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Transplant Immunology

Transplant Immunology. Peter Nickerson Department of Immunology 11 Mar 2010. Y. Y. Transplant Immunology. Transplanted organ. Donor and recipient APCs migrate From the graft to LN. Y. Y. Effector T cell circulates. Y. Y. Naïve T cell. Activated T cell. Effector T cells

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Transplant Immunology

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  1. Transplant Immunology Peter Nickerson Department of Immunology 11 Mar 2010

  2. Y Y Transplant Immunology Transplanted organ Donor and recipient APCs migrate From the graft to LN Y Y Effector T cell circulates Y Y Naïve T cell Activated T cell Effector T cells and alloantibody destroy organ Activated antibody-producing B cell Naïve B cell Lymph Node or Spleen T and B cell activation

  3. MHC“the target”

  4. MHC II MHC I DP DO DM DQ C DR B A Major Histocompatibility Complex (Short Arm Chromosome 6) 21 61 28 # Serologically Defined Alleles (DRB1 loci) 293 464 229 # DNA Defined Alleles Class II Class I Polymorphic Residues a b b2m a HLA Genetics:

  5. HLA Disparity:Effect of Matching on Graft Survival % Graft Survival T1/2 13.4 11.3 9.3 T1/2* 13.0 8.9 8.3 Time Post-Transplant ( Opelz et al, Rev Immunogenetics (1999) 334 ) * (Terasaki, Clinical Transplants (2000) 497) (Takemoto et al, NEJM (2000) 1078)

  6. Y Y Transplant Immunology Transplanted organ Donor and recipient APCs migrate From the graft to LN Y Y Effector T cell circulates Y Y Naïve T cell Activated T cell Effector T cells and alloantibody destroy organ Activated antibody-producing B cell Naïve B cell Lymph Node or Spleen T and B cell activation

  7. MHC –PeptideRestriction of T-cells

  8. Figure 5-17

  9. MHC Alloimmune Responses

  10. Figure 5-18

  11. Th Rejection pathways (antigen recognition) Direct Allo-recognition Indirect Allo-recognition Recipient Th Recipient Th Th Donor MHC II with donor peptide Self MHC II with donor peptide Donor APC Self APC Donor MHC I with donor peptide Shed MHC I+II from donor tissue CTL Recipient CTL

  12. T cell activation occurs in secondary lymphoid organs Lakkis, F.G. et al. Nature Med. 6, 686-688 (2000).  Naïve alloreactive T cells cannot be activated in the absence of LN/spleen (aly/aly mice) and thus do not reject allografts. Primed T cells can reject grafts in the absence of secondary lymphoid organs Daniel Kreisel et al.Nature Medicine 8, 233 - 239 (2002) Endothelial cells can activate naïve alloreactive T cells. Zhou P, Woo Hwang K, Palucki D et al.Am J Transplant 2003; 3: 259-266. Graft rejection can occur in the absence of secondary lymphoid organs

  13. Costimulation is required to activate T cells

  14. Costimulatory molecules i.e CD28 and CD40L IL-2 IL-2R b g TCR complex ab TCR, CD3, CD4, etc a --CD25 (inducible) Calmodulin Kinase activities Apoptosis Calcineurin TOR Phosphatase activity G1 S NFAT AP-1 NFKB G2 IL-2 gene M Nucleus Proliferation and differentiation

  15. T cell differentiation CD62Llo CD44hi Circulates widely Naive T cell 24 h IL-2 CD62Lhi CD44lo Restricted to LN/spleen 2-4 days IFN IL-4 IL-5 GrB Cytotoxicity antigen ? Effector T cell IFN IL-4 IL-5 GrB Cytotoxicity < 24 h antigen Memory T cell

  16. Y Y Transplant Immunology Transplanted organ Donor and recipient APCs migrate From the graft to LN Y Y Effector T cell circulates Y Y Naïve T cell Activated T cell Effector T cells and alloantibody destroy organ Activated antibody-producing B cell Naïve B cell Lymph Node or Spleen T and B cell activation

