Figure 3. Tumor-reactive T cells induce transient cell-in-cell structure.

(A) Mean percentage of cell-in-cell tumor formations following incubation of B16F10 cells with immune cells (n=4). (B) Representative 3D projection and horizontal sections (Z-stack) of B16F10 cells incubated with gp100-reactive CD8⁺ T cells. (C) Mean percentage of cell-in-cell tumor formations and single cells in tumor-bearing NSG^-/- mice treated with DC adjuvant and anti-TRP1 antibodies (n=4). (D) Mean counts of cell-in-cell tumor formations in mouse cancer cell lines cultured overnight with activated CD8⁺ T cells (n=4). (E) Mean counts of cell-in-cell tumor formations in human cancer cell lines cultured overnight with activated allogeneic CD8⁺ T cells (n=4). (F–G) Representative images of freshly-isolated human melanoma cells incubated overnight with autologous tumor-reactive T cells. (H) Mean percentage of cell-in-cell tumor formations following incubation of tumor cells isolated from melanoma patient with autologous tumor-reactive T cells (four technical replicates). (I) Histological section of human melanoma in NSG^-/- mice 7 days after treatment with patient autologous TIL and high-dose IL-2. (J) H&E staining of histological sections of involved lymph node from metastatic melanoma patient. (K) Immunostaining of involved lymph nodes from metastatic melanoma patient. (L) H&E staining of histological sections of primary and sentinel lymph nodes from untreated stage IV melanoma patients. (M) H&E staining of histological sections of untreated primary and relapsed melanoma patient. Statistical significance was calculated using ANOVA with Tukey’s correction for multiple comparisons (* denotes p<0.05, ** denotes p<0.01 *** denotes p<0.001, and **** denotes p<0.0001). Error bars represent standard error. Scale bars = 20 μm.

Figure 3—figure supplement 1. Murine tumor cell lines undergo cell-in-cell formation incubation with reactive T cells, Related to Figure 2 and Figure 3.

(A) Representative images of B16F10 cells following incubation with different immune cell subsets from naïve mice. (B) Mean percentage over time of cell-in-cell and simple cells from B16F10 tumor incubated with gp100-reactive CD8⁺ or TRP1-reactive CD4⁺ T cells (n=4). (C) Representative images of breast epithelial cancer cells (4T1) incubated with allogeneic T cells (D). Representative images of breast epithelial non-transformed cells (EPH4) incubated with allogeneic T cells. (E) Representative confocal images of B16F10 and 4T1 incubated overnight with gp100-reactive or allogenic CD8⁺ T cells, respectively. (E-H). Representative images of murine cancer cell lines incubated overnight with activated allogeneic splenic CD8⁺ T cells. All experiments were repeated at least three times. Scale bars = 20 μm.

Figure 3—figure supplement 2. Human tumor cells undergo cell-in-cell formation following incubation with reactive T cells, Related to Figure 2 and Figure 3.

(A-G) Representative images of human cancer cell lines incubated overnight with allogeneic T cells from healthy donors. (H) H&E staining of involved lymph nodes from metastatic melanoma patients. All experiments were repeated at least three times. Scale bars = 20 μm.

Figure 3—video 1. B16 cell-in-cell induction in vitro following incubation with gp100-reactive T cells, related to Figure 3.

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Figure 3—video 2. HT29 cell-in-cell induction in vitro following incubation with allogeneic T cells, related to Figure 3.

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