ANTI-HLA ANTIBODIES IN KIDNEY TRANSPLANTATION

ANTI-HLA ANTIBODIES IN KIDNEY TRANSPLANTATION Rabat, July 2, 2005 Domenico Adorno National Council of Researches “Institute of Organ Transplantation and Immunocytology” L’Aquila - Italy ANTI-HLA ALLOANTIBODIES Any individual who has experienced sensitizing events, such as transfusion, pregnancy or previous transplant, is at risk for developing antiHLA antibodies. So, it is important to detect and identify these antibodies prior to the patient receiving an organ for transplant or re-transplant. Istituto Trapianti d’Organo PRA (Panel Reactive Antibodies) Pool of well-characterized panel cells in order to detect alloantibody reactivity and specificity in patients’ sera. The PRA represents the percentage of positive cells. Istituto Trapianti d’Organo CROSSMATCH IN KIDNEY TRANSPLANTS Negative Crossmatch Proceed to transplant Positive Crossmatch Controindication Istituto Trapianti d’Organo CDC CROSSMATCH IN KIDNEY TRANSPLANTS Reject (≥ 48h) Positive crossmatch 24 No Reject 6 Negative crossmatch 8 187 Patel R., Terasaki P.I. : N.Engl. J. Med. 1969; 280: 735  Positive crossmatch: high risk (80%) of immediate graft loss.  Prospective crossmatch; if positive, transplant is not allowed.  Some antibodies (not anti-HLA) are directed against donor lymphocytes but are not clinically relevant. Istituto Trapianti d’Organo CROSSMATCH (I) 1969. Standard-, Basic-, direct NIH-CDC. 1969. Extended incubation of T and B CDC. 1970. Amos (3 washes) and Amos-modified (1 wash) T and B CDC: washes eliminate anticomplementary factors. 1972. AHG – CDC T (Johnson A.H.): the addition of human anti-globulin identifies low titer antibodies as well as antibodies that do not fix complement in vitro. 1984. AHG – CDC B (Gebel H.M.): as B cells express surface immunoglobulin, AHG could bind to these and activate the complement = false positive result. Using a two-color immunofluorescent technique, B cells are first marked with a fluorescent anti-immunoglobulin which prevents binding between AHG and surface immunoglobulins. Istituto Trapianti d’Organo CROSSMATCH (II) 1983. T and B Flow crossmatch (FCXM) (Garavoy): after incubation with recipient serum, donor lymphocytes are stained with a fluorochrome-conjugated secondary antibody (anti-human IgG or IgM):  Independent and simultaneous evaluation of anti-T and anti-B antibodies (marked with anti-CD3 and anti-CD19).  Identification of the type of antibody (IgG, IgM).  Identification of antibodies independently by complement activation.  25 - 250 times more sensitive than CDC.  Semi-quantitative measure of antibody binding. Istituto Trapianti d’Organo CHARACTERIZATION OF ALLOANTIBODIES 1976. Positive crossmatch due to auto-antibodies (IgG or IgM) of no clinical rilevance: - absorption of sera with autologous lymphocytes - absorption of sera with DTT 1999. Identification of anti-HLA specificity using microbeads covered with purified HLA antigens as a target, isolated from cell lines (Pei R.). 2001. Identification of anti-HLA specificity using soluble HLA antigens as a target, fixed in the solid phase of the ELISA (Zachary A.A.). 