Peripheral and Local Human Papillomavirus 16–Specific CD8+ T-Cell Expansions Characterize Erosive Oral Lichen Planus

  • Author Footnotes
    13 The first two authors contributed equally to this work.
    Manuelle Viguier
    Footnotes
    13 The first two authors contributed equally to this work.
    Affiliations
    Antiviral Immunity Biotherapy and Vaccine Unit, Institut Pasteur, Paris, France

    Sorbonne Paris Cité Université Paris Diderot, Paris, France
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  • Author Footnotes
    13 The first two authors contributed equally to this work.
    Hervé Bachelez
    Correspondence
    Sorbonne Paris Cité Université Paris Diderot, Paris 75000, France
    Footnotes
    13 The first two authors contributed equally to this work.
    Affiliations
    Sorbonne Paris Cité Université Paris Diderot, Paris, France

    Université Paris Descartes, Imagine Institute, Necker Hospital, Paris, France
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  • Béatrice Poirier
    Affiliations
    Antiviral Immunity Biotherapy and Vaccine Unit, Institut Pasteur, Paris, France
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  • Jérémy Kagan
    Affiliations
    Centre de Physiopathologie de Toulouse Purpan, Toulouse, France

    INSERM, U1043, Toulouse, France

    CNRS, UMR5282, Toulouse, France

    Université Toulouse III Paul-Sabatier, Toulouse, France
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  • Maxime Battistella
    Affiliations
    Antiviral Immunity Biotherapy and Vaccine Unit, Institut Pasteur, Paris, France

    Université Paris Diderot, UMR-S728, INSERM, Paris, France
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  • François Aubin
    Affiliations
    Service de Dermatologie, Centre Hospitalier Universitaire, SFR FED 4234, EA 3181, Université de Franche Comté, Besançon, France
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  • Antoine Touzé
    Affiliations
    UMR 1282 INRA-Université François Rabelais, Tours, France
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  • Maryvonnick Carmagnat
    Affiliations
    Laboratoire d’Immunologie, Hôpital Saint-Louis, Paris, France
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  • Camille Francès
    Affiliations
    Service de Dermatologie-Allergologie, Hôpital Tenon, Université Paris VI, AP-HP, Paris, France
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  • Marie-Lise Gougeon
    Correspondence
    Sorbonne Paris Cité Université Paris Diderot, Paris 75000, FranceAntiviral Immunity Biotherapy and Vaccine Unit, Institut Pasteur, Paris 75724, France
    Affiliations
    Antiviral Immunity Biotherapy and Vaccine Unit, Institut Pasteur, Paris, France
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  • Author Footnotes
    14 The last two authors contributed equally to this work.
    Nicolas Fazilleau
    Correspondence
    Centre de Physiopathologie de Toulouse Purpan, Toulouse 31300, France
    Footnotes
    14 The last two authors contributed equally to this work.
    Affiliations
    Centre de Physiopathologie de Toulouse Purpan, Toulouse, France

    INSERM, U1043, Toulouse, France

    CNRS, UMR5282, Toulouse, France

    Université Toulouse III Paul-Sabatier, Toulouse, France
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  • Author Footnotes
    13 The first two authors contributed equally to this work.
    14 The last two authors contributed equally to this work.
      Erosive oral lichen planus (OLP) is a chronic, disabling mucocutaneous dysimmune rare disease characterized by mucosal inflammatory erosive lesions with pathological evidence for a marked CD8+ cytotoxic T-lymphocyte (CTL) infiltration. However, the specificity of lesional CTL in OLP has never been analyzed. To investigate the molecular mechanisms underlying dysregulation of T-cell immune responses in patients with OLP, we studied the diversity and antigen specificity of the TCR expressed by CD8+ T cells using dextramer staining, spectratyping, and TCR sequencing in 10 OLP patients undergoing extracorporeal photochemotherapy. Expansions of TCRVβ3-bearing CD8+ T cells were found in peripheral blood and in lesional tissues of OLP patients. Spectratyping and sequencing studies identified specific clonotypes in each patient. These expansions were enriched with human papillomavirus 16 (HPV16)–specific CD8+ T cells in HLA-A*0201+ patients as shown by their immune recognition of the E711-20 immunodominant epitope. Under treatment with extracorporeal photochemotherapy, clonotypic CD8+ T-cell expansions decreased in parallel with clinical remission. Altogether, these data establish a link between HPV infection and OLP pathogenesis by identifying a massive clonal expansion of CD8+ T cells with increased frequency of HPV 16–specific CD8+ T cells in OLP patients.

