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Evolution of anti-donor alloimmunity following transplantation - implications for CAN
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Evolution of anti-donor alloimmunity following transplantation -
implications for CAN
Maria Hernandez-FuentesRichard BakerWan Fai Ng
Osquel Barroso-Herrera
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Indirect allorecognitionIndirect allorecognition
IL-2IL-2
II
IIII
Allorecognition: the two pathways
DirectDirect allorecognition allorecognition
allo APC
Shed allogeneic MHC
CD8 CD4
Auto APC
Taken up and processed by auto APC IIII
IL-2IL-2
II
CD8 CD4
allo APC
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HTLp IL-2HTLp IL-2
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CD45RACD45RA++ (Naïve) and CD45RO (Naïve) and CD45RO++ (Memory) T cells (Memory) T cellshave different patterns of recirculation. have different patterns of recirculation.
THYMUSTHYMUS
BLOODSTREAMBLOODSTREAM
HEVHEVLYMPHNODE
THORACIC DUCT
tissue tissue parenchymparenchym
al cell al cell
““RA circuit”RA circuit”
““RO circuit”RO circuit”
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A significant drop in the anti-donor A significant drop in the anti-donor response is only observed in the response is only observed in the
CD4CD4++CD45ROCD45RO++ fraction. fraction.
p>0.05(Wilcoxon)
Ratio of frequency post/pre for CD4+CD45RA+ cells
TH LM AT AL DM JW SA CS SAl
Ratio
10-3
10-2
10-1
100
101
102
103
RA donor RA 3rd party
Ratio of frequency post/pre for CD4+CD45RO+ cells
TH LM AT AL DM JW SA CS SAlR
atio10-3
10-2
10-1
100
101
102
103
p=0.008(Wilcoxon)
RO donor RO 3rd party
33rdrd party ratios close to unity mean that immunosuppression has party ratios close to unity mean that immunosuppression has not significantly affected the lymphocyte response; as can be not significantly affected the lymphocyte response; as can be observed for both fractions.observed for both fractions.
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deletion anergy
Mechanisms of peripheral toleranceMechanisms of peripheral tolerance
deletion anergy
regulation
?
ignorance
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Experimental Design
+ IL2 (30 U/ml) for 3 days
Wash and rest for 1 day
CD4 cells
LDA
LDA
PBMCadherenceon plastic
1 hrCocktails of mAbFollowed by magnetic beads to remove non-CD4 cells
Readout:
IL2 bioassay with CTLL
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Anti-donor frequencies before and after IL-2T.H
.
J.W
.
J.B
.
A.W
.
M.M
.
R.D
.
S.E.
C.S.
K.M
.
P.K
.
G.K
.
D.T
.
100
1000
10000
100000
1000000
1/Freq
Non Hypo-responsive PatientsDonor Specific Hypo-responsive Patients
Patients with Reveral of Hyporesponsiveness in bold
Post IL-2
Pre IL-2
Donor-specific hyporesponsiveness was reversed in Donor-specific hyporesponsiveness was reversed in 5 out of 5 patients with IL-2 5 out of 5 patients with IL-2
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Elevated frequencies of T cells with indirect anti-donor
allospecificity in patients with chronic transplant rejection
Vella et al, ‘97 Transplantation renal transplant recipients
Ciubotariu et al, ‘98 JCI heart transplant recipients
Hornick et al, ‘00 Circulation .. .. ..
SivaSai et al, ‘99 Transplantation lung transplant recipients
Baker et al, 2001 JI renal transplant recipients
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1/f
req
ue
ncy
104
105
106
p<0.05 *
CAN CAN Free
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Lymph Node
Direct pathway
Indirect pathway
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Mechanisms of peripheral toleranceMechanisms of peripheral tolerance
deletion anergy
regulation
?
ignorance
Direct pathway naïve T cells?
Direct pathway - memory T cells
Evolution of anti-donor direct and indirect alloresponses following transplantation using conventional immunosuppression
Indirect pathway T cells - in some patients
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B
AB
Graft acceptance
Tolerant to AA
The T cells that transfer tolerance appear to have indirect indirect allospecificity
CD4+
Tolerance to MHC class I-only mismatched graft transferred by CD4 T cells (Waldmann)
Pre-treatment with donor MHC class I induces tolerance when combined with anti-CD4 mAb(Wood)
Tolerance could not be induced unless the indirect pathway was intact (Auchincloss)
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“Indirect recognition by helper T cells can induce donor-specific cytotoxic T lymphocytes in vivo”
RS Lee, ……. H Auchincloss Jr.
J. Exp. Med. 1994 179:865
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CD4
Immature recipient DC
Mature recipient DC presenting donor MHC indirectly
Donor parenchymal cells
Unlinked help and suppression - the “4 cell problem”
Unlinked help or suppression…?
