1. IMMUNOPHENOTYPE OF INFLAMMATORY CELLS OF RENAL ALLOGRAFT BIOPSIES WITH ACUTE CELLULAR REJECTION Mosquera Reboredo J. M 1.; Vázquez Martul E. 1 ; Fernández Rivera C. 2 ; Filgueira Fernández P. 3 ; and Valdés Cañedo F 2. 1 Pathology Deparment, 2 Nephrology Deparment and 3 Investigation Laboratory. XXVIIth International Congress of International Academy of Pathology Athens 12-17 October 2008

2. 4. T-cell-mediated rejection (TCMR, may coincide with categories 2 and 5 and 6) Acute T-cell-mediated rejection (Type/Grade:) IA. Cases with significant interstitial infiltration (>25% of parenchyma affected, i2 or i3) and foci of moderate tubulitis (t2) IB. Cases with significant interstitial infiltration (>25% of parenchyma affected, i2 or i3) and foci of severe tubulitis (t3) IIA. Cases with mild-to-moderate intimal arteritis (v1) IIB. Cases with severe intimal arteritis comprising >25% of the luminal area (v2) III. Cases with ‘transmural’ arteritis and/or arterial fibrinoid change and necrosis of medial smooth muscle cells with accompanying lymphocytic inflammation (v3) Chronic active T-cell-mediated rejection ‘chronic allograft arteriopathy’ (arterial intimal fibrosis with mononuclear cell infiltration in fibrosis, formation of neo-intima) Banff 97 diagnostic categories for renal allograft biopsies—Banff’07 update American Journal of Transplantation 2008; 8: 753–760 ACUTE CELLULAR REJECTION

3. The composition of the inflammatory infiltrate in acute rejection is dominated by T-lymphocytes (CD4 or CD8) but macrophages, plasma cell, granulocytes or NK cells can be present. The role of T-Lymphocytes in this process is well established but not the involvement and relevance of the other inflammatory cells.

4. IMMUNOPHENOTYPE OF INFLAMATORY CELLS IN ACUTE CELLULAR REJECTION Composition of interstitial cellular infiltrate identified by monoclonal antibodies in renal biopsies of rejecting human renal allogafts. The relation of different inflammatory cell types to the various parenchymal components of rejecting kidney allografts. Reitamo S, Histopathology. 1980 Sep;4(5):517-32.Reitamo S Rejected human renal allografts: recovery and characteristics of infiltrating cells and antibody. Tilney NL Transplantation. 1979 Nov;28(5):421-6. Tilney NL Renal allograft cell infiltrates associated with irreversible rejection. Sanfilippo F Transplantation. 1985 Dec;40(6):679-85.Sanfilippo F The location, as well as the number and type of cell infiltrates are critical in evaluating cellular forms of rejection Hancock WWHancock WW et al. Transplantation. 1983 May;35(5):458-63.

5. T-Lymphocites

6. PLASMA CELLS AND AUTE CELLULAR REJECTION Plasma cell-rich acute renal allograft rejection Charney DACharney DA, Nadasdy T, Lo AW, Racusen LC.Nadasdy TLo AWRacusen LC T ansplantation 1999: 68; 791-797 Plasma cell-rich acute allograft rejection is associated with poor graft survival Is not a manifestation of concomitant chronic allograft nephropathy or viral infection, including posttransplant lymphoproliferative disorder. The clinical and pathologic implications of plasmacytic infiltrates in percutaneus renal allograft biopsies. Meehan SMMeehan SM et al. Hum Pathol. 2001 Feb;32(2):205-15. - Plasmacytic infiltrates in renal allografts comprise a spectrum of lesions from acute rejection to PTLD, with a generally poor prognosis for long-term graft survival. PLASMA CELL RICH REJECTION POOR PROGNOSIS

7. PLASMA CELLS AND AUTE CELLULAR REJECTION Desvaux et al. Nephrol Dial Transplant 2004 19 933-9 Desvaux et al. Nephrol Dial Transplant 2004 19 933-9 - Oedema and plasma cell-rich acute rejections (OPcR) >10% of the graft infiltrating cells. - OPcR rejections are highly resistant to therapy and portend a poor outcome, irrespective of Banff score. - Suggest that an antibody-mediated mechanism was involved in most OPcR ( 9 of 12 patients had sings suggesting antibody mediated rejection).

8. PLASMA CELLS AND ACUTE REJECTION - Plasma Cell-rich infiltrates correlated with C4d (p = 0.0080). Acute rejection in non-compliant renal allograft recipients: a distinct morphology Lerut E. et al Clin Transplant. 2007 May-Jun;21(3):344-51. Implications of Immunohistochemical Detection of C4d along Peritubular Capillaries in Late Acute Renal Allograft Rejection Poduval, Meehan et al. Transplantation 2005: 27:79 228-235 - 52% of AHR (C4d +) showed plasmacytic infiltrates ( 16% Cd4 -) ( p=0,02) - Evidence of acute cellular with occult humoral rejection is identified in more than 40% of late AR episodes.

