Association Alopecia Areata | Molecular Basis of Alopecia Areata

Molecular Basis of Alopecia Areata

Auteur : Pr. Amos GILHAR (Skin Research Laboratory Technion Faculty of Medicine P.O.B. 9649 HAIFA 31096 ISRAEL).

Travail subventionné par l’AAA (subvention 2000-2001) à hauteur de   60 000 F.

Subvention débloquée le 26 juillet 2001.

Compte rendu fourni le 9 juin 2002.

 

COMPTE RENDU DETAILLE

FINAL REPORT FOR FRENCH GRANT : ALOPECIA AREATA

SPECIFIC AIMS

Purpose : We have demonstrated that melanocyte associated T-cell epitopes are capable of functioning as autoantigens to induce alopecia areata (35). These findings were documented with a panel of peptide epitopes and 6 patients. Il is necessary to expand these findings to a larger number of patients to determine both how general this finding is, and the range of peptide autoantigen epitopes. The goal of this project is to identify, and catalogue, the melanocyte asssociated autoantigen peptide T-cell epitopes that drive alopecia areata. These studies will concentrate on melanocyte associated HLA-A,B, C restricted peptide T-cell epitopes previously identified in human melanoma studies. This is an vitro study.

METHODS  

PATIENT SELECTION :
  1. Patients with alopecia totalis (S5), or 76-99 % hair loss (S4)
  2. Donors typed for HLA-A, B, C antigens. HLA-A2 donors selected.

Criteria for inclusion of alopecia areata patients : otherwise healthy subjects with alopecia areata of the scalp. For inclusion in this study, the scalp must have been untreated for a period of at least 4 weeks. Efforts were made to select both men and women. Alopecia areata patients with totalis (S5), or 76-99 % hair loss (S4) was studied. The S4 et S5 designations are derived from the alopecia areata investigational assessment guidelines, which will be used in assessing these patients (30). Activity of alopecia areata was clinically determined by positive hair pull test. Duration, and extent of disease was recorded as per NAAF guidelines for possible correlations with laboratory findings.

INITIAL APPROACH : Isolation of skin homing lymphocytes from peripheral blood by use of skin homing receptor, CLA. It is possible to enrich skin homing lymphocytes by positive selection with the skin homing receptor, cutaneous lymphocyte antigen (CLA), using the HECA-452 (anti-CLA FITC, Pharmingen 35824X) rat IgM kappa monoclonal antibody. CLA+ T-cells comprise approximately 5 % of peripheral blood T-cells. Thus, CLA + selection would greatly enrich for skin homing T-cells. First the T-cells are incubated 30′ on ice with anti-CLA FITC (HECA-452 Pharmingen 35824X), washed, and incubated with mouse anti-Rat kappa Microbeads (MACS) for 15′ at 4°C. The cells are then layered over a MACS column, which is placed in the appropriated magnet. CLA + cells are retained, and the CLA-cells eluted. The column is then removed from the magnet, and the CLA+ cells washed out. CLA+ T-cells will be enriched from the peripheral blood of alopecia areata patients.

We performed the above separations with peripheral blood T-cells from several patients with alopecia areata. Separations were technically successful with enrichment of peripheral blood CLA+ T-cells from 5 % to 73 % using magnetic beads. These CLA+ peripheral blood T-cells were then stimulated in vitro with hair follicle homogenate, or melanoma homogenate along with antigen presenting cells. T-cell lines were generated by addition of IL2. Unfortunately, when these T-cell liness were later assayed for antigen specific proliferation, it was determined they were not specific for either hair follicle, or melanocyte autoantigens. It was decided that the frequency of autoreactive T-cells was below the level of detection for this method, and cytofluorograph technology (below) was then applied to the problem.

CYTOFLUOROGRAPH ANALYSIS FOR IDENTIFICATION OF AUTOANTIGEN RESPONSE

Cytofluorograh analysis of intacellular Interferon-g (INF-g) can determine the proportion (frequency) of antigen specific T-cells responding to antigen by production of the TH1 cytokine INF-g. With appropriate controls, and sufficient repetitions of experiments, this method is sensitive to 1/10,000 cells specific for the antigen of interest. Details of methodology can be found at : www.bdbiosciences.com

  1. Summary of technique :

A – PBMC from alopecia areata patients, and controls incubated with antigens of interest.

  1. Control : Media alone
  2. Hair follicle homogenate
  3. Melanoma homogenate
  4. Melanocyte HLA-A2 restricted peptides

B – Cells will be stained both for cell surface molecules, (e.g. T-lymphocyte markers ; CD4/CD8), and intracellular cytokine (e. g. INF-g)

C – It is also possible to stain with two cell surface antibodies (e.g. CLA +, and CD3), in addition to intracellular INF-g

D – Proportion of T-cells producing INF-g in response to antigen will be determined by comparison with background in the absence of antigen.

