«Allogenicity & Immunogenicity of Stem Cell Therapy : a Cardiovascular Focus »

DOMINIQUE CHARRON,MD,PhD

Dominique.Charron @ sls.aphp.frLaboratoire « Jean Dausset » & INSERM U 940

Hopital Saint-Louis ,IUH ,Université Paris-Diderot , France

VHIR CONFERENCE 2013BARCELONA

XXth Century

HLA, MHC ,Cytokines,Receptors….

…TRANSPLANTATION, AUTOIMMUNITY,INFECTIONS

XXth Century

HLA, MHC ,Cytokines,Receptors….

…TRANSPLANTATION, AUTOIMMUNITY,INFECTIONS

IMMUNOGENETICS & MEDICINE

XXI st Century

HLA & MEDICINE (Schizophrenia,Parkinson … )IMMUNO PHARMACOGENETICS (Abacavir,Carbamazepin ,Allopurinol…)REGENERATIVE MEDICINE/CELL & IMMUNO THERAPIES

TOWARD « SYSTEMS BIOLOGY/SYSTEMS MEDICINE »

.

Complete sequence and gene map

MHC / HLA

HLA CONSORTIUM – Nature 11/1999

# HLA Alleles

HLA DIVERSITY = BIOLOGICAL SELFHLA DIVERSITY = BIOLOGICAL SELF

2013 : 8496 ALLELES

WE ARE THE LIMIT

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-cellsIndirect allorecognitionIndirect allorecognition

Immune CellImmune Cell

Stem CellStem Cell

Allo Recognition/Activation Pathways

Direct Direct vsvs Indirect Indirect

Recognition phase

APC

HLA-peptide

T cell frequency

Donor Recipient

Donor -Donor Recipient - Donor

Pre-existing1/103 – 1/104 1/105 – 1/106

Immediate ResponseEffector phase

Cytokine production(inflammatory response)

B cell activation(Ab production)

+

+++

++

-Direct cytotoxicity(of donor tissue) -++

Th

Tc

Duration of responseShort lived(donor APC)

long lived(donor peptide)

Outcome Acute Rejection Chronic Rejection

CONSEQUENCES OF HLA HISTO- INCOMPATIBILITY IN TRANSPLANTATION ……2012

T CELL MEDIATED : REJECTION (ORGANS) & GVH(HSCT)

ANTI HLA ANTIBODY MEDIATED: CHRONIC REJECTION(ORGANS) & NO ENGRAFTMENT (HSCT)

2013 CHANGE OF PARADIGM

Antibody-mediated vascular rejection of kidney allografts:a population-based study

Carmen Lefaucheur*, Alexandre Loupy*, Dewi Vernerey, Jean-Paul Duong-Van-Huyen, Caroline Suberbielle, Dany Anglicheau, Jérôme Vérine, Thibaut Beuscart, Dominique Nochy, Patrick Bruneval, Dominique Charron, Michel Delahousse, Jean-Philippe Empana, Gary S Hill, Denis Glotz, Christophe Legendre, Xavier Jouven

LANCET Nov 23,2012

Population based study2079 patients(nck/sls)+ 602validation samples(foch)

302 biopsy proven rejection(1998-2008)

CINICAL, HISTO PATHOLOGICAL(including C4d)& IMMUNOLOGICAL(DSA) DATA

Hierarchical cluster analysisunsupervised principal component

4 patterns of rejectionTCMR/V+ :T cell mediated rejection (26 9 °/°)

ABMR/V+ :Antibody mediated rejection(64 21°/°)

TCMR/V- : T cell mediated rejection without vasculitis(139 46°/°)

ABMR/V- : Antibody mediated rejection without vasculitis(73 24°/°)

