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Souris humanisées: Progrès & perspec5ves 4ème Journée Scientifique du Réseau de Pathologie
Expérimentale (Pathex)
Médecine moléculaire et modèles animaux
Pathologie du système lymphoïde
Pu7ng some human pieces into mice……. human 5ssue engra7ment humaniza5on HIV, HBV, HCV, Plasmodium falciparum tumors auto-­‐immune diseases human molecule expression The different Human/Mouse chimera models
of HIV infection
Principle hu TG-­‐mouse = Co-­‐expression through transgenesis of human CD4 and HIV corecepteurs Limits No produc5ve infec5on Hu-­‐PBL-­‐SCID = Xenogra7 of human adult PBMCs in SCID hosts Hu-­‐SCID = Double xenogra7 of human fetal liver and thymus in SCID hosts GVH S.Garcia et al, Blood, 1996, 89;329. No peripheral immune system The second generation of Human/Mouse chimera models
Murine host
RAG-/- γc-/- or
NOD SCID γc-/NOD RAG γc-/-
Human donor
no lymphocytes T, B, NK
limited rejection of
human cells by murine host
=>
Yahata et al, 2002, J Immunol, 169:204 Ito et al, 2002, Blood. 2002;100:3175 Traggiai et al, 2004, Science, 304:104 Gimeno et al, 2004, Blood, 104:388 Ishikawa, I et al, 2005, Blood, 106:1565. Human hematopoietic
precursors derived from:
fœtal liver
cord blood
adult BM
adult blood
Easy to make IgM responses Thymic development benefits HIV, dengue, EBV infec5ons All subsets present at the periphery Diversity of T cell repertoire HSC survival Ig produc5on Myeloid development limits MHC restric5on (HLA, H-­‐2?) Low number of T cells Impaired B cell development few human RBCs La troisième génération de souris hôtes: vers
des souris de plus en plus humanisées…..
Illustra5on: Pascal Gérard Tolérance immunitaire Niche hematopoïé5que Cytokines/MHC adapté de A.P.A. Theocharides et al, heamatologica, 2016 1- le modèle CH1-2hSa :
à propos du MHC et de SIRPα…..
Le modèle CH1-2
Murine host
Human donor
RAG-/- γc-/- = no lymphocytes T, B, NK
C5-/= absence of hemolytic factor C
=> limited rejection of human cells
by murine host
HLA-A2+hß2m
HLA-DR1+
+
murine ß2m-/I-Aßb-/-
Stronger interactions between
MHC and TCR/CD4/CD8
•  higher thymic selection
•  higher peripheral survival
MHC-restriction of human response
by the sole HLA molecules
Human
hematopoietic
progenitors
The CH1-2 murine hosts
RAG-/- γc-/- mß2m-/- I-Aßb-/- C5-/- HLA-DR1+ HLA-A2+
CH1-2
HLA expression in the thymus and spleen of CH1-2 hosts
CH1-2 thymic anlages support better maturation of human
thymocytes than RAG- γc- thymic anlages
Thymic lobes CH1-2
Human CD34+ % of max
CD4+ CD8+
54%
CD8+
CD4+
23%
Control
0
100
1000
10000
1x105
CD3
Thymic lobes RAG- γc% of max
Human CD34+ CD4+ CD8+
CD8+
13%
CD4+
4%
Control
0
100
1000
CD3
10000
1x10 5
Unsuccessful engraftment of human hematopoietic
cells in CH1-2 murine hosts
CB CD34+ -> CH1-2
10
5
0
0
huCD45
103
2
0
0.0319
104
10 3
10
0
huCD45
10 4
5
10
2
10
1.22
0
2
10
98.8
3
10
Ly5
4
10
10
5
0
1.95
0
2
10
98
10
3
Ly5
4
10
5
10
the SIRPα-CD47 pathway matters….
Role for CD47-SIRP signaling in xenograft rejection
by macrophages
Kentaro Ide*, Hui Wang†, Hiroyuki Tahara*, Jianxiang Liu‡, Xiaoying Wang‡, Toshimasa Asahara*, Megan Sykes†,
Yong-Guang Yang†§, and Hideki Ohdan*¶
*Department of Surgery, Division of Frontier Medical Science, Programs for Biomedical Research, Graduate School of Biomedical Science, Hiroshima
University, Hiroshima 734-8551, Japan; †Bone Marrow Transplantation Section, Transplantation Biology Research Center, Massachusetts General
Hospital, Harvard Medical School, Boston, MA 02129; and ‡Neuroprotection Research Laboratory, Department of Radiology and Neurology,
Massachusetts General Hospital, and Program in Neuroscience, Harvard Medical School, Boston, MA 02129
Edited by Charles T. Esmon, Oklahoma Medical Research Foundation, Oklahoma City, OK, and approved February 1, 2007 (received for review
October 31, 2006)
We have previously proven that human macrophages can phagocytose
porcine cells even in the absence of Ab or complement
opsonization, indicating that macrophages present a pivotal immunological
obstacle to xenotransplantation. A recent report indicates
that the signal regulatory protein (SIRP) is a critical
immune inhibitory receptor on macrophages, and its interaction
with CD47, a ligand for SIRP, prevents autologous phagocytosis.
