2. Three major problems that limit the clinical applications of transplantation are: morbidity/mortality associated with longterm immunosuppression “chronic rejection” shortage of organs…….

3. Immunological tolerance would address all three issues…. drug-free transplant survival prevention of CR extend longevity of transplanted organs

4. 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 Allogeneic cell Responder antigen presenting cell CD8 + cytotoxic T cell CD4 + helper T cell Donor MHC- derived peptide CLIP Pathways of MHC allorecognition

5. 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)

6. In context of MHC incompatibility deletion may be required

7. 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…

8. CD4 + CD25 + Tr1 APC The spectrum of regulatory T cells….. IL-10 TGF-  resp. T ? Regulation mediated by soluble factors, acting on APC or neighbouring T cells Regulation mediated by cell:cell contact, involving unknown molecules NKT CD8 + CD28 - ? CD4 - 8 - ?

9. Tolerance is maintained by regulatory T cells

10. Lessons from patients

11. HTLp IL-2

12. 1/frequency 10 4 10 5 10 6 p<0.05 * CANCAN Free

13. Lymph Node Direct pathway Indirect pathway

14. 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… Apparent indirect anti-donor allospecificity

15. Preclinical testing of strategies to promote transplantation tolerance

16. “Negative vaccination” to induce donor-specific (N.B. indirect pathway) regulatory cells in vivo - pre-transplantation….

18. Adoptive therapy with “customised” regulatory cells, selected and expanded ex vivo

19. Methods CBA/Ca H2 k Spleen and LN cells immature DC + K b peptide CD4 + CD25 + CD25 CD4

20. CD4 + CD25 + line cells retain their phenotype while expanding in ex-vivo cultures CD25CD69CD44 i.c. CTLA-4 GITR CD62L CCR7 25+ line 25- line

21. CD4 + CD25 + line cells express high levels of Foxp3 mFoxP3 ßActin freshlines 25 - 25 +

22. In vitro suppressor function of the CD4 + CD25 + T cell-line CD4 + CD25 + line cells are more potent suppressors than freshly isolated CD 4 + 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 + freshCD4 + 25 + line Stimulation with  CD3 and syngeneic APCs

23. CD4 + CD25 + line cells accumulate at the site of antigenic challenge d 0 CBA/Ca d-1 CD4+CD25- and CD4 + CD25 + line-GFP CBK Flow cytometry d+40 draining LN mesenteric LN grafted skin GFP

24. In T-depleted recipients, CD4 + CD25 + line-cells prevent CBK skin graft rejection by CD4 + CD45RB hi cells H2 k + K b CBK donor H2 k + K b days after transplantation 12010080604 0 200 1.0.8.6.4.2. 100 80 60 40 20 RB hi (n=8) RB hi /25 + (n=7) 25 + (n=3) Survival 0

25. … but cannot prevent 3 rd party skin graft rejection by CD4 + CD45RB hi cells 3 rd party B10.A H2 k + D d Survival 100 80 60 40 20 Class I mismatch RB hi (n=5) RB hi /25 + (n=5) RB hi (n=5) 3 rd party BALB/c H2 d Class I and II mismatch 100 80 60 40 20

26. 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 CD4 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-K d peptide with A b ) constructs for retrovirus production Activated with CD3/CD28 beads or APC+antiCD3 and IL-2 (2 days) Viral supernatant TT T TT 3 day after transduction, Functional and Flow cytometric analysis

27. 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: