Fig 2: The Myb -68 enhancer function persists only in basophils and mast cells.a mRNA expression levels of stem/progenitor and lineage marker genes in GFP-Ly6C- GMPs (grey), GFP+Ly6C- GMPs (green), granulocyte progenitors (GP, blue) and monocyte progenitors (MP, yellow) (n = 5 mice for Myb, Gata2, Tal1, Apoe, and Csf3r, n = 4 mice for Csf1r and Irf8, and n = 3 mice for others). FACS gating strategies are shown in Supplementary Fig. 12 and Fig. 2c. Data are presented as mean values ± SD. p = 0.10, 0.018, 0.0016, 0.044, 0.032, 0.040, 0.0066, 8.9 × 10−5, 9.3 × 10−5, 0.27, 0.013, 2.8 × 10−7, and 1.0 × 10−7 from left to right by two-tailed Student’s t-test without adjustments for multiple comparisons. GFP activity in FcεR1α- and FcεR1α+ Ly6C- GMPs (b), basophils in peripheral blood (c), and mast cells in the peritoneal cavity (d) of Myb -68 GFP mice. FACS gating strategies are shown in Supplementary Fig. 12, 13c and 13d. Representative FACS plots of indicated cells from control (grey) and Myb -74 GFP mice (green) are shown. Numbers shown are mean percentages±SD of GFP+ cells in Myb −68 GFP mice. The percentages of GFP+ cells are also shown in the right graphs. Data are presented as mean values±SD. b n = 5 mice. **p = 0.0014 by two-tailed Student’s t-test. c Lin- indicates CD4-CD8-B220-Gr1-Ly6C-CD11c-cKit-Ter119-SiglecF-IL7Ra-NK1.1-. n = 4 mice for basophils and n = 5 mice for others. p = 0.0041 by one-way Welch’s ANOVA. *p < 0.05 by the Games-Howell post hoc test. p = 0.032 between neutrophils (Neu) and basophils (Baso), p = 0.033 between eosinophils (Eo) and Baso, p = 0.033 between monocytes (Mono) and Baso, p = 0.032 between B220+ cells and Baso, and p = 0.033 between CD3+ cells and Baso. d n = 5 mice. e Analysis of published ChIP-seq data demonstrates that multiple transcription factors including MITF bind to the Myb -68 enhancer (red rectangle).

Fig 3: Activation of long-lived memory CD8 + T cells by OBP-702 in a PAN02 subcutaneous tumor model.a–c C57BL/6 mice bearing or not bearing PAN02 subcutaneous tumors were intratumorally treated with OBP-702 (1 × 108 PFU) or PBS once and sacrificed 14 days after treatment (a). Splenocytes collected from the harvested spleen were subjected to flow cytometry for SLECs, MPECs, TEMps, and TCMps. Representative figures of flow cytometry are shown in (b), in which CD3+/CD8+ cells are analyzed in the upper figures, and CD3+/CD8+/CD127+ cells are analyzed in the lower figures. Populations of SLECs, MPECs, TEMps, and TCMps are compared among control, OBP-702 vaccination, tumor inoculation, and OBP-702 treatment to tumor (n = 3–4) (c). d, e C57BL/6 mice bearing PAN02 subcutaneous tumors were intratumorally treated with PBS or OBP-702 (5.0 × 107 PFU) three times in a week and sacrificed 28 days after the initial treatment (d). Tumor volume was monitored after treatment by OBP-702 or PBS (n = 5) (e). f, g PAN02 tumors harvested at 28 days after the initial treatment with OBP-702 were subjected to flow cytometry for CD8+ cells and TRMs. Representative figures of flow cytometry are shown in (f), in which live cells are analyzed in the upper figures, and CD8+/CD45+ cells are analyzed in the lower figures. Populations of CD8+ cells and TRMs are compared between control and OBP-702 (n = 3–5) (g). h, i The spleens harvested 28 days after the initial treatment with OBP-702 were subjected to flow cytometry for TEMs and TCMs. Representative figures of flow cytometry are shown in (h), in which CD3+/CD8+ cells are analyzed. Populations of TEMs and TCMs are compared between control and OBP-702 (n = 5) (i). *P < 0.05, **P < 0.005, ***P < 0.001. SLEC short-lived effector T cell, MPEC memory precursor effector cell, TEMp effector memory precursor cell, TCMp central memory precursor cell, TRM tissue-resident memory T cell, TEM effector memory T cell, TCM central memory T cell, Con control, 702 OBP-702.

