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T-regulatory Cells in Renal Ischemic injury
Alvaro Pacheco-Silva
Laboratory of Clinical and Experimental Immunology Division of Nephrology
Universidade Federal de São Paulo Hospital do Rim e Hipertensão
Hospital Israelita Albert EinsteinSão Paulo, Brasil
Ischemia and Reperfusion InjuryIschemia and Reperfusion Injury
Bonventre & Weinberg, JASN 14:2199-2210, 2003; Schrier et al, J Clin Invest 14(1):5-14, 2004
Acute Renal Failure (ARF) Delayed Graft Funciton (DGF)
Graft Rejection
Very complex
Incompletely
understood
Ischemic phase - blockade of blood influx, oxygen and
nutrients
Reperfusion phase – enhancement of tissue damage
Interaction between vasculature (endothelium), tubular
cells and incoming cells.
Ischemia/Reperfusion Injury
Oxygen deprivation Reactive oxygen species (ROS)
Cellular events Inflammatory response
Haemodynamic events
Cytoskeletal breakdown
Loss of polarityApoptosis and
necrosisDesquamation of
viable and necrotic cells
Tubular obstruction
Endothelial activationLeukocyte activation
and migration – neutrophils,
lymphocytes, macrophages
Cytokines, chemokines
Adhesion molecules
Vasoconstriction VasodilatationEndothelial and
smooth muscle cells structural damage
Endothelial-leukocyte adhesion, vascular obstruction
Immune response in IRIImmune response in IRI
Boros and Bromberg, Am J. Transplantation 2005
Impact of ischemia/Reperfusion
injuryAcute Rejection Renal Fibrosis
P 0,01
P 0,01
Discharge 6 months > 6 months0
5
10
15
20
25
30
35
40
45NO DGF
With DGF
Re
jec
tio
n
(%)
Burne-Tarne al. Kidney Int 2005.Ojo et al. Transplantation 1997.
Prediction of Clinical outcomes (DGF)Variables p valueDonor type <0,001CIT 0,005WIT 0,013TNFα <0,001CD25 <0,001TGF-β <0,001A20 <0,001IL-10 <0,001ICAM 0,006
CD4+T lymphocytes and IRI
A phase I trial of immunossupression with anti-ICAM-1 (CD54) mAb in renal allograft recipients. Haug C. et al. Transplantation 55(4):766-772, 1993.
Graft TreatmentPrimary Non
FunctionGraft Survival
Kidney BIRR1 (anti-ICAM) 0 78 %
Contralateral KidneyCurrent
immunosuppression3 56 %
N= 18 hight risk for delayed graft function patientsFollow up 16 to 30 months
CD4+T lymphocytes and IRI
A prospective, randomized, clinical trial of intraoperative versus postoperative Thymoglobulin in adult cadaveric renal transplant recipients.
Goggins WC. et al. Transplantation. 76(5):798-802, 2003
Post Op Days
Fig. 1 Fig. 2
CD4+T lymphocytes participates on IR injury
Identification of the CD4+T cell as a major pathogenic factor in ischemia acute renal failure. Burne MJ. Et al. J Clin Invest. 2001 Nov;108(9):1283-90.
a
Nu/nu mice
WT type
Nu/nu mice reconstituted wild-type T cells
WT type
CD4-/- reconstituted with wild-type T cells
CD4 -/-
b
Tregs and Innate immune response
Enhanced Regulatory T Cell Activity is an Element od the Host response to Injury Choileain NN. Et al. J immunol 2006 Jan 1;176(1):225-36
Regulatory T cells
Developmental classification for T Reg
Naturally arising T reg cells:
Constitutive expression at higher levels:Interleukin (IL)-2 receptor alpha chain (CD25) (Sakaguchi et al., 2004)
