REGULATION OF MHCII EXPRESSION AND ANTIGEN PRESENTATION Éverton Padilha Gilberto Sabino Isabela 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-termN-term LRR domain Transcription factors binding Chromatin remodeling Nucleus drive and recruitment of enhanceossome

Gene structure mice Walter Reith et al., Nature immunology reviews

CIITA regulation

Enhanceossome

Chromatin remodeling Transcription promoter Walter Reith et al., 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 WT KO March-I Knock in MHC II I-A b immunoprecipitation

How is the expression of MHC class II in immature and mature DC? DC differentiated with GM-CSF WT KO March-I Immature DC Facs analysis Mature DC stimulated with 1  g LPS

Ubiquitination of pMHC-II affects surface expression and intracellular distribution in DCs. DC differentiated with GM-CSF WT KO March-I Immature DC Immunofluorescence microscopy Mature DC stimulated with 1  g 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 WT KO March-I Knock in MHC II I-A b 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 WT KO March-I Knock in MHC II I-A b 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 1  g 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 WT KO March-I Treatment with HEL protein overnight Analyzed by FACS Absence LPS Presence LPS overnight Expression of I-A k HEL 46-61

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 genes qPCR 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 siRMND5B qPCR/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

Nine unrelated candidates proteins might control MHC-II distribution.

MelJuSo siRNA imDCs 6 days on GM-CSF Confocal microscopy Do these genes mimic a mDCs phenotype? Six candidates proteins might control MHC-II distribution.

Monocytes imDCs 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.

Monocytes imDCs 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