Altered phenotype and function of NK cells infiltrating Human Papillomavirus (HPV)-associated genital warts during HIV infection

Clinical Immunology (2014) 150, 210–219

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Clinical Immunology www.elsevier.com/locate/yclim

Altered phenotype and function of NK cells infiltrating Human Papillomavirus (HPV)-associated genital warts during HIV infection☆ Alfred Bere a,1 , Shahila Tayib b,1 , Jean-Mari Kriek a , Lindi Masson a , Shameem Z. Jaumdally a , Shaun L. Barnabas a,c , William H. Carr d , Bruce Allan a , Anna-Lise Williamson a,e , Lynette Denny f , Jo-Ann S. Passmore a,e,⁎ a

Institute of Infectious Disease and Molecular Medicine and Division of Medical Virology, University of Cape Town, Cape Town, South Africa b Dept. Obstetrics and Gynaecology, Jalan Taming Sari, Taiping Hospital, Perak, Malaysia c Desmond Tutu HIV Foundation, Cape Town, South Africa d Department of Biology, Medgar Evers College, The City University of New York, NY, USA e National Health Laboratory Services, Cape Town, South Africa f Dept Obstetrics and Gynaecology, University of Cape Town and Groote Schuur Hospital, Cape Town, South Africa

Received 10 September 2013; accepted with revision 5 December 2013 Available online 14 December 2013 KEYWORDS Genital warts; Mucosal; HPV; HIV; NK cells

Abstract HIV-infected individuals experience more persistent HPV infections and are less likely to resolve genital warts. This study compared phenotype and functions of NK and T cells from genital warts and blood from 67 women. We compared in vitro functional responses of NK and T cells by multiparametric flow cytometry. HIV+ women had significantly lower frequencies of CD4 T cells in warts (p = 0.001) and blood (p = 0.001). While the distribution of NK cell subsets was similar, HIV+ women tended to have lower frequencies of CD56Dim NK cells in both blood (p = 0.0001) and warts (p = 0.006) than HIV− women. Wart NK cells from HIV+ women expressed significantly lower CD107a and produced IFN-γ. HAART status was not associated with differences in NK cell functionality. We

☆ This work was funded by the Cancer Association of South Africa (CANSA) and Poliomyelitis Research Foundation (PRF). AB and JP received training in the USA as part of the Columbia University-Southern African Fogarty AITRP Program. AB is a recipient of the Wellcome Trust Clinical Infectious disease research initiative (CIDRI) fellowship from UCT. NIH FIC K01-TW00703-04A1 grant provided support to WHC. JMK is a recipient of the National Research Foundation (NRF) of SA Scarce Skills Post-Graduate Scholarship. SLB is a recipient of a SHAPe fellowship of the HIV Vacccine Trials Network (HVTN) and the Desmond Tutu HIV Foundation. This work is partially based on research supported by the South African Research Chairs Initiative of the Department of Science and Technology and National Research Foundation (NRF) of South Africa. ⁎ Corresponding author at: Institute of Infectious Disease and Molecular Medicine, Division of Medical Virology, Faculty of Health Sciences, University of Cape Town, Anzio Road, Observatory 7925, Cape Town, South Africa. Fax: + 27 21 4066681. E-mail address: [email protected] (J.-A.S. Passmore). 1 Alfred Bere and Shahila Tayib contributed equally to this work. 1521-6616/$ - see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.clim.2013.12.005

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conclude that wart NK cells from HIV+ women have defects in their ability to degranulate and/or secrete IFN-γ, which may provide insights into why HIV+ women fail to spontaneously resolve genital warts. © 2013 Elsevier Inc. All rights reserved.

