Supplementary Figure 12 from Spatial and Single-Cell Analyses Reveal Heterogeneity of DNAM-1 Receptor–Ligand Interactions That Instructs Intratumoral γδT-cell Activity

Supplementary Figure 12 The effects of CD112 on γδT-cell-cytotoxicity against neuroblastoma, and on proliferation and migration of NB cells. (A-D) rhCD112 is used to treat γδT cells for 24 h. TIGIT, CD96 and CD112R are detected by FACS (left) or Western blot and MFI is labeled in the representative FACS histograms (right). (E) γδT-cell numbers are counted and statistically analyzed during in vitro culture in presence of rhCD112. (F) The CFSE proliferation assays are performed during in vitro culture in presence of rhCD112. γδT cells are detected by FACS (right) and statistically analyzed (left). MFI is labeled in the representative FACS histograms. (G) γδT cells are pre-treated with rhCD112 for 24 h and subjected to calcium flux detection by Fluo-4 AM labeling and low-concentration PMA and ionomycin stimulation. The calcium flux results are used to benchmark the levels of activation. The results are recorded and illustrated by FACS (right) and statistically analyzed (left). (H) The phosphorylation of Akt and ERK1/2 in γδT cells is detected by Western blot after treatment using rhCD112 with or without α-DNAM-1 for 24 h. (I) and (J) The cytotoxicity assays are performed with γδT cells pre-treated with rhCD112 for 24 h and NB cell lines. (K) and (L) The cytotoxicity assays are performed to detected γδT-cell cytotoxicity against SH-SY5Y and CHLA-255 cells in presence of α-CD112. (M-O) CCK-8, Transwell and wound-healing sassays are performed using CHLA-255-NC and CHLA-255-shCD112 cells. Representative images of CHLA-255 cell migration obtained from the Transwell (magnification × 100) and wound-healing (magnification × 100) assays are shown (right). The cell numbers obtained from the Transwell assays are counted, and the relative migration rate obtained from the wound-healing assays is calculated by dividing the change in the distance between the scratch edges by the initial distance (left). (P) CD112 is overexpressed in IMR-32 cells transiently by transfecting constructed CD112 vector and CD112 expression is detected by qPCR, FACS and Western blot. MFI is labeled in the representative FACS histograms. (Q) DNAM-1 reduction is detected by FACS after 24 h co-culture with IMR-32-vector or IMR-32-CD112 cells (left) and MFI is labeled in the representative FACS histograms (right). (R) The phosphorylation of Akt and ERK1/2 in γδT cells is detected by Western blot after CHLA-255-NC and CHLA-255-shCD112 stimulation. (S) The cytotoxicity assays are performed to detected γδT-cell cytotoxicity against IMR-32-vector and IMR-32-CD112 cells. The results are expressed as the means ± SEMs from at least three independent experiments. Significant differences between groups are represented by ns no significance, * p < 0.05, ** p < 0.01 and *** p < 0.001.