PATHOLOGICAL & IMMUNOLOGICAL PHENOTYPES OF THE 4 REJECTION PATTERNS

Endarteritis --

Endarteritis +

Cellular (Tcell) rejection Antibody mediated rejection

TCMR V -

TCMRV+

ABMR V -

ABMR V +

GRAFT SURVIVAL IN THE 4 REJECTION PHENOTYPES

ABMR V+

HLA, MHC AND MUCH MORE….…TRANSPLANTATION, AUTOIMMUNITY AND MUCH MORE

HLA, MHC AND MUCH MORE….…TRANSPLANTATION, AUTOIMMUNITY AND MUCH MORE

HLA in MEDICINEIMMUNOPHARMACOGENETICS REGENERATIVE MEDICINESYSTEMS BIOLOGY

REGENERATIVE MEDICINE AND TRANSPLANTATIONREGENERATIVE MEDICINE AND TRANSPLANTATION

PLURI / MULTIPOTENCY SELF RENEWAL IN VITRO SPECIFIC DIFFERENCIATION IMMUNE PRIVILEGE ?

PLURI / MULTIPOTENCY SELF RENEWAL IN VITRO SPECIFIC DIFFERENCIATION IMMUNE PRIVILEGE ?

BENEFITSBENEFITS

LIMITS OF IN VIVO ENGRAFTMENT AND FUNCTIONALITYLIMITS OF IN VIVO ENGRAFTMENT AND FUNCTIONALITY

IMMUNOGENICITY/ALLOGENICITY/REJECTION/AUTOIMMUNITY ? DISPONIBILITY – TIMELINE AGING SAFETY ETHICAL – REGULATORY ISSUES

IMMUNOGENICITY/ALLOGENICITY/REJECTION/AUTOIMMUNITY ? DISPONIBILITY – TIMELINE AGING SAFETY ETHICAL – REGULATORY ISSUES

THE IMMUNITY FACTORS IN REGENERATIVE CELL THERAPIES

THE IMMUNOGENETIC FACTOR: ALLOGENICITY HLA, MHC and Much More….

THE IMMUNE EFFECTORS: DIRECT vs INDIRECT PATHWAYS OF ALLO RECOGNITION

Cells, Mediators and Allo Antibodies...

THE AGING FACTOR: IMMUNO SENESCENCE

Immune CellImmune Cell

Stem CellStem Cell

Towards an IMMUNOLOGICALLY EDUCATED CHOICE OF SCs

ALLOGENEIC STEM CELLS ARE NOT ALLOGENEIC STEM CELLS ARE NOT IMMUNO PRIVILEGED IMMUNO PRIVILEGED

ALLOGENEIC STEM CELLS ARE NOT ALLOGENEIC STEM CELLS ARE NOT IMMUNO PRIVILEGED IMMUNO PRIVILEGED

MHC EXPRESSION

IMMUNOGENICITY INCREASES UPON DIFFERENCIATION

IN VIVO REJECTION

Immune CellImmune Cell

Stem CellStem Cell

2002 -2008

3 SUPPORTING PAPERS

M. DRUKKER, G. KATZ, A. URBACH, M. SCHULDINER, G. MARKEL, J. ITSKOVITZ-ELDOR, B. REUBINOFF, O. MANDELBOIM, N. BENVENISTY

PNAS, 2002, 99:9864

CHARACTERIZATION OF THE EXPRESSION OF MHC PROTEINS IN HUMAN EMBRYONIC STEM CELLS

2m

HLA-I

HLA-II

721/HLA-G

Fluorescence intensity

Coun

ts

DIFFERENTIATEDUNDIFFERENTIATEDIn vitro In vivo

Immune CellImmune Cell

Stem CellStem Cell

IFN- induction of MHC-I in human ES cells is dose and time dependent

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 MyocardiumR-J Swijnenburg, M. Tanaka, H. Vogel, J. Baker,T. Kofidis, F. Gunawan, D.R. Lebl, A.D. Caffarelli, J.L. de Bruin, E.V. Fedoseyeva, R.C. Robbins

