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.

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.