Fig 4: Abscopal effect of combination therapy of GEM and nab-PTX with OBP-702 in a PAN02 bilateral subcutaneous tumor model.a C57BL/6 mice bilaterally bearing PAN02 subcutaneous tumors were treated with GN (GEM 50 mg/kg, nab-PTX 5 mg/kg) intraperitoneally and/or OBP-702 (5 × 107 PFU) intratumorally once a week for 3 weeks, and sacrificed 34 days after initial treatment. OBP-702 was injected into the bigger tumor. Tumor volume was monitored (n = 7). The left figure shows tumor volume of the OBP-702-injected side, and the right figure shows the tumor volume of the OBP-702-uninjected side. b Anti-CD8α antibody was additionally injected into the peritoneal cavity once a week for 4 weeks, and tumor volume of control and OBP-702 with or without anti-CD8α antibody was monitored until 28 days after the initial treatment (n = 3–7). c, d The spleens harvested 14 days after the initial treatment were subjected to flow cytometry for SLECs, MPECs, TEMps, and TCMps. Representative figures of flow cytometry are shown in (c), in which CD3+/CD8+ cells are analyzed in the upper figures, and CD3+/CD8+/CD127+ cells are analyzed in the lower figures. Populations of SLECs, MPECs, TEMps, and TCMps are compared (n = 5) (d). e, f PAN02 tumors harvested 34 days after the initial treatment were subjected to flow cytometry for CD8+ cells and TRMs. Representative figures of flow cytometry are shown in (e), in which live cells are analyzed in the upper figures, and CD8+/CD45+ cells are analyzed in the lower figures. Populations of CD8+ cells and TRMs in OBP-702-injected tumors and OBP-702-uninjected tumors are compared (n = 3–7) (f). g, h The spleens harvested 34 days after the initial treatment were subjected to flow cytometry for TEMs and TCMs. Representative figures of flow cytometry are shown in (g), in which CD3+/CD8+ cells are analyzed. Populations of TEMs and TCMs are compared (n = 7) (h). *P < 0.05, **P < 0.005, ***P < 0.001. Con control, GN gemcitabine + nab-paclitaxel, 702 OBP-702, SLEC short-lived effector T cell, MPEC memory precursor effector cell, TEMp effector memory precursor cell, TCMp central memory precursor cell, TRM tissue-resident memory T cell, TEM effector memory T cell, TCM central memory T cell.

Fig 5: Activation of long-term anti-tumor immunity by GN + OBP-702 in a neoadjuvant model using PAN02 subcutaneous tumors.a Study protocol of a neoadjuvant model. C57BL/6 mice bearing PAN02 subcutaneous tumors were treated with GN (GEM 50 mg/kg, nab-PTX 5 mg/kg) intraperitoneally and/or OBP-702 (5 × 107 PFU) intratumorally three times in a week, and these tumors were surgically resected 7 days after the initial treatment. PAN02 cells were re-inoculated into the opposite side of the flank to the first tumor 28 days after tumor resection, and mice were monitored with no treatment and sacrificed 28 days after re-inoculation. b Tumor volume was monitored for 7 days until resection (left) (n = 6–7) and for 28 days after re-inoculation with no treatment (right). c Anti-CD8α antibody was added once on the day of the initial treatment in the protocol (a). Tumor volume was monitored the same as in (b) (n = 7). d, e PAN02 tumors harvested 28 days after re-inoculation were subjected to immunohistochemical staining for CD8. Representative images of immunohistochemical staining are shown in (d). Scale bar, 200 µm. The number of CD8+ TILs, which were evaluated in three different, randomly selected fields, were compared (n = 5-7) (e). f, g The spleens harvested 14 days after the initial treatment in the protocol (a) were subjected to flow cytometry for SLECs, MPECs, TEMps, and TCMps. Representative figures of flow cytometry are shown in (f), in which CD3+/CD8+ cells are analyzed in the upper figures, and CD3+/CD8+/CD127+ cells are analyzed in the lower figures. Populations of SLECs, MPECs, TEMps, and TCMps are compared (n = 5) (g). h, i The spleens harvested 35 days after the initial treatment in the protocol (a) were subjected to flow cytometry for TEMs and TCMs. Representative figures of flow cytometry are shown in (h), in which CD3+/CD8+ cells are analyzed. Populations of TEMs and TCMs are compared (n = 5) (i). *P < 0.05, **P < 0.005, ***P < 0.001. Con control, GN gemcitabine + nab-paclitaxel, 702 OBP-702, SLEC short-lived effector T cell, MPEC memory precursor effector cell, TEMp effector memory precursor cell, TCMp central memory precursor cell, TEM effector memory T cell, TCM central memory T cell.