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T+HIV Infection and Outgrowth in CD4Monocyte-Derived Dendritic Cells Boosts

+Bacterial/Fungal Pathogens in CD16RALDH Activity Induced by

Chomont, Jean-Pierre Routy and Petronela AncutaAnnie Gosselin, Eric A. Cohen, Jérôme Estaquier, NicolasRosario, Laurence Raymond Marchand, Jonathan Dias, Amélie Cattin, Vanessa Sue Wacleche, Natalia Fonseca Do

http://www.jimmunol.org/content/206/11/2638doi: 10.4049/jimmunol.2001436May 2021;

2021; 206:2638-2651; Prepublished online 24J Immunol 

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6.DCSupplementalhttp://www.jimmunol.org/content/suppl/2021/05/24/jimmunol.200143

Referenceshttp://www.jimmunol.org/content/206/11/2638.full#ref-list-1

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RALDH Activity Induced by Bacterial/Fungal Pathogens inCD16+ Monocyte-Derived Dendritic Cells Boosts HIV Infectionand Outgrowth in CD4+ T Cells

Am�elie Cattin,*,† Vanessa Sue Wacleche,*,† Natalia Fonseca Do Rosario,†

Laurence Raymond Marchand,† Jonathan Dias,*,† Annie Gosselin,† Eric A. Cohen,*,‡

J�erome Estaquier,§,{ Nicolas Chomont,*,† Jean-Pierre Routy,‖,#,** and Petronela Ancuta*,†

HIV reservoirs persist in gut-homing CD4+ T cells of people living with HIV and receiving antiretroviral therapy, but theantigenic specificity of such reservoirs remains poorly documented. The imprinting for gut homing is mediated by retinoic acid(RA), a vitamin A�derived metabolite produced by dendritic cells (DCs) exhibiting RA-synthesizing (RALDH) activity. RALDHactivity in DCs can be induced by TLR2 ligands, such as bacterial peptidoglycans and fungal zymosan. Thus, we hypothesizedthat bacterial/fungal pathogens triggering RALDH activity in DCs fuel HIV reservoir establishment/outgrowth in pathogen-reactive CD4+ T cells. Our results demonstrate that DCs derived from intermediate/nonclassical CD16+ compared with classicalCD162 monocytes exhibited superior RALDH activity and higher capacity to transmit HIV infection to autologous Staphylococcusaureus�reactive T cells. Exposure of total monocyte-derived DCs (MDDCs) to S. aureus lysates as well as TLR2 (zymosan andheat-killed preparation of Listeria monocytogenes) and TLR4 (LPS) agonists but not CMV lysates resulted in a robustupregulation of RALDH activity. MDDCs loaded with S. aureus or zymosan induced the proliferation of T cells with aCCR5+integrin b7+CCR6+ phenotype and efficiently transmitted HIV infection to these T cells via RALDH/RA�dependentmechanisms. Finally, S. aureus� and zymosan-reactive CD4+ T cells of antiretroviral therapy-treated people living with HIVcarried replication-competent integrated HIV-DNA, as demonstrated by an MDDC-based viral outgrowth assay. Together, theseresults support a model in which bacterial/fungal pathogens in the gut promote RALDH activity in MDDCs, especially in CD16+

MDDCs, and subsequently imprint CD4+ T cells with gut-homing potential and HIV permissiveness. Thus, nonviral pathogensplay key roles in fueling HIV reservoir establishment/outgrowth via RALDH/RA�dependent mechanisms that may betherapeutically targeted. The Journal of Immunology, 2021, 206: 2638�2651.

Antiretroviral therapy (ART) controls HIV-1 replication toundetectable plasma levels and improved the life qualityof people living with HIV (PLWH) (1). However, HIV

cure is not achieved because of the persistence of viral reservoirsthat are relatively stable despite long-term ART (2�4). Long-livedAg-experienced memory CD41 T cells carry such HIV reservoirs,and viral replication-rebound occurs shortly after treatment

interruption (3, 5). The clonal expansion of memory CD41 T cellscarrying viral reservoirs was identified as a key mechanism bywhich HIV persists during ART (3, 6, 7). This clonal expansion isgoverned by the interaction of memory CD41 T cells with dendriticcells (DCs), which are well-established professional APCs (8) witha unique capacity to capture HIV virions and efficiently transfer in-fection to CD41 T cells within immunological infectious synapses

*Facult�e de M�edecine, D�epartement de Microbiologie, Infectiologie et Immunologie,Universit�e de Montr�eal, Montreal, Quebec, Canada; †Centre de Recherche du CentreHospitalier de l’Universit�e de Montr�eal, Montreal, Quebec, Canada; ‡Institut de Re-cherches Cliniques de Montr�eal, Montreal, Quebec, Canada; xUniversit�e Laval, QuebecCity, Quebec, Canada; {Centre de Recherche du Centre Hospitalier de l’Universit�e La-val, Quebec City, Quebec, Canada; ‖Infectious Diseases and Immunity in Global HealthProgram, Research Institute, McGill University Health Centre, Montreal, Quebec, Cana-da; #Chronic Viral Illness Service, McGill University Health Centre, Montreal, Quebec,Canada; and **Division of Hematology, McGill University Health Centre, Montreal,Quebec, Canada

ORCIDs: 0000-0001-6741-5145 (A.C.); 0000-0001-5109-085X (N.F.D.R); 0000-0002-7842-0115 (J.D.); 0000-0003-1269-3791 (E.A.C.); 0000-0001-9747-5018 (N.C.); 0000-0001-9897-7589 (J.-P.R.); 0000-0003-1922-5640 (P.A.).

Received for publication December 22, 2020. Accepted for publication March 30, 2021.

This work was supported by Canadian Institutes of Health Research (CIHR) GrantHOP-120239 to P.A., the Fonds de Recherche du Qu�ebec-Sant�e (FRQ-S)/AIDS andInfectious Diseases Network, the Canadian HIV Cure Enterprise Team Grant(CanCURE 1.0), funded by the CIHR in partnership with Canadian Foundation forAIDS Research and International AIDS Society (CanCURE 1.0 and HIG-133050), andthe Canadian HIV Cure Enterprise Team Grant (CanCURE 2.0), funded by the CIHR(HB2-164064). Core facilities and people living with HIV cohorts were supported bythe Foundation du Centre Hospitalier de l’Universit�e de Montr�eal and the FRQ-S/AIDSand Infectious Diseases Network. The funding institutions played no role in the design,collection, analysis, and interpretation of data. A.C. received a doctoral award from theUniversit�e de Montr�eal (UdeM). J.E., J.-P.R., and E.A.C. hold the CIHR CanadaResearch Chair Tier 1 in Pathophysiology of Cell Death in Host�Pathogen Interactions,

the Louis Lowenstein Chair in Hematology and Oncology, McGill University, and theIRCM-UdeM Chair of Excellence in HIV Research, respectively.

A.C. designed and performed research, analyzed data, prepared figures, and wrote themanuscript. V.S.W. designed and performed research, analyzed results, and preparedfigures. N.F.D.R., L.R.M., J.D., and A.G. performed research related to cell sorting andculture and/or HIV reservoir measurements. E.A.C. provided cell lines and contributedto research design. J.E. and N.C. contributed to research design and manuscript revision.J.-P.R. provided access to clinical samples/information, set up clinical researchprotocols, and contributed to manuscript revision. P.A. conceived the research studyhypothesis, designed research, analyzed data, and wrote the manuscript. All coauthorsrevised the manuscript.

Address correspondence and reprint requests to Dr. Petronela Ancuta, Centre Hospitalierde l’Universit�e de Montr�eal Research Centre, 900 Rue Saint-Denis, Tour Viger R,Room R09.416, Montreal, QC H2X 0A9, Canada. E-mail address: petronela.ancuta@umontreal.ca

The online version of this article contains supplemental material.

Abbreviations used in this article: ART, antiretroviral therapy; ARV, antiretroviral;CHUM, Centre Hospitalier de l’Universit�e de Montr�eal; DC, dendritic cell; DEAB,N,N-diethylaminobenzaldehyde; HKLM, heat-killed preparation of Listeriamonocytogenes; MDDC, monocyte-derived DC; PAMP, pathogen-associated molecularpattern; PGN, peptidoglycan; PLWH, people living with HIV; RA, retinoic acid; RARa,RA receptor a; SEB, staphylococcal enterotoxin B; Treg, regulatory T cell; VOA, viraloutgrowth assay.

