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Universidad  Autónoma  de  Madrid   Facultad  de  Ciencias  

Departamento  de  Biología  Molecular  

Programa  de  Doctorado  en  Biociencias  Moleculares  

Regulation of pre-TCR and TCR function by the transmembrane domain of CD3ζ chain

during T cell development

Tesis  Doctoral  

Elena  Rodríguez  Bovolenta   Madrid,  2018  

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Doctoral  Thesis  

Regulation of pre-TCR and TCR function by the transmembrane domain of CD3ζ chain during T cell development

 

This   thesis   is   submitted   by   Elena   Rodríguez   Bovolenta   in   fulfilment   of   the   requirements  for  the  degree  of  Doctor  in  Molecular  Biology.    

 

Thesis  directors:  

 

Dr.  Hisse  Martien  van  Santen  

Assistant  professor  of  the  Consejo  Superior  de  Investigaciones  Científicas   Centro  de  Biología  Molecular  Severo  Ochoa  (CBMSO-­‐‑CSIC)  

Universidad  Autónoma  de  Madrid    

Dr.  Balbino  Alarcón  Sánchez    

Research  Professor  of  the  Consejo  Superior  de  Investigaciones  Científicas   Centro  de  Biología  Molecular  Severo  Ochoa  (CBMSO-­‐‑CSIC)  

Universidad  Autónoma  de  Madrid    

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This   work   has   been   performed   at   the   Centro   de   Biología   Molecular   Severo   Ochoa   under   the   supervision   of   Drs.   Hisse   Martien   van   Santen   and   Balbino   Alarcón   Sánchez   and   the   work   has   been   funded   by   the   MINECO   project   SAF2013-­‐‑47075-­‐‑R.   Short   term   period   in   Dr.   Wolfgang   Schamel´s   laboratory   at   the  BIOSS  Centre  in  Freiburg  im  Breisgau  was  supported  by  EMBO  Short  Term   Fellowship  program.    

 

   

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A MIS PADRES

                 

   

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CONTENTS

 

LIST OF THE FIGURES AND TABLES 13

ABBREVIATIONS 17

SUMMARY 21

RESUMEN 23

INTRODUCTION 25

A  brief  introduction  to  the  adaptive  immune  system   25  

T  cell  development:  from  the  thymus  to  the  periphery   26  

Pre-­‐‑TCR  and  TCR  complexes   31  

TCR  and  pre-­‐‑TCR  signalling   34  

TCR  triggering  models:  TCR  clustering  and  conformational  change   36   Role  of  the  preTCR-­‐‑CD3  and  associated  signalling  molecules  in  early  T  cell  development   41  

MATERIALS AND METHODS 47

1. MATERIALS 47

  Cells   47  

  Mice   49  

  Reagents   51  

  Antibodies  and  fluorescent  probes   53  

  Vectors   55  

  Oligonucleotides   56  

2. METHODS 59

Generation  of  CD3ζ-­‐‑/-­‐‑  /  TgCD3ζWT-­‐‑GFP  and  CD3ζ-­‐‑/-­‐‑  /  TgCD3ζL19A-­‐‑GFP  mouse  lines   59  

Lentiviral  production  and  cell  line  transduction   60  

Generation  of  a  CD3ζ-­‐‑deficient  pre-­‐‑T  cell  lines   60  

Generation  of  the  SCBζWT  and  SCBζL19A  cell  lines   62  

Obtention  of  cell  suspension  from  mice  lymphoid  organs   62  

FACS  staining  protocol   62  

Annexin  V  staining   63  

BrdU  staining   63  

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Proliferation  and  functional  assays   64  

Mouse  peripheral  blood  collection  for  phenotyping   64  

FACS  analysis   64  

Mouse  genotyping   64  

Electron  Microscopy  (EM)   65  

Western  Blotting   66  

Blue  Native  –PAGE  (BN-­‐‑PAGE)   67  

Total  Internal  Reflection  Fluorescen  Microscopy  (TIRFM)   68  

Statistical  analysis   69  

OBJECTIVES 71

OBJETIVOS 73

RESULTS 77

ANALYSIS OF THE EFFECTS OF THE L19A MUTATION ON EARLY T CELL DEVELOPMENT

I.   The  L19A  mutation  alters  thymus  size  and  cellularity   77   II.   L19A  DN  thymocytes  fail  in  the  upregulation  of  maturation  markers  during  DN3  to  DN4  

transition   81  

III.   Proliferation  and  cell  death  are  altered  in  L19A  DN  thymocytes   83   IV.   L19A  pre-­‐‑TCR  has  a  reduced  proximal  signalling  capacity   86  

V.   Pre-­‐‑TCR  clustering   90  

STUDY OF THE EFFECTS OF THE L19A MUTATION IN TRC-DEPENDENT THYMOCYTE DEVELOPMENT AND MATURE T LYMPHOCYTE PHENOTYPE AND ACTIVATION

I.   Effect  of  L19A  mutation  in  TCRαβ-­‐‑expressing  thymocytes   99  

II.   Generation  of  natural  Treg  cells  in  thymus   101  

III.   Phenotype  of  L19A  immature  thymocytes  in  a  OT-­‐‑I  and  OT-­‐‑II  TCR  repertoire   102   IV.   Phenotype  and  function  of  mature  CD3ζ-­‐‑GFP  transgenic  lymphocytes   106   V.   L19A  CD4  and  L19A  CD8  mature  T  cell  function  upon  TCR  stimulation   109  

DISCUSSION 115

How  is  the  CD3ζ  transmembrane  domain  involved  in  signalling  through  the  pre-­‐‑TCR?   115

 

Pre-­‐‑TCR  in  β-­‐‑selection,  the  importance  of  the  complex  integrity   121

 

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Negative  and  positive  selection  on  a  context  of  reduced  TCR  signalling   123

 

TCR  signal  strength,  T  cell  commitment  and  peripheral  T  cell  repertoire  properties   126

 

CONCLUSIONS 133

CONCLUSIONES 135

BIBLIOGRAPHY 137

ACKNOWLEDGMENTS 151

   

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LIST OF THE FIGURES AND TABLES

INTRODUCTION

Figure  1.  Thymus  structure  and  the  dinamics  of  the  thymocyte  development.   28

 

Figure  2.  Scheme  of  the  αβ  T  cell  development.   30

 

Figure  3.  Scheme  of  the  immature  pre-­‐‑TCR  and  mature  TCR  complexes.   32

 

Table  1.  Comparison  of  pre-­‐‑TCR  and  TCR  characteristics.   33

 

Figure  4.  Pre-­‐‑TCR  signalling.   36  

Table  2.  Summary  of  TCR  characteristics   38  

MATERIALS AND METHODS

• MATERIALS

Table  1.  Material  used  for  culture  cells   48

 

Table  2.  Reagents,  source  and  application   51

 

Table  3.  Buffers   52

 

Table  4.  Primary  Antibodies.   53

 

Table  5.  Secondary  antibodies   55

 

Table  6.  Fluorescent  probes   55

 

Table  7.  Vectors   55

 

Table  8.  Oligonucleotides  used  for  pcr  typing   56

 

Table  9.  Oligonucleotides  used  in  CRISPR/Cas9  System   56

 

• METHODS

Figure  1.  Scheme  of  the  generation  of  CD3ζ-­‐‑/-­‐‑  /  TgCD3ζWT-­‐‑GFP  and  CD3ζ-­‐‑/-­‐‑  /  TgCD3ζL19A-­‐‑

GFP  mouse  lines.   59

 

Figure  2.  Scheme  of  the  generation  of  the  SCBζWTGFP  and  SCBζL19AGFP  pre-­‐‑TCR  cell  lines.60   Figure  3.  Elution  picks  of  the  fragments  amplified  in  CRISPR-­‐‑STAT  method  of  both  clones  

ζKO  obtained.   61

 

