Morelli Lecture 2014
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This document summarizes the role of dendritic cells (DCs) in renal transplant rejection. It discusses how DCs from the donor that migrate to recipient secondary lymphoid organs after transplant present donor major histocompatibility complex (MHC) molecules to recipient T cells via direct and indirect pathways. It also describes how recipient DCs can present donor alloantigens via the indirect pathway. Finally, it mentions that regulatory DCs (DCregs) from the donor that are resistant to maturation can prolong cardiac allograft survival in mice when administered before transplant.
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Morelli Lecture 2014
- 1. Fisiologia de las celulas dendriticas en el rechazo de transplante renal Adrian E. Morelli, M.D., Ph.D. T.E. Starzl Transplantation Institute. Dept. of Surgery. University of Pittsburgh. Pittsburgh, PA. USA.
- 2. T.E. Starzl Transplantation Institute T.E. StarzlJ.E. SalkM.L. Menten
- 4. Que son las celulas dendriticas (CDs) ?
- 5. Caracteristicas de las celulas dendriticas Rare
- 6. Ubiquitous Caracteristicas de las celulas dendriticas Rare
- 7. Powerful Caracteristicas de las celulas dendriticas Rare Ubiquitous
- 8. Celulas dendriticas como reguladoras de linfocitos T Co-stimulation Cytokines (IL-4, IL-6, IL-12p70, IFN-γ, TGF-β1) Signal 1 Co-regulation Signal 3 Signal 2 MHC + peptide (↑ expression, ↑ affinity) Full activation Polarization (Th1, Th2, Th17, Tc1) Immunity T cell Effector + migratory function Co-stimulation Co-regulation Immune-suppression Tolerance T cell Regulatory T cells Cytokines (TGF-β1) Signal 2 Signal 1 Deficient activation/ anergy/apoptosis T cell Signal 3 Signal 2 Signal 1 MHC + peptide (↓ expression, ↓ affinity)
- 9. MHC + peptide MHC + peptide CD86 MHC + peptide CD86 Immature DCs (peripheral tissues) Semi mature/ Quiescent DCs (secondary lymphoid tissues) Mature/Activated DCs Steady-State Inflammation ImmunitySelf-tolerance Celulas dendriticas : Biosensores del sistema immune Microbial products (TLRs) Pro-inflammatory cytokines Necrotic/stressed cells Alarmins DAMPs Cytokines (TGF-β, IL-10, VEGF) Neuropeptides (POMC, ACTH, α-MSH, CGRP) Low UVB Apoptotic cells (ACAMPs) Tumor products
- 10. Morfologia de las celulas dendriticas Immature Mature
- 11. MHC-I MHC-II CD40 CD80 CD11b CD54 Fluorescence intensity (FITC) Numberofcells OX40 LigandCD86 CD11c Estadios de activacion / maduracion de las CDs
- 12. Las CDs activadas / maduras (CD86high ) priman linfocitos T virgenes
- 13. Conventional Dendritic cells Peripheral tissue-resident DCs Langerhans’ cells Dermal dendrocytes Interstitial DCs Migratory DCs Blood DCs Veiled cells (lymph) Lymphoid-tissue-resident DCs Marginal zone DCs T cell area DCs Thymic DCs Pre-Dendritic cells (pre-DCs) Pre-plasmacytoid DCs → Plasmacytoid DCs Monocytes → Myeloid DCs Inflammatory Dendritic cells Inflammatory monocytes → Inflammatory DCs K. Shortman & S. H. Naik. Nat. Rev. Immunol. &:19, 2007. Steady state Inflammatory or microbial stimuli Categorias de CDs
- 14. Linfocitos T (virgenes y de memoria) reconocen peptidos antigenicos dentro de moleculas del complejo mayor de histocompatibilidad CD4+ T cell MHC-II Effector/ memory CD4+ T cells Effector/ memory CD8+ T cells CD8+ T cell MHC-I Extracellular Ag (i.e. alloMHC) DC Intracellular Ag Effector/ memory CD8+ T cells Cross-presentation CD8+ T cellMHC-I