Copyright©2021 byTheAmericanAssociation of Immunologists, Inc. 0022-1767/21/$37.50

www.jimmunol.org/cgi/doi/10.4049/jimmunol.2001436

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(9�11). Although DCs can transfer HIV particles to CD41 T cellsduring the process of Ag presentation (12, 13) in the absence of pro-ductive infection (11), DCs are also able to support covert HIV inte-gration and further transmit de novo�produced virions to CD41 Tcells (14, 15). Thus, HIV highjacks the cross-talk between DCs andCD41 T cells, a process essential for the maintenance of immuno-logical memory (8), to ensure its persistence into the host. There-fore, HIV eradication strategies require a profound understanding ofthese immunological processes that occur in ART-treated PLWH.Bone marrow�derived monocytes represent key precursors

for DCs in vivo and in vitro (16, 17). Monocytes are highlyheterogeneous and classified according to their expression ofthe LPS-binding receptor CD14 and the FcgRIII/CD16 intoclassical CD1411CD16�, intermediate CD1411CD161 andnonclassical CD141CD1611 monocytes (16�18). Previousstudies by our group and others demonstrated that intermedi-ate/nonclassical CD161 versus classical CD16� monocytesdiffer in their biological functions and trafficking potential,consistent with their distinct proteomic and transcriptional pro-files (19�22). Consequently, it is well established that humanCCR2lowCX3CR1highCD161 and CCR2highCX3CR1lowCD16�

monocytes represent precursors for functionally distinct mono-cyte-derived DC (MDDC) subsets (23, 24). Also, comparedwith their CD16� counterparts, CD161 monocytes rapidly dif-ferentiate into DCs upon reverse transendothelial migrationand acquire an increased expression of costimulatory mole-cules when cultured in the presence of GM-CSF and IL-4in vitro (25). Nevertheless, the contribution of human CD161

versus CD16� MDDCs subsets to disease pathogenesis, suchas the HIV infection, remains poorly investigated. This knowl-edge is important to determine the impact of an alteredCD16�/CD161 monocyte ratio, a condition observed in multi-ple pathological conditions, including HIV infection (24, 26,27), on the quality of DC-mediated immune responses.Genome-wide transcriptional profiling performed by our group

demonstrated that CD161 compared with CD16� monocytes differ-entiate in vitro into CD1031 DCs with a unique transcriptional sig-nature; notably, they preferentially express the retinoic acid(RA)�synthesizing enzyme RALDH mRNA (23). RALDH metabo-lizes diet vitamin A into RA (28) and is highly expressed by muco-sal CD1031 DCs (29). RA induces expression of gut-homingmolecules, such as integrin a4b7 and the chemokine receptorCCR9 (30, 31). Moreover, RA plays a major role in the develop-ment and the maintenance of secondary lymphoid organs (32) andthe homeostasis of the intestinal mucosal immunity by controllingthe induction of regulatory T cells (Tregs) (33). Furthermore, ourgroup demonstrated that RA acts on memory CD41 T cells to pro-mote viral replication, mainly in CCR61 Th17 cells, via mTOR-de-pendent mechanisms (34). RALDH activity in DCs is upregulatedby zymosan, a cell wall component of Saccharomyces cerevisiaeand other fungi, via a TLR2-dependent mechanism (35). Interesting-ly, TLR2 and dual TLR2/7 agonists have been shown to reactivatelatent HIV reservoirs via the secretion of TNF-a (36). Notably,CD161 MDDCs, similar to CD161 monocytes, represent majorTNF-a producers in response to LPS stimulation (21�23, 37, 38).Whether CD161 MDDCs play a pathogenic role during HIV infec-tion by promoting HIV reservoir establishment and reactivation inCD41 T cells specific or reactive to certain bacterial/fungal patho-gens in a RALDH-dependent manner remains unknown.In this manuscript, we explored the role of CD161 versus

CD16� MDDCs in transmitting HIV infection and reactivating viralreservoirs in CD41 T cells in the context of Ag-mediated clonal ex-pansion. The Ags used were S. aureus lysates [known to triggerTh17 responses (39)] and CMV [known to trigger Th1 responses

(40)], as well as the staphylococcal enterotoxin B [SEB; a super Agknown to trigger polyclonal T cell activation and proliferation (31)].Our results point to an essential contribution of bacterial/fungalpathogens in fueling HIV reservoir establishment/reactivation inCD41 T cells specific/reactive to such pathogens. This process isdependent on the induction of the RALDH activity in MDDC viabacterial/fungal components that trigger TLR2/TLR4 activation(e.g., peptidoglycan [PGN], zymosan, LPS). These results supportthe existence of a mechanistic link between specific components ofthe microbiota and HIV reservoir persistence/reactivation in ART-treated PLWH. Our findings open the path for further investigationsaimed at exploring whether the RALDH/RA pathway in CD161

MDDCs can be therapeutically targeted in HIV cure/remissionstrategies.

Materials and MethodsStudy participants

PLWH receiving ART (HIV1ART1) as well as HIV-uninfected individuals(HIV�) were recruited at the McGill University Health Centre for leukaphe-resis donation, as previously reported (41). Clinical parameters of study par-ticipants are detailed in Table I. Leukaphereses were collected, and PBMCwere isolated by gradient centrifugation and frozen until use, as we previous-ly described (41).

Ethics statement

This study, using biological samples from HIV1ART1 and HIV� individu-als, was conducted in compliance with the principles included in the Declara-tion of Helsinki and received approval from the Institutional Review Boardof the McGill University Health Centre and Centre Hospitalier de l’Universit�ede Montr�eal (CHUM) Research Centre. All subjects signed written informedconsent for their study participation.

Flow cytometry analysis

The following Abs were used for flow cytometry analysis: CD1c BV786(clone L161; BioLegend), CD1c PA (clone AD5-8E7; Miltenyi Biotec),CD3 FITC (clone BW264/56; Miltenyi Biotec), CD3 Pacific Blue (cloneUCHT1; BD Biosciences), CD3 Alexa Fluor 700 (clone UCHT1; BioLe-gend), CD8 FITC (clone RE734; Miltenyi Biotec), CD14 allophycocyanin(clone M5E2; BD Biosciences), CD16 PE-Cy5 (clone 3G8; Beckman Coul-ter), CD19 FITC (clone LT19; Miltenyi Biotec), CD56 FITC (clone HCD56;BioLegend), CCR5 PE (clone 2D7; BD Biosciences), CCR6 BV421 (clone11A9; BD Biosciences), integrin b7 PE-Cy5 (clone FIB504; BD Bioscien-ces), HIV-p24 PE (clone KC57; Beckman Coulter), IL-17A eFluor 660(clone eBio64DEC17; eBioscience), IFN-g Alexa Fluor 700 (clone B27; BDBiosciences), TNF-a efluor 450 (clone Mab11; Thermo Fisher Scientific).The phenotype of monocytes, MDDCs, and T cells was assessed by flow cy-tometry, as previously described (23, 31, 41). Briefly, cells were stained withfluorescence-conjugated Abs and fixed using PBS 2% paraformaldehyde fix-ation buffer. Cells were analyzed using an LSR II cytometer (FACSDiva,BD Biosciences) and FlowJo (version 10.6.1, Tree Star).

Magnetic and flow cytometry sorting

CD161 and CD16� monocyte subsets were sorted from PBMCs by magnet-ic and flow cytometry sorting, as we previously reported (23, 41). Briefly, to-tal monocytes were first enriched by negative selection using magnetic beads(Pan Monocytes Isolation Kit, human; Miltenyi Biotec). CD161 and CD16�

monocytes were then sorted from total monocytes by flow cytometry (BDFACSAria III) upon staining with fluorescence-conjugated CD3 FITC, CD8FITC, CD56 FITC, CD19 FITC, CD1c PE, and CD16 PE-Cy5 Abs (as listedabove), as previously described (23, 41). The purity of each subset was>98%, as demonstrated by postsort quality controls (data not shown). Mem-ory CD41 T cells were sorted from PBMCs by negative selection usingmagnetic beads (Memory CD41 T Cells Isolation Kit; Miltenyi Biotec) witha purity >95%, as we previously described (41). Briefly, this kit allows theisolation of memory CD41 T cells by depleting naive (CD451) CD41 Tcells, CD81 T cells, B cells, NK cells, g/d T cells, monocytes, DCs, granu-locytes, platelets, and erythrocytes; the enriched memory CD41 T cells ex-hibited a CD45RA�CD3�CD8�CD41 phenotype (data not shown).

Generation of MDDC

To obtain MDDCs, total monocytes, CD161 and CD16� monocytes, werecultured in RPMI 1640 2% FBS in the presence of GM-CSF and IL-4 (20

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ng/ml, R&D Systems) for 6 d, as we previously reported (23). Medium wasrefreshed every 2 d. At day 6, MDDCs were harvested for phenotypic analy-sis and functional studies.

CFSE-based proliferation assay

The dilution of the CFSE dye, indicative of cell proliferation, was measuredby flow cytometry upon culture in the presence of Ag, as we previously re-ported (31). Briefly, memory CD41 T cells were washed twice with PBS1� and were resuspended at 20 � 106 cells/ml in PBS containing CFSE(0.5 mM; Sigma-Aldrich) for 8 min in the dark. Human serum (1 ml/20 �106 cells; Gemini Bio Products;) was added for 1�2 min, and CFSE-loadedcells were washed once with PBS and once with RPMI 1640. Finally, cellswere resuspended in proliferation medium (RPMI 1640, 10% human serum,1% L-glutamine, 1% HEPES, 1% penicillin/streptomycin [Life Technolo-gies]) for subsequent coculture experiments with Ag-loaded MDDCs.

