-13 in HIV� (n � 14, left)and HIV� (n � 43, right) PBMC are shown. B, LPA responses againstHIV-1 p24 (n � 30), PPD (n � 6) or PHA (n � 13), � IL-13 in HIV�

PBMC are shown. � and f in A and B show responses in the absence orpresence of IL-13, respectively. Values of p are shown on top of eachgraph.

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To determine whether the IL-13-mediated increase of recall re-sponses was directed to the expansion of CD3�/CD4� T cellsfollowing a modulation of CD14� monocytes, experiments wererepeated using donor-enriched cell populations of CD3�/CD4� Tcells and matched CD14� cells. Data showed a significant en-hancement in the LPA response or increase in the fraction ofCD3�/CD4� T cells with a CFSE dye change following cocultureof enriched CD3�/CD4� T cells (not exposed to IL-13) with 18 hIL-13 pre-exposed monocytes. Similar data were generated

whether IL-13 was washed out before Ag and T cell introductionor maintained throughout culture (representative LPA and CFSEresponses shown in Fig. 2 for four HIV� and two HIV� subjects).No effect was noted by IL-13 on CFSE changes in either CD14�

or CD3�/CD4� T cells in the absence of coculture (data notshown). Taken together, data support the interpretation that PBMCLPA response increases by IL-13 are due to the expansion of recallmemory CD3�/CD4� T cells following the acute modulation ofthe CD14� cell subset.

FIGURE 2. IL-13 significantly enhances re-call CD4� T cell responses through modulationof CD14� monocytes. Responses in A and B areshown following coculture of Flu Ag with iso-lated CD3�/CD4� T cells (without IL-13) to-gether with either: CD14� monocytes withoutIL-13 (superscript a), IL-13-pre-exposed CD14�

monocytes in which IL-13 was washed out be-fore subsequent coculture (superscript b), orIL-13 was maintained throughout coculture (su-perscript c). A, LPA responses of isolated cellsubset cocultures (top) and CFSE-dye analysis ofCD4� T cells within cocultures (bottom) areshown for four representative HIV� donors. B,LPA responses (top) and CFSE-dye analysis(bottom), as described above, are shown for rep-resentative HIV� donors. In bar graphs, � and f

in A and B show responses (SI against Flu) in theabsence or presence of IL-13, respectively. Dueto cell number limitation, experimental condi-tions with the continuous presence of IL-13 uponcoculture (conditions with superscript c) were notperformed in all donors.

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IL-13 increased CD4 T cell HIV-1-specific and recalllymphoproliferative responses by an IL-12-independentmechanism in association with an acute up-regulation of CD86cell surface expression

Because IL-12 has been reported to enhance cell-mediated Ag re-sponses in HIV-1� PBMC (25) and IL-13 can regulate for pro-duction of IL-12 following TLR stimulation (13), we testedwhether the IL-13-mediated increase in lymphoproliferative re-sponses was dependent on IL-12.

The role of IL-12 in the enhancement of cell-mediated Ag re-sponses in HIV� subjects was confirmed by our findings, becauseaddition of the neutralizing anti-IL-12 Ab resulted in a significantdecrease of recall responses compared with recall responses in theabsence of IL-13 (n � 11, p � 0.01; Fig. 3A). However, a signif-icant increase of recall response was still observed following ex-posure to IL-13 in presence of the neutralizing anti-IL-12 Ab whencompared with PBMC in the presence of neutralizing anti-IL-12Ab only (n � 11, p � 0.007; Fig. 3A). Importantly, no significantdifference was observed between the level of IL-13-mediated in-duction in the presence or absence of neutralizing IL-12 Ab (n �11, p � 0.2). Taken together, although our data do not exclude thatIL-12 may play a role in the total induction of recall responses byIL-13, they suggest that IL-12 is not the predominant factor re-sponsible for the IL-13-mediated effects on recall responses.

