Simultaneous Genetic Ablation of PD-1, LAG-3, and TIM-3 in CD8 T Cells Delays Tumor Growth and Improves Survival Outcome.

Immune checkpoint inhibitors (ICI) represented a step forward in improving the outcome of patients with various refractory solid tumors and several therapeutic regimens incorporating ICI have already been approved for a variety of tumor entities. However, besides remarkable long-term responses, checkpoint inhibition can trigger severe immune-related adverse events in some patients. In order to improve safety of ICI as well as T cell therapy, we tested the feasibility of combining T cell-based immunotherapy with genetic disruption of checkpoint molecule expression. Therefore, we generated H-Y and ovalbumin antigen-specific CD8+ T cells with abolished PD-1, LAG-3, and TIM-3 expression through CRISPR/Cas9 technology. CD8+ T cells, subjected to PD-1, LAG-3, and TIM-3 genetic editing, showed a strong reduction in immune checkpoint molecule expression after in vitro activation, while no relevant reduction in responsiveness to in vitro stimulation was observed. At the same time, in B16-OVA tumor model, transferred genetically edited OT-1 CD8+ T cells promoted longer survival compared to control T cells and showed enhanced expansion without associated toxicity. Our study supports the notion that antigen-specific adoptive T cell therapy with concomitant genetic disruption of multiple checkpoint inhibitory receptors could represent an effective antitumor immunotherapy approach with improved tolerability profile.

Long-term in vitro expansion ensures increased yield of central memory T cells as perspective for manufacturing challenges.

Adoptive T cell therapy (ATT) has revolutionized the treatment of cancer patients. A sufficient number of functional T cells are indispensable for ATT efficacy; however, several ATT dropouts have been reported due to T cell expansion failure or lack of T cell persistence in vivo. With the aim of providing ATT also to those patients experiencing insufficient T cell manufacturing via standard protocol, we evaluated if minimally manipulative prolongation of in vitro expansion (long-term [LT] >3 weeks with IL-7 and IL-15 cytokines) could result in enhanced T cell yield with preserved T cell functionality. The extended expansion resulted in a 39-fold increase of murine CD8+ T central memory cells (Tcm). LT expanded CD8+ and CD4+ Tcm cells retained a gene expression profile related to Tcm and T memory stem cells (Tscm). In vivo transfer of LT expanded Tcm revealed persistence and antitumor capacity. We confirmed our in vitro findings on human T cells, on healthy donors and diffuse large B cell lymphoma patients, undergoing salvage therapy. Our study demonstrates the feasibility of an extended T cell expansion as a practicable alternative for patients with insufficient numbers of T cells after the standard manufacturing process thereby increasing ATT accessibility.

Chaperone protein HSC70 regulates intercellular transfer of Y chromosome antigen DBY.

Recent studies have demonstrated that CD4+ T cells can efficiently reject MHC-II-negative tumors. This requires indirect presentation of tumor-associated antigens on surrounding antigen-presenting cells. We hypothesized that intercellular transfer of proteins is not the sole consequence of cell death-mediated protein release, but depends on heat-shock cognate protein 70 (HSC70) and its KFERQ-like binding motif on substrate proteins. Using human Y chromosome antigen DBY, we showed that mutation of one of its 2 putative binding motifs markedly diminished T cell activation after indirect presentation and reduced protein-protein interaction with HSC70. Intercellular antigen transfer was shown to be independent of cell-cell contact, but relied on engulfment within secreted microvesicles. In vivo, alterations of the homologous KFERQ-like motif in murine DBY hampered tumor rejection, T cell activation, and migration into the tumor and substantially impaired survival. Collectively, we show that intercellular antigen transfer of DBY is tightly regulated via binding to HSC70 and that this mechanism influences recognition and rejection of MHC-II-negative tumors in vivo.

A Transgenic Dual-Luciferase Reporter Mouse for Longitudinal and Functional Monitoring of T Cells In Vivo.

Adoptive T-cell therapy (ATT) efficacy is limited when targeting large solid tumors. The evaluation of ATT outcomes using accessory treatment would greatly benefit from an in vivo monitoring tool, allowing the detection of functional parameters of transferred T cells. Here, we generated transgenic bioluminescence imaging of T cells (BLITC) mice expressing an NFAT-dependent click-beetle luciferase and a constitutive Renilla luciferase, which supports concomitant in vivo analysis of migration and activation of T cells. Rapid transferability of our system to preestablished tumor models was demonstrated in the SV40-large T antigen model via both crossbreeding of BLITC mice into a T-cell receptor (TCR)-transgenic background and TCR transduction of BLITC T cells. We observed rapid tumor infiltration of BLITC CD8+ T cells followed by a burst-like activation that mirrored rejection kinetics. Using the BLITC reporter in the clinically relevant H-Y model, we performed female to male transfers and detected H-Y-specific alloreactivity (graft-versus-host disease) in vivo In an H-Y solid tumor model, we found migration of adoptively transferred H-Y TCR-transgenic CD4+ T cells into the tumor, marked by transient activation. This suggests a rapid inactivation of infiltrating T cells by the tumor microenvironment, as confirmed by their expression of inhibitory receptors. In summary, the BLITC reporter system facilitates analysis of therapeutic parameters for ATT, is rapidly transferable to models of interest not restricted to tumor research, and is suitable for rapid screening of TCR clones for tumor rejection kinetics, as well as off-target effects. Cancer Immunol Res; 6(1); 110-20. ©2018 AACR.