Dendritic cell type-specific HIV-1 activation in effector T cells: implications for latent HIV-1 reservoir establishment Rene´e M. van der Sluisa, Toni M.M. van Capelb, Dave Speijerc, Rogier W. Sandersa,d, Ben Berkhouta, Esther C. de Jongb, Rienk E. Jeeningaa and Thijs van Montforta Background: Latent HIV type I (HIV-1) infections can frequently occur in short-lived proliferating effector T lymphocytes. These latently infected cells could revert into resting T lymphocytes and thereby contribute to the establishment of the long-lived viral reservoir. Monocyte-derived dendritic cells can revert latency in effector T cells in vitro. Methods: Here we investigated the latency activation properties of tissue-specific immune cells, including a large panel of dendritic cell subsets, to explore in which body compartments effector T cells are most likely to maintain latent HIV-1 provirus and thus potentially contribute to the long-lived reservoir. Results: Our results demonstrate that blood or genital tract dendritic cells do not activate latent provirus in effector T cells, whereas gut or lymphoid dendritic cells induce virus production from latently infected effector T cells in our in-vitro model for latency. Toll-like receptor 3-induced interferon production by myeloid dendritic cells abolished the dendritic cells’ ability to induce viral gene expression. Conclusions: In this study, we show that HIV-1 provirus residing in effector T cells is activated from latency by tissue-specific dendritic cell subsets and other immune cells with remarkably different efficiencies. Our new assay system points to an important, neglected aspect of HIV-1 research: the ability of other immune cells, especially dendritic cells, to differentially affect latency establishment as well as virus reactivation. Copyright ß 2015 Wolters Kluwer Health, Inc. All rights reserved.

AIDS 2015, 29:1003–1014 Keywords: dendritic cell disruption of HIV-1 latency, dendritic cell subsetspecific purging, dendritic cell–T-cell interactions, HIV-1, HIV-1 latency establishment, reversion of HIV-1 latency

Introduction Viral latency is a barrier towards eradicating HIV-1 in infected individuals and attempts to purge these reservoirs

remain challenging [1–4]. Recent evidence suggests reservoirs to be larger than previously anticipated [5]. Cells with latent proviruses are established early in infection, forming lifelong sources of proviral DNA

a Laboratory of Experimental Virology, Department of Medical Microbiology, Centre for Infection and Immunity Amsterdam (CINIMA), bDepartment of Cell Biology and Histology, Center for Immunology Amsterdam (CIA), cDepartment of Medical Biochemistry, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands, and dDepartment of Microbiology and Immunology, Weill Medical College of Cornell University, New York, USA. Correspondence to Thijs van Montfort, Laboratory of Experimental Virology, Department of Medical Microbiology, Centre for Infection and Immunity Amsterdam (CINIMA), Academic Medical Centre, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, the Netherlands. Tel: +31 20 56 68571; fax: +31 20 69 16 531; e-mail: [email protected] Received: 9 October 2014; revised: 20 February 2015; accepted: 24 February 2015.

DOI:10.1097/QAD.0000000000000637

ISSN 0269-9370 Copyright Q 2015 Wolters Kluwer Health, Inc. All rights reserved.

Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.

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[6–9]. After years of successful antiretroviral therapy, with viral loads at undetectable levels, HIV-1 can rebound from latently infected cellular reservoirs and re-establish systemic infection upon therapy interruption [10–15]. The long-lived reservoir consists primarily of latently infected resting memory T lymphocytes [16]. Infection of these cells, however, is inefficient due to incomplete HIV-1 reverse transcription and reduced integration efficiency [17,18]. Latent infections occur frequently in short-lived effector T lymphocytes [19,20]. These cells might escape cytotoxic killing (no viral proteins are expressed) and undergo transition into a resting state, thus eventually contributing to the long-lived latent viral reservoir [21,22]. HIV-1 latency in proliferating effector T cells differs from latency in resting T cells. Drugs such as histone deacetylase (HDAC) inhibitors, protein kinase C agonists, T-cell receptor, and nuclear factor-kB (NF-kB) activators that activate HIV-1 from latency in resting memory T-cell lines [19], do ‘not’ work in HIV-1infected effector cells. Of note, using these drugs on latently infected resting cells from HIV-1 patients also did not show significant evidence of antilatency activity ex vivo, with T-cell activators as an exception [23,24]. Monocyte-derived dendritic cells (moDCs) can activate latent virus in effector T lymphocytes; for every virusproducing cell, two to three latently infected cells initiated virus production after 24 h co-culture with moDCs [19]. Viral latency in effector cells was detected up to 9 days postinfection, demonstrating latency in effector cells can be maintained. More importantly, moDCs could induce virus production from these cells after 9 days. Here we studied the capacity of primary dendritic cells and other immune cells to activate provirus in effector T lymphocytes to investigate the likelihood of viral reservoirs being established in various tissues. Immature myeloid dendritic cells obtained from blood and representative gut dendritic cells efficiently activated latent virus, thus potentially reducing reservoir formation. But skin-derived dendritic cells as well as other antigen-presenting cell types, like monocytes, B cells and different macrophage subsets, did not induce activation of the latent virus. Treatment of myeloid or gut dendritic cells with different Toll-like receptor (TLR) ligands, to mimic matured dendritic cells found in lymphoid tissue or Peyer’s patches, yielded diverse antilatency profiles. Lipopolysachariden (LPS)-stimulated dendritic cells efficiently activated HIV-1 from latency, whereas poly(I:C)-stimulated dendritic cells did not revert latency. This suggests that dendritic cell tissue-specificity and dendritic cell activation, which can be influenced by co-infection with other pathogens, may dictate where viral reservoirs in T cells can be established.

