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Exploring  the  function  of  IL -­10,  BTLA  and  
DCs  as  regulators  of  immune  responses  
 
 
 
 
 
 
 
 
 
 
 
 
Dissertation  
Zur  Erlangung  des  Grades  
Doktor  der  Naturwissenschaf  t
 
 
 
 Am  Fachbereich  Biolog  ie
der  Johannes  Gutenberg -­‐Universität  Mainz  
 
   
Nir  Yogev  
 
geb.  am  31.  Dezember  1973  in  Kibutz  Kineret,  Isr  ael
 
 
Mainz,  2010  ABBREVIATIONS................................ ................................ ................................ ................................ ..IV  
1   INTRODUCTION ............................... 1  
1.1   INTERLEUKIN -­‐10 ........... 4  
1.1.  1 IL-­10  protein,  gene  and  expression4  
1.1.  2 IL-­10  receptor  and  signaling ...........................................................................5  
1.1.  3 IL-­10R  signal  transduction...............6  
1.1.  4 IL-­10  Function........6  
1.1.4.1   Antigen  presenting  cells ............ 6  
1.1.4.2   T  cell................................s ................. 7  
1.1.  5 IL-­10  in  infectious  disease8  
1.1.  6 IL-­10  in  autoimmunity.......................9  
1.1.  7 IL-­10  and  experimental  autoimmune  encephalomyelitis.................10  
1.2   B  AND  T  L YMPHOCYTE   ATTENUATOR ................................ ................................ ................................ ....11  
1.2.  1 Co -­receptors  of  the  CD28  and  TNFR  superfamilies.............................12  
1.2.  2 BTLA  structure,  expression  and  signaling...............13  
1.3   DENDRITIC  CELLS ................................ ........15  
2   MATERIALS  AND  METHOD S................................ ................................ ................................ .....17  
2.1   CHEMICALS  AND  BIOLOGI CAL  MATERIAL 17  
2.2   M OLECULAR  BIOLOGY 19  
2.2.  1 Competent  cels  and  isolation  of  plasmid  DNA......19  
2.2.  2 Isolation  of  genomic  DNA  from  ES  cells  and  mouse  organs.............19  
2.2.  3 RT -­PCR  and  quantitative  real -­time  PCR ...................................................20  
2.2.  4 Agarose  gel  electrophoresis  and  DNA  gel  extraction.........................20  
2.2.  5 DNA  sequencing ..................................................................20  
2.2.  6 Quantification  of  DNA......................21  
2.2.  7 Polymerase  Chain  Reaction  (PCR)..............................21  
2.2.  8 Southern  blot  analysis22  
2.3   CELL  BIOLOGY .............. 23  
2.3.  1 Embryonic  stem  cel  culture ..........................................23  
2.3.  2 Tat -­Cre  protein  (HTNC)  treatment.............................................................24  
2.3.  3 EL-­4  cell  line  culture  and  electroporation...............24  
2.3.  4 Preparation  of  Mouse  Embryonic  Fibroblasts  (MEFs).......................25  
2.3.  5 Preparation  of  c ells  from  lymphoid  organs............25  
2.3.  6 Culture  of   ex  vivo  lymphocytes.....26  
2.3.  7 Cel  counting .........................................26  
2.3.  8 Adoptive  T  cel  transfer  and  CFSE  labeling.............26  
2.3.  9 Flow  Cytometry...................................27  
2.3.10   Flow  Cytometry  and  Intracelular  Cytokine  Staining  (ICS)...........28  
2.3.11   Magnetic  cel  sorting  and  FACS  sorting.................29  
2.3.12   Isolation  of  CNS  infiltrates29  
2.3.13   Isolation  of  splenic  DCs..................30  
2.3.14   Induction  of  CD11c -­CreER  activity   in  vivo..........................................30  T
2.3.15   In  vitro  2D2 -­iTreg  differentiation  and  analysis..30  
2.3.16   T  and  iTreg  in  vitro  Differentiation ......................................................30  eff
2.4   IN  VIV  DOEPLETION  OF  CD25+  CELLS .....31  
2.5   IN  VIV  DOEPLETION  OF  DENDRITIC  CELLS .............................. 31  
2.6   TISSUE  PREPARATION  FOR  IMMUNOH ISTOCHEMISTRY ........32  
2.6.  1 Flow -­Cytomix ........................................32  
2.6.  2 ELISA........................32  
II  2.7   M OUSE  E XPERIMENTS ................................ ................................ ................................ ............................... 32  
2.7.  1 Induction  and  assessment  of  EAE................................32  
2.7.  2 Induction  and  assessment  of  Colitis............................33  
2.7.  3 MCMV  and  MHV  Infection ...............................................33  
2.8   STATISTICS ................... 34  
2.9   M ICE ................................ .............................. 34  
3   RESULTS .......... 35  
3.1   INTERLEUKIN -­‐10 ................................ ........35  
3.2   BTLA ............................ 57  
3.2.  1 Generation  of  BTLA  over  expressing  mouse............................................57  
3.2.  2 BTLA  over  expression  by  dendritic  cels...................62  
3.2.  3 BTLA  over  expression  by  T  cels ...................................70  
3.3   DENDRITIC  CELLS ........82  
4   DISCUSSIO................................N ...99  
4.1   INTERLEUKIN -­‐10 ........99  
4.2   B  AND  T  L YMPHOCYTE   ATTENUATOR ................................ .101  
4.3   DENDRITIC  CELLS ................................ .....105  
5   REFERENCES ................................ ................................ 109  
6   SUMMARY ................................ .....130  
7   ZUSAMMENFASSUNG ................ 131  
8   ACKNOWLEDGEMENTS ................................ ................................ ............ 132  
9   LEBENSLAUF 133  
10   PUBLICATIONS ................................ ......................... 134  
11   ERKL ÄRUNG ................................ ................................ .............................. 135  
 
