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
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.
PATIENT SELECTION :
- Patients with alopecia totalis (S5), or 76-99 % hair loss (S4)
- 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
Summary of technique :
A – PBMC from alopecia areata patients, and controls incubated with antigens of interest.
- Control : Media alone
- Hair follicle homogenate
- Melanoma homogenate
- 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)
- First incubate with anti- INF-g NOT labeled with PE
- 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.
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.
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