      Abbreviations

      CDR3
      complementary determining region 3
      CTL
      cytotoxic T lymphocyte
      ECP
      extracorporeal photochemotherapy
      HPV
      human papillomavirus
      OLP
      oral lichen planus
      PBMC
      peripheral blood mononuclear cell

      INTRODUCTION

      Lichen planus is a chronic inflammatory disease of unknown etiology involving the skin and/or mucous membranes, leading to a variety of clinical presentations (
      • Le Cleach L.
      • Chosidow O.
      Clinical practice. Lichen planus.
      ). Erosive oral lichen planus (OLP) is the most prevalent subtype, affecting 0.1 to 4% of the general population, and is characterized by a severely disabling course in its erosive form that often requires immunomodulatory systemic therapies such as oral steroids, immunosuppressants, and more recently extracorporeal photochemotherapy (ECP) (
      • Becherel P.A.
      • Bussel A.
      • Chosidow O.
      • et al.
      Extracorporeal photochemotherapy for chronic erosive lichen planus.
      ;
      • Edwards P.C.
      • Kelsch R.
      Oral lichen planus: clinical presentation and management.
      ;
      • Guyot A.D.
      • Farhi D.
      • Ingen-Housz-Oro S.
      • et al.
      Treatment of refractory erosive oral lichen planus with extracorporeal photochemotherapy: 12 cases.
      ). Pathologically, OLP is characterized by a predominant infiltration of immune cells, mainly CD8+ cytotoxic T lymphocytes (CTLs), associated with apoptosis of epithelial cells and disruption of the basement membrane zone (
      • Zhou X.J.
      • Sugerman P.B.
      • Savage N.W.
      • et al.
      Intra-epithelial CD8+ T cells and basement membrane disruption in oral lichen planus.
      ). Similarly, the presence in OLP lesions of CTL in the vicinity of damaged epithelial cells supports the hypothesis that a dysregulated CTL response of unknown antigen specificity has a key role in the pathogenesis of the disease (
      • Santoro A.
      • Majorana A.
      • Bardellini E.
      • et al.
      Cytotoxic molecule expression and epithelial cell apoptosis in oral and cutaneous lichen planus.
      ).
      However, the nature of these antigens remains controversial. Indeed, although autoreactivity of lesional skin–derived CTL against epithelial cells has been suggested in OLP patients (
      • Sugerman P.B.
      • Satterwhite K.
      • Bigby M.
      Autocytotoxic T-cell clones in lichen planus.
      ;
      • Gorsky M.
      • Epstein J.B.
      Oral lichen planus: malignant transformation and human papilloma virus: a review of potential clinical implications.
      ;
      • Syrjanen S.
      • Lodi G.
      • von Bultzingslowen I.
      • et al.
      Human papillomaviruses in oral carcinoma and oral potentially malignant disorders: a systematic review.
      ), other studies identified the presence of DNA from several human papillomavirus (HPV) subtypes in mucosal lesions of OLP, raising the issue of the self versus non-self nature of the target antigens in OLP (
      • Jontell M.
      • Watts S.
      • Wallstrom M.
      • et al.
      Human papilloma virus in erosive oral lichen planus.
      ;
      • Young S.K.
      • Min K.W.
      In situ DNA hybridization analysis of oral papillomas, leukoplakias, and carcinomas for human papillomavirus.
      ;
      • Miller C.S.
      • White D.K.
      • Royse D.D.
      In situ hybridization analysis of human papillomavirus in orofacial lesions using a consensus biotinylated probe.
      ;
      • Lodi G.
      • Scully C.
      • Carrozzo M.
      • et al.
      Current controversies in oral lichen planus: report of an international consensus meeting. Part 1. Viral infections and etiopathogenesis.
      ;
      • Yildirim B.
      • Senguven B.
      • Demir C.
      Prevalence of herpes simplex, Epstein Barr and human papilloma viruses in oral lichen planus.
      ). So far, the lack of any study using accurate tools to address the specificity of lesional CTL in OLP precludes any conclusion about mechanisms underlying this dysregulation. In this study, using dextramer, spectratyping, and sequencing in 10 patients with severe erosive OLP, we investigated the antigen specificity of the CD8+ T-cell expansions.