Circulation. 2005;112:I-166-I-172

Graft infiltration of immune cells after transplantation of in vivo differentiated ESCs

T cells B cells

DIFFERENCIATION OF ALLOGENEIC MESENCHYMAL STEM CELLS INDUCES IMMUNOGENICITY & LIMITS THEIR LONG-TERM BENEFITS FOR MYOCARDIAL REPAIR

Xi-Ping Huang & coll Circulation .2010 ;122:2419-242• Wistar and lewis rats• MSCs untreated vs MSCs cultured with 5-azacytidine(to induce myogenic

differentiation)Flow cytometric & mRNA evaluation of MHC Ia,II and CD86 is increased by

>30% upon differentiation While MHC Ib is decreased -------GFP+ MSCs Implanted into the infarcted myocardium 3 weeks after MI

express low level of MHC Ia when undifferenciated(alpha-SMA-) at day seven & high level of MHC Ia when differenciated(alpha-SMA+) at Day 14(differenciated)

------ Implanted Allogeneic MSCs induce a local immune reaction after 7 days and are not detected in situ after 5 weeks

-------Allogeneic MSCs restore cardiac function as effectively as Syngeneic MSCs for 3 months but not 6 monts after implantation

While immunoprivileged in their undifferenciated state MSCs become immunogenic in vitro & in vivo when differenciated (biphasic immune response)

2O10

ALLOGENICITY/IMMUNOGENICITY &

IMMUNOMODULATORY PROPERTIES of HUMAN

CARDIAC PROGENITOR/STEM CELLS

Human Cardiac Stem/Progenitor Cells Characterization

•Cells from Different donors : Endomyocardic biopsy Collagenase Treatment ckit purification/enrichment • Pluripotency Transcription factors

• CARDIAC LINEAGE SC MARKERS

hCPC Cardiac differenciation potency(in vitro)

CardiomyocytesEndothelial cells

Smooth muscle cells

hCPC Cardiac differenciation potency(in vitro)

CardiomyocytesEndothelial cells

Smooth muscle cells

STEM CELL MARKERS

Nadal-Ginard B et al. Resident human cardiac stem cells: role in cardiac cellular homeostasis and potential for myocardial regeneration. Nat Clin Pract Cardiovasc Med 2006;3

Immunology of Human Cardiac Stem/Progenitor Cellsfor cardiac repair

Inflammatory (IFN 72h)

Non-Inflammatory

3% O2

low immunogenic profile

IL-10 producing CD4+ cells

Lauden L. et al, Circ Res, 2013

Immunology of Human Cardiac Stem/Progenitor Cellsfor cardiac repair

Down-regulate an ongoing immune response (CD4+, CD8+, IFN, IL-2)

hCPC are Immuno-modulators

Lauden L. et al, Circ Res, 2013

More potent within inflammatory conditions

hCPC & Regulatory T cells

ALLOGENEIC hCPC ACTIVATE AND EXPAND T regs ALLOGENEIC hCPC ACTIVATE AND EXPAND T regs

Characterisation of hCPC activated Regulatory T cells

HLA DR+ PD-1+ Regulatory T CellsHLA DR+ PD-1+ Regulatory T Cells

PD-L1 (1)

anti-PD-L1 anti-PD-L1

Inhibition of Treg expansion

Inhibition of the Immunomodulatory

effect

siRNA Inhibition siRNA Inhibition

PD-L1 (2)

PD-L1 is implicated in Treg s activation & immunomodulation by hCPCsPD-L1 is implicated in Treg s activation & immunomodulation by hCPCs

Conclusions

• hCPC are attractive for clinical Translation- Low immunogenic profile( No immediate rejection ? )-Maintainance of immunologic properties under inflammatory conditions-Immunomodulatory properties

• PD-L1/PD-1 orchestrate immunologic properties of hCPC- Treg generation- hCPC-induced immunomodulation

