Transcript Document

Three major problems that limit the
clinical applications of transplantation are:
• morbidity/mortality associated with
longterm immunosuppression
• “chronic rejection”
• shortage of organs…….
Immunological tolerance would address all
three issues….
• drug-free transplant survival
• prevention of CR
• extend longevity of transplanted organs
Pathways of MHC allorecognition
CD4+
CD8+
cytotoxic T cell cytotoxic T cell
The indirect pathway of
allorecognition
Allogeneic cell
The direct pathway of
allorecognition
CD8+
cytotoxic T cell
CD4+
helper T cell
IL-2
IL-2
TCR
MHC class I
MHC class II
Donor MHCderived peptide
Allogeneic (stimulator)
antigen presenting cell
CLIP
Responder antigen presenting cell
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)
In context of MHC incompatibility deletion may be required
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
The spectrum of regulatory T cells…..
Tr1
resp. T
IL-10
TGF- b
Regulation mediated
by soluble factors,
acting on APC or
neighbouring T cells
NKT
?
?
Regulation mediated by
cell:cell contact,
involving unknown
molecules
APC
?
CD8+CD28-
CD4-8-
resp. T
CD4+CD25+
Tolerance is maintained by regulatory T cells
Lessons from patients
HTLp IL-2
Donor
3rd Party
Chronic Rejectors
2
10
high
3
1/frequency
10
4
10
5
10
6
10
low
7
10
1
2
3
9/9
4
5
6
7
8
9
10
11 12
13 14
15
16 17
Donor specific hyporesponsivness
18 19
20
13/13
21 22
104
1/frequency
p<0.05 *
105
106
CAN
CAN Free
Direct pathway
Indirect
pathway
Lymph Node
Peripheral Tx tolerance is transferable…
A skin
adult B strain
tolerant to A
Tolerance
protocol
B
adoptive transfer
CD4+ T cells
A skin
Apparent
indirect anti-donor
allospecificity
Graft
acceptance
“naive” B
Preclinical testing of strategies to promote
transplantation tolerance
“Negative vaccination” to induce donor-specific
(N.B. indirect pathway) regulatory cells in vivo pre-transplantation….
Adoptive therapy with “customised” regulatory cells,
selected and expanded ex vivo
Methods
Spleen and
LN cells
CD25
CBA/Ca
H2k
CD4+CD25+
CD4
immature DC + Kb peptide
CD4+CD25+ line cells retain their phenotype while
expanding in ex-vivo cultures
25- line
25+ line
CD25
GITR
CD62L
CD69
i.c. CTLA-4
CCR7
CD44
CD4+CD25+ line cells express high levels of Foxp3
mFoxP3
ßActin
25- 25+
25- 25+
fresh
lines
In vitro suppressor function of the CD4+CD25+ T
cell-line
CD4+ CD25+ line cells are more potent suppressors than freshly
isolated CD4+CD25+ T cells
70000
60000
cpm
50000
40000
30000
20000
10000
0
25-/25 +
(1:0.5)
(1:0.25)
(1:0.125)
25-
25-/25-
25+
(1:1)
CD4+25+ fresh
CD4+25+ line
Stimulation with aCD3
and syngeneic APCs
CD4+CD25+ line cells accumulate at the site of
antigenic challenge
draining LN
CD4+CD25- and
CD4+CD25+ line-GFP
d-1
mesenteric
LN
d0
Flow cytometry
d+40
CBA/Ca
grafted
skin
GFP
CBK
In T-depleted recipients,
CD4+CD25+ line-cells prevent CBK skin graft
rejection by CD4+CD45RBhi cells
CBK donor
H2k + Kb
Survival
1001.0
25+ (n=3)
80.8
RBhi/25+
(n=7)
60.6
40.4
RBhi
(n=8)
20.2
0.
0
20
40
60
80
days after transplantation
10 0
120
…
but cannot prevent 3rd party skin graft rejection by
CD4+CD45RBhi cells
3rd party B10.A
3rd party BALB/c
H2k + Dd
H2d
60.6
.2
20
RBhi/25+
(n=5)
Survival
Survival
80.8
40.4
100
1.0
Class I
mismatch
1.0
100
RBhi
(n=5)
80.8
60.6
RBhi/25+
(n=5)
40.4
20.2
RBhi
(n=5)
0.0
0.0
10
Class I and II
mismatch
15
20
days after Tx
25
10
20
15
days after Tx
25
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 CD4+CD25+ T
cells with biotinylated anti-CD25
and streptavidin microbeads Activated with
CD4+CD25-
CD4+CD25+
Viral supernatant
CD3/CD28 beads
or APC+antiCD3
and IL-2 (2 days)
T
CD25
T
T
T
T
3 day after transduction,
Functional and Flow cytometric analysis
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.