Tregulatory Cells in Renal Ischemic injury

1
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 Einstein São Paulo, Brasil
2
Ischemia and Reperfusion Injury

• Ischemic phase - blockade of blood influx,
  oxygen and nutrients

• Reperfusion phase enhancement of tissue damage
• Interaction between vasculature (endothelium),
  tubular cells and incoming cells.

Bonventre Weinberg, JASN 142199-2210, 2003
Schrier et al, J Clin Invest 14(1)5-14, 2004
3
Ischemia/Reperfusion Injury
4
Immune response in IRI
Boros and Bromberg, Am J. Transplantation 2005
5
Impact of ischemia/Reperfusion injury
Acute Rejection
Renal Fibrosis
P ? 0,01
45
NO DGF
40
With DGF
35
30
P ? 0,01
25
Rejection ()
20
15
10
5
0
6 months
gt 6 months
Discharge
Burne-Tarne al. Kidney Int 2005.
Ojo et al. Transplantation 1997.
6
Prediction of Clinical outcomes
(DGF) Variables p value Donor type
lt0,001 CIT 0,005 WIT 0,013 TNFa lt0,001
CD25 lt0,001 TGF-ß lt0,001 A20 lt0,001 I
L-10 lt0,001 ICAM 0,006
7
CD4T 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.
N 18 hight risk for delayed graft function
patients Follow up 16 to 30 months
8
(No Transcript)
9
(No Transcript)
10
CD4T 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
11
CD4T lymphocytes participates on IR injury
Identification of the CD4T cell as a major
pathogenic factor in ischemia acute renal
failure. Burne MJ. Et al. J Clin Invest. 2001
Nov108(9)1283-90.
12
Strategies of treatment IRI
13
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 1176(1)225-36
14
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)
15
CD4T lymphocytes in Renal Ischemia reperfusion
modelStrategies
16
PC61 depleting rat IgG1 anti-CD25 (alpha chain
of IL-2R)
Choileain NN. Et al J. Immunol, 2006
17
DTA-1 agonist rat IgG2a anti-GITR
(Glucocorticoid-induced TNF receptor)
DTA-1 mAb abrogates suppression mediated by
CD25CD4 T cells breaking immunological self-
tolerance
18
DTA-1 mAb abrogates suppression mediated by
CD25CD4 T cells leading to development of
autoimmune gastritis in mice
19
IR antibody treatment Protocol
20
Renal Ischemia Reperfusion Model
Reperfusion times 24 and 72hs Analyses blood
creatinine and urea LN, spleen flow cytometry
Kidneys morphometry, Real Time PCR
21
IR antibody treatment
Effect of Antibody treatment in
TCD4CTLA4Foxp3 cells
PC61 Treatment
DTA-1 Treatment
22
IR antibody treatment
Depletion of TCD4CTLA4Foxp3 cells
23
IR antibody treatment Renal Function Outcome
24
Morphometric analyses Acute tubular necrosis and
tubular regeneration
IR 24 hours
plt 0.001
25
Morphometric analyses Acute tubular necrosis and
tubular regeneration
IR 72 hours
plt 0.001
26
IR antibody treatment Histopathological
analyses HE

I
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.
27
IR antibody treatment Anti-Inflammatory
Genes/TH2 response
28
IR antibody treatment Pro-Inflammatory Genes
29
IR antibody treatment
30
IR antibody treatment Conclusions and
Perspectives
At 24 hours of reperfusion, depletion of
TCD4CTLA-4Foxp3 cells was 30,3 (spleen) and
67,8 (para renal lymphnodes).After 72 hours of
reperfusion, depletion of TCD4CTLA-4Foxp3 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 (plt0.001), comparing
with IgG treated group, suggesting that
TCD4CTLA4FOXP3 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
TCD4CTLA4FOXP3 cells (naturally arising T teg
cells) in renal IRI experimental model.
31
IR antibody treatment Hypothesis
72 hs TRegs GITRhigh stimulated by DTA-1 start
to suppress TCD4 response
32
IR antibody treatment Perspectives
33
(No Transcript)
34
Acknowledgements
Rebecca M. M. Monteiro Marcio J. Damião Giselle
Gonçalves Carla Q. Feitoza Marcos
Cenedeze Nephrology Division - Universidade
Federal de São Paulo Brazil Prof. Dr.Mauricio
M. Rodrigues Fanny Tzelepis Interdisciplinary
Center for Gene Therapy CINTERGEN Universidade
Federal de São Paulo, Brazil Prof. Dr.Niels
Olsen S. Camara Immunology Division Universidade
de São Paulo USP, Brazil
Vicente de Paula A. Teixeira Marlene A. dos
Reis Department of Pathology, Universidade
Federal de Uberaba, Minas Gerais, Brazil Prof
Dr. S. Sakaguchi T. Yamaguchi (DTA-1) H. Uryu
(PC61) K. Nagahama (IRI) M. Ono (Real Time
PCR) Institute for Frontier Medical
Sciences Kyoto University Japan
35
ã