1. Introduction Human Papillomaviruses (HPVs) are a highly diverse group of DNA viruses replicating in differentiating epithelial cells. Some HPV sub-types preferentially infect the genital mucosa. Many of these viruses cause benign genital warts and are referred to as low-risk types (including HPV-11 and -6) [1,2]. Whereas others, such as HPV-16 and HPV-18, have the potential to cause cervical cancer and are referred to as high-risk HPV types [3–5]. In the absence of HIV, infection by either high or low risk HPV sub-types commonly regresses without treatment. Typically, 23% of warts are cleared by two months following infection and 66% are cleared by two years [6]. Host defences that are responsible for resolving such HPV infections rely on an intact immune response involving both T cells and natural killer (NK) cells [7,8]. Because HPV-associated genital warts are typically self-limiting or self-resolving, their persistence is often indicative of immune dysfunction. Impaired NK cell function and skewed NK cell subset distributions have been reported in HIV+ individuals. The ability of these NK cells to control secondary viral infections differs depending on which viral sub-type initiates the secondary infection (9). Chronic HPV infections, which are often characterized by high HPV viral loads, may be facilitated by HIV co-infection that reduces the likelihood of spontaneous HPV clearance [10–12]. Also, it is likely that shared transmission routes via the genital mucosa increase the probability of individuals acquiring HPV and HIV simultaneously. Co-infection with HIV and HPV can present major challenges to an infected individual's immune system as both viruses interfere with innate and adaptive immunity. NK cells can be subdivided into distinct populations based on the expression of CD16 and CD56 cell-surface antigens, with the two main NK cell subsets being defined as CD56Dim CD16 + and CD56Bright CD16 −. During chronic HIV disease, there is an expansion of a functionally-impaired CD56− CD16+ NK cell subset [13]. CD16, the FcRIII receptor, is involved in the antibody-dependent cellular cytotoxic (ADCC) function of NK cells [14]. The CD56Dim NK cells represent the major cytotoxic subset responsible for the direct lysis of virally-infected cells (8, 14). CD107a expression is used as a surrogate marker of cytotoxic degranulation that is upregulated on the surface of NK cells when stimulated [15]. CD56Bright NK cells, in contrast, are reported to have low cytotoxic capacity but produce cytokines abundantly [14,16]. The CD56 − CD16 + NK cell subset, which increases in frequency during HIV disease progression, exhibits low cytotoxicity and poor cytokine secretion potential [13]. While many studies have clearly described the phenotypic and functional characteristics of NK cells circulating in peripheral blood, fewer have compared these to NK cells found at effector sites and in tissues. Because of their role in placental formation and fertility, several important

studies on uterine NK cells in the upper reproductive tract suggest that their phenotypes differ significantly from those of systemic NK cells, and likely depend on their tissue location and the cytokine environment in which they exist [17]. While uterine NK cells express CD56, few express CD16. In comparison, NK cells derived from the upper female genital tract tend to be CD56Bright while those in the lower genital tract tend to be CD56Dim [17]. Better understanding of the specific role(s) of, and defects in, NK cell function in HPV-associated genital wart regression during HIV infection is significantly hindered by a lack of data on NK cell function at sites in the genital mucosae where HPV infection and replication primarily take place. The aim of this study was to compare the phenotypes (CD16 and CD56 expression) and functions (CD107a degranulation and IFN-γ production) of NK cells isolated from paired HPV-associated genital warts and blood obtained from both HIV+ and HIV− women. Since CD4 T cells that produce IFN-γ are the best-characterized correlate of wart regression [18] depletion and loss of function of CD4 T cells during HIV disease progression may influence HPV pathogenesis. Thus, we also compared IFN-γ production of in vitro stimulated CD4 T cells derived from warts and blood. We hypothesised that HIV+ women would have systemic NK and T cell defects that would be evident by HPV-associated genital warts containing wartinfiltrating NK and T cells with reduced function compared to those found in HIV− women.

2. Materials and methods 2.1. Study participants and sample collection Sixty-seven women with genital warts were recruited from the Colposcopy Outpatients clinic at Groote Schuur Hospital. The Human Research Ethics Committee of the University of Cape Town approved all aspects of the study and written consent was obtained from each of the women participating in this study. Genital warts were removed from each woman under speculum examination in surgical theatre and immediately placed into a 15 ml conical tube containing 3 ml of R10 (10% FBS in RPMI-1640 medium (GIBCO®) containing 50 ug/ml Penicillin, 50 mg/ml Streptomycin, 0.8 mg/ml Fungin and 50 mg/ml L-glutamine) for transport to the laboratory. Tubes containing genital warts were transported to the laboratory at UCT within 1–2 h of collection. A portion of each of the warts was placed immediately into a Digene Transport tube for isolation of DNA for HPV genotyping. In addition, 40 ml ACD anti-coagulated whole blood was collected from each woman by venepuncture for the isolation of peripheral blood mononuclear cells (PBMCs).

2.2. Disaggregation of genital warts Genital warts were transferred onto a stainless steel 35 micron cell strainer together with the 3 ml of R10 transport

212 medium. The tissue was cut into 2–3 mm pieces with a sterile, disposable scalpel and the wart was disaggregated manually by agitating the tissue chunks through the stainless steel strainer using a plastic plunger to separate the lymphocytes from the tissue matrix. The strainer was flushed with another 3 ml of R10 medium to remove all the cells and the homogenized suspension was pooled into a 15 ml conical tube and centrifuged at 1200 rpm (280 g) for 10 min. Cells were re-suspended in 6 ml of R10 medium.

2.3. PBMC isolation PBMCs were isolated by density gradient centrifugation using Ficoll-Histopaque (Sigma–Aldrich, Egham, Runnymede, UK) and LeucoSep® centrifuge tubes (Greiner Bio-one, Frickenhausen, Germany). Plasma was stored at −80 °C for measurement of HIV viral loads.

