Supplementary Figure 13 from Spatial and Single-Cell Analyses Reveal Heterogeneity of DNAM-1 Receptor–Ligand Interactions That Instructs Intratumoral γδT-cell Activity
Authors: Wang Xiaolin , Wang Hui , Lu Zhengjing , Liu Xiangjun , Chai Wenjia , Wang Wei , Feng Jun , Yang Shen , Yang Wei , Cheng Haiyan , Chen Chenghao , Zhang Shihan , Sun Nian , Liu Qiaoyin , Li Qiliang , Song Wenqi , Jin Fang , Zeng Qi , Wang Shengcai , Su Yan , Wang Huanmin , Ni Xin , Gui Jingang 
Published: January 2025

<p>Supplementary Figure 13 The effects of CD155 on γδT-cell-cytotoxicity against neuroblastoma, and on proliferation and migration of NB cells. (A) and (B) α-TIGIT is used in the rhCD155 treatment of γδT cells. DNAM-1 is detected by FACS and Western blot. (C-F) rhCD155 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). (G) γδT-cell numbers are counted and statistically analyzed during in vitro culture in presence of rhCD155. (H) The CFSE proliferation assays are performed during in vitro culture in presence of rhCD155. γδT cells are detected by FACS (right) and statistically analyzed (left). MFI is labeled in the representative FACS histograms. (I) γδT cells are pre-treated with rhCD155 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). (J) The phosphorylation of Akt and ERK1/2 in γδT cells is detected by Western blot after treatment using rhCD155 for 24 h. (K) and (L) The cytotoxicity assays are performed with γδT cells pre-treated with rhCD155 or α-TIGIT for 24 h and NB cell lines. (M) and (N) The cytotoxicity assays are performed to detected γδT-cell cytotoxicity against SH-SY5Y and CHLA-255 cells in presence of α-CD155. (O-Q) IFN-γ, perforin and granzyme B expression are detected by FACS upon PMA and ionomycin stimulation after rhCD155 pre-treatment (left) and MFI is labeled in the representative FACS histograms (right). (R-T) CCK-8, Transwell and wound-healing sassays are performed using SK-N-BE2-NC and SK-N-BE2-KoCD155 cells. Representative images of SK-N-BE2 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). (U) The phosphorylation of Akt and ERK1/2 in γδT cells is detected by Western blot after SK-N-BE2-NC and SK-N-BE2-KoCD155 stimulation. (V) TIGIT is detected by FACS after 24 h co-culture with SK-N-BE2-NC and SK-N-BE2-KoCD155 cells (left) and MFI is labeled in the representative FACS histograms (right). 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.</p>