2003. Identification of anti-HLA antibody specificity using single antigens bound to microbeads and studied using Flow cytometry (Pei R.): - more sensitive - makes the analysis of anti-HLA antibodies easier in the presence of high PRA. Istituto Trapianti d’Organo TECHNIQUES FOR EVALUATING ANTI-HLA ANTIBODIES (I) Non Antigen-specific CDC: • % PRA • Specificity (class I & II ?) Antigen-specific ELISA: • Neg/Pos • % PRA (class I & II) • Specificity (class I & II) Flow Cytometry (FlowPRA Beads): • % PRA (class I & II) • Specificity (class I & II) • IgG e IgM Istituto Trapianti d’Organo TECHNIQUES FOR EVALUATING ANTI-HLA ANTIBODIES (II) Analysis of HLA Presensitization (PRA) Method CDC ELISA Pos. (n°)/Paz. (n°) 12/66 32/66 % 18% 48% Flow Istituto Trapianti d’Organo 48/66 73% By W.E. Herczyk 2003 INCIDENCE OF ANTI-HLA SENSITIZATION (I) ENHANCING CHARACTERIZATION OF PRESENSITIZATION STATUS IN KIDNEY TRANSPLANT CANDIDATES USING SENSITIVE TECHNIQUES A. Piazza, E. Poggi, G. Ozzella, P.I. Monaco, C.U. Casciani, D. Adorno Transplant 2001 Meeting, Chicago May 11-16 2001 Istituto Trapianti d’Organo INCIDENCE OF ANTI-HLA SENSITIZATION FlowPRA Screening Results FlowPRA + Patients Ts-group (n. 107) (II) FlowPRA Patients Immunizing events (mean ± SD) Immunizing events (mean ± SD) 2.8% 38.5% 25.3% P<0.00001 9.7±6.0 1 2.8±1.2 P=0.0367 97.2% 61.5% 74.7% 4.3±4.0 1 3.1±2.1 Tx-group (n. 39) Pg-group (n. 75) Istituto Trapianti d’Organo THE ROLE OF Ig-M DONOR-SPECIFIC ANTIBODIES  Few studies.  Contrasting results.  In the absence of definitive studies, these antibodies are not considered in clinical practice. Our laboratory has found anti-HLA IgM antibodies (identified using microbeads coated with single HLA antigens) both preand post- transplant. Istituto Trapianti d’Organo THE ROLE OF NON ANTI-HLA ANTIBODIES The following have been associated with graft rejection:  Anti-phospholipid antibodies  Anti-endothelium antibodies  Organ-specific antibodies These antibodies do not react with donor lymphocytes and are not investigated in clinical practice. Istituto Trapianti d’Organo ANTI-HLA ANTIBODIES The official position of ASHI (approved on June 7th 2003)  Positive crossmatches due to anti-HLA IgG antibodies reacting specifically with donor lymphocytes are clinically relevant.  A positive CDC crossmatch controindication to transplant. is the strongest Istituto Trapianti d’Organo CLINICAL SIGNIFICANCE OF LOW TITER AND/OR NONCOMPLEMENT-FIXING ANTIBODIES (CDC- , FCXM+) (I) Points open to discussion:  do these antibodies represent an unacceptable clinical risk ? Or in other words do they automatically cause antibodymediated hyperacute or accelerated rejection ?  do they affect an individual’s chances of receiving a transplant ? Istituto Trapianti d’Organo CLINICAL SIGNIFICANCE OF LOW TITER AND/OR NONCOMPLEMENT-FIXING ANTIBODIES (CDC- , FCXM+) (II) Incidence : - First transplants 15% - Re-transplants 34% Early Graft Loss (< 3 months): FCXM + FCXM - First transplants Re-transplants 20% 60% 5% 15% (HM Gebel et al. Am J of Transplant 2003; 3: 1488) Istituto Trapianti d’Organo CLINICAL SIGNIFICANCE OF LOW TITER AND/OR NONCOMPLEMENT-FIXING ANTIBODIES (CDC- , FCXM+) (III) Conclusions:  low titer and/or noncomplement-fixing anti-HLA antibodies, only detectable by sensitive methods such as flow cytometry, can have significant clinical consequence.  these antibodies appear to represent a significant risk factor that should be integrated into the patient assessment algorithm. Istituto Trapianti d’Organo POSITIVE CROSSMATCH FOR B-CELLS ONLY (CDC)  Can be caused by anti-class II antibodies or low levels of anticlass I antibodies.  