Analysis of changes in cell surface expression of molecules as-sociated with Ag presentation (CD40, CD80, and CD86) within18 h of a single exposure to IL-13 showed a significant inductionof CD86 cell surface expression (MFI) in adherent cells from bothHIV-1� (n � 10) and HIV-1� PBMC (n � 12) (one-tail t tests:p � 0.001 and p � 0.001, respectively; Fig. 3B), while no 18 hchange in expression (MFI and percent positive) for CD40 and

CD80 was noted in either group (data not shown). Taken together,the IL-13-mediated acute induction of CD86 expression onHIV-1� and HIV-1� monocytes suggests a direct mechanism by

FIGURE 3. Effect of IL-13 on recall response isIL-12 independent, yet associated with an acute CD86up-regulation. A, Flu SI � IL-13 and neutralizing anti-IL-12. Gray area defines negative response (SI �3).Comparisons were performed by Wilcoxon/Kruskal-Wallis nonparametric test. Data shown as interquartilebox plots (median and outliers) with significant p val-ues on top (HIV� subjects, n � 11, 18 h exposure). B,IL-13 modulation of CD86 in adherent cells isolatedfrom HIV-1� (n � 10) and HIV-1� (n � 12) PBMC isshown (left and right panels, respectively). MFI data(�IL-13, 18 h) shown as in A with p values (one-tail ttest) shown on top of each graph.

FIGURE 4. IL-13 decreases TNF-� and IL-10 secretion in HIV�

PBMC within 18 h of exposure. Left panel, Shows constitutive PBMCsecretion of TNF-� (top, n � 8) and IL-10 (bottom, n � 11) � IL-13 (18h) in HIV� measured by in-house RIA. HIV� subjects’ constitutive PBMCsecretion of TNF-� (top, n � 14) and IL-10 (bottom, n � 29) � IL-13 (18h) is shown on the right panel. Data shown as interquartile box plot (me-dian and outliers), with significant p values on the top of each graph.Comparison between HIV� and HIV� for TNF-� and IL-10 secretion(�IL-13) revealed a significantly higher TNF-� secretion in HIV� whencompared with HIV� in the absence of IL-13 (p � 0.0002).

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which IL-13 may augment the activation of Ag-specific CD3�/CD4� T cell responses.

TNF-� and IL-10 secretion by HIV-1� PBMC andcorresponding monocyte gene expression was inhibited by IL-13

We investigated the effect of IL-13 on the constitutive PBMC se-cretion of TNF-� and IL-10 over 18 h after isolation based on theirwell-documented up-regulation in HIV-1 infection (26, 27), theirnegative effects on APC function in association with membrane-bound TNF-�-induced apoptosis (4), and the inhibition of Ag pre-sentation function by IL-10 (28–30). IL-13 decreased significantlythe constitutive high secretion levels of TNF-� (n � 14, p �0.001) and IL-10 (n � 29, p � 0.001) by HIV-1� PBMC over 18 hof culture (Fig. 4, right panel), while having no equally significanteffect in the secretion of TNF-� (n � 8, p � 0.461) and IL-10 (n �11, p � 0.054) by HIV� PBMC (Fig. 4, left panel). Although

unpaired comparison between HIV� and HIV� for constitutiveTNF-� secretion levels showed a significantly higher TNF-� se-cretion by PBMC from HIV� as compared with HIV� in the ab-sence of IL-13 ( p � 0.0002), no difference was observed betweenthe two groups with regard to IL-10 secretion ( p � 0.467). Nosignificant correlation between TNF-� or IL-10 production (prioror after exposure to IL-13) with each other, CD4� T cell count, norplasma HIV-1 RNA was observed (data not shown).

IL-13-mediated regulation of TNF-� and IL-10 gene expressionwas investigated in corresponding adherent monocytes by RPA(Fig. 5A) and RT-PCR (Fig. 5B). For this set of experiments andto increase our ability to detect TNF-� and IL-10 expression inmonocytes, 11 HIV� subjects under no antiretroviral therapy wereused (n � 11, median CD4 count 576 cells/�l (25th-75th IQR:165–677), median plasma HIV-1 RNA 1500 copies/ml (25th-75thIQR: 3370–7500)). RPA analysis indicated a lower constitutive