Methods Cells Human embryonic kidney (HEK) 293T cells were grown as monolayers in Dulbecco’s minimal essential medium (Gibco, BRL, Gaithersburg, Maryland, USA) supplemented with 10% (v/v) fetal calf serum (FCS), 40 U/ml penicillin, 40 mg/ml streptomycin, and nonessential amino acids (Gibco, BRL) at 378C and 5% CO2. Human peripheral blood mononuclear cells (PBMCs), isolated from buffy coats (Central Laboratory Blood Bank, Amsterdam, the Netherlands) using Ficoll gradients, were frozen in multiple vials. PBMCs were activated with phytohemagglutinin (PHA, Remel Lenexa, KS, 2 mg/ml) and cultured in RPMI–1640 medium supplemented with 10% FCS (RPMI-10%) and recombinant interleukin (IL)-2 (rIL-2; Novartis, Emeryville, California, USA; 100 U/ml). CD8þ T lymphocytes were depleted from culture on day 3 using CD8þ immunomagnetic beads (Dynal, Invitrogen, AS, Oslo, Norway), and CD4þ-enriched T lymphocytes were cultured for 3 days. Monocytes (CD14 beads), B lymphocytes (CD19 beads), CD1cþ-myeloid dendritic cells (BDCA-1 Dendritic Cell Isolation Kit), CD141þ-myeloid dendritic cells (BDCA3 MicroBead Kit), and plasmacytoid dendritic cells (Diamond Plasmacytoid Dendritic Cell Isolation Kit II) were isolated from PBMCs with the magnetic bead cell sorting system from Miltenyi Biotec (GmbH, Bergisch Gladbach, Germany) according to the manufacturer’s protocol. Monocytes, cultured in RPMI-10%, were differentiated into immature moDCs by stimulation with 45 ng/ml IL-4 (rIL-4; Biosource, Nivelles, Belgium) and 500 U/ml granulocyte macrophage colony-stimulating factor (GM-CSF; Schering-Plough, Brussels, Belgium) on days 0 and 2, and used on day 6 [25]. Mature moDCs were obtained on day 6 after stimulating immature moDCs on day 5 with indicated compounds. CD103þ retinoic acid (RA)-moDCs were cultured as moDCs, but with the addition of 1 mmol/l retinoic acid (Sigma– Aldrich, St Louis, Missouri, USA) [26]. For monocytederived macrophages (MØ), monocytes, cultured in RPMI-10%, were differentiated with 5 ng/ml GM-CSF for a type I phenotype or with 5 ng/ml macrophage colony-stimulating factor (M-CSF; Immunotools, Friesoythe, Germany) for a type II phenotype on days 0 and 3, and used on day 7. B lymphocytes were cultured in RPMI-10%. Myeloid and plasmacytoid DCs were cultured in RPMI-10% with 500 U/ml GM-CSF or 10 ng/ml IL-3 (Invivogen, Toulouse, France), respectively. Langerhans cells and dermal dendritic cells (dDCs) were isolated from the skin [27]. Briefly, the skin was incubated in 0.2% dispase II (Roche, Mannhein, Germany) overnight at 48C to separate epidermis from dermis. To yield single cells, epidermal sheets were treated with

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DC subsets differentially activate latent HIV-1 van der Sluis et al.

0.25% trypsin (Invitrogen) for 5 min at 378C, and dermal sheets were treated with Iscove’s modified Dulbecco’s media supplemented with 0.5% collagenase D (Roche), 30 U/ml DNase I (Boehringer Mannheim, Mannheim, Germany), and 2% FCS for 2 h at 378C. Langerhans cells and dDCs, labeled with fluorescent-conjugated antibodies, were isolated by fluorescence-activated cell sorting (FACS) sorting [Becton Dickinson (BD) Biosciences, San Jose, California, USA] and cultured in RPMI medium supplemented with 10% FCS. DDCs were cultured with 500 U/ml GM-CSF.