Can be due to non-HLA antibodies.  Can be the result of serum IgG binding to B cells in a nonspecific manner by means of Fc receptors. Istituto Trapianti d’Organo THE CLINICAL SIGNIFICANCE OF A POSITIVE CROSSMATCH FOR B-CELLS ONLY To interpret the crossmatch for B-cells in CDC :  Perform an auto-crossmatch  Analyze serum by solid-phase assays documented to be more sensitive than CDC assay (Flow cytometry or ELISA) Istituto Trapianti d’Organo THE CLINICAL SIGNIFICANCE OF “CURRENT NEGATIVE HISTORIC POSITIVE” (CNHP) CDC CROSSMATCH FOR T-CELLS (I) Strategy currently adopted:  accurate immunological history of the patient  serum testing every 3 months (EFI) or every month (ASHI) for PRA evaluations. Istituto Trapianti d’Organo THE CLINICAL SIGNIFICANCE OF “CURRENT NEGATIVE HISTORIC POSITIVE” (CNHP) CDC CROSSMATCH FOR T-CELLS (II) Evolution:  1970: all historic sera were tested; if only one of the sera tested was positive, then transplant was contraindicated.  1982 (Cardella): no difference in one year graft survival between CNHP and CNHN recipients.  Many centers performed crossmatching only with current serum for sensitized patients without, in general, the onset of hyperacute rejection. Istituto Trapianti d’Organo THE CLINICAL SIGNIFICANCE OF “CURRENT NEGATIVE HISTORIC POSITIVE” (CNHP) CDC CROSSMATCH FOR T-CELLS (III) Istituto Trapianti d’Organo (HM Gebel et al. Am J Transplant 2003; 3: 1488) THE CLINICAL SIGNIFICANCE OF “CURRENT NEGATIVE HISTORIC POSITIVE” (CNHP) CDC CROSSMATCH FOR T-CELLS (IV)  The CNHP recipients showed an increased rate of antibodymediated rejection in the first three months.  However a significant proportion of CNHP recipients have been transplanted successfully. Conclusion The cumulative published data suggests that while a positive crossmatch with an historical serum does not exclude transplantation completely, it is nevertheless a significant risk factor. Istituto Trapianti d’Organo EVALUATION OF RISK FOR ANTIBODY-MEDIATED REJECTION OR EARLY GRAFT LOSS ON THE BASIS OF CROSSMATCH (I) (HM Gebel et al. Am J Transplant 2003; 3: 1488) Istituto Trapianti d’Organo EVALUATION OF RISK FOR ANTIBODY-MEDIATED REJECTION OR EARLY GRAFT LOSS ON THE BASIS OF CROSSMATCH (II) The official position of ASHI (approved on June 7th 2003)  High risk: transplant is normally ruled out. If transplanted, patients require major pre-transplant intervention (IVIG + plasmapheresis to modify risk), post- transplant additional treatment and accurate monitoring.  Intermediate risk: transplant is normally ruled out. If transplanted, patients may require augmented immunosuppression and accurate post-transplant monitoring.  Negligible Risk: transplant takes place with no changes in their normal practice. Istituto Trapianti d’Organo ANTIBODY-MEDIATED REJECTION 1966. Kissmeyer-Nielsen: hyperacute rejection associated with pre-existing anti-HLA antibodies directed against donor cells. 1968. Patel R., Terasaki P.I.: Hyperacute rejection associated with a positive crossmatch in kidney transplantation. 