To gain insights into the parameters that affect the outcome of adoptive T cell therapy (ACT), we have utilized a mouse model that involves treating C57BL/6 mice bearing pre-B cell lymphomas with tumour antigen-specific Cytotoxic T Lymphocytes (CTLs). This model has been used previously to demonstrate that when the CTL are transferred into a host bearing a small tumour load, the tumour cells are efficiently eliminated. Conversely, when the tumour load is large, CTLs cannot eliminate the tumour and become functionally impaired. Antigen presentation by a number of tumour cells above a critical threshold was necessary and sufficient to trigger CTL inactivation. Here, we present our characterization of this mechanism of CTL impairment.
Our results show that level of antigen presentation by the tumour cells is irrelevant for CTL inactivation. Likewise, TCR affinity had no influence on the initiation of the inactivation program. Density of target cells was confirmed as the critical determinant of CTL activity and, consequently, ACT outcome. We employed live two-photon microscopy to analyse in situ the behaviour of the anti-tumour CTL in conditions of successful versus failed therapy (high vs. low tumour burden, respectively). The CTL that encountered high tumour cell density displayed lower speed and abnormal motility. RNA sequencing was utilized to compare the transcriptome of active and inactivated CTL purified from tumour-bearing mice. This analysis revealed a gene signature in the inactivated CTL that included genes related to the induction of both exhaustion and anergy.
Our results suggest that encounter of a high density of target (tumour) cells initiates a novel mechanism of CTL inactivation characterised by impaired cell motility and the induction of a genetic program with hybrid features of anergic and exhausted T cells. Interfering with this genetic program of inactivation may allow us to optimize CTL performance and improve the outcomes of ACT.