Melanocyte-Associated T Cell Epitopes Can Function As Autoantigens for Transfer of Alopecia Areata to Human Scalp Explants on Prkdcscid Mice

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Melanocyte-Associated T Cell Epitopes Can Function as Autoantigens for Transfer of Alopecia Areata to Human Scalp Explants on Prkdcscid Mice

Amos Gilhar, Marina Landau,* Bedia Assy, Raya Shalaginov, Sima Sera®movich, and Richard S. Kalish² Skin Research Laboratories, Flieman Medical Center and Rappaport Faculty of Medicine, Technion Institute of Technology, Haifa, Israel; *Department of Dermatology, Elias Sourasky Medical Center, Tel Aviv, Israel; ²Department of Dermatology, State University of New York at Stony Brook, U.S.A.

Alopecia areata is a tissue restricted autoimmune restricted melanocyte peptide epitopes were then condition affecting the hair follicle, resulting in hair tested with lesional scalp T cells from HLA-A2-posi- loss. The goal of this study was to test the hypothesis tive alopecia areata patients. Melanocyte-peptide- that the autoantigen of alopecia areata is melanocyte activated T cells signi®cantly reduced the number of associated. Potential autoantigens were tested in the hairs regrowing in two experiments with six patients human scalp explant/Prkdscid CB-17 mouse transfer (p < 0.001 by ANOVA). Injected scalp grafts showed system. Scalp T cells from lesional (bald) alopecia histologic and immunochemical changes of alopecia areata scalp were cultured with antigen-presenting areata. The most consistent peptide autoantigens cells, and antigen, along with interleukin-2. The were the Gp100-derived G9-209 and G9-280 T cells were then injected into autologous lesional peptides, as well as MART-1 (27±35). Melanocyte scalp grafts on SCID mice, and hair regrowth was peptide epitopes can function as autoantigens for measured. Hair follicle homogenate was used as an alopecia areata. Multiple peptides were recognized, autoantigen control. T cells cultured with melanoma suggesting epitope spreading. Key words: autoimmu- homogenate induced statistically signi®cant reduc- nity/human/peptides/skin. J Invest Dermatol 117:1357± tion in hair growth (p < 0.01 by ANOVA). HLA-A2- 1362, 2001

lopecia areata is a tissue restricted autoimmune follicular expression of DR and ICAM-1 is induced by interferon-g condition directed at the hair follicle, resulting in hair (INF-g). Lesional scalp from alopecia areata patients grafted onto loss. Patients frequently suffer severe psychiatric nude mice regrows hair coincident with a loss of in®ltrating consequences (Colon et al, 1991; Koo et al, 1994). lymphocytes from the graft (Gilhar and Krueger, 1987). Alopecia AThis is especially true of girls and young women who areata responds to immunosuppressive doses of systemic steroids. become bald. The incidence of alopecia areata in the U.S.A. The condition also has an association with additional autoimmune (Minnesota) is 20.2 per 100,000 person-years, with a lifetime risk of processes such as autoimmune thyroiditis (Milgraum et al, 1987) approximately 1.7% (Safavi et al, 1995). There is no signi®cant and vitiligo (Shellow et al, 1992). Circulating autoantibodies to gender difference. The disease is often chronic with a remitting, follicular structures are present in alopecia areata, but they are also relapsing course. Although it responds to immunosuppression, reported in normal controls (Tobin et al, 1997), and there is no generalized immunosuppression has signi®cant morbidity and consistent pattern of reactivity of these antibodies. Furthermore, it treatment is frequently frustrating and not successful. New is not possible to transfer alopecia areata by injection of IgG into treatment options are essential. With evidence that alopecia areata human skin explants on nude mice (Gilhar et al, 1992). is a T-lymphocyte-mediated autoimmune condition, it has become Severe combined immunode®ciency (SCID) mice do not reject a model system for the study of pathogenesis and treatment of human lymphocytes or skin. SCID mice grafted with human skin T-cell-mediated autoimmunity, and as such is a model for a host and injected with human lymphocytes can be used to study the of additional T-cell-mediated autoimmune conditions. immunopathogenesis of human skin conditions, and have been Hair loss in alopecia areata is associated with a perifollicular used to demonstrate the role of lymphocytes in induction lymphocytic in®ltrate made up primarily of CD4+ cells, with a (Boehncke et al, 1996; Wrone-Smith and Nickoloff, 1996) and CD8+ intrafollicular in®ltrate (Todes-Taylor et al, 1984), along maintenance (Gilhar et al, 1997) of psoriasis lesions. We have since with expression of both HLA-DR and intercellular adhesion extended this model to the use of human scalp explants on SCID molecule 1 (ICAM-1) on the follicular epithelium (Messenger and mice to study alopecia areata. Bleehen, 1985; Khoury et al, 1988). It is presumed that the We have shown that it is possible to transfer alopecia areata to human scalp explants on SCID mice by the injection of scalp- Manuscript received May 31, 2001; revised August 2, 2001; accepted for in®ltrating T lymphocytes (Gilhar et al, 1998). It was necessary to publication August 14, 2001. ®rst activate the T lymphocytes in vitro by culture with hair follicle Reprint requests to: Dr. Amos Gilhar, Laboratory for Skin Research, homogenate in the presence of antigen-presenting cells (APC). Rappaport Building, Technion Faculty of Medicine, P.O.B. 9649, Bat- Culture with nonfollicular scalp homogenate did not induce hair Galim, Haifa 31096, Israel. loss (Gilhar et al, 1999). Thus, alopecia areata is mediated by