FIGURE 5. IL-13 decreases TNF-� and IL-10 gene expression in HIV�

adherent cells within 18 h of exposure. A, Representative RPAs showingthe effect of IL-13 on TNF-� (left) and IL-1r� (right) mRNA induction at5- and 18 h exposure, respectively. Image shows the hck-3 (left) and hck-2(right) probe sets without treatment with RNases (lane 1, P), as well as thecorresponding RNase-protected probes following hybridization with totalRNA derived from HIV-1� adherent cells nonexposed (lane 2) and ex-posed (lane 3) to IL-13. Note that each unprotected probe band (lane 1)migrates slower than its protected band (lanes 2 and 3) due to flankingsequences in the probes that are not protected by mRNA. B, RT-PCRsshowing the effect of IL-13 in TNF-� (top, 5 h exposure) and IL-10 (bot-tom, 18 h exposure) gene expression inclusive of control levels of 18S. Bargraphs at the right panel show pixel intensity of TNF-� and IL-10 geneexpression depicted at left panel, following normalization for 18S (�,TNF-� or IL-10 gene expression, respectively, in the absence of IL-13exposure; f, TNF-� or IL-10 gene expression, respectively, followingIL-13 exposure). Representative experiments of three independent exper-iments are shown.

FIGURE 6. IL-13 enhances endocytic uptake in HIV-1� monocytes. A,Histograms show total HRP uptake (HRP nanogram per microgram of celllysate) by HIV-1� (n � 4, left panel) and HIV-1� (n � 11, right panel)adherent cell populations � IL-13 (72 h). Significant p values are shown atthe top of each graph, respectively. See Results for comparison of theendocytic uptake of HRP by adherent cells between HIV� and HIV�

monocytes. B, Histograms show IL-13-induced increase in endocytosis ofdextran Alexa 647 within CD3�/CD4�/CD14�/HLA-DR� cells from arepresentative HIV� (left panel, dextran Alexa 647 MFI: no IL-13 � 341,IL-13 � 579) and HIV� (right panel, dextran Alexa 647 MFI: no IL-13 �388, IL-13 � 1204) donor following 72 h cytokine exposure before endo-cytic assay. As a negative control for background fluorescence, each panelalso includes the MFI data for endocytosis of dextran amino in the absenceor presence of IL-13 (HIV� left panel, dextran amino MFI: no IL-13 �1.92, IL-13 � 1.61; HIV� right panel, dextran amino MFI: no IL-13 �1.58, IL-13 � 1.79). C, Shows representative single cell fluorescence mi-croscopy photomicrographs of HIV-1� adherent cells (�IL-13, 48 h), fol-lowed by dextran-Texas Red uptake and ApopTag fluorescein staining. Topphotomicrographs are at �40 (bar, 13 mm � 80 �m), while those on thebottom are at �100 (bar, 16 mm � 40 �m).

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TNF-� expression in the presence of IL-13 (Fig. 5A, left panel),while constitutive IL-10 expression levels were not detected bythis method (Fig. 5A, right panel). Interestingly, consistent withthe activity of IL-13 on IL-1 receptor antagonist (IL-1r�) (9, 10)and as a positive control for modulation of gene expression, weconfirm a marked increase in IL-1r� mRNA at 18 h of IL-13 ex-posure (Fig. 5A, right panel). TNF-� and IL-10 gene expressionwere further analyzed by competitive RT-PCR to confirm a 38 and27% decrease in TNF-� and IL-10 expression, respectively, in thepresence of IL-13 (Fig. 5B), consistent with reductions observed inTNF-� and IL-10 protein secretion data described above. Takentogether, IL-13’s inhibition of constitutive secretion levels ofTNF-� and IL-10 in HIV infection is consistent with an indirecteffect on recall responses due to the potential negative effects ofboth cytokines on Ag presentation function.

IL-13 restores significantly impaired monocyte endocytic activityin association with a significant reduction of spontaneousapoptosis by HIV-infected monocytes

Regarding Ag uptake function by adherent APCs, a single 72 hexposure of IL-13 significantly increased the pinocytic activity ofHIV-1� and HIV-1� monocytes, as measured by soluble HRP(n � 4, p � 0.02 and n � 11, p � 0.0005, respectively; Fig. 6A).In general, uptake by HIV� monocytes exposed to IL-13 rose tolevels observed in IL-13-unexposed HIV� monocytes as mono-cytes from HIV-1� subjects had significantly lower endocytic ac-tivity at 72 h of culture as compared with HIV� monocytes (HRP,p � 0.0001). Similar results were obtained by direct flow cytom-etry analysis of dextran uptake by IL-13-exposed vs non-IL-13-exposed adherent cells when analyzed for uptake levels withingated CD14� cell subsets (HIV-1�, n � 5; HIV-1�, n � 3; arepresentative donor from each group is shown in Fig. 6B). En-hanced endocytic uptake in conjunction with measurement of cell

viability in the presence of IL-13 over 48 h was also documentedvia microscopy at a single cell level in cultured monocytes fromHIV-1� subjects coanalyzed with dextran-Texas Red uptake andApoptTag Fluorescein staining (Fig. 6C).