9. MAST CELLS AND ACUTE CELLULAR REJECTION - M ast cell were associated with allograft fibrosis in chronic rejection and chronic CsA toxicity. Might play a pathogenic role in fibrotic process. -No mast cell increase during AR in those case that progressed to chronic rejection. MAST CELLS FIBROSIS Mast cells in human allografted kidney: correlation with interstitial fibrosis. Goto EGoto E et al Clin Transplant. 2002;16 Suppl 8:7-11. Close association between renal interstitial fibrosis and mast cells Important role in the development of CAN

10. MAST CELLS AND ACUTE CELLULAR REJECTION Mast cells in acute cellular rejection of human renal allografts. Lajoie G et al. Mod Pathol. 1996 Dec;9(12):1118-25., Lajoie G Toshiro Ishidaa et al, Clinical Transplantation Volume 19 Page 817 Dec 2005 Biopsy at 100 days: Mast cells were related to decline of long-term graft function and fibrosis Danilewicz M Danilewicz M Med Sci Monit. 2004 May;10(5):BR151-6. Epub 2004 Apr 28. Interstitial mast cell are correlated with interstitial volume, and interstitial expression of alpha-SMA. Role in the development of early interstitial fibrosis - Correlation between number of mast cells and time since transplantation, severity of interstitial fibrosis and with interstitial oedema (all P < 0.005). - Mast cells are increased in moderate and severe A.R. compared with mild and normal kidneys.

11. EOSINOPHILS AND ACUTE CELLULAR REJECTION - M ore than 4% eosinophils in the tissue inflamatory exsudate is a specific (91%) and fairly sensitive (78%) indicator of an severe acute rejections. The importance of eosinophils in kidney allograft rejection. Kormendi F Transplantation. 1988 Mar;45(3):537-9. The prognostic value of the eosinophils in acute renal allograft rejection. Weir MR Transplantation. 1986 Jun;41(6):709-12 - The increased of eosinophils in peripheral blood (p<0.01) and/or renal graft biopsy specimen (P< 0.02) is an adverse prognostic factor in acute rejection outcome.

12. EOSINOPHILS AND ACUTE CELLULAR REJECTION Abundance of interstitial eosinophils in renal allograft is associated with vascular rejection. Meleg-Smith S et al. Transplantation 2005;79:444-450 - Significant interstitial graft eosinophilic infiltrate (>10% of inflamatory infiltrate) is associated with vascular rejection in the patients on high-dose immunosupression (p=0,04) Banff criteria as predictors of outcome following acute renal allograft rejecction. Macdonald FI et al. Nephrol Dial Transplant. 1999 Jul;14(7):1692-7. -Tissue eosinophilia (>10 eosinophils/mm2) is associated with vascular rejection (P= 0.02) but is not of independent prognostic significance. EOSINOPHILS VASCULAR REJECTION

13. EOSINOPHILS AND ACUTE CELLULAR REJECTION - Eosinophilic infiltration is a negative predictor, which can indicate higher grade, more severe course of ARAR and increased resistance to an anti-rejection therapy. - Chronic rejection was seen in 25% patients of EG and in 11% in Control Group (CG) in the first year after transplantation. Biopsy eosinophilia as a predictor of renal graft dysfunction Pol Merkur Lekarski. 2006 Aug;21(122):152-5

14. MACROPHAGES AND ACUTE CELLULAR REJECTION The significance of monocytes in glomeruli of human renal transplant. Harry et al Transplantation. 37(1):70-72, January 1984. Prognosis was significantly worse during the six months after the biopsy Diagnostic and predictive value of an immunohistochemical profile in aymptomatic acute rejecion of renal allograft. Copin MC. Et al. Transpl Immunol. 1995 Sep;3(3):229-39. High number of interstitial macrophages are significantly related to unfavorable graft outcome (p < 0.01). - Macrophages are associated with steroid resistance (p < 0.01). The presence and prognostic importance of glomerular macrophage infiltration in renal allograft. Ozdemir BH. et al. Nephron 2002 Apr 90(4) 442-6 Macrophages worse prognosis. poor graft survival Intraglomerular monocyte/macrophages

15. MACROPHAGES AND ACUTE CELLULAR REJECTION The macrophage is the predominant inflammatory cell in renal allograft intimal arteritis Matheson PJ et al Transplantation. 2005 Jun 27;79(12):1658-62.Matheson PJ - In the intimal arteritis (Banff II or III )the infiltrating cells were predominantly macrophages. T-cell were in the minority (P< 0.01) Univariate analysis shows a trend for a higher infiltration with CD8+ ( P = 0.053 ) and CD68+( P = 0.06 ) cells in clinical rejection. Inflammatory factor-1+-activated macrophages (activation marker) were increased in clinical rejection (P = 0.014) Clinical rejection is distinguished from subclinical rejection by increased infiltration by a population of activated macrophages Grimm PC et al J Am Soc Nephrol. 1999 10(7):1582-9.Grimm PC

