1. Immunophenotyping Intimal Arteritis Anthony Chang, MD University of Chicago Medical Center

2. Immunophenotyping intimal arteritis Is there a difference in the immunophenotype of lymphocytes in the setting of intimal arteritis associated with T cell-mediated rejection (TCMR) versus antibody-mediated rejection (AMR)?

3. Literature Review Intimal arteritis immunophenotype studies –Tuazon TV et al. Am J Pathol 1987 CD8, CD4 –Alpers C, et al. Mod Pathol 1990 CD45, UCHL-1 (CD45RO or memory T cells) L-26 (CD20+ B cells) HAM-56 (macrophages, endothelium) Leu-M1 (CD15 granulocytes), Factor VIII and Ulex uropaeus (endothelium), HHF-35 (smooth muscle actin), S100 (DCs, some macrophages) 22 cases

4. Literature Review Intimal arteritis immunophenotype studies –Matheson P, et al. Transplantation 2005 UCHL-1 (CD45RO T cells) CD68 (macrophages) 8 of 24 bxs with type II or III rejection CD68 > UCHL-1 –Kozakowski N, et al. NDT 2009 CD68/CD3 ratio in 116 arteries from 34 patients –Did not correlate with C4d –Did not correlate with worse graft survival

5. Literature Review Intimal arteritis immunophenotype studies –Sementilli A, et al. Tx Proceedings 2010 CD68, CD4/CD8, CD20, S100 IL-4, IL-10, interferon-gamma 10 of 40 bxs with type II rejection CD8 > CD68

6. Immunophenotyping intimal arteritis Is immunophenotype of lymphocytes in the setting of intimal arteritis associated with T cell-mediated rejection (TCMR) different from the lymphocytes associated with antibody-mediated rejection (AMR)?

7. Our study Intimal arteritis/endarteritis lesions –U of Chicago Med Ctr (n=20) –Cedar Sinai Med Ctr (n=12) 15 – AMR only, 11 – TCMR+AMR, 6 – TCMR only Immunohistochemistry –CD3, CD8, CD4, Foxp3 –CD20 –CD68 and CD163 –CD56

8. CD3+ T cells

10. CD8+ T cells

11. Tregs (CD4+/foxp3+) 1 of 13 cases

12. Tregs (CD4+/foxp3+)

13. CD68

14. CD163 Scavenger receptor Limited to macrophage/monocyte lineage

15. CD163

17. CD68 / CD163 CD68CD163

18. CD20+ B cells

20. 5 of 29 cases with any CD20+ B cells –2 with isolated v lesions and C4d+ 1 DSA+ class II, 1 DSA+ class I+II –1 with isolated v lesion C4d+ 5% of ptc, DSA/PRA-not tested –1 with TCMR and AMR C4d+ 5-10% of ptc, high PRA class I/II, DSA- –1 isolated v lesion C4d-, PRA/DSA- –15 isolated v lesions with AMR only

22. CD56

23. CD56 (NCAM)

25. CD56

27. CD56 (NCAM) 9 CD56+ cases –No more than 4 CD56+ NK cell present –2 cases with CD56 granular staining only; no CD56+ lymphocytes Of 9 isolated v lesions –7 with either CD56+ cell (n=5) or granular staining (n=2) 7 of 10 CD56 staining with AMR alone 0 of 2 CD56 staining in TCMR alone 2 of 10 CD56 staining in TCMR+AMR

28. Summary CD56 may be a useful marker to distinguish intimal arteritis associated with AMR from TCMR –Additional studies CD20 – infrequent CD3, CD8, CD68, CD163 – no significant difference

29. Acknowledgement Collaborators –Mark Haas, Cedars Sinai Medical Center