SIXTH INTERNATIONAL SYMPOSIUM ON STEM CELL THERAPY AND CARDIOVASCULAR INNOVATIONS

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



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



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



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





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





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

SIXTH INTERNATIONAL SYMPOSIUM ON STEM CELL THERAPY AND CARDIOVASCULAR INNOVATIONS

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