  17. Activated T cells, Macrophages and B-cells are Associated with Allograft Rejection Phenotype CD 4 CD 8 CD 68 CD 20 Activation Marker CD 45RO+ CD 69 CD 25 AIF-1 Normal 345 ± 126 165 ± 44 316 ± 145 9 ± 3 180 ± 46 17 ± 6 0.8 ± 0.6 0.3 ± 0.2 Subclinical Rejection 803 ± 372 351 ± 102 626 ± 245 29 ± 9 824 ± 287 38 ± 11 2.5 ± 1.1 0.7 ± 0.5 Clinical Rejection 1222 ± 532 1454 ± 582 1438 ± 473 72 ± 29 1096 ± 491 37 ± 12 1.8 ± 1.1 5.9 ± 2.6 (Grimm et al, J Am Soc Nephrol (1999) 10:1582)

  18. Proinflammatory Cytokine Gene Transcriptsare Associated with Allograft Rejection Transcript TGFb IFNg IL-2 IL-15 Granzyme B Perforin Fas Ligand Normal 10% 10 0 10 0 0 20 Borderline 0% 36 0 45 9 9 27 Subclinical Rejection 0% 45 18 73 27 55 45 Clinical Rejection* 45% 75 8 83 91 83 83 (Lipman et al, Transplantation 1998;66:1673)(*Strehlau et al, PNAS 1997;94:695)

  19. IF/TA (n=10) IF/TA (n=10) IF/TA (n=10) Normal histology (n=24) Normal histology (n=24) Normal histology (n=24) Subclinical borderline tubulitis (n=15) Subclinical borderline tubulitis (n=15) Subclinical borderline tubulitis (n=15) Subclinical tubulitis Ia/Ib (n=22) Subclinical tubulitis Ia/Ib (n=22) Subclinical tubulitis Ia/Ib (n=22) Clinical tubulitis Ia/Ib (n=17) Clinical tubulitis Ia/Ib (n=17) Clinical tubulitis Ia/Ib (n=17) p<0.0001 p=0.06 p=0.54 p=0.24 p=0.82 A B 80 300 p=0.0001 250 p<0.0001 60 200 p=0.0002 p<0.0001 CXCL9 (ng/mmol) CXCL10 (ng/mmol) 40 150 p=0.03 p=0.02 p=0.03 100 p=0.06 20 p=0.02 50 p=0.01 0 0 C 40 30 CXCR3 Chemokines Associated with Rejection CXCL11 (ng/mmol) 20 10 0

  20. CTL Rejection pathways (overview) Antigen recognition Effector phase Proliferation Destruction FasL • Allorecognition • Direct (Donor APC) • Indirect (Recipient APC) IL-2 Granzyme B Perforin Cellular rejection IFNγ Th Th MØ Th Th Th APC IL-2 TNFα etc. IL-5 Tissue injury Fibrosis • Costimuli • B7:CD28 • CD40-CD40L • ICAM-LFA-1 etc. Eo M MBP IFNγ IL-4 IL-5 IL-13 Humoral rejection B-cell anti-HLA Ab  C-activation  ADCC

  21. Fc Receptor mediated binding (ADCC) Neutrophils Mǿ C’ Activation Platelet microthrombi anti-MHC-Ab C4a +C4b MAC C4d Rejection pathways (humoral rejection) Endothelium Donor MHC I/II Histology Neutrophils, Mǿ in vessels Microthrombi C4d staining in immunohistochemistry Effector pathways of humoral rejection Complement activation  cell lysis (necrosis) ADCC  cell death (necrosis/apoptosis)

  22. HLA A2 anti-HLA A2 Ab APC B IFNg Th IL-2 APC “Sensitizing Events” lead to B cell & T cell Memory Blood Transfusion Pregnancy T Prior Transplant

