Non-Conventional T-cells in the Pathogenesis of Alopecia Areata
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Elucidating the role of non-conventional T cells and the innate immune system in the pathogenesis of alopecia areata.
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Non-Conventional T-cells in the Pathogenesis of Alopecia Areata
- 1. Skin Research Laboratory, Technion , Institute of Technology Haifa, Israel Non–conventional T-Cells in Alopecia Areata Amos Gilhar, M.D.
- 2. The pathogenesis of AA Gilhar et al. Autoimmune Review 2016 Trichohyalin peptides IL-13 Pre-existing CD8+cells
- 3. Using the AA humanized SCID mouse model enabled us to find the role of various cells and components such as: • CD8+cells (effector cells) Gilhar A. et al. J Clin Invest 1998 • CD4+ cells (helper functions) Gilhar A. et al. Arch Dermatol 2002 • Melanocytes T cells Gilhar A. et al. J Invest Dermatol 2001 • Collapse of immune privilege Gilhar A. et al. J Invest Dermatol 2013 • Preventive effects of KV1.3 blocker Gilhar A, Etzioni A, Paus R. N Engl J Med. 2012 • and of PDE4 inhibitor Gilhar A, Schrum AG, Etzioni A, Waldmann H, Paus R. Autoimmun Rev. 2016 NKG2D+ cells Gilhar A. et al. . J Invest Dermatol 2013
- 4. NKG2D+ cells represent a rather mixed collection of cells, such as: CD8+ Type 1 innate lymphoid cells (ILC1) NK cells γδ-T cells iNKT cells
- 5. Innate Lymphoid Cells ILCs are important effector cells mediating Ag-independent immune responses and are involved in various aspects.
- 6. Rankin L1, Groom J, Mielke LA, Seillet C, Belz GT. Diversity, function, and transcriptional regulation of gut innate lymphocytes. Front Immunol. 2013;4:22. & NKp44+ ILCs Classification
- 7. Induction of Psoriatic Skin by only ILC3+ cells Normal Skin Graft l Psoriatic Induced Graft Gilhar et al., Submitted
- 8. Humanized Mouse model Human skin Normal skin Psoriasis Induction of Psoriatic Skin by ILC3+ cells
- 9. Gating The presence of ILC1 cells in Alopecia Areata patient (using immunofluorescence confocal microscopy)
- 10. NKp44/T-betNKp44/CD103 NKp44/CD103 ILC1+ cells in AA Patients Normal scalp skin AA scalp skin
- 11. Classical NK cells in Human Alopecia Areata NK triple staining (Perforin,CD161,CD117)
- 12. Distribution of ILC1 and ILC2 around the hair follicles in AA patients
- 13. CD127 CRTH Lineage - CD127+ NKp44- CRTH- C-KIT - CD161+ CRTH- CD127+ Lineage - NKp44 - C-KIT-NKp44 - CD161+ ILC1 Isolation of ILC1 by FACS Gaiting strategy of lin minus
- 14. Control without cells 150 cells 300 cells 300 cells 150 cells 300 cells Per hair follicle ILC1 ILC2 ILC3 Following six days in culture Co-culture of normal hair follicles with ILCs
- 15. Control without cells 150 cells 300 cells 300 cells 150 cells 300 cells Haircyclescore Hair cycle score: 100 – Anagen 200 – Early catagen 300 – Mid catagen 400 – Late catagen ILC1 ILC2 ILC3 Hair Cycle Score Following Culture Of HFs With ILCs Langan EA et al. Exp Dermatol 2015 24:903-11. doi: 10.1111/exd.1283
- 16. Very Preliminary Results Indicate an Effect of ILC1 On Human Hair Follicles
- 17. Pham et al. Front Immunol. 2016 Autoimmunity= Imbalance Between Regulatory and Effector Cell Activities Protective cells Effector T cells
- 18. Types of protective cells: FOXP3 Regulatory T-cells FOXP3 γδ T-cells Regulatory NK-cells FOXP3 iNKT-cells iNKT10. α - GalCer Expansion of NKT10 cells Repeated injections prevented the development of EAE Sag J Clin Invest 124 :3725–3740, 2014
