Malattie Autoimmuni Rappresentano ancora oggi uno dei problemi più spinosi dell’immunologia, sia sul piano sperimentale che su quello clinico. Le conoscenze attuali sui meccanismi coinvolti sono ancora incomplete e di conseguenza l’eziologia è spesso ignota. Inoltre sono per lo più malattie multifattoriali, in quanto nella loro patogenesi intervengono fattori di predisposizione genetica e fattori ambientali.

Manifestazioni Autoimmuni Diabete mellito di tipo I Tiroidite Anemia emolitica Enteropatia Dermatite Alopecia

CD4+ CD25+ Regulatory T cells The immune system Protection from microorganisms Tolerance to the SELF CD4+ CD25+ Regulatory T cells

Principali meccanismi di eliminazione (periferici) di linfociti autoreattivi Delezione clonale (interazione Fas e Fas-L) Anergia clonale Attività delle T regolatorie Indifferenza clonale

Generazione delle Treg Il timo produce la maggior parte delle Treg CD4+CD25+, (5-10% linfociti CD4+T circolanti) come sottopopolazione distinta e funzionalmente matura Lo sviluppo di T reg puo’ avvenire a partire da cellule convenzionali, sotto specifiche condizioni di attivazione

CD4+ CD25+ Regulatory T cells in the immune system maintenance of immunological self-tolerance suppressing the proliferation or function of autoreactive T cells possibility that Treg cells may play a central role in immune homeostasis and regulation of immune responses toward foreign antigens

CASO CLINICO IPEX: Immune Dysregulation, Polyendocrinopathy, Entheropathy, X-linked. Sindrome X-linked recessiva Mortalità elevata entro il primo anno di vita. → Patologia da difetto della tolleranza immune.

CD4+CD25+ Regulatory T cells are involved in P E X mmune dysregulation olyendocrinopathy nteropathy -linked Presents most commonly in early childhood: IDDM Severe enteropathy Skin disorders Variable autoimmune phenomena Autoantibodies against: Thyroid Kidney Pancreatic islets Small Intestine Platelets and others

FoxP3 in regulatory T cells Foxp3 is a regulatory T cell-specific transcriptional factor – FKH family master regulator of the development and function of regulatory T cells As trascrptional factor: Probably regulates/suppresses cytokine expression NFAT and NF-kappaB (nuclear factors) C blocks their ability to induce the endogenous expression of their target genes, including key cytokine genes C

FOXP3 as proteic product 11 esoni – 431 aa functional domains: Zn FKH N C PRR 1 2 3 Proline rich region in N-terminal zone Zinc finger domain Forkhead winged-helyx domain: necessary of DNA-binding and nuclear location

Human IPEX: 13 mutations identified until now  In the FKH domain in leucine zipper domain removing of stop codon  products with C-terminal extensions Every patient with these mutations has classic IPEX, and symptomatic differences are unremarkable

Molecular and proteomical study 1 . Identification and estimation of FOXP3 expression 2 . Identification and estimation of other important and related genes expression 3 . Identification of specific protein-protein interactions of FOXP3 and its different domains

Cloning of FoxP3 gene and its domains Steps of the study Cloning of FoxP3 gene and its domains N PRR Zn FKH C 1 2 3 Cloning into expression vector Recombinant protein Tagged products

Expression of FoxP3 in procariotic cells  pTrcHis  Production in  Purification bacterial cells by His Tag Recombinant  protein  Policlonal sera

Expression of FoxP3 in eukaryotic cells Cloning of FoxP3 in fusion with GFP - N-terminal with StrepTag - C-terminal FoxP3/Dom GFP FoxP3/Dom TAG Expression in culture cells: - HEK293 - T lymphocytes (if possible)

Localization of FoxP3 MICROSCOPY  Confocal  Detection of GFP Use of anti-His (or anti-GFP) antibodies on  Nuclear extracts  Cytoplasmatic extracts Under different conditions of stimulation

FOXP3 interactions Immunoprecipitation of FoxP3 with anti-TAG (anti-His / anti-GFP / Strep-Tag vs Strep-Tactin) Characterization of co-precipitated proteins (mass spectrophotometry…)

Mass spectometry identification Interagent molecule 2D SDS-PAGE IEF detection Mass spectometry identification

Use of Bacterial Two-Hybrid System FOXP3 interactions Use of Bacterial Two-Hybrid System Easy in vivo screening and selection of function interactions between two proteins Analysis of different cDNA libraries of T cells subpopulations under different conditions of stimulation Informations about methabolic pathways in which FoxP3 is involved