Fig 2: FACS gating strategy of OVA-TET+ CD8+ T cellsFACS analysis of OVA-TET+ CD8+ cells from spleen of mice treated with PBS (-) or infected with LmOVA (Figure 1A).(A) Gating strategy of OVA-TET+ CD8+ cells of splenocytes.(B) Shown are the percentage and absolute number of OVA-TET+ CD8+ cells which isolated from spleen analyzed by flow cytometry. All data are the mean ± s.e.m.; n = 5 independent wells per experiment. Two-tailed Student’s t test;∗∗∗∗p < 0.0001.

Fig 3: CD3/CD20 bispecific antibodies activate T-cells through direct conjugation of T-cells and B-cells. (a) Total splenocytes were incubated with CD3/CD20 and CD3/Neg in both mG1KD D265A and mG2aH4 backbones for 24 h. T-cell activation was assessed by CD25 staining followed by flow cytometry (mean ± SD, n = 3). (b) B/T-cell conjugates were captured by flow cytometry following incubation of total splenocytes with CD3/CD20, Neg/CD20, or CD3/Neg in the mG1KD D265A backbone for 1.5 h. The total percentage of conjugates were calculated from Σ(CD19+CD4+, CD19+CD8+, CD19+CD4+CD8+)/total events (mean ± SD, n = 3). Representative flow cytometry plots at (c) 2.26E-4 ug/ml and (d) 40 ug/ml CD3/CD20 antibody depicts the concentration-dependent increase of conjugated CD4+CD19+ B-cells and T-cells (top right quadrant).

Fig 4: Scheme of the adoptive transfer modelPurified OT-1 CD8+ cells are adoptively transferred to CD45.1 mice and then the recipient mice are infected with LmOVA.

Fig 5: GPC3 overexpression induces CD8+ cell infiltration and enhances cellular immune response to GPC3. The percentage of tumor-infiltrating CD8+ cells is shown in A and B. Results are representative of three independent experiments. Splenocytes were isolated on day 7 after inoculation with cancer cells and were stimulated with GPC3-expressing HM-1 cell lysate for 6 h in the presence of monensin. Then, they were immunostained with anti-IFN-γ and CD8 antibodies for FACS (C). Using ELISA kits, IFN-γ levels were measured in the culture medium of splenocytes at indicated times after stimulation with GPC3-expressing HM-1 cell lysate. Results are representative of three independent experiments (D). TUNEL assay of apoptotic cells in tumor tissue. TUNEL labeling of apoptotic cells in tumor-tissue samples obtained from mice 7 and 14 days after inoculation with cancer cells in peritoneal cavities (E). Error bars for B and D, represent SD.