γ9δ2T cell diversity and the receptor interface with tumor cells
Anna Vyborova, … , Zsolt Sebestyen, Jürgen Kuball
Anna Vyborova, … , Zsolt Sebestyen, Jürgen Kuball
Published June 2, 2020
Citation Information: J Clin Invest. 2020;130(9):4637-4651. https://doi.org/10.1172/JCI132489.
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Research Article Immunology Oncology

γ9δ2T cell diversity and the receptor interface with tumor cells

Abstract

γ9δ2T cells play a major role in cancer immune surveillance, yet the clinical translation of their in vitro promise remains challenging. To address limitations of previous clinical attempts using expanded γ9δ2T cells, we explored the clonal diversity of γ9δ2T cell repertoires and characterized their target. We demonstrated that only a fraction of expanded γ9δ2T cells was active against cancer cells and that activity of the parental clone, or functional avidity of selected γ9δ2 T cell receptors (γ9δ2TCRs), was not associated with clonal frequency. Furthermore, we analyzed the target-receptor interface and provided a 2-receptor, 3-ligand model. We found that activation was initiated by binding of the γ9δ2TCR to BTN2A1 through the regions between CDR2 and CDR3 of the TCR γ chain and modulated by the affinity of the CDR3 region of the TCRδ chain, which was phosphoantigen independent (pAg independent) and did not depend on CD277. CD277 was secondary, serving as a mandatory coactivating ligand. We found that binding of CD277 to its putative ligand did not depend on the presence of γ9δ2TCR, did depend on usage of the intracellular CD277, created pAg-dependent proximity to BTN2A1, enhanced cell-cell conjugate formation, and stabilized the immunological synapse (IS). This process critically depended on the affinity of the γ9δ2TCR and required membrane flexibility of the γ9δ2TCR and CD277, facilitating their polarization and high-density recruitment during IS formation.

Authors

Anna Vyborova, Dennis X. Beringer, Domenico Fasci, Froso Karaiskaki, Eline van Diest, Lovro Kramer, Aram de Haas, Jasper Sanders, Anke Janssen, Trudy Straetemans, Daniel Olive, Jeanette Leusen, Lola Boutin, Steven Nedellec, Samantha L. Schwartz, Michael J. Wester, Keith A. Lidke, Emmanuel Scotet, Diane S. Lidke, Albert J.R. Heck, Zsolt Sebestyen, Jürgen Kuball

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Figure 5

Target cell-surface distribution of BTN3A1 with and without γ9δ2T cell engagement.

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Target cell-surface distribution of BTN3A1 with and without γ9δ2T cell e...
(A) Enrichment of the TCR in the IS was quantified as the ratio between CD3ε-AF647 Ab signal intensity inside versus outside the synapse area. Analysis was done on at least 7 independent T cell–tumor cell conjugate images (representative images are shown in B). Significance was determined by 2-way ANOVA with Mann-Whitney U post hoc analysis. A P value of less than 0.05 was considered significant. Dots represent individual images, and bars represent the median values. (B) Representative images of the TEG–tumor cell conjugates and the regions of interest (ROIs) used for quantification. Original magnification, ×63. (C) Topology of BTN3A1 molecules: the number of fluorescence events on the cell membrane (BTN3A1-EmGFP) was classified according to the cluster size based on confocal microscopic analysis of HEK293T cells stably expressing BTN3A1-EmGFP (n = 15). (D) Histogram comparing the Hopkins statistic (H) of BTN3A1 clustering for control and PAM-treated cells, imaged using dSTORM. A total of 275 ROIs across 10 cells were analyzed, and the probability distribution function (PDF) for H was plotted. (E) Cumulative distribution function (CDF) for the nearest neighbor distances (NNDs) for control-treated (HEK resting, green), PAM-treated (HEK PAM, red), and 20.1 Ab–treated (HEK mAb, blue) samples. The CDF plots presented are read as follows: the curve furthest to the right for high values of the CDF represent data that tend to have greater localizations per cluster compared with the curves on the left. (F) Example of BTN3A1-EmGFP polarization to the IS in the presence of 20.1 Ab. Threshold area boxes were used to calculate the percentage of synaptic threshold pixels at time point 0 (left) and 20 minutes after T cell conjugation (right, upper). The lower panel shows target cells (green) and T cells (red) at corresponding time points. Original magnification, ×20. (G) Synaptic BTN3A1-GFP polarization measured on HEK293T cells following engagement with primary γ9δ2T cells with or without 20.1 Ab, NKT cells served as a control. n = 30 BTN3A1-GFP cells analyzed for each condition across 3 experiments. A P value of less than 0.041 was considered significant. *P < 0.041, **P < 0.0041, and ***P < 0.00041, by 2-way ANOVA with Bonferroni’s post hoc correction for multiple comparisons.