      RESULTS AND DISCUSSION

       TCRVβ3+ clonal expansions alter CD8+ T-cell repertoires of OLP patients at flare

      To understand the molecular events that govern the physiopathology of OLP, we investigated the T-cell repertoire of 10 patients with OLP. At flare, the usage of each TCRVβ gene segment, as estimated by quantitative real-time reverse-transcriptase–PCR in CD4+ and CD8+ peripheral blood T cells, revealed striking predominance of the TCRVβ3 gene segment in CD8+ T cells (26.49±4.81%), whereas it was used at a much lower level in the circulating CD4+ counterparts (11.49±2.24%) or in both isolated CD4+ and CD8+ T cells from healthy donors (Figure 1a). Results from quantitative real-time reverse-transcriptase–PCR were confirmed by flow cytometry, showing predominance of TCRVβ3 expression in the peripheral blood CD8+ T lymphocytes (data not shown, 17.27±4.51%).
      Figure thumbnail gr1
      Figure 1TCRVβ3+ clonal expansions skewed peripheral and lesional CD8+ T-cell repertoires of oral lichen planus (OLP) patients at flare. (a) Reverse-transcriptase–PCR (RT–PCR) amplifications of the TCRVβ3 usage in CD4+ and CD8+ T cells from the blood of healthy donors (HDs) or OLP patients. PBMC, peripheral blood mononuclear cell. (b) CDR3β length distributions for CD4+ and CD8+ Vβ3-Cβ rearrangements from a representative HD and a representative OLP patient. The peak of the 11th codon is marked on the abscissa axis. (c) Table of CDR3 clonotypes identified in each OLP patient (nucleotidic and amino-acid (AA) sequences). (d) CDR3β length distributions (left) and clonotype distribution estimated by sequencing (right) in lichen lesions in three representative OLP patients (OLP nos. 2, 4, and 5). *P<0.05, **P<0.01.
      The quantitative PCR products were further used for the amplification of dye-labeled oligonucleotides specific for Cβ and allowed to assess the complementary determining region 3 (CDR3) length distribution. For each TCRVβ gene segment tested and for the TCRVβ3 gene segment from CD4+ and CD8+ T cells from healthy donors or from CD4+ T cells isolated from OLP patients (Figure 1b), CDR3β length distribution profiles displayed Gaussian-like curves, the hallmark of a polyclonal T-cell repertoire. In contrast, CDR3β distribution profiles of the TCRVβ3 gene segment were altered with single expansions in blood CD8+ T cells from all OLP patients, as observed in OLP no. 5 for a CDR3β size of 11 amino acids (Figure 1b). Therefore, we determined the Vβ-Jβ rearrangement corresponding to the observed clonal expansion and further cloned and sequenced it. As shown in Figure 1c, one single CDR3β sequence was found in each patient. Furthermore, we revealed that similar TCRVβ3+ clonal expansions were also present in tissue lesions of three OLP patients (Figure 1d). Overall, neither CDR3β length nor consensus sequence was shared across all OLP patients. However, all detected CD8+ clonal expansions were using the TCRVβ3 gene segment, even in patients not sharing common class I HLA alleles (Supplementary Table S1 online). Interestingly, 5 out of the 10 clonotypic TCRs were composed of the Vβ-Jβ2.7 rearrangement, and one clonotypic sequence was found in two different patients (OLP nos. 2 and 8; Figure 1c). Of note, flow cytometric characterization of TCRVβ3+CD8+ T cells in peripheral blood mononuclear cells (PBMCs) confirmed their activated status as shown by surface expression of HLA-DR (data not shown, 20.99±4.52%, n=7), CD38 (24.85±6.18%, n=4), and perforin content (15.2±4.54%, n=7). Altogether, these data show that clonotypic TCRVβ3+CD8+ T-cell expansions are detected in situ and in the blood of OLP patients at flare. These sets of data extend previous observations reported in two studies, one reporting the increased presence of TCRVβ3- and TCRVα2-positive cells in the oral mucosal T-cell infiltrate of 7 OLP patients (
      • Simark-Mattsson C.
      • Bergenholtz G.
      • Jontell M.
      • et al.
      T cell receptor V-gene usage in oral lichen planus; increased frequency of T cell receptors expressing V alpha 2 and V beta 3.
      ), and the other performed in 12 OLP patients showing that CD8+ T cells had skewed TCR repertoire toward the usage of a restricted set of TCRVβ segments including TCRVβ3 (
      • Gotoh A.
      • Hamada Y.
      • Shiobara N.
      • et al.
      Skew in T cell receptor usage with polyclonal expansion in lesions of oral lichen planus without hepatitis C virus infection.
      ).