A. Bere et al. Biosciences), with approximately 0.5 × 106 events being acquired per sample. Data analysis was performed using FlowJo software v8.5.3 (Tree Star, Inc; Ashland, Oregon, OR, USA). Dead cells (ViVid+), monocytes (CD14 +), and B cells (CD19+) were excluded from the analysis. Fluorescence minus one (FMO) controls were used to set gates. For the gating strategy, doublets were excluded based on forward scatter (FSC) height and FSC area (Fig. 1). A broad PBMC gate was then defined based on FSC height and side light scatter. Monocytes and B cells were excluded based on CD14 and CD19 gating, respectively. NK cells were identified within the CD3 − gate based on the expression of CD16 and CD56. NK cells were then subdivided into CD56Bright , CD56Dim , and CD16+CD56 − populations and subsequently analyzed for the expression of IFN-γ and CD107a as summarised on Fig. 1.

2.5. Statistical analysis 2.4. Flow cytometry Genital wart- and blood-derived NK and T cell phenotypes and functional responses were measured using the following panel of fluorescently-conjugated monoclonal antibodies: CD3-APC-H7, CD4-PerCp-Cy5.5, CD16-PE, CD56-PE Cy7, CD107-FITC and IFN-γ-Alexa fluor 700 (all BD Biosciences, San Diego, CA, USA), CD8-quantum dot 605 (Invitrogen, Carlsbad, CA, USA), CD14-PacBlue (BD Biosciences San Diego, CA, USA), and CD19-PacBlue (Invitrogen, Carlsbad, CA, USA). All antibodies were pre-titered to optimal concentrations. Violet viability reactive dye (“Vivid”; Invitrogen) was included to differentiate live from dead cells. To investigate the function of genital wart and blood NK cells, these were stimulated with (a) the tumour cell line K562 (1 × 105 K562 cells in 100 μl); (b) PMA (0.1 μg/ml)/ionomycin (0.1 μg/ml) [positive control]; and (c) media alone (negative control). To each stimulation, 10 μl of R10 stimulation mix (4.5 ml R10, 500 μl DNase I, anti-CD49d (1 μg/ml), anti-CD28 (1 μg/ml), BFA (0.5 μg/ml) and monensin (10 μg/ml) (Sigma Aldrich) was added. In addition, anti-CD107a-FITC antibody (5 μl) was included in each stimulation tube. For PMA/ionomycin stimulation, cells were incubated for 3 h. For the remaining stimulations with K562 or no antigen (negative control), cells were cultured for 6 h at 37 °C, 5% CO2 (Thermo Electron Corporation). Cells were subsequently washed with FACS wash buffer (1% FBS and 0.01% NaN3, GIBCO® PBS, Invitogen™, Carlsbad, CA, USA) and centrifuged at 838 g (2300 rpm) for 3 min at 4 °C. Cells were re-suspended in 50 μl PBS containing Vivid dye (Invitrogen) and incubated for 20 min at room temperature. For cell-surface staining, a mixture of antibodies, consisting of anti-CD19-Pacific Blue, anti-CD14-Pacific Blue, anti-CD8-Qdot 605, anti-CD4-PerCP-Cy5.5, CD16-PE and CD56-PE Cy7 in PBS, was prepared and cells were stained for 20 min at room temperature. Cells were fixed and permeabilized at room temperature for 20 min using Cytofix/ Cytoperm buffer (BD Biosciences) and stained intracellularly with anti-CD3-APC-H7 and anti-IFN-γ-Alexa fluor 700. All staining was done in PBS at a final volume of 50 μl. Cells were washed, and finally resuspended in 1% paraformaldehyde (Cell fix BD Biosciences) and stored at 4 °C until acquisition (within 24 h). Samples were acquired on a BD Fortessa (BD Biosciences) with FACSDiva software version 6.0 (BD

Statistical analyses were performed using GraphPad Prism 5® (GraphPad Software, San Diego California USA). The Mann–Whitney U test was applied for independent sample comparisons, the Wilcoxon Ranks Test was used for matched non-parametric comparisons and Spearman Ranks correlation was applied for assessing associations. P-values of ≤ 0.05 were considered significant.

