three major problems that limit the clinical applications of transplantation are:
DESCRIPTION
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…. - PowerPoint PPT PresentationTRANSCRIPT
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
The direct pathway of allorecognition
IL-2
MHC class I MHC class II
TCR
Allogeneic (stimulator) antigen presenting cell
CD8+
cytotoxic T cell CD4+
cytotoxic T cell
The indirect pathway of allorecognition
IL-2
Allo
gene
ic c
ell
Responder antigen presenting cell
CD8+ cytotoxic T cell
CD4+
helper T cell
Donor MHC- derived peptide
CLIP
Pathways of MHC allorecognition
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
adoptive transfer of CD4+
T cells
Graft acceptance
A skin
“naive” B
adult B strain tolerant to A
A skin
B
Tolerance protocol
Peripheral Tx tolerance is transferable…
CD4CD4++CD25CD25++
Tr1Tr1
APCAPC
The spectrum of regulatory T cells…..
IL-10TGF-
resp. T
?resp. T
Regulation mediated by soluble factors, acting on APC orneighbouring T cells
Regulation mediated by cell:cell contact, involving unknown molecules
NKTNKT
CD8CD8++CD28CD28--
?
CD4CD4--88--
?
Tolerance is maintained by regulatory T cells
Lessons from patients
HTLp IL-2HTLp IL-2
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
1/fr
eque
ncy
Donor
3rd Party
9/9 13/13Donor specific hyporesponsivness
high
low
102
103
104
105
106
107
Chronic Rejectors
1/fre
quen
cy104
105
106
p<0.05 *
CAN CAN Free
Lymph Node
Direct pathway
Indirect pathway
adoptive transfer CD4+ T cells
Graft acceptance
A skin
“naive” B
adult B strain tolerant to A
A skin
B
Tolerance protocol
Peripheral Tx tolerance is transferable…
Apparentindirect anti-donorallospecificity
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
CBA/Ca H2k
Spleen and LN cells
immature DC + Kb peptide
CD4+CD25+
CD25
CD4
CD4+CD25+ line cells retain their phenotype while expanding in ex-vivo
cultures
CD25 CD69 CD44
i.c. CTLA-4GITR
CD62L CCR7
25+ line25- line
CD4+CD25+ line cells express high levels of Foxp3
mFoxP3
ßActin
fresh lines
25- 25+ 25- 25+
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
0
1000020000
3000040000
5000060000
70000
25-/25+
(1:1)(1:0.5) (1:0.25) (1:0.125) 25- 25-/25- 25+
cpm
CD4+25+ fresh CD4+25+ line Stimulation with CD3 and syngeneic APCs
CD4+CD25+ line cells accumulate at the site of antigenic challenge
d 0
CBA/Ca CBA/Ca
d-1
CD4+CD25- and CD4+CD25+ line-GFP
CBK
Flow cytometry d+40
draining LN
mesenteric LN
grafted skin
GF
P
In T-depleted recipients, CD4+CD25+ line-cells prevent CBK skin graft
rejection by CD4+CD45RBhi cells
CBK donor H2H2kk + K + Kbb
days after transplantation
12010 0806040200
1.0
.8
.6
.4
.2
.
100
80
60
40
20
RBhi (n=8)
RBhi/25+
(n=7)
25+ (n=3)
Surv
ival
0
…but cannot prevent 3rd party skin graft rejection by
CD4+CD45RBhi cells
3rd party B10.A
H2H2kk + D + Ddd
Surv
ival
Surv
ival
days after Tx
25201510
1.0
.8
.6
.4
.2
0.0
100
80
60
40
20
Class I mismatch
RBhi
(n=5)
RBhi/25+ (n=5)
days after Tx
25201510
1.0
.8
.6
.4
.2
0.0
RBhi/25+ (n=5)
RBhi
(n=5)
3rd party BALB/c
H2H2dd Class I and II mismatch
100
80
60
40
20
TCR transduction as a tool to confer the desired specificity to regulatory T cells: methods and efficiency of transduction
Negative selection of CD4+ T cells with antibody cocktail and anti-rat dynal beads
CD4+CD25- CD4+CD25+
CD25
CD
4
Lymph node and spleen from C57BL/6
Positive selection of CD4+CD25+ T cells with biotinylated anti-CD25 and streptavidin microbeads
Transfection of phoenix packaging cells with indirect allospecific TCR (TCR34-Kd peptide with Ab) constructs for retrovirus production
Activated with CD3/CD28 beads or APC+antiCD3 and IL-2 (2 days)
Viral supernatant
T TT
TT
3 day after transduction,Functional and Flow cytometric analysis
• 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.
Conclusions: