Abstract

Blockade
of PD-1/PDL-1 interactions is proving an exciting, durable therapeutic modality
in a range of cancers to release T cells from checkpoint inhibition reviving
their inherent anti-tumour activity. Here we have studied various relevant ways
to model ex vivo T cell function to
compare the impact of the clinically utilised anti-PD-1 antibody, pembrolizumab
(Keytruda^®)
on the activation of human T cells: focussing on the release of
pro-inflammatory IFNγ and anti-inflammatory IL-10 to assess functionality. Firstly,
we investigated the actions of pembrolizumab in an acute model of T-cell
activation with either immature or mature allogeneic dendritic cells (DCs);
pembrolizumab enhanced IFNγ and IL-10 release from purified CD4+ T-cells in the
majority of donors. Next, we modelled the impact of pembrolizumab in settings
of more chronic T-cell activation. In a 7-day antigen-specific response to EBV peptides, the presence of pembrolizumab
resulted in a relatively modest increase in both IFNγ and IL-10 release.  When pembrolizumab was assessed upon long-term
stimulated CD4+ cells that had up-regulated the ‘exhaustion markers’ TIM-3,
LAG-3 as well as the pembrolizumab target PD-1,
there was a highly effective rescue of the otherwise exhausted response to
allogeneic DCs with respect to IFNγ production. By contrast, the restoration of
IL-10 production was considerably more limited. To assess clinical relevance in
a disease context, we investigated the consequence of PD-1/PD-L1 blockade in dissociated
cells of lung and colon carcinomas responding to allogeneic DCs: here,
pembrolizumab also enhanced IFNγ production from the majority of tumour
preparations whereas, again, the increase in IL-10 release was relatively modest
at best. In conclusion, we have shown that the contribution of PD-1 – revealed
by use of a canonical blocking antibody to interrupt its interaction with PD-L1
– to the production of an exemplar pro- and anti-inflammatory cytokine,
respectively, depends in magnitude and ratio on the particular stimulation setting
and activation status of the target T cell. Of note, using an allogeneic DC
stimulation assay we have identified that the response profile of CD4+ T cells
with exhaustion-like features directly mirrors that of dissociated cell
populations from primary tumours thereby indicating a disease-relevant
functional assay for the screening of immune checkpoint inhibitors in current
and future development.