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