The Regulation of Interleukin 7 Receptor Alpha Internalization, Recycling and Degradation by IL-7

Universidade de Lisboa

Faculdade de Medicina

Unidade de Biologia do Cancro, Instituto de Medicina Molecular

The regulation of Interleukin 7 receptor alpha internalization, recycling and degradation by IL-7

- Possible implications in T-cell homeostasis,

migration and leukaemogenesis -

Catarina Martins de Oliveira Henriques

Doutoramento em Ciências Biomédicas

For the degree of Doctor of Philosophy

2009

Universidade de Lisboa Faculdade de Medicina

Unidade de Biologia do Cancro, Instituto de Medicina Molecular

The regulation of Interleukin 7 receptor alpha internalization, recycling and degradation by IL-7

- Possible implications in T-cell homeostasis,

migration and leukaemogenesis -

Catarina Martins de Oliveira Henriques

(Recipient of a scholarship- SFRH7BD/21940/2005 from Fundação para a Ciência e Tecnologia)

Tese orientada pelo Doutor João T. Barata, Prof Doutor.Gerard Graham e Prof. Doutora Leonor Parreira

Doutoramento em Ciências Biomédicas, especialidade em Ciências Biopatológicas

For the degree of Doctor of Philosophy 2009

As opiniões expressas são da exclusiva responsabilidade do seu autor

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Para a Prof. Filomena Mota. Sem o seu apoio e inspiração há muitos anos atrás, eu não seria hoje uma bióloga nem esta tese teria alguma vez existido…

Table of Contents

Table of contents……………………………………………………….…………………...... Aknowledgements…………………………………………………………………………………………….. Abbreviation list………………………………………………………………………………………...... Abstract…………………………………………………………………………………………………...... Resumo…………………………………………………………………………………………………………......

Chapter 1: General Introduction …………………………………………………………...... 1

1.1 Signalling at the cell surface and beyond …...………………...... 3

● The Eukaryotic cell…………………………………………………………....…...... 3

● Receptor-mediated endocytosis, recycling and degradation...... 6

● Receptor targeting and sorting………….…………………………………...... 11

● Receptor trafficking and signalling……………………………...... ……………13

1.2 The immune system and T-cell migration …………………...... ….14

● T-cell polarization and motility………………………………………...... ……….15

● Choosing the right direction………………………………………...... ……………16

● T-cell homing……………………..………………………………………...... ……….….22

● The adhesion cascade……………………………………………………...... ………24

◦ Rolling……………………………………………………………….…...... ……….25

◦ Activation and arrest………………….…….…………...... …………….27

◦ Trans-endothelial migration………………………………...... …………28

1.3 T-cell homeostasis: The role of Interleukin 7………...... …….38

1.4. IL-7 and T-cell acute lymphoblastic leukaemia: Proliferation, survival, growth and dissemination ?...... 37

1.5 Thesis overview …………………………………………………………………………….41

Chapter 2: Assessing IL-7 mediated T-cell motility and migration in vitro ……………………………………………………………………………………………………………….….43

2.1 Abstract…………………………………………………………………………………………..45

2.2 Introduction…………………………………………………………….....……………..…45

2.3 Materials and Methods …….....………………………..………….……………….48

2.4 Results …………………………………………………..………………….....……………….50

2.4.1 IL-7 stimulation activates T-ALL cell motility in vitro ………50

2.4.2 IL-7 attracts and retains T-ALL cells in vitro …………….….…52

2.4.3 IL-7 induces transient up-regulation of key adhesion molecules and CCR5 chemokine receptor in T-ALL cells……...... …….54

2.5 Discussion ……………………………………………………………………...... ……….…56

Chapter 3: Regulation of IL-7Rα internalization, recycling and degradation by IL-7…………………………………………………….…………………..…….…….63

3.1 Abstract …………………………………………………………………………………………..65

3.2 Introduction …………………………………………….....………………………………..65

3.3 Material & Methods……………………………….....…………………………………67

3.4 Results ……………………………………………………….....………………………..…….72

3.4.1 IL-7 induces rapid IL-7Rα internalization in T-cells…………72 3.4.2 IL-7Rα internalization occurs via clathrin-coated pits and is required for IL-7-mediated signalling……………………………………...... …76

3.4.3 A pool of internalized IL-7Rα constitutively recycles back to the cell surface……………………………………..…………………………...... ………………80

3.4.4 IL-7 induces rapid IL-7Rα degradation in a lysosome-dependent manner……………………………………………….…………………...... ………….....…..…82

3.4.5 IL-7-induced IL-7Rα degradation, but not internalization, is partially dependent on JAK3 activity...... 85

3.5 Discussion ...... 87

Chapter 4: General discussion and perspectives ……………………...... ….……91

References …………………………………………...... ………………………………………………..…107

Aknowledgements

Abbreviation list

Abstract

Resumo

in vitro.

