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SIXTH INTERNATIONAL SYMPOSIUM ON STEM CELL THERAPY AND CARDIOVASCULAR INNOVATIONS. Madrid , April 23th-24th , 2009. IS IT TIME TO USE ALLOGENEIC STEM CELLS?. AN IMMUNOLOGICAL PERSPECTIVE. Dominique CHARRON, MD-PhD ([email protected]) - PowerPoint PPT Presentation
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SIXTH INTERNATIONAL SYMPOSIUM ON STEM SIXTH INTERNATIONAL SYMPOSIUM ON STEM CELL THERAPY AND CELL THERAPY AND
CARDIOVASCULAR INNOVATIONSCARDIOVASCULAR INNOVATIONS
SIXTH INTERNATIONAL SYMPOSIUM ON STEM SIXTH INTERNATIONAL SYMPOSIUM ON STEM CELL THERAPY AND CELL THERAPY AND
CARDIOVASCULAR INNOVATIONSCARDIOVASCULAR INNOVATIONS
MadridMadrid, , AprilApril 23th-24th23th-24th, , 20092009
IS IT TIME TO USE ALLOGENEIC STEM CELLS?
AN AN IMMUNOLOGICALIMMUNOLOGICAL PERSPECTIVE PERSPECTIVE AN AN IMMUNOLOGICALIMMUNOLOGICAL PERSPECTIVE PERSPECTIVE
Dominique CHARRON, MD-PhD ([email protected])
Hôpital Saint Louis, IUH - INSERM U 940 – CIB-HOG – Université Paris Diderot
Dominique CHARRON, MD-PhD ([email protected])
Hôpital Saint Louis, IUH - INSERM U 940 – CIB-HOG – Université Paris Diderot
ALLOGENIC IMMUNITY I: MOLECULAR BASISALLOGENIC IMMUNITY I: MOLECULAR BASISALLOGENIC IMMUNITY I: MOLECULAR BASISALLOGENIC IMMUNITY I: MOLECULAR BASIS
SYSTEMSYSTEM STRUCTURESTRUCTURE POLYMORPHISMPOLYMORPHISM EXPRESSIONEXPRESSION AbsAbs CC OUTCOMEOUTCOME
ABO CARBOHYDRATE RESIDUES
GLYCOLIPIDS
LIMITED ALL CELLS PREFORMED + HYPER ACUTE REJECTION
MHC HLA
SCLASS I CLASS II
3000
100
ALL CELLS APC+ ACTIVATED
ENDO/EPITHELIA
PRE T(Pregnancy/Transfusion)
POST TDe novo
+
+-
ACUTE REJECTION
CHRONIC REJECTION
m H AgPRESENTEDBy MHC
VARIABLE + + + Variable ? GVH
MICA
HLA
Nat.Rev.Cancer.2004. 4:371-380
ALLOGENEICALLOGENEIC IMMUNITYIMMUNITY IIII : CELLULAR : CELLULAR RESPONSESRESPONSESALLOGENEICALLOGENEIC IMMUNITYIMMUNITY IIII : CELLULAR : CELLULAR RESPONSESRESPONSES
INDUCTION/TRIGGERING/EFFECTOR PHASE (REJECTION)
Preexisting Allogeneic T Cells (CD8/CD4) Immediate Response
1- 5 % of Circulating T Cells vs< 0.5% for Ag
2 pathways
Direct – Indirect
TcCytotoxic T-cell
ThHelper T-cell
Allogeneic (Donor) APC(stimulator)
Class I Class II
Allo MHC moleculesfrom the Donor are recognized by
Host T-cells
Pathways of allorecognitionPathways of allorecognition
Direct allorecognitionDirect allorecognition
Allogeneic (Donor) Cell
MHC or other moleculesare shed
taken up andprocessed by host APC
Host APC(recipient)
ThHelper T-cell
TcCytotoxic T-cell
Peptide derived from allo moleculespresented on host MHC
to Host T-cells
Indirect allorecognitionIndirect allorecognition
IMMUNE RECOGNITION: SELF VS NON SELF VS ALLO
IMMUNE RECOGNITION: SELF VS NON SELF VS ALLO
+ SELECTION - MHC RESTRICTION - SELECTION - SELF TOLERANCE+ SELECTION - MHC RESTRICTION - SELECTION - SELF TOLERANCE
ALLOGENEIC SC ARE NOT ALLOGENEIC SC ARE NOT IMMUNO PRIVILEGED !!!IMMUNO PRIVILEGED !!!
