The Nuclear Factor of Activated T cells (NFAT) is a transcription factor that has been always associated to T cell elicitation since its activation via calcium and calcineurin (Cn) results in the survival and expansion of lymphocytic clones via NFAT-dependent interleukin (IL)-2 production. Recently, NFATs has emerged as a fundamental transcription factor also in innate immunity, in particular in dendritic cells (DCs), being activated downstream pathogen recognition receptors (PRRs) engagement. Furthermore, IL-2 production and trans-presentation by DCs to naïve T cells represents a crucial event for their elicitation. Therefore, the aim of our study is to evaluate the role of NFAT in innate immunity in the context of mismatched transplantation, in which overt adaptive response occurs via donor’s and recipient’s DCs-mediated antigen presentation. By taking advantage of a minor antigen-based mismatched transplant setting, we grafted NFATc2 KO male-derived skin into C57BL/6 WT female recipients and monitored acute rejection. Compared to the WT male counterpart, recipients of NFATc2 KO male skin exhibited delayed rejection, since NFATc2 activation in donor cells occurs rapidly after transplantation and promote rejection. The absence of NFATc2 in the skin graft revealed to be sufficient to reduce the IFN-γ production in the skin by recipient’s infiltrating immune cells and to limit donor DCs maturation and migration to the graft-draining lymph nodes. Indeed, NFATc2 absence in the skin graft resulted in the reduced expression of both CCR7 and the costimulatory molecule CD86, thus affecting DCs migration and antigen presentation to naïve alloreactive T cells in the lymph nodes, alterated even by the reduced expression of CCL21 by the afferent lymphatic vessels. To deepen the role of the NFAT members in innate immunity, our collaborators of the NanoBioLab provided us with a novel nano-tool to selectively target phagocytes, hence also DCs, with an NFAT specific inhibitor: the VIVIT peptide. Indeed, the conjugation of VIVIT to nanoparticles (NPs) allowed us to exploit the intrinsic capability of phagocytes to engulf external particulates. By treating the animals with VIVIT NPs, even for a limited time window after transplantation, we obtained the complete acceptance of the graft thanks to the expansion of skin graft-specific CD4+ CD25+ Foxp3+ regulatory T cells (Tregs). Therefore, not only VIVIT-NPs constitute a novel approach to study the role of NFAT specifically in innate immunity, but they may also represent an efficacious therapeutic strategy for grafted patients that are currently long-life administered with Cn inhibitors, subjected to several severe side effects.

Il Fattore Nucleare dei linfociti T Attivati (NFAT) è sempre stato associato ai linfociti T poiché la sua attivazione, a valle di un aumento di calcio intracellulare e dell’attivazione di calcineurina (Cn), porta alla sopravvivenza e proliferazione dei cloni linfocitari grazie alla trascrizione di interleuchina (IL-) 2, mediata da NFAT stesso. Recentemente, NFAT è emerso come un fattore cruciale anche per l’immunità innata, attivato a valle di alcuni dei recettori dei profili di patogeni (PRRs). Inoltre, nelle cellule dendritiche (DCs) l’attivazione di NFAT porta alla trascrizione di IL-2 che viene trans-presentata dalle DCs stesse ai linfociti T, evento fondamentale per una corretta risposta adattativa. Pertanto, lo scopo del nostro studio è quello di valutare il ruolo di NFAT nell’immunità innata, in particolar modo nelle DCs, nel contesto dei trapianti non identici, dove si assiste ad una risposta adattativa alloreattiva dovuta alla presentazione dell’antigene da parte delle DCs sia del donatore che del ricevente. Utilizzando un modello di trapianto di cute non identico, abbiamo trapiantato la cute di un topo NFATc2 KO in femmine C57BL/6 WT e abbiamo monitorato il rigetto acuto. Comparando questi animali con quelli riceventi la cute di un maschio WT, abbiamo osservato una cinetica di rigetto ritardata, poiché abbiamo visto che l’attivazione di NFATc2 nel trapianto avviene subito dopo il trapianto stesso e promuove il rigetto. L’assenza di NFATc2 è sufficiente per osservare una riduzione della produzione di IFN-γ da parte di cellule immunitarie del ricevente infiltranti il trapianto e una maturazione e migrazione ai linfonodi drenanti di DCs limitata. Infatti, l’assenza di NFATc2 nel trapianto comporta la riduzione dell’espressione di CCR7 e CD86 nelle DCs, rendendole meno prone alla migrazione e presentazione dell’antigene, anche considerando la ridotta espressione di CCL21 dai vasi linfatici afferenti. Per approfondire il ruolo di tutti i membri NFAT nell’immunità innata, i nostri collaboratori del NanoBioLab ci hanno fornito delle nanoparticelle (NPs) coniugate ad un inibitore specifico di NFAT: il peptide VIVIT. Infatti, sfruttando la proprietà intrinseca dei fagociti di fagocitare particolato estraneo, abbiamo potuto veicolare il VIVIT selettivamente le cellule dell’immunità innata. Trattando i riceventi dei trapianti con le NPs VIVIT, anche se per un breve periodo dopo il trapianto, abbiamo osservato una completa accettazione del trapianto stesso, grazie all’espansione di cellule T regolatorie CD4+ CD25+ Foxp3+ specifiche per il trapianto. Pertanto, non solo le NPs VIVIT rappresentano un metodo innovativo per studiare il ruolo di NFAT nell’immunità innata in molteplici contesti, ma anche potrebbero essere una terapia efficace per i pazienti trapiantati che, al giorno d’oggi, vengono trattati con inibitori della Cn per tutta la vita e con considerevoli effetti avversi.