Considering the limited compatibility (73%) in amino acid sequences
between pig and human CD47, we hypothesized that the
interspecies incompatibility of CD47 may contribute to the rejection
of xenogeneic cells by macrophages. In the present study, we
have demonstrated that porcine CD47 does not induce SIRP
tyrosine phosphorylation in human macrophage-like cell line, and
soluble human CD47-Fc fusion protein inhibits the phagocytic
activity of human macrophages toward porcine cells. In addition,
we have verified that manipulation of porcine cells for expression
of human CD47 radically reduces the susceptibility of the cells to
phagocytosis by human macrophages. These results indicate that
the interspecies incompatibility of CD47 significantly contributes to
the rejection of xenogeneic cells by macrophages. Genetic induction
of human CD47 on porcine cells could provide inhibitory
signaling to SIRP on human macrophages, providing a novel approach
to preventing macrophage-mediated xenograft rejection.
xenotransplantation phagocytosis reticuloendothelial system
Manipulating the SIRPα-CD47 pathway
SIRPα
CD47
Ubiquitous
No phagocytosis
macrophages, DC, neutrophils
5
0.2
94.9
mCD11b1b
mCD1
10
10
4
10
3
10
2
0
hu-SIRPA
3.11
0
Murine cfms- promoter
CH1-2hSa
1.75
10
2
10
3
10
4
huSirpα
hCD172
10
5
huSirpα expression promotes human hematopoietic
progenitor engraftment
6 weeks Blood
CH1-2
non Tg mice
2
10
1.95
0
2
10
10
3
Ly5
4
10
10 4
huCD45
3
10
Ly5
4
10
0
10
5
10
2.53
0
2
10
97.2
3
10
Ly5
4
10
10
5
2
0
10
2.17
0
0.413
10 3
2
0
5
2
10
10 4
10 3
10
0
0
99.4
5
10
2
10
10
3
Ly5
4
10
2.87
5
10
56.7
104
103
0
2
10
3
10
Ly5
4
10
10
5
0
0.621
3.08
0
10
5
2
10
65.1
3
10
Ly5
4
10
1.86
10
5
62.5
10 4
10 3
2
99
31.2
2
0
58.5
10
0.55
5
10 3
2
0.481
10
10 4
10
5
10
0.134
103
10
0.234
39.2
104
huCD172
0.0138
5
10
2
0
98
huCD172
5
0.0688
10 3
10
10
0.0688
huCD45
103
0
5
10 4
huCD45
104
10
huCD45
0.0319
huCD172
0
huCD172
5
10
CH1-2hSa+ Tg
mice
10
38.5
0 10
2
1.97
3
10
Ly5
4
10
5
10
2
0
3.35
0
2
10
3
10
Ly5
4
10
10
5
Human lymphocyte subsets in the spleen of
engrafted CH1-2hSa host
T cells
B cells
NK cells
10 5
10 5
10 4
10 4
10 4
10 2
10 2
0
0
0
10 3
0
10 2
10 3
10 4
10 5
10
0
10 2
77.7
10 5
5.87
59.7
10 3
2
12.7
0
10 2
0
0
10 2
10 3
CD8
10 4
10 5
11.4
0
10 2
23
10 3
IgD
10 4
97
0
0.32
10 2
10 3
CD16
IgM
10 3
10
10 4
10 4
CD4
10 4
10 3
CD45
10 5
0.17
10 3
CD45
5
2.47
CD56
10 2
CD3
10 3
CD19
10 5
10 5
10 4
10 5
Human T and NK cells in the thymus of
reconstituted CH1-2hSa host
Treg cells
T cells
10 5
10 5
10
CD25
0
0
0
10 2
10 3
10 4
100
% of max
DP
80
60
SPCD8+
40
SPCD4+
20
0 102
103
1.4
10 2
94.3
10 3
CD4
CD8
0
0
0
10 5
104
TCRαβ
105
2.19
10 2
10 2
17.2
2
2.03
10 3
10 3
10 3
10 5
10 4
10 4
CD4
10
23.4
4.25
CD56
40.9
4
0.04
NK cells
10 4
10 5
93.4
0
10 2
2.35
10 3
CD16
10 4
10 5
Human lymphocytes in the BM of
engrafted CH1-2hSa
T cells
B cells
IgM
CD19
10 3
10 5
104
10 4
103
102
10 2
0
0
0
10 2
10 3
CD45
10 4
10 5
0.72
10 3
10 2
0
10 5
0
102
103
IgD
104
105
0.41
0.17
10 4
CD56
74.4
10 4
105
CD3
10 5
NK cells
10 3
10 2
75.9
0
10 2
10 3
CD45
10 4
10 5
0
98.9
0
10 2
0.52
10 3
CD16
10 4
10 5
Human myeloid cells and HSPC in the BM of
reconstituted CH1-2hSa host
macrophages
and DC cells
0.94
10 5
1.97
10 5
9.21
4.24
10 4
CD34
CD11c
10 4
HSPC cells
10 3