0022-202X/01/$15.00 ´ Copyright # 2001 by The Society for Investigative Dermatology, Inc. 1357 1358 GILHAR ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

T lymphocytes that recognize a hair follicle antigen. We have since Table I. Clinical summary of alopecia areata patients found that optimal transfer of hair loss requires both CD4+ and CD8+ T cells (Gilhar et al, in press). Depletion of either CD8+ T cells (McElwee et al, 1996) or CD4+ T cells (McElwee et al, 1999) Patient Age Sex Alopecia subtype Duration can reverse alopecia areata in the Dundee experimental bald rat, A 31 F Severea 8 y supporting a synergy or cooperation between CD8+ and CD4+ T B 32 M Severe 4 y cells. In¯ammatory T cells of alopecia areata are cytotoxic and C 26 F Severe 10 y possess both the Fas/Fas ligand and granzyme B cytotoxic D 26 F Severe 10 y mechanisms (Bodemer et al, 2000). It is proposed that the CD4+ 1 23 M Severe 7 y T cells provide help for the effector function of the CD8+ T cells. 2 39 F Severe 2 y It is hypothesized that the autoantigen of alopecia areata is 3 36 M Severe 2 y melanocyte derived (Paus et al, 1994). The initial basis for this is the 4 32 M Totalis 6 y frequent clinical observation that with disease activity pigmented 5 22 F Severe 10 y hairs are lost preferentially to nonpigmented (e.g., white) hairs. 6 31 M Totalis 10 y Furthermore, with regrowth, there is a tendency for the initial regrowing hairs to be white. Melanocytes are a signi®cant aSevere alopecia areata is de®ned as large areas of alopecia with small residual areas of hair, or nearly complete alopecia totalis (76%±99% hair loss). Donors A±D component of the hair bulb, which is the site of immunologic were studied in the melanoma homogenate experiment. Donors 1±6 are HLA-A2 attack. Furthermore, there is an association of alopecia areata with positive and were studied in the melanomo peptide experiments. vitiligo (Shong and Kim, 1991; Shellow et al, 1992), and hair bulb melanocytes in alopecia areata show both histologic and ultrastructural abnormalities (Tobin et al, 1990). It is possible to induce Punch biopsies (2 mm) from involved scalp were obtained from each lymphocyte-mediated alopecia in mice immunized against mela- patient. Four biopsies from each patient were used for the isolation of nocyte-associated (e.g., melanoma) antigens (Becker et al, 1996). cutaneous T cells. Two biopsies were snap-frozen in liquid nitrogen for There is a large literature on melanocyte-associated T cell immunoperoxidase staining. One frozen biopsy was sectioned vertically, epitopes generated by the search for melanoma-associated antigens and the other was sectioned horizontally. An additional biopsy from each (Bakker et al, 1995; Rivoltini et al, 1995; Parkhurst et al, 1996; patient was submitted for routine histologic examination with hematox- Salgaller et al, 1996; Fleischhauer et al, 1997; Salazar-Onfray et al, ylin and eosin and horizontally sectioned in its entirety. Punch biopsies 1997; Bettinotti et al, 1998; Kittlesen et al, 1998). Most interest has (2 mm) were grafted to SCID mice in sets of three biopsies per mouse, focused on peptides presented by HLA-A2, which is present in one mouse per group per subject. approximately half the population1. HLA-A2 may also be associated Animals CB-17 Prkdcscid (SCID) mice (Charles River, U.K.), 2± with alopecia areata (Hordinsky et al, 1984). Many of the antigenic 3 mo of age, were used in this study. The mice were raised in the peptides are derived from melanosome-associated proteins such as pathogen-free animal facility of the B. Rappaport Faculty of Medicine, gp100, MART-1/Melan-A, melanocortin 1 receptor (MC1R), or Technion-Israel Institute of Technology. Animal care and research tyrosinase, and are present in both normal melanocytes and protocols were in accordance with institutional guidelines. melanomas. MAGE antigens tend to be preferentially expressed Skin grafting Graft transplantation to SCID mice was performed as on melanoma cells, and are less likely to function as autoantigens for previously described (Gilhar and Krueger, 1987; Gilhar et al, 1988, alopecia areata. 