(2019). Long-term graft tolerance induction by NFATc pathway inhibition in innate immune cells. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2019).

Long-term graft tolerance induction by NFATc pathway inhibition in innate immune cells

GORNATI, LAURA
2019

Abstract

The Nuclear Factor of Activated T cells (NFAT) is a transcription factor that has been always associated to T cell elicitation since its activation via calcium and calcineurin (Cn) results in the survival and expansion of lymphocytic clones via NFAT-dependent interleukin (IL)-2 production. Recently, NFATs has emerged as a fundamental transcription factor also in innate immunity, in particular in dendritic cells (DCs), being activated downstream pathogen recognition receptors (PRRs) engagement. Furthermore, IL-2 production and trans-presentation by DCs to naïve T cells represents a crucial event for their elicitation. Therefore, the aim of our study is to evaluate the role of NFAT in innate immunity in the context of mismatched transplantation, in which overt adaptive response occurs via donor’s and recipient’s DCs-mediated antigen presentation. By taking advantage of a minor antigen-based mismatched transplant setting, we grafted NFATc2 KO male-derived skin into C57BL/6 WT female recipients and monitored acute rejection. Compared to the WT male counterpart, recipients of NFATc2 KO male skin exhibited delayed rejection, since NFATc2 activation in donor cells occurs rapidly after transplantation and promote rejection. The absence of NFATc2 in the skin graft revealed to be sufficient to reduce the IFN-γ production in the skin by recipient’s infiltrating immune cells and to limit donor DCs maturation and migration to the graft-draining lymph nodes. Indeed, NFATc2 absence in the skin graft resulted in the reduced expression of both CCR7 and the costimulatory molecule CD86, thus affecting DCs migration and antigen presentation to naïve alloreactive T cells in the lymph nodes, alterated even by the reduced expression of CCL21 by the afferent lymphatic vessels. To deepen the role of the NFAT members in innate immunity, our collaborators of the NanoBioLab provided us with a novel nano-tool to selectively target phagocytes, hence also DCs, with an NFAT specific inhibitor: the VIVIT peptide. Indeed, the conjugation of VIVIT to nanoparticles (NPs) allowed us to exploit the intrinsic capability of phagocytes to engulf external particulates. By treating the animals with VIVIT NPs, even for a limited time window after transplantation, we obtained the complete acceptance of the graft thanks to the expansion of skin graft-specific CD4+ CD25+ Foxp3+ regulatory T cells (Tregs). Therefore, not only VIVIT-NPs constitute a novel approach to study the role of NFAT specifically in innate immunity, but they may also represent an efficacious therapeutic strategy for grafted patients that are currently long-life administered with Cn inhibitors, subjected to several severe side effects.
GRANUCCI, FRANCESCA
ZANONI, IVAN
NFAT; Dendritic cells; Transplantation; Nanoparticles; Tolerance
NFAT; Dendritic cells; Transplantation; Nanoparticles; Tolerance
MED/04 - PATOLOGIA GENERALE
English
5-feb-2019
MEDICINA TRASLAZIONALE E MOLECOLARE - DIMET - 76R
31
2017/2018
open
(2019). Long-term graft tolerance induction by NFATc pathway inhibition in innate immune cells. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2019).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/241247
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