• Allogenic-driven benefit?- hCPC-induced allogenic response is biased towards Treg- Immune properties of hCPC are controlled by PD-L1/PD-1 pathway

• hCPC are attractive for clinical Translation- Low immunogenic profile( No immediate rejection ? )-Maintainance of immunologic properties under inflammatory conditions-Immunomodulatory properties

• PD-L1/PD-1 orchestrate immunologic properties of hCPC- Treg generation- hCPC-induced immunomodulation

• Allogenic-driven benefit?- hCPC-induced allogenic response is biased towards Treg- Immune properties of hCPC are controlled by PD-L1/PD-1 pathway

ALLOGENICITY

Allogenic-driven-risk versus Allogenic-driven-benefit

Beneficial Detrimental

Optimization of ALLOGENIC STEM CELLS for use in Regenerative Medicine

Immunologically educated choice of ALLOGENIC STEM CELLS

Immune behavior of other stem cells (iPSC and ESC- and iPSC-derived progenitors)

Regulation of MHC expression in stem cells and their progenitors

Reactivity with allo-antibodies (risk or benefit?)

Markers of selection (PD-L1?)

Allogeneic ESCs are Immunogenic : alloimmunity

2010 - 2012

Reprogrammed iPSCs are immunogenic :autoimmunity

Gene Transduced cells are immunogenic: autoimmunity

Allogeneic MSCs are immunogenic:alloimmunity

Endomyocardiac stem cells are allogeneic and Immunomodulatory

Immune CellImmune Cell

Stem CellStem Cell

2002 - 2010

• IMMUNOGENETIC SELECTION/BANK OF SC/MATCHING

• INDUCTION OF TOLERANCE

• IMMUNOMODULATION• IMMUNOMONITORING

Immunogenetic selection– HLA MATCHING - REDUCING HLA MISMATCHING

• Pre TX Screening for anti HLA Characterizing anti HLA specificities(SAB) + MIC-A? C1Q? C4d? Cross-Matching

Post TX monitoring anti HLA antibodies

STEM CELL BANKING Autologous Cells (anticipatory) Cells derived from Homozygous individuals (frequent haplotypes) Allogenic Cells

A transplant immunologist point of view

• Minimize immunogenetic differences

• Assay the immunization status of the recipient prior to SCT injection

• Monitor allogenic & autoimmunity post SCT

• Be pragmatic (…immunosupression) and

• Utopic( …hope for tolerance one day )

AcknowledgementAcknowledgement

Luis BorladoMiguel Mulet ParadaCoretherapix (Madrid, Spain)

Bernardo Nadal-GinardGeorgina EllisonLiverpool John Moores University (Liverpool, UK)

FP7 – CARE-MI Consortium

, INSERM; EU FP7 – CARE-MI

Reem AL DACCAK

Khaoussou Sylla Isabelle Martins Kiran RamgolamLaura LaudenWahid Boukouaci

Hopital Saint-Louis

ICIC

TC&SCTC&SC

UMRS940

Ryad Tamouza

Caroline Suberbielle

Pascale Loiseau Emeline Masson 

NOW THIS IS NOT THE ENDIT IS NOT EVEN THE BEGINNING OF THE END

BUT IT IS PERHAPS,

THE END OF THE BEGINNING…THE END OF THE BEGINNING…

Hopital Saint-Louis

HLA IMMUNITY & STEM CELL THERAPY 2013

HLA-class I(850)

HLA-DR(7)

HLA-DQ(0)

HLA-DP(18)

CD40(0)

CD80(0)

CD86(33)

HLA-DR(405)

HLA-DQ

(540)

HLA-DP

(540)

HLA-class I

(18400)

Immune phenotype: HLA expressionImmune phenotype: HLA expressionImmune phenotype: HLA expressionImmune phenotype: HLA expression

hCSC-IFN

MHC II… inductiononly seen when cells are maintained under

hypoxia (3% O2)

physiological inflammatory conditions…?

