Morelli Lecture 2014

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


Rare

Powerful


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


CD8+ T cell

MHC-I

Extracellular Ag (i.e. alloMHC)

DC

Intracellular Ag


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


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


p < 0.001

CD11c-DTR-eGFP B6 → wt B6 (n=5)

wt B6 → wt B6 (n=5)



wt B6 → wt B6 (n=8)


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.

Health & Medicine

Morelli Lecture 2014