1970. The predominant role of T mediated rejection: the posttransplant study of antibodies appears to be of little clinical relevance (antibodies are only a marker for T-mediated sensitization) 1990. Re-evaluation of rejection mediated by anti-HLA antibodies. Istituto Trapianti d’Organo ANTIBODY-MEDIATED REJECTION (AMR) (I) Consensus Opinion from the Antibody Working Group (Transplantation 2004; 78: 181) AMR = Antibody mediated rejection Hyperacute AMR : high titer of DSA; alteration of graft function and, nearly always, graft loss within 24 hours post-transplant due to preformed DSA. Accelerated AMR: low titer of DSA; rejection occurs after 24 hours; no response to standard therapy. Acute AMR: “de novo” production of DSA or post-desensitization restart of DSA production; rejection from 1 week to 3-6 months posttransplant; no response to standard therapy. Late or chronic AMR: “de novo” production of DSA; usually 1 year post-transplant; few symptoms but progressive; poor or no response to standard therapy. Istituto Trapianti d’Organo AMR (II) Istituto Trapianti d’Organo AMR (III) Consensus Opinion from the Antibody Working Group (Transplantation 2004; 78: 181) HYPERACUTE AMR:  high levels of pre-formed DSA rapidly endothelium and activate the complement attack graft  endothelial cells are stimulated to secrete Von Willebrand factor which mediates platelet adhesion and aggregation  platelet activation following exposure to subendothelial proteins  thrombosis and vascular occlusion Istituto Trapianti d’Organo AMR (IV) Consensus Opinion from the Antibody Working Group (Transplantation 2004; 78: 181) ACCELERATED-ACUTE AMR:  altered graft function similar to acute tubular necrosis  biopsy shows inflammatory cells in the peritubular capillaries; no tubulitis which is the most significant symptom of T-cell rejection; no immunoglobulins are detectable; C4d deposits present in peritubular capillaries.  circulating DSA  may coexist with cell-mediated rejection (CMR). Istituto Trapianti d’Organo AMR (V) Consensus Opinion from the Antibody Working Group (Transplantation 2004; 78: 181) DELAYED or CHRONIC AMR:  circulating DSA  C4d deposits in peritubular capillaries (not always)  Histological findings of glomerulopathy  May occur with CMR Istituto Trapianti d’Organo AMR DIAGNOSIS (I) Consensus Opinion from the Antibody Working Group (Transplantation 2004; 78: 181)  Altered graft function.  Typical histological findings and peritubular C4d deposits.  Presence of DSA. Istituto Trapianti d’Organo AMR DIAGNOSIS (II) Consensus Opinion from the Antibody Working Group (Transplantation 2004; 78: 181) Histological findings  Polymorphonucleate/macrophage infiltration.  Presence of thrombi in capillaries and/or fibrinoid necrosis and/or acute tubular damage. Istituto Trapianti d’Organo AMR DIAGNOSIS (III) Consensus Opinion from the Antibody Working Group (Transplantation 2004; 78: 181) Immunopathological findings Immunofluorescence reveals peritubular Cd4 deposits = class 2 of the Banff classification C4 C4a C4c Istituto Trapianti d’Organo C4b C4d (binds stably to the tissue) AMR DIAGNOSIS (IV) Consensus Opinion from the Antibody Working Group (Transplantation 2004; 78: 181) Clinical situations at high risk with stable graft function:  Presence of typical histological findings and DSA = preclinical process, mediated by antibodies, which may or may not develop into AMR (preventive treatment aimed at eradicating DSA is useful).  