ALDEFLUOR-ALDH activity assay

The ALDEFLUOR assay (STEMCELL Technologies) was used to measurethe activity of the RALDH enzyme activity in MDDCs, using the man-ufacturer’s protocol. In this assay, the activated ALDH substrate (BODIPYaminocetaldehyde) diffuse into the cells and get converted into the ALDHproduct (BODIPY aminocetate) by the ALDH enzyme. The product accumu-lation in the cells causes the ALDH-expressing cells to be highly fluorescentcompared with the ALDH cells. These cells can be identified by FACS asGFP1. Briefly, MDDC were resuspended in ALDEFLUOR assay buffer at1 � 106 cells/ml, and 5 ml of the activated ALDEFLUOR reagent wasadded. Half of the suspension was immediately transferred into a controltube containing the RALDH inhibitor N,N-diethylaminobenzaldehyde(DEAB). Cells were incubated at 379C for 1 h and washed with ALDE-FLUOR Assay Buffer before proceeding to the surface staining protocol andFACS acquisition. To prevent the efflux of the ALDEFLUOR product, cellswere stained in ALDEFLUOR Assay Buffer and kept at 4�C. The negativitygates were defined based on the fluoresce signal obtained for each sampleupon culture in the presence of DEAB. In specific experiments, the ALDHactivity was measured in MDDCs upon stimulation (48 h) with the followingstimuli: S. aureus lysate (5 � 106 cells per assay, LAM strain; a gift fromDr. L.de Repentigny, Universit�e de Montr�eal, Qc, Canada), CMVpp65 pep-tide pool (1 mg/ml; Miltenyi Biotec), zymosan (10 mg/ml; InvivoGen), andHuman TLR1-9 Agonist Kit (InvivoGen); also used were Pam3CysSerLys4(Pam3CSK4; 1 mg/ml), heat-killed preparation of Listeria monocytogenes(HKLM; 1 � 108 cells/ml), Poly I:C (high and low m.w.; 1 mg/ml), LPSfrom Escherichia coli K12 (1 mg/ml), flagellin from Salmonella typhimurium(FLA-ST; 1 mg/ml), FSL-1 (Pam2CGDPKHPKSF, 1 mg/ml), imiquimod(1 mg/ml), ssRNA40 (1 mg/ml), and ODN2006 (5 mM). The TLR ligandconcentrations used are those recommended in the manufacturer’s protocol(InvivoGen).

MDDC-based Ag-presentation assay

MDDCs were loaded with S. aureus lysate (5 � 106 cells per assay; a giftfrom the Laboratoire de Microbiologie et Immununologie, Universit�e deMontr�eal, Qc, Canada), Candida albicans lysate (5 � 106 cells per assay;LAM strain; a gift from Dr. L. de Repentigny, Universit�e de Montr�eal, Qc,Canada), CMV-pp65 peptide pool (1 mg/ml; Miltenyi Biotec) or SEB (25ng/ml; Toxin Technology) for 1 h. For all Ags, the optimal dose (defined asthe dose inducing the highest levels of T cell proliferation and viability) wasestablished based on preliminary dose-response experiments for each batchof Ags produced or purchased (data not shown), as previously reported (31,39, 42). Ag-loaded MDDCs were cocultured with autologous CFSE-loadedmemory CD41 T cells for 5 d at 37�C. At day 5, cells were harvested andstained with the LIVE/DEAD dye (Invitrogen) and fluorochrome-conjugatedAbs against lineage markers to distinguish MDDC (CD1c1CD3�) from Tcells (CD1c�CD31) as well as other surface markers (CCR5, CCR6, and in-tegrin b7), as specified in the figure legends. In specific experiments, cellswere permeabilized using the BD Cytofix/Cytoperm kit (BD Biosciences)and intracellularly stained with IL-17A, IFN-g, and/or TNF-a Abs afterstimulation with PMA (50 ng/ml) and ionomycin (1 mg/ml) in the presenceof brefeldin A (2 mg/ml). In specific coculture experiments, the RA receptorantagonist LE540 (0.5 mM; Wako Pure Chemicals) and the ALDH inhibitorDEAB (10 mM; STEMCELL Technologies) was used to interfere with theRA signaling pathway. The LE540 concentration used was identified in pre-liminary dose-response experiments (data not shown), as we previously re-ported (31).

MDDC-based HIV transmission assay

Matched CD161 and CD16� MDDCs or total MDDCs from HIV� individ-uals were exposed to the replication-competent NL4.3BaL HIV-1 strain (10ng HIV-p24/1 � 106 MDDCs, an HIV molecular clone expressing the R5-

tropic BaL env on the NL4.3 backbone and gfp in place of nef; a gift fromDr. D. Gabuzda, Dana Farber Cancer Institute, Boston, MA) for 3 h at37�C. Unbounded viruses were removed by extensive washing. MDDCswere loaded in Ags, as described above, and then cocultured with autologousmemory CD41 T cells. In one set of experiments, cells were harvested atday 5 postcoculture and stained with fluorescence-conjugated CD3 PB andHIV-p24 PE Abs, and the frequency of CD41 T cells productively infectedwith HIV (CD31HIV-p241) was quantified by FACS. In another set of ex-periments, MDDCs were cocultured with autologous memory CD41 T cellsfor 12 d in the presence or the absence of LE540 or DEAB. Supernatantswere refreshed every 3 d, and levels of HIV-p24 in cell culture supernatantswere quantified by ELISA, as we previously reported (41, 43).

HIV-DNA quantification

Integrated HIV-DNA quantification was performed on cell lysates from in-fected cells using nested real-time PCR (LightCycler 480; Roche) and specif-ic HIV long terminal repeat and Alu primers, as we previously described(34, 41). HIV copy numbers were normalized relative to cell numbers uponsimultaneous CD3 amplification with specific primers (two CD3 copies percell). ACH2 cells containing one copy of proviral HIV-DNA was used togenerate a standard curve (30,000; 3,000; 300, 30, and 3 cells per reaction).The limit of detection of the assay was three copies per test for both HIVand CD3. Each PCR was performed in triplicates, using 30,000�100,000cells per reaction.

MDDC-based viral outgrowth assay

To perform a MDCC-based viral outgrowth assay (VOA), MDDCs were ob-tained from total monocytes of ART-treated PLWH, as detailed above. Atday 6, MDDCs were loaded with S. aureus, CMV-pp65, SEB, or zymosanand cocultured with autologous memory CD41 T cells at a 1:4 MDDC/Tcell ratio (e.g., 0.25 � 106 MDDCs for 1 � 106 CD41 T cells) up to 18 d.In parallel, a classical VOA was performed using matched mCD41 T cells,as we recently reported (44). Briefly, memory CD41 T cells (1 � 106 cells)were activated with CD3/CD28 Abs (1 mg/ml) for 3 d, when cells werewashed with culture media. For both MDDC-based VOA and the classicalVOA, cells were split into two new wells, with media (RPMI 1640, 10%FBS, 1% penicillin/streptomycin) containing IL-2 (5 ng/ml; R&D Systems)being refreshed every 3 d until day 18 postculture, as previously reported(41). Each condition was performed in triplicate. Levels of HIV-p24 in thecell culture supernatant were quantified by a homemade ELISA, as we previ-ously reported (41).

Type I IFN quantification

To quantify type I IFN activity in cell culture supernatants, the HEK-BlueIFN-a/b indicator cells carrying a reporter gene for secreted embryonic alka-line phosphatase (SEAP) induced in response to type I IFN were used (In-vivoGen; gift from Dr. E. Cohen laboratory, Institut de Recherches Cliniquesde Montr�eal, Montr�eal, QC, Canada). The SEAP levels were measured usinga spectrophotometer. The cells were cultured in DMEM, 10% FBS contain-ing blasticidin (30 mg/ml), Normocin (100 mg/ml), and Zeocin (100 mg/ml).When the cells were at confluence, 5 � 105 cells (in 180 ml) were culturedin the presence of the cell culture supernatant to be tested (20 ml) in 96-wellplate wells at 37�. After 24 h, 20 ml of the cell culture supernatant was addedto 180 ml of QUANTI-Blue (InvivoGen) and incubated at 37� for up to 3 hin 96-well plate wells. In parallel, a standard curve was prepared using 15 se-rial dilutions of rIFN-b (500-0.015 ng/ml). The OD was quantified 620 nmon a spectrophotometer.

TNF-a quantification by ELISA

TNF-a levels in cell culture supernatants were quantified by ELISA, accord-ing to the manufacturer’s protocol (Human TNF-a DuoSet; R&D Systems).

Statistical analysis

Statistical analyses were performed using the GraphPad Prism 8 software(GraphPad Software). The Wilcoxon test was used to determine statisticalsignificance for differences between two matched groups. The Friedman testwith Dunn multiple comparisons were used to evaluate statistical significancebetween multiple matched groups. The Mann�Whitney U test was used todetermine statistical significance for differences between unmatched groups.The use of nonparametric tests is justified by the fact that data sets did notpass the normal distribution Kolmogorov�Smirnov test. Statistical tests withp values <0.05, a classic threshold of significance, were considered statisti-cally significant, whereas p values <0.1 were considered marginally signifi-cant. The statistical tests applied are specified in the figure legends.

