Figure 5: CD8⁺ T-cells are required for synergy between SMC and immune checkpoint inhibitors for the treatment of glioblastoma.

(a) The expression of the indicated immune factors was detected by ELISA from cell culture supernatants of CT-2A cells that were co-incubated for 48 h with splenocytes derived from naive mice or mice previously cured of intracranial CT-2A tumours by SMC and anti-PD-1 cotreatment (1:20 ratio of CT-2A cells to splenocytes). Data are plotted as heat maps using normalized scaling. Box and whisker plots of the data are shown in Supplementary Fig. 13a. (b) Quantification of the indicated factor was determined by ELISA from CT-2A cells that were cocultured with splenocytes derived from naive or cured mice (1:20 ratio) and treated with vehicle or 5 μM LCL161 (SMC) for 48 h. Box and whisker plots of the data are shown in Supplementary Fig. 13b. (c) Splenocytes from naive or cured mice were cocultured with mKate2-tagged CT-2A cells (CT-2A-mKate2) in the presence of 20 μg ml⁻¹ control IgG or anti-PD-1 and 5 μM of the indicated SMC. Enumeration of CT-2A-mKate2 cells was performed using the Incucyte Zoom. Crosses depict mean, solid horizontal lines depict median, boxes depict 25th to 75th percentile, and whiskers depict min–max range of the values. Significance was compared with naive splenocytes as assessed by ANOVA with Dunnett’s multiple comparison test. Significance is reported as * when P<0.0001. n=6 for naive mice and n=6 for cured mice. Scale bar, 100 μm. (d) C57BL/6 mice harbouring intracranial CT-2A tumours were treated at the indicated date with combinations of either IgG (i.p.) and vehicle (oral) or α-PD-1 (i.p) and 75 mg kg⁻¹ LCL161 (oral) and i.p. administration of either IgG, α-CD4 or α-CD8 (all antibodies were 250 μg). (e) CD-1 nude mice bearing intracranial CT-2A tumours were treated at the indicated times with combinations of vehicle or 75 mg kg⁻¹ LCL161 orally and PBS or 250 μg of IgG or α-PD-1 i.p. (d,e) Data represent the Kaplan–Meier curve depicting mouse survival. Log-rank with Holm–Sidak multiple comparison: *P<0.05; **P<0.01. Numbers in parentheses represent number of mice per group.