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Three major problems that limit the clinical applications of transplantation are:

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….

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Three major problems that limit the clinical applications of transplantation are:

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

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

  3. The indirect pathway of allorecognition The direct pathway of allorecognition CD8+ cytotoxic T cell CD4+ helper T cell CD4+ cytotoxic T cell CD8+ cytotoxic T cell Allogeneic cell IL-2 IL-2 TCR MHC class I MHC class II Allogeneic (stimulator) antigen presenting cell Donor MHC- derived peptide CLIP Responder antigen presenting cell Pathways of MHC allorecognition

  4. 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)

  5. In context of MHC incompatibility deletion may be required

  6. Peripheral Tx tolerance is transferable… A skin adult Bstrain tolerant to A Tolerance protocol B adoptive transfer of CD4+ T cells A skin Graft acceptance “naive” B

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

  8. Tolerance is maintained by regulatory T cells

  9. Lessons from patients

  10. HTLp IL-2

  11. 4 1 0 p < 0 . 0 5 * 1/frequency 5 1 0 6 1 0 C A N C A N F r e e

  12. Direct pathway Indirect pathway Lymph Node

  13. Apparent indirect anti-donor allospecificity Peripheral Tx tolerance is transferable… A skin adult Bstrain tolerant to A Tolerance protocol B adoptive transfer CD4+ T cells A skin Graft acceptance “naive” B

  14. Preclinical testing of strategies to promote transplantation tolerance

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

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

  17. CD25 CD4 Methods Spleen and LN cells CBA/Ca H2k CD4+CD25+ immature DC + Kb peptide

  18. CD25 CD69 CD44 i.c. CTLA-4 GITR CCR7 CD62L CD4+CD25+ line cells retain their phenotype while expanding in ex-vivo cultures 25- line 25+ line

  19. mFoxP3 ßActin 25- 25+ 25- 25+ fresh lines CD4+CD25+ line cells express high levels of Foxp3

  20. In vitro suppressor function of the CD4+CD25+ T cell-line + + C D 4 C D 2 5 l i n e c e l l s a r e m o r e p o t e n t s u p p r e s s o r s t h a n f r e s h l y + + c e l l s 4 C D 2 5 T i s o l a t e d C D 7 0 0 0 0 6 0 0 0 0 5 0 0 0 0 4 0 0 0 0 cpm 3 0 0 0 0 2 0 0 0 0 1 0 0 0 0 0 - - + 2 5 / 2 5 ( 1 : 0 . 5 ) ( 1 : 0 . 2 5 ) ( 1 : 0 . 1 2 5 ) 2 5 2 5 - / 2 5 - 2 5 + ( 1 : 1 ) Stimulation with aCD3 and syngeneic APCs + + + + C D 4 2 5 f r e s h C D 4 2 5 l i n e

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

  22. In T-depleted recipients, CD4+CD25+ line-cells prevent CBK skin graft rejection by CD4+CD45RBhi cells CBK donorH2k + Kb 100 1 . 0 25+ (n=3) 80 . 8 Survival RBhi/25+ (n=7) 60 . 6 40 RBhi (n=8) . 4 20 . 2 0 . 0 2 0 4 6 0 8 0 1 0 0 1 2 0 0 d a y s a f t e r t r a n s p l a n t a t i o n

  23. 100 100 80 80 60 60 40 40 20 20 …but cannot prevent 3rd party skin graft rejection by CD4+CD45RBhi cells 3rd party B10.A H2k + Dd 3rd party BALB/c H2d Class I mismatch Class I and II mismatch Survival Survival RBhi/25+ (n=5) RBhi/25+ (n=5) RBhi (n=5) RBhi (n=5)

  24. CD4+CD25- CD4+CD25+ CD4 CD25 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 CD3/CD28 beads or APC+antiCD3 and IL-2 (2 days) Viral supernatant T T T T T 3 day after transduction, Functional and Flow cytometric analysis

  25. 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.

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