Studi di immunogenicità in modelli murini

Laura Pisapia

Giovanna Del Pozzo

Napoli, 27 Giugno 2013

Corso C. et al. EBP1 and DRBP76/NF90 binding proteins are included in the Major Histocompatibility Complex Class II RNA operon. 2011 N.A.R Sep 1;39(16):7263-75.

Pisapia L. et al. Co-regulated expression of alpha and beta mRNAs encoding HLA-DR surface heterodimers is mediated by the MHCII RNA operon. 2013 N.A.R. Apr1;41(6):3772-86

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MHC class II RNA Operon

EBP1

Identified as ErbB3 binding protein, is

the human homologue of mouse cell-cycle regulated

protein p38-2G4

In carcinoma cell lines p48 isoform functions as tumor

suppressor

In PC12 and glioblastoma p48 works as oncogene, while p42 isoform is a tumor suppressor

Translational control:• in the cytoplasm is associated to

80S ribosomes and polysomes• inhibits translation of AR mRNA

Transcriptional repressor:Inhibits transcription of E2F regulated promoters

by recruiting HDAC2/Rb/Sin3A complex

RNA binding protein:• influences bcl2 stability,

promotes decay of androgen receptor mRNA,• binds UTRs of MHCII

p48 and p42 isoforms

• p48 isoform binds 5’ and 3’UTR of MHCII mRNAs

• The over expression of p48 isoform determines an up- regulation of MHC Class II in different cancer cell lines

Ctrl EBP1 p48 EBP1 p420

4

8

12

16 M14

HLA

-DR

MFI

fold

var

iatio

n

Ctrl EBP1 p48 EBP1 p420

4

8

12

16U87

HLA

-DR

MFI

fold

var

iatio

n

Ctrl EBP1 p48 EP1 p420

1

2

3MCF7

HLA

-DR

MFI

fold

var

iatio

n

Ctrl EBP1 p48 EP1 p420

1

2

3

B16 OVAHL

A-DR

MFI

fold

var

iatio

n

Over-expression of p42 and p48 EBP1 influences MHCII amount

OT-II MOUSEOT-I MOUSE

Spleen Spleen

T CD4+ magnetic selectionT CD8+ magnetic selection

B16-OVAa and CD8+ co-culture

B16-OVA and CD4+ co-culture

Melanoma B16-OVACD8+

T cell

CD4+ T cell

CD8 OVA257–264(SIINFEKL) peptide

CD4 OVA323–339 (ISQAVHAAHAEINEAGR)

peptide

CD8+ CFSE labelling

CD4+ CFSE labelling

Ctrl EBP1 p48 EBP1 p420

0.5

1

T CD8+ proliferation assay

CFSE

MFI

fold

var

iati

on

Ctrl EBP1 p48 EBP1 p420

0.5

1

T CD4+ proliferation assay

CFSE

MFI

fold

var

iati

on

T cells activation and proliferation are

demonstrated by the decrease of CFSE labelling

CFSE