Acute rejection after kidney transplantation Pr Lionel ROSTAING Département de Néphrologie et Transplantation d’Organes (DNTO) CHU Toulouse-Rangueil, France rostaing.l@chu-toulouse.fr Tbilisi, April 8th2016

Prevalence of acute rejection: 10 to 20% [acute cellular rejection : 90% antibody-mediated rejection: 10%] -2-

Hyperacute rejection It is mediated by preformed circulating antibodies that do bind complement : they can be directed against Donor HLA antigens ABO antigens (in the setting of ABO incompatible kidney transplantation) Xeno-antigens. Complement activation results in platelet aggegation and ultimately vascular thrombosis organ necrosis This type of acute rejection should NEVER be observed -3-

Homoral/vascular rejection It is mediated by alloantibody (ies) already present at transplantation but however, at very low titer : thereby the cross-match (microlymphocytotoxicity) is negative. Conversely, these alloantibody (ies) can be synthetized at posttransplant anamnestic response). Anti-HLA alloantibody Anti-endothelial cell antibody Mostly observed in patients transfused in the past or in women (pregnancy, miscarriage) Histology : Vascular lesions (endarteritis ; C4d deposits on vascular walls) Withoit specific treatment results in organ necrosis This type of rejection represents less than 10% of acute rejections. -4-

Acute cellular rejection Acute cellular rejection : this is the most frequent acute rejection type This is a T-cell mediated process Differed process (a few days to a few months) If no treatment is implemented, this will result in organ destruction Acute cellular rejection occurs in 10 to 15% of kidney transplant patients -5-

Targets of acute rejection Kidney  tubular cells, endothelium (glomeruli) Prevalence of acute cellular rejection : 10 to 15% Prevalence of acute humoral rejection : a few % What are the mediators of acute rejection? T activated cytotoxic lymphocytes (CD8+, CD45 RO+, DR+, Perforine+, granzyme B+, Fas L+) Donor-specific alloantibodies (DSA); complement activation. -6-

Rejection description according to Banff classification -7-

Banff 07 diagnostic categories for renal allograft biopsies (1); revised in 2013 T-cell-mediated rejection (TCMR)  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) Solez K et al. Am J Transplant 2008;8:753-760 -8-

Banff 07 diagnostic categories for renal allograft biopsies (2); revised in 2013 Borderline changes: ‘Suspicious’ for acute T-cell-mediated rejection This category is used when no intimal arteritis is present, but there are foci of tubulitis (t1, t2 or t3) with minor interstitial infiltration (i0 or i1) or interstitial infiltration (i2, i3) with mild (t1) tubulitis Solez K et al. Am J Transplant 2008;8:753-760 -9-

Banff 07 diagnostic categories for renal allograft biopsies (3); revised in 2013 Acute/active AMR; all three features must be present for diagnosis 1. Histologic evidence of acute tissue injury, including one or more of the following: Microvascular inflammation (g > 0 and/or ptc > 0) Intimal or transmural arteritis (v > 0) Acute thrombotic microangiopathy, in the absence of any other cause Acute tubular injury, in the absence of any other apparent cause 2. Evidence of current/recent antibody interaction with vascular endothelium, including at least one of the following: Linear C4d staining in peritubular capillaries (C4d2 or C4d3 by IF on frozen sections, or C4d > 0 by IHC on paraffin sections) At least moderate microvascular inflammation ([g + ptc] ≥ 2) Increased expression of gene transcripts in the biopsy tissue indicative of endothelial injury, if thoroughly validated 3. Serologic evidence of donor-specific antibodies (DSAs) (HLA or other antigens) Haas M et al. Am J Transplant 2014;14:272-283 -10-