Virus Plasmid DNA encoding the C-X-C chemokine receptor type 4-using HIV-1 Lai (LAI) primary isolate [28] was transiently transfected into HEK 293T cells with calcium phosphate [29]. Virus supernatant was harvested after 2 days, filtered by passage through a 0.2 mm filter, and stored in aliquots at
808C. Concentrations of virus stocks were determined by CA-p24 ELISA. Extracellular capsid-p24 ELISA Culture supernatant was heat-inactivated at 568C for 30 min in the presence of 0.05% Empigen-BB (Calbiochem, La Jolla, California, USA). Capsid (CA)-p24 concentration was determined by twin-site ELISA with D7320 (Biochrom, Berlin, Germany) as capture antibody (Ab), and alkaline phosphatase-conjugated anti-CA-p24 (EH12-AP) as detection Ab. Quantification was performed with the lumiphos plus system luminescence reader (Lumigen, Southfield, Michigan, USA) in a LUMIstar Galaxy (BMG Labtechnologies, Offenburg, Germany). Recombinant CA-p24 was used as standard. Reagents Fusion inhibitor T1249, used at a work concentration of 0.1 mg/ml, came from Pepscan (Therapeutics BV, Lelystad, the Netherlands). To mature dendritic cells, 0.1 mg/ml LPS (Invivogen), 20 mg/ml Poly(I:C) (Sigma– Aldrich), 10 mg/ml PAM3/CSK4 (Invivogen), 10 mg/ml peptidoglycan (PGN) (Invivogen), 1 mg/ml flagellin (Invivogen), 5 mg/ml CLO97 (Invivogen), 5 mg/ml R848 (Invivogen), 10 mg/ml muramyl dipeptide (MDP) (Invivogen), or 2000 U/ml interferon (IFN)-g (Life Technologies, Carlsbad, California, USA) was used. CD1c, CD3, CD11c, CD14, CD19, CD83, CD86, CD45, CD123, human leukocyte antigen-D related (HLA-DR), and a-lineage antibodies came from BD Pharmingen. Other antibodies used were: DC-SIGN (R&D Systems, Minneapolis, Minnesota, USA); CD163 and CD206 (BioLegend, Fell, Germany); BDCA-1, BDCA-3, and CD304/BDCA-4 (Miltenyi Biotec); and CAp24-RD1 (Coulter, Woerden, the Netherlands) (clone KC57). R-hIFN-a (Roferon-A; Hoffmann La Roche, Basel, Switzerland), r-hIFN-b (Roferon-A; Hoffmann La Roche), or r-hIFN-g (Life Technologies) was used at indicated concentrations. Polyclonal antibodies against the IFN-receptor (hIFN-a/bR1, R&D

Systems), IFN-a, and IFN-b (Pbl Interferon source) were used at 10 mg/ml, 2000 U/ml, and 400 U/ml, respectively.

HIV-1 latency assay HIV-1-infected cells were used in the latency assay as described [19,30,31]. Briefly, PHA-activated CD4þ T lymphocytes were infected with HIV-1 (20 ng CA-p24). Virus was washed away after 4 h and cells were cultured with fusion inhibitor T1249, blocking new infections. Twenty-four hours after infection, cells (1.5  105/well) were either mock-treated or co-cultured with different immune cells in ratios indicated. After 24 h co-culture, cells were harvested, stained for CD3 and intracellular CA-p24, and analyzed by flow cytometry. Percentage of CA-p24-positive cells in treated cultures was divided by percentages of CA-p24 cells in mocktreated cultures to measure the proviral latency (fold activation). Flow cytometry For CA-p24 analyses, cells fixed in 4% formaldehyde, 10 min, room temperature (RT), were washed with 2% FACS buffer (PBS supplemented with 2% FCS). Cells were permeabilized and stained for CA-p24 and CD3 for 1 h in BD Perm/Wash buffer (BD Pharmingen, San Diego, California, USA) at 48C. Unbound antibody was removed and cells were analyzed on a BD Canto II flow cytometer with BD FACSDiva Software v6.1.2 (BD Biosciences) in 2% FACS buffer. The T-lymphocyte population was defined based on forward/sideward scatter and CD3 (T-cell receptor) expression. Virus production on the gated Tlymphocyte population was determined by measuring intracellular viral CA-p24 protein. Gate settings were fixed between samples for each experiment. Cell morphology analysis Monocyte-derived dendritic cells and MØs type I and II were cultured on 10-mm glass coverslips (VWR, Germany). Cells were fixed with 3.7% paraformaldehyde (PFA) for 20 min. PFA was quenched with 50 mmol/l NH4Cl and cells were permeabilized with 0.1% saponin (Riedel de Haen, Germany), 10 mmol/l NH4Cl, and 1% BSA in PBS for 30 min. Subsequently, cells were stained with Hoechst 33258 (Sigma–Aldrich). Excess Hoechst was removed by washing with permeabilization buffer (twice), PBS (once), and water (twice). Cells embedded in Vectashield were analyzed by confocal microscopy. Fluorescent images with a line average of four scans per image and pixel size of 1024  1024 were made with a Leica DM SP2 AOBS confocal microscope with an X63 HCX PL APO 1.32 oil objective. Statistical analysis One-way analysis of variance (ANOVA) and Student’s t test (two-tailed) were used to evaluate if observed differences between groups or pairs were significant (Graphpad Prism, version 5) (P values: , P < 0.05; , P < 0.01; , P < 0.001).