RESULTS

ANALYSIS OF THE EFFECTS OF THE L19A MUTATION ON EARLY T CELL DEVELOPMENT

Figure  1.  Thymus  size  and  cellularity  in  CD3ζWT-­‐‑GFP  and  CD3ζL19A-­‐‑GFP  transgenic  mice.   77

 

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Figure  2.  Percentage  and  size  of  the  thymocytes  subsets  in  CD3ζWT-­‐‑GFP  and  CD3ζL19A-­‐‑GFP  

transgenic  mice.   78

 

Figure  3.  DN  thymocyte  populations  in  CD3ζWT-­‐‑GFP  and  CD3ζL19A-­‐‑GFP  transgenic  mice.   79

 

Figure  4.  Intracellular  TCRβ  levels  in  DN3  subpopulation.   80

 

Figure  5.  Levels  of  pre-­‐‑TCR  expression  on  L19A  and  WT  DN  CD44neg  thymocytes.   82

 

Figure  6.  Protein  expression  levels  of  the  CD27,  CD98  and  CD71  maturation  markers  on  the  

surface  of  the  DN3  and  DN4  thymocytes.   83

 

Figure  7.  FCS  levels  in  DN  thymocytes.   84

 

Figure  8.  Ki67  levels  in  DN  thymocytes.   85

 

Figure  9.  BrdU  incorporation  assay  in  DN  thymocytes.   86

 

Figure  10.  Annexin  V  staining  in  DN  thymocytes.   87

 

Figure  11.  TCR  expression  levels  in  SCB.29  and  derived  cell  lines.   88

 

Figure  12.  Steady  state  phosphorylation  levels  of  CD3ε  in  SCIET.27,  SCB.29,  ζKO  and  

reconstituted  cell  lines.   89

 

Figure  13.  Phosphorylation  levels  in  SCBζGFPWT  and  SCBζGFPL19A  cell  lines  after  

stimulation  with  anti-­‐‑CD3ε  antibody.   89

 

Figure  14.  Pre-­‐‑TCR  distribution  on  the  cell  surface  of  the  SCB.29,  the  SCBζWT  21.1  and  the  

SCBζL19A  21.1  cells.   91

 

Figure  15.  Analysis  of  the  pre-­‐‑TCR  clustering  distribution  using  BN-­‐‑PAGE.   92

 

Figure  16.  TIFRM  analysis  of  the  pre-­‐‑TCR  on  CD44neg  primary  thymocytes.   94

 

STUDY OF THE EFFECTS OF THE L19A MUTATION IN TRC-DEPENDENT THYMOCYTE DEVELOPMENT AND MATURE T LYMPHOCYTE PHENOTYPE AND ACTIVATION.

Figure  17.  Single  positive  thymocyte  populations  in  WT  and  L19A  thymus  of  transgenic  mice.99

 

Figure  18.  Single  positive  thymocytes  populations  in  WT  and  L19A  thymus  of  transgenic  

mice.   100

 

Figure  19.  CD69  and  CD5  expression  levels  in  DP  thymocytes.   101

 

Figure  20.  Treg  cells  in  WT  and  L19A  mice  thymus.   102

 

Figure  21.  Thymic  populations  in  CD3ζ-­‐‑GFP  OT-­‐‑I  double  transgenic  mice.   103

 

Figure  22.  Number  of  cells  in  CD3ζ-­‐‑GFP  OT-­‐‑I  double  transgenic  thymuses.   104

 

Figure  23.  Thymic  populations  in  CD3ζ-­‐‑GFP  OT-­‐‑II  double  transgenic  mice.   105

 

Figure  24.  Number  of  cells  in  CD3ζ-­‐‑GFP  OT-­‐‑II  double  transgenic  thymuses.   106

 

Figure  25.  Mature  lymphocytes  in  LNs  of  WT  and  L19A  mice.   107

 

Figure  26.  TCR  expression  levels  in  peripheral  CD4  T  and  CD8  T  lymphocytes.   108

 

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Figure  27.  Treg  cells  in  peripheral  LNs  of  WT  and  L19A  mice.   109

 

Figure  28.  In  vitro  proliferation  and  functional  assay  in  mature  CD4  and  CD8  T  polyclonal  

lymphocytes   112

 

Figure  29.  In  vitro  stimulation  and  proliferation  assay  in  mature  OT-­‐‑II  CD4  T  and  OT-­‐‑I  CD8  T  

lymphocytes.   112

 

DISCUSSION

Figure  1.  CRAC  and  CARC  cholesterol  binding  motifs  in  the  transmembrane  domain  of  the   pTα,   TCRα,   TCRβ,   CD3ε,   CD3γ,   CD3δ,   CD3ζ   chains   of   the   pre-­‐‑TCR   and   TCR  

complexes.   118

 

Figure  2.  Transmembrane  domain  sequence  of  the  CD3ζ  in  different  mamal  species.   119

 

Figure  3:  Proposed  cell  model  based  in  the  data  obtained  in  this  work.   130

 

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ABBREVIATIONS

4SP  Single  positive  CD4   8SP  Single  positive  CD8  

ACK  Erythrocytes  lysis  buffer  BCR  B  cell  receptor  BSA  Bovine  Serum  Albumin  B2  B   cell  subtype    

BN-PAGE Blue  Native  Polyacriladide  gel  electroforesis   bp  base  pairs  

BRS Basic  amino  acid-­‐‑Rich  Sequence BSA  Bovine  Serum  Albumin  

CRISPR Clustered  Regularly  Interspaced  Short  Palindromic  Repeats CS  Cholesterol  Sulfate  

DMEM Dulbecco'ʹs  Modified  Eagle  Medium DN  Double  Negative    

DNA  Deoxyribonucleic  acid     DP  Double  positive  

EM  Electron-­‐‑mycroscopy  

ERK  Extracellular  signal–regulated  kinase     ETPs  Early  T  cell  progenitors  

FACS  Fluorescent  activated  cell  sorting   FBS  Fetal  Bovine  Serum    

FSC  Forward  Scatter    

GFP  Green  Flourescent  protein   HRP  Horseradish  peroxidase     I.p Intraperitoneal  

IP  Immunoprecipitation  

ISP8  Immature  Single  Positive  CD8  

ITAM  Immunoreceptortyrosine-­‐‑Based  activation  motif   ITIM  Immunoreceptortyrosine-­‐‑Based  inactivation  motif   kD  Kilodalton  

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KO  Knock  Out  

L19A  Mutation  Leucine  19  to  Alanine   L9A  Mutation  Leucine  9  to  Alanine   LAT  linker  for  activation  of  T  cells   MFI  Mean  Fluorescence  Intensity    

MHC  Molecular  Histocompatibility  complex     OVA  Ovalbumin  

PBS  Phosphate  buffered  saline       PFA  Paraformaldehyde    

pre-TCR pre  T  Cell  Receptor   PRS Proline  Rich  Sequence     PTKs  Protein  Tyrosine  Kinases    

PTPases  Protein  Tyrosine  Phosphatases     RPM  Revolutions  Per  Minute  

RPMI  Roswell  Park  Memorial  Institute  Medium   S1P1  Sphingosine  1-­‐‑phosphate  receptor  type  1   SD  Standard  Deviation  

SDS-PAGE Sodium  Dodecyl  Sulfate-­‐‑PAGE   SEM  Standard  Error  of  Mean  

T-ALL T-­‐‑cell  Acute  Lymphoblastic  Leukemia TCR  T  cell  receptor    

TIRFM  Total  Internal  Reflection  Fluorecent  Microscopy   TM  Transmembrane  domain    

WB  Western  Blot     WT  Wild-­‐‑Type    

ZAP70  Zeta-­‐‑Associated  Protein  of  70  KD    

 

   

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SUMMARY

Signalling   through   pre-­‐‑T   Cell   Receptor   (pre-­‐‑TCR)   and   the   αβ   T   cell   Receptor   (TCR)   complexes  are  key  events  during  thymic  T  cell  development,  which  assure  the  correct   generation  of  a  functional  and  self-­‐‑tolerant  mature  T  cell  repertoire.  Previous  work  of   the  laboratory,  using  a  transmembrane  point  mutant  of  the  TCR-­‐‑associated  CD3ζ  chain   (L19A),   has   shown   that   ligand-­‐‑independent   pre-­‐‑clustering   of   the   TCR   plays   an   important   role   in   providing   sensitivity   to   mature   T   cells,   possibly   by   facilitating   receptor-­‐‑proximal   cooperative   signalling   mechanisms.   Dimerization   of   the   pre-­‐‑TCR   has   been   proposed   as   necessary   for   the   correct   function   of   the   pre-­‐‑TCR   during   the   earliest  steps  of  T  cell  development  in  the  thymus,  although  its  effect  on  signalling  has   not  been  directly  addressed.  Given  the  relevance  of  T  cell  development  in  determining   the  protective  and  pathogenic  capacities  of  the  mature  T  cell  repertoire,  it  is  important   to  understand  the  molecular  mechanisms  of  pre-­‐‑TCR  and  TCR  function  and  signalling   underlying  the  formation  of  this  repertoire.  