3. Results 3.1. Clinical characteristics of women Sixty seven women with genital warts were included in this study to investigate the impact of HIV-infection on the phenotype and function of NK and T cells resident in HPVassociated genital warts and how these compare with cells circulating in blood. Of the 67 women included, 51/67 (76.1%) were HIV+ while 16/67 (23.9%) were HIV− (Table 1). Of the 51 HIV+ women, 37/51 (72.5%) were on HAART while 14/51 (27.5%) were not. HIV+HAART+ women tended to be older than both HIV+HAART− and HIV− women (p b 0.01), had lower HIV viral loads than HIV+HAART− women (p b 0.001), were more likely to have normal Pap smear results (p = 0.04), and were more likely to have milder and smaller warts (p = 0.006) compared to HIV+HAART− women. HIV+HAART+ women did not differ significantly from HIV− women in terms of their Pap smear results and the sizes of their genital warts. Despite HIV+HAART + women having a higher proportion of mild warts than HIV+HAART − women, CD4 counts among HIV + women, irrespective of HAART status, did not differ. Irrespective of HIV or HAART status, HPV-11 and then HPV-6, were found to be the most common HPV types associated with genital warts in this study, with HPV-16 and -45 being the next most common types detected.

3.2. Comparison of NK cell phenotype and function in genital warts and blood The frequencies of different NK cell subsets in genital warts were compared to those found in blood (Fig. 2). Irrespective of HIV status, CD56Dim CD16 + NK cells were the most

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Singlets

FITC CD56 -PECy7

Total NK cells

CD107a -FITC

CD4-PerCPCy5.5

CD8 QD605

CD56-PECy7

SSC

CD3 APC -Cy7

CD3+ T cells

CD56 bright

Cd56 Dim

CD4+ T cells

IFN -g AF700

CD16+CD56CD107a -FITC

CD107a -FITC

CD16-PE

CD107a -FITC

CD3 APC-Cy7

NK cell subsets

CD16-PE

IFN-g AF700

ViVid/CD19/CD14

SSC

FSC-H

FSC-A

CD3

Live cells

Lymphocytes

FSC-H

Time

IFN-g AF700

IFN-g AF700

Figure 1 Gating scheme used to evaluate NK cells and CD4 T cells isolated from genital warts and blood. Cells were first gated on singlets, lymphocytes and then live cells. For NK cell analysis, CD3 − cells were gated, and then further gated on total NK cells or NK cell subsets (CD56BrightCD16 −, CD56dimCD16 + and CD16+CD56 −). From these subsets, functional gates were then set. For CD4 T cells, CD3 + cells were gated and then further gated on CD4 and CD8 populations. Functional gates for CD107a and IFN-γ were set based on FMOs and negative control and were kept constant for all samples analysed.

frequent NK cell subset found in both genital warts and blood. In HIV − women, genital warts and blood showed similar proportions of CD56Dim, CD56Bright and CD16+ CD56− NK cells. Differences in NK cell populations between genital warts and blood were evident in HIV + women in whom CD56Dim NK cells were detected at significantly higher frequencies in genital warts than in blood (p b 0.0001). In contrast, CD56Bright and CD16+CD56 − NK cell subsets were significantly elevated in blood compared to genital warts (p = 0.0009 and p = 0.0004, respectively). Among HIV + women no correlation was observed between the NK cell frequencies found in blood and those found in genital warts (data not shown). Since we identified differences in the frequencies of NK cell subsets between the blood and genital warts, we wanted to investigate whether the same compartmental differences are evident with respect to the function of these NK cell subsets. The ability of NK cells to degranulate (CD107a) and produce IFN-γ in responses to stimulation with

the tumour antigen cell line K562 was compared in genital warts and matched blood (Fig. 3). CD56Dim NK cells have previously been reported to exhibit the highest cytolytic ability compared to the other NK cell subsets, while CD56Bright NK cells produce cytokines better than other subsets. Generally, the predominant NK cell function was CD107a degranulated rather than IFN-γ production in both compartments, irrespective of NK cell subset (Fig. 3).

3.3. Impact of HIV infection on NK cell subset frequencies and function We investigated the impact of HIV infection on both the frequency and function of NK cell subsets derived from genital warts and blood (Figs. 2–4). In both blood and genital warts from HIV + women we found significantly higher frequencies of CD16+CD56 − NK cells (p = 0.03) and lower frequencies of CD56Dim NK cells (p = 0.006) compared to

214 Table 1

A. Bere et al. Clinical characteristics of women involved in this study.

Characteristic

HIV+HAART +

HIV+HAART −

HIV −

N Age [years; median (IQR)] CD4 count [cell/μl; median (IQR)] Plasma viral load [RNA copies/ml; median (IQR)] Pap smear results Normal LSIL HSIL Size of genital warts Mild Moderate Severe Weight of genital wart (g)

37 28 (24–36) 328 (218–481) 0 (0–360)

14 23 (21–25) 299 (299–415) 33 356 (11 000–75 000) a

16 24 (20–35) N/A N/A

9/30 a (30.0%) 19/30 a (63.3%) 2/30 a (6.7%)

8/14 (57.1%) 6/14 (42.9%) 0/14 (0.0%)

6/9 a (66.7%) 2/9 a (22.2%) 1/9 a (11.1%)

25/31 a (80.6%) 5/31 a (16.1%) 1/31 a (3.2%) 0.10 (0.06–0.3)

5/14 (35.7%) 8/14 (57.1%) ⁎ 1/14 (7.1%) 0.35 (0.13–0.36) ⁎

12/16 (75.0%) 3/16 (18.8%) 1/16 (6.3%) 0.16 (0.08–0.22)

a

Pap smear cytology and wart size data were only available for a subset of the women in the study. ⁎ p b 0.01.

