Fernandez 120509
13
e from Ken Shortman and Yong-Jun Liu, Nature Reviews Immunology 2, 151-161 (2002). Luis P. Fernández December 5, 2009 “Embryonic stem cell-based therapy for transplantation tolerance"
description
I presented my ongoing research from Massachusetts General Hospital at the American Society of Transplantation Annual Scientific Exchange meeting in Orlando, FL.
Transcript of Fernandez 120509
Image from Ken Shortman and Yong-Jun Liu, Nature Reviews Immunology 2, 151-161 (2002).
Luis P. FernándezDecember 5, 2009
“Embryonic stem cell-based therapy for transplantation
tolerance"
Relevant Financial Relationship Disclosure Statement
Luis P. Fernández, PhDTransplantation Biology Research Center/
Massachusetts General Hospital
I have no financial relationships to disclose within the past 12 months relevant to my presentation
AND
My presentation does not include discussion of off-label or investigational use
Goals
• Circumvent DC immunogenicity by introducing allogeneic DC in the form of poorly immunogenic progenitors
• Characterize the differentiation potential of these progenitors in vitro and in vivo.
• Assess the spectrum of tolerogenicity induced by ES cell-derived DC.
Experimental Design
ES cells (ESC)
ES-DC precursors (ES-DCp)
Immature ES-DCs
C57BL/6
Balb/c
Immunomodulation
In vitro In vivo
Hypothesis
• Similar to ESC, ES-DCp are weakly immunogenic and will be accepted by mildly conditioned allogeneic hosts.
• ES-DCp will continue to differentiate in vivo and colonize lymphoid organs.
• It is predicted that allogeneic DCs will "redefine self" and downmodulate T cell responses to allogeneic antigens.
Mild conditioning
Harvest EBs
GMCSF+IL3
Non-adherent plate + PHM Adherent plate
-LIF
Gelatinized flask
M-CSFR
Actin
Oct3/4
Nanog
Eto2
Lmo2
PU.1
CX3CR1
CD11b
CD11c
Flt3
F4/80
I-Ab
Kb
M-CSFR
(-)EB EBB6BM
B6BMDC (-)
B6BM
B6BMDC (-)
+LIF
Nanog
Oct3/4
Actin
Eto2
PU.1
Kb
Lmo2
Flt3
F4/80
CD11b
CD11c
EB EB
In vitro ES-DCp differentiation by PCR
Week 1 Week 2 Week 3 Week 4 Week 5
40x 400x800x
I-Ab-FITC
Dd-FITC Dd-PE
CD11c-PE I-Ab and CD11c
CD11c-PE
I-A
b-F
ITC
100
101
102
103
104
100
101
102
103
104
5.0 40.0
100
101
102
103
104
100
101
102
103
104
ESC
ES-DCp Week 3
ES-DCp Week 5
BM-DC (GM-CSF)
MHCI
100
101
102
103
104
0
20
40
60
80
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101
102
103
104
0
20
40
60
80
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104
0
20
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100
MHCII CD45 CD11b CD11c CD80 CD86 c-kit Flt3
100
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104
0
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M-CSFR
In vitro ES-DCp differentiation by FACS
Lack of allostimulatory capacity of ES-DCp
-10 0 10 20 30 40 50 60 70 80
Balbc
B6
CBA
Week 3 ES-DCp
Week 5 ES-DCp
B6 BM-DC
Stimulation index
StimulatorsResponders
Balbc
100 101 102 103 104
100
101
102
103
104
16.1 50.5
14.918.5
100 101 102 103 104
100
101
102
103
104
3.98 56.1
20.519.4
100 101 102 103 104
100
101
102
103
104
4.46 5.3
1476.2
4 100 101 102 103 104
100
101
102
103
104
4.88 1.94
2.2890.9
CD
80-
PE
CD11c-APC
100 101 102 103 104100
101
102
103
104
10.8 47.5
20.521.1
100 101 102 103 104100
101
102
103
104
3.58 48.3
27.720.4
100 101 102 103 104100
101
102
103
104
1.46 4.76
17.476.4
100 101 102 103 104100
101
102
103
104
2.54 1.4
2.593.6
CD
86-
PE
CD11c-APC
PIlow FSChigh gate
ES-DCp are amenable to maturation
LPS LPS + IL-4 IL-4 No cytokine
H&E
CD11c, I-Ab
Ig isotype
400x
100x
kd cap
100x
kd cap
100x
kd cap
capkd
P15
ES
C
Imp
lant
ed
ES
-DC
p
BM
-DC
ME
F
sple
en
PU.1
CX3CR1
CD11b
CD11c
Oct3/4
β-Actin
wat
er
GM-CSFIL-3
ES-DCp
Follow up on weeks 1, 2, 3, 6
ES-DCp in immunosuppressed mice: test of sustained commitment
1 mg anti-CD4 & -CD8
ESC
Kb
I-Ab
actin
LN Thy Spn
(anti-CD4)
LN Thy Spn
(anti-CD4 &-CD8)
LN Thy Spn
(anti-CD4)
LN Thy
(anti-CD8)
0.5 mg 0.25 mg
Spn
ES-DCp ESC
Thymus, week 3CD11c (red)I-Ab (green)
I-Ab α
β−actin
I-Ab β
β-actin
Kb
ES
C
ES
-DC
p
BM
-DC
Wat
er
Responder mice
injected with:ES-DCp ESC
% o
f E
SC
-tre
ate
d B
AL
B/c
resp
on
se t
o B
6
p= 0.001
25
50
75
100
Summary
• Immunocompetent hosts: rejection of derivatives of ES-DCp and other cells does not permit further study
• Immunocompromised host: ES-DCp differentiate in situ in a narrow window before teratomas take over
• Modulation of immunosuppression to allow ES-DCp differentiation and T cell recovery
• Thymic migration of ES-DCp derivatives and potential to downmodulate alloimmune response.
• The need to purify ES-DCp
PU.1 promoter
Transfect C57BL/6 ES cells
ES-DCp
Balb/c
LIF withdrawal
Expand G418-resistant colonies
RFP+ B220- SSEA1- cells
Strategy to enrich ES-DCp for implantation
(pDsRed1-1™, Clontech)
Three-week culture
GMCSF+IL3Non-adherent plate
RFP NeoR
• Eliminate teratogenic cells/purify ES-DCp.
• In the ES-DCp-treated animal:– Detect donor-derived cells in lymphoid organs by IHC.
• Determine phenotype of donor DCs in lymphoid organs and establish correlates between phenotype and tolerance induction.
• Assess host immunity to donor antigens over a longer time period.– Examine signs of regulation (T regs)
• Test the impact of ES-DCp therapy on transplantation tolerance.
ACKNOWLEDGEMENTS
TBRC/Massachusetts General HospitalChristian LeguernPaulo MartinsSharon GermanaKaela GoldsteinYuanto WangGuotong ManAkira Shimizu
Childrens Hospital BostonGeorge Q. Daley