Lymph Node
CD8
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CFSE-R1
R1 x R4Acceptor DC only
R1
R4
0h
20h
0h
20h3.5%13.8%48.5%
2.9%2.8%3.0%
0.1%1.1%0.5%
M1
2.5%3.7%2.3%
M1
0.4%1.5%0.8%
3.7%10.1%41.2%
4.5%13.7%19.4%
1.8%15.4%23.1%
H-2Ab
immatureBALB/c DCs
matureBALB/c DCs
H-2Kb
C57BL/6DCs
Co-culture of MHC-mismatched DC leads to class I and II exchange
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1.3%7.6%7.5%
1.6%23.9%17.4%
1.2%19.0%18.3%
1.7%5.0%7.2%
H-2AbH-2Kb
MHC transfer between DCs can occur in the absence of cell contact
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0
5 0 0 0 0
1 0 0 0 0 0
1 5 0 0 0 0
2 0 0 0 0 0
2 5 0 0 0 0
3 0 0 0 0 0
3 5 0 0 0 0
S t i m u l a t o r c e l l s
T : D C = 1 0 : 1
CPM
0
5 0 0 0 0
1 0 0 0 0 0
1 5 0 0 0 0
2 0 0 0 0 0
2 5 0 0 0 0
3 0 0 0 0 0
3 5 0 0 0 0
S t i m u l a t o r c e l l s
T : D C = 1 0 : 1
Responders:BALB/c CD4+ T cells
Responders:BALB/c CD8+ T cells
Stimulators:
BALB/c DCs alone.
BALB/c DCs from mixe d cultures .
C57BL/6 DC s-CFS E from mixed culture s .
BALB/c DCs from trans -well cultures .
0
2 0 0 0 0
4 0 0 0 0
6 0 0 0 0
8 0 0 0 0
1 0 0 0 0 0
1 : 1 5 : 1 1 0 : 1
T : D C r a t i o
CPM
0
2 0 0 0 0
4 0 0 0 0
6 0 0 0 0
8 0 0 0 0
1 0 0 0 0 0
1 : 1 5 : 1 1 0 : 1
T : D C r a t i o
Respo nder s:F5 CD8+ T ce lls
Respo nder s:OT-II CD4+ T ce lls
Stimula tors:
o BALB/c DCs alone.
æBALB/c DCs from mixed cultures.
= C57BL/6 DCs-CFSE from mixed cultures.
◊ BALB/c DCs from trans-well cultures.
99.9%
99.5%
Mixed cultureCFSE+veC57BL/6 DCs
CFSE-veBALB/c DCs
FSC
FSC
FSC
Transferred MHC:peptide complexes are recognised by T cells
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0.260.5 37.5
1.8
4.360.3 4.4
34.9
0.157.5 29.8
2.6
4.856.8 3.2
35.2
0.242.1 57.2
0.5 0.142.2 57.4
0.3
CFSE
ImmatureBALB/c DCs
MatureBALB/c DCs
(CBA ECs+ IFNγ) -CFSE
C57BL/6 ECs -CFSE
(C57BL/6 ECs+ IFNγ) -CFSE
DCs acquire MHC class I from γ-IFN-treated ECs
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1.020.4 25.1
53.5 0.620.7 22.7
56.0
0.120.9 34.7
44.3 0.122.1 31.7
46.1
1.820.8 27.8
49.5 1.421.3 29.1
48.2
CBA ECs + IFNγCBA ECs
CFSE
CFSE-labelled C57BL/6 DCs
MHC transfer between ECs and DCs is unidirectional
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DCs acquire MHC class I and II molecules in vivo
(+PBS) (+IFNγ) (+IFNγ)
(+ PBS) (+IFNγ)immatur e DCs matur e DCs
(+IFNγ) (+PBS)
(+PBS)
78 .6
0.6 7.5
13 .3 92 .2
3.1 1.2
3.4 96 .7
2.8 0
0.5 99 .0
0.6 0
0.4
97 .9
2.0 0
0.196 .2
3.8 0
0.191 .5
3.5 2.0
3.076 .0
2.1 12.0
9.9
CFSE
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B H-2Ab CFSE + H-2AbCFSE
a
PBS + immatureDCs-CFSE
b c
d
IFNγ + immatureDCs-CFSE
fe
h ig
+ PBS matureDCs-CFSE
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d
g
IFNγ + immatureDCs-CFSE
+ PBS matureDCs-CFSE
ih
fe
a
+ PBS immatureDCs-CFSE
C
cb
H-2Kb CFSE + H-2K bCFSE
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CD4CD8
Immature recipient DC
Mature recipient DC presenting donor MHC directly and indirectly
Donor parenchymal cells
Linking direct and indirect pathway T cells
Help or suppression
Lymph Node
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Conclusions
• Dendritic cells acquire MHC class I and class II molecules from other DCs and from ECs
• The acquired MHC molecules are recognised by T cells
• MHC acquisition by DCs occurs in vivo, as well as in vitro
• This raises the possibility that direct alloresponses are maintained after donor DCs
are deleted
• This phenomenon may provide a mechanism whereby T cells with indirect allospecificity
can help or suppress T cells with direct anti-donor allospecificity.
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Speculation….. the acquisition of MHC:peptide complexes by DCs traffickingthrough tissues provides a failsafe mechanism to ensure the presentation in lymphoid tissue of the viral peptide:MHC complexes that are most highly represented in the infected tissue…
an alternative to cross-priming….?
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CD4CD8
Immature recipient DC
Mature recipient DC presenting viral peptide:class I complexes to CD8+ T cells
infected parenchymal cells
DC MHC acquisition as an alternative to cross-priming
Help
Lymph Node