Fernandez 111309
31
n Shortman and Yong-Jun Liu, Nature Reviews Immunology 2, 151-161 (2002). Luis P. Fernández November 13, 2009 "Controlled dendritic cell development for modulation of T cell reactivity"
Transcript of Fernandez 111309
Image from Ken Shortman and Yong-Jun Liu, Nature Reviews Immunology 2, 151-161 (2002).
Luis P. FernándezNovember 13, 2009
"Controlled dendritic cell development for modulation
of T cell reactivity"
Dendritic cell therapy for transplantation tolerance
• Promising but not yet realized
• Phenotype of tolerogenic DCs in context of transplantation is not clearly defined
• Prolongation of graft not remarkable
Graft survival of untreated (no DST/anti-CD40L) mice after adoptive transfer of 2x106 naive pDCs or YAe+ PDCA-1+ cells from LN of “10-week tolerized” mice.
Nature Immunology (2006) 7:652
Balb/c
B6
d0 wk10d-7
DSTanti-CD40L Yae+ pDC (LN)
Balb/c
B6
BMC57/Bl6 + GM-CSF FACS analysis 7 days
Phenotype of iDCs and mDCs derived from Bone Marrow in GM-CSF
C57BL/6 Bone Marrow
GM-CSFiDCs
mDCs7 days
GM-CSF + IL-4
SORTING Mixed Lymphocyte Reactions
8383
31701
34054
304736
0 50000 100000 150000 200000 250000 300000 350000
A-CD4+
A-CD4+ B6-CD90- splenocytes
A-CD4+ B6-iDC
A-CD4+ B6-mDC
avg cpm
iDCs
mDCs
Isotype control
CD2510 0 10 1 10 2 10 3 10 4
0
20
40
60
80
100
CD4010 0 10 1 10 2 10 3 10 4
CD8010 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4
0
20
40
60
80
100
DEC20510 0 10 1 10 2 10 3 10 4
Mac110 0 10 1 10 2 10 3 10 4
CD86
A-L. Blanc
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.
Directed DC differentiation from ES cells
DCs for immunotherapy : primary, untransformed
Overexpression or functional ablation of candidate genes
Resistance to genetic modificationProblems in efficiency, stability of gene expression, immunogenicity, effect on DC maturation
“Differentiation on demand”: ES cells differentiate to stable lines of DC with defined phenotype.
Graphic version
Exploiting the unique properties of ES cells:pluripotency, self-renewal, tractability for genetic
modification
Paucity of stable DC lines
ES-DCs remain immature for prolonged time periods.
Transfect ES cells Directed differentiation
ES cells (ESC)
ES-DC precursors (ES-DCp)
Immature ES-DCs
C57BL/6
Balb/c
Immunomodulation
Experimental Design
When do they commit to the DC lineage? Is the commitment
sustainable in vivo?
In vitro In vivo
Mild conditioning
Hypotheses
• 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.