       Tracking of CD8+TCRVβ3+ clonal expansions following disease activity in OLP patients

      We next reasoned that the CD8+ clonal expansions could actively participate in the physiopathology of OLP and tested this hypothesis by performing a longitudinal study of the TCR repertoire in all OLP patients. PBMCs were isolated at different time points corresponding to distinct clinical status: flare (before starting ECP), partial remission, complete remission following ECP, and relapse after ECP withdrawal. For all collected samples, TCRVβ usage and CDR3β length distribution were performed as well as sequencing of Vβ-Jβ rearrangements corresponding to the clonal expansions at flare (Figure 1c). This allowed a follow-up of peaks corresponding to TCRVβ3+ clonal expansions in the T-cell compartment (Figure 2 and Supplementary Figure S1 online). For three patients (OLP nos. 5, 6 (Figure 2) and 8 (Supplementary Figure S1 online)), we observed a disappearance of the peaks corresponding to the TCRVβ3+ clonal expansions that strictly correlated with a clinical improvement (either partial remission or complete remission). However, for the seven remaining OLP patients (as an example OLP no. 4, Figure 2), CDR3 distribution profiles were all disturbed irrespective of the clinical status of the patients. Nevertheless, we found a strong correlation between the clonotype usage and the clinical status of OLP patients for 8 out of 10 patients. A pronounced decrease or disappearance of the frequency of TCRVβ3+ clonotype was observed in OLP patients 1, 4, 5, 6, and 8, whereas a slight decrease was found in OLP cases 2, 3, and 7 (Figure 2 and Supplementary Figure S1 online). Moreover, the clonotype identified at initial flare was also found at high frequency at the time of clinical relapse (OLP nos. 4, and 5 (Figure 2) and nos. 9 and 10 (Supplementary Figure S1 online)). Overall, these data support that clinical remission obtained with ECP in OLP is associated in most cases with a decrease or disappearance of TCRVβ3 clonotypic CD8+ T-cell expansions, suggesting the pathogenic relevance of these clonotypic expansions.
      Figure thumbnail gr2
      Figure 2Decrease or disappearance of TCRVβ3+ clonal expansions over the clinical course of the disease in oral lichen planus (OLP) patients. CDR3β length distributions (left column) for different CD8+ Vβ3-Jβ rearrangements over the clinical course of the disease following extracorporeal photochemotherapy (ECP) and clonotype distribution estimated by sequencing (right column) for three different representative OLP patients (OLP nos. 4, 5, and 6). CR, complete remission; PR, partial remission.

