MalattieMalattie AutoimmuniAutoimmuni

Rappresentano ancora oggi uno dei problemi più spinosi Rappresentano ancora oggi uno dei problemi più spinosi dell’immunologia, sia sul piano sperimentale che su quello dell’immunologia, sia sul piano sperimentale che su quello clinico.clinico.

Le conoscenze attuali sui meccanismi coinvolti sono ancora Le conoscenze attuali sui meccanismi coinvolti sono ancora incomplete e di conseguenza l’eziologia è spesso ignota. incomplete e di conseguenza l’eziologia è spesso ignota.

Inoltre sono per lo più malattie multifattoriali, in quanto nella Inoltre sono per lo più malattie multifattoriali, in quanto nella

loro patogenesi intervengono fattori di predisposizione genetica loro patogenesi intervengono fattori di predisposizione genetica

e fattori ambientali.e fattori ambientali.

Manifestazioni AutoimmuniManifestazioni Autoimmuni

Diabete mellito di tipo I

Tiroidite Anemia emolitica

EnteropatiaDermatite•Alopecia

The immune systemThe immune system

Protection from Protection from microorganismsmicroorganisms

Tolerance to the Tolerance to the SELFSELF

CD4+ CD25+ CD4+ CD25+ Regulatory T cellsRegulatory T cells

Principali meccanismi di eliminazione (periferici)Principali meccanismi di eliminazione (periferici)di linfociti autoreattividi linfociti autoreattivi

Indifferenza clonale

Delezione clonale(interazione Fas e Fas-L)Anergia clonale

Attività delle T regolatorie

Generazione delle TregGenerazione delle Treg

Lo sviluppo di T reg puo’ avvenire a partire da cellule convenzionali, sotto specifiche condizioni di attivazione

Il timo produce la maggior Il timo produce la maggior parte delle Treg CD4parte delle Treg CD4++CD25CD25++, , (5-10% linfociti CD4 (5-10% linfociti CD4++T T circolanti) come circolanti) come sottopopolazione distinta e sottopopolazione distinta e funzionalmente maturafunzionalmente matura

CD4+ CD25+ Regulatory T CD4+ CD25+ Regulatory T cells in the immune systemcells in the immune system

• maintenance of immunological maintenance of immunological self-tolerance self-tolerance

• suppressing the proliferation or function suppressing the proliferation or function of autoreactive T cellsof autoreactive T cells

• possibility that Treg cells may play a central role in immune homeostasis possibility that Treg cells may play a central role in immune homeostasis and regulation of immune responses toward foreign antigensand regulation of immune responses toward foreign antigens

CASO CLINICOCASO CLINICO

IPEX: Immune Dysregulation, Polyendocrinopathy, IPEX: Immune Dysregulation, Polyendocrinopathy,

Entheropathy, X-linked. 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 inCD4+CD25+ Regulatory T cells are involved in

IIPPEEXX

mmune dysregulationmmune dysregulation olyendocrinopathyolyendocrinopathy nteropathynteropathy -linked-linkedPresents most commonly in early Presents most commonly in early

childhood:childhood:

- IDDMIDDM- Severe enteropathySevere enteropathy- Skin disordersSkin disorders- Variable autoimmune Variable autoimmune phenomenaphenomena

Autoantibodies Autoantibodies against:against:

ThyroidThyroidKidneyKidneyPancreatic isletsPancreatic isletsSmall IntestineSmall Intestine

Platelets and othersPlatelets and others

FoxP3 in regulatory T cellsFoxP3 in regulatory T cells

• Foxp3Foxp3 is a regulatory T cell-specific is a regulatory T cell-specific transcriptional factortranscriptional factor – – FKH familyFKH family

• master regulatormaster regulator of the development and function of regulatory T cells of the development and function of regulatory T cells

As trascrptional factor:As trascrptional factor:

• Probably regulates/suppresses cytokine expressionProbably regulates/suppresses cytokine expression

NFAT NFAT andand NF-kappaB NF-kappaB (nuclear factors)(nuclear factors)

C

blocks their ability to induce the endogenous expression of their target genes, blocks their ability to induce the endogenous expression of their target genes, including including key cytokine geneskey cytokine genes