hCSC

hCPC IMMUNOPHENOTYPE

RESTING & UNDER INFLAMMATORY CONDITION (INF g treated)

No change in morphology & pulripotency markers after IFNγ treatment

Induction of HLA-class IIIncrease expression of HLA-class I & PD-L1

hCPC display a low immunogenic profile

+ INF G

Immunology of Human Cardiac Stem/Progenitor Cellsfor cardiac repair

Low immune risk even within inflammatory environment

Reparatory by promoting Treg and by their expression of PD-L1*Regulatory T cells are implicated in cardiac repair after Myocardial infarction

Tang TT, et al. Cell Physiol Biochem. 2010; Dobaczewski M, et al. Am J Pathol. 2010; Tang TT, et al. Basic Res Cardiol. 2012

*Myocardial PD-L1/PD1 control immune-mediated cardiac injury and polymorphonuclear inflammation Grabie N, et al. Circulation. 2007;116:2062

Allogenic Human Cardiac Progenitor Cells

PD-L1-Dependent Allogenic-Driven Benefit

PromotesClinical Translation

Highlights PD-L1

marker Identify & Select

Low-Risk/High-Benefit allogenic cardiac repair cells

NK cells response Reactivity with anti-HLA antibodies

CD4+ T cells

hCSCs-triggered Allogenic Response

PHA alone Allo PBMC CSCs

CSCs + IFNγ MSCs

CD

4C

D8

89.1% 95.3% 71.6% 70.4% 28.1%

90.7% 97.1% 71.6% 19.6%69.6%

T c

ell

pro

life

rati

on

CFSE

hCSCs immune-modulation of an ongoing T cell response

CSCs

PBMC

CSCs can be recognized by anti HLA allo-antibodies

SUMMARYSUMMARYSUMMARYSUMMARY

CSCs express MHC I and MHC II under physiological & inflammatory situation

They display higher ALLOGENICITY than MSCs and are less powerful in down regulating an ongoing immune response

CSC capacity to induce or modulate an immune response is variable depending on both donor and recipient

CSCs are recognized by allo-anti-HLA sera, which can

lead to in their elimination but could also be part of their paracrine effect

Circulation Research 2012(in press)

Immunosuppressive Therapy Mitigates Immunological Rejection of Human Embryonic Stem Cell XenograftsR.J SWIJNENBURG, S. SCHREPFER, J.A. GOVAERT, F. CAO, K. RANSOHOFF, A.Y SHEIKH, M. HADDAD, A.J CONNOLLY, M.M DAVIS, R.C ROBBINS, J.C WU

PNAS, 2008,105:12991

IN VIVO VISUALIZATION OF HESC SURVIVAL

Immune CellImmune Cell

Stem CellStem Cell

hCPC induced T Cell Response

Allogeneic co culture: CFSE Labelled PBMC/ Mitomycin-C treated hCPC Allogeneic co culture: CFSE Labelled PBMC/ Mitomycin-C treated hCPC

Low response similar to MSC induced Low IFNγ & IL-2 productionHigh IL10 Production

hCPC induce low allogeneic T-cell responsehCPC induce low allogeneic T-cell response

Oct4

Sox2

Nanog

-actin

PBM

Cs

CSC-

IFNγ

CSC

MSC

DAPI NKx2.5

DAPI oct4

DAPI SSEA-

4

DAPI FITC-

IgG

DAPI PE-IgG

DAPI FITC-IgG

Cel

l co

un

ts

Fluorescence intensity

c-kit(48)

CD90(2880)

SSEA-1(269)

SSEA-4(1779)

CD166(100)

CD44(14955)

Human cardiac Stem Cells characterization

Cells from three different donors

CH1, CH3, CH4

Cardiac differenciation potency

(In vitro)