HIV− women (Fig. 2). Interestingly, the distribution of genital wart NK cell subsets did not differ significantly between HIV+ women who were HAART therapy naïve compared to those using HAART (data not shown). We investigated whether blood- and genital wart derived NK cells from HIV+ women had detectable functional defects relative to HIV− women. Based on CD107a expression (as a surrogate for degranulation) and intracellular IFN-γ production, NK cells from HIV− women were generally more functional than those from HIV+ subjects (Fig. 3). Warts from HIV+ women (naïve to HAART) contained significantly lower frequencies of CD56Bright and CD56Dim NK cells that expressed CD107a (p = 0.001 for CD56Bright and p = 0.003 for CD56Dim) and produced IFN-γ (p = 0.001 for CD56Bright and p = 0.0002 for CD56Dim) than warts from HIV− women. HAART did not improve these functional defects in wart NK cells because HIV+ women on HAART had similar frequencies of NK cells expressing CD107 or producing IFN-γ as HIV+ women naïve to HAART. Although HIV+ women on HAART tended to have smaller warts than their HAART naïve counterparts (Table 1), the NK cells of HAART+ women had similar functionality (based on CD107a expression and IFN-γ production) to those of HIV+HAART− negative women (Figs. 3A-B). Functional defects (CD107a expression or IFNproduction) were not similarly evident in blood derived NK cells from these same HIV+ women, who had higher CD56Bright and CD56Dim NK responses than HIV− women (Figs. 3C–D). We stratified these functional responses based on the clinical characteristics of genital warts (mild, moderate and severe; Table 2). Among HIV − women, moderate warts from HIV− women had 9-fold higher frequencies of CD107a + CD56Dim (p = 0.005) and 7-fold higher frequencies of CD56Bright NK cells (p = 0.008) than mild warts. While CD56Dim CD107a + NK cells were similarly 12-fold elevated in moderate compared to mild warts of HIV+ women, CD56Bright NK cells were absent from both moderate and mild found in these women. IFN-g + NK cell subsets were present at similarly low frequencies in the moderate and mild warts of both HIV− and HIV+ women, and were not obviously associated with wart severity (Table 2). The ability of blood NK cells to produce IFN-γ was lower than their ability to express CD107a. Despite this, during

HIV-infection, IFN-γ production by all subsets of NK cells in blood was significantly higher than those detected in genital warts, with genital wart IFN-γ production only being evident at low frequencies in a few HIV + women (Fig. 4).

3.4. Defects in CD4 T cell frequencies rather than function during HIV disease CD4 T cell frequencies (of CD3 + cells) were significantly lower in the blood of HIV + women than they were in the blood of HIV − women (Fig. 5A; p = 0.002 for HAART − and p = 0.007 for HAART +). There were lower frequencies of CD4 T cells in genital warts than in blood. Similarly, CD4 T cell frequencies were also significantly lower in the genital warts of HIV + women than HIV − women, regardless of treatment status (p b 0.01). HAART + women had significantly higher CD4 T cell frequencies in warts compared to HAART − women (p = 0.0001), although these still present at lower frequencies than those measured in HIV − women. While CD4 T cells accounted for ~17% of all CD3 T cells isolated from the genital warts of the HIV− women, this helper T cell subset was almost completely absent from the genital warts of HIV+ women, in which CD8 T cells made up the major population. Despite reduced numbers, their ability to produce IFN-γ in response to PMA/ionomycin stimulation was similar (Fig. 5B). This indicates that reduced numbers of CD4 T cells rather than these cells being functionally defective in the genital warts and blood of HIV+ women may underlie their observed inability to spontaneously clear HPV-associated genital warts.