 
 
 
 
 
 
III  Abbreviations    
 
Ab       antibody    
ALT              al  a  ni  ne  transaminase  
APC     antigen  presenting  cell  or  allophycocyanin    
approx.     approximately    
BAC     bacterial  artificial  chromosome  
Bio     biotinylated    
β-­‐ME     β-­‐mercaptoethanol    
bp       base  pair    
BMDCs          bone  marrow  derived  dendritic  cells    
BSA       bovine  serum  albumin    
°C       temperature  in  celsius  degrees    
CD       cluster  of  differentiation    
CFA       Complete  Freund’s  Adjuvant    
CFSE              c  arboxyfluorescein  diacetate  succinimidyl  ester  
cDNA     complementary  DNA    
CNS       central  nervous  system    
cpm       counts  per  minute  
Cre       site-­‐specific  recombinase  (causes  recombination)    
d       day/s    
d.p.i              d  ay    s    post  immunization  
DC       dendritic  cel  l  
DMEM     Dulbecco’s  modified  Eagle  medium    
DN              d    oub  l  e  nega   tive  
DNA     desoxyribonucleic  acid    
dNTP     desoxynucleotide  triphosphate  
DP              dou    b  l  e    positive    
DTA     Diphtheria  toxin    A  
DTT                      dithiothritol  e  
DTR     Diphtheria  toxin  receptor    
Dtx     Dipht  toxin    
EAE       experimental  autoimmune   encephalomyelitis    
EDTA     ethylene-­‐diaminetetraacetic  acid    
ELISA     enzyme -­‐linked  immuno -­‐sorbent  assay    
ES     embryonic  stem    
EtOH     ethanol    
FACS     fluorescence  activated  cell  sorting    
FCS       fetal  calf  seru  m  
Fig.     Figure    
FITC     fluorescein  isothiocyanate    
Flp     site-­‐specific  recombinase,  product  of  yeast  FLP1-­‐gene    
FoxP3     forkhead  box  protein  3    
FRT       Flp  recombination  target    
IV  gr       gram  
hr       hour/s    
HEPES     N-­‐2-­‐hydroxyethylpiperazine-­‐N’-­‐2-­‐ethansulfonic  acid    
iDTR     inducible  Diphtheria  toxin  receptor    
i.p.       intraperitoneally    
i.v.       intravenously    
ICS                         intracellular  staining  
IFN-­‐γ   interferon-­‐γ  
Ig       immunoglobulin    
IL     interleukin    
kb       kilobase  pair  
l       liter    
LN       lymph  node/s    
P     recognition  sequence  for  Cre  (locus  of  -­‐ing  over  of  phaX ge  P1 )    
LPS     lipopolysaccharide    
Ly6C     lymphocyte  antigen  6  complex,  locus  C    
M       molar    
MACS     magnetic  activated  cell  sorter    
MFI       mean  fluorescence  intensity    
MgCl2   magnesium  chloride    
MHC     major  histocompatibility  complex    
min       minute    
ml       millilite  r  
mM       millimolar  
MOG     myelin  oligodendrocyte  glycoprotein    
mRNA     messenger  RNA    
MS       multiple  sclerosi  s  
M Φ     macrophage/s    
NaCl       sodium  chloride    
n       nano    
NaOH     sodium  hydroxide    
neo       neomycin  resistance  gene  
ng       nanogram    
o/n       over  night  
OD       optical  density  
pDC       Plasmacytoid   dendritic  cell      
PBS       phosphate  buffered  saline    
PCR       polymerase  chain  reaction  
PE       phycoerythrine    
Ptx     Pertussis  toxin    
RAG       recombinase  activating  gene    
RNA     ribonucleic  acid    
rpm       revolutions  per  minute    
RT       room  temperature    
sec       seconds    
SA       streptavidine    
V  
loxs.c.       subcutaneously    
sc       spinal  cord    
SDS       sodium  dodecyl  sulfate    
SN       supernatant    
SP                         single  positive    
SSC     sodium  chloride/sodium  citrate  buffer  
TAE       Tris -­‐acetic  acid-­‐EDTA  buffer    
TAM     Tamoxifen    
Taq       polymerase  from   Thermus  aquaticus  
TCR       T  cell  receptor    
TE       Tris -­‐EDTA  buffer    
TEC       thymic  epithelial  ce  ll  
tg       transgenic    
TGF-­‐β   transforming  growth  factor-­‐β  
Th       helper  T  cel  ls  
T     effector  T  cel  ls  eff
Tregs     regulatory  T  cell  s  
Tris       2-­‐amino -­‐2-­‐(hydroxymethyl-­‐)1,3 -­‐propandiole    
U       units    
UV       ultraviolett    
V       volts    
vs       versus    
v/v       volume  per  volume    
w/v       weight  per  volume    
WT       wild  type  
µg       microgram  
µl       microliter    
µM       micromolar    
3’       three  prime  end  of  DNA  sequences    
5’     five  prime  end  of  DNA  sequences  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
VI  1 INTRODUCTION  
 