- 15. IL-6 MHC CD80/86 IL-12p70 + + Activated T cell CD154 + T cell activation/ proliferation Danger signals (i.e. TLR ligands) CD40 + T-cell Immunity T-cell homeostasis/tolerance IFN-γ + IFN-γR (CD119) CD80/86 ↑ IDO + CTLA4-Ig CD152 TReg cell FasL (CD178) + Deficient T cell activation T cell apoptosis/anergy Tryptophan catabolism Expansion/generation of TReg cells ? ↓ T cell proliferation ↑ T cell apoptosis Fas (CD95) MHC CD80/86 Plasticidad de las CDs
- 16. Post-transplant surgery Graft Parenchymal cells Donor DC Inducible (high) migration of mature DCs (passenger leukocytes) Signal 1: Allo-MHC + X-peptide Signal 2: High Frequency of responder T cells: high T cell proliferation: high T cells Direct pathway MHC + peptide Rol de las CDs en el rechazo de transplante •Uptake of necrotic cells •Vesicular exchange •Uptake of soluble Ag Signal 1: Self-MHC + allo-peptide Signal 2: High Frequency of responder T cells: low T cell proliferation: high Recipient DC precursors Recipient DC Th1/Th2 Indirect pathway MHC + peptide Semi-direct pathway
- 17. Antigen Transporting Cells: Uptake/transport of Ag from periphery to secondary lymphoid tissues. Link the “conserved” PRR of the innate immune system (i.e.TLRs, PAMPs) to the “variable” PRR of the adaptive immune system (TCR). Stimulation of naïve and memory T cells. Presentation of peptides derived from extra-cellular Ag to CD8+ T cells via MHC-I molecules (cross-presentation). CDs como celulas presentadoras de Ag profesionales
- 18. Celulas dendriticas renales TJ Soos, et al. Kidney Int 70:591-596, 2006
- 19. Modelo experimental de transplante cardiaco en el raton Corry RJ et al. Transplantation . 16 : 343-350, 1973
- 20. Pocas CDs del donante migran al bazo despues del transplante cardiaco 0 200 400 600 800 1000 1200 1400 1600 1 2 3 7 Donor cells per spleen Days after transplantation p < 0.05 N.D. 0 2 4 6 8 10 12 14 16 1 2 3 7 Donor cells per 106 splenocytes Days after transplantation p < 0.05 N.D.
- 21. Rol de las CDs del receptor en la generacion de DSA T T T B B Recipient DC Donor Alloantigen Plasma cell DSA Internalization and processing Donor Alloantigen
- 22. Sumario (I) • Las CDs inician la sensibilizacion contra Ags del MHC del donante. • El pacientes previamente sensibilizados, CPAs no profesionales (i.e. celulas B) son igualmente importantes. • Imediatamente despues del transplante, CDs del donante se activan y migran hacia los organos linfaticos secundarios del receptor. • En los organos linfaticos secundarios, CDs del donante presentan moleculas del MHC del donante (via directa) y transfieren moleculas intactas del MHC al las CPAs del receptor (via semi-directa) que son presentadas a los linfocitos T. • En los organos linfaticos secundarios, CDs del receptor presentan peptidos derivados de las moleculas del MHC del donante (via indirecta) a los linfocitos T.
- 23. Fisiologia de las celulas dendriticas reguladoras (CDreg) en la induccion de immunosupresion en el transplante renal Adrian E. Morelli, M.D., Ph.D. T.E. Starzl Transplantation Institute. Dept. of Surgery. University of Pittsburgh. Pittsburgh, PA. USA.
