Evolution of anti-donor alloimmunity following transplantation - implications for CAN

1. Evolution of anti-donor alloimmunity following transplantation - implications for CAN Maria Hernandez-Fuentes Richard Baker Wan Fai Ng Osquel Barroso-Herrera

2. Allorecognition: the two pathways Indirect allorecognition Direct allorecognition CD8 CD4 I allo APC CD8 CD4 Shed allogeneic MHC IL-2 II IL-2 Taken up and processed by auto APC I II allo APC Auto APC

3. HTLp IL-2

4. CD45RA+ (Naïve) and CD45RO+ (Memory) T cells have different patterns of recirculation. THYMUS BLOODSTREAM THORACIC DUCT “RA circuit” HEV LYMPH NODE tissue parenchymal cell “RO circuit”

5. A significant drop in the anti-donor response is only observed in the CD4+CD45RO+ fraction. R a t i o o f f r e q u e n c y p o s t / p r e f o r R a t i o o f f r e q u e n c y p o s t / p r e f o r C D 4 + C D 4 5 R A + c e l l s C D 4 + C D 4 5 R O + c e l l s 3 3 1 0 1 0 2 2 1 0 1 0 1 1 0 1 1 0 R O d o n o r R A d o n o r 1 0 0 0 1 0 r d r d R A 3 p a r t y R O 3 p a r t y - 1 1 0 - 1 1 0 Ratio Ratio - 2 1 0 - 2 1 0 p = 0 . 0 0 8 p > 0 . 0 5 ( W i l c o x o n ) ( W i l c o x o n ) - 3 1 0 - 3 1 0 T H L M A T A L D M J W S A C S S A l T H L M A T A L D M J W S A C S S A l 3rd party ratios close to unity mean that immunosuppression has not significantly affected the lymphocyte response; as can be observed for both fractions.

6. Mechanisms of peripheral tolerance deletion deletion anergy anergy ? regulation ignorance

7. Readout: IL2 bioassay with CTLL Experimental Design LDA adherence PBMC on plastic Cocktails of mAb CD4 cells 1 hr Followed by magnetic beads to remove non-CD4 cells + IL2 (30 U/ml) for 3 days Wash and rest for 1 day LDA

8. R.D. Donor-specific hyporesponsiveness was reversed in 5 out of 5 patients with IL-2 A n t i - d o n o r f r e q u e n c i e s b e f o r e a n d a f t e r I L - 2 1 0 0 D o n o r S p e c i f i c H y p o - r e s p o n s i v e P a t i e n t s N o n H y p o - r e s p o n s i v e P a t i e n t s 1 0 0 0 1/Freq 1 0 0 0 0 P o s t I L - 2 1 0 0 0 0 0 P r e I L - 2 1 0 0 0 0 0 0 C.S. T.H. S.E. J.W. J.B. A.W. D.T. G.K. P.K. K.M. M.M. P a t i e n t s w i t h R e v e r a l o f H y p o r e s p o n s i v e n e s s i n b o l d

9. Elevated frequencies of T cells with indirect anti-donor allospecificity in patients with chronic transplant rejection Vella et al, ‘97 Transplantation renal transplant recipients Ciubotariu et al, ‘98 JCI heart transplant recipients Hornick et al, ‘00 Circulation .. .. .. SivaSai et al, ‘99 Transplantation lung transplant recipients Baker et al, 2001 JI renal transplant recipients

10. 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

11. Direct pathway Indirect pathway Lymph Node

12. Direct pathway naïve T cells? Mechanisms of peripheral tolerance Evolution of anti-donor direct and indirect alloresponses following transplantation using conventional immunosuppression Direct pathway - memory T cells deletion anergy ? regulation ignorance Indirect pathway T cells - in some patients

13. Tolerance to MHC class I-only mismatched graft transferred by CD4 T cells (Waldmann) Pre-treatment with donor MHC class I induces tolerance when combined with anti-CD4 mAb (Wood) Tolerance could not be induced unless the indirect pathway was intact (Auchincloss) Tolerant toA B The T cells that transfer tolerance appear to have indirect allospecificity CD4+ A B Graft acceptance

14. “Indirect recognition by helper T cells can induce donor-specific cytotoxic T lymphocytes in vivo” RS Lee, ……. H Auchincloss Jr. J. Exp. Med. 1994 179:865

15. Unlinked help and suppression - the “4 cell problem” Donor parenchymal cells Immature recipient DC CD8 Unlinked help or suppression…? Mature recipient DC presenting donor MHC indirectly CD4 Lymph Node

16. R4 • R1 CFSE-R1 R1 x R4 Acceptor DC only Co-culture of MHC-mismatched DC leads to class I and II exchange

17. MHC transfer between DCs can occur in the absence of cell contact

18. Transferred MHC:peptide complexes are recognised by T cells

19. DCs acquire MHC class I from g-IFN-treated ECs

20. MHC transfer between ECs and DCs is unidirectional CFSE-labelled C57BL/6 DCs

21. DCs acquire MHC class I and II molecules in vivo

25. Linking direct and indirect pathway T cells Donor parenchymal cells Immature recipient DC Help or suppression CD8 CD4 Mature recipient DC presenting donor MHC directly and indirectly Lymph Node

26. Conclusions • Dendritic cells acquire MHC class I and class II molecules from other DCs and from ECs • The acquired MHC molecules are recognised by T cells • MHC acquisition by DCs occurs in vivo, as well as in vitro • This raises the possibility that direct alloresponses are maintained after donor DCs are deleted • This phenomenon may provide a mechanism whereby T cells with indirect allospecificity can help or suppress T cells with direct anti-donor allospecificity.

27. Speculation….. the acquisition of MHC:peptide complexes by DCs trafficking through tissues provides a failsafe mechanism to ensure the presentation in lymphoid tissue of the viral peptide:MHC complexes that are most highly represented in the infected tissue… an alternative to cross-priming….?

28. DC MHC acquisition as an alternative to cross-priming infected parenchymal cells Immature recipient DC Mature recipient DC presenting viral peptide:class I complexes to CD8+ T cells Help CD8 CD4 Lymph Node