REGULATION OF MHCII EXPRESSION AND ANTIGEN PRESENTATION
Éverton PadilhaGilberto SabinoIsabela Fontoura
Expression in:
• B cells
• Dendritic cells
• Macrophages
• Thymus epitelial cells
MHCII structure
Antigen processing and presentation by MHCII
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
Gene structuremice
Walter Reith et al., 2005. Nature immunology reviews
CIITA regulation
Enhanceossome
Chromatin remodelingTranscription promoter
Walter Reith et al., 2005. Nature immunology reviews
Pol I
Kenneth L. and Jenny P-Y. Ting, 200. Trends in immunology
Factors that regulates positively MHCII expression:
IFN-γ
Factors that regulates negatively MHCII expression:
↑MARCH-1
Éverton
Gilberto
Éverton Padilha
Role of ubiquitination in pMHCII complex recycling in DCs
What is the role of March-I in pMHC-II ubiquitination and surface expression in both immature
and mature DCs?
Validation of methodology
immunoblotting
C57BL/6
DC with GM-CSF
WTKO
March-IKnock in
MHC II I-Ab
immunoprecipitation
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
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?
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!
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?
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
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
Berger A C , Roche P A J Cell Sci 2009;122:1-4
Adapted of Lysosome
Immature dendritic cells
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.
What regulates MHC-II transcription?
Fundamental questions…
What controls MHC-II transport in dendritic cells?
Effects on MHC-II expression and peptide loading
Method validation
13 candidates with function already known in literature.
8% (21 of 276) were associated with autoimmune diseases
genetic association
Method validation
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.
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.
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 +/-
What about intracellular distribution of MHC-II?
267
Quantitative microscopy analysis
Quantitative microscopy analysis
Nine unrelated candidates proteins might control MHC-II distribution.
MelJuSo siRNAimDCs
6 days on GM-CSFConfocal microscopy
Do these genes mimic a mDCs phenotype?
Six candidates proteins might control MHC-II distribution.
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.
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
MHC-II transport to the plasma membrane
ARL14/ARF7 connect to the actin network via ARF7EP-MYO1E to control export of MHC-II
Conclusions
Transcriptional networks
Conclusions
MHC-II distribution pathway to the plasma membrane