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Results Monocyte-derived macrophages have reduced anti-latency potency compared to monocyte-derived dendritic cells Previously, we described that moDCs induced gene expression of latent HIV-1 provirus in effector T lymphocytes [19]. To determine whether monocytederived macrophages (MØ) with type I or type II phenotypes have this ability, monocytes were isolated from peripheral blood of healthy donors and differentiated into MØs, or, as positive control, into moDCs. Confocal microscopy and flow cytometry confirmed specific MØ and moDC morphology and marker expression (additional file 1, http://links.lww.com/ QAD/A669). MØ type I and II only marginally reverted latency by 1.4-fold compared to the untreated culture, whereas moDCs induced a robust 2.3-fold increase in virus-producing cells (Fig. 1a and b). Blood-derived myeloid dendritic cells activate latent HIV-1 provirus Two different subsets of myeloid dendritic cells were isolated from peripheral blood. Purity of conventional (CD1cþ) and cross-presenting (CD141þ) myeloid dendritic cells was determined by flow cytometry (additional file 2a and b, http://links.lww.com/QAD/ A669). Both CD1cþ and CD141þ myeloid dendritic cells reverted viral latency (average of 1.9 and 1.8-fold, respectively) (Fig. 1c). In contrast, blood-isolated plasmacytoid dendritic cells (additional file 2c, http:// links.lww. com/QAD/A669) did not affect latent proviral gene expression (Fig. 1c). Blood-derived monocytes, B cells, natural killer or T cells do not activate latent provirus To investigate whether other primary immune cells can revert HIV-1 latency, undifferentiated CD14þ monocytes (additional file 3, http://links.lww.com/QAD/A669), CD19þ B lymphocytes (additional file 4, http:// links.lww.com/QAD/A669), and bulk ‘left-over’ T lymphocytes, containing unstimulated CD4þ/CD8þ T lymphocytes and NK cells derived from PBMCs (additional file 5, http://links.lww.com/QAD/A669), were tested for HIV-1 antilatency properties. None of them could activate dormant HIV-1 in effector T lymphocytes (Fig. 1d–f). Table 1 shows whether immune cells revert latency strongly (green), moderately (orange), or not at all (red). Skin-derived dendritic cells cannot revert HIV-1 latency Thus far, we tested different peripheral blood cell types; next, we tested two types of dermal dendritic cells, CD14þ and CD14
, and skin-derived Langerhans cells (additional file 6, http://links.lww.com/QAD/A669). Co-culturing of HIV-1-infected T lymphocytes with

these cells did not induce HIV-1 production, showing that dermal dendritic cells and Langerhans cells do not induce HIV-1 expression in latently infected effector T cells (Fig. 1g).

Lymph node homing dendritic cells activate HIV-1 provirus, depending on the kind of maturation stimulus Dendritic cells can undergo maturation upon recognizing pathogen-associated molecular patterns (PAMPs) derived from pathogens such as viruses or bacteria, by pathogen recognition receptors (PPRs) and TLRs. To investigate whether dendritic cell maturation affects viral latency in T lymphocytes, moDCs were matured by stimulating TLR4 with bacterial lipopolysaccharide (moDCLPS) or TLR3 with poly(I:C), a mimic of double-stranded RNA (moDCpoly(I:C)). Increased expression of maturation markers CD83, CD86, and HLA-DR on matured moDCs was compared to untreated moDCs by flow cytometry (additional file 7, http://links.lww.com/ QAD/A669). MoDCsLPS or moDCspoly(I:C) displayed significant reductions in reverting viral latency, yielding 1.8 and 1.6-fold activation, respectively, whereas untreated moDCs displayed 2.8-fold activation (Fig. 1h). During HIV-1 infection, primary immature dendritic cells in tissue or blood can encounter other pathogens or stimuli inducing maturation. Maturing dendritic cells then migrate to the lymph nodes to exert an immune response. To investigate whether lymphoid homing dendritic cells affect HIV-1 latency, primary bloodderived CD1cþ and CD141þ-myeloid dendritic cells were matured with poly(I:C) or LPS and compared with untreated myeloid dendritic cells (Fig. 1i, j). Surprisingly, LPS-stimulated myeloid dendritic cells were significantly more potent in activating latent HIV-1 provirus than poly(I:C)-treated myeloid dendritic cells. Thus, dependent on the maturation stimulus, mature myeloid dendritic cells, as encountered in local lymph nodes, can differently modulate viral latency in effector T lymphocytes. To further unravel the influence of maturation signals on dendritic cell-mediated activation of viral latency, we selected the following TLR agonists: PAM3CSK4 (a ligand for TLR1/2); peptidoglycan (PGN, ligand for TLR2); poly(I:C) (TLR3); LPS (TLR4); flagellin (TLR5); CLO97 (TLR7/8); R848 (TLR8); and muramyldipeptide [MDP, nucleotidebinding oligomerization domain-containing protein 2 receptor] (Fig. 2a and additional file 8, http://links. lww.com/QAD/A669). Differently matured moDCs and CD1cþ-myeloid dendritic cells exhibited latencyactivation efficiencies comparable to immature moDCs or myeloid dendritic cells. Only poly(I:C)-stimulated myeloid dendritic cells lost the ability to activate latent virus (Fig. 2b, c). No effect on proviral latency was observed when T lymphocytes were cultured with the different stimuli in absence of dendritic cells (Fig. 2d).

Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.

DC subsets differentially activate latent HIV-1 van der Sluis et al.

2.4%

0.0%

101

102

103

+ moDC 3.3%

0.0%

101

1.4%

103

102

5.8%

101

101

1.2%

90.8%

102

102

95.3%

103

+ MØ type II 3.6%

102

95.2%

101

101

CD3

102

2.5%

103

+ MØ type I

95.0%

103

Mock 103

(a)

0.0%

101

102

3.3%

103

0.0%

101

102

103

CA-p24 (b)

(c) *** * *

Fold activation

3

2

2

1

1

1

0 Mock

M1

M2

MoDC

(e)

Fold activation

0 Mock

CD1c+ CD141+ mDC mDC

pDC

3

2

2

1

1

1

B cells

moDC

Mock

(h)

Bulk

CD1c+ mDC

(i) 4

***

(j) 4

** *

4

*** ***

2

2

2

1

1

1

***

0 LPS moDC

Poly (I:C)

CD14+ dDC

CD14– dDC

*** **

LC

* **

3

Mock Immature

Mock

**

3

0

MoDC

0

0 Mock

Mono

3

***

2

0

Mock

(g)

(f)

***

3

***

3

*** ***

2

0

Fold activation

(d) 3

3

0 Mock Immature

LPS

Poly (I:C)