E – Experiments will be performed with blood from both alopecia areata patients, and controls

2. Summary of staining protocol with use of BD Pharmingen reagents :

A – Activate PBMC with antigen, incubate 37°C for 48 hours in presence of Brefeldin A (Golgi Plug)

B – Block Fc receptors by incubating on ice with human serum, wash

C – Stain for cell surface molecule (e.g. CD3). Must use direct conjugated FITC antibody for cell surface molecule, as FITC is less sensitive than PE (which is used for intracellular molecule) ; wash

D – Fix and Permeablize ; wash

E – Stain for intracellular cytokine with PE conjugated antibody (e.g. anti- INF-g PE) ; wash

F – Controls for intracellular INF-g (both are used)

  1. First incubate with anti- INF-g NOT labeled with PE
  2. Isotype contol mouse IgG-PE

G – Two color analysis on cytofluorograph (FACS) : Need to analyze at 100,00 cells per tube rather than the usual 10,000 cells/tube

 

Frequencies of responding T-cells will be compared in the presence, and absence of the test peptides. The above comparisons will be performed for both control subjects, and subjects with alopecia areata. The cut-off for statistically significant T-cells response in the above system needs to be determined in part by analysis of the data for control subjects, as well as the background level of INF-g producing T-cells in the absence of test antigen. This will require consultation with a statistician.

RESULTS

CYTOFLUOROGRAPH DETERMINATION OF INF-g PRODUCING T-CELLS.

Peripheral blood T-cells producing INF-g in response to follicular homogenate, or melanocyte peptides was determined by cytofluorograph analysis using the methodology above. T-cells producing INF-g were selected by gating on cells double stained for anti CD3, and INF-g. Ten alopecia areata donors were studied by this technique. The first 2 donors were used in preliminary experiments. The final 8 donors gave promising results which suggest that this technique has valu for the identification of alopecia areata autoantigen peptides. Most of the donors (5/8) showed an increased proportion of T-cells producing INF-g following incubation with hair follicle homogenate. This is the same antigen preparation used previously to activate T-cells in vitro for the transfer of alopecia areat to human scalp grafts on SCID mice. Suggestive response of T-cells to melanocyte peptides was also observed. However, this preliminary data is from only 3 donors.

PERCENTAGE OF INF-g PRODUCING CD3+ T-CELLS FOLLOWING ANTIGENIC STIMULATION WITH HAIR FOLLICLE HOMOGENATE

 

DONOR NO ANTIGEN HAIR FOLLICLE HOMOGENATE SPECIFIC T-CELL RESPONSE
1 0,16 % 0,38 % 0,22 % (1/454)
2 1,6 % 2,53 % 0,93 % (1/107)
3 0,0 % 0,11 % 0,11 % (1/909)
4 0,05 % 0,13 % 0,08 % (1/1,250)
5 0,09 % 0,07 % 0,0 %
6 0,15 % 0,14 % 0,0 %
7 0,0 % 0,08 % 0,08 % (1/1,250)
8 0,0 % 0,0 % 0,0 %

 

Data Interpretation : The above data will require confirmation by continuing studies with additional donors. However, we are confident that we now have a system capable of detecting low frequencies of autoreactive T-cells in the peripheral blood. The frequencies of cells detected as per the above table are biologically significant. Using limiting dilution techniques, we previously reported that the frequency of urushiol specific T-cells in the peripheral blood of subjects with allergic contact dermatitis to urushiol was generally less than 1/10,000 (36).

Future investigations. We plan to use cytofluorograph detection of INF-g producing T-cells to screen larger number of patients with alopecia areata, for the purpose of defining the array of autoantigens recognized. The obvious starting point, based on our published data (35), would be HLA-A2 restricted melanocyte peptides. Potential autoantigen peptides will be confirmed for pathogenicity using the human scalp graft/SCID mouse transfer system (35). Once relevant autoantigen peptides are identified, work will focus on developing desensitization protocols directed at these autoantigens. Options including induction of immune deviation from TH1 to TH2, and development of altered peptide ligands that act as pratical T-cell agonists to induce tolerance.