ALLOGENEIC SC ARE NOT ALLOGENEIC SC ARE NOT IMMUNO PRIVILEGED !!!IMMUNO PRIVILEGED !!!
1. MHC EXPRESSION2. IMMUNOGENEICITY INCREASES UPON
DIFFERENCIATION3. IN VIVO REJECTION
THREE SUPPORTING PAPERS
Micha Drukker, Gil Katz, Achia Urbach, Maya Schuldiner, Gal Markel, Joseph Itskovitz-Eldor, Benjamin Reubinoff, Ofer
Mandelboim, and Nissim Benvenisty
PNAS, 2002, 99:9864
1
Characterization of the expression of MHC proteins in human embryonic stem cells
Expression of MHC proteins in undifferentiated and differentiated human ES cells
2m
HLA-I
HLA-II
721.221
Fluorescence intensity
Co
un
ts
IFN- induction of MHC-I in human ES cells is dose and time dependent
Embryonic Stem Cell Immunogenicity Increases Upon Differentiation After
Transplantation Into Ischemic Myocardium
Rutger-Jan Swijnenburg, MS; Masashi Tanaka, MD; Hannes Vogel, MD; Jeanette Baker, PhD; Theo Kofidis, MD; Feny Gunawan, BS;
Darren R. Lebl, MS; Anthony D. Caffarelli, MD; Jorg L. de Bruin, MD; Eugenia V. Fedoseyeva, PhD; Robert C. Robbins, MD
Circulation. 2005;112:I-166-I-172
2
Graft infiltration of immune cells after transplantation of in vivo differentiated ESCs
Immunosuppressive therapy mitigates immunological rejection of human embryonic stem cell xenografts
Rutger-Jan Swijnenburg, Sonja Schrepfer, Johannes A. Govaert, Feng Cao, Katie Ransohoff, Ahmad Y. Sheikh, Munif Haddad, Andrew J.
Connolly, Mark M. Davis, Robert C. Robbins, and Joseph C. Wu
PNAS, 2008,105:12991
3
In vivo visualization of hESC survival
MEDICAL CONSEQUENCES OF ALLORECOGNITION
MEDICAL CONSEQUENCES OF ALLORECOGNITION
1
1,37
1,61*
1,78*
1
1,12
1,37
1
1,5
0143
1655
22015
33288
42576
51049
6177
RR
Incompatibilities HLA-A-B-DR
Failu
re :
R
R
Multivariate Analysis (Cox)
Three-year graft survival rates (A) and frequency of rejection treatment during first year (B) in relation to number of HLA-A, -B, and -DR mismatches in patients transplanted from 2000 to 2004. p<0.001
PRE IMPLANT Ab (TRANSFUSION/PREGNANCY)
PRE IMPLANT Ab (TRANSFUSION/PREGNANCY)
Ab DETECTIONCROSS MATCH
Terasaki et al.
