Transcript Immature DC

REGULATION OF MHCII EXPRESSION AND
ANTIGEN PRESENTATION
Éverton Padilha
Gilberto Sabino
Isabela Fontoura
MHCII structure
Expression in:
• B cells
• Dendritic cells
• Macrophages
• Thymus epitelial cells
Antigen processing and presentation by MHCII
Regulation of MHCII expression
CIITA – a master regulation of MHCII expression
( Transactivator of MHCII genes)
C-term
CIITA gene
N-term
LRR domain
Transcription factors binding
Chromatin remodeling
Nucleus drive and
recruitment of enhanceossome
Gene structure
mice
Walter Reith et al., 2005. Nature immunology reviews
CIITA regulation
Enhanceossome
Pol I
Chromatin remodeling
Transcription promoter
Walter Reith et al., 2005. Nature immunology reviews
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
Role of ubiquitination in pMHCII complex
recycling in DCs
Éverton Padilha
What is the role of March-I in pMHC-II
ubiquitination and surface expression in both immature
and mature DCs?
Validation of methodology
C57BL/6
DC with
GM-CSF
WT
KO
March-I
Knock in
MHC II I-Ab
immunoprecipitation
immunoblotting
How is the expression of MHC class II in
immature and mature DC?
DC differentiated with GM-CSF
WT
Immature
DC
KO
March-I
Mature DC
stimulated with
1mg LPS
Facs analysis
How is the
of
Ubiquitination
of intracellular
pMHC-II affectslocalization
surface expression
and
distribution
in DCs.
MHC
II intracellular
in mature and
immature
DCs?
DC differentiated with GM-CSF
WT
Immature
DC
KO
March-I
Mature DC
stimulated with
1mg LPS
Immunofluorescence
microscopy
Does ubiquitination
be regulating
Does
ubiquitination be
endocytosis
in DCs?
endocytosis
in HeLa
cells?
DC HeLa-CIITA
with GM-CSF
WT
KO
March-I
Transfected
GFP
alone
Knock in
MHC II I-Ab
GFP+
March I
Absence
Presence
LPS
LPS
Immunoprecipitation
overnight
Isotype
Anti-MHC II acontrol IgG
chain mAb
The cells were incubated
on ice with mAb Y3P
Immunoblotting
(anti-ubiquitin or
anti class IIwere
b)
Endocytosis
measured for MFI
The ubiquitination
Increase fourfold!
Does ubiquitination be regulating endocytosis
in HeLa cells?
HeLa-CIITA
Ubiquitination
does not
affect the
kinetics of
pMHC-II
Facs analyses
endocytosis.
Transfected
GFP
alone
GFPMarch I
Incubated with mAb
against pMHC II
How does March-I regulate the MHCII
expression on DC surface?
DC with GM-CSF
WT DC differentiated with GM-CSF
KO
Knock in
WT
March-I MHC II I-Ab
Ubiquitination
m
by March-I
regulates
pMHC-II
degradation.
Mature DC
stimulated with
Absence LPS
1 g LPS
Immature
DC
(Immature DC)
surface
biotinylating
The cells were
Recultured
biotinylated
on ice. 8h
Immunoprecipitation
Immunoprecipitation
SDS/Page
and avidin
SDS/Page
and avidin
Does March-I regulate MHCII expression in immature
DCs by promoting especific degradation?
DC with GM-CSF
KO
March-I
WT
Treatment with HEL
Ubiquitination
protein overnight
regulates degradation
Absence
Presence
of internalized
pMHC-II
LPS
LPS
complexes in immature
DCs.
Expression of I-A
overnight
k
HEL46-61
Analyzed
by FACS
Immature dendritic cells
Adapted of
Berger A C , Roche P A J Cell Sci 2009;122:1-4
Lysosome
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.
Fundamental questions…
What regulates MHC-II transcription?
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.
Method validation
genetic association
8% (21 of 276) were associated
with autoimmune diseases
Which genes do affect MHC-II transcription?
MelJuSo
siRNA 276 candidates genes
qPCR
HLA – DR mRNA
CIITA mRNA
Ii mRNA
The silencing of nine candidates proteins may affect transcription of one or
several of the tested genes.
Is there any interconnection between these nine candidates?
MelJuSo
Silencing of each 9 candidates genes
qPCR
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
siRMND5B
qPCR/confocal
TGF-β +/-
RMND5B might act as an inhibitor of SMADs by a probably interaction, once
RMND5B also translocates to nucleus with SMAD4 upon TGF-β exposure.
What about intracellular distribution of MHC-II?
267
Quantitative microscopy analysis
Quantitative microscopy analysis
Nine unrelated candidates proteins might control MHC-II distribution.
Do these genes mimic a mDCs phenotype?
MelJuSo
siRNA
imDCs
Confocal microscopy
6 days on GM-CSF
Six candidates proteins might control MHC-II distribution.
MHC-II transport to the plasma membrane
Monocytes
imDCs
6 days on GM-CSF
Confocal
microscopy/qPCR
PSD4 is a GEF that may upregulate MHC-II expression by activating ARL14/ARF7.
MHC-II transport to the plasma membrane
Monocytes
imDCs
6 days on GM-CSF
Confocal
microscopy/IP and
WB
lipid biding-assay
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
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