CD1c+ myeloid DC

Mock Immature

LPS

Poly (I:C)

CD141+ myeloid DC

Fig. 1. Latency activation properties of different, blood or skin derived, primary antigen presenting cells. (a) Typical dot-plots of HIV-1-infected T lymphocytes positive for CA-p24 and CD3 after co-culture with MØ type I (M1), MØ type II (M2), immature moDCs (at ratios of 1 : 3 T cells), or mock treatment. (b) Percentages CA-p24-positive T lymphocytes in co-cultures, normalized to percentages CA-p24 upon mock treatment to determine viral latency, shown as fold activation (FA) (mean value: 12 replicates, 2 donors  SEM). FA of T lymphocytes with or without (c) CD1cþ-myeloid DCs (CD1cþ-mDC), CD141þ-myeloid DCs (CD141þmDC), or plasmacytoid DCs (pDC) (ratio mDC : T-cell 1 : 15, ratio pDC : T-cell 1 : 5; mean values of 10 (CD1cþ- and CD141þmDCs) or 12 replicates (pDC), 4 donors, SEM); (d) CD14þ monocytes or immature moDCs (mean value: 6 replicates, 2 donors, SEM); (e) CD19þ B cells or immature moDCs (mean value: 12 replicates, 4 donors, SEM); (f) bulk ‘left-over’ lymphocytes (NK and/or T cells) or CD1cþ-myeloid DCs (mean value: 4 replicates, 2 donors, SEM); (g) skin-derived CD14þ-dermal DCs (CD14þdDC), CD14--dermal DCs (CD14--dDC), or Langerhans cells (LC) (ratio DC : T-cell 1 : 3, mean value: 4 (CD14þ-dDC), 6 (CD14-dDC) or 5 (LC) replicates, 2 donors, SEM); (h) immature moDC, LPS, or poly(I:C)-matured moDC (mean value: 6 replicates, 2 donors, SEM); (i) immature CD1cþ-myeloid DC, LPS, or poly(I:C)-matured CD1cþ-myeloid DC (mean value: 9 replicates, 3 donors, SEM); (j) immature CD141þ-myeloid DC, LPS, or poly(I:C)-matured CD141þ-myeloid DC (mean value: 4 replicates, 2 donors, SEM) was determined. P values (one-way ANOVA) indicate statistically significant differences; () P < 0.05, () P < 0.01, () P < 0.001. All (co)-culturing was performed for 24 h. ANOVA, analysis of variance; DC, dendritic cell; mDC, myeloid dendritic cell; moDCs, monocyte-derived dendritic cells; pDC, plasmacytoid dendritic cell.

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Table 1. HIV-1 latency activation properties of tissue specific immune cells. Compartment

Blood

Tissue/skin

Lymph node

Cell type

MDDC CD1c + myeloid DC CD141+ myeloid DC T lymphocyte Monocyte B lymphocyte Plasmacytoid DC MDMØ type I MDMØ type II CD14+ Dermal DC CD14- Dermal DC Langerhans cell MDDC

Stimulus

CD1c mDC

CD141+ mDC Intestine

+

CD103 RA-MDDC

Mock culture

Co-culture

(% CA-p24 positive cells)

(% CA-p24 positive cells)

(± SEM)

average

(± SEM)

average

4.7 3.9

0.7 0.4

10.1 8.0

1.8 0.8

4.4

0.3

7.7

0.8

5.2

0.3

4.9

0.4

5.9

1.1

6.8

1.3

3.6

0.5

4.5

0.8

4.0

0.4

4.4

0.4

6.2 6.2

1.1 1.1

8.9 8.9

1.7 1.7

4.7

0.6

5.9

0.6

4.8

0.5

5.8

0.5

4.5

0.5

5.4

0.8

1/2

3.3

0.8

7.3

1.8

PGN Poly(I:C)

2 3

3.3

0.8

9.8

2.5

3.0

0.6

3.2

0.4

LPS

4

3.0

0.6

4.9

0.9

Flagellin

5

3.3

0.8

5.5

1.1

7/8 8 NOD2

3.3

0.8

8.3

1.7

3.3

0.8

9.5

1.9

3.3

0.8

10.2

2.6

1/2

5.1

0.3

14.0

1.1

PGN Poly(I:C)

2 3

5.1

0.3

14.0

0.3

3.6

0.4

4.5

0.6

LPS

4

3.6

0.4

9.5

1.2

Flagellin

5

5.1

0.3

12.9

0.7

CLO97 R848 MDP Poly(I:C) LPS Poly(I:C) LPS R848

7/8 8 NOD2 3 4 3 4 8

5.1 5.1 5.1

0.3 0.3 0.3

11.1 11.1 11.4

0.8 0.3 1.0

5.4

0.2

5.7

0.4

5.4

0.2

10.6

1.8

6.4 6.4 6.4 6.4

0.5 0.5 0.5 0.5

14.4 10.4 17.6 21.2

2.0 0.9 1.4 2.1

2.2 2.1 1.8 0.9 1.2 1.2 1.2 1.4 1.4 1.3 1.2 1.2 2.2 2.8 1.2 1.7 1.7 2.6 3.0 3.0 2.8 2.8 1.3 2.6 2.6 2.2 2.2 2.2 1.1 2.0 2.2 1.6 2.8 3.3