       CD8+ T cells expressing clonal TCRVβ3+ in OLP patients are specific for HPV16

      Lesional CTLs from OLP patients are suspected to recognize an antigen associated with major histocompatibility complex class I molecule on lesional keratinocytes (
      • Sugerman P.B.
      • Satterwhite K.
      • Bigby M.
      Autocytotoxic T-cell clones in lichen planus.
      ). Furthermore, several studies have shown the presence of several HPV subtypes in mucosal OLP lesions (
      • Jontell M.
      • Watts S.
      • Wallstrom M.
      • et al.
      Human papilloma virus in erosive oral lichen planus.
      ;
      • Young S.K.
      • Min K.W.
      In situ DNA hybridization analysis of oral papillomas, leukoplakias, and carcinomas for human papillomavirus.
      ;
      • Miller C.S.
      • White D.K.
      • Royse D.D.
      In situ hybridization analysis of human papillomavirus in orofacial lesions using a consensus biotinylated probe.
      ;
      • Lodi G.
      • Scully C.
      • Carrozzo M.
      • et al.
      Current controversies in oral lichen planus: report of an international consensus meeting. Part 1. Viral infections and etiopathogenesis.
      ;
      • Yildirim B.
      • Senguven B.
      • Demir C.
      Prevalence of herpes simplex, Epstein Barr and human papilloma viruses in oral lichen planus.
      ). Detection of current or past HPV infection could be performed in 6 out of our 10 patients either on tissue biopsies or by serology. Three out of six OLP tested patients were found HPV positive and, among them, HPV16 DNA and/or HPV16-specific IgG were found in 2 out of the 3 HPV-positive patients (OLP nos. 2 and 5). To test whether TCRVβ3+ clonal expansions might exhibit HPV specificity, we stained PBMCs from the five HLA-A*0201+ OLP patients of the cohort with peptide-containing major histocompatibility complex class I dextramer. For all these patients, a distinct population of CD8+ T cells expressing a TCRVβ3 specific for the E711-20 immunodominant peptide of HPV16 was detected, as shown for OLP no. 2 at flare or OLP no. 4 under ECP (Figure 3a; mean percentage of CD8+Dext-HPV+ in TCRVβ3+CD3+ cells: 2.62±1.7%, n=5). As expected, almost no TCRVβ3+ T cells were stained with HLA-A2/HIV-p17 Gag dextramer in these HIV-negative patients (Figure 3a; CD8+Dext-HIV+: 0.64±0.3%, n=5). We then sorted out HPV-specific CD8+ TCRVβ3+ cells as well as their CD8+ TCRVβ3+ Dext-HPV- counterparts from OLP nos. 3, 4, and 7. Spectratyping analysis and CDR3β sequencing were performed, revealing a striking enrichment of Dext-HPV+ populations with the clonotype in contrast with Dext-HPV- subset that showed no enrichment (Figure 3b). In the last series of experiments, we performed in situ immunostaining on OLP lesion from OLP no. 2. The presence of cells expressing CD3, CD8, and the cytotoxic marker TIA1 (T-cell-restricted intracellular antigen 1) were detected (Figure 4a), some of these bearing TCR specific for HLA-A2/HPV E711-20 peptide, whereas staining with the irrelevant Dext-HIV yielded negative results in the same patient (Figure 4b).
      Figure thumbnail gr3
      Figure 3A subpopulation of peripheral CD8+ T cells from oral lichen planus (OLP) patients is human papillomavirus 16 (HPV16) specific and is enriched in TCRVβ3+ clonal expansions. (a) Flow cytometric detection using dextramer of HIV- and HPV-specific CD8+ T cells in CD3+ Vβ3+ cells from the blood of two representative HLA-A*0201+ OLP patients (OLP nos. 2 and 4). (b) CDR3β length distributions for Vβ3-Cβ rearrangements on unsorted T cells or isolated T cells with HPV-Dextramer (left column) and clonotype distribution estimated by CDR3 sequencing (right column) for three different representative HLA-A*0201+ OLP patients (OLP nos. 7, 3, and 4).
      Figure thumbnail gr4
      Figure 4Human papillomavirus 16 (HPV16)–specific CD8+ T cells infiltrate lichen lesions. (a) The in situ immunostainings of CD3, CD8 (original magnification × 200, scale bars=100 μm), and TIA-1 (T-cell-restricted intracellular antigen 1) (original magnification × 400, scale bar=50 μm) on a biopsy taken from mucosal lesions in oral lichen planus (OLP) patient no. 2. (b) The in situ immunostainings using either dextramer HIV-1 (left) or dextramer HPV (right) ( × 630, scale bars=25 μm; inset original magnification × 1,000, scale bars=10 μm).
      Overall, these data demonstrate that a notable proportion of clonal CD8+ blood T cells of OLP patients are HPV16 specific, also infiltrating mucosal and/or skin lesions, and decreasing or disappearing following clinical remission, suggesting their pathogenic role in OLP. These results are reminiscent of results from previous in vitro studies showing the capacity of this E711-20 HPV16 epitope to favor the stimulation and expansion of CTL clones expressing TCRVβ3, without evidence of HLA class I restriction (
      • Nilges K.
      • Hohn H.
      • Pilch H.
      • et al.
      Human papillomavirus type 16 E7 peptide-directed CD8+ T cells from patients with cervical cancer are cross-reactive with the coronavirus NS2 protein.
      ;
      • Schreurs M.W.
      • Scholten K.B.
      • Kueter E.W.
      • et al.
      In vitro generation and life span extension of human papillomavirus type 16-specific, healthy donor-derived CTL clones.