To further address IL-13 effects on monocyte viability as a po-tential factor that may impact Ag uptake and function based on thedescribed effects of type 2 cytokines such as IL-4 on monocyteapoptosis (31), quantification of spontaneous apoptosis of HIV-1�

monocytes by flow cytometry in the presence or absence of IL-13over 48 h showed that IL-13 exposure resulted in a significantdecrease in CD14� cell apoptosis as reflected by changes in MFIand percent positive of adherent cells incorporating fluoresceindUTPs within DNA strand breaks (n � 8, p � 0.008 and p �0.004, respectively) (Fig. 7, top panel). The effect by IL-13 wasspecific to the adherent cell fraction as no significant differencewas detected in IL-13-exposed donor-matched PBL (Fig. 7, bottompanel). Taken together, we interpret that IL-13 effects on mono-cytes from HIV� and HIV� monocytes include an increased ca-pacity for Ag uptake, which is associated, yet independent of adecrease in spontaneous apoptosis.

DiscussionWe show IL-13 as the first anti-inflammatory cytokine with amechanism of action centered on increasing Ag presentation func-tion in HIV infection, leading to a significant increase of cell-mediated CD4� T cell responses in vitro. Although the activity byIL-13 to augment recall responses is not restricted to HIV infec-tion, the mechanism of action for IL-13 is directly associated withactivity on dysregulated APC functions in HIV-1 infection that arebroadly associated with decreased cell-mediated immune re-sponses (1, 2, 4, 28, 32–34).

A mechanism of action for IL-13 as primarily acting to modu-late the CD14� subset was directly shown following isolation andIL-13 exposure of this subset before washout and coculture withdonor-matched CD3�/CD4� T cells (Fig. 2). Results do not indi-cate IL-13 to have a single mechanism of action in augmentingrecall responses, but rather a cumulative effect on multiple mono-cyte functions (CD86, apoptosis, endocytosis, cytokine regulation)that culminate in activation of CD4� T cell recall responses. Ourdata document the normalization by IL-13 of potentially suppres-sive mechanisms by monocytes on CD3�/CD4� T cell recall re-sponse activation such as an increased constitutive IL-10 andTNF-� secretion, lower endocytic uptake, and decreased cell sur-vival (Figs. 4–7). Constitutive TNF-� and IL-10 PBMC secretionas well as monocyte viability have been associated with inhibitorymechanisms on the activation of CD4� T cells in HIV infectiondue to TNF-� receptor-mediated apoptosis (4, 34) and IL-10-me-diated inhibition of Ag uptake and T cell activation (28, 29). How-ever, our data do not exclude a role for IL-13 in independentlyaugmenting positive mechanisms of Ag presentation function bymonocytes, as shown by its significant increase of CD86 and Aguptake in uninfected monocytes or its effect on activation of recallresponses in uninfected PBMC. To our knowledge, this is the firstreport of an anti-inflammatory or Th2 cytokine acting to augmentT cell-mediated responses by targeting the reversal of inhibitorymechanisms on Ag presentation in HIV-1 infection.

Cell-specific activity by IL-13 in increasing Ag presentation po-tential within HIV-infected PBMC has been shown with regard toits role in differentiating in vitro dendritic cell subsets when com-bined with GM-CSF after a prolonged culture with both cytokinesover 7 days (35, 36). Our data now show that IL-13 can acutelyand without GM-CSF, or a requirement for long-term culture con-ditions, act to quickly modulate monocytes from HIV-infectedsubjects, allowing for a greater potential to activate memory T cell

FIGURE 7. IL-13 decreases spontaneous apoptosis in HIV-1� mono-cytes. Effect of IL-13 over 48 h of exposure in the apoptosis of adherentcells (top panel) and matching PBL (bottom panel) from HIV� subjects(n � 8). Results measured by the TUNEL assay are expressed by MFI (left)and percentage of positive cells (right). Data shown as interquantile boxplots (median and outliers), with significant p values on the top of eachgraph.