16. MACROPHAGES AND ACUTE REJECTION Monocytes and peritubular capillary C4d deposition in acute renal allograft rejection Magil y Tinckam K. International 2003 63: 1888 Transplant Mac attack:Humor the macrophages Colvin K. International 2003; 63:1953 Macrophages are a part of the pathogenesis of acute humoral rejection - Glomerular and interstitial monocyte/ macrophages(MO) infiltrations closely associated with C4d+ (p<0.0001) -Confirm the correlation of C4d+ and PMN. -Sensitivity 91% and Specificity 93% (MO/glomerulus >1 as threshold) Propose add to the histological criteria of Acute Humoral Rejection. C4d

17. MATERIALS AND METHODS Review 6 month post-transplant kidney biopsies with acute cellular rejection Grade and tabulate the histological findings according Banff score. Measure the average per high power field (hpf) of plasma cell, macrophages, mast cells, T- lymphocytes (CD4 and CD8) and eosinophils using inmunohistochemistry. All biopsies were stained with C4d antibody by inmunohistochemistry. Clinical parameters like HLA mismatch, cytotoxic cross-match, type of immunosupression, creatinine level, response to anti-rejection therapy and evolution of allograft were reviewed. Identify follow-up biopsies if available. Statistical analysis: we used SPSS statistical software. Comparisons of different data were performed with t-Student, Man-Whitney and X 2 test. Kaplan-Meier: Logistic regression for survival analysis

18. IMMUNOPHENOTYPE OF INFLAMATORY CELLS OF RENAL ALLOGRAFT BIOPSIES WITH ACUTE CELLULAR REJECTION The aim of this study is: -Try to find relation betwen type of cells in acute rejection and: - Are really useful do it in allograft biopsies with acute rejection - Clinical parameters - Outcome and survival rates of allograft. - Response to antirejection therapy. - HLA. - Evolution to chronic rejection

19. RESULTS 76 biopsies from 70 kidney transplants (51 men and 26 women) Age: 46,34 ( 20-68). Donor age: 43,46 ±16,91 (2-71) Ischemic time: 21,7±5,37 (5-33) Time to biopsy: 37,28± 48,24 days (4-180d) Creatinine: 6,47± 2,27 (1,70-12,30) Proteinuria: 2,04±2,36 (0-12)

20. Cellular Infiltrate Cells/hpfMean ±SD Plasma cells4,08±5,37 (0-47,4) Macrophages48.53±41,39 (0-250) Mast-cells1,22±2,56 (0-16,6) Lymphocytes (CD4)19,98±24,1 (0-124) Lymphocytes (CD8)42,80±35,1 (1,1-141) Eosinophils0,68±2,88 (0-23,8)

21. BANFF GRADE 40,8% 17,1% 26,3% 10,5% 2,6% 57,9% 39,4% Banff grade Vascular rejection T-test: More monocyte/macrophages in vacular rejection compared with tubulo-interstitial (60,6 vs 40,6) (p=0,039) No correlation with U-Mann Withney (p=0,09). No correlation with other type of cells

22. Glomerulitis T-test : High number of macrophages in glomerulitis group (>g1) (55,5vs 35,7) (p=0,04). No correlation with U-Mann Withney. No correlation with other type of cells Log rank= 5,07 p=0,024 g0 >g1

23. Fibrosis T-test: more plasma cells and Cd8 lymphocytes in cases with fibrosis compared with those without (plama cell 6,2 vs 1,1, p=0,01) (Cd8 38 vs 29,2, p=0,03) No correlation with U-Mann Withney. RESULTS

24. InDR 1 p Plasma cells 1,6 ± 3 5,1±10,4 0,03 Macrophages 45,7±33 50,7 ±45 ns Mast cells 0,7 ±1,2 1,5 ±3 ns CD4 24,1±31 17,7 ±20 ns CD8 43,1±39 42,9±34 ns Eosinophils 1,3 ±5 0,3±0,8 ns Nº HLA mismaches Incompatible DR > o < 1 T-Student U-Mann Whitney no correlations. Plasma cells (p=0,58)

25. RESULTADOS 2 In 3 p Plasma cells 3,8±8,1 4,1±9,9 ns Macrophages 42,3±36,2 59,1±47 0,09 Mast Cells 1,3±2,2 1,1±3,1 ns CD4 23,5±27,5 14,4±18,3 ns CD8 45,4±35 39,5±35,8 ns Eosinophils 0,9±3,8 0,2±0,4 ns Nº HLA mismaches Incompatible > O < 3 U-Man Withney : No correlation with any type of cell but macrophages p=0,07 T-test