In   this   thesis   we   have   addressed   whether   and   how   the   L19A   mutation   affects   T   cell   differentiation   and   activation,   focussing   on   the   early   pre-­‐‑TCR-­‐‑dependent   differentiation   steps,   TCR-­‐‑dependent   positive   and   negative   selection   as   well   as   activation  of  mature  T  cells  and  we  have  also  investigated  to  what  extent  this  is  due  to   alterations  in  the  organization  of  these  receptors  at  the  cell  surface.  To  address  these   questions,   we   generated   transgenic   mice   expressing   recombinant   CD3ζWT   or   CD3ζL19A   chains   coupled   to   GFP   in   an   endogenous   CD3ζ-­‐‑deficient   mouse   background.   Our   data   demonstrate   that   the   L19A   mutation   partially   arrests   T   cell   development  at  the  Double  Negative  (DN)  stage,  more  specifically  at  the  DN3  to  DN4   transition.   Data   obtained   with   pre-­‐‑T   cell   lines   expressing   the   WT   or   L19A   pre-­‐‑TCR   support  the  notion  that  this  impairment  in  the  DN3-­‐‑DN4  transition  is  most  likely  due   to  an  alteration  in  the  transmission  of  the  outside-­‐‑in  signal  mediated  by  the  pre-­‐‑TCR.  

L19A   DN   thymocytes   present   a   more   immature   phenotype,   proliferate   less   and   are   more   prone   to   die   compared   to   WT   DN   thymocytes.   This   defect   is   translated   into   a   mild   but   significant   peripheral   lymphopenia,   probably   aggravated   by   a   poorer   efficiency  of  thymic  selections  and/or  lineage  commitment  at  the  Double  Positive  (DP)   stage.  Notably,  CD4  T  lymphocytes  are  more  affected  than  CD8  T  lymphocytes.  From  a   molecular  perspective,  TIRFM  analysis  provides  evidence  that  the  mutant  pre-­‐‑TCR  on   the  plasma  membrane  of  CD44-­‐‑negative  DN  primary  thymocytes  are  more  mobile  and   less  prone  to  clustering  that  the  WT  pre-­‐‑TCR,  suggesting  a  role  of  the  Leu19  in  the  pre-­‐‑

TCR  cluster  formation.    

Together  these  data  indicate  that  pre-­‐‑TCR  clustering  optimizes  the  receptor  signalling   capacities  and  that  the  transmembrane  region  of  the  CD3ζ  chain  has  an  important  role   in  pre-­‐‑TCR  clustering  formation.  

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RESUMEN  

 

 

RESUMEN

La   correcta   generación   de   linfocitos   T   maduros,   funcionales   y   tolerantes   a   péptidos   propios  requiere  una  señalización  adecuada  a  través  de  la  forma  inmadura  y  madura   del  Receptor  para  Células  T  (pre-­‐‑TCR  y  TCR  respectivamente)  durante  su  desarrollo  en   el  timo.  Investigaciones  previas  en  nuestro  laboratorio  con  una  cadena  CD3ζ  del  TCR   mutada   puntualmente   en   su   región   transmembrana   (L19A),   mostraron   que   los   clústeres   de   TCR   independientes   de   ligando   juegan   un   papel   importante   en   la   sensibilidad  de  las  células  T  maduras  por  el  antígeno,  posiblemente  debido  a  que  estos   clústeres   favorecen   mecanismos   cooperativos   durante   la   señalización   temprana.   A   pesar   de   la   falta   de   evidencias   directas,   actualmente   se   cree   que   la   dimerización   del   pre-­‐‑TCR  es  necesaria  para  su  correcta  función  durante  las  primeras  fases  del  desarrollo   de   los   linfocitos   T.   Ya   que   las   capacidades   protectoras   y   patogénicas   del   repertorio   periférico   de   células   T   maduras   se   establecen   durante   su   desarrollo   en   procesos   dependientes   de   los   complejos   pre-­‐‑TCR   y   TCR,   es   esencial   conocer   el   mecanismo   molecular  de  la  señalización  y  función  de  éstos  complejos.  

En   esta   tesis   hemos   abordado   la   caracterización   del   impacto   de   la   mutación   L19A   durante  el  desarrollo  de  los  linfocitos  T.  Nos  hemos  centrado  en  las  etapas  tempranas   dependientes   de   pre-­‐‑TCR,   en   las   selecciones   negativa   y   positiva   y   en   el   fenotipo   y   activación   de   los   linfocitos   maduros,   además   de   investigar   hasta   que   punto   estas   alteraciones  son  debidas  a  la  organización  de  éstos  receptores  en  la  superficie  celular.  

Para   ello,   hemos   generado   ratones   transgénicos   donde   las   variantes   CD3ζWT-­‐‑GFP   y   CD3ζL19A-­‐‑GFP   de   la   cadena   CD3ζ   del   TCR   fueron   reintroducidas   en   ratones   deficientes   para   CD3ζ.   Nuestros   datos   demuestran   que   la   mutación   L19A   bloquea   parcialmente   el   desarrollo   de   linfocitos   T   en   el   estadio   Doble   Negativo   (DN).   Más   específicamente,  en  la  transición  entre  los  estadios  DN3  y  DN4.  Los  datos  obtenidos  en   líneas   celulares   pre-­‐‑TCR   sugieren   que   este   bloqueo   se   debe   a   una   alteración   en   la   transmisión  de  la  señal  a  través  de  éste  receptor.  Los  timocitos  DN  mutantes  presentan   un   fenotipo   más   inmaduro,   proliferan   menos   y   son   más   sensibles   a   sufrir   apoptosis   que   los   timocitos   DN   silvestres.   Éste   efecto   se   traduce   posteriormente   en   una   ligera   pero   significativa   linfopenia   en   los   órganos   linfoides   periféricos,   posiblemente   agravada   por   la   pobre   eficiencia   de   las   selecciones   tímicas   y/o   un   defecto   en   el   establecimiento  de  linaje  en  el  estadio  Doble  Positivo  (DP).  Es  interesante  destacar  que   los  linfocitos  CD4  están  más  afectados  que  los  linfocitos  CD8.  Desde  un  punto  de  vista   molecular,  los  análisis  mediante  TIRFM  sugieren  que  el  pre-­‐‑TCR  mutado  es  más  móvil   y  menos  propenso  a  la  oligomerización  en  la  membrana  celular  de  timocitos  primarios   que  el  pre-­‐‑TCR  silvestre,  indicando  un  papel  de  la  Leu19  en  la  formación  de  clústeres   de  pre-­‐‑TCR.  En  conjunto,  estos  datos  indican  que  el  la  presencia  de  clústeres  del  pre-­‐‑

TCR  optimiza  la  capacidad  de  señalización  del  receptor  y  que  la  región  transmembrana   de  la  cadena  CD3  ζ  tiene  un  papel  importante  en  este  proceso.  