- 24. In vitro-generated DCreg (+/- pharmacological immunosuppression) • Immature DCs • Maturation-resistant DCs • Alternatively-activated DCs Donor-derived DCs Recipient-derived DCs + alloAg Donor-recipient DC-DC hybrids Graft Secondary lymphoid organ In vitro analysis: FACS, MLC Ex vivo analysis: MLC, ELISPOT Allograft survival: MST, Histopathology Vacunacion negativa con CDreg en el transplante X X ↓ Systemic anti-donor response Prolongation of allograft survival (I.v., day -7)
- 25. DCreg origin Type of DCreg Reference MST (days) Donor- derived DC IM-DC Fu F et al (Transplantation; 1996) 22 IM-DC (TGFβ-DC) Lu L et al (Transplantation; 1997) 30 MR-DC Lutz MB et al (Eur. J. Immunol.; 2000) 100 BM-DC-tgFasL Min W-P et al. (J. Immunol. 2000) 20 IM-DC (NF-κB ODN + Ad CTLA4Ig) Bonham CA (J. Immunol. 2002) 71 Splenic DC O’Connell PJ et al. (J. Immunol.; 2002) 20, 29, 26 IM-DC DePaz HA et al (Transplantation; 2003) 19 Alternative activated DC (Dexametasone) Emmer PM et al. (Transplantation; 2006) 20 Alternative activated DC (TGFβ1, IL-10, LPS) Lan YY et al. (J. Immunol.; 2006) 30 MR-DC (LF15-0195) Zhang X et al. (Clin. Immunol. 2008) @ 40 Recipient- derived DC IM-DC Xu DL et al. (Scand J. Immunol.; 2004) 36 MR-DC (Rapamycin) Tanner T et al. (Am. J. Transplant.; 2005) 24 IM-DC Peche H et al (Am. J. Transplant.; 2005) 23 MR-DC (Rapamycin) Turnquist HR et al. (J. Immunol.; 2007) 40 Sobrevida de transplantes cardiacos en ratones tratados exclusivamente con CDreg
- 26. DC Monocytes (humans, NHP) BM DC precursors (rodents) Cytokines, growth factors • ↓ GM-CSF • ↑ IL-10 (mammalian, viral) • ↑ TGF-β1 • ↑ VEGF Drugs, soluble mediators • Immunosuppressive/ anti-inflammatory drugs: Cyclosporine, rapamycin, tacrolimus, deoxyspergualin, mycophenolate mofetil, sanglifehrin A, costicosteroids, aspirin • Vitamins: 1α,25-dihydroxy-vitamin D3 • Antioxidants: N-acetyl-L-cysteine • Cyclic AMP inducers: prostaglandin E2, histamine, β2 agonists, neuropeptides • Glucosamine • Cobalt protoporphyrin • Ligands for ILT receptors (HLA-G) Genetic engineering • Recombinant viral vectors or naked DNA: FasL, CTLA-4Ig, IL-10, TGF-β1, IDO, soluble TNF-R, CCR7, dominant negative IκB kinase • Oligodeoxyribonucleotides (ODNs): NFkB-specific decoy ODNs • RNA interference: RelB, IL-12 ↓ MHC ↓ Costimulatory molecules ↓ IL-12p70 ↑ Functional IDO ↑ T cell death-inducing molecules (i.e. FasL) Regulatory DC Expansion of TReg cells X ↑ IL-10 ↑ TGFβ1 X ↑ Inhibitory molecules (i.e. PDL-1) ↓ Ag internalization and processing↓ NFκB ↓ DC maturation ↑ Migration to lymphoid organs X X ↑ CCR7 Produccion de DCreg in vitro A. Morelli & A. Thomson Nat. Rev. Immunol 7:610, 2007.
- 27. Generation in vitro de CDreg resistentes a la maduracion GM-CSF + IL-4 2 4 6 Media change + cytokines & 1α,25(OH)2 VD3 5 x 106 DCreg / 200 ml PBS / mouse / i.v. Day 7 day DCreg purification C57Bl/6 (B6) mouse (H2b ) BALB/c mouse (H2d )
- 28. Control-DCs CD80 CD86 CD40 DCreg CD40 DCs + DC-maturation cocktail (CpG + IL-1β + TNF-α + IFN-γ) DCs control-DCs control-DCs + DC maturation cocktail DCreg DCreg + DC maturation cocktail 640 320 160 80 40 20 10 5 0 10000 20000 30000 40000 50000 60000 # T cells : 1 DC [3H]TdRIncorporation(c.p.m.) 0 250 500 750 1000 1250 1500 control-DC MR-DC ND ND ND - DC1c - DC1c IL-12p70(pg/ml) CDreg del donante resistentes a la maduracion
- 29. 0 25 50 75 100 0 25 50 75 100 Non-treated controls BALB/c VD3-DC third party VD3-DC Days post-transplantation Percentsurvival i.v. injection (day -7) BALB/c DCreg Tx (day 0) BALB/c heart B6 recipient Allograft survival CDreg del donante prolongan la sobrevida del transplante de corazon en ratones