Chapter 1: General Introduction

General Introduction

1.1 Signalling at the cell surface and beyond

The Eukaryotic cell

General Introduction

c c c . Adapted from Hausman (2006).

General Introduction

Receptor-mediated endocytosis, recycling and degradation

General Introduction

c c

General Introduction

Receptor targeting and sorting

General Introduction

Receptor trafficking and signalling

General Introduction

Drosophila

1.2 The immune system and T-cell migration

General Introduction

T-cell polarization and motility

General Introduction

Choosing the right direction

General Introduction

discoideum in vitro D. discoideum

General Introduction

A B

General Introduction

Adapted from Barber and Welch (2006). in vitro in vivo

General Introduction

c Adapted from Hogg, Laschingner et al. (2003)

General Introduction

ccc

General Introduction

Adapted from Ladi, Yin et al. (2006). T-cell homing

General Introduction

c c

Adapted from Kruger and Mooren (2007).

General Introduction

The adhesion cascade

ccc

General Introduction

Adapted from Ley, laudanna et al. (2007).

Rolling `

General Introduction

Adhesion molecule Distribution Binding partner c c c c c c Adapted from Smith (2008).

General Introduction

Activation and Arrest

General Introduction

Adapted from Ley, Laudanna et al. (2007). Trans-endothelial migration

General Introduction

Adapted from Ley, laudanna et al. (2007).

1.3 T-cell homeostasis: The role of Interleukin-7

General Introduction

Receptor Level of expression: chain Naïve T-cells Effector T-cells Memory T-cells γc IL-2Rα IL2-Rβ IL-4Rα IL-7Rα IL-15Rα IL-21R c c c c Adapted from Rochman, Spolski et al. (2009).

General Introduction

ccc Adapted from Rochman, Spolski et al. (2009).

General Introduction

IL-7RA

General Introduction

. `

General Introduction

Adapted from (Barata, Cardoso et al. 2005) 1.4 IL-7 and T-cell acute lymphoblastic leukaemia: Proliferation, survival, growth and dissemination?

General Introduction

γ in vitro

General Introduction

1.5 Thesis overview in vitro

Chapter 2: Assessing IL-7- mediated T-cell motility and migration in vitro

Assessing IL-7 mediated T-cell mobility and migration in vitro

2.1 Abstract

2.2 Introduction

Assessing IL-7 mediated T-cell mobility and migration in vitro per se. Pseudomonas

Assessing IL-7 mediated T-cell mobility and migration in vitro in vivo . in vitro

Assessing IL-7 mediated T-cell mobility and migration in vitro in vivo

2.3 Materials & Methods

c cc

Assessing IL-7 mediated T-cell mobility and migration in vitro

cc α ( − c

Assessing IL-7 mediated T-cell mobility and migration in vitro 2.4 Results

2.4.1 IL-7 stimulation activates T-ALL cell motility in vitro

in vitro ´` in vitro

Assessing IL-7 mediated T-cell mobility and migration in vitro

c c versus versus

Assessing IL-7 mediated T-cell mobility and migration in vitro 2.4.2 IL-7 attracts and retains T-ALL cells in vitro

c c c

Assessing IL-7 mediated T-cell mobility and migration in vitro versus cccc

Assessing IL-7 mediated T-cell mobility and migration in vitro c c c c 2.4.3 IL-7 induces transient up-regulation of key adhesion molecules and CCR5 chemokine receptor

Adhesion CD11α CD18 CD29 CD44 CD49a CD49e CD49f CD50 CD58 CD62L Molecules Chemokine CCR5 CXCR3 CXCR4 CXCR5 Receptors ccc c

Assessing IL-7 mediated T-cell mobility and migration in vitro

cc α,

Assessing IL-7 mediated T-cell mobility and migration in vitro

c c

2.5 Discussion

Assessing IL-7 mediated T-cell mobility and migration in vitro ´`

Assessing IL-7 mediated T-cell mobility and migration in vitro α ; , ,

Assessing IL-7 mediated T-cell mobility and migration in vitro in vivo in vivo . in vitro our in-vitro

Assessing IL-7 mediated T-cell mobility and migration in vitro cc

Assessing IL-7 mediated T-cell mobility and migration in vitro

Chapter 3: Regulation of IL-7Rα internalization, recycling and degradation by IL-7

cc Blood.

