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

HLA AND PEPTIDES

PLAY

IN A QUARTET

IMMUNE RECOGNITION: SELF VS NON - SELF VS ALLO

IMMUNE RECOGNITION: SELF VS NON - SELF VS ALLO

+ SELECTION MHC RESTRICTION- SELECTION SELF TOLERANCE

MEDICAL CONSEQUENCES OF ALLORECOGNITION

MEDICAL CONSEQUENCES OF ALLORECOGNITION

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

PRE TRANSPLANT Abs (TRANSFUSION/PREGNANCY)

Ab DETECTIONCROSS MATCH

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

THE IMMUNE CLOCKTHE IMMUNE CLOCK

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 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 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

RESETRESET

THE IMMUNE CLOCKTHE IMMUNE CLOCK

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

- Immediately operational- usable for in vitro

engineering- source of younger stem cells

D. CHARRON HUMAN IMMUNOL 2007, 68,805-812.

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