EBAG9 controls CD8+ T cell memory formation responding to tumor challenge in mice.

Insight into processes that determine CD8+ T cell memory formation has been obtained from infection models. These models are biased toward an inflammatory milieu and often use high-avidity CD8+ T cells in adoptive-transfer procedures. It is unclear whether these conditions mimic the differentiation processes of an endogenous repertoire that proceed upon noninflammatory conditions prevailing in premalignant tumor lesions. We examined the role of cytolytic capacity on CD8+ T cell fate decisions when primed by tumor cells or by minor histocompatibility antigen-mismatched leukocytes. CD8+ memory commitment was analyzed in Ebag9-deficient mice that exhibited enhanced tumor cell lysis. This property endowed Ebag9-/- mice with extended control of Tcl-1 oncogene-induced chronic lymphocytic leukemia progression. In Ebag9-/- mice, an expanded memory population was obtained for anti-HY and anti-SV-40 T antigen-specific T cells, despite unchanged effector frequencies in the primary response. By comparing the single-cell transcriptomes of CD8+ T cells responding to tumor cell vaccination, we found differential distribution of subpopulations between Ebag9+/+ and Ebag9-/- T cells. In Ebag9-/- cells, these larger clusters contained genes encoding transcription factors regulating memory cell differentiation and anti-apoptotic gene functions. Our findings link EBAG9-controlled cytolytic activity and the commitment to the CD8+ memory lineage.

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.

The sorting receptor Sortilin exhibits a dual function in exocytic trafficking of interferon-γ and granzyme A in T cells.

Immunological control of infections or tumors depends on the release of effector cytokines and polarized secretion of cytotoxic granules from T cells and natural killer cells. Here we show that the sorting receptor Sortilin controlled both processes. In murine Sortilin-deficient cytotoxic T lymphocytes, regulated secretion of granzyme A and cytotoxic killing was enhanced and correlated with increased vesicle-associated membrane protein 7 availability. In contrast, loss of Sortilin reduced the release of interferon-γ upon infections and in autoimmune colitis. Exit of interferon-γ from the Golgi apparatus required the presence of Sortilin. Furthermore, we tracked the transport route of interferon-γ beyond this Sortilin-dependent Golgi to early endosome step. In wild-type T cells, trafficking of interferon-γ from the endosomal sorting platform to the plasma membrane proceeded independently of recycling endosomes, and interferon-γ remained excluded from late endosomes. Our results suggest that Sortilin modulates systemic immune responses through exocytic sorting of immunological effector molecules.