C

• 11 esoni – 431 aa11 esoni – 431 aa

• functional domains:functional domains:

FOXP3 as proteic productFOXP3 as proteic product

1.1. Proline rich regionProline rich region in N-terminal zone in N-terminal zone

2.2. Zinc finger domainZinc finger domain

3.3. Forkhead winged-helyx domainForkhead winged-helyx domain: necessary of DNA-binding and : necessary of DNA-binding and nuclear locationnuclear location

Zn FKHNN CCPRR

1 2 3

Human IPEXHuman IPEX: 13 mutations identified until now : 13 mutations identified until now

• In the FKH domainIn the FKH domain

• in leucine zipper domainin leucine zipper domain

• removing of stop codon removing of stop codon products with C-terminal products with C-terminal extensionsextensions

Every patient with these mutations has classic IPEX,Every patient with these mutations has classic IPEX,

and symptomatic differences are unremarkableand symptomatic differences are unremarkable

Molecular and proteomical studyMolecular and proteomical study

1 . Identification and estimation of FOXP3 expression1 . Identification and estimation of FOXP3 expression

2 .2 . Identification and estimation of other important andIdentification and estimation of other important and

related genes expressionrelated genes expression

3 . Identification of specific protein-protein interactions of3 . Identification of specific protein-protein interactions of

FOXP3 and its different domainsFOXP3 and its different domains

Steps of the studySteps of the study

Cloning of FoxP3 gene and its domainsCloning of FoxP3 gene and its domains

Zn FKH

Cloning into expression vectorCloning into expression vector

N CPRR

11 2 3 2 3

Recombinant proteinRecombinant protein Tagged productsTagged products

pTrcHis pTrcHis Production in Production in Purification Purification bacterial cells by His Tag bacterial cells by His Tag

Recombinant Recombinant protein protein

Policlonal seraPoliclonal sera

Expression of FoxP3 in Expression of FoxP3 in procariotic cellsprocariotic cells

Expression of FoxP3 in Expression of FoxP3 in eukaryotic cellseukaryotic cells

Cloning of FoxP3 in fusion Cloning of FoxP3 in fusion

with GFP with GFP - N-terminal - N-terminal

with StrepTag - C-terminalwith StrepTag - C-terminal

Expression in culture cells: Expression in culture cells:

- HEK293- HEK293

- T lymphocytes (if possible) - T lymphocytes (if possible)

FoxP3/Dom GFP

FoxP3/Dom TAG

Localization of FoxP3Localization of FoxP3

MICROSCOPY MICROSCOPY

ConfocalConfocal

Detection of GFPDetection of GFP

Use of anti-His (or anti-GFP) antibodies on Use of anti-His (or anti-GFP) antibodies on

Nuclear extractsNuclear extracts

Cytoplasmatic extractsCytoplasmatic extracts

Under different conditions of stimulationUnder different conditions of stimulation

Immunoprecipitation of FoxP3 with anti-TAG Immunoprecipitation of FoxP3 with anti-TAG (anti-His / anti-GFP / Strep-Tag vs Strep-Tactin) (anti-His / anti-GFP / Strep-Tag vs Strep-Tactin)

FOXP3 interactionsFOXP3 interactions

Characterization of co-precipitated Characterization of co-precipitated

proteins (mass spectrophotometry…)proteins (mass spectrophotometry…)

Interagent molecule 2D SDS-PAGE IEF2D SDS-PAGE IEF detectiondetection

Mass spectometry identificationMass spectometry identification

Collegamento a opr02PQ31.lnk

FOXP3 interactionsFOXP3 interactions

Use of Bacterial Two-Hybrid SystemUse of Bacterial Two-Hybrid System

• Easy Easy in vivo in vivo screeningscreening and and selectionselection ofof functionfunction interactionsinteractions between two between two proteinsproteins

• Analysis of different Analysis of different cDNA librariescDNA libraries of of T cellsT cells subpopulations under subpopulations under different conditions of stimulationdifferent conditions of stimulation

Informations about methabolic pathways in which Informations about methabolic pathways in which FoxP3 is involvedFoxP3 is involved