9/30/2016 Every cell expresses a HLA to present antigens to T lymphocytes In peripheral HLA class I Infection blood CD8 Perforine, Granzyme, Granulysin Cytotoxic T cell 23% The ABC’s of HLA: TCR Cell Beginners to Advanced CD4 IL2, IFN- γ Helper � Rajalingam Raja, Ph.D, D(ABHI) 48% T cell TCR � Professor of Clinical Surgery � Director of Immunogenetics and Transplantation Laboratory � University of California, San Francisco B cell � Phone: 415-476-0647 11% APC HLA � Email: Rajalingam.Raja@ucsf.edu HLA Antibody class II BCR 1 2 HLA is the Challenging Barrier to Transplantation Consequences of Pre-formed Donor-Specific HLA Antibodies HLA mismatched Recipient Induction Maintenance Allograft Therapy Therapy T T T T NK NK NK NK T T T T Immunosuppression B B B B Lymphocytes Depletion • Hyperacute rejection NK NK Anti-Thymoglobulin → T & NK cells Cyclosporine NK NK B B B B T T T T Anti-CD3 → T cells MMF NK NK NK NK T T T T Anti-CD25 → Activated T cells Steroids • Delayed graft function T T T T Anti-CD52 → mature lymphocytes B B B B Anti-CD20 → B cells • Accelerated acute rejection Rejection Lymphocytes Antibody Depletion • Chronic rejection Plasmaphoresis Antibody Blocking IVIG • Prolonged waiting times Plasma cell Anti-C5a Unacceptable Antigens HLA antibodies • No transplantation • Transplantation • Pregnancy • Transfusion 3 4 1
9/30/2016 Means of HLA Antibody-Mediated Rejection Histocompatibility Testing for Solid Organ Transplantation HLA DSA Class II 1. Activation of complement Recipient Donor cascade C1q Organ HLA Typing HLA Mismatch HLA Typing Allograft 2. Antibody-dependent HLA Antibodies Preformed-DSA Endothelium Cell-mediated Pre- cytotoxicity (ADCC) Transplant Cells Serum FcR Donor-Specific Crossmatch Compatibility HLA HLA Antibodies Class I NK Cell FcR Post- 4. Activation of Donor-specific HLA Antibodies 3. Opsonization & Endothelial Cell Transplant APC APC Antibodies (DSA) increased antigen presentation 5 6 Complement Dependent Cytotoxicity (CDC) Crossmatch Donor Recipient Paul Terasaki Lymphocytes Serum + Fluorescein Diacetate + Ethidium Bromide Complement Hyperacute or Functional Accelerated Graft CDC xM Rejection (n=225) Positive 24 6 (n=30) Specificity Problem Dead cells Live cells Negative 8 187 Membrane Attack (n=195) Complex (MAC) Negative Sensitivity Problem Positive 7 8 2
9/30/2016 Flow Cytometry Crossmatch Donor Recipient • Median Chanel Shift (MCS) – a quantitative readout (Ag+Ab) Detects only IgG antibodies • Non-specific reactivity can be • reduced by Pronase digestion Lymphocytes Serum + T cell B cell Negative Control Anti-human IgG F(ab’) 2 FITC Patient Measure FITC intensity Serum by flow cytometry T cell MCS > 50 B cell MCS > 120 9 10 Virtual Flow Cytometry Crossmatch Donor • Median Chanel Shift (MCS) – Recipient Flow Crossmatch - problems a quantitative readout (Ag+Ab) • Detects only IgG antibodies • Non-specific reactivity can be reduced by Pronase digestion • ~8% of flow crossmatches are + Lymphocytes Serum false positive – unneccessary B cell T cell exclusion Negative Control • ~7% of flow crossmatches are Anti-human IgG F(ab’) 2 FITC false negative – risk to patient Patient Measure FITC intensity Serum by flow cytometry T cell MCS > 50 B cell MCS > 120 11 3