1998). Each 2 mm graft was inserted through an incision in the skin into The goal of this study was to test the hypothesis that melanocyte- the subcutaneous tissue over the lateral thoracic cage of each mouse, and associated antigens can function as autoantigens to induce hair loss covered with a standard Band Aid dressing. The dressing was removed on day 7. in alopecia areata. For this purpose, HLA-A2-positive patients with alopecia areata were selected for studies of HLA-A2-restricted Isolation of T lymphocytes from scalp punch biopsies Four melanocyte peptide epitopes. Scalp T cells were cultured with punch biopsies from each patient were used for T cell isolation. Tissue- autologous APC and either hair follicle homogenate (positive in®ltrating lymphocytes were isolated from scalp punch biopsies with the control) or melanocyte T cell epitopes. The cells were then use of collagenase (Sigma, St Louis, MO), as described previously (Gilhar et al, 1998). transferred to autologous scalp explants on SCID mice, inducing hair loss. Isolation of human peripheral blood mononuclear cells (PBMC) PBMC were isolated from heparinized whole blood by centrifugation over Hypaque 1077 (Amersham Pharmacia Biotech, MATERIALS AND METHODS Uppsala, Sweden). Patients Ten patients with either alopecia totalis or severe alopecia Hair follicle homogenate preparation Anagen hair follicles were areata were studied. Specimens from four patients were used for isolated under a stereodissecting microscope from normal scalp biopsies experiments with melanoma homogenate, and specimens from six obtained from healthy subjects who underwent hair transplantation patients were used for experiments with melanocyte-associated peptides. procedures. The follicles were homogenized and processed as previously HLA-A2-positive alopecia areata patients were selected for the peptide described (Gilhar et al, 1998). component of this study. The four donors used for the melanoma Peptides Peptides were selected based on identi®cation in the homogenate experiment were not HLA typed. Severe alopecia areata literature as HLA-A2-restricted melanocyte (e.g., melanoma) associated T was de®ned as large areas of alopecia with small residual areas of hair. cell epitopes. The following peptides were commercially synthesized These patients would be categorized as S4 (76%±99% hair loss) by the (Chiron Technologies, Raleigh, NC) with free amino and carboxylic alopecia areata investigational assessment guidelines (Olsen et al, 1999). acid termini: Gp100/G9-154, KTWGQYWQV (Bakker et al, 1995; Duration, age, sex, and clinical characteristics of all donors are listed in Parkhurst et al, 1996; Salgaller et al, 1996); Gp100/G9-209, Table I. There were no other preselection criteria, and data on these 10 ITDQVPFSV (Bakker et al, 1995; Parkhurst et al, 1996; Salgaller et al, patients were previously unpublished. Informed consent was obtained 1996); Gp100/G9-280, YLEPGPVTA (Bakker et al, 1995; Parkhurst et after the nature and possible consequences of the studies were explained. al, 1996; Salgaller et al, 1996); MC1R 291, AIIDPLIYA (Salazar-Onfray Protocols were approved by the Institutional Review Board. None of et al, 1997); MC1R 244, TILLGIFFL (Salazar-Onfray et al, 1997); the patients had any therapy in the 60 d before obtaining the scalp MC1R 283, FLALIICNA (Salazar-Onfray et al, 1997); MART-1 (27± biopsy specimens. HLA-A2 expression was determined by 35), AAGIGILTV (Bettinotti et al, 1998; Rivoltini et al, 1995); and microlymphocytotoxicity assay (Immunology Allergy and AIDS Institute, Tissue Typing Laboratory, B. Rappaport Faculty of Medicine, Technion tyrosinase, AFLPWHRFL (Kang et al, 1995). Peptides were negative for and Rambam Medical Center, Haifa). endotoxin by the Limulus Amebocyte Lysate method (Bactochem, Nes Ziona, Israel). 1Antigen frequencies from Histocompatibility Testing 1980. Los Angeles: Human melanoma cells MeWo cells were obtained from Dr. Z. UCLA Tissue Typing Laboratory, 1980. Smetana (Central Virology Laboratory, Chaim Sheba Medical Center, VOL. 117, NO. 6 DECEMBER 2001 ALOPECIA AREATA MELANOCYTE AUTOANTIGENS 1359