LITERATURE CITED

 

  1. Tobin DJ, Hann SK, Song MS, Bystryn JC. Hair follicle structures targeted by antibodies in patients with alopecia areata. Arch Dermatol 133:57-61, 1997.
  1. Gilhar A, Pillar T, Assy B, David M. Failure of passive transfer of serum from patients with alopecia areata and alopecia universalis to inhibit hair growth in transplants of human scalp skin grafted on to nude mice. Br J Dermatol 126:166-171, 1992.
  1. Gilhar A, Ullmann Y, Berkutzki T, Assy B , Kalish RS : Alopecia areata transferred to human scalp explants on SCID mice with T-lymphocytes injections. J Clin Invest 101:62-67, 1998.
  1. Gilhar A, Shalaginov R, Assy B, Serafimovich S, Kalish RS : Alopecia areata is a T-lymphocyte mediated autoimmune disease : Lesional human T-lymphocytes transfer alopecia areata to human skin graft on SCID mice. J Invest Dermatol Symp Proc 4:207-210, 1999.
  1. Gilhar A, Landay M, Assy B, Shalaginov R, Serafimovich S, Kalish RS : Alopecia areata is a mediated by cooperation between CD4+ and CD8+ T-lymphocytes : Transfer to human scalp explants on Prkdcscid mice. (In Press : Archives of Dermatology).
  1. Mc Elwee KJ, Spiers EM, Oliver RF : In vivo depletion of CD8+ T-cells restores hair growth in the DEBR model of alopecia areata. Br J Dermatol 135:211-217, 1996.
  1. Mc Elwee KJ, Spiers EM, Oliver RF. Partiol restoration of hair growth in the DEBR model for alopecia areata after in vivo depletion of CD4 + T cells. Br J Dermatol 140:432-437, 1999.
  1. Bodemer C, Peuchmaur M, Fraitaig S, Chatenoud L, Brousse N, de Prost Y. Role of cytotoxic T cells in chronic alopecia areata. J Invest Dermatol 114:112-116, 2000.
  1. Paus R, Slominski A, Czarnetzki BM. Is alopecia areata an autoimmune-response against melanogenesis-related proteins, exposed by abnormal MHC class I expression in the anagen hair bulb ? Yale J Biol Med 66:541-554, 1994.
  1. Shong YK, Kim JA. Vitiligo in autoimmune thyroid disease. Thyroidology 3:89-91, 1991.
  1. Shellow WV, Edward JE, Koo JY. Profile of alopecia areata : a questionnaire analysis of patient and family. Intl J Dermatol 31:186-189, 1992.
  1. Tobin DJ, Fenton DA, Kendall MD. Ultrastructural observations on the hair bulb melanocytes and melanosomes in acute alopecia areata. J Invest Dermatol 94:803-807, 1990.
  1. Heldstrand H, Perheentupa J, Ekwall O, Gustafsson J, Michaelsson G, Husebye E, Rorsman R, Kampe O. Antibodies against hair follicles are associated with alopecia totalis in autoimmune polyendocrine syndrome Type I. J Invest Dermatol 113:1054-1058, 1999.
  1. Becker JC, Varki N, Brocker EB, Reisfels RA. Lymphocyte-mediated alopecia in C57B1/6 mice following successful immunotherapy for melanoma. J Invest Dermatol 107:627-632, 1996.
  1. Brocker EB, Echternach-Happle K, Hamm H et al. : Abnormal expression of class I and class II major histocompatibility antigens in alopecia areata : modulation by topical immunotherapy. J Invest Dermatol 88:564-568, 1987.
  1. Bettinotti MP, Kim CJ, Lee KH, Roden M, Cormier JN, Panelli M, Parker KK, Marincola FM. Stringent allele/epitope requirements for MART-1/Melan A immunodominance : implications for peptide-based immunotherapy. J Immunol 161:877-889, 1998.
  1. Rivoltini L, Kawakami Y, Sakaguchi K, Southwood S, Sette A, Robbins PF, Marincola FM, Salgaller ML, Yannelli JR, Apella E, et al. Induction of tumor-reactive CTL from peripheral blood and tumor-infiltrating lymphocytes of melanoma patients by in vitro stimulation with an immunodominant peptide of the human melanoma antigen MART-1. J Immunol 154:2257-2265, 1995.
  1. Valmori D, Fonteneau JF, Lizana CM, Gervois N, Lienard D, Rimoldi D, Jongeneel V, Jotereau F, Cerottini JC, Romero P. Enhanced generation of specific tumor-reactive CTL in vitro by selected Melan-A/MART-1 immunodominant peptide analogues. J Immunol 160:1750-1758, 1998.
  1. Mukherji B, Chakraborty NG, Yamasaki S, Okino T, Yamase H, Sporn JR, Kurtzman SK, Ergin MT, Ozols J, Meehan J, et al. Induction of antigen-specific cytolytic T cells in situ in human melanoma by immunization with synthetic peptide-pulsed autologous antigen presenting cells. Proc Natl Acad Sci USA 92:8078-8082, 1995.
