In order to probe the efficacy of new immune-intervention strategies in alopecia areata, the field can now choose from two mutually complementary mouse models.

1. Skin Research Laboratory,
The B. Rappaport Faculty of Medicine, Technion-Israel Institute of Technology,
Haifa, Israel
Human Scalp Skin Xenotransplants in Androgenetic
AlopeciaPre-clinical research in Vivo: Pros and Cons of the
C3H/HeJ versus Humanized Mouse Model
Human skin grafts on animal models
Amos Gilhar, M.D.

2. In our opinion both models should be usedBochenska K et al. J. Mol. Sci. 2017
Murine models
of Psoriasis
According to the literature there are 11 models for vitiligo
19 for Atopic Dermatitis, more than 20 for psoriasis
but 2 for Alopecia Areata
1. The C3H/HeJ is used widely, whereas
2. The humanized mice (no NK , T and B cells) is used in only one Lab.

3. Normal Human
scalp skin/SCID*
mice injected
with IL-2
enriched PBMCs
C3H/HeJ
with skin
graft-
induced
hair loss
Animal
Model
+Regrowth
<3%
Sudden hair loss with temporarily hair regrowth.
++Spontaneous hair growth
++Mononuclear infiltrate around and within the hair bulb.
++Peri- and intrafollicular- CD8+ T-cells
+NANKG2D and NKG2D- ligands
++Major histocompatibility complex (MHC) I and II
-+The hair loss pattern is categorize to focal and diffuse pattern
Table 1
AA criteria in the animal models
Similarities and differences between the C3H/HeJ & humanized mouse model
Gilhar A et al Autoimmun Rev. 2016

4. C3H/HeJ with skin graft-induced hair loss AA humanized mouse model
Easy to use Housed together. Do not require specific pathogen-free
conditions.
Technical expertise is required, complicated
Convenient and cheap Relatively inexpensive, convenient model of AA. Expensive
Grooming induced hair loss Grooming and scratching behaviors None
Potential for pre-clinical drug
screening
With limited value due to murine-specific disease
pathology.
Can be used widely due to its reliance on healthy human donor tissue
and human disease pathology
Disadvantages The histological feature is not specific for AA:
The inflammatory cells are also observed above the hair
bulb, CD4+ cells dominants over CD8 + cells
CTLA4 polymorphisms, is not adequately represented.
MICA is strikingly absent in mice (only 27% amino-acid
identity with human MICA).
Difficulty in obtaining sufficient clinical material
Require a pathogen-free environment for maintenance
Absence of genetic background
Advantages Has yielded tremendous insight into the pathogenesis and
treatment of AA
Specific histological feature: around and within the hair bulb as in
human AA
CD8+ cells dominants over CD4 + cells as in human AA

5. Differences between non-conventional T cells populations among human versus mice should be taken into
account when using mice as preclinical models of human disease.
There are several distinct subsets of γδ T cells in mice and humans, but mouse and human subsets notably have different TCR use, antigen reactivity and
patterns of tissue homing
Godfrey et al. Nature Immunology, 2015
Mouse models have failed to account for the natural diversity in human immune responses. As a result,
insights gained in the lab may be lost in translation
Konrad Buscher et al. Nature Communications, 2017
Differences between immune system of human & mice
There is growing concern that laboratory mice do not reflect relevant aspects of the human immune system,
which may account for failures to translate disease treatments
Beura et al. Nature , 2016 from bench to bedside

6. Foxp3
Vδ2TCR
‫ע‬37%
Increased level of γδTreg
γ/δTregs
γδTregs are the predominant
regulatory T cells and have more
potent immunosuppressive activity
than CD4+ or CD8+ Tregs
Hu G, et al. Oncoimmunology 2017
Gu Y, et al. J Immunol Methods 2014
Feng Y, et al. Nature 2015
Hu y, et al. Hematol Oncol. 2017
Humanized
model
TGF-β INF-γ
Gilhar et al. unpublished
High levels of TGF-β and low of IFN-ƴ in γδTreg
Humanized
model
Human
AA
Human
Scalp
Skin
Humanized
AA
model
Green –Keratin 15
Normal HF of
normal scalp skin
AA HF
Presence of γ/δTregs
among stem cells in the bulge area