- 30. Non-treated Treated with DCreg CDreg del donante prolongan la sobrevida del transplante de corazon en ratones Days post-transplantation Cumulative graft survival 0 25 50 75 100 0 25 50 75 100 p < 0.05 p < 0.001 Non-treated (n =15) BALB/c (donor) DCref (n = 9) C3H (third-party) DCreg (n = 6) BALB/c (donor) DCreg(n = 5) B6 (syngeneic) DCreg (n = 4) S.J. Divito. Blood 116: 2694-2705, 2010
- 31. Como testear el uso terapeutico de CDreg en el transplante • Small animal models • Clinical trials • Non human primates
- 32. Administracion de CDreg del donante prolonga la sobrevida del transplante renal en primates no humanos M.B. Ezzelarab et al. Am J Transplant 13: 1989-2005, 2013
- 33. M.B. Ezzelarab et al. Am J Transplant 13: 1989-2005, 2013 Administracion de CDreg del donante prolonga la sobrevida del transplante renal en primates no humanos
- 34. Sobrevida de CDreg del donante en organos linfaticos secundarios BALB/c DCreg (MR-DC) (IgG2aa ) B6 (IgG2ab ) I.v. S15 (control) (1231bp) Spleen BALB/c IS-DC : B6 splenocyte ratio 1h 6h 24h - + - + 1:1 1:102 1:103 1:104 1:105 1:106 -NK1.1 Ab DNAladder Time after BALB/c IS-DC injection No M R-DCNo DNABALB/c M R-DC IgG2aa (BALB/c) (111 bp) DNAladder IgG2aa (BALB/c) (111 bp) S15 (control) (1231bp) S.J. Divito. Blood 116: 2694-2705, 2010
- 35. CDreg del donante son internalizadas por las CDs convencionales del receptor 6 24 48 0 10 20 30 hours post injection %PKH26+ eGFP+ cells Confocal eGFP- PKH26BALB/c DCreg (PKH26) B6 CD11c-eGFP I.v . BALB/c DCreg (CD45.2) B6 (CD45.1) I.v. S.J. Divito. Blood 116: 2694-2705, 2010
- 36. 2.6 83.3 12.9 3.1 1.6 -1 day -3 days -7 days -14 days Non-treated Injected (i.v.) with BALB/c DCreg CFSE Cellnumber BALB/c DCreg injection (i.v.) CFSE-labeled 1H3.1 CD4 T cells -14d -7d -3d -1d 0d +3d CFSE dilution (by FACS) Spleen CDreg del donante son re-procesadas por la CDs convencionales del en los organos linfaticos secundarios del receptor S.J. Divito. Blood 116: 2694-2705, 2010
- 37. En el transplante, las CDreg terapeuticas injectadas sistemicamente son: •CPAs tolerogenicas, como es considerado clasicamente? •O simplemente funcionan como Celulas Transportadoras de Ags, tranfiriendo Ag del donante al las CPAs del receptor, la cuales en condiciones normales mantienen tolerancia T periferica ?
- 38. Interaccion in vivo entre CDreg del donante y linfocitos T allo-reactivos B6 MHC-II -/- (Thy1.2) 24 h B6, IEα52-68 pulsed DCreg (i.v.) Presentation of IEα52-68-IAb to TCRtg CD4+ T cells B6 (Thy1.1) CFSE-labeled 1H3.1 TCRtg naïve CD4+ T cells specific for IEα52-68-IAb 1H3.1 CD4 Tcells in MHC class-IIKO-/- B6 hosts 41.61.71.6 9.9 Cellnumber CFSE B6 DCreg + IEα52-68No DC (5 x 106 ) (15 x 106 ) (5 x 106 ) B6 LPS-matured DC + IEα52-68 0 250 500 750 1000 1250 1500 NS NS # of 1H3.1 T cells per spleen (x 103 ) p < 0.05 No DCs 5 x 106 15 x 106 LPS-DCs DCreg S.J. Divito. Blood 116: 2694-2705, 2010
- 39. CDreg del donante son procesadas por CD convencionales del receptor en organos linfaticos secundarios BALB/c DCreg wt B6 CD11chi CD8α+ APC CD11chi CD8- APC CD11cint CD45RA+ APC CD11c- APC Spleen 20h 1H3.1 TCRtg CD4 T cells specific for IEα52-68-IAb FACS-sorting I.v. DC plus IEα52-68No APC CD11chi CD8- DC CD11chi CD8α+ DC CD11cint CD45RA+ Plasmacytoid DCCD11c- splenocytes 80.71.1 8.1 5.8 1.1 1.6 1.0 1.1 1.2 1.8 B6 injected i.v. with BALB/c DCreg B6 (non-treated) Thy1.1 CFSE S.J. Divito. Blood 116: 2694-2705, 2010