 
Immune   homeostasis   depends   on   the   existence   of   equilibrium   between  
responses  that  control  infection  and  tumour   growth,  and  reciprocal  responses  that  
prevent   inflammation   and   autoimmune   diseases.   Protection   against   infection   is  
fundamental  to  the  survival  of  all  multicellular  organisms  and  is  mediated  by  the  
immune  system,  which  has  evolved  both  innate  and  adoptive   mechanisms  to  deal  
with  invading  microorganisms.  The  effector  mechanisms  used  by  the  host  to  control  
infection   include   production   of   proinflammatory   cytokines   and   chemokines,  
recruitment  of  inflammatory  cells  to  the  site  of  infection  and  activation  of  cytotoxic  
T  lymphocytes  (CTL)  and  natural  killer  (NK)  cells,  which  lyse  infected  host  cells.  
Although  these  responses  help  to  eliminate  or  slow  the  spread  of  the  pathogen,  if  
they  are  not  tightly  controlled,  they  can  result  in  severe  infon  lammand  ticollatera l  
tissue  damage   (Artavanis -­‐Tsakonas  et  al.,  2003) .  
As  the  cells  and  molecules  of  thimmue  ne  system  that  respon d  to  pathogen-­‐
derived  antigens  might  also  respond  to  self-­‐antigens,  autoimmune  disease  can  result  
if   this   reactivity   is   not   tightly   cont(von  rolled  Herrath   and   Harrison,   2003).  
Therefore,   inflammation   and   immune   response   to   pathogens   are   regulated   by  
various  host  suppressor  mechanisms,  including  the  production  of  anti -­‐inflammato ry  
cytokines,  co -­‐inhibitory  signals  and  regulatory  cel  ls.
 