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INTRODUCTION

A brief introduction to the adaptive immune system

Each   individual   has   an   intrinsic   capacity   to   defend   itself   against   environmental   pathogens.  In  vertebrates,  this  capacity  is  known  as  the  immune  system  and  it  consists  of   an  organization  of  cells  and  molecules  with  specialized  roles  that  will  trigger  an  immune   response  against  potential  pathogens,  such  as  bacteria  and  viruses,  or  in  order  to  prevent   the  uncontrolled  growth  of  cells  that  may  form  a  tumour.  

Two   interacting   defences   form   the   immune   system:   the   innate   and   the   adaptive   immunity.   The   innate   immunity   provides   an   earliest   line   of   defence.   It   is   activated   by   pattern  recognition  receptors  that  identify  unique,  pathogen-­‐‑derived  molecules  (i.e.  not   shared  with  eukaryotic  cells),  which  are  well  conserved  among  different  pathogens.  The   principal   components   of   this   defence   system   are   physical   and   chemical   barriers,   phagocytic  cells  (neutrophils,  macrophages),  dendritic  cells,  natural  killer  cells  and  blood   proteins,  such  as  the  molecules  of  the  complement.  All  of  them  are  in  place  even  before   infection   and   are   poised   to   response   rapidly.   In   contrast,   activation   of   the   adaptive   immune   system   enables   antigen-­‐‑specific   recognition   of   pathogens   and   tumoral   cells   resulting   in   long-­‐‑term   immunity,   including   immunological   memory;   however,   the   adaptive  immune  response  requires  time  to  arise  and,  hence,  it  is  not  as  effective  as  the   innate  immune  response  at  the  onset  of  a  new  antigenic  challenge.  The  adaptive  immune   system   is   further   differentiated   into   a   humoral   component,   which   is   mediated   by   antibody-­‐‑producing   B   lymphocytes,   and   a   cellular   response,   which   is   mediated   by   T   lymphocytes  (Fayard  et  al.,  2010).  

• B   CELLS:   Express   on   their   membrane   the   B   Cell   Receptor   (BCR)   that   is   able   to   bind   intact   antigens.   They   have   to   principal   roles:   to   produce   specific   antibodies/immunoglobulins,  and  to  function  as  antigen-­‐‑presenting  cells  (APCs).  

For   this   second   role,   the   bound   antigen   molecules   are   engulfed   by   the   B   cells   where  it  is  then  digested  into  fragments  and  displayed  at  the  cell  surface  nested   inside  a  class  II  major  histocompatibility  complex  molecule  (MHC-­‐‑II).  Helper  T  

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cells  bind  the  B  cell  through  the  T  Cell  Receptor  (TCR)  and  secrete  cytokines  that   stimulate   the   B   cell   to   become   and   effector-­‐‑antibody-­‐‑producer   B   cell   (Cantrell,   2015).  

• T  CELLS:  Express  on  their  surface  the  T  Cell  Receptor  (TCR).  According  to  their   TCR,  it  is  possible  to  distinguish  gamma/delta  (γδ)  and  alpha/beta  (αβ)  T  cells.  

The   vast   majority   of   αβ   T   cells   recognize   antigen   peptides   presented   by  

“classical”  class  I  or  class  II  major  histocompatibility  complex  molecules  (MHC-­‐‑I   and   MHC-­‐‑II   respectively),   and   are   referred   to   as   “conventional”   T   cells.  

Conventional  T  cells  are  distributed  into  two  subsets  that  differ  by  their  function   and  their  expression  of  CD4  or  CD8,  two  surface  glycoproteins  that  contribute  to   recognition  of  MHC  molecules  and  signalling:  

> CD8+  T  cells,  also  known  as  cytotoxic  T  lymphocytes  (CTLs).  These  cells   recognize  epitopes  presented  by  the  MHC-­‐‑I  molecules  and  are  involved   in  the  immune  defence  against  intracellular  pathogens.    

> CD4+   T   cells,   that   bind   epitopes   presented   by   the   MHC-­‐‑II   and   are   essential   for   both   cell-­‐‑mediated   and   antibody-­‐‑mediated   branches   of   the   immune   system.   They   are   also   known   as   helper   T   cells   because   they   produce   regulatory   cytokines   and   chemokines   that   contribute   in   the   activity  and  function  of  other  immune  cells.    

Establishment  of  a  functional  and  competent  CD4  T  and  CD8  T  cell  repertoire  endowed   with  the  capacity  to  respond  to  pathogens  and  tolerate  self-­‐‑antigens,  happens  during  the   lymphocyte  development.  This  process  take  place  most  efficiently  in  the  thymus,  which   provides  a  complexity  of  discrete  cellular  microenvironments  specialized  in  supporting   each  of  the  steps  of  this  developmental  process  (Cantrell,  2015).  

T cell development: from the thymus to the periphery

The  formation  of  a  functional  T  cell  population  with  a  broad  TCR  repertoire  depends  on   a   precisely   controlled   developmental   process   that   takes   place   in   the   thymus,   in   which   epithelial,   mesenchymal   and   hematopoietic   stromal   cells   provide   a   unique  

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microenvironment  and  the  signals  necessary  for  proper  thymocyte  differentiation.  There   are  several  observations  supporting  the  importance  of  this  organ  in  the  generation  of  the   T   cell   repertoire;   in   the   absence   of   the   thymus,   a   reduction   of   peripheral   T   cells   is   observed,  resulting  in  severe  immunodeficiency  (Bosma  et  al.,  1983;  Miller,  2002)  .  Also,   contrary   to   the   B   cells   that   can   be   easily   differentiated   in   vitro   from   bone   marrow   precursors,  the  in  vitro  generation  of  T  cells  require  complex  thymic  organ  cultures  for   their   successful   differentiation   (Jenkinson   and   Owen,   1990).   T   cell   development   and   differentiation  is  also  a  dynamic  process;  developing  thymocytes  moves  through  defined   thymic   regions,   with   lymphoid   progenitors   entering   the   thymus   at   the   cortico–

medullary   junction,   then   migrating   to   the   outer   cortex,   and   finally   returning   to   the   medulla   (Figure   1)   (Lind   et   al.,   2001).   These   coordinated   movements   are   directed   by   chemokines,   and   mediated   by   interactions   between   integrins   expressed   on   T   cell   progenitors   and   their   ligands   on   stromal   cells.   In   summary,   T   cell   development   is   a   complex,   thymic-­‐‑dependent   process   that   involves   multiple   and   precisely   coordinated   differentiation   and   proliferation   events.   During   this   process,   hematopoietic   precursors   give   rise   to   T   cells   able   to   respond   to   antigen   stimulation   and   ready   for   effector   differentiation  (Fayard  et  al.,  2010;  Zuniga-­‐‑Pflucker,  2004).  

The  development  of  T  cells  can  be  separated  in  three  broad  steps:  Thymic  colonization   by  bone  marrow  precursors  followed  by  the  divergence  of  αβ  and  γδ  lineages.  Finally,   αβ   and   γδ   lineage   cells   complete   their   differentiation   and   acquire   immunological   properties   (Carpenter   and   Bosselut,   2010).   The   γδ   T   cell   is   a   very   heterogeneous   population   with   a   not   yet   fully   understood   developmental   process   that   takes   place   in   waves  (Prinz  et  al.,  2013).  The  work  presented  in  this  thesis  has  been  performed  with  αβ   T  cells  and  their  precursors  therefore  from  here  on  I  will  exclusively  focus  on  this  T  cell   type.    

Blood-­‐‑borne  T  cell  precursors  continuously  access  the  vascularized  microenvironment  of   the   thymus   at   its   cortico-­‐‑medullary   junction.   Once   situated   within   the   thymus,   these   precursors   begin   to   proliferate   and   give   rise   to   cells   designated   as   early   T   lineage   progenitors  (ETPs).  The  immediate  progeny  of  ETPs  is  phenotypically  characterized  by   the  lack  of  CD4  and  CD8  proteins  at  the  cell  surface,  known  as  Double-­‐‑Negative  (DN)    

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Figure  1.  Thymus  structure  and  the  dinamics  of  the  thymocyte  development.    