Pam3/CSK 4

Pam3/CSK 4

Latency activation potential

Fold activation

average

CLO97 R848 MDP +

TLR

(± SEM) #donors

n=

0.1 0.1

6 5

22 13

0.1

6

18

0.1

1

6

0.0

2

12

0.1

4

12

0.1

4

12

0.1 0.1

2 2

12 12

0.1

2

4

0.0

2

6

0.1

2

5

0.2

3

8

0.3

3

8

0.1

4

11

0.1

4

11

0.2

3

8

0.2

3

8

0.3

3

8

0.3

3

8

0.1

2

4

0.2

2

4

0.1

4

10

0.1

4

10

0.1

2

4

0.1 0.1 0.1

2 2 2

4 4 4

0.1

4

10

0.3

4

10

0.2 0.1 0.1 0.2

3 3 3 3

9 9 8 8

strong strong strong weak weak weak weak intermediate intermediate weak weak weak strong strong weak intermediate intermediate strong strong strong strong strong weak strong strong strong strong strong weak strong strong intermediate strong strong

Fold latency activation less than 1.3, weak (red); 1.4–1.7, intermediate (orange); greater than 1.8, strong (green). (a) Monocyte-derived DC; (b) type I macrophages; (c) type II macrophages; (d) RA-stimulated moDC representing CD103þ gut DC [26]. DC, dendritic cell; moDCs, monocyte-derived dendritic cells.

Gut-residing dendritic cells efficiently revert HIV-1 provirus from latency The intestine is richly inhabited by CD103þ dendritic cells, which function in oral tolerance to food antigens and commensal bacteria, but also control infection by mucosal pathogens [32–34]. A major site of HIV-1 replication is the lamina propria of the gastrointestinal tract. Especially in the acute phase of HIV-1 infection, massive CD4þ T-cell depletion occurs in the gut [35,36]. These dendritic cells may purge HIV from the T-cell reservoirs, thereby contributing to virus production in the gut and possibly T-cell depletion. In-vitro generated CD103þ RA-moDCs, closely resembling natural gut dendritic cells [26], were matured with either poly(I:C), LPS, or R848, or were left untreated for an immature phenotype. Immature untreated RA-moDCs reverted viral latency by 2.2-fold (Fig. 2e). Proviral activation was more efficient with RA-moDCsLPS or RA-moDCsR484 (2.8 and 3.3-fold, respectively). In contrast, RAmoDCsPoly(I:C) activated only 1.6-fold. Thus, CD103þ RA-moDC, resembling gut dendritic cells, can activate latent HIV-1 efficiently. Especially LPS-matured RAmoDCs are potent in the activation of latent provirus. This may contribute to the massive T-lymphocyte depletion in damaged gut epithelia of HIV-1 patients upon exposure to LPS-containing bacteria [35,36].

Myeloid dendritic cells maintain latency activation properties upon antiviral kinase protein kinase R induction Poly(I:C) activates protein kinase R (PKR) and TLR3 [37]. Active PKR phosphorylates eukaryotic translation initiation factor 2 alpha, inhibiting mRNA translation, thus protecting against viral infection by blocking viral protein synthesis [38,39]. Additionally, PKR induces cytokine expression to signal the antiviral response to bystander cells [40]. To investigate whether signaling via PKR rather than TLR3 prevents dendritic cell-mediated activation from latency, CD1cþ-myeloid dendritic cells were stimulated with IFN-g to trigger PKR, but not TLR3 (Fig. 2f). IFN-g-matured CD1cþ-myeloid dendritic cells induced viral gene expression from latently infected T cells as effectively as immature or LPS-matured CD1cþ-myeloid dendritic cells. Thus, poly(I:C)-induced TLR3 signaling reduces antilatency properties of CD1cþ-myeloid dendritic cells. Type I, but not type II, interferon abolishes dendritic cell-mediated latency activation Most TLRs signal via the adaptor protein MyD88 and activate NF-kB and mitogen-activated protein kinase (MAPK) pathways [41]. TLR3, however, signals via TIR-domain-containing adaptor-inducing interferon-b

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DC subsets differentially activate latent HIV-1 van der Sluis et al. (a)

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Fig. 2. Lymph node homing dendritic cells revert HIV-1 latency depending on the kind of maturation stimulus. Myeloid and monocyte-derived dendritic cells were matured with different Toll-like receptor ligands to mimic matured lymph node residing dendritic cells. (a) Different agonists and their receptors used to induce DC maturation. (b) HIV-1-infected T lymphocytes were mock-treated, co-cultured with immature moDCs or moDCs stimulated with different TLR ligands and MDP, as indicated under (a) and fold activation (FA) was calculated as a measure for viral latency activation (mean value: 7 replicates, 3 donors,  SEM). (c) FA of T lymphocytes with or without immature CD1cþ-myeloid DCs or with CD1cþ-myeloid DCs stimulated with the ligands indicated under (a) (mean value: 4 replicates, 2 donors, SEM). (d) To investigate possible direct TLR ligand effects, FA was determined of control T lymphocytes treated with the TLR ligands depicted under (a) (mean value: 9 replicates, 3 donors, SEM). (e) Retinoic acid stimulated monocyte-derived DCs (RA-moDC) closely resemble primary gut CD103þ DCs [26]. FA of T lymphocytes with or without immature, poly(I:C), LPS, or R848-matured RA-moDCs (mean value: 7 replicates, 3 donors, SEM). (f) To investigate whether induction of the antiviral protein kinase R (PKR) reduces DC-mediated purging of latent virus, blood-derived CD1cþ-myeloid DCs were matured with LPS (triggering TLR4), poly(I:C) (triggering TLR3 and PKR), IFN-g (triggering PKR) or left immature. FA of T lymphocytes with or without the indicated CD1þ-matured myeloid DCs (mean value: 4 replicates, 2 donors, SEM) was determined. P values (one-way ANOVA) indicate statistically significant differences; () P < 0.001. All (co)-culturing was performed for 24 h. ANOVA, analysis of variance; DC, dendritic cell; moDCs, monocyte-derived dendritic cells; TLR, Toll-like receptor.

