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Dominique CHARRON, MD-PhD (Dominique.Charron@sls.aphp.fr)
Hôpital Saint Louis, IUH - INSERM U 662 – CIB-HOG – Université Paris Diderot
UNIVERSITE DE PARIS 7 DENIS DIDEROT
MADRID, April 24th-25th, 2008
« IMMUNITY AND ALLOGENEICITY » STEMCELLNESS - EDUCATION VS DIVERSITY
FIFTH INTERNATIONAL SYMPOSIUM ON STEM CELL THERAPY AND APPLIED CARDIOVASCULAR BIOTECHNOLOGY
I – Immunogenetics
II – Alloreactivity/Tolerance Direct AllorecognitionIndirect
Allorecognition
III – Medical impact in Transplantation RejectionCancer
IV – Immunosenescence Immune reconstitution
HLA, MHC andMUCH MORE
ExtendedClass II
HS
ET
DP
B1
DP
A1
DQ
B1
DQ
A1
DR
B1
DR
A
Chromosome 6
C4 C4 TNF B C E A G F HFE
D6S73 D6S223
Extended HLA complex
Class II Class III Class I ExtendedClass I
0.3Mb 0.9Mb 0.7Mb 1.8Mb 4Mb
Map of human MHC lociMap of human MHC lociMap of human MHC lociMap of human MHC loci
NUMBER OF HLA ALLELES – Feb. 2008NUMBER OF HLA ALLELES – Feb. 2008
HLA CLASS I ALLELES
A B C
630 (44) 979 (33) 33 (7)
HLA CLASS II ALLELES
DRA DRB DQAI DQBI DPAI DPBI
3 633 (8) 34 (1) 90 (1) 24 128 (2)
2047
944
2047
944
2991
HLA Null alleles ( ) 6506
IMMUNE RECOGNITION: SELF VS NON - SELF VS ALLO
IMMUNE RECOGNITION: SELF VS NON - SELF VS ALLO
+ SELECTION MHC RESTRICTION- SELECTION SELF TOLERANCE
Graft survival rates in relation to the number of HLA-A, -B, -DR mismatches for transplants performed 1985–1994 (left) and 1995–2004 (right).
Effect of Human Leukocyte Antigen Compatibility on Kidney Graft Survival
Effect of Human Leukocyte Antigen Compatibility on Kidney Graft Survival
p<0.001 p<0.001
G. Opelz and B. Dohler, Transplantation, 84:137, 2007G. Opelz and B. Dohler, Transplantation, 84:137, 2007
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
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
Two Pathways of AllorecognitionTwo Pathways of Allorecognition
RENAL TRANSPLANTATION De Novo ANTI HLA ANTIBODIES
RENAL TRANSPLANTATION De Novo ANTI HLA ANTIBODIES
- OCCURRENCE 26 %- PROLONGED ABSENCE OF ANTIBODY CORRELATES WITH
GOOD FUNCTION
- WHILE 86% OF GRAFT FAILURE HAVE HLA AND/OR MICA Abs
PROSPECTIVE STUDY HEARTLUNG
RENAL TRANSPLANTATIONANTIBODIES AGAINST MICA ANTIGENS
1910 PATIENTS 217 + (11.4%)
RENAL TRANSPLANTATIONANTIBODIES AGAINST MICA ANTIGENS
1910 PATIENTS 217 + (11.4%)
1 YEAR ALLOGRAFT SURVIVAL
ALL CASES 88.3 vs 93 p0.01
FIRST TRANSPLANT 87 vs 93.5p0.005
WELL HLA MATCHED 83.2 vs 95.1 p0.002
ALL CASES 88.3 vs 93 p0.01
FIRST TRANSPLANT 87 vs 93.5p0.005
WELL HLA MATCHED 83.2 vs 95.1 p0.002
Zou et al. NEJM 357, 1293, 2008
POST TRANSPLANT MALIGNANCIESPOST TRANSPLANT MALIGNANCIES
RISK OF MALIGNANCY X 4 TO 500RISK OF MALIGNANCY X 4 TO 500
- EARLY EVENT (1Y) PT LYMPHOPROLIFERATIVE DISEASE- LATE EVENT SKIN CANCER
IN 200 000 TRANSPLANTS OVER 10 YEARS FOLLOW UP 11.8 FOLD HIGHER RISK (P0.0001)
HIGHEST RISK OF LYMPHOMA AT 1 YEARRR 239.5 IN HEART-LUNG TRANSPLANT
IN 200 000 TRANSPLANTS OVER 10 YEARS FOLLOW UP 11.8 FOLD HIGHER RISK (P0.0001)
HIGHEST RISK OF LYMPHOMA AT 1 YEARRR 239.5 IN HEART-LUNG TRANSPLANT
Robson et al. 2005 - Am J Transplant 5, 2954
CTS
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
IMMUNOSCENESCENCEIMMUNOSCENESCENCE
DECREASES LYMPHOPOEISIS THYMIC INVOLUTION AND DECLINE IN T CELL FUNCTIONS SUBSTANTIAL CHANGES IN ALL B CELL COMPARTMENTS
AGE-RELATED HSC DEFECT ???
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 POTENTIALQUANTITATIVE IN VITRO ANALYSIS OF HSC DIFFERENTIATION POTENTIAL
HSC : DECREASED B BUT NOT MYELOID POTENTIAL WITH AGEHSC : DECREASED B BUT NOT MYELOID POTENTIAL WITH AGE
Young
Old
YoungOld
CLP : DECREASED B CELL POTENTIAL IS ASSOCIATED WITH A REDUCTION IN EBF AND PAX5 EXPRESSION
CLP : DECREASED B CELL POTENTIAL IS ASSOCIATED WITH A REDUCTION IN EBF AND PAX5 EXPRESSION
Re
lativ
e e
xpre
ssio
n (
a.u
.)Ebf Pax5 Gata-3
*
**
Young
Old
1/285
1/15
Young
Old
*
% n
egat
ive
wel
ls
B Potential
number CLP / well
TRANSDUCTION OF EBF AND PAX5 IN CLP FROM OLD MICE
TRANSDUCTION OF EBF AND PAX5 IN CLP FROM OLD MICE
Measure B cell potential
In 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
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 GFPIRES
LTR
+EBFLTR P GFP
IRESLTREBF
Measure B cell potential
In 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
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 GFPIRES
LTR
LTR P GFPIRES
LTRPax5
Measure B cell potential
In 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
HSC CLP Pro-B Pre-B immatureB cell
T/NKprgenito
r
EBF
Pax5
Ag
ing
o Adaptative Immunotherapy (curative)
o Immune reconstitution (preventive) Vaccine optimization
Infection Cancer
Autologous – Immunocompetent cells (T, B, APC, DC…)Immediately available and operationnal
But IMMUNOSENESCENCE
PARADIGM SHIFTPARADIGM SHIFT
DEFFERED USEDEFFERED USE
ALSO TECHNOLOGICAL LEAP LONG TERM CRYOPRESERVATION – LOGISTICS FOR DELIVERY
VS STEM CELLS Immature and non educated Immunologically naive Not immediately operational G-CSF mobilization
iatrogenic risk Also age
VS CORD BLOOD CELLS Naive cells (lacking Memory) Once a life time collection Limited number/quantity Septic collection risk
WBC BENEFIT : EDUCATED, INTELLIGENT : NAIVE + MEMORY IMMEDIATELY OPERATIONAL (THERAPEUTICALLY)
UNLIMITED, LIFE TIME OPPORTUNITY, STERILE COLLECTION
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
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