2640 BACTERIAL/FUNGAL PATHOGENS FUEL HIV RESERVOIR PERSISTENCE

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ResultsCD161 versus CD16� MDDCs exhibit a higher capacity to transmitHIV infection to CD41 T cells proliferating in response to S. aureus

Studies by our group and others previously reported that the fre-quency of intermediate/nonclassical CD161 compared with classicalCD16� monocytes is increased in the blood of ART-treated PLWH,with CD161 monocytes differentiating into DCs with unique tran-scriptional profiles (23, 45). In this study, we investigated functionaldifferences between CD161 and CD16� MDDCs in terms of immu-nogenic potential and HIV trans-infection capacity. Highly pureCD16� and CD161 monocytes isolated by flow cytometry fromPBMCs of HIV� individuals were differentiated into MDDCsin vitro (data not shown), as we previously reported (23). MDDCsubsets were exposed or not to the CCR5-tropic replication-compe-tent NL4.3BaL HIV-1 strain and loaded or not with Ags. Precisely,SEB, CMV-pp65, and S. aureus lysates were used to induce poly-clonal, Th1, and Th17 proliferative CD41 T cell responses, respec-tively, consistent with previous reports (39, 46). Subsequently,MDCCs were cocultured with autologous CFSE-loaded CD41 Tcells for 5 d (Fig. 1A). In preliminary experiments, we demonstratedthat the exposure to the conjugated CD16 Abs used for flow cytom-etry sorting of CD161 and CD16� monocytes did not affect the im-munogenic potential of MDCCs (n = 5; data not shown) nor theirability to transmit HIV to autologous CD41 T cells proliferating inresponse to SEB, CMV-pp65, or S. aureus lysates (n = 4; data notshown).In a first set of experiments performed in the absence of HIV,

differences between CD161 and CD16� MDDCs in terms of abili-ty to induce the proliferation of CD41 T cells and their production

of Th1/Th17�lineage cytokines was tested. Results, shown inSupplemental Fig. 1A�C, demonstrate that SEB induced higherlevels of T cell proliferation in comparison with S. aureus lysate,whereas the frequency of CMV-specific T cells was the lowest.Noteworthy, the proliferation magnitude was higher in T cells co-cultured with CD161 compared with CD16� MDDCs in responseto SEB (p = 0.04) and S. aureus lysate (p = 0.04) but not CMV(Supplemental Fig. 1C). Among proliferating T cells, the highestlevels of IL-17A and IFN-g expression were observed in T cellsproliferating in response to S. aureus lysate (Th17 profile) andCMV-pp65 (Th1 profile), respectively, whereas TNF-a expressionwas similarly high in response to all three Ags used (SupplementalFig. 1D�F). In CD161 compared with CD16� MDDC:T cell co-cultures, IL-17A levels were lower in response to SEB(Supplemental Fig. 1D), IFN-g levels were lower in response toSEB and S. aureus (Supplemental Fig. 1E), and TNF-a levelswere lower and higher in response to SEB and CMV, respectively(Supplemental Fig. 1F). Of note, a similar Th17 profile was ob-served for T cells proliferating in response to C. albicans�loadedMDDCs (data not shown), consistent with previous reports (46).Together, these results reveal the presence of S. aureus�reactive Tcells at higher frequencies compared with CMV-specific T cells inHIV� individuals, with T cells proliferating in response to S. aure-us exhibiting a Th17 polarization profile in the presence of bothCD161 and CD16� MDDCs.Upon HIV exposure, the CD161 and CD16� MDDCs subsets

exhibited a similar permissiveness to integrative infection, as re-flected by comparable levels of integrated HIV-DNA detected atday 3 post-HIV exposure (n = 4; data not shown). Nevertheless,

CD16

-MDDC

CD16

+MDDC

HIV-p24

CFSE

BCMVpp65 SEBS. aureus

ACD16- MDDC

andCD16+ MDDC

Read outs:- Proliferation- HIV transmission

+ HIV-1

+ Antigens+ CFSE-loadedmCD4+ T cells

3h 5 days

C

FIGURE 1. CD161 compared with CD16� MDDCs exhib-it a superior capacity to transmit HIV infection to CD41 Tcells proliferating in response to S. aureus. (A) Shown is theexperimental flowchart. Briefly, MDDCs derived fromCD16� or CD161 monocytes isolated from HIV-uninfected(HIV�) individuals were exposed to the replication-competentNL4.3BaL HIV strain for 3 h. Unbounded virus was removedby extensive washing. MDDCs were further loaded with Ags(S. aureus, CMVpp65, SEB) and cocultured with autologousCFSE-loaded CD41 T cells (MDDC/T cell ratio of 1:4) for 5d. (B) Shown are representative flow cytometry plots of intra-cellular HIV-p24 expression and CFSE dilution on CD41 Tcells cocultured with CD16� or CD161 MDDCs. Surfacestaining using CD3 and CD1c Abs identified T cells(CD31CD1c� phenotype) in MDDC:T cell cocultures. (C)Shown is the frequency of HIV-p241 T cells proliferating(CFSElow) in response to S. aureus, CMV, and SEB after co-culture with CD161 or CD16� MDDCs. Results were gener-ated using matched CD161/CD16� MDDCs, as well asautologous CD41 T cells from n = 5 different donors. Pairedt test p values are indicated on the graphs.

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CD161 versus CD16� MDDC exhibited a superior capacity totransfer HIV infection to autologous CD41 T cells proliferating inresponse to S. aureus lysate, as reflected by their intracellular HIV-p24 protein expression (Fig. 1B�C). Noteworthy, the frequency ofHIV-p241 cells within the pool of T cells proliferating in responseto S. aureus exceeded 30% in the presence of CD161 MDDCs(Fig. 1C), indicative of an extremely high permissiveness of thesecells to HIV infection in vitro.

CD161 versus CD16� MDDC exhibit superior RALDH2 activity

We previously reported that CD161 MDDCs are distinguished fromCD16� MDDCs by their higher expression of RALDH2 mRNA(23). The activity of RALDH2, an enzyme metabolizing vitamin Ainto RA (28), is induced by the GM-CSF (47) used to differentiatemonocytes into DCs. Such differences between CD161 and CD16�

MDDCs may have tremendous consequences in the context of HIVinfection, consistent with our previous studies demonstrating thatRA increases HIV permissiveness in CD41 T cells (34, 48). To ex-plore the functional relevance of such differences, we used the AL-DEFLUOR assay to measure the constitutive ALDH activity inCD161 and CD16� MDDCs. The ALDH enzyme inhibitor DEABwas used as a negative control. Results, shown in Fig. 2, demon-strate that CD161 MDDCs exhibited a higher ALDH activity com-pared with their CD16� counterparts (Fig. 2A, 2B), suggesting thatCD161 MDDCs may preferentially facilitate HIV trans infection ofCD41 T cells via a RALDH2/RA�dependent mechanism.

The frequency of CD161 monocytes correlate with the ALDHactivity in total MDDCs

Considering the superior RALDH2 activity in CD161 versusCD16� MDDCs (Fig. 2), we hypothesized that the frequency ofCD161 monocytes predicts the ALDH activity in bulk MDDCs.Consistent with the documented expansion of CD161 monocytesduring ART-treated HIV infection (24, 26, 27), results generated inour cohort of ART-treated PLWH (HIV1ART1) and HIV� individ-uals confirm an increased frequency of CD161 monocytes duringHIV infection (Fig. 3A); this is especially the case for the

intermediate CD1411CD161 monocytes (Supplemental Fig. 2). Asexpected, in HIV� individuals, the ALDH activity in bulk MDDCswas negatively correlated with the frequency of CD16� monocytes(p = 0.0603, r = �0.6242; n = 11) and positively correlated with thefrequency of CD161 monocytes (p = 0.0280, r = 0.6697; n = 11)(Fig. 3B�D). Thus, the frequency of CD161 monocytes predicts themagnitude of constitutive ALDH activity in bulk MDDCs.

