Abstract Book 2010 - CIMT Annual Meeting

057 Foerster | Cellular therapy
Generation of multivirus-specific CD4+ and CD8+ T cells
for adoptive immunotherapy
Anna Foerster, Verena Lasmanowicz, Olaf Brauns, Sven Kramer, Petra Jekow, Wolfgang Rönspeck,
Jürgen Schmitz, Mario Assenmacher, Anne Richter
Department of Research & Development, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany
Adoptive transfer of T lymphocyte populations
with specificities for several antigens is an attractive
strategy for the treatment of multiple infections
in immunocompromised patients or of cancer. We
have established a protocol for the generation of
multiantigen-specific CD4+ and CD8+ T cells
using newly developed pools of peptides for in vitro
T cell stimulation and a subsequent magnetic enrichment
of functional T cells according to IFN-γ
secretion.Peptide pools consist of synthetic peptides
of mainly 15 amino acid (aa) length with 11
aa overlap covering the complete antigenic protein.
We have analyzed the efficiency of peptide pools
compared to recombinant proteins and immunodominant
HLA-A2 and –B7-restricted peptides for
short-term in vitro restimulation and induction of
IFN-γ in cytomegalovirus (CMV) pp65 and IE-1 specific
CD4+ and CD8+ T cells derived from CMVinfected
healthy blood donors.
The results show that the peptide pools as well as
the 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 peptide epitopes of 8-10
amino acids. The latter are well defined only for
a limited panel of antigens and are restricted to
certain HLA alleles.
To test the usability of peptide pools to generate
multivirus-specific T cells for adoptive immunotherapy
we stimulated PBMC from leukapheresis of
healthy donors with a combination of four peptide
pools selected from CMV pp65 and IE-1, adenovirus
(AdV) hexon, and Epstein-Barr-Virus (EBV) EBNA-1
and BZLF-1 for four hours. The Large Scale IFN-γ
Secretion Assay Enrichment Kit was used to magnetically
enrich IFN-γ secreting T cells to a purity of
>90%. Antigen specificity and functionality of the
enriched T cells were controlled after expansion.
Cells expanded between 4 and 745 fold within 9-14
days. Expanded cells contained high frequencies
of pp65495-503/A2 tetramer+ and pp65417-426/B7
tetramer+ CD8+ T cells. Additionally after restimulation
of the expanded cells with the mixture
of peptide pools and intracellular IFN-γ staining,
21-53% of CD4+ T cells and 53-87% of CD8+ T
cells produced IFN-γ. Moreover comparing the stimulation
of PBMC with reactivation of the T cell
lines with each peptide pool separately showed that
the specificity for each antigen sustained during
the enrichment and expansion phase.
The concomitant addition of four peptide pools increases
the competition of peptides to be loaded on
MHC molecules, which might decrease the efficient
activation of each antigen-specific T cell. Therefore
we included in our study the separation of PBMC
in four samples, separate loading of the samples
with one peptide pool for two hours, and recombination
of PBMC for T cell stimulation for four
hours. We found comparable results in the number
of enriched cells, expansion rate of T cells, and the
antigen-specificity of the T cell lines for concomitant
and separate antigen loading.
In summary, we have established a protocol for
rapid in vitro generation of multivirus-specific
CD4+ and CD8+ T cells using a combination of
peptide pools from several antigens for restimulation
and subsequent magnetic selection of IFN-γ
secreting T cells.
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