  23. Y Y Transplant Immunology Transplanted organ Donor and recipient APCs migrate From the graft to LN Y Y Effector T cell circulates Y Y Naïve T cell Activated T cell Effector T cells and alloantibody destroy organ Activated antibody-producing B cell Naïve B cell Lymph Node or Spleen T and B cell activation

  24. Overview • Where have we been …… …..how did we get here? • What is the problem facing us today? • Where do we need to go…… …..how might we get there? Disclaimer: Perspective nephrocentric is used as a model system of transplantation

  25. Part I • Where have we been …… …..how did we get here? • What is the problem facing us today? • Where do we need to go…… …..how might we get there?

  26. Cadaveric Renal Allograft Survival (A Clinical Perspective) • Radiation • Prednisone • 6-MP • CsA Emulsion • Tacrolimus • CY-A • MMF • OKT3 • Dicluzimab • Basiliximab • AZA • Thymoglobulin • Sirolimus • ATGAM ‘60 ‘65 ‘70 ‘75 ‘80 ‘85 ‘90 ‘95 ‘00 Year Adapted from Stewart F, Organ Transplantation, 1999

  27. Costimulatory molecules i.e CD28 and CD40L IL-2 mAb IL-2R mAb b g TCR complex ab TCR, CD3, CD4, etc a --CD25 (inducible) Calmodulin Kinase activities Apoptosis RAPA CsA/FK Calcineurin TOR Phosphatase activity G1 S NFAT AP-1 NFKB G2 IL-2 gene M PRED Imuran/MMF Nucleus Proliferation and differentiation

  28. CsA / Immuran / Pred n=93 57% CsA / MMF / Pred n=50 38% IL2mAb / CsA / MMF / Pred n=46 17% FK / MMF / Pred n=54 11% Manitoba ExperienceImpact of Drug Therapy on Early Rejection (<1 mo)in Primary Renal Allografts(June ’92 to June ’03)

  29. Cadaveric Renal Allograft Survival (A Lab Perspective) • Serologic Typing • DNA SSP Typing • ELISA Ab Screen • Flow Screening • Beads • CDC CXM • Flow Specificity • Beads • AHG CXM • Flow Single • Ag Beads • Flow CXM ‘60 ‘65 ‘70 ‘75 ‘80 ‘85 ‘90 ‘95 ‘00 Year

  30. Recipient Sera anti- Class I HLA Ab C’ MHC I MAC Donor T-cell Dye In Vitro Testing for Donor Reactive HLA antibodies CDC T-cell crossmatch

  31. Dilution anti-HLA Ab 1:1 1:2 1:4 1:8 1:16 1:32 AHGCDC 8 8 8 8 6 2 NIH CDC 8 2/4 1 1 1 1 In Vitro Testing for anti-Donor HLA antibodies AHG-CDC T-cell crossmatch Enhances Sensitivity Anti-Human IgG (AHG) Recipient Sera anti- Class I HLA Ab C’ MHC I MAC Donor T-cell

  32. Flow Crossmatch (FCXM) Recipient Sera anti- Class I HLA Ab Anti-Ig FITC MHC I Anti-CD3 PerCP Donor T-cell ( Garovoy et al, Tran Proc (1983) 15:2939)

  33. Transplantation Outcomes Based on Crossmatch TechniqueManitoba Experience (Primary Grafts) % Survival (6 mo) 89.1+4.6 98.6+1.7 Method AHG-CDC T-cell, CDC B-cell AHG-PRA FCXM T- and B-cell Flow bead Years 1993-1999 2000-2003 p<0.003 Note: Surgical causes of early graft loss excluded from analysis (i.e. vascular thrombosis)

  34. Purified HLA Antigens Solid Phase, Antigen Specific Assays Extract and Purify HLA Antigens EBV Transformed Cell Line HLA Transfected Cell Line Class I or II Phenotype or Individual Alleles Flow Cytometry Microparticles Luminex ELISA

