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.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.

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

7. 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

8. Donor-specific hyporesponsiveness was reversed in 5 out of 5 patients with IL-2q 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

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

11. Direct pathway
Indirect
pathway
Lymph Node

12. 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

13. 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)

14. “Indirect recognition by helper T cells can induce
donor-specific cytotoxic T lymphocytes in vivo”
RS Lee, ……. H Auchincloss Jr.
J. Exp. Med :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. Co-culture of MHC-mismatched DC leads to class I and II exchange
CFSE-R1
R1 x R4
Acceptor DC only R1 R4

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