10 3
10 2
10 2
0
0
0
10 2
10 3
CD14
10 4
10 5
84.7
0
10 2
1.8
10 3
10 4
CD117 (cKit)
10 5
Induction of splenic HBV-specific HLA-A2 restricted CD8 T cell
response upon vaccination of CH1-2hSa chimera
Vaccinated
98.4
2.78
104
10
2
10
CD8
103
CD8
10
10
0
10
0
102
103
104
105
HBV pentamer
99.4
3
2
102
0
10
102
10
CD8
103
10
0
0
0
10
10
3
10
4
HCV pentamer
0
0
0
10
10
5
5
0.286
103
10
2
10
3
10
4
10
0
5
99.2
0.813
4
0
0
HBV pentamer
0.501
2
99.7
104
105
4
0
0.917
4
0
CD8
10
99.1
0
0
105
5
CD8
5
Cord blood
105
102
103
104
HBV pentamer
100
105
0
104
CD8
10
non vaccinated
3
103
2
102
0
0
0
0
10
2
10
3
10
4
HCV pentamer
10
0
0
0
5
0
102
103
104
pentamer HCV
105
% tetramers+ / human CD8 T cells
Induction of splenic HBV-specific HLA-A2 restricted CD8 T
cell response upon vaccination of CH1-2hSa chimera
3
2,5
HLA-A2+
2
HBV specific tetramers
1,5
HCV specific tetramers
1
HLA-A20,5
0
Vaccinated
Non vaccinated
Cord blood
Only CH1-2HuSa chimera reconstituted with human CD34+ CB from HLA-A2+ donor
are able to mount HBV-specific HLA-A2 restricted CD8 T cell response upon
vaccination
Conclusions 1
-  L’expression des molécules HLA permet une meilleure maturation des précurseurs
thymiques.
- L’expression de Sirpα humain par les macrophages/DC/neutrophiles murins suffit à
permettre la greffe et la différenciation de précurseurs hématopoïétiques humains
dans les souris hôtes CH1-2 hosts.
- Les souris hôtes CH1-2hSa permettent le développement et la différenciation à la
fois des populations myéloïdes et lymphoïdes humaines (macrophages, Dcs, cellules
NK, T CD4, T CD8, B).
-  Les souris hôtes CH1-2hSa permettent le développement de réponses immunitaires
Ag-specifiques HLA-restreintes.
2- Introduction de cytokines humaines
Hématopoièse: les cytokines impliquées
from Pharmacology today 2015 Quand les hôtes murins expriment des cytokines humaines…
LD. Shultz, Nature Immunol. reviews, 2012 TPO Effect on human cells technology Human cytokine KI ↗ human myelopoiesis ↘ lymphopoiesis ↗ HSC self-­‐renewal membrane SCF CMV Tg ↗ engra7ment, no need ⚡︎ ↗ mast cells IL-­‐3/GM-­‐CSF KI Replacement of alveolar macrophages M-­‐CSF KI ↗ mono/macro SCF/GM-­‐CSF/IL-­‐3 CMV Tg ↗ myeloid in BM ↘HSC, B cells Flt3l/GM-­‐CSF/IL4 hydrodynamic DNA ↗ DC IL-­‐15/IL15-­‐IL-­‐15Rα
rprot, adeno ↗ NK, ↗ T?, ↗ DC? IL-­‐3+EPO hydrodynamic DNA or rprot IL-­‐7 rprot, Fc-­‐prot, len5viral ↗ Thymocytes, periph? MISTRG KI, BAC Tg ↗ fonc5onal myeloid cells, NK cells, tumor engra7s NBSGW cKit mutant W41 ↗ myeloid cells, IIary HSC transplants PiEall for hosts Thrombocytopenia in host mice Pulmonary alveolar proteinose in host NR mice " 3 weeks survival MISTRG versus c-Kit mutant…
MISTRG A. Rongvaux et al, Nature Biotech, 2014 cKit W41 K.N. Cosgun et al, Cell Stem Cell 2014 MISTRG contre c-Kit mutant…
MISTRG A. Rongvaux et al, Nature Biotech, 2014 cKit W41 K.N. Cosgun et al, Cell Stem Cell 2014 cKit deficiency may provide a suitable environment for human HSC
engraftment, development and differentiation w/o the need of
introducing human factors. Conclusions 2
-  L’expression de cytokines humaines par les hôtes murins permet d’augmenter le
développement/survie de sous-populations humaines, les souris MISTRG étant pour
le moment les plus « abouties » pour la reconstitution myéloïde.