Predictive Value of HLA Antibodies and SerumCreatinine in Chronic Rejection: Results of a 2-yearProspective Trial
Transplantation 2005;80: 1194–1197
De Novo ANTI HLA ANTIBODIES (+ CD 4d deposit) DEVELOP AFTER RENAL
TRANSPLANTATION
De Novo ANTI HLA ANTIBODIES (+ CD 4d deposit) DEVELOP AFTER RENAL
TRANSPLANTATION
PROLONGED ABSENCE OF ANTIBODY CORRELATES WITH GOOD FUNCTION
WHILE 86% OF GRAFT FAILURE HAVE ANTI HLA AND/OR MICA Abs
PROLONGED ABSENCE OF ANTIBODY CORRELATES WITH GOOD FUNCTION
WHILE 86% OF GRAFT FAILURE HAVE ANTI HLA AND/OR MICA Abs
PROSPECTIVE STUDY HEARTLUNG
1910 RENAL TRANSPLANTATIONANTIBODIES AGAINST MICA ANTIGENS
217 + (11.4%)
1910 RENAL TRANSPLANTATIONANTIBODIES AGAINST MICA ANTIGENS
217 + (11.4%)
1 YEAR ALLOGRAFT SURVIVAL
ALL CASES 88.3 vs 93 p0.01
FIRST TRANSPLANT 87 vs93.5 p0.005
WELL HLA MATCHED 83.2 vs 95.1p0.002
ALL CASES 88.3 vs 93 p0.01
FIRST TRANSPLANT 87 vs93.5 p0.005
WELL HLA MATCHED 83.2 vs 95.1p0.002
Zav et al. NEJM 357, 1293, 2008
HSCTHSCT
HLA MATCHED SIBLINGS GVHD 30 % UPHLA MATCHED SIBLINGS GVHD 30 % UP
PERFECT HLA MATCH # OPTIMAL GENETIC MAKE UPPERFECT HLA MATCH # OPTIMAL GENETIC MAKE UP
GENES OUTCOMES
HLA
NON HLA MHC
NON MHC
GVHD - REJECTION
INFECTIONS
TRM – RELAPSE
SURVIVAL
INTEGRATED GENETICSINTEGRATED GENETICS
DONOR SELECTION
PREVENTION / PROPHYLAXISADAPTED TREATMENT
Survival according Survival according to number of HLA to number of HLA
disparitiesdisparities
11 (n=48)
12 (n=14)
10 (n=34)
6-9 (n=25)
6-10 (n=59)
11-12 (n=62)
Unrelated BMTUnrelated BMT
0 50 100 150
Days
0.0
0.2
0.4
0.6
0.8
1.0
Cu
mu
lative
in
cid
en
ce
of
se
ve
re
ba
cte
ria
l in
fectio
ns
HLA-E 101/103 or 103/103HLA-E 101/101
5318% vs 2512; PGray test = 0.035
0 1 2 3 4 5 6
months
0.0
0.2
0.4
0.6
0.8
1.0
Cu
mu
laitve
in
cid
en
ce
of
tra
nsp
lan
tatio
n-re
late
d m
orta
lity
HLA-E 101/103 or 103/103HLA-E 101/101
Risk of transplant-related mortality: HLA-E*0101/0101 donor genotype:
Risk of of severe bacterial infections: HLA-E*0101/0101 donor genotype:
5318% vs 2714% PGray test = 0.08
CYTOKINES - INFLAMMATIONCYTOKINES - INFLAMMATION
ALLELES/GENOTYPE
PHENOTYPE
(LEVEL PRODUCTION) HSCT OUTCOMES
PROINFLAMMATORY• IL6-174 G HIGH
a GVHD
c GVHD
• TNF
----------------------------• , , R
d 3
------------------------------------
…
HIGH
-------------------------------
-
a GVHD
(- mCB)
---------------------------------
C GVHD
• INT 2/2
3/3
HIGH
LOW
GVHD
GVHD
ANTI INFLAMMATORY
•IL 10 PROMOTOR
GCC
ATA
ACC
HIGH
INTERMEDIATE
LOW
a GVHD
c GVHD
SURVIVAL
(- mCB)
• TGF promotor -509
codon 25
C/T
-
-
HIGH
0
GVHD
HSCT : INNATE IMMUNOGENETICSHSCT : INNATE IMMUNOGENETICS
Frequency/Severity of infectious (viral, bacterial, fungal) events Frequency/Severity of infectious (viral, bacterial, fungal) events
Intricated with GVHD TRM - Survival
GENESGENES GENOTYPESGENOTYPESINFECTIONS/GVHD
TRM
INFECTIONS/GVHD
TRM
MPO AG or AA (non CC)
MBL MBL2
Fc RII a (CD 32) R 131
Fc RIII b HNA-1a
TLR 4 -
NOD2/CARD 15 SNP 8, 12, 13
Oestrogen Receptor intron 1
Vitamin D Receptor intron 8
Fas - 670 G
STEM CELL/RECIPIENT COMPATIBILITYSTEM CELL/RECIPIENT COMPATIBILITY
PREDICTION ANTI- HLA/MIC A DETECTION (quarterly), IDENTIFICATION
+ CROSS MATCHING PRE TRANSPLANT (FINAL TEST)
Best acheviable HLA A, B, DR, DQ, TypingC, DP mandatory if antibodies present
Post transplantation FOLLOW UP
Donor specific AbHLA - MIC
IMMUNOGENETIC MATCHINGIMMUNOGENETIC MATCHING
ABO COMPATIBILITY feasible
+
HLA COMPATIBILITY Level 1 Generic matching (OT)Level 2 Intermediate matching (CB)Level 3 Full matching (UD BMT)
+m Hag COMPATIBILITY !!!