  1. Fleischhauer K, Tanzarella S, Russo V, Sensi ML, van der Bruggen P, Bordignon C, Traversari C. Functional heterogeneity of HLA-A*02 subtypes revealed by presentation of a MAGE-3-encoded peptide to cytotoxic T cell clones. J Immunol 159:2513-2521, 1997.
  1. Salazar-Onfray F, Nakazawa T, Chhajlani V, Petersson M, Karre K, Masucci G, Gelis E, Sette A, Southwood S, Apella E, Kiessling R. Synthetic peptides derived from the melanocyte-stimulating hormone receptor MC1R can stimulate HLA-A2-restricted cytotoxic T lymphocytes that recognize naturally processed peptides on human melanoma cells. Cancer Res 57:4348-4355, 1997.
  1. Castelli C, Mazzochi A, Rini F, Tarsini P, Rivoltini L, Maio M, Gallino G, Belli F, Parmiani G. Immunogenicity of the ALLAVGATK (gp100[17-25]) peptide in HLA-A3.1 melanoma patients. Euro J Immunol 28:1143-1154, 1998.
  1. Skipper JC, Kittlesen DJ, Hendrickson RC, Deacon DD, Harthun NL, Wagner SN, Hunt DF, Engelhard VH, Slingluff CL Jr. Shared epitopes for HLA-A3-restricted melanoma-reactive human CTL include a naturally processed epitope from Pmel-17/gp 100. J Imunol 157:5027-5033, 1996.
  1. Parkhurst MR, Salgaller ML, Southwood S, Robbins PF, Sette A, Rosenberg SA, Kawakami Y. Improved induction of melanoma-reactive CTL with peptides from the melanoma antigen gp 100 modified at HLA-A*0201-binding residues. J Immunol 157:2539-2548, 1996.
  1. Salgaller ML, Marincola FM, Cormier JN, Rosenberg SA. Immunization against epitopes in the human melanoma antigen gp100 following patient immunization with synthetic peptides. Cancer Res 56:4749-4757, 1996.
  1. Bakker AB, Schreurs MW, Tafazzul G, de Boer AJ, Kawakami Y, Adema GJ, Figdor CG. Identification of a novel peptide derived from the melanocyte-specific gp 100 antigen as the dominant epitope recognized by an HLA-A2.1-restricted anti-melanoma CTL line. Intl J Cancer 62:97-102, 1995.
  1. Kittlesen DJ, Thomson LW, Gulden PH, Skipper JC, Colella TA, Shabanowitz JA, Hunt DF, Engelhard VH, Slingluff CL Jr. Human melanoma patients recognize an HLA-A1-restricted CTL epitope from tyrosinase containing two cysteine residues : implications for tumor vaccine development. J Immunol 160:2099-2016, 1998.
  1. Kang X, Kawakami Y, el-Gamil M, Wang R, Sakaguchi K, Yannelli JR, Appella E, Rosenberg SA, Robbins PF. Identification of a tyrosinase epitope recognized by HLA-A24-restricted, tumor-infiltrating lymphocytes. J Immunol 155:1343-1348, 1995.
  1. Antigen frequencies from : Histocompatibility Testing , 1980, Los Angeles, UCLA Tissue Typing Laboratory, 1980, p962.
  1. Olsen E, Hordinsky M, McDonald-Hull S, Price V, Roberts J, Shapiro J, Stenn K. Alopecia areata investigational assessment guidelines. J Am Acad Dermatol 40:242-246, 1999.
  1. Picker LJ, Martin RJ, Trumble A, Newman LS, Collins PA, Bergstresser PR, Leung DY. Differential expression of lymphocyte homing receptors by human memory/effector T cells in pulmonary versus cutaneous immune effector sites. Euro J Immunol 24:1269-1277, 1994.
  1. Picker LJ. Regulation of tissue-selective T-lymphocyte homing receptors during the virgin to memory/effector cell transition in human secondary lymphoid tissues. Amer Rev Resp Dis 148:S47-54, 1993.
  1. Bos JD, de Boer OJ, Tibosch E, Das PK, Pals ST. Skin homing T lymphocytes : detection of cutaneous lymphocyte-associated antigen (CLA) by HECA-452 in normal human skin. Arch Dermatol Res 285:179-183, 1993.
  1. Brosterhus H, Brings S, Manz RA, Miltenyi S, Radbruch A, Assenmacher M, Schmitz J. Rapid enrichment and characterization of lime antigen-specific T-cells using cellular affinity matrix technology. 10th International Congress of Immunology pp 1469-1473, 1998.
  1. Gilhar A, Landau M, Assay B, Shalaginov R, Serafimovich S, Kalish RS : Melanocyte associated T-cell epitopes can function as autoantigens for transfer of alopecia areata to human scalp explants on Prkdc scid mice. J Invest Dermatol 117:1357-1362, 2001.
  1. Kalish RS, Johnson KL : Enrichment and function of urushiol (poison ivy) specific T-lymphocytes in lesions of allergic contact dermatitis to urushiol. J Immunol 145:3706-3713, 1990.