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responses. Consistent with the activity of IL-13 to modulate mono-cytes into dendritic-like cells after exposure, IL-13 has been shownto induce expression of dendritic cell-specific intracellular adhe-sion molecule-3-grabbing nonintegrin in human monocyte-derivedmacrophages (20, 37). Interestingly though, IL-13-mediated up-regulation of dendritic cell-specific intracellular adhesion mole-cule-3-grabbing nonintegrin was also observed to be an acute ef-fect of exposure in the absence of GM-CSF and was not associatedwith an increased transmission of CXCR4 HIV-1 (20). In sum-mary, in contrast to long-term differentiation of dendritic cell sub-sets in the presence of IL-13 and GM-CSF, our data show thatshort-term IL-13 effects can modulate monocyte function within18 h of cytokine exposure and stress its association to activation ofrecall responses in HIV infection. Our results on IL-13 modulationof HIV-infected PBMC in vitro are also in accordance with in vivoimmunoregulatory outcomes of decreased IL-10 expression in ma-caques infected with SIV for 22 mo and given daily IL-13 doses of10 �g/kg/day, for 21 days before euthanasia (38). Interestingly, theprevious descriptive in vivo study showed an increased recruit-ment of inflammatory cells and IFN-� expression in the gut inassociation with an inflammation-induced shedding of villi ob-served in IL-13-treated SIV-infected, but not in IL-13-treated un-infected or untreated SIV-infected macaques (38). Based on this invivo evidence for immune activation following IL-13 administra-tion and the prominent role of gut-associated viral replication inSIV and HIV-1 pathogenesis (39–42), further investigation wouldneed to define the IL-13-dependent mechanisms in vivo that mayhave contributed to the activation of an otherwise down-regulatedinflammatory response in the gastrointestinal track of SIV-infectedanimals.

Although we show that the effects of IL-13 were not associatedwith viral load or CD4 count in our cohort, it is important to high-light that the majority of our subjects were on therapy, and thosethat were not had a median CD4 count above 500 cells/�l to sug-gest that the level of immune function within the cohort tested maybe at a higher level than otherwise observed if we had equal rep-resentation of subjects in end-stage disease. We interpret this to bethe predominant reason that despite the presence of IL-13-medi-ated changes, no differences were noted at baseline values in thelevel of recall responses against Flu (Fig. 1), median IL-10 levels(Fig. 4), or apoptotic rates among lymphocytes between uninfectedand infected groups despite significant differences in TNF-� se-cretion (Fig. 4) levels and endocytic activity (Fig. 6). As our datareflect the patient cohort currently under medical care in center-city Philadelphia, where it is highly uncommon that subjects witha high viral load and a low CD4 count would be followed in aclinical setting in the absence of therapy, further analysis in sub-jects at end-stage disease would be needed to confirm the expec-tation that IL-13 would modulate monocytes at end-stage diseaseand augment recall responses in the presence of underlying Ag-specific CD4� T cells.

In conclusion, the activity by IL-13 in increasing T cell LPAresponses against HIV-1 and other recall Ags is shown to be as-sociated with multiple changes in monocyte cell surface expres-sion, function, and viability that are interpreted to collectively actto increase T cell-mediated responses. Based on IL-13’s lack ofdirect T cell modulation, further investigation will need to addresswhether IL-13 effects on monocytes could complement approachestargeted to expand T cells and cell-mediated responses in HIVinfection (i.e., IL-2, IL-12, IL-15) (7, 25). Together with data al-ready available on the antiviral activity of IL-13 (16), impairedsecretion in HIV-1 infection (15), and its effect on augmenting Tollreceptor-mediated IL-12 secretion by HIV-infected monocytes(13), our data further support IL-13 as a candidate antiviral cyto-

kine of potential benefit to Ag presentation function and activationof cell-mediated T cell responses in HIV-1 infection.

AcknowledgmentsWe thank each of the HIV� and HIV� participants; Matthew Farabaughand Maxwell Pistilli for technical assistance; the Board and Staff of theWistar Institute (D. D. Davis, J. S. Faust, and J. E. Hayden) and the Im-munodeficiency Program Clinic at the Hospital of the University of Penn-sylvania; and C. Gallo, J. Shull, and the Philadelphia Field Initiating Groupfor HIV-1 Trials.

DisclosuresA. Minty is employed by Sanofi-Synthelabo Recherche, which holds claimrights to IL-13, studied in the present work.

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