Ekspresija i biohemijska i funkcionalna karakterizacija R-MC 17 antigena pacova

Abstract

Razvoj T limfocita u timusu je rezultat niza dvosmernih komunikacija između timocita i ćelija mikrosredine, posredovanih pretežno adhezivnim molekulima. U Institutu za medicinska istaživanja VMA je napravljen panel monoklonskih antitela (mAt) R-MC serije, koja prepoznaju timusne epitelne ćelije (TEC). Cilj ovog rada je bio biohemijska, imunohistohemijska i funkcionalna karakterizacija antigena koga detektuje R-MC 17 mAt. Ovo mAt detektuje antigen od 205 kDa u timusu pacova. Na osnovu poređenja biohemijskih karakteristika anti-DEC 205 mAt (HD83) i unakrsne imunoprecipitacije antigena iz lizata timusa pacova pomoću ova dva mAt zaključeno je da R-MC 17 mAt detektuje pacovski DEC-205 molekul. R-MC 17 antigen je jako ispoljen na kortikalnim TEC, medularnim timusnim dendritskim ćelijama (TDC), DC u parakorteksu i interfolikularnim zonama limfnog čvora i DC u periarteriolarnom limfnom omotaču slezine. Slaba imunoreaktivnost je detektovana na limfocitima u folikulima limfnog čvora i slezine i makrofagama u crvenoj pulpi slezine. Takođe, ekspresija DEC-205 molekula je detektovana na oko 40% ćelija TEC linije sa karakteristikama ćelija „dadilja“. R-MC 17 mAt podstiče vezivanje i emperipoezu timocita od strane ove epitelne linije. Istovremeno, ekspresija R-MC 17 antigena je podjednaka na subpopulacijama TDC pacova (CD11b+ i CD11b-), i ne menja se pod uticajem GM-CSF-a. Oba anti-DEC 205 mAt (R-MC 17 mAt i HD83 mAt) stimulišu apoptozu timocita u kokulturi sa TDC i ovaj proces je povezan sa povećanim vezivanjem timocita za TDC. R-MC 17 mAt inhibira proliferaciju singenih timocita i alogenih T limfocita pacova u kokulturi sa TDC u prisustvu anti-TCR (R7.3) mAt. Intratimusna aplikacija R-MC 17 mAt dovodi do smanjenja celularnosti timusa pacova što je povezano sa indukcijom apoptoze timocita. Fenotipska analiza subpopulacija timocita nakon intratimusne aplikacije R-MC 17 mAt pokazuje smanjenje ukupnog broja CD4+CD8+ i CD4+CD8-, a povećanje ukupnog broja CD4-CD8+ timocita, dok je ukupan broj zrelih CD5+TCRjako+ timocita nepromenjen. Sveukupno, ovi rezultati prvi put opisuju potencijalnu funkciju R-MC 17 antigena u timusu.Opisani mehanizmi mogu biti povezani sa procesima intratimusne diferencijacije timocita i prezentacije vi autoantigena u timusu od strane kortikalnih TEC i medularnih TDC, a što u krajnjem ishodu ima uticaj na procese selekcije (delecije autoreaktivnih klonova timocita).

Intrathymic development of T lymphocyte is mainly governed by an array of bidirectional interactions between thymocytes and thymic microenvironment, which are mostly provided by the adhesive molecules. The monoclonal antibody (mAb) panel of R-MC series, designed against thymic epithelial cells (TEC), was developed at the Institute of medical research (MMA). The aim of this doctoral thesis was focused on biochemical, immunohistochemical and functional characterization of the R-MC 17 antigen, detected by R-MC 17 mAb. R-MC 17 mAb recognizes the 205 kDa molecule expressed in the rat thymus. Based on similarity in biochemical features with an anti- DEC-205 mAb (HD83), and cross-immunoprecipitation of antigen from the thymic lysate with these two mAbs, R-MC 17 was considered to be the antibody specific for the rat DEC-205 molecule. Cortical TEC, medullary thymic dendritic cells (TDC), lymph node dendritic cells (DC) in paracortex and interfollicular zones, and spleen paraarteriolar-lymphoid sheaths DC, are highly positive with R-MC 17. Lymphocytes in lymph node and spleen follicles and red pulp macrophages in spleen, show weak R-MC 17 reactivity. About 40% of TEC-line cells with nursing characteristics express R-MC 17 antigen as well. R-MC 17 stimulates thymocyte binding and emperipoesis displayed by this cell line. Concomitantly, R-MC 17 antigen is equally expressed on two rat TDC subpopulations (CD11b+ and CD11b-), and cultivation with GM-CSF has no effect. RMC 17, as well as HD83, stimulates thymocyte apoptosis in co-culture with TDC, and this process is associated with the increased binding of thymocytes to TDC. R-MC 17 mAb inhibits proliferation of syngeneic thymocytes and allogeneic T lymphocytes in anti-TCR (R7.3)-stimulated cocultures with TDC. Intrathymic injection of R-MC 17 mAb decreases thymic cellularity, which was related to the induction of thymocyte apoptosis. Phenotypic analysis of thymocyte populations after intrathymic application of R-MC 17 mAb, indicates decrease of the absolute number of CD4+CD8+ and CD4+CD8- , and increase of CD4-CD8+ thymocyte subpopulations, while the absolute number of mature CD5+TCRhigh thymocytes stays unchanged. Taken together, these results show, for the first time, a potential function of R-MC 17 antigen in rat thymus. Mechanisms viii described, are potentially involved in the processes of intrathymic differentiation and the presentation of autoantigen in the thymus by the cortical TEC and medullary TDC, having final implications in the processes of thymic selection (deletion of autoreactive thymocytes).