CTLA-4 (CD152) (Sakaguchi et al., 2004)
Glucocorticoid induced TNF receptor (GITR)(McHugh et al., 2002, Shimizu et al., 2002)
FOXP3 (Schubert and Ziegler et al, 2001, Fontenot et al, 2003; Hori et al, 2003)
Adaptative T regs:
Induced from naive T cells by antigenic stimulation and cytokine millieu
IL-10 (Roncarolo et al, 2006)
TGF-beta (Nakamura et al, 2004)
CD4+T lymphocytes in Renal Ischemia reperfusion model
Strategies
CD4+ T cells Post ischemic injury
CD4+CD25+Foxp3+
T CellsCD4+ effector T Cells
Post ischemic injury
?Decrease number
(Anti-CD25 depleting antibody)
Decrease suppressor activity(Anti-GITR)
Increase CD4+ effector T cells activity
PC61: depleting rat IgG1 anti-CD25 (alpha chain of IL-2R)
Choileain NN. Et al J. Immunol, 2006
DTA-1: agonist rat IgG2a anti-GITR (Glucocorticoid-induced TNF receptor)
DTA-1 mAb abrogates suppression mediated by CD25+CD4+ T cells breaking immunological self- tolerance
DTA-1 mAb abrogates suppression mediated by CD25+CD4+ T cells leading to development of autoimmune gastritis in mice
IR antibody treatmentProtocol
Day 0 Day 1 Day 2 (24 hs) Day 4 (72hs)
Sacrifice: Blood, kidneys, spleen, lymph nodes harvested
Treatment PC61 200 gDTA-1 400 gIgG 400 g Isquemia 45 min
Microclamps for renal artery and vein (renal pedicle)
cm
Background: C57 BL/6 male
IRI model adapted from KELLY et al., 1996
Reperfusion times : 24 and 72hs
Analyses: blood: creatinine and urea
LN, spleen: flow cytometry
Kidneys: morphometry,
Real Time PCR
Renal Ischemia Reperfusion Model
Effect of Antibody treatment in TCD4+CTLA4+Foxp3+ cells
IR antibody treatment
32,240,2
69,6
90,2
0
20
40
60
80
100
% d
ep
leti
on
SPl LN
Organ
Depletion of TCD4+CTLA4+FOXP3+ cells by PC61 treatment
IR24
IR72
14,0
7,6
13,2
50,1
0
10
20
30
40
50
60
% d
ep
leti
on
SPl LN
Organ
Effect of by by DTA-1 treatment in the TCD4+CTLA4+Foxp3+ population
IR24
IR72
DTA-1 Treatment PC61 Treatment
IR antibody treatment
Depletion of TCD4+CTLA4+Foxp3+ cells
12.6210.52 8.08
18.22 3.11 1.57
Spleen
pRenal LN
IgG PC61 IR 24 PC61 IR 72
IR antibody treatmentRenal Function Outcome
Blood Urea
0
20
40
60
80
100
120
140
160
180
200
24 72
Hours
Blo
od U
rea (
mg/dL)
I gGPC61DTA-1
weight loss after IR24 + antibody treatment
00,5
11,5
22,5
33,5
4
IgG PC61 DTA-1
Treatment
Wei
ght L
oss
(g)
P=0,0018
P=0,0002
weight loss after IR 72 + antibody treatment
0
1
2
3
4
5
6
7
IgG PC61 DTA-1
TreatmentW
eigh
t Lo
ss (
g)
P=0,0001
P=0,0033
* p< 0.001
Morphometric analysesAcute tubular necrosis and tubular regeneration
IR 24 hours
Morphmetric analyses IR 24 groups
0
10
20
30
40
50
60
70
80
IgG IR 24 PC61 IR 24 DTA-1 IR 24
Groups
Sco
re necrosis
regeneration
*
*
*
* p< 0.001
Morphometric analysesAcute tubular necrosis and tubular regeneration
IR 72 hoursMorhmetric analyses IR72 groups
0
10
20
30
40
50
60
70
IgG IR 72 PC61 IR 72 DTA-1 IR 72
Groups
Score necrosis
regeneration
*
*
*
Kidney Tissue HE. A, B, C, D: IR 24 hs. A: Sham, B: IgG treated, 400 micrograms C: PC61 treated, 200 micrograms. D: DTA-1 treated, 400 micrograms, E, F, G, H: IR 72 hs. E: Sham, F: IgG treated, 400 micrograms, G: PC61 treated 200 micrograms mg, H: DTA-1 treated, 400 microgams.