Bruce4 ES cell line(from C57BL/6)
Day 13C57BL/6embryo
Mouse embryonic fibroblasts (MEFs)
Irradiate MEFs
ES cell culture
Deriving DC precursors from ES cells
Embryoid body (EB)culture
Harvest EBsat different timepoints
GMCSF+IL3
Non-adherent plateLong-term observationChange media weeklyAdd fresh cytokines
Adherent plate
+LIF2 passageswithout MEFs
-LIF
Gelatinized flask
Low density plating
Fairchild/Waldmann protocol for ES-DC derivation
Week 1 Week 2 Week 3 Week 4
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
cytokines
cytokines (d0)
day 1 2 3 4 5 6 7
RT-PCR
week2week1
LIF withdrawal
ESC
Oct3/4
PU.1
-Actin
CX3CR1
CD11c
CD11b
d1
- +d2
- +d3
- + - +d4
- - -d5 d6 d7
L ce
lls
ME
Fs
BM
-DC
s
wat
er
RT-PCR
Oct3/4
-Actin
PU.1
CX3CR1
ESC
RT-PCR
0.5d
- +1d
- +2d
- +3d
- +4d
- +
cytokines (d0)
day1 2 3 4
RT-PCR
week2week1
LIF withdrawal
ESC
Week 1 cytokine addition
Weeks 1 and 2 cytokine addition
Week 1 Week 2 Week 3
Immunocompetent mice reject ES-DCp
Conditioning regimen is necessary to realize the differentiation potential of ES-DCp
P15
ES
C
Impl
ante
d E
S-D
Cp
BM
-DC
ME
F
sple
en
PU.1
CX3CR1
CD11b
CD11c
Oct3/4
-Actin
wat
er
H&E
CD11c, I-Ab
Ig isotype
400x
100x
kd cap
100x
kd cap
100x
kd cap
capkd
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
CD11c (red)I-Ab (green)
ES-DCp Control Balb/c
Teratomas take over kidney by week 6
Week 3 Week 6
H&E
CD11c (red)I-Ab (green)
100x 100x
200x 200x
Setting conditions for in vivo differentiation of ES-DCp
• Use lower dose of mAbs to:
– allow engraftment, differentiation, and migration of ES-DCp
– allow quick recovery of T cell response
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
Detection of donor-derived cells by rt-PCR
Confirmation of donor-derived cells in thymus by immunohistochemistry
Lesser alloresponse to donor antigens suggests tolerogenic potential of ES-DCp.
I-Ab
actin
I-Ab
-actin
Kb
ES
C
ES
-DC
p
BM
-DC
Wat
er
ES-DCp ESC
Thymus, week 3
CD11c (red)I-Ab (green)
Detection of donor-derived cells in thymus
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• Further characterization and enrichment of ES-DCp
Further characterization and enrichment of ES-DCp
• OP9 coculture. • Minimize adherence of weeks 1 and 2 EBs
using PHM.• Week 3: time of implantation. Switch culture
to adherent plates.• Pulse EB cultures with cytokines on week 3.
Actin
Oct3/4
Nanog
Eto2
Lmo2
M-CSFR
PU.1
CX3CR1
CD11b
CD11c
Flt3
F4/80
I-Ab
Kb
1 2 3 4 5
1 2 3 4 5
1 = ESC2 = d7 Step1 OP9 coculture3 = d5 Step 2 OP9 coculture4 = OP95 = H2O
1 = d7 Step 3 adherent2 = d7 Step 3 floating3 = B6 BM unsorted4 = B6 BM-DC5 = H2O
Adherent plate
-LIF OP9 coculture
Non-adherent plate
Step 1OP9+GMCSF-
Step 2OP9+GMCSF+
Step 3OP9-GMCSF+
GMCSF1000 U/ml
6 days 7 days 5-6 days 7-14 days
GMCSF500 U/ml
Gelatinized flask
+LIF
M-CSFR
Nanog
Oct3/4
Actin
Eto2
PU.1
Kb
Lmo2
Flt3
F4/80
CD11b
CD11c
Harvest EBs
GMCSF+IL3
Non-adherent plateLong-term observationAdherent plate
-LIF
Gelatinized flask
Revisiting the Fairchild/Waldmann protocol for ES-DC derivation
Week 1 Week 2 Week 3 Week 4
Harvest EBs
GMCSF+IL3
Non-adherent platecontaining PHM
Long-term observationAdherent plate
Week 5
200x
200x100x
+LIF
Harvest EBs
GMCSF+IL3
Non-adherent plate +/- PHMAdherent plate
-LIF
Gelatinized flask
Week 1 Week 2 Week 3 Week 4 Week 5
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
ESC
ES-DCp Week 3
ES-DCp Week 5
BM-DC (GM-CSF)
MHCI
100
101
102
103
104
0
20
40
60
80
100
100
101
102
103
104
0
20
40
60
80