7. High levels of intracellular IL-10 & TGF-β in γδTreg
Co-culture of CD8+NKG2D+ cells with γδTreg cells
Human HF organ culture
Effect of IL-10 and TGFβ on HFs co-culture
with CD8/NKG2D cells
IFN-γTGF-β
%Catagen
Control CD8/
71%
44% %43%43
p<0.01
CD8/NKG2D cells
TGFβ IL-10 TGFβ/
IL10

8. Induction of psoriatic skin by ILC3+ cells
Normal Skin Graft
l
Psoriatic Induced Graft
Keren ….Gilhar et al . JACI , 2018
PSORIASIS CAN BE INDUCED by
pure ILC3 without the
involvement TH17
Normal
scalp skin
AA scalp skin graft
Normal scalp
skin graft
AA
scalp skin
Normal
scalp skin
N
o
r
m
a
l
s
Increased number of ILC1 in AA patients
A study to determine the role of ILC1 in AA

9. Kv1.3 blocker,
PAP-1
suppresses AA
development in
human skin
grafts
Ecopic HLA-DR
Non - responder graft Responder graft
No R
No DR
Gilhar et al. J Invest Dermatol. 2013
Kv1.3 blockers
preferentially
suppress
autoreactive
CCR7− effector
memory T cells
Additional Therapeutic Targets for AA Need to be Identified and Explored
Gilhar, Keren. Paus . Lancet- accepted for publication
The Humanized mice can be used as a pre clinical drug screening

10. Schafer PH, et al . Br J Pharmacol. 2010
Apremilast in a model of psoriasis
Preclinical modeling of atopic dermatitis
Apremilast on Humanized Mice for Psoriasis and Atopic Dermatitis
Remission of Established Atopic Dermatitis
Predicting the exact efficacy of
apremilast as was observed later on
in clinical trials

11. A comparative study: Therapeutic Effect of Apremilast versus
Tofacitinib
Mice were randomly divided into three groups:
(i) Control (0.5% methylcellulose, bid) (n=5)
(ii) Treated orally with Apremilast (5 mg/kg/day,bid) (n=5).
(iii) Treated orally with Tofacininib (10 mg/kg, bid) (n=5).
Control Apremilast Tofacitinib
Therapeutic effect
Apremilast Versus Tofacitinib
A comparative study
Treatments of Apremilast and Tofacitinib were given to
humanized mice with ongoing alopecia from day 45 after
injections of NKG2D enriched cells, till day 105. .
Tofacitini
b
Control
Gilhar et al. unpublished

12. Agent
1
Montarolo et al. demonstrated protective but not therapeutic effect in EAE mice. PLOS ONE, 2014
Protective but not Therapeutic Effect of Apremilat in Mice
Preventive Non-therapeutic
Potent enough
Treatment
Not potent enough
t

13. Increased number of NKT/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
Ghraieb … Gilhar , J Autoimmun. 2018

14. 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 NKT10 cells in lesional areas of AA patients and humanized mouse model
α-GalCer Induced Expansion of
NKT10
Ghraieb … Gilhar , J Autoimmun. 2018
o

15. 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
Ghraieb … Gilhar , J Autoimmun. 2018

16. Meanhairnumber(pergraft)
Before
injection
30-40d after
injection
50-60d after
treatment
Treatment
starts
Treatment
ends
16-30d after
stopping
treatment
Before injection 50- 60d of treatment
Treatment starts Treatment ends
Normal scalp skin
grafts bearing hair
AA-Induction
Therapeutic effect
of α-GalCer
Reappearance
of AA
30-40d after injection 16-30 after stopping treatment
Reappearance of AA Following Sopping Treatment With α-GalCer
Reappearance
of AA
conclusion
The study demonstrated
the role of NKT and
NKT10 in AA
Ghraieb … Gilhar , J Autoimmun. 2018 Ghraieb … Gilhar , J Autoimmun. 2018
The regulatory effect is
achieved not only by
Tregs

17. Our very preliminary experiments demonstrated
•Effects of Kv1.3 blocker and α-GaLcer (NKT10)
•A significant difference between therapeutic effect of Tofacitinib VS Apremilast
•Non-conventional T cells may play a role in AA and thus
may serve as future therapeutic targets
Summary

18. Laboratory Staff
Aviad Keren , PhD
Nadia Smirnov , B A
Natalia Kaplun, M D
Gil Kaufman, PhD
Rimma Laufer, MD
Amal Ghraieb , MSc
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
& University of Miami, USA
Nira Goldstein, BA
Prof. A. Schrum, PhD
Columbia, Missouri