Fig 2: Characterization of Delta-24-ACT functionality in DIPG.(A) 4-1BBL protein expression in NP53, XFM, SU-DIPG IV, and TP54 cells infected with Delta-24-ACT at the indicated MOIs, as determined by Western blotting. C+, 4-1BBL recombinant protein. (B) Representative immunofluorescence images of 4-1BBL expression in NP53- and XFM-infected cells compared with mock-infected cells. Scale bar: 20 µm. (C) 4-1BBL protein expression in the membranes of murine and human cells infected with Delta-24-ACT at the indicated MOIs, as determined by flow cytometry. The percentage of 4-1BBL+ cells is shown. One-way ANOVA was performed (n = 3, each group), and P values are shown above respective bars. Data are shown as the mean ± SEM. (D) Schedule of the experiment for the in vivo determination of 4-1BBL expression, and evaluation of 4-1BBL protein expression in NP53 tumors from control- or Delta-24-ACT–treated mice (n = 3), as determined by Q-PCR. Data are shown as the mean ± SEM. (E) IFN-? and (F) granzyme B production by CD8+ lymphocytes. CD8+ T cells from PMEL mice were cocultured with NP53 cells infected with Delta-24-RGD, Delta-24-ACT (MOI = 100), or the mock control. CD8+ lymphocytes activated with CD3 and CD28 but not NP53 cells, and CD8+ lymphocytes activated with CD3, CD28, and 4-1BB antibody were used as negative and positive controls for the experiment, respectively. One-way ANOVA was performed (n = 3, each group), and P values are shown above respective bars. Data are shown as the mean ± SEM.

Fig 3: Modulation of the immune compartment in GL261 tumor-bearing mice after treatment with Delta-24-ACT in combination with PD-L1 antibody. Analysis of tumor-infiltrating lymphocyte populations in the brains of mice bearing GL261 tumors treated with PBS (control), aPD-L1, Delta-24-ACT or Delta-24-ACT/aPD-L1. The studied populations were CD45+ cells (A), myeloid cell populations, including macrophages, DCs and microglia (B), and lymphoid cell populations, including CD8+, CD4+, Tregs and NK cells (C). The analyses of the functional status of the myeloid cells described above included analyses of PD-L1 (D), CD86+MHCI+I (E), CD137L (F) markers and the percentage of M1 macrophages (G). Percentage of DCs present in the lymph nodes. (H) DC functional status. The CD40, CD86 and PD-L1 markers were analyzed (I). Phenotype of CD8+ and CD4+ T cells (J) based on studies of the CD137, GITR, PD-1 and GrzB markers. (K) Quantification of splenocyte IFN-gamma production by ELISPOT after the indicated treatment. Top panel: mean counted spots. Lower panel: mean spot size (one-way ANOVA).