Cardiomyocyte

Endothelial

Smooth muscle

Endomyocardic BiopsyCollagenase treatementC-kit purification/enrichment

Nadal-Ginard B et al. Resident human cardiac stem cells: role in cardiac cellular homeostasis and potential for myocardial regeneration. Nat Clin Pract Cardiovasc Med 2006;3

2012

Immunology of Human Cardiac Stem/Progenitor Cellsfor cardiac repair

Lauden L. et al, Circ Res, 2013

hCPC in allogenic settings have the ability to induce tolerance, by promoting allo-stimulation and a contact and PD-L1-dependent

regulatory response

HLA-DR+ PD1+ Regulatory T cells

2011

• INDUCED PLURIPOTENT STEM CELLS(iPS) ARE IMMUNOGENIC & RESULT IN AUTO-IMMUNITY

IN THE NEWS

Immunogenicity of induced pluripotent stem cellsT. Zhao, Z-N Zhang, Z. Rong and Y. Xu

Nature, 2011, doi:10.1038/nature10135

Immune CellImmune Cell

Stem CellStem Cell

ESCs from C57BL/6

ESCs from 129/SvJ

B6

B6

Teratomal Immune Rejection/Tumor Regression

(IR/TR)0

+++

* Proof of Principle

IR/TR

* Reprogramming of of B6 Embryonic Fibroblast (MEF)Oct4,Sox2,Klf4/Oct4,Sox2,Myc, Klf4

Retroviral Approach

ViPSCs Episomal Approach

EiPSCs

B6 mice+++ ++ CD4 T cell infiltration/Cell necrosis

Mechanisms??Mechanisms??* Gene profiling of EiPSCs

Major findings* episomal vector is deleted* abnormal overexpression of endogenous genes

- Hormad 1 (tumor specific Ag)- Spt1 (tumor specific Ag)- Zg16

* Immune Rejection/Tumor Rejection

Major findings* CD4 CD8 mediated immune rejection through Ag specific (Zg16, Hormad1) T cells

- confirmed by in vivo purified T cells (Ag specific) detected by (IFN release

assay) upon co-culture with Ag-transfected DC from B6 mice

ConclusionConclusion

Break of Peripheral Tolerance/Expression of Minor AntigensBreak of Peripheral Tolerance/Expression of Minor AntigensImmune CellImmune Cell

Stem CellStem Cell

Clinical translation can only be successful if good knowledge of biological processes linked to the therapeutic effect exists

Cancer Stem Cells Stem Cells

Factors promoting their Immune Behavior

PluripotencySelf-renewal

PlasticityImmune-modulation

Common Distinct

How these cells are Tolerated to persist

EngraftmentRepair

EliminateCombat

BiomarkersImmune protocols

Targets

(PD-L1?, MHC II?)

hCPC Immunomodulatory property

hCPC are capable to modulate an ongoing Immune response hCPC are capable to modulate an ongoing Immune response

PHA polyclonal stimulation PHA polyclonal stimulation

the function of MHC II molecules is not limited to their role as antigen-presenting structures; they are receptors that by triggering a variety of signaling pathways can regulate cells activities from proliferation and maturation to apoptosis

Could a signal via MHC II or I contribute to the Regenerative PARACRINE effect of allogenic CSCs???

SUMMARYSUMMARYSUMMARYSUMMARY

CSCs express MHC I and MHC II under physiological & inflammatory situation

They display higher ALLOGENICITY than MSCs and are less powerful in down regulating an ongoing immune response

CSC capacity to induce or modulate an immune response is variable depending on both donor and recipient

CSCs are recognized by allo-anti-HLA sera, which can

lead to in their elimination but could also be part of their paracrine effect

Circulation Research 2012(in press)

Additional Challenges

• Injured myocardium is inflammatory:Immune reactivity increase

• Presence of APC within the differenciated cell population(Endothelial ,Dentritic cells …)

• MHC class I expression :NK cells susceptibility vs Cytotoxic T lymphocytes