The absence of histological findings but the presence of DSA = latent immunological response (if this occurs in the immediate post-operative period, therapy needs to be changed or increased). Istituto Trapianti d’Organo AMR THERAPY Treatment must be early and aggressive:  Anti-thymocyte globulins  Mycophenolate Mofetil (MMF)  Plasmapheresis  Intravenous administration of Ig (IVIG)  Immunoadsorbment with protein A with or without cyclophosphamides  Anti-CD20 Istituto Trapianti d’Organo PROTOCOLS FOR HYPERIMMUNE PATIENTS Save Our Souls (SOS) U.K. The sera of Highly Immunized Trial hyperimmune Switzerland, Czech Republic, patients are tested Slovakia, Poland, Spain with all ABO compatible donors. Regional Organ Procurement (ROP) USA Istituto Trapianti d’Organo ACCEPTABLE MISMATCH PROGRAM (EUROTRANSPLANT 1985) (Hyperimmune patients = PRA ≥ 85%)  Identification of acceptable mismatches (MMs) by analyzing the phenotype of negative panel donors; or testing patient sera with one MM donors (20,000 blood donors typed for HLA).  The crossmatch is “forecasted” to be negative and the kidney is sent to the transplant center.  Crossmatch is performed on current and historical sera. (FH Claas et al. Transplantation 2004; 78: 190) Istituto Trapianti d’Organo HLA MATCHMAKER (RJ Duquesnoy)  A molecularly based algorithm developed to identify “acceptable” HLA antigens in hyperimmune patients without the need for extensive serum screening.  Based on the concept that immunogenic epitopes are represented by amino acid triplets on exposed parts of HLA class I molecules, accessible to alloantibodies.  In many cases this program permits the identification of mismatched HLA antigens that share all of their polymorphic triplets with the patient’s HLA antigens and, therefore, could be considered fully compatible.  HLA Matchmaker provides an assessment of donor/recipient HLA compatibility at a structural level in contrast to conventional methods based on the counting of numbers of mismatched HLA antigens or CREGs. Istituto Trapianti d’Organo STUDY OF THE EPITOPES INVOLVED IN THE PRODUCTION OF CLASS I ANTIHLA ANTIBODIES IN KIDNEY TRANSPLANTS A. Piazza, E. Poggi, L. Borrelli, A. Scornajenghi, G. Ozzella, P.I. Monaco e D. Adorno Istituto CNR Trapianti d’Organo – Sezione di Roma THE “TYPE” OF HLA-A EPITOPES INVOLVED IN THE PRODUCTION OF DS-Abs (I) 7 6 5 4 3 2 1 0 116-D 127-K 144-K 145-H 151-H 166-D 114-Q/R 167-G 193-A 65-R 66-N 74-D 76-A 77-N 81-A 83-R 90-D 62-G/Q 70-H/Q 253-Q 79-G 142-T 294-F 80-T 82-L 299-A/T  32 different epitopes (with a total of 76 amino-acid residues)  Highest frequency: 127-K (Lys)=9,2%; 116-D (Asp)=7,9%; Istituto Trapianti d’Organo 62-G (Gly)/Q (Gln)=6,6% 334-M 97-M 105-S 194-V 207-S 246-S 276-P 67-V 298-I 95-I THE “TYPE” OF HLA-A EPITOPES INVOLVED IN THE PRODUCTION OF DS-Abs (II) Istituto Trapianti d’Organo THE “TYPE” OF HLA-B EPITOPES INVOLVED IN THE PRODUCTION OF DS-Abs (I) 8 7 6 5 4 3 2 1 0 163-E/L/T 113-H 152-E 67-Y 9-H 80-I/N 156-D 11-A 81-A 83-R 77-N/S 131-S 305-T 45-T 12-M 82-L 194-I  16 different epitopes (with a total of 45 amino-acid residues)  Highest frequency: 77-N (Asn)/S (Ser)=17,8%; 81-A (Ala)=13,3%; 80-I (Ile)/N (Asn)=11,1%; 82-L (Leu)=11,1%; 83-R (Arg)=11,1%; 163-E (Glu)/L (Leu)/T (Thr)=11,1% Istituto Trapianti d’Organo THE “TYPE” OF HLA-B EPITOPES INVOLVED IN THE PRODUCTION OF DS-Abs (II) Istituto Trapianti d’Organo CLINICAL RELEVANCE OF ANTI-HLA ANTIBODIES DEVELOPED AFTER KIDNEY TRANSPLANTATION A. Piazza, E. Poggi, L. Borrelli, G. Ozzella, P.I. Monaco, D. Settesoldi, D. Fraboni, S. Scornajenghi, C. Cortini, A. Iacona, C.U. Casciani, D. Adorno 