Efficient and Rapid In Vitro Generation of Multi-Virus-Specific CD4+ and CD8+ T Cells for Adoptive Immunotherapy.
Authors: Richter Anne , Foerster Anna , Lasmanowicz Verena , Brauns Olaf , Kramer Sven , Jekow Petra , Rönspeck Wolfgang , Assenmacher Mario , Schmitz Juergen 
Published: October 2019

Abstract
Abstract 1175
Adoptive transfer of T cells with specificity for one or several viruses is a promising strategy for the treatment or prevention of multiple opportunistic infections after allogenic stem cell transplantation. Here we have established a protocol for the generation of CMV, EBV and adenovirus (tri-virus) specific T cells. Virus-specific CD4+ and CD8+ T cells are stimulated in vitro using pools of peptides and subsequently magnetically enriched by virtue of their IFN-g secretion.
The peptide pools are composed of synthetic peptides of mainly 15 amino acid length with 11 amino acid overlap covering the complete protein sequence of a viral antigen. We have analyzed the efficiency of short-term in vitro restimulation of cytomegalovirus (CMV)-specific CD4+ and CD8+ effector/memory T cells from CMV-infected healthy donors by CMV pp65 and IE-1 peptide pools as compared to single MHC class I restricted immunodominant pp65 and IE-1 9-mer peptides and CMV pp65 and IE-1 recombinant proteins based on flow cytometric analysis of the IFN-g response. The results show that peptide pools as well as recombinant proteins are useful for efficient activation of CD4+ T helper cells. In contrast, efficient stimulation of CD8+ T cells is achieved only using either the overlapping 15-mer peptide pools or the immunodominant 9-mer peptides.
To test the usability of peptide pools to generate multi-virus-specific CD4+ and CD8+ T cells for adoptive immunotherapy we stimulated PBMC of several healthy donors with a combination of 5 peptide pools selected from CMV pp65, CMV IE-1, adenovirus hexon, EBV EBNA-1 and EBV BZLF-1 for 4 hours. Concomitant addition of 4 peptide pools to a single PBMC sample might decrease the activation efficiency for each peptide pool due to competition of peptides for MHC binding. Thus, as a control, we have divided PBMC into 4 aliquots, incubated each aliquot with a single peptide pool for 2 hours, and recombined the aliquots for T cell stimulation for another 4 hours. Afterwards, we used the IFN-g secretion assay technology to magnetically select for antigen-activated IFN-γ-secreting CD4+ and CD8+ T cells to a purity of > 90%. Antigen specificity and functionality of the enriched T cell population was examined after expansion. Within 9 – 14 days, T cells expanded between 4 and 745 fold. In addition, 21–53% of CD4+ T cells and 53–87% of CD8+ T cells re-expressed IFN-γ upon re-stimulation with the mixture of 4 peptide pools, confirming the high antigen specificity and functionality of the expanded T cells. Enrichment and expansions of T cells works equally well independent on whether the T cells are specific for CMV pp65, CMV IE-1, adenovirus hexon, EBV EBNA-1 or EBV BZLF-1. This can be deduced from the fact that the relative frequencies of T cells specific for each single antigen are about the same before and after the coenrichment and coexpansion process. For both approaches, the concomitant and separate antigen loading, we found comparable results with respect to recovery and purity of enriched IFN-γ-secreting T cells, expansion rate and antigen specificity of the expanded T cells.
In summary, our results show that the combined usage of several peptide pools enables simultaneous and efficient activation of CMV, EBV and adenovirus (tri-virus) specific CD4+ and CD8+ T cells. Activated tri-virus specific T cells can be coenriched using the IFN-g secretion assay technology and used either directly or after in vitro coexpansion for adoptive immunotherapy.

Disclosures:
Richter: Miltenyi Biotec GmbH: Employment. Foerster:Miltenyi Biotec GmbH: Employment. Lasmanowicz:Miltenyi Biotec GmbH: Employment. Brauns:Miltenyi Biotec GmbH: Employment. Kramer:Miltenyi Biotec GmbH: Employment. Jekow:Miltenyi Biotec GmbH: Employment. Rönspeck:Miltenyi Biotec GmbH: Employment. Assenmacher:m: Employment. Schmitz:Miltenyi Biotec GmbH: Employment.