4. Discussion HIV + women generally have more persistent HPV-infections than those occurring in HIV − women. This increased persistence places HPV-infected women at greater risk of developing cervical disease [10–12]. Although HPV types that cause genital warts are typically benign, failure to spontaneously clear such warts during HIV infection has important socio-economic consequences. In this study, we have investigated the impact of HIV-infection on both the

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215

A) Blood

B) Genital warts

p=0.0001

p=0.006

%NK cell expression

100

100

80

p=0.0007

80

60

60

40

40

20

20

0

HIV+ HIVp=0.03

0 CD56Bright

CD56Dim

CD56Bright

CD16+CD56-

C) HIV-

CD56Dim

CD16+CD56-

D) HIV+

Genital warts Blood

P<0.0001

100

100

80

80

60

60

40

40

20

20

0

0

% of NK cells

P=0.001

P<0.0001

Figure 2 Comparison of NK cell phenotypes in (A) blood versus (B) genital warts. HIV − women (grey boxes) and HIV + women (white boxes). Each box and whisker plot shows the median (central line), IQR (outer lines of box) and 10–90% range (error bars) of the individuals. Wilcoxon Rank Test was used to compare groups and p b 0.05 was considered significant.

phenotype and function of NK and T cell subsets isolated from HPV-associated genital warts and blood of women. Genital warts were larger in HIV-infected than uninfected women, containing similar frequencies of CD56Dim, CD56Bright and CD16+CD56− NK cells despite containing significantly lower frequencies of CD4 T cells. Despite being present at similar frequencies in HIV-infected and HIV negative warts, significantly lower frequencies of CD56Dim and CD56Bright NK cell subsets from HIV-infected women expressed CD107a and produced IFN-γ compared to HIV negative women, suggesting important functional defects in these anti-viral cells. While CD4 T cells were present at significantly lower frequencies in both blood and genital warts in HIV infected compared to uninfected women, the ability of these cells to produce IFN-γ in response to PMA/ionomycin stimulation was similar. The best known correlate of HPV-associated wart regression, described in the canine oral papillomavirus model, is CD4 T cell infiltration into warts [7,19]. Nicholls et al. (7) reported an influx of lymphocytes just prior to the regression of lesions, and that this influx reached a peak during the resolution of lesions. Nicholls et al., [7] noted that the dense infiltration of T cells happened at the basal region of the epidermis, and that the most abundant population was that of intraepithelial CD4 T cells. They hypothesized that the infiltration of CD4 T cells would facilitate lesion clearance through cytokine-induced activation of macrophages that subsequently kill HPV-infected keratinocytes. However,

Nicholls et al. [7] did not address how HPV-associated genital warts are resolved during CD4 T cell lymphopenia, such as that which occurs during chronic HIV disease. To our knowledge, no comparable studies identifying correlates of wart clearance or persistence have been carried out in humans. Whereas our cross-sectional study cannot infer causality, we discovered differences in functional responses of NK cells derived from genital warts of HIV + women compared to HIV − women. We also found lower frequencies of CD4 T cells among warts of HIV + women compared to those of HIV − women. Taken together, these results imply that it is entirely plausible that these defects may underlie the failure of HIV + women to clear warts. Nicolls et al. [7] suggested that CD4 T cells trafficked into canine oral warts and produced IFN-γ to resolve wart lesions. We found that among HIV + women lower frequencies of CD4 T cells in genital warts, rather than intrinsic decreased functionality, were associated with larger warts. Nicolls et al. [7] did not measure NK cell frequencies nor functionality, thus they could not comment on the role of NK cells in HPV pathogenesis during HIV co-infection. Our finding that HIV + women have multiple NK cell subset and functional deficiencies suggests that multiple factors may underlie wart persistence. It was interesting to note that many of the HIV − women developing warts in this study had CD4 + T-cells percentages (of CD3+ cells) in blood and genital warts of ~50% (suggesting a

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A) CD107a

B) IFN-

30

30

p=0.0008

p=0.01 p=0.0007

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20 p=0.006

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p=0.004

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10

0

0 CD56Bright

30

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CD56Dim CD16+CD56-

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CD56Dim CD16+CD56-

D) IFN30 p=0.02 p=0.0009

20

Blood

20 p=0.01

10

10

0

0 CD56Bright

CD56Dim CD16+CD56-

CD56Bright

CD56Dim CD16+CD56-

Figure 3 Comparison of distinct NK cell subset functions in genital warts (A–B) and blood (C–D) genital warts. PBMC and disaggregated wart-infiltrating cells were stimulated by the tumour cell line K562 to measure NK cell function (CD107a degranulation or IFN-γ production). HIV + women (HAART −: white boxes; HAART +: black boxes) and HIV − women (grey boxes). Each box and whisker plot shows the median (central line), IQR (outer lines of box) and 10–90% range (error bars) of the individuals. Wilcoxon Rank Test was used to compare groups and p b 0.05 was considered significant.

