|Tipo di tesi||Tesi di laurea magistrale|
|Titolo||Generazione in vitro dei precursori delle plasmacellule|
|Titolo in inglese||In vitro generation of plasma cell precursors|
|Struttura||Dipartimento di Scienze della Vita|
|Corso di studi||BIOTECNOLOGIE MEDICHE (D.M. 270/04)|
|Data inizio appello||2020-03-07|
|Disponibilità||Accessibile via web (tutti i file della tesi sono accessibili)|
Nelle risposte immunitarie sistemiche ha luogo l'attivazione delle cellule B dipendente dalle cell T e le cell B attivate si differenziano in plasmablasti(PB) migratori secernenti anticorpi. I PB di nuova formazione migrano nel midollo osseo(MO) seguendo un gradiente di CXCL12. Una volta arrivati i PB competono con le vecchie plasmacellule della memoria(mPC) per entrare in una nicchia di cell stromali(CS) dove si differenziano in mPC di lunga durata. Apparentemente 1 CS può ospitare solo 1 mPC e il nuovo PB deve quindi competere con la vecchia mPC. Le PC resting appena generate sono mantenute nel MO in un numero costante che riflette il 10-20% della popolazione di PB originariamente generata. Rimane da stabilire quale sia la competenza molecolare richiesta dai nuovi PB per diventare mPC. Il differenziamento di PB in mPC dipende da segnali ricevuti nella nicchia di sopravvivenza o ci sono meccanismi intrinseci all'interno del PB che determinano il differenziamento a mPC? Come viene regolata la sopravvivenza del PB fino a quando non raggiunge la sua nicchia nel MO?
In systemic immune responses T-cell-dependent B-cell activation takes place and the activated B cells differentiate into migratory antibody-secreting plasmablasts(PB). Newly formed PB migrate into the bone marrow(BM) by following mainly a CXCL12 gradient, once arrived in the BM, PB compete with old memory plasma cells(mPC) for habitation in a stromal cell(SC) niche where they differentiate into longlived mPC. Apparently 1 SC can host only 1 mPC and new PB have to compete with old PC. The newly generated resting PC are maintained in the BM at a constant number reflecting 10-20% of the originally generated PC population. How the survival of only this amount of PC is regulated and the molecular competence required by newly formed PC to become mPC in the BM still needs to be determined. Is the differentiation of PB into mPC dependent on signals received in the survival niche or are there intrinsic mechanism within the PB that determine differentiation into PC? How is PB survival regulated until the PB reaches its survival niche in the BM? The aim of this thesis was to lay the groundwork to be able to answer these questions by establishing an in vitro system to generate PB from primary naïve B cells according to the publication of Nojima et(2011). Nojima et al established a culture system(iGB) in which murine naïve B cells acquire a germinal center(GC) phenotype due to the activation with BAFF, CD40L and IL-4 followed by the addition of IL-21 which directs the GC-phenotype towards a PC-phenotype. To establish this in vitro system a feeder layer of 40LB cells(3T3 fibroblasts modified to express BAFF and CD40L)was prepared. Naïve B cells were isolated from C57BL/6J or C57BL/6J Blimp-1:GFP mice and cultured on the 40LB cell feeder layer for 4days with IL-4 and then for up to 10days with IL-21 to induce class switch recombination(CSR) to IgG and Blimp-1 expression in B cells. By flow cytometric evaluation of GCmarkers the generation of GC reaction and the maturation of naïve B cell towards a PB phenotype was monitored. After 10-14days PB were isolated by FACS or MACS. The isolated in vitro generated PB were cultured together in an in vitro niche for PC (ST2 SC and rAPRIL) previously established in the group. In the in vitro culture the survival of PB was assessed and the upregulation of markers of PC, like Prdm-1, was monitored together with entrance into quiescent state by staining for Ki-67. In vitro generated CD138+B220-PB survived in the niche for up to 5/7days in numbers reflecting 10% of the originally generated PB, thus resembling the in vivo PB behaviour. In vitro generated PB also expressed CXCR4 and thus were used to perform a chemotaxis assay where they showed the ability to migrate towards a CXCL12 gradient like they do in vivo. To confirm these findings mice were immunized 3 times with NP-CGG in IFA and in vivo generated PB were isolated at different time points following the last immunization from SP and BM to compare the capacity of PB from different time points of the immune reaction to become mPC. Noteworthy, PB isolated 14days after the last immunization showed improved survival. Around 40% of them survived in the in vitro niche, in line with findings that most mPC are generated late in the GC reaction. In summary, the in vitro generated PB show CSR to IgG, Blimp-1 and CXCR4 expression and they are able to migrate towards a CXCL12 gradient, similar to PB in vivo. Once in the in vitro niche with ST2 and rAPRIL the B220-CD138+PB/PC population survives for up to 7days and enter in a quiescent state. In conclusion, these data show that a system was established that lays the groundwork for the further molecular characterization of the differentiation and the intrinsic competence of B220-CD138+ to become longlived mPC.