- 19. CD4/6B11 iNKT cells In Alopecia Areata Lesions Hair Bulb DP
- 20. Humanize AA Mouse Model Gilhar et al. J Invest Dermatol 133:844-7,2013 Pre-incubation PBMCs/IL-2/α-GalCer Prevented the induction of AA
- 21. α-GalCer Induced Expansion of NKT10 PBMC’s/IL-2 PBMC’s/IL-2+ αGalCer NKT10= 41.6% NKT10= 1.3% αGalCer Without With CD197-, CD3+, CD200+, CD49d+, CD45RA+, CD279+, CD304+
- 22. The production levels of IL-10, IL-4, TGF-β and IFN-γ cytokines by PBMCs were measured by ELISA j j
- 23. Regulatory role of iNKT/IL-10 in humanized AA mice following treatment with α-GalCer IL-10 IL-10 IL-10 IL-10 Double staining Double staining NKT cells Double staining NKT cells NKT cells NKT cells Double staining
- 24. MeannumberofIL10+/NKT+cells (per0.5mm2) AA patient Human alopecia areata AA likeNormal skin Humanized mouse model AA like/GalCer treatment Healthy volunteer p=0.05 P<0.05 P<0.05 P<0.005 Mean number of IL-10/iNKT cells in lesional areas of AA patients and humanized mouse model
- 25. PBMC’s/IL-2 Depletion of NKT cells(6B11) Vα24 isotype control (NKT cells (Vα24 blocking Abs IL-10 isotype control IL-10 blocking Abs Meanhairnumber(pergraft) Number of Hair in Humanized AA mice PBMC’s/IL -2 +α -Gal-Cer Injection day End of the study
- 26. IL-10 blocking Abs/ Ongoing AAIL-10 isotype Abs. Pre-incubation/iNKT blocking Abs.NKT(Vα24) isotype control Abs. CD8+ HLA-DR expression and in AA Induced Grafts/α-GalCer HLA-DR- HLA-DR+ HLA-DR+ HLA-DR+ HLA-DR+ HLA-DR+ DP DP Hair BulbHair Bulb DP DP Hair Bulb CD8+ iNKT Depletion CD8+ DPCD8+ Hair Bulb DP CD8+ DP CD8+ DP Hair Bulb α-GalCer & controls α-GalCer & blocking Abs. α-GalCer & iNKT Depletion
- 27. Normal scalp skin before induction of hair loss Induction of AA skinday 60))graft Hair growth on AA induced graft /rIL-10 day day105))treatment Therapeutic Effect of IL-10 and α-Gal-Cer in AA mouse Model Hair growth on AA induced graft /α-Gal-Cer
- 28. iNKT/IL10/CD1d in humanized AA mice following treatment with α-GalCer AA skin graft/α-GalCer AA skin graft
- 29. Correlation between increased number of NKT10 and normalization of scalp skin grafts j
- 30. CONCLUSION • α-Gal-Cer seems to possesses therapeutic and preventive effects in the humanized mouse model
- 31. Laboratory Staff Aviad Keren , PhD Nadia Smirnov , B A Natalia Kaplun, M D Gil Kaufman, PhD Rimma Laufer, MD Amal Ghraieb , MSc Prof. R. Bergman Rambam Medical Center, Israel Prof. Y. Ullmann Rambam Medical Center, Israel Prof. A. Shemer Sheba Medical Center, Israel Prof. Y. Refaeli, University of Colorado, Denver Prof. M. David Beilinson Medical Center, Israel Prof. R. Paus University of Manchester,UK & Münster,Germany Nira Goldstein, BA Prof. A. Schrum, PhD Mayo Clinic College, Rochester, MN Dr. A. Ginzburg
- 32. PBMC’s were incubated with IL-12, IL-18 and IL-33 for two weeks (Silver JS et al.,2016). Following incubation, ILC1 cells were isolated using FACS sorter (Lineage - , CD127+, CRTH-, NKp44 -, c-KIT- and CD161+ cells). ILC2 cells (Lineage - , CD127+, CRTH+, c-KIT- and CD161+ cells). ILC3 cells (Lineage - , CD127+, CRTH-, c-KIT+ and NKp44+ cells). Autologous ILC1/ILC2/ILC3 cells were subsequently co- cultured for six days with microdissected hair follicles (150/300 cells per hair follicle). Silver JS et al. Nat Immunol. 2016;17:626-35. Protocol for Isolation of ILC1