Banff 07 diagnostic categories for renal allograft biopsies (4); revised in 2013 Chronic, active AMR; all three features must be present for diagnosis 1. Morphologic evidence of chronic tissue injury, including one or more of the following : Transplant glomerulopathy (TG) (cg > 0), if no evidence of chronic thrombotic microangiopathy> 0) Severe peritubular capillary basement membrane multilayering (requires EM) Arterial intimal fibrosis of new onset, excluding other causes 2. Evidence of current/recent antibody interaction with vascular endothelium, including at least one of the following: Linear C4d staining in peritubular capillaries (C4d2 or C4d3 by IF on frozen sections, or C4d > 0 by IHC on paraffin sections) At least moderate microvascular inflammation ([g + ptc] ≥ 2) Increased expression of gene transcripts in the biopsy tissue indicative of endothelial injury, if thoroughly validated 3. Serologic evidence of DSAs (HLA or other antigens) Haas M et al. Am J Transplant 2014;14:272-283 -11-

Banff 07 diagnostic categories for renal allograft biopsies (5); revised in 2013 Interstitial fibrosis and tubular atrophy, no evidence of any specific etiology (may include nonspecific vascular and glomerular sclerosis, but severity graded by tubulointerstitial features)  Grade : I. Mild interstitial fibrosis and tubular atrophy (<25% of cortical area) II. Moderate interstitial fibrosis and tubular atrophy (26–50% of cortical area) III. Severe interstitial fibrosis and tubular atrophy/loss (>50% of cortical area)    Solez K et al. Am J Transplant 2008;8:753-760 -12-

Antibody-mediated vascular rejection of kidney allografts : a population-based study (1) Identification of four distinct rejection patterns according to clinical, histological, and immunological variables The unsupervised principal component analysis examined kidney recipients with acute biopsy-proven rejection with seven variables: glomerulitis, peritubular capillaritis, donor-specific anti-HLA antibodies, C4d deposition, interstitial inflammation, tubulitis, and endarteritis. The horizontal axis opposes cellular rejection (interstitial inflammation and tubulitis) and antibody-mediated rejection (donor-specific anti-HLA antibodies, glomerulitis, peritubular capillaritis and C4d), as recognised by the international Banff classification. The vertical axis defines the presence or absence of lesions of endarteritis (appendix). -13- Lefaucheur C. et al.The Lancet 2013;381:313-319

Antibody-mediated vascular rejection of kidney allografts : a population-based study (2) Comparison of morphological and immunological variables in the four rejection patterns Bars represent SD. NS=not significant. TCMR/V– =T cell-mediated rejection without vasculitis. TCMR/V+ =T cell-mediated vascular rejection. ABMR/V+ =antibody-mediated vascular rejection. ABMR/V– =antibody-mediated rejection without vasculitis. -14- Lefaucheur C. et al.The Lancet 2013;381:313-319

Antibody-mediated vascular rejection of kidney allografts : a population-based study (3) Kaplan-Meier curves for kidney graft survival by acute rejection phenotype Initial diagnoses as per (A) Banff classifications and (B) our new approach. Graft survival in patients without rejection is purely illustrative; graft survival in these individuals starts at time of transplantation. TCMR/V– =T cell-mediated rejection without vasculitis. TCMR/V+ =T cell-mediated vascular rejection. ABMR/V– =antibody-mediated rejection without vasculitis. ABMR/V+ =antibody-mediated vascular rejection. -15- Lefaucheur C et al.The Lancet 2013;381:313-319

Antibody-mediated vascular rejection of kidney allografts : a population-based study (4) Multivariate analysis of factors associated with graft loss in patients with antibody-mediated vascular rejection, Number of patients Numbers of events Hazard ratio (95% CI) P value Intersistial inflammation and tubulitis score* 3 32 7 1 … >3 14 4.33 (1.5-12.1) 0.005 Endarteritis score <3 52 15 3 12 6 5.17 (1.8-14.6) 0.002 DSAmax MFI <3000 41 9 3000 23 3.88 (1.5-9.8) 0.004 Treatment strategy Steroids and intravenous immune globulin 13 Steroids plus munomonab-CD3 or rabbit antithymocyte globulin 29 11 0.4 (0.2-1.3) 0.1 Steroids, plasmapheresis, intravenous immune globulin, and rituximab 22 0.16 (0.04-0.66) 0.01 Hazard ratios were estimated in a single Cox proportional hazards model. DSAmax MFI=maximum mean intensity of fluorescence of donor-specific anti-HLA antibodies. *Interstitial inflammation and tubulitis score was defined as the sum of interstitial inflammation and tubulitis, and was graded from 0 to 6. -16- Lefaucheur C et al.The Lancet 2013;381:313-319