ABO COMPATIBILITY feasible
+
HLA COMPATIBILITY Level 1 Generic matching (OT)Level 2 Intermediate matching (CB)Level 3 Full matching (UD BMT)
+m Hag COMPATIBILITY !!!
STEM CELL BANKS
IMMUNOGENETIC SELECTION
LOOKING FORWARDLOOKING FORWARD
MATCHING - REDUCING MISMATCHING
• STEM CELL BANKS
• STEM CELL INGENEERING
INNOVATIVE IMMUNOSUPPRESSION/INDUCTION OF TOLERANCE
T CELLS COSTIMULATION BLOCKADE
REGULATORY T CELLS
B CELLS
IV Ig
CD 40 L BY ANTI CD 40 L
B7 BY CTLA 4 Ig
ANTI LFA1…CD 20
VELCADE
GENERATION OF MIXED CHIMERISM/ THYMIC INGENEERING
STANDARD CHRONIC IMMUNO SUPPRESSION:
INFECTIONS/CANCER, AGENT SPECIFIC KIDNEY FAILURE, DIABETES, OSTEOPOROSIS, HYPERTENSION…
THE STORY BOARD OF MEDICINE
PREDICTIVE MEDICINE
PREDICTIVE MEDICINE
HIPPOCRATES OF KOS
HIPPOCRATES OF KOS
PREVENTIVE MEDICINE
PREVENTIVE MEDICINE
GENETICS/PHARMACOGENETICS
INDIVIDUALIZED SUSCEPTIBILITY
GENETICS/PHARMACOGENETICS
INDIVIDUALIZED SUSCEPTIBILITY
1st SCHOOL OF MEDICINE1st SCHOOL OF MEDICINE
EMPIRISM ECONOMICS
GOD OF HEALINGGOD OF HEALING
PUBLIC HEALTHINDIVIDUAL + POPULATION
PUBLIC HEALTHINDIVIDUAL + POPULATION
ANTICIPATIVEANTICIPATIVE
INTEGRATIVE
MEDICINEMEDICINE
1st HOSPITAL1st HOSPITAL
SCIENCE
MYTH
HOLISTIC
Immunogenicity and Allogenicity: A Challenge of Stem Cell Therapy
Dominique Charron.Caroline Suberbielle-Boissel.Reem Al-Daccak
J.of Cardiovasc.Trans.Res.2009,8- 9062-9
AGING OF HSCAGING OF HSC
Aging and HSCAging and HSC
Increased number of HSC with age in certain strains of mice
(Morrison et al 1996, Nature Med)
Increased number of HSC in cycle(Rossi et al 2005, PNAS)
Expression of senescence marker (p16)(Janzen et al 2006, Nature,)
Decreased reconstitution capacity - lymphoid compartment.
(Sudo et al 2000, JEM)
Intrinsic changes in the lymphoid potential of HSC ?