-  L’utilisation des souris cKit W41 a montré que « l’ouverture de la niche murine » et
la disponibilité accrue de cytokines murines pour les cellules humaines peut
compenser l’absence de cytokines analogues humaines.
-  La survie d’érythrocytes humains reste un problème majeur.
- 
La représentation des sous-populations humaines reste différente de la
représentation dans les hôtes murins (granulocytes/lymphocytes).
La souris hôte du futur: une combinaison de toutes les souris existantes et
plus?......
CSEH: vers de nouvelles cellules donneuses….
The HESCRIM model Murine host
RAG-/- γc-/- = no lymphocytes T, B, NK
C5-/= absence of hemolytic factor C
=> limited rejection of human cells
by murine host
HLA-A2+hß2m
HLA-DR1+
+
murine ß2m-/I-Aßb-/-
Human donor
Human hematopoie5c precursors derived from human embryonic stem cells Stronger interactions between
MHC and TCR/CD4/CD8
•  higher thymic selection
•  higher peripheral survival
MHC-restriction of human response
by the sole HLA molecules
⇒  unlimited and reproducible source of donor cells Obtention of CD34+ human cells from H9 HLA-A2/DR1 hESC
Controls
0.051
104
0
103
103
102
102
101
99.8
101
99.9
0.1
100
100
100
101
102
103
104
100
SSEA4 APC
H9+OP9
104
0.4
38.4
103
103
102
102
101
101
100
60.4
100
0.88
101
CD34 PE
102
101
102
0
103
104
OP-9 / ES d8
OP-9 / ES d4
104
0
0.094
103
100
104
100
1.69
0
96.8
1.49
101
102
103
104
10
SSEA-4+ cells
8
CD34+ cells
50
40
6
30
4
20
2
10
0
d4
d5
d6 d7 d8 d9 d10
days of co-culture
RAG-/- γc-/- H-2-/HLA-DR1 HLA-A2
….and MSC, thymic epithelial cells…….derived from hESC or hIPCs…..
0
% of SSEA-4+ cells
104
OP-9 alone
% of CD34+ cells
MEF alone
Liver precursor cells
Brain precursor cells
Pancreatic precursor cells
EB 8d
Neural cells
BFU-E
EB 14d
Epith cells
Granulo/Mφ
Neural epith
Bone
Cartilage
Striated
muscle
Kydneys
Gut
Multi-tissue chimeric models for human diseases: HESCRIM
syngeneic hESCderived nonhematopoietic
tissue precursors:
HLA+ mice
liver
pancreas
Human immune
system
brain
====> models for HIV, HCV/HBV, prion, type I diabetis, tumors….
Towards best adapted chimeras ……..
- CH1-2hSa
- hESC, hIPCs
from A.P.A. Theocharides et al, heamatologica, 2016
UPBL
C. RAMOND, Unité de
A.A. FREITAS
I. AMADO
Hospital, London
A. BANDEIRA
J-H. COLLE
F. LEMONNIER, Cochin
F. HUETZ
D. HUME, Glasgow, UK
M. SERRA
UBIHP
A. SCHERF
S. MECHERI
M.SERRA
And all members
B. STOCKINGER, NMIR, London
D. ALTMANN, Hammersmith
M. ANSON
M-C. VOUGNY
Lymphopoïèse, IP
M-L. MICHEL, IP, PVHB
M BOURGINE, IP, PVHB
S. DION, IP, PVHB
A. ULLRICH, Martinsried, Germany
F. LANGA, IP
Auteur
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