Influence of acute rejection type upon kidney allograft survival cellulaire P < 0,001 humoral Everly MJ & al. Am J Transplant. 2009;9:1063-1071 -17-

Influence of de novo DSA on kidney allograft De novo DSA at the time of acute rejection :  graft survival Rapid decrease in DSA  Improvement of allograft survival Everly MJ & al. Am J Transplant. 2009;9:1063-1071 -18-

Influence of de novo DSA at the time of acute rejection on allograft survival p= 0,001 Everly MJ & al. Am J Transplant. 2009;9:1063-1071 -19-

Diagnosis of acute rejection (1) Two situations: early posttransplant, and after a few weeks: Immediately posttransplant acuet rejection has to be ruled out in the following settings: Prolonged acute tubular necrosis (> 5 days), specially in the setting of living kidney transplant A rapid drop in urine outpout A stable but impaired serum creatinine(i,e, above the level at which it should be) An increase in serum creatinine above its baseline value Otherwise, we have to rule out acute rejection wherever there is an unexplained rise in serum creatinine. -20-

Diagnosis of acute rejection (2) Positive diagnosis: To rule-out other causes of allograft dysfunction, such as obstruction of urinary tract, or ureteral leaking, or acute pyelonephritis, or vascular thrombosis (artery or vein), or toxic levels of ciclosporin /tacrolimus Kidney allograft biopsy : Lymphocytic interstitial infiltrate (CD3 +); apply Banff classification C4d deposits? Endarteritis ? Glomerulitis? Interstitial haemorrhage and/or interstitial edema? -21-

Elementary lesions -22-

Elementary lesions Acute Glomerulitis Inflammatory infiltrate Tubulitis Capillaritis Vasculitis Chronic Chronic allograft nephropathy Interstitial fibrosis Tubular atrophy Allograft vasculopathy Arteriolar hyalinosis Others : Edema Interstitial hemorraghae C4d

Acute lesions

CD3

C4d

Diagnosis Arteritis Tubulitis + inflammatory infiltrate Capillaritis + glomerulitis + edema + C4d (+) Moderate acute rejection (grade II) Severe AR (grade III) Borderline acute rejection Grade I acute rejection (mild) Humoral rejection -36-

Differential diagnosis Infection : Acute pyelonephritis BK-virus infection Vascular problem : Arterial stenosis Venous stenosis Lymphoproliferative disorder -37-

Treatment of acute rejection First line therapy: IV Methylprednisolone: 10 mg/kg/d for 3 consecutive days = proapoptotic effect In case of steroid-resistant acute rejection (less tha 10% of cases): antilymphocyte preparations (Thymoglobulines®) In case of humoral rejection: plasmapheresis +/- IVIg +/- Rituximab (anti-CD20 monoclonal antibody) -38- 38

How to prevent acute rejection? (1) To perform pretransplant a cross-match (microlymphocytotoxicity and when possible by flow cytometry) in order to rule-out donor-specific alloantibody (ies) T-cell cross-match (assess anti-class I antibodies) B-cell cross-match (assess anti-class I and anti-class II antibodies) Dithiotrietol (DTT) which enables to get rid out of IgM alloantibodies (i,e, they are not pathogenic) -39-

How to prevent acute rejection? (2) How to perform the cross-match? By micro-lymphocytotoxicity (CDC cross-match) lymphocytes + serum + complement --> Patient’s T-cell lysis? If yes --> positive cross-match Flow cytometry Results : If T-cell cross-match is positive : NO kidney transpalntation If B-cell cross-match is positive: * transplant day serum: NO transplantation * on historical serums only: kidney transplantation IS possible but high risk of antibody-mediated rejection -40-