100
100
101
102
103
104
0
20
40
60
80
100
MHCII CD45 CD11b CD11c CD80 CD86 c-kit Flt3
100
101
102
103
104
0
20
40
60
80
100
100
101
102
103
104
0
20
40
60
80
100
100
101
102
103
104
0
20
40
60
80
100
100
101
102
103
104
0
20
40
60
80
100
100
101
102
103
104
0
20
40
60
80
100
100
101
102
103
104
0
20
40
60
80
100
100
101
102
103
104
0
20
40
60
80
100
100
101
102
103
104
0
20
40
60
80
100
100
101
102
103
104
0
20
40
60
80
100
100
101
102
103
104
0
20
40
60
80
100
100
101
102
103
104
0
20
40
60
80
100
100
101
102
103
104
0
20
40
60
80
100
100 101 102 103 1040
20
40
60
80
100
100
101
102
103
104
0
20
40
60
80
100
100
101
102
103
104
0
20
40
60
80
100
100
101
102
103
104
0
20
40
60
80
100
100
101
102
103
10
0
20
40
60
80
100
100
101
102
103
104
0
20
40
60
80
100
100
101
102
103
104
0
20
40
60
80
100
100
101
102
103
104
0
100
101
102
103
104
0
20
40
60
80
100
100
101
102
103
104
0
20
40
60
80
100
100
101
102
103
104
0
20
40
60
80
100
100
101
102
103
104
0
20
40
60
80
100
100
101
102
103
104
0
20
40
60
80
100
100
101
102
103
104
0
20
40
60
80
100
100
101
102
103
104
0
20
40
60
80
100
100
101
102
103
104
0
20
40
60
80
100
100
101
102
103
104
0
20
40
60
80
100
100
101
102
103
104
0
20
40
60
80
100
100
101
102
103
104
0
20
40
60
80
100
100
101
102
103
104
0
20
40
60
80
100
100
101
102
103
104
0
20
40
60
80
100
100
101
102
103
104
0
20
40
60
80
100
100
101
102
103
104
0
20
40
60
80
100
100
101
102
103
104
0
20
40
60
80
100
100
101
102
103
104
0
20
40
60
80
100
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
0 200 400 600 800 1000
SSC
0
200
400
600
800
1000
FS
C
0 200 400 600 800 1000
0
200
400
600
800
1000
0 200 400 600 800 1000
0
200
400
600
800
1000
0 200 400 600 800 1000
0
200
400
600
800
1000
ES-DCp are amenable to maturation
LPS LPS + IL-4 IL-4 No cytokine
100 101 102 103 104100
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 104100
101
102
103
104
4.46 5.3
1476.2
4 100 101 102 103 104100
101
102
103
104
4.88 1.94
2.2890.9
CD
80-P
E
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-P
E
CD11c-APC
ES-DCp gated on PIlow FSChigh
Conclusion
• OP9 coculture did not generate ES-DCp in my experience.
• Preventing adherence of EBs increased frequency of EBs as source of ES-DCp
• Using more primers for PCR and surface markers for FACS validates timing of commitment and implantation.
• Pulsing EBs with cytokines on week 3 does not change kinetics of commitment and differentiation.
• ES-DCp accomplish in vitro differentiation but remain non-stimulatory.
ES-DCp
Next steps
• Eliminate teratogenic cells/purify ES-DCp before implantation.
• Detect donor-derived cells in lymphoid organs by immunohistochemistry.
• Assess quality of immune response:– TH1, TH2, TH17– Examine signs of regulation (T regs)
• Test tolerance by heart transplants.
• Determine phenotype of donor DCs in lymphoid organs and establish correlates between phenotype and tolerance induction.
PU.1 promoter NeoRRFP
C57BL/6 ES cells
ES-DCp Balb/c
Begin LIF withdrawal
Transfect
Expand G418-resistant colonies
SortRFP+ B220- SSEA1-
cells
Strategy to enrich ES-DCp for implantation
(pDsRed1-1™, Clontech)
Three-week ES-DCp culture
GMCSF+IL3
Non-adherent plate + PHM
Next steps
• 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.
National Orchid Garden of SingaporeApril 26, 2009
AcknowledgementsChristian Leguern
Paulo MartinsSharon GermanaKaela GoldsteinYuanto WangGuotong ManAkira Shimizu
George Daley labOlaia Naveiras
Nalu Navarro-Alvarez
