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URL: https://dx.doi.org/DOI:10.17504/protocols.io.jn4cmgw

Authors: Aleksey Pichugin, Alexander Bagaev, Ravshan Ataullakhanov

Summary: ELISPOT allows detection of single IFN-γ-secreting cell, but the use of unseparated spleen cells removes the advantages of the method due to a high back ground of IFN-γ-production and uncertainty of the phenotype of secreting cells (CD4, CD8 or NK).We used high purity sorted CD4 and CD8 cells.For in vitro restimulation of CD8 T-cells, we used dendritic cells that present antigenic epitopes  of  M2,  NP  or  of  adenovirus  in  the  context  of  MHC  class  I.  In  this  instance,  dendritic  cells  were  transduced  with  Ad5-tet-M2,  Ad5-tet-NP  or  Ad5-null, respectively.For restimulation of CD4 T-cells in vitro, we used dendritic cells that present antigenic M2 or NP epitopes in the context of MHC class II. For this purpose, dendritic cells were additionally activated by lipopolysaccharide E. coli (1 μg/ml) and were loaded with recombinant protein NP or M2e peptide..

Associated Publications: Tutykhina I, Esmagambetov I, Bagaev A, Pichugin A, Lysenko A, Shcherbinin D, Sedova E, Logunov D, Shmarov M, Ataullakhanov R, Naroditsky B, Gintsburg A (2018) Vaccination potential of B and T epitope-enriched NP and M2 against Influenza A viruses from different clades and hosts. PLoS ONE 13(1): e0191574. doi: 10.1371/journal.pone.0191574

Affiliations: NRC Institute of Immunology FMBA of Russia, Moscow, Russia, NRC Institute of Immunology FMBA of Russia, Moscow, Russia, NRC Institute of Immunology FMBA of Russia, Moscow, Russia

External URL: https://doi.org/10.1371/journal.pone.0191574

Version: 1

Publication Date: 2018

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