Tel Hashomer, Israel) (Sauvaigo et al, 1986). They were propagated in RPMI-1640 supplemented with 10% fetal bovine serum (FBS), 1% glutamine, 1% sodium pyruvate, plus penicillin/streptomycin. Culture of T lymphocytes Lymphocytes were plated at 1 3 105 cells per well in RPMI-FCS, along with irradiated (5000 R) PBMC (1 3 105 cells per well) in 24 plate wells (Greiner, Lake Mary, FL), as feeders. After 3 d of culture, recombinant interleukin-2 (rIL-2, 10 U per ml; Genzyme Diagnostics, San Carlos, CA) was added. Every 7 d the T cells were re-stimulated with feeders, for a total of 21 d culture. As indicated for experimental groups, lymphocytes were stimulated with either follicular homogenate (10 mg per ml) or peptide (10 mg per ml), along with the feeder cells at each stimulation. Phenotypic characterization of cultured T lymphocytes Phenotype of the cultured T cell lines was determined by immunocytochemistry on cytospin preparations, using procedures we have previously published (Gilhar et al, 1997). This approach was Figure 1. Hair loss induced by melanoma antigen. Signi®cant preferred over cyto¯uorograph analysis because of the small numbers of (p < 0.01 by ANOVA) hair loss was noted in grafts injected with T cells cells available. The following monoclonal antibodies were used: anti- incubated either with human melanoma homogenate (MeWo cells) CD3 FITC (clone SK 7, Becton Dickinson), anti-CD4 (IgG2a, S3.5), 30 mg per ml or human hair follicle homogenate. Data represent four anti-CD8 (IgG2a, 3B5) (Caltag, Burlingame, CA), anti-HLA-DR donors. (IgG2a, DK22, Dako, Glostrup, Denmark). Subtype matched mouse IgG-FITC (Becton Dickinson) was used as a negative control. Injection of cultured T lymphocytes into grafts on SCID mice The SCID mice bearing grafts of lesional scalp were divided into autoreactive T cells would be enriched in lesional skin relative to groups as indicated for each experiment. Between the twenty-third and peripheral blood (Kalish and Johnson, 1990). thirtieth days, the grafts were injected intradermally (0.1 ml) with For transfer experiments, lesional scalp lymphocytes from six autologous lymphocytes as noted for each experiment. On day 90 biopsies were obtained for immunohistochemical and histologic analysis. HLA-A2-positive donors were cultured with autologous APC, plus Both the percentage of grafts with hair and the number of hairs per graft either IL-2 medium alone, hair follicle homogenate (positive were recorded. control), or one or two melanocyte T cell epitope peptides. Table II shows the lymphocyte recovery after 24 d. The initial Immunohistochemical staining Staining was performed as reported number of lymphocytes per group was 5 3 105. Cell recovery previously (Gilhar et al, 1998). Monoclonal antibodies to human antigens from peptide and follicular homogenate groups varied between used were as follows: anti-HLA-DR, anti-CD54 (ICAM-1) (Biodesign, 6 6 Carmel, NY), anti-CD3, anti-CD4, anti-CD8, anti-CD25 (Dako), and 5.8 3 10 and 19 3 10 . The responding T cells were phenotyped anti-HLA-class 1 (Dako). Each specimen was coded at the time of by immunohistochemistry analysis of cytospin preparations biopsy and evaluated by two observers who were blind to the coding. (Table III). The T cells were shown to be activated as judged by expression of CD25 (19%±30% positive). CD4+ T cells Statistical analysis Statistical analysis was carried out using ANOVA for multiple comparisons. predominated for all donors. Lesional scalp T cells from the above cultures (Table II) were RESULTS injected into autologous scalp grafts on SCID mice as previously described. Six donors, all HLA-A2 positive, were used in two Melanocyte-associated antigens can function as experiments. The