HSC fraction CLP fraction
(Lin-Sca1hicKithiIL7R-) (Lin-Sca1locKitloIL7R+)
Old 18-24m Young 1-2m
Bone marrow
Plate in 96 wells at limiting dilution
OP9 + IL7 OP9 + IL7
Facs sort wells
Frequency CD19+ (B cells) Frequency CD11b+ (Myeloid)
QUANTITATIVE IN VITRO ANALYSIS OF HSC DIFFERENTIATION POTENTIAL
QUANTITATIVE IN VITRO ANALYSIS OF HSC DIFFERENTIATION POTENTIAL
HSC : decreased B but not myeloid potential with age
Young
Old
YoungOld
Transduction of EBF and Pax5 in CLP from old mice
Measure B cell potentialIn vitro differentiation OP9 + IL-7, c-KitL, Flt3
Frequency of B cell clones(CD19+)
Transduction CONT±EBF,Pax5
Young(1-2m)
Old(18-24m)
CONT LTR P GFP
IRESLTR
CONT+EBF LTR P GFP
IRESLTREBF
CONT+Pax5 LTR P GFP
IRESLTRPax5
Isolate CLP Lin-IL-7R+Sca-1+c-Kit+
EBF expression restores B cell potential of CLP from old mice
B potential
1/570
Young1/18
Old
% n
eg
ati
ve w
ells
Old+EBF
1/43
Number of cells / well
CONT
LTR P GFP
IRESLTR
+EBFLTR P GF
P
IRESLTREB
F
Measure B cell potentialIn vitro differentiation OP9 + IL-7, c-KitL, Flt3
Frequency of B cell clones(CD19+)
Transduction withEBF vector
Young(1-2m)
Old(18-24m)
Isolate CLP Lin-IL-7R+Sca-1+c-
Kit+
Pax5 expression restores B cell potential of CLP from old mice
1
10
100
0 50 100 150 200 250 300
1/285
Young1/9
Old
B Potential
% n
eg
ati
ve w
ells
Old+Pax5
1/41
Number of cells / well
+Pax5
CONT
LTR P GFP
IRESLTR
LTR P GFP
IRESLTRPax5
Measure B cell potentialIn vitro differentiation OP9 + IL-7, c-KitL, Flt3
Frequency of B cell clones(CD19+)
Transduction withPAX5 vector
Young(1-2m)
Old(18-24m)
Isolate CLP Lin-IL-7R+Sca-1+c-
Kit+
ConclusionConclusion
Intrinsic loss of B cell potential of HSC with age
Due to decreased expression of EBF and Pax5
Intrinsic loss of B cell potential of HSC with age
Due to decreased expression of EBF and Pax5
HSC CLP Pro-B Pre-B
immatureB cell
T/NKprgenit
or
EBF
Pax5
Ag
ing
IMMUNOSCENESCENCEIMMUNOSCENESCENCE
DECREASES LYMPHOPOEISIS THYMIC INVOLUTION AND DECLINE IN T CELL
FUNCTION SUBSTANTIAL CHANGES IN AN B CELL COMPARTMENTS
AGE-RELATED HSC DEFECT ??
DECREASES LYMPHOPOEISIS THYMIC INVOLUTION AND DECLINE IN T CELL
FUNCTION SUBSTANTIAL CHANGES IN AN B CELL COMPARTMENTS
AGE-RELATED HSC DEFECT ??