9/30/2016 Virtual Crossmatch - Essentials Donor Recipient Crossmatch Methods Crossmatch Sensitivity Specificity Cost Turnaround method (US $) time Lymphocytes Serum CDC Low Low 600 3.5 hours HLA Antibody Testing Flow Intermediate Intermediate 600 5 hours Anti HLA-A2 antibodies Pronase >Intermediate >intermediate 600 6.5 hours Virtual Crossmatch Virtual 100% 100% 0 10 min Positive HLA Typing A2, A24, B7, B18, DR1, DR4 13 14 Virtual crossmatch by listing Virtual Crossmatch - Advantages Unacceptable Antigens in UNet • Eliminates the physical crossmatch - Saves 4-6 hours – cut downs cold ischemic time X - No samples required - Reduces laboratory & OPO workload - Reduces laboratory, OPO, and Tx program cost • Adds precision to actual crossmatch - CDC/flow XM prediction - DSA identification • Improves allocation efficiency • Increased rate of transplantation for sensitized patients • Risk of memory response can be accounted: Previous transplants & pregnancies 15 16 4
9/30/2016 Unacceptable HLA Antigens & Virtual Crossmatch Potential Donors, >12,000 A B DR DQ Candidate: 1 68 8 13 4 15 2 5 anti-A2 48% cPRA 2 24 7 18 1 10 5 5 2 29 13 51 8 14 4 8 + anti-DR4 61% cPRA 23 26 49 62 1 17 2 5 + anti-DQ5 76% cPRA 2 68 39 71 15 16 5 6 1 36 7 44 9 17 4 9 69 74 55 60 4 7 7 8 3 24 18 39 1 4 4 4 11 33 51 64 15 18 5 7 24 43 27 45 4 8 4 8 2 25 39 65 9 17 4 9 2 23 44 45 13 18 7 8 1 2 8 62 4 17 4 7 2 34 57 61 11 14 2 4 66 68 27 39 4 15 8 5 17 3 29 35 44 1 11 7 6 KAS Priority Points KAS: Major Allocation Components CPRA (%) Priority points for Old New 0–19 0 CPRA>19% 20–29 0 • Increase priority for sensitized 30–39 0 40–49 0 candidates/CPRA sliding scale 50–59 0 60–69 0 • Replace SCD/ECD with KDPI 70–74 0 75–79 0 • Add longevity matching 80–84 4 • Include pre ‐ registration dialysis time 85–89 4 90–94 4 • Incorporate A2/A2B to B 95 4 96 4 • Base pediatric priority on KDPI 97 4 98 4 • Remove payback system 99 4 Regional Sharing • Remove variances 100 4 National Sharing 19 20 5
9/30/2016 Sequence-Specific Oligonucleotide ( SSO ) Hybridization Method Distribution of CPRA scores in UCSF Kidney Transplant Waitlist (n=5461) 1.6% 0.8% 4% 5% Points #Patients Polystyrene Microspheres Polystyrene Microspheres CPRA 0-19% 0 3974 15.9% 20-84% 0.08-2.46 867 85-97% 4.05-17.3 273 24.4 46 98% 99% 50.09 85 100% 202.1 216 72.7% 21 22 Luminex: rSSO Method Luminex technology Cell A1 A2 Denatured Amplified PCR DNA products A11 A23 A3 DNA A2 A1 A2 A1 + A11 A23 A11 A23 * A3 A3 PCR amplification Hybridization using florochrome-tagged locus-specific primers Detection & Interpretation • 100 Color-coded Polystyrene beads using a blend of different fluorescent intensities of two dyes • Each bead is conjugated with oligonucleotide probe specific for a HLA allele (s) 23 24 6