9° Congresso Nazionale AIBT, Pesaro 19-21 Settembre 2002 Preliminary data in: A. PIAZZA, E. POGGI, L. BORRELLI., S. SERVETTI , P.I. MONACO, O. BUONOMO, M. VALERI, N. TORLONE, D. ADORNO and C.U. CASCIANI. Impact of donor-specific antibodies on chronic rejection occurrence and graft loss in renal transplantation. Transplantation 2001; 71: 1106-1112. A. PIAZZA, E. POGGI, L. BORRELLI., M. VALERI, O. BUONOMO, S. SERVETTI, C.U. CASCIANI, D. ADORNO. Relevance of post-transplant HLA class I and class II antibodies on renal graft outcome. Transplant Proc 33(1-2): 478-480, 2001 Istituto Trapianti d’Organo Anti-DSA Analysis using FlowPRA 60,0% 51,4% 40,0% 27,0% 20,0% 21,6% 0,0% anti-HLA class I & II anti-HLA class II anti-HLA class I Istituto Trapianti d’Organo Anti-DSA and Transplant Outcome 51,3% 60% 30,8% 40% P = 0,0001 20% P = 0,0001 Anti-DS Positive Patients 12,8% 6,0% 0% Anti-DS Negative Patients Acute Rejection Graft Loss Istituto Trapianti d’Organo CONTROL OF ANTIDONOR ANTIBODY PRODUCTION WITH TACROLIMUS AND MYCOPHENOLATE MOFETIL IN RENAL ALLOGRAFT RECIPIENTS WITH CHRONIC REJECTION T.P. Theruvath, S.L. Saidman, S. Mauiyyedi, F.L. Delmonico, W.W. Williams, N. Tolkoff-Rubin, A.B. Collins, R.B. Colvin, A.B. Cosimi and M. Pascual Transplantation 2001; 71(1): 77-83 Istituto Trapianti d’Organo Figure 2: Kinetics of circulating donor-specific antibodies (DSA) and renal allograft function in four patients with chronic humoral rejection (CHR). Istituto Trapianti d’Organo SUPPRESSION OF ANTIDONOR ANTIBODY PRODUCTION BY MYCOPHENOLATE MOFETIL IN KIDNEY TRANSPLANTED PATIENTS A. Piazza, E. Poggi, O. Buonomo, F. Pisani, S. Servetti, C.U. Casciani , D. Adorno Transplant 2002 Meeting, Washington April 26-May 1 2002 Istituto Trapianti d’Organo Immunological and Clinical data * All before therapy switch ** All but two before therapy switch ** 50% 40% 30% 20% 10% 0% P=0.0281 * P=0.0005 MMF-switched-group Aza-group MMF-group Anti-donor antibodies ARj DFG Viral infections Graft failure Istituto Trapianti d’Organo PREDICTING KIDNEY GRAFT FAILURE BY HLA ANTIBODIES: A PROSPECTIVE TRIAL Paul I. Terasaki and Miyuki Ozawa Am J Transplant 2004; 4: 438 Istituto Trapianti d’Organo Istituto Trapianti d’Organo Istituto Trapianti d’Organo CONCLUSIONS (I) INCIDENCE AND ROLE OF ANTIBODY IN GRAFT INJURY Junchao Cai and Paul I. Terasaki Transplant. Reviews 2004, 18: 192-203 Istituto Trapianti d’Organo CONCLUSIONS (II) …. Antibody-mediated rejection in allograft recipients is a complicated pathological process…..not yet completely clear. …. The following factors may determine how fast rejection develops: 1. The level of donor-specific antibodies in blood….. 2. The capability of transplanted organ tissue repair……… this regeneration capability is tissue dependent. 3. Immunosuppressive and anticoagulation therapy…… Istituto Trapianti d’Organo CONCLUSIONS (III) …. Alloantibodies, either preexisting or de novo developed, are associated with hyperacute, acute, and chronic rejection. …..Post-transplantation antibody monitoring becomes extremely critical in transplant clinics, not only because it can help determine the extent of a patient’s humoral response to allograft but also, and perhaps more importantly, it will direct clinicians to optimize immunosuppressive therapy. Istituto Trapianti d’Organo