TLR2 and TLR4 triggering promotes ALDH activity in MDDCs

Previous studies in mice reported that ALDH activity in DCsis induced by zymosan, a component of the fungal walls, in aTLR2-dependent manner (35, 47). In addition to zymosan, S.aureus lysate can also trigger TLR2 activation via the PGN(49), a component of both Gram-positive/negative bacterialwalls. Thus, we hypothesized that the superior ability ofCD161 MDDC to trans infect CD41 T cells proliferating inresponse to S. aureus lysate was dependent on the TLR2-medi-ated induction of RALDH activity in MDDCs. To test this hy-pothesis, MDDCs from HIV� and HIV1ART1 individualswere stimulated with S. aureus lysate, zymosan, CMV-pp65,CMV lysate, and SEB. The ALDH inhibitor DEAB (50) wasused as negative control for the gating strategy (Fig. 4A). Theuse of CMV-pp65 and CMV lysate in parallel is justified bythe fact that peptides and whole pathogen lysates are not pro-cess similarly by DCs, with lysates, including pathogen-associ-ated molecular patterns (PAMPs), to be sensed by DCs viapathogen recognition receptors, such as TLRs (51). AlthoughS. aureus lysate and zymosan induced a statistically significantincrease in ALDH activity of MDDCs from HIV� individuals,CMV-pp65, CMV lysates, and SEB did not upregulated thisactivity (Fig. 4B). The ability of S. aureus lysate and zymosanto upregulate the ALDH activity in MDDCs was observed inMDDCs from both HIV� (Fig. 4C, 4D) and HIV1ART1 indi-viduals (Fig. 4E, 4F).Further, a TLR ligand functional screening was performed to

identify modulators of ALDH activity in MDDCs. In preliminaryexperiments, we tested the functionality of this TLR1�9 agonist kitby measuring the TNF-a and IFN-I levels on PBMC (SupplementalFig. 3A, 3B). Noteworthy, the ability of specific TLR ligands to in-duce TNF-a (e.g., FSL-1) and/or IFN-I (e.g., imiquimod) productionin PBMC (Supplemental Fig. 3A, 3B) did not coincide with theability of these ligands to induce ALDH activity in MDDCs(Supplemental Fig. 3C). The highest levels of ALDH activity inMDDCs was observed upon exposure to TLR2 (HKLM, zymosan)and TLR4 (LPS) ligands, whereas TLR1, 3, 5, 7, 8, and 9 did notsignificantly affect the ALDH activity (Supplemental Fig. 3C). Ex-periments performed in the presence of polymyxin B, an inhibitorof TLR4 triggering (52), confirmed the capacity of LPS to induceALDH activity in MDDCs via TLR4-dependent mechanisms (datanot shown). Given the documented capacity of S. aureus to triggerTLR2 pathway activation (53), these results support the idea that S.aureus lysate stimulates a TLR2-dependent ALDH activity inMDDCs, likely preferentially in CD161 MDDCs. Similar effectsare mediated by Gram bacteria rich in LPS.

MDDC ability to transmit HIV infection to S. aureus�reactive CD41

T cells is dependent on RALDH activity and RA signaling

To investigate the role of RA produced by RALDH21 MDDCs ontheir HIV trans-infection potential, two different inhibitors of theRA pathway were used: the ALDH enzyme inhibitor DEAB (50) orthe RA receptor antagonist LE540 (54). Briefly, MDDCs derivedfrom bulk monocytes of HIV individuals were exposed to theNL4.3BaL HIV strain, loaded with zymosan, S. aureus lysate, orSEB, and cocultured with autologous memory CD41 T cells in the

RALDH

SSC-A

Substrate+ DEAB

Substrateonly

A

B

CD16

-MDDCCD16

+MDDC

FIGURE 2. CD161 compared with CD16� MDDCs exhibit superiorALDH activity. The ALDH activity in CD161 and CD16� MDDCs ofHIV� individuals was measured using the ALDEFLUOR assay. Briefly,CD161 and CD16� monocytes were isolated by flow cytometry fromPBMCs of HIV-uninfected donors and differentiated into MDCCs. CD161

and CD16� MDDCs were incubated with the ALDH substrate in the pres-ence or the absence of the ALDH inhibitor DEAB. The ALDH activity(GFP1) was quantified by flow cytometry. (A) Shown are representativeflow cytometry dot plots of side scatter area (SSC-A) and GFP1 ALDHproduct in CD16� and CD161 MDCCs. (B) Shown are results generatedusing matched CD161 and CD16� MDDCs from n = 5 different donors.Paired test p values are indicated on the graphs. Bars represent medianvalues.

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presence or the absence of LE540 or DEAB (Fig. 5A�D). In the ab-sence of LE540 and DEAB, HIV replication levels were remarkablyincreased when T cells were cocultured with MDDCs loaded withzymosan (data not shown) and S. aureus lysates compared withSEB (Fig. 5A�D), and this occurred despite superior T cell prolifer-ation in response to SEB compared with S. aureus (SupplementalFig. 4A, 4B). LE540 and DEAB significantly reduced HIV

replication in both S. aureus� and SEB-loaded MDDC:T cell cocul-tures (Fig. 5C, 5D). In contrast, LE540 or DEAB did not affect HIVreplication in CD3/CD28�activated T cells cultured alone (Fig. 5E,5F), indicative that RA produced by RALDH21 MDDCs plays animportant role in the process of HIV trans infection. WhereasLE540 had no impact on T cell proliferation, DEAB significantlydecreased T cell proliferation in cocultures with MDDCs loadedwith S. aureus but not SEB (Supplemental Fig. 4A, 4B), a super Agthat does not require processing for Ag presentation. This points toa potential impact of DEAB on the capacity of MDDCs to capture,process, and/or present S. aureus, thus reducing the subsequent HIVreplication in T cells.Further, we observed that T cells proliferating in response to S.

aureus compared with SEB expressed higher levels of the HIV cor-eceptor CCR5 (Supplemental Fig. 4C, 4D), thus explaining in-creased HIV replication. In line with previous evidence generatedby our group that RA promotes integrin b7 and CCR5 expressionon T cells (31, 34, 48), both LE540 and DEAB significantly reducedthe expression of CCR5 (Supplemental Fig. 4C, 4D) and integrinb7 (Supplemental Fig. 4E, 4F) but not CCR6 (in which expressionis independent of the RA signaling) (Supplemental Fig. 4G, 4H),with LE540 having the highest capacity to decrease CCR5 and in-tegrin b7 expression (Supplemental Fig. 4C�F). These results sug-gest that T cells proliferating in response to bacterial/fungalpathogens able to promote ALDH activity in MDDCs are prone tosupport HIV replication, whereas the SEB-mediated polyclonal stim-ulation results in the proliferation of a variety of T cell clones withheterogeneous permissiveness to HIV infection.

S. aureus�reactive CD41 T cells carry integrated HIV-DNA inART-treated PLWH

The results obtained upon HIV infection in vitro (Figs. 1, 5) raisedthe possibility that HIV reservoirs may be established and persist inS. aureus�reactive CD41 T cells of ART-treated PLWH. In con-trast, CMV-specific T cells were reported to be relatively protectedagainst HIV infection via their autocrine production of CCR5-bind-ing chemokines (40, 55�57) and low CCR5 expression (31). To testthis possibility, MDDCs derived from monocytes of HIV1ART1

individuals were loaded with S. aureus lysate, CMV-pp65, or SEBand cultured with CFSE-loaded autologous memory CD41 T cells.Cocultures were performed in the presence of antiretrovirals(ARVs) to prevent HIV cell-to-cell spreading in culture. After 5 d,proliferating CFSElow T cells (identified as CD1c�CD31) weresorted by FACS (Fig. 6A). In preliminary experiments, we investi-gated the frequency of T cells proliferating in response to S. aureuslysate, CMV-pp65, and SEB and observed no significant differencesbetween the HIV� (n = 7) and HIV1ART1 (n = 7) individualsstudied (data not shown). As expected, a higher frequency of T cellsproliferated in response to SEB compared with the fraction of Tcells proliferating in response to S. aureus lysate or CMV-pp65(Fig. 6B). T cells proliferating in response to S. aureus lysate,CMV-pp65, and SEB (CFSElow) sorted by flow cytometry from n =8 HIV1ART1 individuals were assessed for HIV-DNA integrationby nested real-time PCR. The sample size exceeded 5000 cells perPCR test for the majority of samples, except for two samples (Fig.6C) indicated in red (Fig. 6D). In two of the eight HIV1ART1 indi-viduals, integrated HIV-DNA levels were undetectable in all anti-genic specificities (triangle and diamond symbols), whereas >100HIV-DNA copies per 1 � 106 cells (>0.01% cells carrying integrat-ed HIV-DNA) were detected in positive samples from six of theeight HIV1ART1 individuals (Fig. 6D). Proviral DNA was detectedin T cells proliferating in response to S. aureus, CMV, and SEB infive of eight, two of eight, and four of eight individuals, respectively(Fig. 6D). These results demonstrate that T cells proliferating in

B

C

D

ALDH

SSC-A

Substrate+DEAB

Substrateonly

A

TotalMDDCs

FIGURE 3. The frequency of CD161 monocytes correlates with the ALDHactivity in bulk MDDCs in HIV-uninfected individuals. Total monocytes werepurified from the PBMCs of HIV� and ART-treated PLWH (HIV1ART1) bynegative selection using magnetic beads. (A) Shown is the frequency of classi-cal CD16� and intermediate/nonclassical CD161 monocytes in HIV� andHIV1ART1 individuals (subjects 1�13; subjects 15�19; Table I). (B) Shownare representative flow cytometry dot plots depicting the ALDH activity in thepresence or the absence of the ALDH inhibitor DEAB in total MDDCs ofHIV� individuals (n = 11) (subjects 1�11; Table I). (C�D) Shown are correla-tion between the frequency of classical CD16� or intermediate/nonclassicalCD161 monocytes and the ALDH activity in total MDDCs of HIV� individu-als. Spearman correlation p and r values are indicated on each graph.