26. C4d - Inmunohistochemistry -Ac. Polyclonal anti-C4d.

27. Antibody-mediated changes (may coincide with categories 3, 4 and 5 and 6) Due to documentation of circulating antidonor antibody, and C4d3 or allograft pathology C4d deposition without morphologic evidence of active rejection C4d+, presence of circulating antidonor antibodies, no signs of acute or chronic TCMR or ABMR (i.e. g0, cg0, ptc0, no ptc lamination). Cases with simultaneous borderline changes or ATN are considered as indeterminate Acute antibody-mediated rejection C4d+, presence of circulating antidonor antibodies, morphologic evidence of acute tissue injury, such as (Type/Grade): I. ATN-like minimal inflammation II. Capillary and or glomerular inflammation (ptc/g >0) and/or thromboses III. Arterial—v3 Chronic active antibody-mediated rejection4 C4d+, presence of circulating antidonor antibodies, morphologic evidence of chronic tissue injury, such as glomerular double contours and/or peritubular capillary basement membrane multilayering and/or interstitial fibrosis/tubular atrophy and/or fibrous intimal thickening in arteries Banff 97 diagnostic categories for renal allograft biopsies Banff’07 update American Journal of Transplantation 2008; 8: 753–760

28. MIXED REJECTION CELULAR AND HUMORAL - Halloran Ab Antidonor in 23-38% of. A.C R Transplantation 1992; 53: 550-555. Transplantation 1996; 61: 1586-1592. - Collins et al. 20-25% of Acute Rejection have Ac. Antidonor J. Am Soc. Nephrol. 1999; 10: 2208-2214. - Terasaki Ab. in 96% of Rejections Am. J. Transplant. 2003 3:6; 665-673 - Recommend C4d in all cases of acute graft failure - Evidence of acute cellular with occult humoral rejection is identified in more than 40% of late AR episodes. Poduval, Meehan et al. Transplantation 2005: 27:79 228-235

29. C4d Positive Negative Log Rank=1,15 p=0,28

30. C4d AND CELLULAR INFILTRATE NEGATIVE POSITIVE U-MANN WITHNEY p C4d Plasma cells 36 41 612 0,25 Macrophages 36,6 40,3 652 0,46 Mast cells 37,8 39,1 696 0,78 CD4 Lympho. 29,3 31,7 412 0,59 CD8 Lympho. 32,1 42,9 487 0,049 Eosinophils 38,1 35,9 626 0,64 U-Mann Withney

31. Steroid Therapy NO Yes Steroid resistants 58,4% 41,6% Yes

32. Corticosteroids Resistence YESNOTMann Whitneyp Plasma cells33,227,83430,23 Macrophages35,925,92770,027 Mast cells26,632,33390,2 Lymphocytes CD428,520,91800,06 Lymphocytes CD833,5252630,05 Eosinophils27,230,23540,47 Univariate analysis In multivariate analysis /logistic regression adjusted for macrophages, CD4 and CD8 lymphocytes, a high number of monocyte/ macrophages /hpf is associated with steroid resistance (Odds= 1,021 (ci95% 1-1,041) p=0,04).

33. AliveDeadU-Mann Whitney p Plasma Cells30,140,84370,030 Macrophages31,739,44890,11 Mast cells37,135,15880,66 Lympho. CD42728,73490,7 Lympho CD829,139,94070,04 Eosinophils34,734,15590,89 Univariate Analysis In multivariate analysis /logistic regression adjusted for plasma cells, CD4 and CD8 lymphocytes, a low number of CD8 lymphocytes /hpf is associated with better allograft survival rates (Odds= 0,96 (ci95% 0,93-0,99) p=0,039). ALLOGRAFT SURVIVAL

34. Time of biopsy (< o 30 days) T-test: higher number of macrophages in biopsies de 30 days ( 6,9 vs 2,9)(p=0,071 ). U Mann Whitney higher number of macrophages < 30 days (P=0,043) (41,7-30,4) (U Mann Whitney 417) U-Mann-Withney: higher nº of CD8 before MMF (p=0,025) (39,4-28,4) U-Mann Withney 391, RESULTS Immunosupression

35. CONCLUSIONS CD8 are associated with poor prognosis (univariate and multivariate analysis), steroid resistance (univariate) and C4d. Macrophages are associated wih steroid resistance (univariate and multivariate analysis). Number of macrophages is higher in < 30 days biopsies. Plama cell rich rejections are associated with worse allograft survival (univariate). High percentege of humoral rejection associated with acute cellular rejection

36. ACKNOWLEDGEMENTS - Eduardo Vázquez Martul Pathology Department - Constantino Fernandez Rivera Nephrology Department - Purificacion Filgueira and Investigation Unit