      ). These sets of data also raise the HPV infection status in OLP patients. Similarly, a large set of published HPV detection studies support a pathogenic contribution of some HPV subtypes in OLP (
      • Gorsky M.
      • Epstein J.B.
      Oral lichen planus: malignant transformation and human papilloma virus: a review of potential clinical implications.
      ;
      • Syrjanen S.
      • Lodi G.
      • von Bultzingslowen I.
      • et al.
      Human papillomaviruses in oral carcinoma and oral potentially malignant disorders: a systematic review.
      ). Notably, detection of viral DNA mainly derived from HPV6/11/16/31/33 has been reported in mucosal oral and/or genital lesions from OLP patients in several studies, providing a rationale for their role as putative antigenic stimuli (
      • Lodi G.
      • Scully C.
      • Carrozzo M.
      • et al.
      Current controversies in oral lichen planus: report of an international consensus meeting. Part 1. Viral infections and etiopathogenesis.
      ;
      • Mattila R.
      • Rautava J.
      • Syrjanen S.
      Human papillomavirus in oral atrophic lichen planus lesions.
      ). This latter hypothesis has been reinforced by a recent exhaustive systematic review of HPV detection in 39 selected cross-sectional studies, showing that HPV DNA is significantly more frequently detected in OLP lesions compared with controls (
      • Syrjanen S.
      • Lodi G.
      • von Bultzingslowen I.
      • et al.
      Human papillomaviruses in oral carcinoma and oral potentially malignant disorders: a systematic review.
      ). Indeed, the positive detection of previous or current infection with HPV16 in 2/6 of our patients, including 1 patient who showed clear expansion of HPV16 E711-20–specific cells in blood and lesional mucosa, is a strong support for the contribution of HPV as an antigenic stimulus of CTL expansion that characterizes severe erosive OLP. Finally, the presence of HPV16 E711-20–specific CD8+ T cells in the vicinity of dying epithelial cells in a studied patient correlates with their potential involvement in tissue damage of OLP, even though this latter observation does not rule out the deleterious role of other subtypes than HPV16 that were not addressed in our patients. As some of our patients did not show either the presence of HPV16 DNA or the serological evidence of previous HPV16 infection, it remains to be determined whether other HPV subtypes or even alternative viral agents would be able to trigger similar CTL expansions in OLP (
      • Lodi G.
      • Scully C.
      • Carrozzo M.
      • et al.
      Current controversies in oral lichen planus: report of an international consensus meeting. Part 1. Viral infections and etiopathogenesis.
      ). Nevertheless, HPV serology is a poor marker of current infection and HPV DNA detection can be rendered difficult, as most HPV infections are transient (
      • Ho G.Y.
      • Bierman R.
      • Beardsley L.
      • et al.
      Natural history of cervicovaginal papillomavirus infection in young women.
      ;
      • Vaccarella S.
      • Franceschi S.
      • Clifford G.M.
      • et al.
      Seroprevalence of antibodies against human papillomavirus (HPV) types 16 and 18 in four continents: the International Agency for Research on Cancer HPV Prevalence Surveys.
      ).
      As most adult individuals are infected at some point with one or several HPV subtypes, the present results also raise the mechanisms underlying dysimmunity in OLP patients. Supporting the autoimmune hypothesis and its possible link with HPV is the previously established presence among lesional T lymphocytes of two OLP patients of CTL clones reacting toward autologuous keratinocytes immortalized with HPV16 E6 and E7 (
      • Sugerman P.B.
      • Satterwhite K.
      • Bigby M.
      Autocytotoxic T-cell clones in lichen planus.
      ). It is worth noticing that a causal role for HPV infection in OLP does not necessarily require constant tissue viral replication, as an initial viral stimulation might be sufficient to trigger the founder immune response, with following autoimmune T-cell expansions related to molecular mimicry, unsequestration of masked self epitopes, or both (
      • Selmi C.
      • Leung P.S.
      • Sherr D.H.
      • et al.
      Mechanisms of environmental influence on human autoimmunity: a National Institute of Environmental Health Sciences expert panel workshop.
      ). As such, it might explain why we were able to detect antibodies to HPV16 in the sera of only two patients of our cohort. In line with the former of these hypotheses, molecular mimicry between HPV16 E7 protein and human self has been revealed by computer-based analyses, providing a rational basis for further investigations of OLP lesional CTL regarding their immunoreactivity toward HPV versus self-candidate antigens (
      • Natale C.
      • Giannini T.
      • Lucchese A.
      • et al.
      Computer-assisted analysis of molecular mimicry between human papillomavirus 16 E7 oncoprotein and human protein sequences.
      ). However, alternative mechanisms such as alterations of the TCRVβ3+/HLA-peptide synapse by some viral products cannot be ruled out. Finally, it also remains to be determined whether HPV and/or other viral stimuli are involved in other forms of lichen planus. Our data also suggest that prophylactic HPV16 vaccine in addition to preventing cervical dysplasia and cancer may also protect against OLP.