THE IMMUNE CLOCKTHE IMMUNE CLOCK
Nat.Rev.Cancer.2004. 4:371-380
Dominique CHARRON, MD-PhD ([email protected])
Hôpital Saint Louis, IUH - INSERM U 662 – CIB-HOG – Université Paris Diderot
Dominique CHARRON, MD-PhD ([email protected])
Hôpital Saint Louis, IUH - INSERM U 662 – CIB-HOG – Université Paris Diderot
UNIVERSITE DE PARIS 7 DENIS DIDEROT
MADRID, April 24th-25th, 2008MADRID, April 24th-25th, 2008
« IMMUNITY AND ALLOGENEICITY : STEMCELLNESS EDUCATION VS AND DIVERSITY »
FIFTH INTERNATIONAL SYMPOSIUM ON STEM CELL THERAPY AND APPLIED CARDIOVASCULAR BIOTECHNOLOGY
HSCTHSCT
HLA MATCHED SIBLINGS GVHD 30 % UPHLA MATCHED SIBLINGS GVHD 30 % UP
PERFECT HLA MATCH # OPTIMAL GENETIC MAKE UPPERFECT HLA MATCH # OPTIMAL GENETIC MAKE UP
GENESGENES OUTCOMESOUTCOMES
HLA
NON HLA MHC
NON MHC
HLA
NON HLA MHC
NON MHC
GVHD - REJECTION
INFECTIONS
TRM – RELAPSE
SURVIVAL
GVHD - REJECTION
INFECTIONS
TRM – RELAPSE
SURVIVAL
INTEGRATED GENETICSINTEGRATED GENETICS
DONOR SELECTIONDONOR SELECTION
PREVENTION / PROPHYLAXISPREVENTION / PROPHYLAXIS ADAPTED TREATMENTADAPTED TREATMENT
Human HLA and non HLA ImmunogeneticsHLA
HLA Class I A B CClass II DRB 1, 3, 4, 5
DQA1/DQB1DPB1
HLA – E DMA/BMIC – A/B …
HLA Related systems Minor Histocompatibility antigensCD1 a-e HYKIR/ NKR/ HSP 70 HA. 1.2.3 …
Cytokines Chemokines ReceptorsTNFIL1 IL6 IL10 IFN CCR5 CTL4
IL1 - R IL4 R IL4 …IL1 – R antagonist
Innate Immunity
Toll receptors TLR2, TLR4 E selectin/ICAM-1/PECAM MBL MPO NOD2…
IMMUNOGENETIC INDEX INDIVIDUALIZED MEDICINEPREDICTION PREVENTION
Human HLA and non HLA ImmunogeneticsHLA
HLA Class I A B CClass II DRB 1, 3, 4, 5
DQA1/DQB1DPB1
HLA – E DMA/BMIC – A/B …
HLA Related systems Minor Histocompatibility antigensCD1 a-e HYKIR/ NKR/ HSP 70 HA. 1.2.3 …
Cytokines Chemokines ReceptorsTNFIL1 IL6 IL10 IFN CCR5 CTL4
IL1 - R IL4 R IL4 …IL1 – R antagonist
Innate Immunity
Toll receptors TLR2, TLR4 E selectin/ICAM-1/PECAM MBL MPO NOD2…
IMMUNOGENETIC INDEX INDIVIDUALIZED MEDICINEPREDICTION PREVENTION
HUMAN AGINGHUMAN AGING
• EXPONENTIAL INCREASE IN CANCER INCIDENCE WITH AGE
GENETIC ALTERATION IMMUNO SENESCENCE
Tumor growth
Dissemination
INFECTIONS INCIDENCE – SEVERITY INFECTIONS INCIDENCE – SEVERITY
LESS RESPONSES TO VACCINES (30 to 50 % of individuals over 65 do not respond to flu vaccine) LESS RESPONSES TO VACCINES (30 to 50 % of individuals over 65 do not respond to flu vaccine)
INCREASE INCIDENCE OF AUTO IMMUNE DISEASES INCREASE INCIDENCE OF AUTO IMMUNE DISEASES
Dominique CHARRON, MD-PhD ([email protected])
Hôpital Saint Louis, IUH - INSERM U 662 – CIB-HOG – Université Paris Diderot
Dominique CHARRON, MD-PhD ([email protected])
Hôpital Saint Louis, IUH - INSERM U 662 – CIB-HOG – Université Paris Diderot
UNIVERSITE DE PARIS 7 DENIS DIDEROT
MADRID, April 24th-25th, 2008MADRID, April 24th-25th, 2008
« IMMUNITY AND ALLOGENEICITY » STEMCELLNESS - EDUCATION VS DIVERSITY
FIFTH INTERNATIONAL SYMPOSIUM ON STEM CELL THERAPY AND APPLIED CARDIOVASCULAR BIOTECHNOLOGY