In vitro eradication of citrullinated protein specific B-lymphocytes of rheumatoid arthritis patients by targeted bifunctional nanoparticles

Abstract

BackgroundAutoreactive B cells are crucial players in the pathogenesis of rheumatoid arthritis (RA). Autoantibodies specific for citrullinated proteins (ACPA), present in the serum of approximately 60–70 % of patients, have a pathogenic role in the disease. B cell depleting therapies may result in a transient immunosuppression, increasing the risk of infections. Our aim was to develop a new therapeutic approach to selectively deplete the ACPA producing autoreactive B cells.MethodsTo target B cells synthetic citrullinated peptide derived from the β chain of fibrin, β60-74Cit 60,72,74 (β60-74Cit), the predominant epitope recognized by ACPA was used. Complement dependent cytotoxicity (CDC) was induced by a modified peptide derived from gp120 of HIV-1. To trigger CDC both the targeting peptide and the complement activating peptide were covalently coupled in multiple copies to the surface of poly (DL-lactic-co-glycolic acid) nanoparticles (NPs). Ex vivo antibody synthesis was examined by ELISA and ELISpot. CDC was tested after dead cell staining by flow cytometry.ResultsThe β60-74Cit peptide was selectively recognized by a small subset of B cells from RA patients having high level of peptide specific serum antibody, suggesting that the peptide can target diseased B cells. The modified gp120 peptide covalently coupled to NPs induced the formation of the complement membrane attack complex, C5b-9 in human serum. We show here for the first time that bifunctional NPs coupled to multiple copies of both the targeting peptide and the complement activating effector peptide on their surface significantly reduce β60-74Cit peptide specific ex vivo ACPA production, by inducing complement dependent lysis of the citrullinated peptide specific B cells of seropositive RA patients.ConclusionsBifunctional NPs covalently coupled to autoantigen epitope peptide and to a lytic peptide activating complement may specifically target and deplete the peptide specific autoreactive B-cells.

Autoreactive B‐lymphocytes in SLE and RA patients: Isolation and characterisation using extractable nuclear and citrullinated antigens bound to immunobeads

“…There are very few techniques enabling detection or isolation of autoreactive B lymphocytes in autoimmune diseases [18][19][20][21][22][23][24]. In this work, we purified ENA-and citrullinated peptide-specific ABLs by means of magnetic immunoselection of B cells from the blood bearing specific receptors for ENA and citrullinated peptides in SLE and RA patients, respectively (Supporting Information Fig.…”

“…Humoral immune and autoimmune responses are carried out by two main mechanisms: (i) long-lived ASCs residing in specialised niches where they receive survival signals that promote Ab production, and (ii) stimulation of mature B lymphocytes that differentiate into ASCs. In the blood of SLE and RA patients, both ABLs and ASCs/plasmablasts producing specific autoAb have been detected, although the relative contribution of each population to autoAb levels is unknown [2,6,12,13,[18][19][20][21][22][23][24]31]. Clarification of this point could have therapeutic implications because treatments targeting either plasma cells or B cells would be more efficient depending on the predominant type of cell in a certain patient.…”

“…ABLs are poorly studied, primarily because there are very few techniques enabling precise detection of these populations, which are present at low frequencies [18][19][20][21][22][23][24].…”

Biokonjugátumok – összefogni érdemes

“…29 Azokat a B-sejteket vettük tehát célba, amelyek a fibrin b-láncból származó 60 RPAPPPISSGGYXAX 74 (b60-74) epitóp-specifikus és a RA autoimmun betegség kialakulásában szerepet játszó ellenanyag fehérjéket termelik. Ezen sejtek felszínén ugyanis -a célsejtre jellemzõ -receptorok vannak, amelyek ezt az epitóp peptidet ismerik fel, kötõdnek hozzá.…”

“…Összegezve megállapítható 29 , hogy a háromkomponensû, biodegradábilis poli(tejsav-glikol-sav) + pluronic sav kopolimer nanorészecske konjugátum nagy számban és azonos orientációban tartalmaz sejtfelismerõ epitóp peptidet ( 60 RPAPPPISSGGYXAX 74 ) és sejtpusztulást elõidézõ komplement aktiváló peptidet (Ac-233 C(Acm)NNQTFNGTGPC (Acm)TNV 247 K-CONH 2 ) (7. ábra). A háromkomponensû, biodegradábilis konjugátum a) kötõdik fibrin b-lánc ß60-74 epitóp peptid specifikus IgG ellenanyaghoz és RA betegbõl szá rmazó B-sejtekhez, b) aktiválja a komplement rendszert és ennek kö-vetkezményeként, c) elpusztítotja in vitro azokat a B-sejteket, amelyek fibrin b-lánc b60-74 epitóp peptid specifikus ellenanyagot (autoellenanyag) termelnek.…”