CD4:CD8 ratio of 1:1) which is lower than CD4+ cell frequencies which report typical healthy HIV− CD4:CD8 of N 2:1 [20,21]. This finding possibly suggests that HIV− women developing genital warts in our setting had lower CD4 counts than

Genital wart Blood

% NK cell response

30 p=0.01

20

p=0.01

p=0.01

10

0 CD107a+ IFN- + CD107a+ IFN- + CD107a+ IFN- +

CD56Bright

CD56Dim

CD16+CD56-

Figure 4 Comparison of genital wart (white boxes) and matched blood responses (grey boxes) by distinct NK cell subsets during HIV infection. Each box and whisker plot shows the median (central line), IQR (outer lines of box) and 10–90% range (error bars) of the individuals. Wilcoxon Rank Test was used to compare NK cells in warts to those in blood and p b 0.05 was considered significant.

previously published studies that may account for their inability to control wart development. It would be interesting in future studies to include genital tissue from healthy women without warts for comparison, to further investigate the impact of non-HIV associated CD4 deficiency on susceptibility to HPV wart progression. Impaired NK cell function and the expansion of dysfunctional NK cell subsets have been noted in HIV + individuals, with the extent of impairment being associated with plasma HIV loads [9,13,14]. While several studies have reported a significant reduction in the frequencies of CD56 + NK cells during HIV infections [22–24], the emergence of CD56 − CD16 + NK cells (particularly in individuals with high HIV loads in blood) has been shown to account for this apparent reduction in CD56 + cells [13,25]. We also noted both that CD56 − CD16 + NK cells were significantly higher in frequency in HIV + than HIV − women (particularly in blood, but also in genital warts), and we found that enrichment of this subset was associated with reduced frequencies of CD56Dim NK cells. Interestingly, as others have shown, we did not find that initiation of HAART normalized CD16+CD56 − NK cell subset frequencies in either blood or warts to those seen in HIV− women [26]. In our cohort, the insignificant differences in CD4 count between HAART-experienced and HAART-naïve HIV-infected women suggest either full immuno-competence was not restored by HAART or that the duration of HAART was not sufficient to enable restoration of full immunocompetence.

Defects in genital wart NK cells associated with HIV Table 2

NK subset function according to wart size. NK phenotype and function

HIV −

HIV +

217

Frequency of NK function according to wart size [Median % (IQR)]

N CD56Bright/Freq. CD107a CD56Bright/Freq. IFN-γ CD56Dim/Freq. CD107a CD56Dim/Freq. IFN-γ N CD56Bright/Freq. CD107a CD56Bright/Freq. IFN-γ CD56Dim/Freq. CD107a CD56Dim/Freq. IFN-γ

Mild

Moderate

Severe

12/16 (75.0%) 0.7 (0–1.5) 0.4 (0–0.8) 0.3 (0–0.7) 0.01 (0–0.03) 30/45*** (66.7%) 0.0 (0.0–1.2) 0.0 (0.0–0.3) 0.2 (0.0–6.0) 0.2 (0.3–1.2)

3/16 (18.8%) 4.8 (1.9–6.8)* 0.3 (0.03–0.4) 2.7 (1.0–4.0)** 0.03 (0.0–0.09) 13/45 (28.9%) 0.0 (0.0–1.7) 0.0 (0.0–0.4) 2.4 (0.6–14.0) 0.0 (0.0–1.0)

1/16 (6.3%) 0.0 (0.0–0.0) 0.0 (0.0–0.0) 0.0 (0.0–0.0) 0.0 (0.0–0.0) 2/45 (4.4%) 0.0 (0.0–0.02) 1.4 (0.2–3.5) 0.4 (0.0–1.4) 0.0 (0.0–0.2)

*p = 0.008; **p = 0.005; ***Wart size data was available for 45 HIV + women (31/37 HIV + HAART + and 14/14 HIV + HAART −).

immune reconstitution despite almost complete viral suppression [30–32] and this variability is dependent on the duration of HAART [33]. Poorer recovery of CD4+ T cell counts is associated with delayed initiation of HAART (especially in those with CD4+ T cell counts b 200 cells/ml). The frequency distribution of NK cell subsets found at effector sites such as the genital mucosa is likely to differ from those found in blood because of both their tissue location and the cytokine environment in which they exist [17]. CD56Dim NK cells are the predominant NK cell subset in the spleen and peripheral blood, whereas CD56Bright NK cells are predominant in lymph nodes and tonsils [34,35]. Previous studies have shown that NK cells are relatively abundant in the uterus during pregnancy, expressing CD56 but not CD16 [36,37]. Sentman et al. [17] have further differentiated NK cell phenotypes based on their position within the female