Figure  is  a  modification  of  (Germain,  2002).  

thymocytes.   In   mouse,   four   successive   DN   subpopulations   have   been   distinguished   based   on   the   expression   of   the   surface   molecules   CD25   and   CD44   (inset   in   Figure   2):  

DN1   (CD44posCD25neg),   DN2   (CD44posCD25pos),   DN3   (CD44negCD25pos)   and   DN4   (CD44negCD25neg)   (Godfrey   et   al.,   1993).   It   is   normally   at   the   DN2   stage   where   the   thymocytes   become   firmly   committed   to   the   αβ   T   lineage   fate   and   begin   to   rearrange   their   Tcrβ   locus.   Rearrangement   of   Tcrβ   is   a   mechanism   of   genetic   recombination   that   randomly  selects  and  assembles  V,  D  and  J  segments  of  the  Tcrβ  locus,  resulting  in  the   generation  of  a  diverse  repertoire  of  TCRβ  chains  (Mallick  et  al.,  1993).  DN3  cells  can  be   further  subdivided  into  DN3a  and  DN3b:  DN3a  cells  that  have  productively  rearranged   the  Tcrβ  locus  become  DN3b  cells  that  express  at  the  cell  surface  a  functional  TCRβ  chain   together   with   the   invariant   pre-­‐‑TCRα   chain   (pTα)   and   the   CD3   components.   Whereas   thymocytes  that  fail  to  form  the  pre-­‐‑TCR  die  through  apoptosis,  signalling  through  this   receptor  rescues  DN3  cells  from  programmed  cell  death,  inhibits  further  rearrangement   of  the  Tcrβ  locus  (resulting  in  allelic  exclusion,  a  process  by  which  only  one  allele  of  a   gene   is   expressed   while   the   other   allele   is   silenced),   initiates   cell   proliferation   and   enables   the   developmental   progression   to   the   DN4   phenotype   (Hoffman   et   al.,   1996).  

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These   events,   from   the   successful   TCRβ   rearrangement,   are   referred   to   as  β-­‐‑selection   (Hoffman   et   al.,   1996)   and   this   process   is   also   companied   by   changes   in   molecular   expression  of  various  membrane  receptors  such  as  CD98,  CD71,  CD25,  CD27  and  CD28   (Kelly  et  al.,  2007;  Teague  et  al.,  2010).  At  the  DN4  stage,  thymocytes  may  also  begin  to   express   CD8   on   their   cell   surface,   becoming   cells   known   as   immature   single   CD8   positive  (ISP8).  DN4-­‐‑ISP8  cells  progress  to  a  developmental  stage  characterized  by  the   concomitant  presence  of  CD4  and  CD8  and  hence  called  Doube-­‐‑Positive  (DP)  population   (Figure   2).   Rearrangement   of   the   Tcrα   locus   occurs   in   DP   thymoyctes   and   eventually   enables  the  expression  of  a  mature  αβTCR-­‐‑CD3  complex  where  the  pTα  is  replaced  by  a   TCRα   chain.   The   replacement   of   the   pTα  chain   is   important   to   ensure   the   further   development  of  thymocytes  beyond  the  DN4  stage.  It  has  been  seen  that  overexpression   of  pTα  accelerates  the  transition  from  DN3  to  DN4  but  reduces  the  rate  of  assembly  of   αβTCR,   increasing   the   apoptosis   of   DP   thymocytes   (Lacorazza   et   al.,   2001).   At   the   DP   stage,   thymocytes   are   subjected   to   a   strictly   TCR-­‐‑dependent   selection   process   in   the   thymic  cortex  and  medulla  that  is  aimed  to  test  the  specificity  of  their  TCR  (Figure  1).  

Thymocytes  with  a  TCR  able  to  bind  with  sufficient  affinity  to  self-­‐‑peptide-­‐‑MHC  present   on  epithelial  cells  of  the  cortex,  leads  to  positive  selection  and  continue  their  intrathymic   maturation.   In   contrast,   DP   thymocytes   that   have   a   non-­‐‑functional   TCR   fail   to   receive   any   further   survival   signal   and   are   thus   eliminated   in   a   process   referred   as   “death   by   neglect”.   In   a   subsequent   stage,   which   typically   but   not   exclusively   takes   place   in   the   medulla,   positively   selected   thymocytes   are   submitted   yet   to   another   selection   step   known  as  negative  selection,  which  it  is  the  result  of  strong  TCR  ligation  of  self-­‐‑peptide-­‐‑

MHC.  Here,  thymocytes  with  a  TCR  that  binds  to  self-­‐‑peptide  MHC  complexes  with  an   affinity   above   a   critical   threshold   are   removed   through   induction   of   programmed   cell   death.   This   selection   process   thus   eliminates   potentially   self-­‐‑reactive   thymocytes   that   otherwise  may  elicit  a  harmful  autoimmune  response  as  peripheral  T  cells.  The  affinity   window  for  positive  versus  negative  selection  is  narrow  and,  remarkably,  small  changes   in  the  affinity  of  the  TCR  signal  re-­‐‑direct  the  fate  of  immature  thymocytes.  For  instance,   impairment  of  TCR  signal  strength  results  in  a  shift  from  negative  to  positive  and  from   positive  selection  to  neglect  (Hogquist,  2001;  Moran  and  Hogquist,  2011).  

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Figure  2.  Scheme  of  the  αβ  T  cell  development.    

Characteristic  protein  markers  for  each  cell  at  the  specific  stage  of  development  are  shown.  The   development  of  the  DN  subpopulations  is  presented  in  more  detail  in  the  inset.  ETP:  Early  T  cell   Progenitors,  DN;  Double  Negative,  ISP:  Intermediate  Single  Positive,  DP:  Double  Positive,  4SP:  

Single  Positive  CD4,  8SP:  Single  Positive  CD8.  

Finally,   selected   thymocytes   that   have   a   TCR   restricted   to   MHC   class   I   molecules   will   develop  into  single  positive  CD4negCD8pos  phenotype  (8SP),  whereas  cells  with  an  MHC   class  II-­‐‑restricted  TCR  will  accomplish  a  single  positive  CD4posCD8neg  phenotype  (4SP).  

SP   cells   exit   the   thymus   to   the   periphery   as   naïve   T   cells   through   a   mechanism   that  

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involves  signalling  by  the  sphingosine  1-­‐‑phosphate  receptor  type  1  (S1P1)  (Allende  et  al.,   2004;  Fayard  et  al.,  2010).  

Pre-TCR and TCR complexes

The  TCR  consists  of  six  different  types  of  transmembrane  (TM)  proteins  that  assemble  in   dimers:   the   ligand-­‐‑binding   TCRαβ   heterodimer   and   the   signal-­‐‑transducing   dimers   of   CD3εγ,  CD3εδ,  and  CD3ζζ  (Figure  3,  right).  The  variable  immunoglobulin  (Ig)  domains   of  the  TCRα  and  TCRβ  heterodimer  form  the  binding  surface  for  its  ligand,  the  MHC-­‐‑

peptide,   while   the   constant   Ig   and   TM   regions   couple   to   the   CD3   dimers.   The   CD3   subunits   contain   an   extracellular   Ig   domain   (except   for   CD3ζ),   a   TM   region   and   a   cytoplasmic  tail  that  contains  one  or  more  copies  of  a  conserved  sequence  motif,  known   as  the  Immunoreceptor  Tyrosine-­‐‑based  Activation  Motif  (ITAM).    

The   TCRβ  chain   of   the   pre-­‐‑TCR   is   bound   to   an   invariant   pre-­‐‑T   cell   receptor   α   (pTα)   chain  instead  of  to  the  rearranged  TCRα  chain  (Groettrup  et  al.,  1993)  (Figure  3,  left).  The   pre-­‐‑TCR  complex  can  be  viewed  as  a  variant  of  the  mature  αβTCR,  but  the  pTα  chain   provides  the  pre-­‐‑TCR  with  a  unique  function  due  to  its  different  capabilities  compared   with  the  TCRα  chain  as  has  been  demonstrated  over  the  years  (Table  1).  The  pTα  chain   possesses  a  single  immunoglobulin-­‐‑like  domain  that  is  more  structurally  similar  to  the   constant   domain   of   an   antibody   light   chain   than   to   the   constant   domain   of   the   TCRα.  

