regulation of mhcii expression and antigen presentation Éverton padilha gilberto sabino isabela...
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REGULATION OF MHCII EXPRESSION AND ANTIGEN PRESENTATION
Éverton PadilhaGilberto SabinoIsabela Fontoura
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Expression in:
• B cells
• Dendritic cells
• Macrophages
• Thymus epitelial cells
MHCII structure
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Antigen processing and presentation by MHCII
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Regulation of MHCII expression
CIITA – a master regulation of MHCII expression( Transactivator of MHCII genes)
CIITA geneC-term N-term
LRR domain
Transcription factors bindingChromatin remodeling
Nucleus drive and recruitment of enhanceossome
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Gene structuremice
Walter Reith et al., 2005. Nature immunology reviews
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CIITA regulation
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Enhanceossome
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Chromatin remodelingTranscription promoter
Walter Reith et al., 2005. Nature immunology reviews
Pol I
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Kenneth L. and Jenny P-Y. Ting, 200. Trends in immunology
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Factors that regulates positively MHCII expression:
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IFN-γ
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Factors that regulates negatively MHCII expression:
↑MARCH-1
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Éverton
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Gilberto
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Éverton Padilha
Role of ubiquitination in pMHCII complex recycling in DCs
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What is the role of March-I in pMHC-II ubiquitination and surface expression in both immature
and mature DCs?
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Validation of methodology
immunoblotting
C57BL/6
DC with GM-CSF
WTKO
March-IKnock in
MHC II I-Ab
immunoprecipitation
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How is the expression of MHC class II in immature and mature DC?
DC differentiated with GM-CSF
WTKO
March-I
Immature DC
Facs analysis
Mature DC stimulated with
1g LPS
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Ubiquitination of pMHC-II affects surface expression and intracellular distribution in DCs.
DC differentiated with GM-CSF
WTKO
March-I
Immature DC
Immunofluorescence microscopy
Mature DC stimulated with
1g LPS
How is the intracellular localization of MHC II in mature and immature DCs?
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Does ubiquitination be regulating endocytosis in DCs?
DC with GM-CSF
WTKO
March-IKnock in
MHC II I-Ab
Absence LPS
Presence LPS
overnight
The cells were incubated on ice with mAb Y3P
Endocytosis were measured for MFI
HeLa-CIITA
GFP alone
GFP+ March I
Isotype control IgG
Immunoprecipitation
Anti-MHC II -chain mAb
Transfected
Immunoblotting(anti-ubiquitin or
anti class II
Does ubiquitination be regulating endocytosis in HeLa cells?
The ubiquitination Increase fourfold!
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Ubiquitination does not affect the kinetics of pMHC-II
endocytosis.
HeLa-CIITA
GFP alone
GFP-March I
Facs analyses
Transfected
Incubated with mAb against pMHC II
Does ubiquitination be regulating endocytosis in HeLa cells?
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How does March-I regulate the MHCII expression on DC surface?
Ubiquitination by March-I regulates pMHC-II
degradation.
DC with GM-CSF
WTKO
March-IKnock in
MHC II I-Ab
Absence LPS (Immature DC)
The cells were biotinylated on ice.
SDS/Page and avidin
Immunoprecipitation
WT DC differentiated with GM-CSF
Immature DC
SDS/Page and avidin
Mature DC stimulated with
1g LPS
surface biotinylating
Immunoprecipitation
Recultured 8h
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Does March-I regulate MHCII expression in immature DCs by promoting especific degradation?
Ubiquitination regulates degradation of internalized pMHC-II complexes in immature
DCs.
DC with GM-CSF
WTKO
March-I
Treatment with HEL protein overnight
Analyzed by FACS
Absence LPS
Presence LPS
overnight
Expression of I-Ak HEL46-61
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Berger A C , Roche P A J Cell Sci 2009;122:1-4
Adapted of Lysosome
Immature dendritic cells
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Goal: Investigate the regulation of MHC class II antigen presentation in three processes: peptide loading, transcriptional regulation, and general cell biology which consists in the assembly, intracellular transport, processing in the MIIC, and endo- and exocytosis.
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What regulates MHC-II transcription?
Fundamental questions…
What controls MHC-II transport in dendritic cells?
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Effects on MHC-II expression and peptide loading
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Method validation
13 candidates with function already known in literature.
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8% (21 of 276) were associated with autoimmune diseases
genetic association
Method validation
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MelJuSo
siRNA 276 candidates genes
qPCR
HLA – DR mRNA CIITA mRNA
Ii mRNA
Which genes do affect MHC-II transcription?
The silencing of nine candidates proteins may affect transcription of one or several of the tested genes.
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MelJuSo Silencing of each 9 candidates genesqPCR
Is there any interconnection between these nine candidates?
Most of siRNA affected the expression of one or more other candidate genes, suggesting that they act in complex networks.
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What is the role for RMND5B?
MelJuSo siRMND5BqPCR/
confocal
RMND5B might act as an inhibitor of SMADs by a probably interaction, once RMND5B also translocates to nucleus with SMAD4 upon TGF-β exposure.
TGF-β +/-
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What about intracellular distribution of MHC-II?
267
Quantitative microscopy analysis
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Quantitative microscopy analysis
Nine unrelated candidates proteins might control MHC-II distribution.
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MelJuSo siRNAimDCs
6 days on GM-CSFConfocal microscopy
Do these genes mimic a mDCs phenotype?
Six candidates proteins might control MHC-II distribution.
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MonocytesimDCs
6 days on GM-CSF
Confocal microscopy/qPCR
MHC-II transport to the plasma membrane
PSD4 is a GEF that may upregulate MHC-II expression by activating ARL14/ARF7.
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MonocytesimDCs
6 days on GM-CSF
Confocal microscopy/IP and
WB
MHC-II transport to the plasma membrane
Collectively, this reveals part of a pathway where PIP5K1A and PIP3KR2 creates PIPs that are requires for recruitment or activation of GEF PSD4, which activates
ARL14/ARF7
lipid biding-assay
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MHC-II transport to the plasma membrane
ARL14/ARF7 connect to the actin network via ARF7EP-MYO1E to control export of MHC-II
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Conclusions
Transcriptional networks
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Conclusions
MHC-II distribution pathway to the plasma membrane