      Materials and Methods

       Patients and healthy donors

      Ten patients with OLP treated with ECP were enrolled. They consisted of 8 women and 2 men (mean age: 56.5 years (range: 25–78)) (Supplementary Table S1 online). The characteristics of the eight patients have been previously reported (
      • Guyot A.D.
      • Farhi D.
      • Ingen-Housz-Oro S.
      • et al.
      Treatment of refractory erosive oral lichen planus with extracorporeal photochemotherapy: 12 cases.
      ). Previous treatment with glucocorticoids and immunosuppressive agents was stopped 4 weeks before ECP initiation. All patients reached remission under ECP, either complete remission or partial remission (defined by regression of lesions of at least 50%) (
      • Guyot A.D.
      • Farhi D.
      • Ingen-Housz-Oro S.
      • et al.
      Treatment of refractory erosive oral lichen planus with extracorporeal photochemotherapy: 12 cases.
      ). Clinical statuses were determined at the time of blood collection. The Institutional Review Board (Comité de Protection des Personnes Ile de France IV, 2009/10NI) approved the study that was qualified as noninterventional. The patients were fully informed and had no objection to participate in the study. Written patient consent was not required because French law considers that it is not mandatory for noninterventional biological collection for immunological and molecular research, with the exception of genetic studies on germinal DNA. Healthy donors were blood donors from the Etablissement Français du Sang.

       Preparation of PBMCs

      Blood was collected at baseline before any ECP treatment (“flare”) and at several time points. PBMCs were prepared by Ficoll-Hypaque density gradient centrifugation and cryopreserved in liquid nitrogen in 8% dimethylsulfoxide, 42% fetal calf serum, and 50% RPMI-1640 medium (Invitrogen, Carlsbad, CA).

       Antibodies and flow cytometric analysis

      Anti-CD3 FITC (Clone BW264/56, Miltenyi Biotec, Bergisch Gladbech, Germany), anti-CD8-APC (SK1, BD Pharmingen, Franklin Lakes, NJ), Dextramer-HPV E711-20-PE (Dext-HPV PE; HLA-A*0201; YMLDLQPETT, Immudex, Copenhagen, Denmark), Dextramer-HIV-1 P17 Gag 77-85-PE (Dext-HIV-PE; HLA-A*0201, Bayport, MN; SLYNTVATL, Immudex), and anti-TCR-Vβ3-FITC (JOVI-3, Ancell, Bayport, MN) were used. Cells were incubated with dextramer for 10 minutes at room temperature, and then antibodies were added for 30 minutes at 4 °C. Stained cells were washed with phosphate-buffered saline/0.5% BSA/0.1% NaN3 and analyzed using a FACSCalibur flow cytometer (Becton Dickinson,Franklin Lakes, NJ).

       Purification of T-lymphocyte subsets

      Dead cells were removed using the MACS Dead Cell Removal kit (Miltenyi Biotec). T cells were isolated using anti-CD8 or anti-CD4 antibody-coated immunomagnetic beads and stabilized in PrepProtect buffer (Miltenyi Biotec). Cell purities exceeded 85%. CD3+CD8+TCRVβ3+Dext-HPV+ or Dext-HPV- populations were isolated using a MoFlo Astrios cytometer cell sorter (Beckman Coulter, Villepinte, France).

       TCR repertoire analysis

      RNA was extracted using RNAeasy kit (Qiagen, Venlo, The Netherlands) either from sorted PBMCs or from total skin or mucosal OLP lesions and reverse-transcribed into complementary DNA using Superscript II (Invitrogen Life Technologies, Carlsbad, CA). Quantitative PCR amplifications were performed with TaqMan Universal PCR Master Mix (Applied Biosystems, Carlsbad, CA), TCRVβ gene segment–specific oligonucleotides, TCRCβ-specific antisense primer, and a fluorescent probe specific for the TCRCβ gene segment (
      • Fazilleau N.
      • Bachelez H.
      • Gougeon M.L.
      • et al.
      Cutting edge: size and diversity of CD4+CD25high Foxp3+ regulatory T cell repertoire in humans: evidence for similarities and partial overlapping with CD4+CD25- T cells.
      ).