Regulation of IL-7Rα internalization, recycling and degradation by IL-7

3.1 Abstract IL-7RA

3.2 Introduction

in vitro

Regulation of IL-7Rα internalization, recycling and degradation by IL-7

Regulation of IL-7Rα internalization, recycling and degradation by IL-7 IL-7RA 3.3 Materials & Methods c

Regulation of IL-7Rα internalization, recycling and degradation by IL-7

cccc

Regulation of IL-7Rα internalization, recycling and degradation by IL-7 cc

Regulation of IL-7Rα internalization, recycling and degradation by IL-7 c cc cc

Regulation of IL-7Rα internalization, recycling and degradation by IL-7 (50mM Tris Ph 7.6-8 ; 150 mM NaCl; 5mM EDTA; 1mM NaOVa; 10mM NaF; 10mM Napyrophosphate; 1% NP-40; 10 ηg/ml Aprotinin; 1µg/ml Pepstatin and 10 µg/ml of Leupeptin (Sigma) in water) 10mM Tris-HCL and 0.15M NaCl (Sigma), pH 8.0) with 0.05% of Tween-20 c

Regulation of IL-7Rα internalization, recycling and degradation by IL-7 3.4 Results

3.4.1 IL-7 induces rapid IL-7Rα internalization in T-cells

c c α α α

Regulation of IL-7Rα internalization, recycling and degradation by IL-7

c c α α

Regulation of IL-7Rα internalization, recycling and degradation by IL-7 IL -7 (ng/ml) c c

# Cells α α α

Regulation of IL-7Rα internalization, recycling and degradation by IL-7

c α α

Regulation of IL-7Rα internalization, recycling and degradation by IL-7 3.4.2 IL-7Rα internalization occurs via clathrin-coated pits and is required for IL-7-mediated signalling

Regulation of IL-7Rα internalization, recycling and degradation by IL-7

cccc

Regulation of IL-7Rα internalization, recycling and degradation by IL-7

Regulation of IL-7Rα internalization, recycling and degradation by IL-7 cc c

Regulation of IL-7Rα internalization, recycling and degradation by IL-7 3.4.3 A pool of internalized IL-7Rα constitutively recycles back to the cell surface

Regulation of IL-7Rα internalization, recycling and degradation by IL-7

c cc c c

Regulation of IL-7Rα internalization, recycling and degradation by IL-7 3.4.4 IL-7 induces rapid IL-7Rα degradation in a lysosome and proteasome- dependent manner.

Regulation of IL-7Rα internalization, recycling and degradation by IL-7

c cc versus versus

Regulation of IL-7Rα internalization, recycling and degradation by IL-7 c

Regulation of IL-7Rα internalization, recycling and degradation by IL-7 cccc 3.4.5 IL-7-induced IL-7Rα degradation, but not internalization, is partially dependent on JAK3 activity

Regulation of IL-7Rα internalization, recycling and degradation by IL-7 c

Regulation of IL-7Rα internalization, recycling and degradation by IL-7 3.5. Discussion

IL-7RA

Regulation of IL-7Rα internalization, recycling and degradation by IL-7 de novo de novo

Regulation of IL-7Rα internalization, recycling and degradation by IL-7

IL-7RA

Regulation of IL-7Rα internalization, recycling and degradation by IL-7

Chapter 4: General discussion and perspectives

General discussion and perspectives

Integrating cell motility, regulation of IL-7 receptor trafficking and IL-7-mediated signalling: Implications for T-cell and T-ALL biology

General discussion and perspectives ´`

General discussion and perspectives α ; α, α, α α α/

General discussion and perspectives in vivo . macaques .

General discussion and perspectives per se in vitro in vivo

General discussion and perspectives IL- 7RA

General discussion and perspectives

General discussion and perspectives per se de novo

General discussion and perspectives

de novo

General discussion and perspectives

References