Fig 2: Chemotherapeutic agents, but not MEK inhibitors, impair T cell proliferation and activation in vivo.a C57BL/6-Ly5.1 (CD45.1+) mice infused with CFSE-labeled OT-I T cells (CD45.2+) were treated for 6 days with the indicated doses of MEK inhibitor GDC-0623 or vehicle. Mice were immunized with OVA protein admixed with anti-CD40 Ab. After 3 days, splenocytes were analyzed. Upper row: dot plots of gated OT-I T cells. Lower row: CFSE dilution of gated OT-I T cells; differentially colored histograms represent data from individual mice. Bar charts to the right: cumulative data from six experiments (total mice: vehicle n = 23, GDC-0623 (10 mg kg-1) n = 19, GDC-0623 (30 mg kg-1) n = 21), showing quantification of OT-I T cell numbers and divided fraction. One-way ANOVA with post hoc Tukey’s test. Significance levels are indicated by asterisks (*p = 0.05; **p = 0.01; ***p = 0.001; ****p = 0.0001). b Intracellular IFN? levels of OT-I T cells after ex vivo restimulation with SIINFEKL peptide. Bar charts to the right: cumulative data of four experiments (total n = 12 mice per group). c CD45.1+ mice infused with naive CD45.2+ OT-I T cells were immunized with OVA protein admixed with anti-CD40 Ab. Three days later, mice were infused with CFSE-labeled peptide pulsed target cells (CD45.2+ splenocytes pulsed with 10 µg ml-1 SIINFEKL; 0.3 µm CFSE) admixed with control cells (CD45.2+ splenocytes pulsed with 10 µg ml-1 P53 control peptide AIYKKSQHM; 5 µm CFSE); spleens were analyzed for target cell killing after 24 h. GDC-0623 was administered at indicated doses for 3 days after immunization. Bar charts to the right: cumulative data of two experiments (vehicle n = 8, GDC-0623 (10 mg kg-1) n = 6, GDC-0623 (30 mg kg-1) n = 6) with specific killing normalized to the T cell only group. d As described in a, mice were treated on days 0 and 3 with indicated doses of temozolomide (TEM), gemcitabine (GEM), or vehicle. Upper row: dot plots of gated OT-I T cells. Lower row: CFSE dilution of gated OT-I T cells. Bar charts to the right: quantification of OT-I T cell numbers and divided fraction (three mice/group).

Fig 3: MEKi/anti-CD40 Ab combination therapy increases CD8/Treg ratio and effector function of tumor-infiltrating T cells.a Flow cytometric analysis of the impact of MEKi GDC-0623 and/or anti-CD40 Ab treatment on the T cell infiltrate in B16-OVA, MC-38, and PDA30364 tumors, as performed on four mice per treatment group of the experiments shown in Fig. 3a–c. Representative dot plots are shown. Cells were pre-gated on living, CD45+, CD3+, Thy-1.2+ cells. b–d Quantitation of CD8+ T cell to CD4+ CD25+ Foxp3+ regulatory T cell ratio, regulatory T cell frequencies among CD4+ T cells, and CD8+ T cell frequencies among living cells. #Two animals without Tregs. One-way ANOVA with post hoc Dunnett’s test (treatment groups vs. control). Mean ± s.e.m, n = 4. e TILs isolated from PDA30364 tumors were restimulated ex vivo with PMA/Ionomycin in presence of GolgiPlug and stained for intracellular IFN? and TNFa. Mean ± s.e.m., n = 4. One-way ANOVA with post hoc Tukey’s test. Significance levels are indicated by asterisks (*p = 0.05; **p = 0.01; ***p = 0.001; ****p = 0.0001).

Fig 4: Combination of MEK inhibition with agonist anti-CD40 Ab mediates control of established tumors.a–c Outgrowth of B16-OVA, MC-38, and PDA30364 tumors upon treatment with MEKi GDC-0623 and/or anti-CD40 Ab. Mice were treated daily with 30 mg kg-1 GDC-0623 or vehicle for ~2 weeks (gray rectangle). Anti-CD40 or control Ab were administered on treatment days 3, 5, 7 as well as 1 day prior to biomarker analyses (green arrows). Each group consisted of at least six mice (nB16-OVA = 10, nMC38 = 10, nPDA30364 = 10). Four mice per group were killed at end of treatment for biomarker analyses. Two-way ANOVA with post hoc Dunnett’s test; treatment groups vs. control. Significance levels are indicated by asterisks (*p = 0.05; **p = 0.01; ***p = 0.001; ****p = 0.0001). d Cumulative data of several experiments with the PDA30364 tumor model. e PDA30364 tumor growth in mice treated with monotherapies (anti-CD40 Ab, anti-PD-1 Ab, or GDC-0623) and combination therapies (anti-CD40 Ab + GDC-0623 and anti-PD-1 Ab+GDC-0623). Mice were treated daily with 30 mg kg-1 GDC-0623 or vehicle for ~5 weeks. Anti-CD40, anti-PD-1, and control Ab were administered twice weekly (arrows). Each group consisted of at least nine mice (nControl = 9, nCD40 = 9, nGDC-0623 = 12, nCD40+GDC-0623 = 10, nPD-1 = 10, nPD1-GDC-0623 = 11). Four mice per group were killed 2 weeks after treatment start for biomarker analyses. f Impact of T cell depletion on efficacy of GDC-0623 and/or anti-CD40 Ab treatment against PDA30364 tumors. Where indicated, groups received depletory anti-CD4, anti-CD8 or anti-CD4+ anti-CD8 Abs twice per week. Each group consisted of at least five mice.

Fig 5: 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).