Our observation that higher proportions of functionally impaired NK cells are present in the genital warts of HIV + women has important biological implications. Previously CD56 − CD16 + NK cells have been shown to lack most NK cell functions [27]. Although we did observe that CD56 − CD16 + NK cells from HIV + women had significantly reduced IFN-γ producing capacity compared to HIV − women, they retained the ability to degranulate (i.e. they were CD107a +). We also found that wart-derived CD56dimCD16 + NK cells from HIV + women showed impaired degranulation compared to those from HIV − women. Following HAART initiation, most HIV + individuals show a gradual rise in CD4 T cell counts, although many do not attain pre-infection CD4 counts and remain immune-suppressed [28,29]. Approximately 7–20% of HIV+ individuals who have initiated HAART have been reported to have suboptimal

p=0.007

A

B

p=0,002 p=0.009

20

p=0.001

Genital wart

p=0.0001

% CD4 T cells IFN-

% CD4 T cells (of CD3)

80

60

40

20

10

Blood

5 4 3 2 1 0

0 HIV-

HIV+ HAART-

HIV+ HAART+

HIV-

HIV+ HAART-

HIV+ HAART+

Figure 5 Comparison of CD4 T cell frequencies and the abilities of these cells to produce IFN-γ in genital warts (white boxes) and blood (grey boxes) taken from HIV − and HIV + women (HAART − or HAART +). Each box and whisker plot shows the median (central line), IQR (outer lines of box) and 10–90% range (error bars) of the individuals. Wilcoxon Rank Test was used to compare groups and p b 0.05 was considered significant.

218 genital tract: upper genital tract NK cells were described as being CD56Bright while those in the lower genital tract being CD56Dim. We found that NK cells infiltrating genital warts were predominantly CD56Dim and that this reflected the dominant subset in blood. CD56Dim NK cells, which predominated in our study in both genital warts and blood, irrespective of HIV status, have previously been shown primarily to be cytotoxic killers rather than cytokine producers [14]. Consistent with this, we found both that NK cell degranulation (as evidenced by CD107a expression) was the predominant effector mechanism by this NK cell subset, and that frequencies of CD107a + NK cells were similar in genital warts to those observed in blood. We showed that CD107a expression by CD56Dim NK cells was positively associated with the severity of warts in both HIV+ and HIV− women, with NK cells from moderate warts degranulating more than those from smaller warts. Unlike CD56Dim NK cells or CD56Bright cells from HIV− women, we found that wart CD56Bright cells from HIV+ women lacked CD107a expression and were therefore apparently lacking in their ability to degranulate in response to stimulation with K562 cells. This is interesting because CD56Bright NK cells are reported primarily to produce cytokines rather than exhibit cytolytic activity [14]. Among HIV+ women in general, IFN-γ production was significantly lower in all wart-derived NK cell subsets relative to that observed by blood-derived NK cells. However, following in vitro stimulation, frequencies of IFN-γ-expressing NK cells derived from blood of HIV+ women were similar to or greater than those derived from warts of HIV− women. Others have also found that HIV infection reduces the likelihood of spontaneous HPV clearance and wart regression [10,11]. Thus, based on our data we conclude that this enables the establishment of chronic HPV infections through multiple dysfunctions in cytolytic activity and cytokine production by several NK cell subsets at the genital mucosa. This finding provides new insights into potential causes of HPV persistence. Although the initiation of HAART in HIV + women has been reported to offer some reconstitution of functional deficits among dysfunctional NK subsets (CD56 − CD16 +) [14,26], we did not observe this in our study. We observed no quantitative differences in NK cell function among women on HAART compared to those who were HAART −. Although we do not know the duration of HAART in this cohort or the CD4 counts at the time of HAART initiation, the fact that CD4 counts in women on HAART was not significantly higher than HAART naïve women suggests that CD4 reconstitution in HAART + women was not complete. It is possible that prolonged HAART or earlier HAART initiation would result in reversal of some of the NK cell defects we report in this study. The finding that IFN-γ responses by CD4 T cells were similar while absolute frequencies of CD4 T cells were significantly lower in warts from HIV + compared to HIV − women suggests that wart size or regression was not dependent on CD4 + IFN-γ producing cells. It is clear that women with persistent HPV infections who are unable to contain growth of potentially disfiguring genital warts are at the highest risk of developing cervical disease progressing to cancer. Given that HIV + women frequently fail to contain the proliferation of HPV-associated warts, it is imperative that we understand the underlying reasons for persistent HPV infections in these woman so that we can reduce the likelihood of them developing cervical cancer. The multiple

A. Bere et al. defects we have identified within the genital wart associated NK cell subsets of HIV + women is a small but important step towards the development of strategies combatting persistent secondary high risk HPV infections in immunecompromised women.

Conflict of interest statement The author(s) declare that there no conflicts of interest.

Acknowledgments This study was supported by the Cancer Association of South Africa (CANSA) and Poliomyelitis Research Foundation (PRF).

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