Three major problems that limit the clinical applications of transplantation are: morbidity/mortality associated with longterm immunosuppression “chronic.

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/f

req

ue

nc

y

Donor

3rd Party

9/9 13/13Donor specific hyporesponsivness

high

low

102

103

104

105

106

107

Chronic Rejectors

1/f

req

ue

ncy

104

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+

CD

25

CD4

CD4+CD25+ line cells retain their phenotype while expanding in ex-vivo

cultures

CD25 CD69 CD44

i.c. CTLA-4GITR

CD62L CCR7

25+ line

25- 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

10000

20000

30000

40000

50000

60000

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)

Sur

viva

l

0

…but cannot prevent 3rd party skin graft rejection by

CD4+CD45RBhi cells

3rd party B10.A

H2H2kk + D + Ddd

Sur

viva

l

Sur

viva

l

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: