Journal  Club  20-­‐June-­‐2014  

Background  

Victora,  G.  D.,  &  Nussenzweig,  M.  C.  (2012).  Germinal  Centers.  Annu.  Rev.  Immunol.,  30(1),  429–457  

Are  the  extent  of  clonal  expansion  and  hypermuta@on  regulated  druing  interzonal  germinal  center  cycles?  

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Does  the  amount  of  AG  internalized  by  GC  B  cells  steer  clonal  expansion?  

HC  specific  for  4-­‐hydroxy-­‐3-­‐nitrophenyl  acetyl  (NP)  with  Igλ  LC.    Transferred  #  1.5-­‐5x106  (about  10%  NP  specific)  

10ug   popliteal  lymph  node  

from  spleen  

Ova  

αDEC205-­‐OVA  

B1-­‐8hi  

DEC205+/+  (CD45.1+)  

B1-­‐8hi  

DEC205-­‐/-­‐  (CD45.1+  

CD45.2+)  

5% 95% α  DEC205-­‐CS  Plasmodium  falciparum  circumsporozite  protein-­‐>  irrelavant  AG  

CS  

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50ug  

25  ug  

10ug  into  footpads  

AG  amount  and  capture  regulates  GC  B  cell  expansion  

Fig1  

10ug   lymph  node  

from  spleen  

Suppl  Fig1  B1-­‐8hi  DEC205+/+  

B1-­‐8hi  DEC205-­‐/-­‐  

Fig1  

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AG  amount  and  capture  regulates  GC  B  cell  expansion  

Fig1  

GC  

DZ  

LZ  

Increased  amount  of  cognate  AG  presented  by  GC  B  cell  subset  to  Tb  cells  leads  to  their  selec@ve  expansion  at  expense  of  GC  B  cells  that  present  less  AG.  

B220+ FAS+ CD38- CD86- CXCR4+

B220+ FAS+ CD38- CD86+ CXCR4-

B220+ FAS+

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T-­‐cell  help  regulates  the  number  of  GC  B-­‐cell  divisions  

Suppl2  

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B1-8hi tTA-H2B-mCh

36h or 84h DOX 1.6mg ip and 0.2mg in hind footpad 2mg/ml in drinking water (+10mg/ml sucrose)

B220+ lymphocytes of peripheral blood

T-­‐cell  help  regulates  the  number  of  GC    B-­‐cell  divisions  

Fig  2  

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tTA-­‐H2B-­‐mCh  can  be  used  to  monitor  cell  division  in  GC  (only  prime/boost,  no  αDEC205-­‐OVA)  

B1-­‐8hi  tTA-­‐H2B-­‐mCh  

60h  post  DOX  

! AG  capture  +  presenta@on"  !cell  division  by  GC  B-­‐cells.  Change  in  zonal  distribu@on:  aDEC-­‐OVA  targeted  GC  B  cells  almost  exclusively  in  DZ.  

72h αDEC205-Ova 36h DOX Control cells 2:1 in DZ αDEC205-Ova: 90% DZ

control=αDEC-CS and PBS

Higher  AG  capture  increased  S  phase  ini@a@on  in  DZ  of  GC  B-­‐cells  

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Fig  3  

!AG  captured  and  presented  to  TFH"  !propor@on  of  cells  ini@a@ng  S  phase  in  DZ.  GC  B  cells  that  express  !!  AG-­‐>    ini@ate  addi@onal  cell  divisions  in  DZ  before  returning  to  LZ.      

αDEC205-Ova PBS

Longer  DZ  residence  @me  during  selec@ve  expansion  of  GC  B-­‐cells  

Fig  3  

20.06.2014   9  DZ: B220+ FAS+ CD38- CD86- CXCR4+

LZ: B220+ FAS+ CD38- CD86+ CXCR4-

Increased  cell  division  in  polyclonal  GCs  -­‐>  !Ig  affinity  and  SHM  

Fig  4  

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tTA-H2B-mCh NP OVA 50ug ip + 12.5ug hind foot pad

DOX

d12.5

FACS sort mChHi + mChLo GC B cells

36h

VH 186.2 family genes analysed for high affinity anti-NP W33L mutation

!  rates  of  prolifera@on  and  !muta@on  rate  of  high-­‐affinity  GC  B  cells  in  a  polyclonal  response.  

Analyze intron downstream of JH4 for SHM (region is target for SHM but not subject to selection)

Summary  

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least  prolifera@ve  +  lowest  affinity  B  cells  are  mainly  in  LZ  

!AG  captured    -­‐> #!cell  division-­‐>    ! SHM  (!AG  affinity)  

!AG  captured  -­‐>    !   T  cell  help  to  return  to  DZ  

variable  GC  B  cell  divisions  per  DZ  cycle  (1-­‐6)  -­‐>  regulated  by  amount  of  AG  captured  in  LZ  (high  affinity  clones  outcompete  low  affinity  clones)  

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Background  

•  At  20  weeks  of  gesta@on  T  cells  in  fetus  begin  to  colonize  the  periphery  (blood,  secondary  lymphoid  organs.)  

•  So  far  the  neonatal  T  cell  compartment  was  supposed  to  only  contain  naive  T  cells  (TN)  and  Treg  for  feto-­‐maternal  tolerance  (tolerant  to  noninherited  maternal  AG  -­‐>NIMA).    

•  Fetal  T  cell  compartmet  was  thought  to  be  devoid  of  memory  T  cells  TEM  (adults:  50%  of  T  cells  in  blood  are  TEM)  

•  The  placenta  harbors  a  nonpathogenic  microbiota  and  is  not  sterile  as  previously  thought.  Aagaard,  K.,  et  al  Science  TranslaMonal  Medicine,  6(237),  237ra65–237ra65  

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Cord  blood  contains  CD4  TEM  cells  

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Fig1

Tn CD25-CD127hi

Treg CD25+CD127lo activated Tcells CD25loCD127lo

no  recent  thymic  emigrants  

CD161  -­‐>NKT  cell  marker  and  early  TH17  cell  marker  

matura@on  associated  molecules  

CD25loCD127hiCD3+CD4+

-> no maternal oringin

Neonatal  TEM  are  TCRαβ  T  cells  with  a  polyclonal  TCR  repertoire  

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Fig2

Neonatal  TEM  are  TCRαβ  T  cells  with  a  polyclonal  TCR  repertoire  

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Fig2

TCRBV  germline  genes  cluster  in  24  families  according  to  level  of  homology-­‐>  they  selected  10  for  analysis  (ca  70%  of  adult  T  cell  repertoire).  Wide  expression  of  10  Vb  genes  and  variable  CDR3  region-­‐>  polylonal  TCR  repertoire.    

TCRBVb repertoire analysis

+Immunoscope technology for CD3 length

CD3 length

fluor

esce

nce

in

tens

ity qPCR (cDNA)

Run Off (PCR product)

DNA Sequencer separated acc. to length

Neonatal  TEM  display  TH1  and  TH2  func@ons  

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Fig 3 A+B FACS sorted, 8h PMA/Ionomycin

20h anti-CD3/CD28

72h anti-CD3/CD28

n=13

TEM  cells  can  readily  secrete  TH1/TH2  cytokines.  Under  the  same  condi@ons  this  was  not  true  for  Treg  and  TN  cells  

TN

TEM

TREG

TEM-> same in adults

TEM in adults also IL-17

Chemokine  receptor  analysis  shows  a  large  variety  of  neonatal  CD4  T  cells  

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Fig4

- CXCR3 on TH1 cells - CRTH2 onTH2 cells - CCR6 on TH17 cells -  CCR4 not specific for TH2 but associated with CRTH2 in adults

9  combinatorial  phenotypes  of  chemokine  receptors  in  neonates.  Chemokine  rec.  expression  confirms  TH1  popula@on  but  does  not  discriminate  other  TH  func@ons.  

CXCR3+  35%  CCR6+  20%  (1/3  alsoCXCR3+)    

Chemokine  receptor  expression  paoern  defines  molecularly  different  cord  blood  TEM  subsets  

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Fig 5 n=3 male donors

sequential gating strategy for different chemokine rec. Rare CRTH2 population was not included

35%

10-15%

45-50%

CD45RA+

Func@onal  studies  (before)  show  Th1  and  Th2  func@ons  of  TEM.    Microarray  analysis  of  four  TEM  phenotypes,  show  close  affilia@on  of  CXCR3+  TEM  to  TH1  and  CCR6+  to  TH17.  

B+C) 24h anti-CD3/CD28

fold change rel. to TN

trend for TH2

Microarray  analysis  discriminates  between  different  TEM  subsets  

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Fig 6 24h anti-CD3/CD28

Dis@nct  gene  clusters  between  TEM  subgroups.  CCR-­‐  uplregulate  several  genes-­‐>  poten@ally  s@ll  capable  of  acquiring  different  phenotypes  (intermediate  between  TN  and  TEM?).  

Assessment  of  TH17  poten@al  for  CCR6+  TEM  cells  

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Fig7

fold change of CCR6+ TEM compared to TN

Receptor for IL-1 and IL-23

4-­‐6d  an@-­‐CD3/CD28  +/-­‐  IL-­‐1  and  IL-­‐23  

IL-­‐22  secre@on  indep.  of  IL-­‐1  and  IL-­‐23-­‐>  indicates  no  associa@on  with  TH17  response.  

Neonatal  TEM  cells  can  develop  into  TH17  cells.  

Discussion  •  Iden@fica@on  of  memory  type  CD4  T  cells  in  neonatal  cord  blood  

(CD25loCD127hi),  1-­‐3%  of  total  CD4  T  cells.    

•  Func@onal  studies:  Upon  ac@va@on  TH1  and  TH2  like  func@ons  and  also  poten@al  for  TH17  when  s@mulated  with  IL-­‐1  and  IL-­‐23.  

•  Microarray:  CXCR3+  TEM  cells  express  IFNγ  transcripts-­‐>  inflammatory  TH1  cells  early  in  life  w/o  infec@on  at  steady  state.  

•  CCR6+    TEM  cells  express  TH17  related  genes  w/o  the  secre@on  of  IL-­‐17  

•  Neonatal  TEM  are  highly  diverse.  

•  Do  TEM  develop  in  response  to  maternal  AG?  -­‐>  no  response  to  NIMA  (non  inherited  maternal  AG)  in  vitro.  

•  Do  TEM  develop  in  response  to  mild/asympoma@c  infec@on  or  vaccina@on  of  mother?  

•  Signal  for  immune  matura@on  from  commensal  bacteria  in  placenta?  

•  An@gen  Specificity/Self-­‐reac@vity?-­‐>  origin  and  role  of  those  cells?  Role  in  Vaccines  and  infec@on?  

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