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response to S. aureus harbor integrated HIV-DNA at levels/frequen-cies higher compared with CMV-specific CD41 T cells but similarto those in SEB-specific T cells.

S. aureus� and zymosan-reactive CD41 T cells carry replication-competent HIV reservoirs

To further investigate the replicative competence of HIV-DNA inte-grated in specific CD41 T cell subsets of ART-treated PLWH, weused an MDDC-based VOA, previously described by our group(41). Briefly, MDDCs derived from monocytes of HIV1ART1 indi-viduals were loaded or not with S. aureus lysate, CMV-pp65, SEB,or zymosan and cocultured with autologous memory CD41 T cells.In parallel, memory T cells activated with CD3/CD28 Abs wereused as positive controls, using a simplified VOA we recently de-scribed (44), to promote HIV outgrowth in all T cells regardless oftheir antigenic specificity. Although in all four HIV1ART1 individ-uals tested, HIV outgrowth was observed at relatively high levels inCD3/CD28�activated CD41 T cells, there was a large donor-to-do-nor variability in terms of HIV outgrowth in response to differentAgs (Fig. 7A�D). For HIV1ART1 subject 1, viral outgrowth wasdetected in T cells proliferating in response to all Ags but also inthe absence of Ag stimulation (Fig. 7A), indicative of a relatively largeHIV reservoir size in this individual. For HIV1ART1 subject 2, viraloutgrowth was detected in response to S. aureus, SEB, and zymosanbut not CMV (Fig. 7B). Consistently, for this individual, integratedHIV-DNA levels ex vivo were high in S. aureus but undetectable inCMV-specific T cells (Fig. 6D, square symbols). For HIV1ART1

subject 3, viral outgrowth was detectable at very low levels upon SEBand CMV stimulation starting only at day 15 in culture, whereas viraloutgrowth was undetectable in response to S. aureus (Fig. 7C). Theseresults are also in line with the relatively high integrated HIV-DNAlevels in SEB- and CMV-specific T cells compared with T cells prolif-erating in response to S. aureus lysate (Fig. 6D, circle symbols). Final-ly, for HIV1ART1 subject 4, viral outgrowth was detected at verylow levels, only at day 18 in culture in response to S. aureus andCMV but not zymosan nor SEB (Table I) (Fig. 7D).Because zymosan was the stronger inducer of ALDH activity in

MDDCs (Fig. 4, Supplemental Fig. 3C) and facilitated HIV transinfection in vitro as efficiently as S. aureus lysate (data not shown),in parallel, the MDDC-based VOA was performed in the presenceor the absence of the RA recptor a (RARa) antagonist LE540. Theviral outgrowth observed in the presence of zymosan-loadedMDDCs for HIV1ART1 subject 1 and 2 was strongly inhibited byLE540 (Fig. 7E�H), indicative that viral outgrowth is dependent onthe RA pathway. Together, these results reveal the presence of repli-cation-competent HIV reservoirs in CD41 T cells proliferating in re-sponse to S. aureus and zymosan of certain HIV1ART1 individuals

A

RALDH

SSC-A

Zymosan

No Ag

S. aureus

CMV-pp65

Substrateonly

Substrate

+DEAB

C D

B

No Ag

E F

FIGURE 4. S. aureus and zymosan promote ALDH activity in MDDCsof HIV� and HIV1ART1 individuals. MDDCs differentiated from totalmonocytes of HIV� and HIV1ART1 individuals were stimulated with S.aureus lysate (5 � 106 cells per assay), the TLR2 agonist zymosan(10 mg/ml), CMV-pp65 (1 mg/ml), CMV lysate (1 mg/ml), or SEB

(25 ng/ml) for 48 h. The ALDH activity was measured by flow cytometryusing the ALDEFLUOR assay. (A) Shown are flow cytometry dot plots de-picting the side scatter area (SSC-A) and ALDH activity in total MDDCsof one representative HIV� individual upon exposure to S. aureus, zymo-san, and CMV-pp65 in the presence (negative control, upper dot plot) or inthe absence of the ALDH inhibitor DEAB (middle and bottom dot plots).(B) Shown is statistical analysis of ALDH activity in total MDDCs uponexposure to S. aureus lysate, zymosan, CMV-pp65, CMV lysate, or SEB.Bars represent the mean ± SD values. Friedman matched-pairs test p valuesand Dunn multiple comparisons significance are indicated on the graphs.(C�F) Shown is the ALDH activity in total MDDCs of HIV� (C and D)and HIV1ART1 individuals (n = 8�11, subjects 1�4, subjects 6�12, sub-ject 14, subjects 18�20; Table I) (E and F) upon MDDCs exposure to S. au-reus lysate (C and E) or zymosan (D and F). Bars represent the mean ± SDvalues. Wilcoxon matched-pairs test p values are indicated on the graphs.

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and point to PAMPs able to trigger TLR2 signaling as potent in-ducers of ALDH activity in MDDCs and RA production and subse-quent RA-dependent HIV outgrowth in CD41 T cells (Fig. 8).

DiscussionIn this study, we report that 1) intermediate/nonclassical CD161 com-pared with classical CD16� monocytes are precursors for DCs, witha higher capacity to transmit HIV infection to S. aureus�reactiveCD41 T cells, consistent with their superior RALDH activity; 2) S.aureus lysates and other TLR2 (zymosan, HKLM) and TLR4 (LPS)agonists upregulate RALDH activity in bulk MDDCs; 3) S. aureus�and zymosan-loaded MDDCs transmit HIV infection to CD41 T cellsvia RALDH/RA�dependent mechanisms; 4) integrated HIV-DNAreservoirs persist in S. aureus� and zymosan-reactive CD41 T cellsof ART-treated PLWH; and 5) viral outgrowth is promoted by zymo-san-loaded MDDCs in a RALDH/RA�dependent manner. Together,these results identify the RALDH activity as a new pathogenic featureof DC derived from CD161 monocytes, a proinflammatory subsetcontributing to HIV pathogenesis (27, 38, 58�63), with abnormalitiespersisting despite virally suppressive ART (26, 27, 38). These resultsalso reveal that specific bacterial/fungal components of the microbiotacontribute to HIV reservoir persistence during ART in CD41 T cells

specific/reactive to such components in a RALDH-dependent manner(Fig. 8).Pioneering studies demonstrated that HIV-specific CD41 T

cells are highly permissive to HIV infection, thus explaining theirpreferential depletion and altered anti-HIV responses in PLWH(12, 40). Similarly, CD41 T cells specific/reactive to other patho-gens, such as Mycobacterium tuberculosis and C. albicans, werealso reported to have an increased permissiveness to HIV infec-tion, with their HIV-mediated depletion explaining opportunisticinfections caused by these pathogens (12, 56, 57, 64). In contrast,CMV-specific CD41 T cells are relatively resistant to HIV infec-tion because of their capacity to secrete MIP-1b, a CCR5 ligandblocking CCR5-mediated HIV entry; consequently, CMV-specificCD41 T cell responses are preserved until CD41 T cell countsdrop below 100 cells/ml of blood in PLWH (40, 55). In this con-text, studies of the antigenic specificity of CD41 T cells carryingHIV reservoirs in ART-treated PLWH represent a research topicof increased interest (40, 65, 66). This knowledge is important toidentify Ags/pathogens able to promote the clonal expansion ofmemory CD41 T cells carrying HIV reservoirs, one key mecha-nism by which HIV is considered to persist during ART (3, 6, 7).The Ag-dependent clonal expansion of memory CD41 T cells isdriven by their interaction with DCs in the context of the Ag-

A B

C D

E F

FIGURE 5. MDDC ability to transmit HIVinfection to S. aureus�specific/reactive CD41

T cells is dependent on RA signaling andRALDH activity. Total MDDCs exposed toreplication-competent NL4.3BaL HIV-1 strainwere loaded with Ags (S. aureus, SEB) and co-cultured with autologous CD41 T cells(MDDC/T cell ratio 1:4) for 12 d in the pres-ence or the absence of the RA receptor antago-nist LE540 or the ALDH inhibitor DEAB. (Aand B) Shown are results from one representa-tive donor depicting HIV-p24 levels quantifiedby ELISA in cell culture supernatants at days3, 6, 9, and 12 postinfection in cocultures per-formed with MDDCs in the absence of Ags(A) or MDDC loaded with S. aureus lysate (B).Shown are HIV-p24 levels in cell culturesupernatants at day 6 postinfection in cocul-tures performed with MDDCs loaded or not inAgs (S. aureus lysate, SEB) in the presence orthe absence of LE540 (C) or DEAB (D). Barsrepresent the mean ± SD values. Wilcoxonmatched-pairs test significance is indicated onthe graphs. (E and F) Memory CD41 T cellswere sorted from PBMC of HIV� individualsby negative selection using magnetic beads, ac-tivated using CD3/CD28 Abs, and exposed toNL4.3BaL HIV-1 strain in the presence or theabsence of LE540 and DEAB. The HIV-p24levels were measured by ELISA in cell culturesupernatants collected every 3 d. Shown arerepresentative HIV replication results generatedwith T cells from one donor, cultivated in thepresence or the absence of LE540 or DEAB(E), and results on HIV-p24 quantification atday 9 post infection in n = 3 donors (F). Barsrepresent the mean ± SD. Friedman test wasused to determine the significance between ex-perimental conditions. **p # 0.01, ***p #

0.001.

