TY - JOUR

T1 - The murine DCs transfected with DNA-plasmid encoding CCR9 demonstrate the increased migration to CCL25 and thymic cells in vitro and to the thymus in vivo

AU - Tereshchenko, Valeriy

AU - Bulygin, Aleksei

AU - Zavodskii, Roman

AU - Maksyutov, Amir

AU - Kurilin, Vasiliy

AU - Fisher, Marina

AU - Semenyuk, Nikita

AU - Aladev, Stanislav

AU - Sennikov, Sergey

N1 - Funding Information: The study was supported by the Russian Science Foundation [Agreement No. 16-15-00086]. The authors are grateful to the staff of the Laboratory of molecular immunology, RIFCI, for all the assistance provided. We thank Edanz Group (https://en-author-services.edanzgroup.com/ac) for editing a draft of this manuscript. The datasets generated during the current study are available from the corresponding author on request. All experimental protocols and procedures were approved by the institutional review board of the Research Institute of Fundamental and Clinical Immunology, Novosibirsk, Russian Federation (Protocol No. 122/2020-02-27). The study followed the principles outlined in the Declaration of Helsinki for all human and animal experimental investigations. Funding Information: The study was supported by the Russian Science Foundation [Agreement No. 16-15-00086]. Publisher Copyright: © 2021 Elsevier Ltd Copyright: Copyright 2021 Elsevier B.V., All rights reserved.

PY - 2021/6

Y1 - 2021/6

N2 - Background: B220+CD11c+plasmacytoid DCs(pDCs) are known to participate in the negative selection and central tolerance induction by the capturing of self-antigens in peripheral tissues and further migration to the thymus using the CCL25-CCR9 chemotaxis axis. Aim: Here we investigate the possibility of DCs migration stimulation to the thymus by the transfection with plasmid DNA-constructs encoding CCR9(pmaxCCR9) to develop a system for desired antigen delivery to the thymus for central tolerance induction. Methods: Dendritic cells(DCs) cultures were generated from UBC-GFP mice bone marrow cells expressing green fluorescent protein using the rmFlt3-L. DCs cultures were transfected with pmaxCCR9 by electroporation. The efficiency of electroporation was confirmed by RT-qPCR and flow cytometry. The migration of electroporated DCs was assessed in vitro and in vivo. Results: Dendritic cells(DCs) cultures obtained from UBC-GFP mice contained both B220+pDCs and SIRPa+cDC2. According to the RT-qPCR assay, the electroporation of obtained DCs cultures with pmaxCCR9 resulted in a 94.4-fold increase of RNA encoding CCR9 compared with non-electroporated cultures. Flow cytometry data showed that DCs cultures electroporated with pmaxCCR9 contained a significantly higher frequency of DCs carrying significantly higher levels of surface CCR9. Migration dynamics of obtained DCs analyzed in vitro showed that pmaxCCR9 electroporated DCs migrated significantly more active to CCL25 and thymic cells than non-electroporated and mock-electroporated DCs. In vivo, 30 days after injection, the relative amount of the DCs electroporated with pmaxCCR9 and pmaxMHC encoding antigenic determinants in the mice thymuses was 2.02-fold higher than the relative amount of the DCs electroporated with control plasmid. Conclusion: Thus, the electroporation of murine DCs with pmaxCCR9 stimulated its migration to CCL25 and thymic cells in vitro as well as to the thymus in vivo. The obtained DCs loaded with a desired antigen may be suggested for further evaluation of central tolerance induction ability in in vivo models of autoimmune diseases and transplantation.

AB - Background: B220+CD11c+plasmacytoid DCs(pDCs) are known to participate in the negative selection and central tolerance induction by the capturing of self-antigens in peripheral tissues and further migration to the thymus using the CCL25-CCR9 chemotaxis axis. Aim: Here we investigate the possibility of DCs migration stimulation to the thymus by the transfection with plasmid DNA-constructs encoding CCR9(pmaxCCR9) to develop a system for desired antigen delivery to the thymus for central tolerance induction. Methods: Dendritic cells(DCs) cultures were generated from UBC-GFP mice bone marrow cells expressing green fluorescent protein using the rmFlt3-L. DCs cultures were transfected with pmaxCCR9 by electroporation. The efficiency of electroporation was confirmed by RT-qPCR and flow cytometry. The migration of electroporated DCs was assessed in vitro and in vivo. Results: Dendritic cells(DCs) cultures obtained from UBC-GFP mice contained both B220+pDCs and SIRPa+cDC2. According to the RT-qPCR assay, the electroporation of obtained DCs cultures with pmaxCCR9 resulted in a 94.4-fold increase of RNA encoding CCR9 compared with non-electroporated cultures. Flow cytometry data showed that DCs cultures electroporated with pmaxCCR9 contained a significantly higher frequency of DCs carrying significantly higher levels of surface CCR9. Migration dynamics of obtained DCs analyzed in vitro showed that pmaxCCR9 electroporated DCs migrated significantly more active to CCL25 and thymic cells than non-electroporated and mock-electroporated DCs. In vivo, 30 days after injection, the relative amount of the DCs electroporated with pmaxCCR9 and pmaxMHC encoding antigenic determinants in the mice thymuses was 2.02-fold higher than the relative amount of the DCs electroporated with control plasmid. Conclusion: Thus, the electroporation of murine DCs with pmaxCCR9 stimulated its migration to CCL25 and thymic cells in vitro as well as to the thymus in vivo. The obtained DCs loaded with a desired antigen may be suggested for further evaluation of central tolerance induction ability in in vivo models of autoimmune diseases and transplantation.

KW - CCL25

KW - CCR9

KW - conventional DC type 2

KW - Migration

KW - plasmacytoid DCs

KW - Thymus

UR - http://www.scopus.com/inward/record.url?scp=85101551724&partnerID=8YFLogxK

U2 - 10.1016/j.cyto.2021.155473

DO - 10.1016/j.cyto.2021.155473

M3 - Article

C2 - 33647585

AN - SCOPUS:85101551724

VL - 142

JO - Cytokine

JF - Cytokine

SN - 1043-4666

M1 - 155473

ER -