Even  so,  there  is  some  homology  between  the  Ig-­‐‑like  domain  of  the  pTα  and  the  two  Ig-­‐‑

like  constant  domain  of  the  TCRα.  The  mode  of  association  between  pre-­‐‑Tα  and  TCRβ   resembles   that   mediated   by   the   constant   domains   of   the   heterodimer   TCRαTCRβ,   although  a  cystein  residue  in  pTα,  responsible  for  interchain  disulphide  bond  formation,   is   in   a   different   position   from   that   in   the   TCRα   chain   and   allows   less   efficient   heterodimerization   with   the   TCRβ   chain   as   well   as   only   weak   association   with   the   CD3ζζ   homodimer   (Boehmer   and   Fehling,   1997).   In   addition,   both   pTα   and   TCRα   transmembrane   domains   present   polar   residues   in   the   same   position   and   orientation,   allowing  the  association  with  the  signal-­‐‑transducing  CD3  molecules.  

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Figure  3.  Scheme  of  the  immature  pre-­‐‑TCR  and  mature  TCR  complexes.    

CD3  chain  names  are  displayed  bellow  each  chain  while  the  TCR  chain  names  are  shown  above   them.    

The   requirement   of   the   engagement   of   mature   TCRs   by   agonist   pMHC   for   signal   transduction   is   very   well   established.   It   is   also   well   described   that   after   TCR   ligation,   there   is   an   accumulation   of   TCRs   in   the   area   of   contact   where   an   immune   synapse   structure  is  formed  (Dustin  and  Shaw,  1999;  Monks  et  al.,  1998).  In  addition,  there  are   several  studies  demonstrating  that  at  least  part  of  the  TCRs  in  resting  T  cells,  i.e.  before   interaction   with   pMHC   agonist   pMHC   ligands,   are   organized   in   clusters   of   nanoscale   size  known  as  nanoclusters  (Lillemeier  et  al.,  2010;  Schamel  et  al.,  2005;  Sherman  et  al.,   2011;   Zhong   et   al.,   2009).   Unlike   for   the   mature   αβTCR   and   despite   its   structural   similarities,   it   has   been   suggested   that   the   pre-­‐‑TCR   signals   in   a   ligand-­‐‑independent   manner,  even  though  the  molecular  mechanism  underlying  the  autonomous  signalling  is   still  unclear.  Some  publications  proposed  that  this  ligand-­‐‑independent  signalling  is  not   attributable  to  the  pre-­‐‑TCR  per  se;  instead,  it  is  a  property  of  the  DN  cell  in  which  pre-­‐‑

TCR  signalling  occurs.  This  was  illustrated  by  studies  in  which  retroviral  transduction  of   the  TCRα  chain  into  pTα-­‐‑deficient  thymocytes  restored  T  cells  development  in  fetal    

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Table  1.  Comparison  of  pre-­‐‑TCR  and  TCR  characteristics.  

thymus  organ  culture  (Haks  et  al.,  2003).  However,  in  vivo  experiments  showed  that  the   replacement  of  the  pTα  coding  sequence  by  a  rearranged  TCRα  chain  using  a  knock-­‐‑in   approach,  impaired  the  efficiency  of  thymocyte  development  form  DN  to  DP  meaning   that  pTα  has  an  advantage  over  the  TCRα  in  β-­‐‑selection  (Borowski  et  al.,  2004)  but  in  DP   the  advantage  is  inverted.  This  highlights  that  preTα  and  TCRα  are  not  interchangeable   and   execute   different   genetic   programs.   Other   evidence   supporting   the   autonomous   signalling  of  the  pre-­‐‑TCR  was  obtained  using  transgenic  mice  expressing  a  mutant  pTα   and   TCRβ   lacking   extracellular   immunoglobulin   (Ig)-­‐‑like   domains   thereby   impeding   any   potential   extracellular   receptor-­‐‑ligand   interaction.   Thymocytes   expressing   these   truncated   chains   could   still   bypass   the   β-­‐‑selection,   suggesting   that   no   extracellular   ligand  binding  was  necessary  (Irving  et  al.,  1998).  On  other  hand,  (Yamasaki  et  al.,  2006)   observed  that  charged  amino  acids  in  the  extracellular  domain  of  pTα  are  essential  for   pTα  self-­‐‑oligomerization  and  critical  for  optimal  traversal  of  the  β-­‐‑selection  checkpoint,   suggesting   that   the   extracellular   domain   is   necessary   for   oligomerization   although   not  

pre-­‐‑TCR   TCR  

       

TCRβ  bound  to  pTα  invariant  chain.   TCRβ  bound  to  a  rearranged  TCRα  chain.  

pTα:  weak  association  with  TCRβ  and  CD3ζζ  

homodimer  (Boehmer  and  Fehling,  1997)     TCRα:  high  efficiency  of  dimerization  with   TCRβ.  

Low  levels  of  expression  at  the  plasma   membrane.  

Peripheral  and  SP  cells  present  high  levels  of   expression  on  cell  surface  while  in  DP  is  lower   but  still  more  than  the  pre-­‐‑TCR.  

No  ligand  binding  to  promote  signalling   (Irving  et  al.,  1998;  Saint-­‐‑Ruf  et  al.,  2003;  

Yamasaki  et  al.,  2006)   Ligand  binding  needed  to  promote  signalling.  

At  least  forming  dimers  (Yamasaki  et  al.,   2006),  but  BN  unpublished  data  suggest  that  is   highly  clustered  (Thesis  of  Dr.  Gina  Fiala  on   Prof.  Wolfgang  Schamel’s  Laboratory)  

TCR  in  clusters  of  diverse  size  (Schamel  et  al.,   2005).    

Constitutively  active.   Ligand-­‐‑dependent  activation.  

Degradation  of  TCR  due  to  the  continuous   signalling.  Similar  phenotype  as  activated   mature  T  cells  (Carrasco  et  al.,  2003;  Panigada   et  al.,  2002;  Valitutti  et  al.,  1997)  

In  resting  T  cells,  TCR  is  recycled  back  to  the   membrane  and  very  few  TCR  are  degraded.  

TCR  in  activated  T  cells  is  internalized  and   degraded  (Carrasco  et  al.,  2003;  Panigada  et   al.,  2002;  Valitutti  et  al.,  1997).  

Localized  in  raft  domains  (Saint-­‐‑Ruf  et  al.,   2003).  

Resting  TCR  localized  in  non-­‐‑raft  domains   (Beck-­‐‑García  et  al.,  2015);  can  move  to  rafts   upon  stimulation  (Burack  et  al.,  2002).  