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presentation process (8). Results included in this manuscript dem-onstrate that CD161 MDDCs transmit HIV infection to S. aur-eus�specific CD41 T cells with higher efficacy compared withtheir CD16� MDDC counterparts. These results are not explainedby differences between CD161 and CD16� MDDCs in terms ofpermissiveness to integrative HIV infection, in the expression ofDC-SIGN (23), nor by differences in the Th17 polarization ofCD41 T cells cocultured with S. aureus�loaded CD161 versusCD16� MDDCs. This justified our search for new mechanismsmediating functional differences between CD161 and CD16�

MDDCs, mechanisms likely linked to their activation by S. aur-eus�specific PAMPs.It is documented that components from the S. aureus walls, such

as PGN, trigger DC activation in a TLR2-dependent manner (49)and that TLR2 signaling leads to RALDH activity in DCs (35). Wepreviously reported that CD161 compared with CD16� MDDCs ex-hibit increased RALDH2 mRNA expression (23). In this manu-script, we further demonstrate that CD161 compared with CD16�

monocytes differentiate into DCs with increased RALDH activityupon culture for 6 d in the presence of IL-4 and GM-CSF, a knowninducer of RALDH activity (47). Consistently, the frequency ofCD161 monocytes predicted the magnitude of RALDH activity inDCs derived from bulk monocytes. In addition to S. aureus lysates,we demonstrate that other stimuli, such as the TLR2 ligands zymo-san and HKLM, as well as the TLR4 ligand LPS, markedly in-creased RALDH activity in MDDCs. These results point to the roleplayed by bacterial/fungal pathogens, able to induce RALDH

activity in MDDCs via TLR2/TLR4�dependent mechanisms, incontrolling the RA production at the mucosal level, with implica-tions for both mucosal immune homeostasis and HIV pathogenesis.This later possibility is in line with recent findings that pre-ART vi-tamin A deficiency is associated with a superior CD4 count recoveryupon 96 weeks of ART (67), likely by reducing HIV permissivenessand the subsequent HIV-mediated depletion of CD41 T cells. Thus,HIV exploits the RALDH/RA pathway, which is an important path-way involved in the development of the immune system, the mainte-nance of mucosal homeostasis and immune tolerance, as well asinflammatory processes (32, 47, 68�70). These findings emphasizenew challenges to HIV cure efforts.The RALDH/RA pathway plays a central role in the maintenance

of gut tolerance to commensal pathogens by promoting Treg differ-entiation to the detriment of proinflammatory Th17 cells (30, 33,35). However, a recent study demonstrated that an environment oflow RA levels favor Th17 polarization, whereas high RA levels pro-mote Treg/Th1 differentiation (71). Our results demonstrate that Tcells cocultured with S. aureus�loaded MDDCs produced high lev-els of IL-17A, indicative of Th17 polarization, as previously docu-mented by other groups (39). Moreover, we demonstrated that highRALDH activity levels in MDDCs, induced upon exposure to S. au-reus and C. albicans lysates as well as zymosan, were associatedwith efficient transmission of HIV from MDDCs to autologousCD41 T cells. Furthermore, we demonstrated that HIV transmissionfrom MDDCs to CD41 T cells was significantly reduced in thepresence of the RALDH inhibitor DEAB and the RARa antagonist

CFSE

SSC-A

S. aureus SEBCMV

BA

C D

FIGURE 6. S. aureus�reactive CD41 T cells carry integrated HIV-DNA in ART-treated PLWH. MDDCs derived from total monocytes of HIV1ART1 in-dividuals (n = 8; subjects 1�5; subjects 18�20; Table I) were loaded with Ags (S. aureus, CMV-pp65, or SEB) and cocultured with autologous CFSE-loadedCD41 T cells (MDDC/T cell ratio 1:4) for 5 d in the presence of ARV drugs (raltegravir, 0.2 mM; saquinavir, 5 mM) to prevent cell-to-cell transmission ofHIV in culture. CD41 T cells proliferating (CFSElow) in response to Ags were sorted by flow cytometry, and integrated HIV-DNA levels were quantified bynested real-time PCR. Shown are representative flow cytometry dot plots for side scatter area (SSC-A) and CFSE expression used to sort proliferating(CFSElow) CD41 T cells (A); the frequency of CFSElow T cells in cocultures performed with MDDCs loaded with S. aureus lysate, CMV-pp65, or SEB(n = 8; subjects 1�5; subjects 18�20; Table I) (B); and levels of integrated HIV-DNA in CFSElow T cells in cocultures performed with MDDC loaded with S.aureus, CMV, or SEB (n = 6; HIV1ART subject 1, subjects 3�5, subject 18, subject 20; Table I) (C�D). The limit of detection of the assay is threeHIV-DNA copies per test. Red symbols correspond to sample with <5000 cells (n = 3).

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A

B

E

F

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D H

FIGURE 7. HIV outgrowth in CD41 T cells specific/reactive to S. aureus and/or zymosan of ART-treated PLWH. An MDDC-based VOA was used to re-activate HIV reservoirs in CD41 T cells of HIV1ART1 individuals with high (n = 2; HIV1ART1 #3 and #18; Table I) and undetectable/low (n = 2;HIV1ART1 #1 and #19; Table I) HIV-DNA reservoirs, as quantified in Fig. 6. MDDCs were loaded with Ags (S. aureus lysate, CMV-pp65, SEB, and zy-mosan) and cocultured with autologous memory CD41 T cells for up to 18 d at an MDDC/T cell ratio of 1:4 (0.25 � 106 MDDCs and 1 � 106 T cells perwell). In parallel, a VOA was performed with CD3/CD28�activated memory CD41 T cells and used as positive controls for viral outgrowth (1 � 106 T cellsper well). Media was harvested every 3 d for HIV-p24 quantification by ELISA, and fresh media was added. Cells (Figure legend continues)

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LE540. These results are consistent with previous findings by ourgroup that RA renders CCR61 Th17 cells highly permissive to HIVreplication via mechanisms involving, in part, the activation ofmTOR (34), a signaling pathway that plays an important role inHIV transcription/replication (43, 72). Our previous transcriptionalprofiling demonstrated similar levels of RARa mRNA expression inCD161 and CD16� MDDC (23) and CCR61 and CCR6� CD41 Tcell subsets (34). Thus, functional differences in HIV trans infectionare, rather, explained by an increased RALDH activity in CD161

versus CD16� MDDC. It is noteworthy that RA-treated CCR61

compared with CCR6� T cells express lower levels of IFN-inducedgenes (e.g., IRF8, IFITM1, IFITM2, IFITM3) (34). WhetherRALDH1CD161 MDDCs dampen the antiviral effects of IFN andIFN-induced genes in T cells via the production of RA will be animportant aspect to explore in future studies.By using an MDDC-based CFSE dilution assay we demon-

strated that S. aureus�reactive CD41 T cells carry integratedHIV-DNA. Alternative methods for the identification of Ag-

specific/reactive T cells in view of HIV reservoir studies includetheir phenotypic identification upon activation using, for example,staining with CD40L/CD154 Abs (31) or the Ag-induced mole-cules assay (66). Nevertheless, the CFSE-based assay performedin the presence of ARVs allowed an efficient clonal expansion ofAg-specific/reactive CD41 T cells while preventing HIV cell-to-cell transmission in vitro. The fact that integrated HIV-DNA isdetected in T cells that proliferate in response to specific Ags isindicative that such HIV reservoir cells may also persist in vivoin ART-treated PLWH. Proliferating T cells sorted this way arehighly likely to be polyclonal and functionally heterogeneous,with only a fraction of them carrying HIV reservoirs. Therefore,single-cell transcriptional profiling is further needed to revealmechanisms by which Ag-specific/reactive T cells carrying HIVreservoirs survive in ART-treated PLWH.Finally, another important finding of our study is that HIV out-

growth from CD41 T cells of ART-treated PLWH was induced byS. aureus lysate and zymosan. Consistent with differences in the

Table I. Clinical information for ART-treated PLWH study participants

Participant ID Age (y) Sex CD4a CD8a CD4/CD8 Ratios PVLb Time of Infection (mo) ART Time on ART (mo)

HIV1ART1 subject 1 47 M 569 462 1.23 <40 119 Ritonvavir3TC

DarunavirRaltegravir

101

HIV1ART1 subject 2 30 F 833 445 1.87 <40 216 ViraceptTruvada

192

HIV1ART1 subject 3 51 M 841 1322 0.64 <40 150 SustivaTruvada

149

HIV1ART1 subject 4 49 M 458 899 0.51 <40 227 TruvadaViramune

203

HIV1ART1 subject 5 47 M 425 1156 0.37 <40 182 Atripla 62HIV1ART1 subject 6 62 M 847 944 0.90 <40 189 Kivexa