       Cloning and sequencing of CDR3β rearrangements

      Vβ-Jβ PCR products were cloned in pCR 4Blunt TOPO vector (Invitrogen Life Technologies). Sequencing reactions were performed using the ABI PRISM Big Dye Terminator Reaction Kit (Applied Biosystems). Reaction products were analyzed on an ABI 3130XL 16 capillaries (Applied Biosystems).

       Detection of HPV subtypes

      Tissue samples were obtained either atraumatically using a cytobrush (DNAPAP Cervical Sampler; Digene, Qiagen) secondarily placed in Cervical Specimen Transport Medium (Digene, Qiagen) or by tissue biopsy secondarily frozen. DNA extraction was performed using the QIAGEN BioRobot EZ1 with the EZ1 DNA Tissue Kit (Qiagen). Human papillomavirus detection was performed using two highly sensitive and specific assays combining a specific multiplex PCR with DNA microarray primer extension as described previously (
      • Schmitt M.
      • Bravo I.G.
      • Snijders P.J.
      • et al.
      Bead-based multiplex genotyping of human papillomaviruses.
      ;
      • Ruer J.B.
      • Pepin L.
      • Gheit T.
      • et al.
      Detection of alpha- and beta-human papillomavirus (HPV) in cutaneous melanoma: a matched and controlled study using specific multiplex PCR combined with DNA microarray primer extension.
      ). Multiplex PCR reactions were performed for the amplification of α-HPV, β-HPV, γ-HPV, and μ-HPV types, respectively. The assays were combined in one single chip to detect 27 HPV types from genus α, 25 cutaneous HPV types from genus β, 16 cutaneous HPV types from genus γ, and one from genus μ. Following PCR amplification, 10 μl of each reaction mixture was analyzed using the Luminex technology (Luminex, Austin, TX). The detection limit was determined using the cutoff of 10 median reporter fluorescence intensity and varied between 100 and 800 pg of PCR products (
      • Schmitt M.
      • Bravo I.G.
      • Snijders P.J.
      • et al.
      Bead-based multiplex genotyping of human papillomaviruses.
      ). For each sample, three multiplex PCR reactions were performed. Search for serum IgG antibodies against HPV16 major capsid protein L1 was performed using ELISA (
      • Combita A.L.
      • Bravo M.M.
      • Touze A.
      • et al.
      Serologic response to human oncogenic papillomavirus types 16, 18, 31, 33, 39, 58 and 59 virus-like particles in colombian women with invasive cervical cancer.
      ). Briefly, serum samples were tested in triplicate in a 1:20 dilution, and virus like particle-bound antibodies were detected using anti-human IgG conjugated to peroxidase diluted at 1:20,000 (Southern Biotech, Birmingham, AL). Cutoff value for positivity based on sample from young children (mean reactivity plus 3 SD) was 0.2. Previously tested samples were retested on the assay plate to confirm ensibility and consistency of the assay (
      • Ferguson M.
      • Heath A.
      • Johnes S.
      • et al.
      Results of the first WHO international collaborative study on the standardization of the detection of antibodies to human papillomaviruses.
      ).

       In situ immunostaining

      Acetone-fixed cryosections were incubated with dextramer for 75 minutes at room temperature and mounted with VECTASHIELD Mouting Medium with 4',6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA). Slides were studied using an Axiovert 200M microscope with MRm camera (Zeiss, Oberbochen, Germany).

       Statistical analysis

      Unpaired t-tests were calculated using Prism software (Irvine, CA).

      Acknowledgments

      We are indebted to Laure Buisson for her help in sequencing analyses (UDEAR, CNRS, France), Pierre-Henri Commère for cell sorting (Flow Cytometry Facility, Pasteur Institute), Nathalie Parquet (Saint-Louis Hospital, Paris, France) and Claire Rabian (Saint-Louis Hospital, Paris, France) for their help in cell collection and cell preparation, and to Tarik Gheit (International Agency for Research in Cancer, Lyon, France) for HPV genotyping. This work was supported by Institut Pasteur, the Société Française de Dermatologie, AVENIR INSERM (NF), Association pour la Recherche sur le Cancer (ARC AO 2009, NF), la Ligue Nationale Contre le Cancer (NF), Conseil Régional Midi-Pyrénées (NF), Institut National contre le Cancer (PLBIO10-195 and INCA-6530, NF), Collery Grant from Académie Nationale de Médecine (MB), and International Re-integration Grant Marie Curie.

      SUPPLEMENTARY MATERIAL

      Supplementary material is linked to the online version of the paper at http://www.nature.com/jid

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