Three major problems that limit the

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Three major problems that limit the clinical
applications of transplantation are
morbidity/mortality associated with
longterm immunosuppression chronic
rejection shortage of organs.
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Immunological tolerance would address all three
issues. drug-free transplant survival
prevention of CR extend longevity of
transplanted organs
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Pathways of MHC allorecognition
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Lessons from rodents
Tolerance more easily achieved when MHC
incompatibility absent or limited
Tolerance impeded if T cell death prevented -
(Bcl-xL-transgenics or IL-2 KO Turka 99)
Tolerance favoured by deliberate deletion -
(IL-2-Fc Rapamycin Strom 06)
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In context of MHC incompatibility deletion may be
required
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Peripheral Tx tolerance is transferable
A skin
adult B strain tolerant to A
Tolerance protocol
B
adoptive transfer of CD4 T cells
A skin
Graft acceptance
naive B
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The spectrum of regulatory T cells..
NKT
resp. T
?
IL-10 TGF-
?
b
Regulation mediated by cellcell contact,
involving unknown molecules
Regulation mediated by soluble factors, acting
on APC or neighbouring T cells
APC
?
CD8CD28-
CD4CD25
resp. T
CD4-8-
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Tolerance is maintained by regulatory T cells
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Lessons from patients
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HTLp IL-2
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A
N
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N

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Direct pathway
Indirect pathway
Lymph Node
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Peripheral Tx tolerance is transferable
A skin
adult B strain tolerant to A
Tolerance protocol
B
adoptive transfer CD4 T cells
A skin
Graft acceptance
naive B
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Preclinical testing of strategies to promote
transplantation tolerance
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Negative vaccination to induce donor-specific
(N.B. indirect pathway) regulatory cells in vivo
- pre-transplantation.
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Adoptive therapy with customised regulatory
cells, selected and expanded ex vivo
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Methods
Spleen and LN cells
CBA/Ca H2k
CD4CD25
immature DC Kb peptide
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CD4CD25 line cells retain their phenotype while
expanding in ex-vivo cultures
25- line
25 line
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CD4CD25 line cells express high levels of Foxp3
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In vitro suppressor function of the CD4CD25 T
cell-line


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cpm
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Stimulation with aCD3 and syngeneic APCs




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CD4CD25 line cells accumulate at the site of
antigenic challenge
draining LN
CD4CD25- and CD4CD25 line-GFP
CBK
mesenteric LN
d-1
GFP
d 0
Flow cytometry d40
grafted skin
CBA/Ca
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In T-depleted recipients, CD4CD25 line-cells
prevent CBK skin graft rejection by CD4CD45RBhi
cells
CBK donor H2k Kb
100
1
.
0
25 (n3)
80
.
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Survival
RBhi/25 (n7)
60
.
6
40
RBhi (n8)
.
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but cannot prevent 3rd party skin graft
rejection by CD4CD45RBhi cells
3rd party B10.A H2k Dd
3rd party BALB/c H2d
Class I mismatch
Class I and II mismatch
Survival
Survival
RBhi/25 (n5)
RBhi/25 (n5)
RBhi (n5)
RBhi (n5)
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TCR transduction as a tool to confer the desired
specificity to regulatory T cells methods and
efficiency of transduction
Lymph node and spleen from C57BL/6
Negative selection of CD4 T cells with antibody
cocktail and anti-rat dynal beads
Transfection of phoenix packaging cells with
indirect allospecific TCR (TCR34-Kd peptide with
Ab) constructs for retrovirus production
Positive selection of CD4CD25 T cells with
biotinylated anti-CD25 and streptavidin microbeads
Activated with CD3/CD28 beads or APCantiCD3 and
IL-2 (2 days)
Viral supernatant
T
T
T
T
T
3 day after transduction, Functional and Flow
cytometric analysis
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Conclusions
Tregs with indirect anti-donor allospecificity
can be generated ex vivo by repeated
stimulation with cognate peptide
Indirect allospecificity can be conferred on
Tregs by gene transfer
Allospecific Tregs traffic to the draining
lymph node and to the allograft following i.v.
injection
Adoptive therapy with Tregs with indirect
allospecificity prolongs allograft survival
Combining Tregs with indirect allospecificity
with short term immunosuppresssion induces
longterm graft survival.