9/30/2016 Single Antigen Bead-based HLA Antibody Testing: Luminex Technology Single HLA Antigen Beads A2 A66 B55 Polystyrene Polystyrene Microspheres Microspheres 25 26 HLA class I antibody test results: Antibodies to A2 CREG Single Antigen Bead-based HLA Antibody Testing: Luminex Technology patient’s serum Single Antigen beads • Specificities: A2, A68, A69, B57, B58 A2 A2 • CPRA: 62% + A66 B55 A66 • One Antibody B55 Detection & Interpretation 27 28 7
9/30/2016 α 1-domain α 2-domain Public and Private Epitopes (antigenic determinants) A2 A68 A69 B57 B58 A1 A36 Public Epitopes Private Epitopes 29 30 No antibodies to self-HLA are made. HLA class I antibody test results: Antibodies to A2 CREG Individuals alloimmunized by a specific HLA type can make antibodies to many HLA types. • Specificities: A2, A68, A69, B57, B58 • CPRA: 62% • One Antibody CPRA: 62% Allograft A1, A2, B7, B8 A23 A68 Anti-A2 A28 B57 A69 A24 B58 Recipient A1, A1, B7, B8 Cross-REactive groups (CREG) 31 32 8
9/30/2016 Bw6 Antibodies Cross-REactive Groups (CREG) CREG HLA Specificities CPRA value A B Cw DR DR DQ A1 A1,A3,A11,A29,A30,A31,A36,A80 65% A2 A2,A23,A24,A68,A69,B57,B58 75% 2 13(Bw4) 10 15 51 5 A10 A25,A26,A32,A33,A34,A43,A66,A74 22% 33 38(Bw4) 7 16 51 5 Bw4 A23,A24,A25,A32,Bw4 74% B5 B18,B35,B46,B49,B50,B51,B52,B53,B62,B63,B71,B72,B73,B75,B76,B77,B78 56% Bw6 Bw6 85% B7 B7,B8,B13,B27,B41,B42,B47,B48,B54,B55,B56,B59,B60,B61,B67,B81,B82 59% B8 B8,B18,B38,B39,B59,B64,B65,B67 36% B12 B13,B37,B41,B44,B45,B47,B49,B50,B60,B61 48% C1 Cw1,Cw7,Cw8,Cw9,Cw10,Cw12,Cw14,Cw16,B46,B73 77% C2 Cw2,Cw4,Cw5,Cw6,Cw15,Cw17,Cw18 66% DR1 DR1,DR10,DR103 21% DR51 DR51,DR15,DR16 29% DR52 DR52,DR11,DR12,DR13,DR14,DR17,DR18 62% DR53 DR53,DR4,DR7,DR9 50% DQ1 DQ5,DQ6 64% DQ2 DQ2 37% DQ3 DQ7,DQ8,DQ9 56% DQ4 DQ4 10% DP1c DP2,DP3,DP4,DP6,DP9,DP10,DP11,DP14,DP17,DP18.DP20,DP28 ---- CPRA=85% DP2c DP1,DP5,DP13,DP15,DP19,DP23 ---- 33 34 Women alloimmunized by Bw6 motif can make antibodies to 2/3 of HLA-B types Weak Bw6 Antibodies – Risk of memory response Spouse HLA A2-B61(Bw6)-DR4 A2-B39(Bw6)-DR4 B7, B8, B14, B18, B22, B35, B39, B40, B4005, B41, B42, B45, B46, B48, B50, B54, B55, B56, B60, B61, B62, B64, B65, B67, B70, B71, B72, B73, B75, B76, B78, B81, B82 Self HLA A2-B44(Bw4)-DR4 A2-B52(Bw4)-DR4 CPRA=85% 35 36 9
9/30/2016 Bw4 & Bw6 Antibodies Bw4 Antibodies A B Cw DR DR DQ 2 46 1 9 53 9 2 46 1 14 52 5 A B Cw DR DR DQ 2 35(Bw6) 4 4 53 8 31 35(Bw6) 4 11 52 7 CPRA=61% 37 38 Antibodies to all HLA except to self-HLA Allele-specific Antibodies 39 40 10
9/30/2016 HLA Antibody Report LABXpress™ Pipettor 41 Page-1 of 2 Page-2 of 2 HLA lab updates VXM & PXM qualification weekly VXM or PXM HLA Lab 43 44 11
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