negative control scalp grafts were not injected autoantigens in alopecia areata Lesional scalp T cells were with T cells. Additional negative control scalp grafts were injected cultured with human melanoma cell homogenates (MeWo cells) in with T cells cultured with APC and IL-2 in the absence of two concentrations (10 mg per ml and 30 mg per ml), along with exogenous antigen, or PBMC activated with IL-2 alone. Positive autologous APC and IL-2 for 24 d. The positive control was control grafts were injected with T cells cultured with follicular provided by incubation of scalp T cells with hair follicle homogenate. Melanoma peptide was added, along with APC, to homogenate as a source of autoantigen. Scalp T lymphocytes the T cells cultured in the ®nal group as indicated in Table II. recovered from the cultures with melanoma homogenate (30 mg Melanoma-peptide-activated T cells were able to signi®cantly (p per ml) were then injected into autologous scalp grafts on SCID < 0.001 by ANOVA) reduce the numbers of hairs regrowing in mice, as were scalp T cells incubated with hair follicle homogenate, both experiments (Table IV). T cells incubated with follicular or APC alone with IL-2. Signi®cant (p < 0.01 by ANOVA) hair homogenate also resulted in signi®cant reduction in hair numbers. loss was noted in grafts injected with T cells incubated either with There was no reduction of hairs in grafts injected with T cells human melanoma homogenate (MeWo cells) or human hair follicle cultured with IL-2 and APC, or PBMC. For all six donors, no hairs homogenate. Figure 1 shows results from four patients. This were present at day 90 in any of the three grafts per donor from demonstrates that it was possible to transfer alopecia areata to scalp grafts injected with T cells activated by either melanocyte peptides, explants on SCID mice by activation of lesional T cells with or hair follicle homogenate. This demonstrates that melanocyte melanoma antigens. peptide epitopes are capable of activating T cells to induce hair loss in alopecia areata. HLA-2-restricted melanoma peptide epitopes can act as autoantigens for transfer of hair loss in the human scalp Melanocyte-peptide-induced transfer of alopecia areata explant/SCID mouse system HLA-A2-restricted induces the histologic and immunologic features of melanocyte peptide epitopes were tested as potential autoantigens alopecia areata Biopsies were taken from the mice in the for alopecia areata. HLA-A2-positive alopecia areata patients were above experiment after 90 d and were analyzed by both histology selected for this study. In a preliminary study, PBMC from these and immunohistochemistry. Noninjected grafts showed growth of patients were incubated with a panel of melanoma peptides at normal terminal anagen hairs. Injected grafts, by contrast, showed 10 mg per ml, and then cultured with IL-2. Fresh APC and peptide dystrophic hair follicles with a dense in®ltrate of CD4+ T cells, as were added to the cultures every 7 d. At the end of 24 d of culture well as an intrafollicular in®ltrate of CD8+ T cells (Fig 2). In the viable cell recovery was determined by trypan blue count. The contrast to noninjected grafts, the follicular epithelium of the results from this preliminary experiment were used to determine injected grafts expressed HLA-A, HLA-B, HLA-C, HLA-DR, and the choice of peptide for each donor in the subsequent experiment ICAM-1 (Fig 2). These are known features of active alopecia using lesional scalp T cells. It would be expected that the areata. 1360 GILHAR ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Table II. Recovery of scalp T cells after culture with melanocyte peptide epitopes, and hair follicle homogenate HLA-A2 alopecia areata patients

