Fig 2: Characterization of PTP1B deficient moDCs. (A) Therapeutic effect of PTP1B deficient moDCs in a mouse model of lymphoma E.G7-OVA: Tumor volume before moDC treatments (10 d after implantation of 5 × 105 E.G7-OVA cells) and (B) 18 d after tumor implantation and 8 d after intraperitoneal (IP) injections of 5 × 106 1B-deficient moDCs compared with control group (tumor-bearing mice without moDC treatment). (C) In vivo images of tumor-bearing mice 8 d after moDCs treatments (n = 5). (D) Quantification of IFN? produced by activated OT-II CD4+ T and OT-I CD8+ T cells respectively co-cultured with mature and OVA-pulsed 1Bfl/fl, 1B fl/wt or wt moDCs during 48 h (n = 4–5). The amount of IFN? produced by mature PTP1B-deficient or wt moDCs was subtracted for both OT-II CD4+ and OT-I CD8+ co-cultures. Production of Th1 polarizing cytokines (n = 5): (E) IL-12 and (F) IFN?. (G) Mean Fluorescent Intensity (MFI) values of the expression of MHC class I, MHC class II. CD86 and CD40 (n = 3). The comparisons were determined using One-Way ANOVA (Holm–Sidak multiple comparison test) for parametric and Dunn's multiple test for non-parametric. The results are representative of at least three independent experiments. p values are indicated.

Fig 3: Pharmacological inhibition of both PTP1B and TC-PTP with specific inhibitor. (A) Specificity of 1B/TC inh (50 µM) to inhibit PTP1B and TC-PTP dephosphorylation (n = 3). (B) Titration of 1B/TC inh in mouse moDCs using IL-12 production as indicator of activation (n = 3). (C) IFN? production by 1B/TC inh-treated mature moDCs (4 µM dose) (n = 4). (D) Expression levels (MFI) of CD40, CD80, CD86, MHC class I and II on 1B/TC inh-treated mature moDCs (4 µM dose) (n = 3). (E) Activation status STAT1 and STAT4 (n = 3). (F) Activation status of Src kinase and IkBa (n = 4). Therapeutic properties of 1B/TC inh-treated moDCs in a mouse model of E.G7 lymphoma: (G) Tumor volume before moDC treatment (10 d after implantation of 5 × 105 E.G7-OVA cells) and (H) after intraperitoneal (IP) injections of 5 × 106 1B/TC inh-treated or non-treated moDCs compared with control group (tumor-bearing mice without moDC treatments). The IP injections with moDCs were given 18 d after tumor implantation. (I) Images represent tumor-bearing mice 8 d after of moDC treatment. The results are representative of at least three independent experiments. The differences in more than two groups were determined using One-Way ANOVA (Holm–Sidak multiple comparison test) for parametric and Dunn's multiple test for non-parametric. The differences between two groups were determined with unpaired t test (two tails of distribution). Significant differences are represented by p values <0.05.

Fig 4: EnAd expressing FRß-targeting BiTEs activates endogenous ascites T cells to kill ascites macrophages. a DLD-1 cells were infected with parental or armed EnAd, and viability assessed four days later by MTT assay. % Live cells was calculated relative to “Mock”-treated cells. b DLD-1 cells were infected with armed EnAd at 100 vp/cell. Supernatants were harvested 72 h later and analysed for BiTE expression by western blotting using anti-His primary antibody and an HRP-conjugated anti-mouse secondary antibody. c-g Total unpurified ascites cells from five patients were infected with 100 vp/cell parental or BiTE-expressing EnAd for five days with or without autologous fluid. c Activation of endogenous CD4+ and CD8+ ascites T cells was assessed by flow cytometric measurement of CD25 expression. d CD4+ and CD8+ cell numbers were determined by adding counting beads to samples immediately prior to antibody staining. Fold-changes in CD4+ and CD8+ cell count were calculated relative to “Mock”-treated samples. e, g Cells were stained with anti-CD11b, anti-CD64, anti-CD80 and anti-CD86 antibodies, and a LIVE/DEAD fixable stain, then analysed by flow cytometry. e % Live residual CD11b+CD64+ cells were calculated relative to “Mock”-treated samples. f IFN-? levels in the supernatants were determined by ELISA. g Fold-changes in geometric MFI values of CD64, CD80 and CD86 on live CD11b+CD64+ ascites cells were calculated relative to “Mock”-treated samples for each patient sample. c-f Data show the grand mean ± SD of five individual patient means (calculated from biological triplicate). c-f Statistical significance was assessed by two-way ANOVA followed by Bonferroni post-hoc analysis, with each treatment being compared to the relevant “Mock” condition (c-f) (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (c-g) (*, P < 0.05; **, P < 0.01; ***, P < 0.001)

Fig 5: Characterization of TC-PTP deficient moDCs. (A) Therapeutic effect of TC-PTP deficient moDCs in a mouse model of lymphoma E.G7-OVA: Tumor volume before moDC treatment (10 d after implantation of 5 × 105 E.G7-OVA cells) and (B) after IP injections of 5 × 106 TC-deficient moDC compared with control group (tumor-bearing mice without moDC treatment). The IP injections with moDCs were given 18 d after tumor implantation. (C) In vivo images of tumor-bearing mice 8 d after moDC treatment. (D) Quantification of IFN? produced by activated OT-II CD4+ T and OT-I CD8+ T cells respectively co-cultured with mature and OVA-pulsed TCfl/fl, TC fl/wt or wt moDCs during 48 h (n = 3–5) and (n = 6–4). The amount of IFN? produced by mature TC-PTP-deficient or wt moDCs was subtracted for both OT-II CD4+ and OT-I CD8+ co-cultures. Production of Th1 polarizing cytokines (n = 5): (E) IL-12 and (F) IFN?. (G) MFI values of the expression of MHC class I and II, CD86 and CD80 (n = 3). The comparisons were determined using One-Way ANOVA (Holm–Sidak multiple comparison test) for parametric and Dunn's multiple test for non-parametric. The results are representative of three independent experiments. p values are indicated in the figures.