IR antibody treatment Histopathological analyses HE
I
Sham PC61 200 gIgG
72 H
24 H
DTA-1 400 g
A B C
GFE
D
H
IR antibody treatment Anti-Inflammatory Genes/TH2 response
GATA-3 Relative Expression PC61
00,5
11,5
22,5
33,5
4
NR IgG IR24
PC61IR 24
IgG IR72
PC61IR 72
Groups
Gata
-3
GATA-3 Relative ExpressionDTA-1
012345678
NR IgG IR24
DTA-1IR24
IgG IR72
DTA-1IR72
Groups
Gata
-3
HO-1 Relative Expression PC61
0
1
2
3
4
5
6
NR IgG IR24
PC61 IR24
IgG IR72
PC61 IR72
Groups
HO
-1
HO-1 Relative Expression DTA-1
05
10152025303540
NR IgG IR24
DTA-1IR24
IgG IR72
DTA-1IR72
Groups
HO
-1
IR antibody treatment Pro-Inflammatory Genes
I L6 Relative ExpressionPC61
0
1
2
3
4
5
NR IgG IR24
PC61 IR24
IgG IR72
PC61 IR72
Groups
IL6
I L6 Relative ExpressionDTA-1
00,20,40,60,8
11,21,4
NR IgG IR24
DTA-1IR24
IgG IR72
DTA-1IR72
Groups
IL6
I L-1b Relative ExpressionPC61
012345
NR IgGIR 24
PC61IR 24
IgGIR 72
PC61IR 72
Groups
IL-1
b
I L-1b Relative Expression DTA-1
0
5
10
15
20
NR I gG IR24
DTA-1I R24
I gG IR72
DTA-1I R72
Groups
IL-1
b
IR antibody treatment
Foxp3 Relative Expression PC61
0
1
2
3
4
5
NR I gGI R 24
PC61I R 24
I gGI R 72
PC61I R 72
Groups
Foxp3
TGFb Relative ExpressionDTA-1
01530456075
NR I gGI R 24
DTA-1
I R24
I gGI R 72
DTA-1
I R72
Groups
TG
Fb
TGFb Relative Expression PC61
01530456075
NR IgGIR24
PC61IR24
IgGIR72
PC61IR72
Groups
TG
Fb
Foxp3 Relative Expression DTA-1
0
2
4
6
8
10
NR IgGIR 24
DTA-1
IR24
IgGIR 72
DTA-1
IR72
Groups
Fo
xp
3
IR antibody treatment Conclusions and Perspectives
At 24 hours of reperfusion, depletion of TCD4+CTLA-4+Foxp3+ cells was 30,3% (spleen) and 67,8% (para renal lymphnodes).After 72 hours of reperfusion, depletion of TCD4+CTLA-4+Foxp3+ was 43,1% (spleen) and 90,22% (para renal lymphnodes). This depletion was efficient in generate significant responses in both 24 hours and 72 hours if reperfusion Depleted mice presented similar renal function to control animals at 24 hours, but 72 hours after IRI, PC61 treated mice presented significant worst renal function compared to the group that received IgG. DTA-1 treated animals presented significant protection at the same timepoint, indicating that different subsets of cells can be acting at these timepoints.
Furthermore, histopathological analyses showed that there was a pronounced incidence of necrosis for both PC61 treated and IgG in IR 24 hours experiments. On the other hand, in IR 72 hours experiments we observed a regeneration pattern in both PC61 and IgG treated animals, but in the PC61 treated group there was a significant necrosis index (p<0.001), comparing with IgG treated group, suggesting that TCD4+CTLA4+FOXP3+ cell population could be important in a late phase of injury recover.
It is known that the stress and tissue damage associated with IRI influence the development of a immune response to protect the tissue damage. Thus, our results suggests a role for TCD4+CTLA4+FOXP3+ cells (naturally arising T teg cells) in renal IRI experimental model.
IR antibody treatment Hypothesis
CD4+CD25+Foxp3+
T CellsCD4+ effector T Cells
Post ischemic injury
24 hs:TCD4+ effectorStimulated by DTA-1escape from T Reg suppression and make injury worst
Proinflamatory cytokinesProinflamatory cytokinesIL-1b and IL-6IL-1b and IL-6
72 hs:TRegs GITRhigh stimulated by DTA-1 start to suppressTCD4+ response
Anti inflamatory Anti inflamatory genesgenesSuch as HO-1 and Such as HO-1 and polarizationpolarizationtoward Th2 (GATA-3) toward Th2 (GATA-3) transcription factortranscription factor
72 hs:If there is no T Reg at this point (PC61 treatment), there is no recovery from injury
Persistence of necrosisPersistence of necrosis
Acknowledgements
Rebecca M. M. MonteiroMarcio J. DamiãoGiselle GonçalvesCarla Q. FeitozaMarcos CenedezeNephrology Division - Universidade Federal de São PauloBrazil
Prof. Dr.Mauricio M. RodriguesFanny TzelepisInterdisciplinary Center for Gene Therapy CINTERGENUniversidade Federal de São Paulo, Brazil
Prof. Dr.Niels Olsen S. CamaraImmunology Division Universidade de São Paulo USP, Brazil
Vicente de Paula A. TeixeiraMarlene A. dos ReisDepartment of Pathology, Universidade Federal de Uberaba, Minas Gerais, Brazil
Prof Dr. S. SakaguchiT. Yamaguchi (DTA-1)H. Uryu (PC61)K. Nagahama (IRI)M. Ono (Real Time PCR)Institute for Frontier Medical SciencesKyoto University Japan