+CD4  T  cells,  also  known  as  T  helper  (Th)  cells,  play  an  important  role  in  
orchestrating  adaptive  immune  responses  to  various  infectious  agents.  They  are  
also  involved  in  the  induction  of  autoimmun e  and  allergic  diseases.  Upon  T  cell  
receptor  (TCR)-­‐mediated  cell  activation,  naïve  CD4  T  cells  can  differentiate  into  at  
least   four   major   lineages,   Th1,   Th2,   Th17   and   iTregwh   cielchls  par,  ticipate   in  
different   types   of   immune   responses.   Networks   of   cytokines   and   transcription  
+factors   are   critical   for   determining   C  TD4  cell  fates  and  effector  cytokine  
production.  The  major  determinant  for  Th  cell  differentiation  is  the  cytokine  milieu  
1  at  the  time  of  antigen  encounter,  although  the  nature  of  cognate  antign  e and  its  
affinity  to  the  TCR  as  well  as  the  available -­‐st  icomulants,  many  of  which  regulate  
initial  cytokine  production,  can  influence  Th  cell  fa   te.
Interleukin  10  -­‐(IL10)  is  a  cytokine  that  modulates  both  innate  and  adoptive  
immunity,  primarily  by  exertng  i anti-­‐inflammatory  effects.  IL-­‐10  is  produced  by  a  
+variety  of  cell  types  of  both  innate  and  adoptive  origin.  During  many  infections,  CD4  
T  cells  produce  both  IFNγ  and  IL -­‐10,  as  the  -­‐10  IL produced  by  effector  Th1  cells  
helps  limit  the  collateral  damagcea  used  by  exaggerated  inflammation.  However,  
this  control  may  limit  the  effectiveness  of  the  immune  response,  resulting  in  a  
failure  to  fully  eliminate  pathoge  ns.
 
The   specific   function   of   an   individual’s   immune   system   in   different  
physiological  and  pathological  setting  is  regulated  by  the  action  of  opposing  factors  
or  systems.  Common  examples  are  the  polarization  of  T  helper  cells  into  Th1  and  
Th2  subsets,  and  the  balance  between  effector  T-­‐cell  (T)  activation  and  regulatory  eff
T-­‐cell  (T)  activation.  At  the  molecular  level,  -­‐sctoimulatory  members  of  the  B7  and  reg
TNF   superfamilies   can   have   both   stimulatory   and   inhibitory   effect   on  -­‐Tcell  
activation.  
Co -­‐signalling   molecules   are   ce-­‐lsulrface   glycoproteins   that   can   direct,  
modulate   and   fine -­‐tune   T-­‐cell   receptr  o signals.   On   the   basis   of   their   functional  
outcome,   co -­‐signalling   molecules   can   be   divided   into   co-­‐stimulators   and   co -­‐
inhibitors,  which  promote  or  suppress  T  cell  activation,  respectively.  By  expression  
at  the  appropriate  time  and  location,  co -­‐signalling   molecules  control  the  priming,  
growth,  differentiation  and  functional  maturation  of  a  T  cell  response  .
Traditionally,  co-­‐signalling  molecules  were  termed  as  receptors  or  ligands  to  
distinguish   the   direction   of   signal   transmission.   However   this   nomenclatu  ire s  
entirely  operational  and  does  not  reflect  the  intrinsic  nature  of  these  molecules.  A  
ligand  could  be  either  a  -­‐csotimulator  or  co-­‐inhibitor,  depending  on  the  specific  
receptor  it  interacts  with:  for  example,  the  binding  of  CD80/CD86  to  CD28  transmit  
a  co -­‐stimulatory  signal,  whereas  the  binding  of  CD80/CD86  to  CTLA4  transmit  a-­‐  co
2  inhibitory  signal.  Moreover,  a  ligand  could  become  a  receptor  (reverse  signalling)  as  
it   receives   a  -­‐coinhibitory   or   -­‐costimulatory   signal,   a   process   that   had   been  
described  for  CTLA4,  P-­‐D1  and  BTLA  (Dong  et  al.,  2003;  Fallarino  et  al.,  2003;  
Grohmann  et  al.,  2002;  Nguyen  et  al.,  200.  2)
Dendritic  cells  (DCs)  considered  are   as  the  most  potent  antigen  presenting  
cells  (APCs)  and  as  suc,  hplay  a  central  role  in  the  orchestration  of  the  various  forms  
of   immunity   and   tolerance.   Their   immune -­‐regulatory   role   relies   mainly   on   the  
ligation  of  specific  receptors  that  initiate  and  modulate  DC  maturation,  resulting  in  
the   development   of   functionall y   different   effector   DC   subsets   that   selectively  
promote  Th -­‐1,  Th-­‐2,  Th-­‐17  or  Treg  cell  responses.  
 
