"Novel  role  for  galec-n-­‐1  in  

CTL  killing"  Gustavo  P.  Amarante-­‐Mendes  

UofT-­‐USP  Oncology  Conference  November  2013  

MOLECULAR  CELL  BIOLOGY  LABORATORY  DEPARTMENT  OF  IMMUNOLOGY  

INSTITUTE  OF  BIOMEDICAL  SCIENCES  UNIVERSITY  OF  SÃO  PAULO  

Ins-tute  for  Inves-ga-on  in  Immunology  Na-onal  Ins-tutes  for  Science  and  Technology  (INCT)  

RESEARCH  INTERESTS  

Molecular  Control  of  Cell  Death  

Cell  Death  Signaling  in  Cancer  

Cell  Death  in  the  Immune  System  

Cell  Death  Signaling  in  infec-ons  

TRAIL

RESEARCH  INTERESTS  

Molecular  Control  of  Cell  Death  

Cell  Death  Signaling  in  Cancer  

Cell  Death  in  the  Immune  System  

Cell  Death  Signaling  in  infec-ons  

Cytotoxic  cell  killing  

Proteomic  analysis  of  cytotoxic  granules  

5 8 pI

42

3

6

7

8

10 11

12

14

13

31

18

26 25

28

21

22

16 17

19 20

27

5

29

30

9

23

15

24

1

10%

SD

S-P

AG

E

Histone  H2B  40S  ribosomal  protein  S16  

Histone  H2A  Profilin  I  Myosin  light  chain  alkali  (MLC3nm)  Cysta-n  C  

Galec1n-­‐1  

Cofilin  40s  ribosomal  protein  S10  

Ubiqui-n  carboxyl-­‐terminal  hydrolase  isozyme  L1  (UCH-­‐L1)  Cyclophilin  B  

Cyclophilin  A  superoxide  dismutase  [Cu-­‐Zn]  

Peroxiredoxin  1  Adenine  phosphoribosyltransferase/APRT  

Ferri-n  light  chain  1  

60S  ribosomal  protein  L9  Rab-­‐2A  

Transla-onally  controlled  tumor  protein  (TCTP)  

Granzyme  C  

Granzyme  A  Granzyme  B  Granzyme  E  Granzyme  C  

Alpha  enolase    

Granzyme  A  Granzyme  E  Granzyme  G  Granzyme  F  Granzyme  B  Purine  nucleoside  phosphorylase  Phosphomannomutase  2  DNAse  gamma/DHP2/LS-­‐DNAse/DNAse  I-­‐like  3  Ras-­‐related  Rab-­‐17  

Protein  dissulfide  isomerase  A3  precursor  (ERp60)    

Phosphoglycerate  kinase  1  Calneuron  1  

Alpha  enolase  

Pyruvate  kinase,  M2  isozyme  Protein  dissulfide  isomerase  A3  precursor  (ERp60)  T-­‐complex  protein  1  (TCP-­‐1-­‐teta)    

Perforin  Zinc  finger  protein  94  (Zfp-­‐94)  

Heat  shock  cognate  71kDa  protein  78kDa  glucose-­‐regulated  protein  precursor  (BIP)  Heat  shock  cognate  71kDa  protein  

NEDD1  protein  

Heat  shock  protein  HSP90-­‐beta  (TSTA)  

Perforin  

5 8 pI

4 2

3

6 7

8

10 11 12

14

13

31

18

26 25

28

21 22

16 17

19 20

27

5

29 30

9

23

15

24

1

10%

SD

S-P

AG

E

1D  and  2D  Gel-­‐Analysis  

Galec-ns  •  β-­‐galactoside  binding  lec-ns.  •  Evolu-onarily  conserved.  •  15  mammalian  galec-ns  iden-fied.  

•  Wide  -ssue  distribu-on.  

•  Have  at   least  one  conserved  carbohydrate  recogni-on  domain  (CRD)   of   approximately   130   amino   acids   that   mediates  carbohydrate  binding.  

•  Involved   in   several   biological   processes,   such   as   T   cell  homeostasis,  cell  signaling,  prolifera-on  and  apoptosis.  

Galec-ns  

(Rabinovich  &  Toscano  –  Nat.  Rev.  Immunol,2009)  

Galectin-1 modulates the early stages of T cell activation by limiting the "membrane raft" coalescence and promote incomplete TCR ζ chain phosphorylation allowing only the response of T cells that require partial TCR signaling.

Galectin-1 expression decreases the TCR avidity and regulates the in vivo TCD8+ cells expansion. It is secreted by activated TCD8+ effector cells.

Galec-n-­‐1  

Valida-on  by  Electron  and  Confocal  Microscopy  

Gal-­‐1   Granzyme  B   Hoechst  

Gal-­‐1  Perforin  Hoechst  

LS  

NL  

NL  

LS  

In  vivo  cytotoxicity  assay  

Experimental  Design  and  Results  

Immuniza-on  with  Ad5βgal  or  Ad5ASP2  

Day  0  

i.v.  injec-on  of  syngeneic  splenocytes  labeled  with  1mM  

(non  target)  or  10mM  (target)  CFSE  (1:1  ra-o)    

Day  7  

Spleen  were  analyzed  by  Flow  

Cytometry  

Day  8  

Target  cells  are  pulsed  with  cognate  

pep-de

An-gen-­‐pulsed  target  cells  are  specifically  killed  in  vivo  

In  vivo  elimina-on  of  specific  targets  depends  on  the  presence  of  CD8  T  cells  

Gal-­‐1KO  mice  are  less  efficient  at  killing  pep-de-­‐pulsed  target  cells  than  WT  mice  

Med

ium  

ICP  

Wild  Type   Gal-­‐1  KO  non  immunized   non  immunized  immunized   immunized  

Gal-­‐1KO  mice  displayed  a  higher  frequency  of  an-gen-­‐specific  T  cells  compared  to  WT  mice.  

Gal-­‐1  +/+ Gal-­‐1  -­‐/-­‐

Gal-­‐1  +/+ Gal-­‐1  -­‐/-­‐

A.

B.

CD8  T  cells  from  Gal-­‐1KO  mice  degranulates  at  least  as  well  as  WT  T  cells  

11.3  

B.  

TCD8-­‐mediated  killing  

Cellular and Molecular Immunology, 6th Edition

Target cell Apoptosis

Apoptosis

perforin and granzymes release

Granzymes

FAS-FASL interaction

Gal-­‐1KO  CD8  T  cells  are  as  efficient  at  killing  pep-de-­‐pulsed  FAS-­‐deficient  target  cells  

v v v v

Working  hypothesis  

Galec-n-­‐1

ACKNOWLEDGMENTS  

Molecular  Cell  Lab  Departamento  de  Imunologia  (ICB-­‐USP)  

Tiago  C.  Machado  Narciso  Vieira  Junior  

 Trinity  College  Dublin  (Ireland)  

Seamus  J.  Mar-n  Colin  Adrain  

Alexander  Luthi    

Universidade  Federal  de  São  Paulo  (UNIFESP)  

Mariana  Dominguez  Mauricio  Mar-ns  Rodrigues  

 

Faculdade  de  Medicina  Ribeirão  Preto  (USP)  

Marcelo  Dias  Baruffi  

 Université  de  Lausanne  

Jürg  Tschopp    

Ins-tut  Curie  -­‐  CNRS  (France)  

Sebas-an  Amigorena