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Fig. 3. Type I, but not type II, interferons produced by poly(I:C)-stimulated dendritic cells reduce dendritic cell-mediated latency activation. To study the influence of type I and II IFNs on DC-mediated activation of latent HIV-1 provirus, T cells were cultured alone or co-cultured with immature moDCs in the presence of increasing concentrations of (a) IFN-a (0, 2.5, 25, 250, or 2500 U/ml); (b) IFN-b (0, 1, 10, 100, or 1000 U/ml); or (c) IFN-g (0, 5, 50, 500, or 5000 U/ml) (mean value: 7 (IFN-a and IFN-g) or 9 (IFN-b) replicates, 3 donors, SEM). To study IFN-a inhibition of DC-mediated latency activation, HIV-1-infected T cells were cultured with or without immature moDCs in the presence or absence of (d) recombinant IFN-a (500 U/ml) and/or anti-IFN-a specific antibodies (mean value: 5 replicates, 2 donors, SEM) and (e) recombinant IFN-b (50 U/ml) and/or anti-IFN-b specific antibodies (mean value: 8 replicates, 3 donors, SEM). (f) Type I IFN production by TLR3 matured DCs inhibits HIV-1 latency activation. HIV-1-infected T cells were cultured with or without poly(I:C)-matured moDCs (moDCpoly(I:C)) in the presence or absence of a mixture of polyclonal antibodies targeting soluble IFN-a, IFN-b, and the IFN receptor (anti-IFN cocktail) (mean value: 11 replicates, 4 donors, SEM). P values (one-way ANOVA) indicate statistically significant differences; () P < 0.05, () P < 0.01, () P < 0.001. ANOVA, analysis of variance; DC, dendritic cell; IFN, interferon; moDCs, monocyte-derived dendritic cells; TLR, Toll-like receptor.

(TRIF), which, apart from NF-kB and MAPK signaling routes, also activates the IRF3 pathway, leading to production of type I IFNs [42]. Increasing concentrations of recombinant type I IFNs – IFN-a or IFN-b, but not type II IFN-g – in the dendritic cell–T cell co-culture reduced moDC-mediated proviral activation (Fig. 3a–c). Subsequent blocking with IFN-a or IFN-b polyclonal antibodies restored dendritic cell-mediated virus activation (Fig. 3d, e). Finally, we determined whether poly(I:C)-matured moDCs displayed increased latencyactivation properties when induction of the IFN type I pathway was blocked. Partial activation by 2.1-fold was

observed when the IFN receptor (IFN-a/bR1) was blocked and the activity of soluble IFN-a and IFN-b was neutralized with specific polyclonal antibodies in the coculture of HIV-1-infected T cells and poly(I:C)-matured moDCs (Fig. 3f). Thus, type I IFNs, locally produced by dendritic or other primary immune cells, may inhibit dendritic cell-mediated proviral activation, enabling HIV-1 to remain dormant. In conclusion, we show that latent HIV-1 provirus in effector T lymphocytes can be activated by CD1cþ and CD141þ-myeloid dendritic cells, moDCs, CD103þ

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DC subsets differentially activate latent HIV-1 van der Sluis et al.

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Fig. 4. Schematics representing possible diverse fates of latently infected proliferating T-lymphocyte reservoirs. NB: though immature myeloid dendritic cells can activate latent HIV-1 in cell cultures, low myeloid dendritic cell numbers in blood (0.3–0.9% of total PBMCs) suggest that HIV-1 likely remains dormant in blood circulating T cells. DCs, dendritic cells; PBMCs, peripheral blood mononuclear cells.

RA-moDCs, and marginally by MØs, but not by monocytes, B-lymphocytes, plasmacytoid dendritic cells, Langerhans cells, and CD14
or CD14þ dermal dendritic cells.

Discussion Dendritic cells are important antigen-presenting cells that activate the immune system against pathogens [43]. In blood, lymph nodes, spleen, and (inflamed) mucosal tissues, dendritic cells interact with T lymphocytes [43,44]. Different dendritic cell subsets have been implicated in sexual HIV-1 transmission [45,46]. Mucosal dendritic cells capture HIV-1 and transmit it to permissive T lymphocytes [47]. Here, we show that latent HIV-1 provirus residing in the effector T cells is activated by

dendritic cell subsets and other immune cells with remarkably different efficiencies. Our results allow us to speculate about the fate of the latently infected proliferating T-lymphocyte reservoir in blood, genital tract, lymphoid organs, or gut (Fig. 4). Primary myeloid dendritic cells isolated from blood can induce virus production from latently infected effector T lymphocytes. In contrast, monocytes, B lymphocytes, plasmacytoid dendritic cells, or other immune cells in blood cannot. Using our novel primary cell-based assay to quantify HIV-1 latency in proliferating cells, we previously showed that antilatency properties of moDCs were lost below 1 : 100 dendritic cell : T-cell ratios [19]. The low percentage of myeloid dendritic cells in blood (0.3–0.9% of the PBMCs) suggests that once a latent provirus is established in an effector T cell, it is likely to remain dormant in peripheral blood (Fig. 4).