 
This  thesis  focuses  on  different  aspects  of  immune  regulation,  both  at  the  
cellular   and   molecular   levels.   More   specifically,   this   work   concesn  otraten  the  
importance  of  Interleukin-­‐10,  B  and  T  Lymphocyte  Attenuator  (BTLA),  and  dendritic  
cells  in  respect to    immune  regulation,  with  special  emphasis  on  autoimmunity.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
3  1.1 Interleukin -­10  
 
Interleukin -­‐10  (I-­‐L10)  is  a  cytokine  that  modulates  both  innate  nd  a adaptive  
immunity,  primarily  by  exerting  ant-­‐iinflammatory  effects.  Originally  identified  as  a  
Th2  cell  derived  fact(Fiorentor   ino  et  al.,  1989),  IL-­‐10  was  later  found  to  be  secreted  
by  a  variety  of  haemopoietic  cell  types  including  activated  macrophages   (de  Waal  
Malefyt  et  al.,  1991;  Maeda  et  al.,  ,  19dendr95)itic  cells  (Anderton  et  al.,  2002;  
Iwasaki  and  Kelsall,  1999;  Khanna  et  al.,  2000;  McGuirk  et  al.,  2002;  Mizoguchi  et  al.,  
2002;  O'Garra  et  al.,  1992;  Stumbles  et  al.,  1B9  9ce8l)ls  and  mast  cel(Mlsasuda    et  
al.,  2002.  )IL-­‐10  is  also  produced  by  regulatory  T  cells  (Tregs),  Th1  (O'Gacelrral  s  
and  V ieira,  2007;  Trinchieri,   ,  and2007)  Th17  cells(  Awasthi  et  al.,  2007;  Fitzgerald  
et  al.,  2007;  McGeachy  et  al.,  2007;  Stumhofer  et  al.,  as2  0we07)ll  as  Th9  ce  lls
(Dardalhon  et  al.,  2008;  Veldhoen  et  a.  lIn  .,  2addition,  008) keratinocytes  can  be  
induced  to  secrete  IL-­‐10  by  contact  allergen  or  UV  irradiati(Pinton   o  et  al.,  2006) .  
1.1.1 IL-­10  protein,  gene  and  expression  
IL-­‐10  protein  is  a  homodimer  composed  of  two  interpenetrating  polypeptide  
chains,   similar   to   interferon   gamma   (IFγN)  (Syto   et   al.,   1998;   Walter   and  
Nagabhushan,  1995).  IL-­‐10  open  reading  frames  (ORF)  encode  a  secreted  protein  of  
178  amino  acids  with  rather  a  we-­‐lclonserved  sequence  of  about  73%  homology  
shared  by  human  and  mice.  
The   murine   IL-­‐10  (mIL -­‐10)   gene   is   encoded   in   five   exons,   located   on  
chromosome  1   (Kim  et  al.,  199.  2Ac)tivation  of  IL-­‐10  gene  expression  results  in  a  1.4  
kb  mRNA,  which  can  be  regulated  by  the  transcription  factors  Sp1  and  Sp(Tone  3   et  
al.,  2000  a)s  well  ast    athe  posttranscriptional  levels  (Powell  et  al.,  2000),  indicating  
that  the  IL-­‐10  gene  is  transcribed  to  some  degree  constitutively  and  subject  to  
control  by  alteration  of  posttranscriptional  RNA  degradation  mechanis ms.  
It  has  been  suggested  that  Toll  Like  Receptor  (TL-­‐2  R)agonists  are  specialized  
in  inducing  IL-­‐10  expression  by  antigen  presenting  cell(s  Agrawal  et  al.,  2003;  Dillon  
et  al.,  2004;  Hu  et  al.,  2006;  Netea  et  a)l.  .,  IL-­‐200410  production  is  also  induced  by  
TLR4,  TLR9  and  TLR3  ligands   (Boonstra  et  al.,  2006) .  Following  TLR  stimulation,  
4