Antigen P-1 P-2 P-3 P-4 P-5 P-6

APC + IL-2, no antigen 3.9a 5.2 3.5 4.5 11.0 3.0 Follicle homogenate 6.9 8.1 5.8 6.9 14.0 6.9 Peptide A 10.0 11.0 6.9 9.4 12.0 7.0 Peptide A Gp100/G9-280 MC1R 291 Gp100/G9-280 Gp100/G9-280 Gp100/G9-280 Gp100\G9-154 Peptide B NA NA NA 6.7 19.0 6.5 Peptide B NA NA NA MART-1 27±35 MART-1 27±35 Gp100/G9-209

aCell recovery3106 after 24 d culture with APC, and antigens as shown. Data are shown for a total of six patients. Initial cell number was 5 3104 for all groups. All patients were HLA-A2 positive. Donors 1±6 correspond to the donor numbers in Table I.

Table III. Phenotype of scalp T cells from HLA-A2+ donors responding to peptide epitopesa

Antigen P-1 P-2 P-3 P-4 P-6

Peptide Gp100/G9-280 MC1R 291 Gp100/G9-280 MART-1 27±35 Gp100\G9-154 CD4+ 54% 85% 39% 54% 55% CD8+ 36% 15% 26% 40% 36% CD25+ 25% 21% 19% 30% 25%

aPhenotype was determined by immunohistochemistry on cytospin preparations.

Table IV. Effects of melanocyte peptide stimulation on hairs per grafta

Experiment 1 Experiment 2 Donors 1, 2, 3 Donors 4, 5, 6 Group Antigen stimulus Hairs per graft (STD) Hairs per graft (STD)

1 No T cells injected 3.8 6 0.5 3.5 6 1.7 2 Hair follicle homogenate 0.0 6 0 0.0 6 0 3 Autologous PBMC 3.2 6 0.5 3.4 6 1.6 4 Scalp T cells in IL-2, no homogenate 3.5 6 0.5 ND 5 Peptide A 0.0 6 0 0.0 6 0 6 Peptide B ND 0.0 6 0

aScalp T cells or PBMC were incubated with either irradiated autologous PBMC and IL-2 alone or antigen provided by either hair follicle homogenate or melanocytic peptides for 24 d. The T cells were then injected into autologous scalp explants on SCID mice, and hair growth was observed. The above data were obtained at 90 d. The patients and peptides studied above correspond to numbers in Tables I±III. The following comparisons are all signi®cant for both experiments (p < 0.001 by ANOVA), peptide A versus PBMC, peptide B versus PBMC, hair follicle homogenate versus PBMC, hair follicle homogenate versus no T cells.

screening, many alopecia areata patients were HLA-A2 negative, DISCUSSION however. It is likely that other melanocyte-associated T cell The goal of this study was to test the hypothesis that melanocyte- epitopes, restricted by additional HLA alleles, can function as associated antigens can function as autoantigens in alopecia areata. autoantigens in alopecia areata. This was tested by isolating T cells from lesional scalp of alopecia Five HLA-A2-restricted melanocyte peptides were identi®ed areata patients and culturing them with autologous APC, IL-2, and with the ability to act as autoantigens in alopecia areata (Gp100/ potential autoantigens. The T cells were then injected into G9-154, Gp100/G9-280, Gp100/G9-209, MC1R 291, MART-1 autologous scalp explants on SCID mice. Hair regrows on these 27±35). The initial choice of peptides tested was biased by those scalp explants unless they are injected with autologous T cells that previously reported in the melanoma literature. It is likely that the have been appropriately activated. Previously we have demon- most immunodominant epitopes were preferentially identi®ed, strated that homogenized hair follicle provides an autoantigen that however. Obviously, there is no single melanocyte antigen, or can activate T cells for the transfer of alopecia areata. It was found epitope, which is generally associated with alopecia areata. Epitope that melanoma homogenate and melanocyte T cell epitopes can spreading is likely to have a role in the broad nature of the likewise activate scalp T cells to prevent hair growth and induce antimelanocyte response. hair loss. The observation that alopecia areata can be transferred by scalp T The melanocyte-associated T cell epitopes used for this study cells activated by hair follicle homogenate has proven to be highly were originally identi®ed as HLA-A2-restricted melanoma-associ- reproducible. The effect has been observed to date in a total of 27 ated T cell epitopes recognized by CD8+ T cells. We have patients. This includes the initial published series of six patients previously demonstrated that transfer of alopecia areata requires an (Gilhar et al, 1998), 11 patients studied in CD4/CD8 separation interaction between CD8+ and CD4+ T cells, which was the experiments (Gilhar et al, in press), as well as a positive control for reason for our choice of these peptides. HLA-A2 may be weakly the 10 patients in this report. Aside from the six patients in this associated with alopecia areata (Hordinsky et al, 1984). In our study selected for HLA-A2 positivity, the patients were not VOL. 117, NO. 6 DECEMBER 2001 ALOPECIA AREATA MELANOCYTE AUTOANTIGENS 1361

