Three-Dimensional Modeling of Solid Tumors and Their Microenvironment to Evaluate T Cell Therapy Efficacy In Vitro.

Immunotherapy development for solid tumors remains challenging, partially due to a lack of reproducible, cost-effective in vitro three-dimensional (3D) models to mimic the heterogeneous and complex tumor microenvironment. Here, we investigate the cellular anti-tumor reactivity of αß T cells engineered to express a defined γδ TCR (TEG A3). For that purpose, we developed a 3D cytotoxicity assay targeting cell line-derived spheroids or patient-derived tumor organoids formed in serum-free media. Tumor cell lysis by TEG A3 was monitored using the Incucyte S3 live-cell imaging system with the apoptosis marker caspase 3/7 green and endpoint readouts of IFN-γ secretion in the supernatant. The 3D cytotoxicity assay model system was able to adequately demonstrate TEG A3 reactivity toward targets expressing an isoform of CD277 (CD277J). To obtain a more complex heterogeneous tumor microenvironment, patient-derived organoids were mixed with unmatched patient-derived fibroblasts or matched cancer-associated fibroblasts. In all assays, we demonstrated the tumor target specificity of TEG A3, lysing tumor cells within 48 h. Our study demonstrates the utility of complex 3D cytotoxicity assay model systems incorporating the tumor microenvironment in the functional evaluation of T cell-based adoptive immunotherapy, providing a useful platform for early-stage preclinical development of immunotherapies.

BTK Isoforms p80 and p65 Are Expressed in Head and Neck Squamous Cell Carcinoma (HNSCC) and Involved in Tumor Progression.

Here, we describe the expression of Bruton's Tyrosine Kinase (BTK) in head and neck squamous cell carcinoma (HNSCC) cell lines as well as in primary HNSCC samples. BTK is a kinase initially thought to be expressed exclusively in cells of hematopoietic origin. Apart from the 77 kDa BTK isoform expressed in immune cells, particularly in B cells, we identified the 80 kDa and 65 kDa BTK isoforms in HNSCC, recently described as oncogenic. Importantly, we revealed that both isoforms are products of the same mRNA. By investigating the mechanism regulating oncogenic BTK-p80/p65 expression in HNSSC versus healthy or benign tissues, our data suggests that the epigenetic process of methylation might be responsible for the initiation of BTK-p80/p65 expression in HNSCC. Our findings demonstrate that chemical or genetic abrogation of BTK activity leads to inhibition of tumor progression in terms of proliferation and vascularization in vitro and in vivo. These observations were associated with cell cycle arrest and increased apoptosis and autophagy. Together, these data indicate BTK-p80 and BTK-p65 as novel HNSCC-associated oncogenes. Owing to the fact that abundant BTK expression is a characteristic feature of primary and metastatic HNSCC, targeting BTK activity appears as a promising therapeutic option for HNSCC patients.

αß-T Cells Engineered to Express γδ-T Cell Receptors Can Kill Neuroblastoma Organoids Independent of MHC-I Expression.

Currently ~50% of patients with a diagnosis of high-risk neuroblastoma will not survive due to relapsing or refractory disease. Recent innovations in immunotherapy for solid tumors are highly promising, but the low MHC-I expression of neuroblastoma represents a major challenge for T cell-mediated immunotherapy. Here, we propose a novel T cell-based immunotherapy approach for neuroblastoma, based on the use of TEG002, αß-T cells engineered to express a defined γδ-T cell receptor, which can recognize and kill target cells independent of MHC-I. In a co-culture killing assay, we showed that 3 out of 6 neuroblastoma organoids could activate TEG002 as measured by IFNγ production. Transcriptional profiling showed this effect correlates with an increased activity of processes involved in interferon signaling and extracellular matrix organization. Analysis of the dynamics of organoid killing by TEG002 over time confirmed that organoids which induced TEG002 activation were efficiently killed independent of their MHC-I expression. Of note, efficacy of TEG002 treatment was superior to donor-matched untransduced αß-T cells or endogenous γδ-T cells. Our data suggest that TEG002 may be a promising novel treatment option for a subset of neuroblastoma patients.

Towards a surrogate system to express human lipid binding TCRs.

BACKGROUND: Previously we reported that natural nut lipids were necessary for sensitization and that natural killer T cells (NKTs) must play a critical role in the development of food allergic responses. A major bottleneck in further understanding the interaction of nut lipids with the cells of the human immune system is the lack of well-characterized lipid responsive human cell lines. OBJECTIVE: In the present study, we engineered human T cell receptor (TCR) sequences TRAV10 and TRBV25 responsive to α-GalCer into a stable murine iNKT hybridoma and surrogate human T cell lines. RESULTS: The murine hybridoma system has shown to be problematic. To overcome this limitation, the expression of human TCR α/ß sequences has been achieved driven by a bidirectional promoter on a plasmids or a lentivirus system, employing stable DC cell lines as lipid presenting cells, and a stable T cell line as a surrogate system. Further, a commercial human Jurkat T cell line containing an inducible secreted luciferase reporter construct was shown to be functional and can be used for a transient expression of human TCRs in a lipid screening program. The transfection efficiencies were improved using the lentivirus polycistronic constructs containing the P2A sequence in a TCR αß/γδ null cell line (Jurkat 76). CONCLUSIONS: The results suggest that the mis-pairing of the endogenous α/ß TCR during ER folding in the presence of the new human TCR sequences could be impairing the functionality of the TCR lipid receptors. The surrogate systems presented here are important first steps in the establishment of human cell-specific lipid responsive libraries for the study of natural lipid substances.