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.
T.E. Starzl Transplantation Institute
T.E. Starzl J.E. Salk M.L. Menten
Que son las celulas dendriticas (CDs) ?
Caracteristicas de las celulas dendriticas
Rare
Ubiquitous
Caracteristicas de las celulas dendriticas
Rare
Powerful
Caracteristicas de las celulas dendriticas
Rare
Ubiquitous
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)
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 Immunity Self-tolerance
Celulas dendriticas : Biosensores del sistema immune
Microbial products (TLRs)Pro-inflammatory cytokinesNecrotic/stressed cells AlarminsDAMPs
Cytokines (TGF-β , IL-10, VEGF)Neuropeptides (POMC, ACTH, α-MSH, CGRP)Low UVBApoptotic cells (ACAMPs)Tumor products
Morfologia de las celulas dendriticas
Immature Mature
MHC-I MHC-II CD40
CD80 CD11b CD54
Fluorescence intensity (FITC)
Num
ber
of c
ells
OX40 LigandCD86
CD
11c
Estadios de activacion / maduracion de las CDs
Las CDs activadas / maduras (CD86high) priman linfocitos T virgenes
Conventional Dendritic cellsPeripheral 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 cellsInflammatory monocytes → Inflammatory DCs
K. Shortman & S. H. Naik. Nat. Rev. Immunol. &:19, 2007.
Steady state
Inflammatory or
microbial stimuli
Categorias de CDs
Linfocitos T (virgenes y de memoria) reconocen peptidos antigenicos dentro de moleculas del complejo mayor de histocompatibilidad
CD4+ T cell
MHC-II
Effector/ memoryCD4+ T cells
Effector/ memoryCD8+ T cells
CD8+ T cell
MHC-I
Extracellular Ag (i.e. alloMHC)
DC
Intracellular Ag
Effector/ memoryCD8+ T cells
Cross-presentation
CD8+ T cellMHC-I
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 activationT 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
Post-transplant surgery
Graft
Parenchymal cells
Donor DC
Inducible (high) migration of mature DCs
(passenger leukocytes)
Signal 1: Allo-MHC + X-peptideSignal 2: HighFrequency of responder T cells: highT 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-peptideSignal 2: HighFrequency of responder T cells: lowT cell proliferation: high
Recipient DC precursors
Recipient DC
Th1/Th2
Indirect pathway
MHC +peptide
Semi-direct pathway
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
Celulas dendriticas renales
TJ Soos, et al. Kidney Int 70:591-596, 2006
Modelo experimental de transplante cardiaco en el raton
Corry RJ et al. Transplantation . 16 : 343-350, 1973
Pocas CDs del donante migran al bazo despues del transplante cardiaco
0200
400
600
8001000
1200
1400
1600
1 2 3 7
Donor cellsper 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.
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
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.
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.
In vitro-generated DCreg(+/- pharmacological immunosuppression)• Immature DCs• Maturation-resistant DCs• Alternatively-activated DCs
Donor-derived DCs
Recipient-derived DCs+ alloAg
Donor-recipientDC-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)
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
DCMonocytes
(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.
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)
Control-DCs
CD80 CD86 CD40
DCreg
CD40
DCs + DC-maturation cocktail (CpG + IL-1β + TNF-α + IFN-γ)
DCs
control-DCscontrol-DCs + DC maturation cocktail
DCregDCreg + DC maturation cocktail
640320
160 80 40 20 10 50
10000
20000
30000
40000
50000
60000
# T cells : 1 DC
[3H
]TdR
Inco
rpor
atio
n (c
.p.m
.)
0
250
500
750
1000
1250
1500
control-DC MR-DC
ND ND ND
- DC1c - DC1c
IL-1
2p
70 (
pg
/ m
l)
CDreg del donante resistentes a la maduracion
0 25 50 75 1000
25
50
75
100 Non-treated controls
BALB/c VD3-DCthird party VD3-DC
Days post-transplantation
Per
cen
t su
rviv
al
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
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 1000
25
50
75
100
p < 0.05p < 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
Como testear el uso terapeutico de CDreg en el transplante
• Small animal models
• Clinical trials
• Non human primates
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
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
Sobrevida de CDreg del donante en organos linfaticos secundarios
BALB/c DCreg (MR-DC) (IgG2aa)
B6 (IgG2ab)
I.v.
S15 (control) (1231bp)
SpleenBALB/c IS-DC : B6 splenocyte ratio
1h 6h 24h
- + - +
1:1
1:10
2
1:10
3
1:10
41:
105
1:10
6
-NK1.1 Ab
DN
A la
dd
er
Time after BALB/c IS-DC injection
No
MR-D
C N
o DNA
BALB/c
MR-D
C
IgG2aa (BALB/c)(111 bp)
DN
A la
dder
IgG2aa (BALB/c)(111 bp)
S15 (control) (1231bp)
S.J. Divito. Blood 116: 2694-2705, 2010
CDreg del donante son internalizadas por las CDs convencionales del receptor
6 24 480
10
20
30
hours post injection
% P
KH
26+
eG
FP
+ cel
ls
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
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
Ce
ll n
um
ber
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
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 ?
Interaccion in vivo entre CDreg del donante y linfocitos T allo-reactivos
B6 MHC-II -/-
(Thy1.2)
24 h
B6, IEα52-68 pulsedDCreg (i.v.)
Presentation of IEα52-68-IAb
to TCRtg CD4+ T cellsB6 (Thy1.1)
CFSE-labeled1H3.1 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
Cel
l nu
mbe
r
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
perspleen (x 103)
p < 0.05
No DCs 5 x 106 15 x 106 LPS-DCs
DCreg
S.J. Divito. Blood 116: 2694-2705, 2010
CDreg del donante son procesadas por CD convencionales del receptor en organos linfaticos secundarios
BALB/c DCreg
wt B6
CD11chi CD8α+ APCCD11chi CD8- APCCD11cint CD45RA+ APCCD11c- APC
Spleen
20h
1H3.1 TCRtgCD4 T cells specific
for IEα52-68-IAb
FACS-sorting
I.v.
DC plus IEα52-68 No APC
CD11chiCD8- DC CD11chiCD8α+ DCCD11cintCD45RA+ 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)
Th
y1.1
CFSE S.J. Divito. Blood 116: 2694-2705, 2010
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
CD
69
CD
62
L
0 2
1 97
21 1
75 3
0.5 0.5
97 2
An
ne
xin
-V
S.J. Divito. Blood 116: 2694-2705, 2010
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
BALB/c → (CD11c-DTR-eGFP B6 → wt B6 ) chimera
Cum
ulat
ive
gra
ft s
urvi
val
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
Sumario (II)
Targeting of recipient’s DCs
Donor-derived DCreg
Recipient-derived DCregpulsed 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 DCregnot exposed to donor-Ag
before injection
I.V. administration of DCreg
Immunoregulatory monoclonal Ab directed against DC marker
Car
ryin
g d
on
or-
Ag
Wit
ho
ut
do
no
r-A
gC
arrying
do
no
r-Ag
With
ou
t do
no
r-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
XQuiescent
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)
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
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 BiologyDonna 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.