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necessarily  for  interaction  with  a  ligand.  However,  a  more  recent  publication  suggests  a   self-­‐‑peptide  MHC  interaction  with  the  pre-­‐‑TCR.  The  authors  of  this  work  propose  that  in   addition  to  the  autonomous  signalling  mechanism,  there  is  also  an  interaction  of  the  pre-­‐‑

TCR   with   the   pMHC   or   other   ligands,   that   tunes   selection   of   β-­‐‑chains   to   be   used   by   αβTCR-­‐‑expressing  DP  thymocytes  (Mallis  et  al.,  2015).  In  agreement  with  a  continuous   signalling  mode  of  the  pre-­‐‑TCR,  it  has  been  shown  that  the  receptor  is  internalized  and   routed  constitutively  to  lysosomes  in  absence  of  deliberate  stimulation,  similar  to  ligand-­‐‑

induced  internalization  of  αβTCRs  on  mature  T  cells  (Carrasco  et  al.,  2003;  Panigada  et   al.,   2002;   Valitutti   et   al.,   1997).   This   observation   can   also   explain   the   low   surface   expression   of   pre-­‐‑TCRs   in   DN   thymocytes.   Finally,   there   are   others   mechanisms   proposed   for   the   ligand-­‐‑independent   signalling   such   a   low   threshold   signalling   of   the   DN  thymocytes  compared  with  DP  thymocytes  (Haks  et  al.,  2003)  or  their  constitutive   localization   in   raft   domains   (Saint-­‐‑Ruf   et   al.,   2003).   Lipid   rafts   are   membrane   microdomains,   which   are   enriched   in   sphingolipids,   cholesterol   and   numerous   signalling  molecules,  including  Lck  or  LAT.  It  is  suggested  that  the  lipid  raft  content  is   elevated   in   DN   thymocytes,   which   could   facilitate   pre-­‐‑TCR   signalling   through   increasing   the   frequency   or   duration   of   the   interaction   of   the   pre-­‐‑TCR   complex   with   signalling   molecules   (Yamasaki   and   Saito,   2006).   As   has   been   exposed   above,   many   hypotheses  have  been  proposed  but,  unfortunately,  the  complete  structural  basis  for  the   oligomerization   of   pre-­‐‑TCRs   and   the   mechanisms   allowing   continuous   signalling   are   still   unclear   and   further   studies   are   required.   One   of   the   main   objectives   in   the   work   presented  in  this  thesis  is  the  better  understanding  of  the  pre-­‐‑TCR  clustering  and  its  role   in  pre-­‐‑TCR  function.    

Altogether,  correct  temporal  regulation,  stoichiometry  and  assembly  of  the  pre-­‐‑TCR  and   TCR  are  processes  required  to  generate  functional  pre-­‐‑TCR  and  TCR  complexes  that  are   essential  in  the  T  cell  development  and  T  cell  function.    

TCR and pre-TCR signalling

The   biochemical   events   activated   downstream   of   the   TCR   after   its   engagement   were   initially  identified  in  mature  T  cells  and  involve  not  only  the  regulation  of  a  number  of   protein  tyrosine  kinases  (PTKs)  and  the  phosphorylation  of  their  substrates,  but  also  the  

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activation  of  several  protein  tyrosine  phosphatases  (PTPases)  (Fayard  et  al.,  2010).  

The  earliest  step  in  intracellular  signalling  following  TCR  ligation  is  the  activation  of  Src   (Lck  and  Fyn)  PTKs,  leading  to  phosphorylation  of  the  CD3  ITAMs.  The  CD45  receptor   tyrosine  phosphatase  modulates  the  phosphorylation  and  activation  of  Lck  and  other  Src   kinases  that  in  turn  promote  the  recruitment  of  the  Zeta-­‐‑chain  associated  protein  kinase   (Zap-­‐‑70)   to   the   TCR/CD3   complex   where   it   becomes   active   and   phosphorylates   the   adaptor  proteins  LAT  and  SLP-­‐‑76.  These  two  adapters  form  the  backbone  of  the  complex   that  organizes  effector  molecules  in  the  correct  spatiotemporal  manner  to  allow  for  the   activation  of  multiple  signalling  pathways  (Figure  4).    

Phosphorylation  of  SLP-­‐‑76  promotes  recruitment  of  Vav  (a  guanine  nucleotide  exchange   factor),   the   adaptor   proteins   NCK   and   GADS,   and   an   inducible   T   cell   kinase   (Itk).  

Phosphorylation   of   phospholipase   Cγ1   (PLCγ1)   by   Itk   results   in   the   hydrolysis   of   phosphatidylinositol   4,5-­‐‑bisphosphate   (PIP2)   to   produce   the   second   messengers,   diacylglycerol   (DAG)   and   inositol   trisphosphate   (IP3).   These   two   messengers   are   essential   for   T   cell   function.  The   production   of   DAG   results   in   the   activation   of   two   major  pathways:  the  PKCθ  and  the  MAPK/Erk  pathways,  both  promoting  transcription   factor  NF-­‐‑κB  activation.  NF-­‐‑κB  is  translocated  into  the  nucleus,  where  it  activates  genes   involved   in   the   function,   survival,   and   homeostasis   of   T   cells.  On   the   other   hand,   IP3   triggers  the  release  of  Ca2+  from  the  endoplasmic  reticulum  (ER),  which  promotes  entry   of  extracellular  Ca2+  into  cells  through  calcium  release-­‐‑activated  Ca2+  (CRAC)  channels.  

Calcium-­‐‑bound  calmodulin  (Ca2+/CaM)  activates  the  phosphatase  calcineurin,  which  in   turn  promotes  the  activation  of  the  nuclear  factor  of  activated  T  cells  (NFAT).  Feedback   regulation   at   several   points   within   these   pathways   allows   for   different   outcomes,   depending   on   the   cell   type   and   environment.   The   incorporation   of   signals   from   additional   cell   surface   receptors   (such   as   CD28   or   LFA-­‐‑1)   further   regulates   cellular   response  (Malissen  and  Bongrand,  2015;  Smith-­‐‑Garvin  et  al.,  2009).  

The  pre-­‐‑TCR  signalling  in  immature  T  cells  occurs  through  pathways  that  are  similar  to   signalling   after   ligation   of   the   αβTCR    in   mature   T   cells   (Michie   and   Zuniga-­‐‑Pflucker,   2002).   However,   despite   activation   of   the   same   transcription   factors,   the   consequences  

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are  different  and  this  probably  reflects  the  differential  accessibility  of  specific  genes  in   lymphocytes  that  are  at  different  stages  of  maturation.    

Figure  4.  Pre-­‐‑TCR  signalling.  

Only  signalling  via  the  pre-­‐‑TCR  complex  has  been  represented,  but  signalling  through  the  mature   TCR  is  similar  to  pre-­‐‑TCR  signalling  as  discussed  in  the  main  text.  Asterisk  (*)  close  to  protein   names,  indicates  that  in  the  absence  of  that  particular  molecule,  T  cell  development  is  partially  or   totally  arrested  

TCR triggering models: TCR clustering and conformational change  

TCRs  on  the  membrane  of  T  cells  are  able  to  bind  to  pMHC  on  the  surface  of  the  antigen   presenting   cells   (APC).   The   T   cells   are   able   to   identify   a   small   numbers   of   foreign   antigenic  peptides  in  a  context  of  thousands  of  self-­‐‑pMHC  in  a  rapid,  highly  sensitive   and  selective  way.  Moreover,  each  TCR  is  cross-­‐‑reactive;  i.e,  it  is  capable  to  interact  with   a   small   spectrum   of   pMHC   ligands   with   different   affinities   in   the   1-­‐‑200   µμM   range.   A   given  TCR  is  capable  of  triggering  a  diverse  range  of  biological  responses  according  to   the  affinity  for  the  pMHC  ligand  that  it  encounters  (Hogquist  and  Jameson,  2014).  This  is   at  the  basis  of  self-­‐‑nonself  discrimination  in  both  the  thymus  and  the  periphery.    

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In   the   periphery,   naive   T   cells   are   continuously   subjected   to   weak   chronic   TCR   interactions   with   endogenous   self   pMHC   that   maintain   the   T   cells   in   a   state   of   heightened   antigenic   reactivity,   whereas   stronger   interactions   with   foreign   agonist   pMHCs  induce  the  T  cells  full-­‐‑fledged  activation  (Malissen  and  Bongrand,  2015).  A  T  cell   also   needs   to   discriminate   between   foreign   pMHCs   and   self   pMHC   molecules   even   though  the  affinity  and  the  kinetics  of  binding  to  self  and  foreign  pMHCs  and  also  the   differences   in   the   molecular   sequence   of   the   peptides   are   not   large   (Chakraborty   and   Weiss,  2014).    