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Dermal dendritic cells or Langerhans cells, found in genital tract mucosal tissues, and plasmacytoid dendritic cells, found in the endocervical epithelium, cannot abrogate latency. We hypothesize that reservoirs in effector T lymphocytes could be established in these tissues. Additionally, local production of IFN-a and/or IFN-b could prevent viral activation by other dendritic cell subsets (see Fig. 3). The only mucosal cells with antilatency capacity, albeit moderate, are type I and II macrophages. Thus, reversion of HIV-1 latency by cells in mucosal tissues of the genital tract is not likely to occur and could allow the establishment of a viral reservoir directly after infection (Fig. 4). Reversion of HIV-1 latency in effector T lymphocytes could happen in lymph nodes and lymphoid organs where intensive contact with immature and matured myeloid dendritic cells occurs. Most dendritic cellmaturation stimuli, except TLR3 triggers, did not change anti-latency properties of dendritic cells (see Fig. 2). Dendritic cell maturation by pathogens, such as bacteria, fungi, or viruses, could thus impact the HIV-1 reservoir (Fig. 4). The effects of numerous pathogens on HIV-1 replication have been studied [48], but ‘not’ in the context of viral latency. It has clinical relevance to assess whether certain co-infections influence HIV-1 reservoir size and thus whether rapid treatment of the co-infection should be initiated. Whether dendritic cells can reactivate virus production in resting T lymphocytes is not clear [21,49]; thus the precise role of dendritic cells in reverting latent HIV-1 in resting T cells needs further study. CD4þ T lymphocytes in gut-associated lymphoid tissue (GALT) play a major role in the persistence of HIV-1 [50–52]. In chronically HIV-infected individuals and in simian immunodeficiency virus (SIV)-infected rhesus macaques, damage of gut mucosa was associated with increased LPS levels due to microbial infestation of the intestine [53]. In-vivo LPS administration to chronically SIVagm-infected African green monkeys, which have a remarkably stable nonpathogenic disease course, enhanced viral replication and caused intestinal CD4þ T-cell depletion [54]. Immature dendritic cells residing in the lamina propria may mature via LPSinduced triggering of the TLR4 receptor. Subsequent contact with latently infected T lymphocytes in the intestinal lamina propria, Peyers patches, or local lymph nodes would enable activation of latent HIV-1. LPSstimulated or untreated CD103þ RA-moDCs, a substitute for graft dendritic cells residing in the gut mucosa, were very efficient in reverting HIV-1 latency (see Fig. 2e). The high density of CD103þ dendritic cells in the gut mucosa or Peyer patches, combined with strong latency activation properties, may contribute to the high viral replication and extreme CD4þ T-cell depletion observed in the gastrointestinal tract [55–59].

The finding that type I IFN prevents dendritic cellmediated activation of latent HIV-1 is interesting. Sandler et al. [60] recently demonstrated that type I IFN responses in SIV infection have detrimental effects. They observed that excessive immune preactivation by IFN-a suppresses acute viral infection, but later on exacerbates aspects of disease progression. Studies have also shown transmitter/ founder viruses isolated from HIV-infected patients to be relatively resistant to IFN-a [61,62]. Thus, one might speculate that the mixed effects of IFN are due to initial viral replication suppression, while at the same time allowing reservoir establishment. Collectively, our results show that different dendritic cells and their maturation state impact HIV-1 latency in effector T cells. Further study is needed to investigate whether dendritic cells have an impact on latent HIV-1 in resting T cells. However, the differential activation of HIV-1 provirus by dendritic cells adds another layer of complexity to viral reservoirs. Thus, our observations essentially open up a new field of study: how do other immune cells affect establishment or maintenance of latent HIV-1 infection?

Acknowledgements We thank S. Heijnen for performing CA-p24 ELISA; A.A.M Thomas for providing textual changes in the manuscript; J.A. Dobber for maintenance of the BD Canto II; L.T.C. Vogelpoel for the kind gift of M-CSF, CD163-APC, and CD206-PE antibodies and expert advice on MØ culturing; L. Jachimowski for the kind gift of the CD19-APC antibody; and J. den Dunnen for the kind gift of PAM2, R848, MDP, PAM3, CLO97, flagellin, and PGN. Research of Rvd.S., Tv.M., B.B., and R.E.J. was supported by the Dutch AIDS Fund (AIDS Fonds 2007028, 2008014, and 2013021; http:// www.aidsfonds.nl/about/organisation). This research was also supported by a Vidi grant from NWO (RWS), and an ERC Starting Investigator grant (RWS). R.M.S., T.C., and T.M. performed experiments; R.M.S. analyzed results and made the figures; E.J., R.J., and T.M. designed the research; D.S., R.W.S., and B.B. provided essential input to the discussion; R.M.S., D.S., B.B., R.J., and T.M. wrote the paper. Part of the research presented in this study was supported by the Dutch AIDS Fund (AIDS Fonds 2007028, 2008014, and 2013021; http://www.aidsfonds.nl/about/ organisation) and a Vidi grant from NWO, and an ERC Starting Investigator grant.

Conflicts of interest The authors have no conflicting financial interests.

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DC subsets differentially activate latent HIV-1 van der Sluis et al.

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