Reyataz165

HIV1ART1 subject 7 31 M 824 900 0.92 <40 57 Atripla 47HIV1ART1 subject 8 21 M 796 399 1.99 <40 8 Stribild 4HIV1ART1 subject 9 23 M 277 909 0.30 <40 96 Complera 8HIV1ART1 subject 10 30 M 598 605 0.99 <40 68 Stribild 64HIV1ART1 subject 11 54 M 886 579 1.53 <40 60 Truvada

Isentress53

HIV1ART1 subject 12 46 M 391 620 0.63 <40 165 KivexaDelavirdine

160

HIV1ART1 subject 13 54 M 617 1272 <40 156 KivexaEfavirenz

150

HIV1ART1 subject 14 47 M 269 282 0.95 <40 109 SustivaTruvada

109

HIV1ART1 subject 15 47 M 581 1060 0.55 <40 96 Isentress/Kivexa 96HIV1ART1 subject 16 62 M 498 531 0.94 <40 213 Raltegravir

IntelenceRitonavir

134

HIV1ART1 subject 17 24 M 776 478 1.62 <40 288 Complera 140HIV1ART1 subject 18 45 M 318 431 0.74 <40 149 Delaviridine

Kivexa148

HIV1ART1 subject 19 29 F 433 240 1.8 <40 184 ViraceptTruvada

160

HIV1ART1 subject 20 55 M 963 644 1.49 <40 124 PrevistaKivexaNorvir

108

aCells/ml.bHIV-RNA copies/ml.F, female; ID, identity designed; M, male; PVL, plasma viral load.

were split into two new wells every 3�6 d, when cell proliferation exceeded optimal cell density (> 2 � 106 cells per well). Thus, each original VOA wellgenerated 8�16 wells at day 18. (A�D) Shown are results from n = 4 HIV1ART individuals one by one (HIV1ART1 subject 1, subject 3, subject 18, subject19; Table I). (E�H) Shown are the effects of LE540 on HIV outgrowth in cocultures performed with MDDC-loaded in zymosan, with HIV-p24 quantificationperformed at day 15 postreactivation. Each VOA was performed in triplicate, and each symbol represents one original culture well. Bars represent themean ± SD values.

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history of infection in the human population, we observed donor-specific viral outgrowth profiles in response to Ags. Importantly,HIV outgrowth induced by zymosan was decreased when the RApathway was blocked, showing an important role played by theRALDH activity in HIV outgrowth. By inducing RALDH activityin MDDCs, such bacterial/fungal pathogens likely fuel residual HIVtranscription in ART-treated PLWH. Of particular notice, the RAderivative acitretin, a drug approved by the U.S. Food and Drug Ad-ministration, was demonstrated to facilitate HIV transcription andthe subsequent viral reservoir purging in ART-treated PLWH via anRA-induced gene (RIG-I)�dependent mechanism (73). Future stud-ies are needed to explore the potential links between intestinal dys-biosis, microbial translocation, RALDH activity in mucosal DCs,mucosal/plasma RA levels, and residual HIV transcription in CD41

T cells, a process observed in PLWH, despite adherence to ART(74�76). Given our previous findings that mTOR1CCR61 CD41 Tcells are located in the colon (34, 77) and carry HIV reservoirs in

ART-treated PLWH (78), future studies are needed to determinewhether such colon-infiltrating T cells are specific/reactive to bacteri-al/fungal pathogens and persist in the proximity of RALDH1 DCs.In conclusion, our results support a model in which the interplay

between DCs and CD41 T cells is modulated by specific compo-nents of the microbiota able to trigger RALDH activity in a TLR2/TLR4�dependent manner. This RALDH activity in specific DC sub-sets subsequently leads to RA production that will transcriptionallyreprogram CD41 T cells for increased permissiveness to HIV infec-tion and ability to support viral outgrowth (Fig. 8). Futures studiesare needed to explore the possibility to target the RALDH/RA path-way for HIV cure/remission interventions.

AcknowledgmentsWe thank Dr. Dominique Gauchat and Philippe St Onge (Flow CytometryCore Facility, CHUM Research Center, Montreal, QC, Canada) for experttechnical support with polychromatic flow cytometry sorting; Olfa Debbeche(Biosafety Level 3 Core Facility CHUM Research Center, Montreal, QC,Canada) and Mario Legault (FRQ-S/AIDS and Infectious Diseases Network,Montreal, QC, Canada) for help with ethical approvals and informed con-sents; Jos�ee Girouard and Angie Massicotte (McGill University Health Cen-tre, Montreal, QC, Canada) for a key contribution to blood collection fromHIV-infected study participants and clinical information from HIV-infectedand uninfected donors; and Nathalie Brassard, Elsa Brunet-Ratnasingham,and Dr. Daniel Kaufmann for providing expertise with CMV lysate function-al titration. Finally, the authors acknowledge the key contribution of all studyparticipants for their precious gift of leukapheresis, essential for this study.

DisclosuresE.A.C. is a member of the Scientific Advisory Board of Theratechnologies.N.C. received research funding from EMD Serono and served on AdvisoryBoards for Gilead Sciences. J.-P.R. performed contract research and/orserved on advisory boards for Gilead Sciences Canada, Merck Canada, Abb-vie, ViiV Healthcare, Bristol Myers Squibb, Janssen, Argos Pharmaceuticalsfrom InnaVirVax, and Theravectys. P.A.’s laboratory receives research fund-ing from GlaxoSmithKlein/NeoMed for projects not related to the presentstudy. P.A. served as a consultant at Merck Canada, relative to research proj-ects different from the present study. The other authors have no financialconflicts of interest.

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Supplemental Figure 1: The cytokine profiles of CD4+ T-cells co-cultured with Ags-loadedCD16+/CD16- MDDCs. (A) Shown is the experimental flow chart. Briefly, MDDCs derived from CD16-

or CD16+ monocytes of HIV- donors were loaded with antigens (SEB, CMV-pp65, S. aureus) andimmediately co-cultured with autologous CFSE-loaded CD4+ T-cells (MDDC:T-cell ratio of 1:4) for 5days. Cytokine expression was measured by intracellular staining with IL-17A, IFN- and TNF- Absafter PMA/Ionomycin stimulation. Surface staining with CD3 and CD1c Abs identified T-cells(CD3+CD1c-) in MDDC:T-cell co-cultures. (B) Shown are representative dot plots of the cytokines andCFSE expression in CD4+ T-cells co-cultured with CD16- or CD16+ MDDCs, as well as the statisticalanalysis of (C) T-cell proliferation and the intracellular expression of (D) IL-17A, (E) IFN- and (F)TNF- in T-cells proliferating in response antigens. Results were generated using matched CD16+ andCD16- MDDC, as well as autologous CD4+ T cells, from n=4-7 different donors. Paired t-test valuesare indicated on the graphs.

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Supplemental Figure 2: Frequency of monocyte subsets in HIV- and HIV+ART+ individuals.Monocytes were from the PBMCs of HIV negative (HIV-) or HIV-infected on ART individuals(HIV+ART+) by negative selection using magnetic beads. Shown is the gating strategy for theidentification of classical (CD14+CD16-), intermediate (CD14+CD16+), and non-classical (CD14-CD16+) monocytes within total monocytes (A) and the frequency of classical (B), intermediate (C)and non-classical (D) monocytes in HIV- (n=13) as well as in HIV+ART+ individuals (n=18). Mann-Whitney test p-values are indicated on each graph. Bars represent mean±SD.

B

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Supplemental Figure 3: TLR2 and TLR4 triggering up-regulates ALDH activity in total MDDCs. (A-B)PBMC from HIV- individuals were stimulated with TLR agonists (Pam3CSK4, HKLM, Poly(I:C), LPS, FLA-ST, FSL-1, Imiauimod, ssRNA, ODN2006 and zymosan) for 48 hours. To determine the functional potency ofthe TLR agonists, TNF- production was quantified by ELISA (A), while the type I IFN production werequantified by ELISA and QUANTI-BlueTM (B), in the cell-culture supernatants. Finally, (C) MDDCs generatedfrom total monocyte of HIV- individuals were stimulated with the listed TLR agonists for 48 hours and theALDH activity was quantified using the ALDEFLUOR Assay. Bars represent the mean±SD values.

A C

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Supplemental Figure 4: + T-cells proliferating in response to S. aureus. Total MDDCs generated from monocytes of HIV- individuals were loaded with Ags (S. aureus, SEB) and co-cultured with autologous CFSE-loaded CD4+ T-cells (MDDC:T-cell ratio 1:4) for 5 days in the presence or the absence of LE540 or DEAB. Shown is the proliferation of CD4+ T-cells (CFSElow) (A-B), as well as the expression of CCR5 (C-D) (E-F), and CCR6 (G-H) onproliferating CD4+ T-cells from co-cultures performed in the presence of the absence of LE540 (A, C, E, and G) and DEAB (B, D, F, and H). Bars represent the mean±SD values (n=6-8). Wilcoxon test p-values are indicated on the graphs.