Fig 2: Organotypic epithelial raft cultures (OERCs) of primary human keratinocytes and tumor cells. (A) Hematoxylin and eosin (H&E)-stained section of an OERC of primary human keratinocytes (PHKs). The dermal equivalent is composed of collagen, and contains murine fibroblasts used as feeder cells. On top of the dermal equivalent, the PHKs form a differentiated epithelium. (B) The MCPyV- tumor cell line MCC14/2 proliferates on top of the dermal equivalent, showing a dysplastic phenotype, without the presence of PHKs, as observed in the H&E-stained section. These cells express the MCC marker NCAM. (C) MCC14/2 cells also proliferate in OERCs when embedded into the dermal equivalents, as indicated by the arrow, while the PHKs form a differentiated epithelium on top. MCC14/2 cells growing into the dermal equivalent also express NCAM. All images were taken at an overall 200× magnification, and the bars equal 100 µm.

Fig 3: Distribution correlation among various TIL subsets. (a) The higher the densities of CD8+ T cells, the more TIM-3 expression was presented. (b) No significant correlation was presented between TIM-3 expression and CD56 distribution. (c, d) The lower the degrees of CD8 (c) and CD56 (d), the more Gal-9 expression was presented.

Fig 4: Identification and validation of NCAM1 as a potential host receptor for ZIKV.a, b Flow cytometry (a) and immunofluorescence (b) analyses of NCAM1 expression in U-251 MG, Vero or 293T cells. 293T cells were transfected with NCAM1 for 48 h. The length of scale bars in (b) is 10 µm. c, d Immunoblot (IB) and immunoprecipitation (IP) of lysates of ZIKV ENV (c) or EGFR (d) and Flag-NCAM1 or Flag-GFP overexpressed 293T cells. e Schematic representation of the viral binding and entry assays. f, g NCAM1 ECD protein and anti-NCAM1 antibody inhibit ZIKV attachment to U-251 MG cells (f). *P = 0.0168; **P = 0.0047. NCAM1 ECD protein and anti-NCAM1 antibody inhibit ZIKV internalized to U-251 MG cells (g). Data from four experiments (n = 4). NCAM1 ECD protein to mock, **P = 0.0013; Anti-NCAM1 antibody to mock, **P = 0.0026. Significantly different from Mock cells (two-tailed Student’s t test). h, i RT-qPCR revealed an increase in viral attachment (h) and entry (i) following NCAM1 overexpression in 293T cells. Data from three experiments (h n = 3; i NC treated n = 10, NCAM1 treated n = 7). *P = 0.0133 (h); *P = 0.0195 (i). Sgnificantly different from NC (negative control) treated 293T cells (two-tailed Student’s t test). j Flow cytometry analyses of NCAM1 expression in NCAM1-KO and WT U-251 MG cells. k Immunofluorescence (IF) staining analyses of ZIKV infection. Images shown are representative of three independent experiments. Scale bars, 50 µm. l Statistical analyses. Each biological replicate (n = 4) contains 3000 analyzed cells. **P = 0.0044. Significantly different from WT cells (two-tailed Student’s t test). Quantitative data in this figure are shown as the mean ± s.e.m. DAPI, blue; ZIKVE, red; NCAM1, green. Source data are provided as a Source Data file.

Fig 5: Conjugation of natural killer (NK) cells with CD19-expressing cells in the presence of 161519 trispecific killer engager (TriKE). (A) Joining of NK cells and tumor cells in the presence of 161519 TriKE. Purified NK cells and Raji cells were mixed with phosphate-buffered saline (PBS) or 161519 TriKE (10 µg/mL) and incubated at 37 °C, followed by staining of CD56 (red), CD19 (green), and DAPI (blue). Accumulation of cells was observed by confocal immunofluorescence microscopy in the presence of 161519 TriKE, scale bar = 50 µm. (B-D) Purified NK cells (CD56+) and CFSE-labeled CD19+ (Namalwa, Daudi, or Raji) cells were mixed with PBS or 161519 TriKE (10 µg/mL) and incubated at 37 °C for 0, 15, or 30 min. Cells were stained with 647-CD56 and analyzed by flow cytometry. The conjugation ratio was calculated by the proportion of FITC/Alexa-647 double-positive events within Alexa-647-positive events. (B) The representative percentage of NK cells binding to Namalwa cells is shown. The cumulative percentage of NK cells conjugated with (C) Namalwa, (D) Daudi, and (E) Raji cells is shown. Data are representative of 3 independent experiments and were analyzed by the Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.