WT1-Specific T Cells Exhibiting a Non-Exhausted, Functional Phenotype Can Be Generated From The Natural Repertoire Of Healthy Donors For Clinical Use
Authors: Schmied Sabine , Richter Anne , Assenmacher Mario , Schmitz Juergen 
Published: October 2019

Background
The Wilms tumor antigen 1 (WT1) is a self-antigen expressed at high levels in leukemic cells, but not in healthy tissue. As WT1 expression in leukemic cells drives leukemogenesis, it is a favorable target antigen for immunotherapy, e.g. adoptive transfer of allogeneic T cells, to prevent or treat leukemic relapse after stem cell transplantation (Cheever et al., Clin Cancer Res 2009;15(17)). WT1-specific CD8+ T cells have been detected in healthy individuals at low frequencies (Rezvani et al., Blood 2003;102). However, a comprehensive characterization 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 rapid method for the generation of functionally potent, polyclonal CD4+ and CD8+WT1-specific T cells for clinical use.

Methods
For direct ex vivo analysis of CD4+ WT1-specific T cells peripheral blood mononuclear cells (PBMC) of healthy blood donors were in vitro stimulated with a pool of overlapping peptides spanning the WT1 protein for 7 hours. Subsequently CD154 (CD40L)-expressing cells were magnetically enriched and flow cytometrically examined for expression of effector cytokines and their differentiation status. Presence and phenotype of CD8+ WT1-specific T cells have been studied after stimulation of presorted naïve and memory T cell populations with WT-1 peptide pool for 30 hours, magnetic enrichment of CD137+ (4-1BB) cells and subsequent staining using pMHCI-Tetramers.
For the generation of polyclonal WT1-specific CD4+ and CD8+ T cells PBMC were in vitro activated with WT-1 peptide pool for 30 hours. CD137+cells were magnetically selected and expanded for 9 days in the presence of the cytokines IL-7, IL-15 and IL-21 at low doses. Expanded T cells were analyzed for their phenotype, the expression of co-stimulatory and exhaustion markers and were tested for their functionality and cytotoxicity by restimulation experiments with antigen-loaded target cells.

Results
Ex vivo frequencies of WT1-specific T cells are low, 1 to 10 WT1-specific CD154+ CD4+ T cells can be detected within 1x106 CD4+ T cells. In about 80% of healthy donors (n=15) a CD4+ memory response, accompanied by production of effector cytokines like IFNγ, TNFα and IL-2, against WT1 peptides is present. Additionally, in all donors naïve WT1-specific CD4+ T cells can be detected. In contrast, detected CD137+CD8+ WT1-reactive T cells exhibit a naïve phenotype (CD45RA+CCR7+) in all donors (n=5), no WT1-reactive CD8+T cells could be enriched from presorted memory T cells.
To evaluate the usefulness of our improved short-term expansion protocol to generate potent WT1-specific T cell cultures for clinical use, we characterized CD137 enriched and expanded T cells. Notably, a high frequency of CD4+ and CD8+ T cells show specific reactivity against WT1-presenting autologous cells as detected by production of effector cytokines like IFNγ, TNFα and IL-2 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. Stainings using multiple WT1-MHCI-tetramers 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) which has been shown to mark cells with stem T cell like properties. Furthermore exhaustion markers like CD279 (PD-1), CD178 (FasL) and CD57 are scarcely expressed.

Conclusions
Functional, polyclonal, CD4+ and CD8+ WT1-specific, reactive T cells can be efficiently enriched directly ex vivo from the natural repertoire by magnetic separation of T cells after antigen-specific stimulation. Phenotypic and functional characterization revealed a non-exhausted phenotype of expanded WT1-specific T cells, thereby suggesting good persistence and functionality of the obtained T cell product in vivo.
Thus, our approach holds great potential for the GMP-compliant generation of WT1-specific T cells for future clinical use.

Disclosures:
Schmied: Miltenyi Biotec GmbH: Employment. Richter:Miltenyi Biotec GmbH: Employment. Assenmacher:Miltenyi Biotec GmbH: Employment. Schmitz:Miltenyi Biotec: Employment.