Volume 2 Supplement 2
P66. Generating and characterising WT1-specific T cells – research towards adoptive tumour therapy
© Schmied et al; licensee BioMed Central Ltd. 2014
Published: 12 March 2014
The Wilms tumour antigen 1 (WT1) is a self-antigen expressed at high levels in leukaemic cells, but not in healthy tissue. WT1, therefore, is a favourable target antigen for allogeneic T cell therapy to prevent leukaemic relapse after stem cell transplantation. However, a comprehensive characterisation of CD4+ and CD8+ WT1-specific T cells is missing and the efficient expansion of a polyclonal WT1-reactive T cell population for clinical use has remained a major challenge.
In this study we aim to directly ex vivo characterize WT1-specific T cells present in the blood of healthy donors at high-resolution and to develop a method for the rapid generation of functionally potent, polyclonal CD4+ and CD8+ WT1-specific T cells for clinical use.
We utilise the magnetic enrichment of activation marker expressing cells after antigen-specific stimulation, as low frequencies of WT1-specific T cells in healthy donors do not allow direct detection.
Ex vivo frequencies of WT1-specific T cells range between 10-6 and 10-5 WT1-specific T cells within CD4+ T cells. In about 80% of healthy donors (n=15) a CD4+ memory response, accompanied by production of effector cytokines like IFN-γ and TNF-α against WT1 peptides is present. In contrast, detected CD137+CD8+ WT1-reactive T cells exhibit a naïve phenotype (CD45RA+CCR7+) in all tested donors (n=5).
An improved short-term expansion protocol to generate potent WT1-specific T cell cultures for clinical use was established utilising a CD137+ cell enrichment step. Notably, a high frequency of expanded CD4+ and CD8+ T cells show specific reactivity against WT1-presenting autologous cells as detected by production of effector cytokines after antigen-specific restimulation. Cytotoxic activity against antigen-loaded target cells could be shown by direct flow-cytometry-based cytotoxicity assays and antigen-specific upregulation of the degranulation marker CD107a. WT1-MHCI-Tetramerstainings furthermore confirmed antigen-specificity and suggested polyclonality within the CD8+ T cell population. In contrast to previous expansion protocols our polyclonally expanded T cells exhibit a favourable, unexhausted memory phenotype, express co-stimulatory markers CD27 and CD28 and the IL7R-a chain (CD127).
Functional, polyclonal CD4+ and CD8+ WT1- reactive T cells can be efficiently enriched directly ex vivo from the natural repertoire by magnetic separation of T cells after antigen-specific stimulation. Thus, our approach holds great potential for the GMP-compliant generation of WT1-specific T cells for future clinical use.
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.