Figure 2. Histology and immunocytochemistry of human scalp grafts. Hematoxylin and eosin stained histology of alopecia areata grafts (A) before grafting, (B) after grafting but not injected, (C) grafted and injected with T cells cultured with melanocyte peptide. Immunohistology of noninjected graft stained for HLA-A, HLA-B, HLA-C (D) (black is melanin). Grafts injected with T cells cultured with melanocyte peptide and stained for (E) HLA-A, HLA-B, HLA-C; (F) HLA-DR; (G) CD4; (H) CD8; (I) ICAM-1. screened, or selected for any criteria other than the clinical ®ndings allows an autoaggressive response by melanocyte-reactive CD8+ T listed. cells. The authors suggested that the CD8+ cells induce HLA-DR The ability of T cells to transfer alopecia areata is not a on the affected hair follicles by production of IFN-g, resulting in a nonspeci®c effect of activated T cells, but is speci®c for T cells second wave of CD4+ cells. We have previously reported the activated by culture with hair follicle homogenate or melanocyte- presence of CD4+ autoreactive T cells in the in®ltrate of alopecia associated antigens. Both scalp and peripheral blood T cells areata (Kalish et al, 1992), as well as a need for both CD4+ and activated by IL-2 alone were unable to transfer hair loss, as were CD8+ T cells to transfer alopecia areata (Gilhar et al, in press). This peripheral blood T cells activated with phytohemagglutinin (Gilhar report supports the hypothesis of Paus on the role of melanocyte et al, 1998). Furthermore, scalp T cells incubated with nonfollicular autoantigens in alopecia areata. Future work will concentrate on scalp homogenate were not effective (Gilhar et al, 1999). The identifying the range of melanocyte-associated autoantigens in requirement for scalp T cells is believed to re¯ect a greater multiple HLA backgrounds, as well as translating this knowledge to precursor frequency in lesional skin than peripheral blood. Thus, therapeutic bene®t. transfer of hair loss is not a nonspeci®c effect of injection of activated T cells, but appears to require activation of T cells by the relevant autoantigen. This work was supported by a grant from the National Alopecia Areata Foundation, It was necessary to use lesional (bald) scalp for the explants as well as the Japanese Society for Investigative Dermatology International because transplantation of noninvolved scalp or scalp from normal Fellowship Shiseido Award 1999. RSK is supported by National Institutes of donors results in loss of hair with inadequate regrowth (Gilhar et al, Health grant 1PO1NS3409201A2. We would like to thank Prof. Shimon 1988). Hair loss following grafting occurs with grafting of human Pollack, M.D., Director of Immunology and Allergy, and Osnat Gideoni, M.Sc., scalp to both humans (e.g., hair transplants) and mice. In our of the Tissue Typing Laboratory, B. Rappaport Faculty of Medicine, Technion and experience with grafts of human scalp to immunode®cient mice, Rambam Medical Center, Haifa, for their generous donation of HLA typing. We the number of hairs regrowing from a 2 mm graft is insuf®cient for wish to thank David Col¯esh and the University Imaging Center, SUNY at Stony analysis, whereas there is suf®cient hair regrowth from grafts of Brook, for expert assistance with the ®gures. involved (bald) alopecia scalp (Gilhar and Krueger, 1987). For this reason, it was not possible to perform these experiments with noninvolved or normal scalp explants. 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