The   mechanisms   by   which   information   is   passed   from   the   TCR   antigen   binding   site   through   the   plane   of   the   membrane   and   results   in   the   earliest   events   of   T   cell   activation   is   termed   TCR   triggering   (Malissen   and   Bongrand,   2015).   These   earliest   events   must   reflect   the   remarkable   and   above-­‐‑described   features   of   the   TCR:  

high   sensitivity   and   selectivity   for   agonist   pMHC   (summarised   in   Table   2).   A   number   of   competing   models   for   receptor   triggering   has   been   proposed,   driven   in   part   by   persisting  uncertainties  about  the  stoichiometry  of  the  complex  and  the  arrangement  of   its  components.  From  our  point  of  view,  there  are,  so  far,  two  main  mechanisms  that  are   able   to   enclose   the   specific   characteristics   of   the   TCR.   These   triggering   models   are   the   conformational  change  and  the  TCR  clustering  and  both  are  at  the  basis  of  the  kinetic   segregation   model,   in   which   T   cell   activation   is   made   possible   by   the   segregation   of   proteins   with   large   extracellular   domain,   such   as   CD45,   from   areas   of   the   membrane   where  the  TCR  and  other  proteins  with  shorter  extracellular  domains  are  accumulating,   and  the  mechanoreceptor  model  in  which  a  force  tangential  to  the  cell  surface  is  applied   to  the  ligand  binding  site  of  the  TCR  promoting  a  rotation  of  the  complex  that  in  turns   allows  the  exposure  of  the  ITAMs  of  the  CD3ε  ectodomains  in  a  piston-­‐‑like  manner.  

CONFORMATIONAL  CHANGE   TCR  CHARACTERISTICS  

   

Very  sensitive.  TCR  can  recognize  1–10   agonist  pMHC  in  a  sea  of  103-­‐‑104  times   more  of  self  pMHC    

High  molecular  specific  

Wide  dynamic  range  in  their  response  to   antigen  (1-­‐‑200uM  range)  

Low  affinity  for  MHC  (1-­‐‑10µμM)   Short  half-­‐‑life  of  the  TCR-­‐‑pMHC   interaction  (t1/2≈15-­‐‑0.1s)  

Table   2.   Summary   of   TCR   characteristics.  

 

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The  conformational  change  model  is  based  on  the  allosteric  control,  in  which  binding  of   a  ligand  at  one  site  of  a  protein  affects  a  distant  functional  site  through  a  conformational   change.  Studies  focused  on  the  understanding  of  the  triggering  mechanism  of  the  TCR   have   documented   that   conformational   changes   could   be   implicated   in   the   outside-­‐‑in   transmission   of   the   TCR   signal.   Three   conformational   changes   have   been   proposed   as   mechanisms  for  TCR-­‐‑inducible  ITAM  phosphorylation:  Two  separate  but  not  mutually   exclusive  conformational  changes  within  the  CD3  cytoplasmic  tails  and  a  third  involving   the  TCRαβ  heterodimer.    

The  first  mechanism  stems  from  the  observation  that  the  cytoplasmic  tails  of  CD3ε  and   CD3ζ   fold   or   interact   with   the   lipid   of   the   inner   leaflet   of   the   plasma   membrane   preventing   the   phosphorylation   of   the   ITAMs.   The   cytoplasmic   tail   of   the   CD3ζ   chain   might  convert  from  a  lipid-­‐‑bound  helical  structure  to  an  unfolded  structure  upon  TCR   triggering   (Aivazian   and   Stern,   2000;   Sigalov   et   al.,   2006;   Xu   et   al.,   2008;   Zhang   et   al.,   2011).   Therefore,   upon   assembly   of   the   TCR-­‐‑CD3   complex,   the   CD3ζ   juxtamembrane   regions   are   forced   apart   adopting   an   inactive   conformation.   TCR   engagement   then   triggers  a  conformational  change,  where  the  CD3ζ  cytosolic  juxtamebrane  regions  move   together  acquiring  an  active  conformation  (Gagnon  et  al.,  2012).  The  group  of  Michael  S.  

Kuhns   (Lee   et   al.,   2015)   suggested   that   the   structural   features   most   likely   to   facilitate   these   changes   are   the   transmembrane   domain   charge   interactions   between   CD3ζζ   and   TCRαβ    and  that  this  region  could  serve  as  a  pivot  point  around  which  the  interacting   subunits  could  move  without  risk  of  separation    

A   second   mechanism   involves   the   CD3ε   chain.   Upon   TCR   ligation   a   proline   rich   sequence  (PRS)  in  CD3ε  is  exposed  and  is  available  to  recruit  the  adaptor  protein  Nck   via  one  of  its  SH3  domains  (Gil  et  al.,  2002).  This  so-­‐‑called  active  conformation  can  also   be  detected  by  a  monoclonal  antibody  (Apa1/1)  specific  for  the  intracellular  domain  of   CD3ε  (Salmerón  et  al.,  1991).  In  relation  with  this  data,  some  amino  acids  residues  were   identified  in  the  membrane-­‐‑proximal  stalk  region  of  the  CD3ε,  in  close  proximity  to  or   part  of  an  evolutionary  conserved  CXXC  motif,  that  were  critical  for  the  formation  of  the   rigid  stalk  conformation  (Wang  et  al.,  2009).  Single  mutation  of  two  amino  acid  residues   (Lys76  and  Cys80)  where  seen  to  change  particularly  their  conformation  in  the  bound  vs  

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unbound   status.   Mutation   in   both   residues   inhibited   or   abrogated   the   adoption   of   the   active  conformation  by  the  PRS  of  CD3ε  and  prevented  T  cell  activation  upon  antibody   or   pMHC   triggering   (Martinez-­‐‑Martin   et   al.,   2009)   thus   suggesting   that   these   residues   are  important  in  the  transmission  of  outside-­‐‑in  information.    

Finally,  the  crystal  structure  of  the  αβLC13  TCR  showed  a  shift  in  the  AB  loop  of  the  Cα   domain   induced   by   the   pMHC   ligand   binding.   These   data   lead   to   propose   that   the   transmission   of   the   outside-­‐‑in   signalling   may   rely   on   conformational   changes   that   reoriented  αβTCR  in  relation  to  the  CD3  components  (Beddoe  et  al.,  2009;  Kjer-­‐‑Nielsen  et   al.,  2003).  Importantly,  all  these  conformational  changes  occur  prior  to  CD3ζ  and  CD3ε   ITAM  phosphorylation  and  the  maintenance  of  the  conformational  change  depends  on   continued  ligand  binding  (Minguet  et  al.,  2007).  

During   T   cell   development,   only   high   affinity-­‐‑negative   selection   inducing   interactions   gave  rise  to  a  conformational  change  (Risueño  et  al.,  2006).  This  discriminating  capacity   is  developmentally  regulated  as  in  preselection  thymocytes  both  low  and  high  affinity   pMHC   ligands   induced   the   conformational   change,   whereas   in   positive   selected   thymocytes   and   mature   T   cells   only   high   affinity   ligands   induced   the   conformational   change  (Gil  et  al.,  2008;  2005).  In  this  regard,  the  work  of  (Blanco  et  al.,  2014)  make  clear   that  the  conformational  change  is  also  a  necessary  step  during  αβ  T  cell  development.  A   mutation   in   the   Cys80   (C80G)   of   the   cytoplasmic   tail   of   the   CD3ε   abrogates   the   T   cell   development  at  the  DN3  level.  These  data  reflect  the  absolute  dependence  of  pre-­‐‑TCR   signalling  on  adopting  the  active  conformation.    

TCR  CLUSTERING  

Independent   groups,   using   biochemical,   high   resolution   light   microscopy   and   electron   microscopy   techniques,   have   reported   that   TCR   clusters   are   not   only   induced   upon   ligand  binding  but  that  they  are  already  present  before  antigen  encounter  (Lillemeier  et   al.,  2010;  Schamel  et  al.,  2005;  Sherman  et  al.,  2011;  Zhong  et  al.,  2009).  These  oligomers   may   reach   a   size   of   up   to   20   units   and   will   be   referred   to   as   TCR   nanoclusters.   A   functional   explanation   for   the   existence   of   these   nanoclusters   is   that   the   TCR   oligomerization  allows  the  propagation  of  the  activating  signalling  from  ligand  bound  

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