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

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

HTLp IL-2

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”

A significant drop in the anti-donor response is only observed in the CD4+CD45RO+ fraction. 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 1 3 1 3 1 2 1 2 1 1 1 1 R A d o n o r R O d o n o r 1 1 R A 3 r d p a r t y R O 3 r d p a r t y Ratio 1 - 1 Ratio 1 - 1 1 - 2 1 - 2 p > . 5 p = . 8 ( W i l c o x o n ) ( W i l c o x o n ) 1 - 3 1 - 3 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.

Mechanisms of peripheral tolerance deletion deletion anergy anergy ? regulation ignorance

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

Donor-specific hyporesponsiveness was reversed in 5 out of 5 patients with IL-2 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 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 R.D. 1/Freq 1 P o s t I L - 2 1 P r e I L - 2 1 T.H. A.W. M.M. S.E. C.S. J.W. J.B. K.M. P.K. G.K. D.T. 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

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

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

Direct pathway Indirect pathway Lymph Node

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 Direct pathway naïve T cells? Indirect pathway T cells - in some patients

Tolerant to A Graft acceptance B The T cells that transfer tolerance appear to have indirect allospecificity CD4+ A B Graft acceptance 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)

“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

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

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

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

Transferred MHC:peptide complexes are recognised by T cells

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

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

DCs acquire MHC class I and II molecules in vivo

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

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.

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….?

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