«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
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