- 40. CDreg del donante promueve activacion deficiente y apoptosis de linfocitos T allo-reactivos (via indirect pathway) Spleen (day 3) BALB/c DCreg BALB/c DCreg + agonistic CD40 Ab B6 DCreg 0 8 1 91 51 3 45 1 7 3 87 3 2 6 0 2 40 2 57 1 89 5 5 1 CFSE CD69 CD62L 0 2 1 97 21 1 75 3 0.5 0.5 97 2 Annexin-V S.J. Divito. Blood 116: 2694-2705, 2010
- 41. CD11c Green fluorescence + DT Wt B6 CD11c DTR-eGFP B6 → wt B6 Tx (BALB/c heart) - 8 - 6 - 4 - 2 0 DT DT DT DT Monitoring graft survival Days: - 7 BALB/c DCreg Total body irradiation B6 CD11c-DTR-eGFP BM cells WT B6 8 weeks 8 weeks Modelo de deplecion transitoria de CD convencionales del receptor Z. Wang et al. Am J Transplant 12: 1398-1408, 2012
- 42. BALB/c → (CD11c-DTR-eGFP B6 → wt B6 ) chimera Cumulativegraftsurvival Days after transplantation p < 0.001 CD11c-DTR-eGFP B6 → wt B6 (n=5) wt B6 → wt B6 (n=5) CD11c-DTR-eGFP B6 → wt B6 (n=5) CD11c-DTR-eGFP B6 → wt B6 (n=5) wt B6 → wt B6 (n=8) CD11c-DTR-eGFP B6 → wt B6 (n=7) Recipient DC-therapy (BALB/c - DCreg) DT - + + - + + - - - + + + CD convencionles del receptor son clave para el efecto terapeutico de CDreg del donante Z. Wang et al. Am J Transplant 12: 1398-1408, 2012
- 44. Sumario (II) Targeting of recipient’s DCs Donor-derived DCreg Recipient-derived DCreg pulsed with donor-Ag before injection Donor leukocyte-derived vesicles (i.e. apoptotic cell vesicles, exosomes) Donor-Ag coupled to monoclonal Ab directed against DC marker Allograft Secondary lymphoid organ Recipient-derived DCreg not exposed to donor-Ag before injection I.V. administration of DCreg Immunoregulatory monoclonal Ab directed against DC marker Carryingdonor-AgWithoutdonor-Ag Carryingdonor-AgWithoutdonor-Ag ? ? ? ? ↑ indirect pathway CD4 Treg Indirect pathway T cells Indirect pathway T cell deletion Anti-donor B cells Allo-Ab Impaired CD4 T-B cell helpX Impaired activation of direct pathway T cells ? X X Quiescent conventional DC Donor-Ag transfer Cross-presentation to indirect pathway CD8 Treg Acquisition of donor-Ag ? Acquisition of donor-Ag ? A. Morelli & A Thomson. Curr. Opin. Organ Transpl (in press)
- 45. Problemas pendientes en vacunacion negativa con CDreg para generar tolerancia donante-specifica • Variante de CDreg generada in vitro • CDreg generadas de leucocitos de donante vs. receptor • Dosis de CDreg • Timing de administracion de CDreg (una vs. multiples dosis) • Es la administracion de CDreg realmente util? • Tipo de (sub-optimal) immunosupression farmacologica • Injeccion de CDreg vs. in situ-targeting of CD convencionales del receptor
- 46. Supported by grants from the NIH and the T.E. Starzl Transplantation Institute Dept. of Dermatology: Adriana T. Larregina, M.D., Ph.D. Geza Erdos, Ph.D. Olga Tkacheva, R.S. C.B.I. Dept. of Physiology & Cell Biology Donna Beer Stolz, Ph.D. Mara L.G. Sullivan, R.S. Katy Baty, Ph.D. Jenny M. Karlsson, Ph.D. Gregory Gibson, R. S. Kevin Alber, R.S. Simon C. Watkins, Ph.D. Acknowledgments Dept. of Surgery: Quan Liu, M.D. Darling M. Rojas, Ph.D. Angela Montecalvo, Ph.D. Sherrie J. Divito, M.D., Ph.D. William Shufesky, R.S. Andrea Gambotto, M.D., Ph.D. Kaori Okada, R.S.
Editor's Notes
- #2: Today I am presenting data from two projects, the first aimed at elucidating the in vivo mechanisms by which therapeutic DC prolong allograft survival, and the second at unearthing the role played by inflammatory DC in initiating/mediating allograft rejection
- #24: Today I am presenting data from two projects, the first aimed at elucidating the in vivo mechanisms by which therapeutic DC prolong allograft survival, and the second at unearthing the role played by inflammatory DC in initiating/mediating allograft rejection