  35. ELISA Anti-IgG Luminex Array Flow Cytometry HLA alloantibody Anti-IgG-PE Anti-IgG-FITC PRA = 78% Gebel and Bray. Transplantation Reviews 20: 189-194, 2006

  36. Independent Risk Factors Associated with Early Clinical Rejection (<1 month) Odds Ratio • Donor Reactive Antibody 10.81 (3.15 - 37.1) • HLA DR Mismatch • 2 DR mismatch 9.02 (3.20 – 25.4) • 1 DR mismatch 3.01 (1.80 - 5.04) • Treatment (vs. CsA / Aza / Pred) • CsA / MMF / Pred 0.46 (0.21 - 1.00) • IL-2 mAb / CsA / MMF / Pred 0.12 (0.05 - 0.30) • FK506 / MMF / Pred 0.10 (0.03 - 0.31)

  37. Deceased Donor Adult Kidney Transplants 1990-2008(Uncensored) IL2RmAb/CsA/MMF/Pred (n=38) CsA/MMF/Pred (n=63) Graft Survival CsA/Aza/Pred (n=170) FK/MMF/Pred (n=106) Years

  38. Deceased Donor Adult Kidney Transplants 1990-2008(Censor for Primary Non-Function, Moved, and Death) IL2RmAb/CsA/MMF/Pred (n=30) CsA/MMF/Pred (n=39) Graft Survival CsA/Aza/Pred (n=98) FK/MMF/Pred (n=96) Years

  39. Part I Where have we been….. • Trying to identify immune memory pre-transplant • Battling the immune response to the graft with HLA matching and immunosuppressive drugs ……how did we get here? • Constantly improving HLA antibody detection techniques • Constantly improving HLA typing techniques • Optimizing organ sharing to accommodate HLA matching • Developing more potent immunosuppressive agents/combinations

  40. Part II • Where have we been, and how did we get here? • Optimizing identification of immune memory pre-transplant • Optimizing HLA matching pre-transplant • Improving immunosuppressive drug therapy • What is the problem facing us today?

  41. Immunity still causes kidney graft failureEl-Zoghby et al AJT (2009) 9:527-535 1317 kidney transplants 1996-2006  318 grafts lost (25%) • Death with Function (138) 10.4% • Primary Non-Function (39) 2.9% • Graft Loss (153) 11.6% • Acute Rejection (11.8%) • Glomerular Pathology (36.6%) • Recurrent disease (15%) • Transplant glomerulopathy (15%) • De novo disease (6.6%) • IF/TA (30.7%) • Polyoma nephropathy (7.1%) • Recurrent rejection (8.5%) • Pyelonephritis (4.5%) • Poor quality donor (2.6%) • Medical/Surgical Conditions (16.3%) 35% Alloimmunity 21% Autoimmunity

  42. Case Study 37 year old female • Recipient of a LRD 3/6 MM graft • Flow Crossmatch negative, Flow Bead I / II = 0/0 • Rx: Simulect, Neoral, MMF, prednisone • No clinical rejections in the first 3 months post-transplant • Protocol Bx at 1 & 2 months (SCr: 1.0-1.2 mg/dL): Normal • Protocol Bx at 3 months (SCr: 1.1 mg/dL)

  43. 37 year old female • Subclinical rejection @ 3 month Rx corticosteroids • No clinical rejections between 3-6 months • 6 month serum creatinine: 1.0 mg/dL • Protocol Bx at 6 months

  44. 37 year old female • 6 month protocol Bx • Serum creatinine: 1.0 mg/dL (stable) • Acute tubulointerstitial rejection (Banff IB: g0,i3,t3,v0) • Chronic tubulointerstitial damage (CAN II: cg0,ci2,ct2,cv0)

  45. A B Determination of Interstitial Fibrosis Volume: Sirius Red Sirius Red-stained tissue (collagen I and III) is birrefringent under polarized light and the volume of interstitial fibrosis can be quantified by Image Analysis.

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