Human c-SRC kinase (CSK) overexpression makes T cells dummy
Adoptive cell therapy with T-cell receptor (TCR)-engineered T cells represents a powerful method to redirect the immune system against tumours. However, although TCR recognition is restricted to a specific peptide–MHC (pMHC) complex, increasing numbers of reports have shown cross-reactivity and off-target effects with severe consequences for the patients. This demands further development of strategies to validate TCR safety prior to clinical use. We reasoned that the desired TCR signalling depends on correct pMHC recognition on the outside and a restricted clustering on the inside of the cell. Since the majority of the adverse events are due to TCR recognition of the wrong target, we tested if blocking the signalling would affect the binding. By over-expressing the c-SRC kinase (CSK), a negative regulator of LCK, in redirected T cells, we showed that peripheral blood T cells inhibited anti-CD3/anti-CD28-induced phosphorylation of ERK, whereas TCR proximal signalling was not affected. Similarly, overexpression of CSK together with a therapeutic TCR prevented pMHC-induced ERK phosphorylation. Downstream effector functions were also almost completely blocked, including pMHC-induced IL-2 release, degranulation and, most importantly, target cell killing. The lack of effector functions contrasted with the unaffected TCR expression, pMHC recognition, and membrane exchange activity (trogocytosis). Therefore, co-expression of CSK with a therapeutic TCR did not compromise target recognition and binding, but rendered T cells incapable of executing their effector functions. Consequently, we named these redirected T cells “dummy T cells” and propose to use them for safety validation of new TCRs prior to therapy.
KeywordsT-cell receptor TCR TCR signalling CSK Immunotherapy
Antigen presenting cells
Fetal calf serum
Green fluorescent protein
Good manufacturing practice
Immunoreceptor tyrosine-based activation motifs
Melanoma-associated antigen 3
Melanoma antigen recognized by T cells 1
Mean fluorescence intensity
Peripheral blood mononuclear cells
Transforming growth factor β II
The authors would like to thank the members of the Smeland lab and of the Department of Cellular Therapy for their positive input and support. We are also thankful to Dr. Pierre Dillard for commenting on this manuscript.
EMI, SW, GES, LEF, and JHM conceived and designed the experiments. EMI, NM, MPO, AF, GS, CP, and JHM performed the experiments. SW, OB, GK, and JHM interpreted the data. SW, EMI, NM, and JHM wrote the manuscript and all authors edited the manuscript.
This work was supported by the Gene Therapy program of the Radium Hospital to Sébastien Wälchli and Anne Fåne, The Norwegian Research Council to Else Marit Inderberg (#244388), and partly supported by an Innovation Grant from Southern and Eastern Norway Regional Health Authority to Nadia Mensali (#13/00367-88).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
The study was approved by the Regional Committee for Medical and Health Research Ethics (REC South-East, Norway) (Approval no. 2013/624).
Informed consent from healthy donors was given.
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