Fig 2: Cytotoxicity and proliferation of CD38 CAR-T cells in vitro. (A) The ability of CAR-T cells to kill tumor cells was measured by luciferase assay. CD38 CAR-T cells or pan-T cells were incubated with RPMI-gfp-luc cells or Raji-luc cells, and the luciferase signal was determined after 12 hours. (B) The ability of CAR-T cells to kill tumor cells was measured by an IncuCyte S3 system. RPMI-gfp-luc cells were incubated with CD38 CAR-T cells or pan-T cells at an E:T ratio of 1:1. Total integrated GFP intensity per well was assessed as a quantitative measure of live, GFP+ tumor cells. The values were normalized to the starting values. (C) The ability of CD38 CAR-T cells to kill tumor cells was determined by flow cytometry. CD38 CAR-T cells or pan-T cells were incubated with RPMI-gfp-luc cells, Raji-luc cells, Daudi cells or K562-hBCMA cells for 12 hours. (D) CD38 CAR-T cells were incubated with RPMI-gfp-luc cells for 12 hours, and the secretion of cytokines was measured with a flow cytometry-based CBA kit. The graph shows the secretion of TNFα, IFN-γ, IL-2 and granzyme B. (E) The proliferation of CD38 CAR-T cells. The proliferation ability was measured by the CFSE-based assay. Data were obtained from three replicates and are presented as the mean ± SEM (n=3). *indicates p value < 0.05; **< 0.01; ***< 0.005 and ****< 0.001, comparison of two groups was performed by Student’s t-test. TNF-α, tumor necrosis factor α; IFN-γ, interferon γ; IL-2, interleukin 2.

Fig 3: CD38 CAR-T cells exhibited significant antitumor activities in a mouse xenograft tumor model. (A) Experimental design indicating tumor cells, timeline, doses, and imaging time points. Mice were randomized after tumor engraftment. T cells from two independent donors were used (n=5). (B) Mice were assessed by bioluminescence imaging once a week starting 3 days after the first injection of CAR-T cells. (C) Tumor burden was monitored weekly and presented as the average intensity. (D) Survival analysis of mice after tumor cell inoculation.n= 5 mice per group.

Fig 4: Abnormal Splenic B Cell Differentiation and B Cell Response in Mice Lacking MTA2 or Both MTA2 and OCA-B(A) Staining of total splenic B (B220+), marginal zone B (MZB) (B220+CD21hiCD23lo), and follicular B (FOB) (B220+CD21−CD23hi) cells in Vav-Cre Mta2fl/fl and control mice. Frequencies of different cell populations in parental cells are indicated.(B) Staining of germinal center B (GCB) (B220+CD95+CD38−CD4−) cells in Vav-Cre Mta2fl/fl and control mice immunized by sheep red blood cells (SRBCs).(C) Numbers of GCB cells and frequencies of GCB cells in splenic B cells are plotted (n = 8 for each genotype; t test; *p = 0.02).(D) Staining of splenic B, MZB, and FOB cells in mice with indicated genotypes.(E) Cell numbers of different B cell subsets are plotted (n = 10 for control, n = 9 for Vav-Cre Mta2fl/fl, n = 5 for Oca-B−/− and DKO mice; t test; *p < 0.05, **p < 0.01, ***p < 0.001).

Fig 5: Germinal Center B-cell responses to P. chabaudi infection are abrogated in Bcl6fl/flCD4-cre+/− mice, and reduced in SAP-deficient mice. Bcl6fl/flCD4-cre+/− and Sh2d1a−/− mice were infected with P. chabaudi via intra peritoneal injection of 105 iRBC, and the occurrence of GC B cells in the spleen was determined by flow cytometry at days 0, 15 and 35 post-infection, and by histology at days 0 and 35 post-infection. (a) Representative plots of naïve (top row) and infected (35 days post-infection, bottom row) Bcl6fl/flCD4-cre+/− and wt control mice. Gates show frequency of B220+ CD19+ IgDneg B cells expressing low levels of CD38 and high levels of GL-7. (b) Total numbers of GC B cells in Bcl6fl/flCD4-cre+/− and wt control mice. (c) Representative plots of naïve and infected Sh2d1a−/− and wt C57BL/6 control mice. (d) Total numbers of GC B cells in Sh2d1a−/− and wt C57BL/6 control mice. Bars represent median values. (e) Representative spleen sections of infected (d35) Sh2d1a−/− and wt C57BL/6 control mice; the white arrows point at individual examples of PNA+ GC structures. (f) Cumulative data displaying area, perimeter and number of GC structures determined by histology in spleen sections of infected (d35) Sh2d1a−/− and wt C57BL/6 control mice. Data are representative or pooled from at least two independent experiments and were obtained in groups of 3–8 mice per time point. Statistical significance was obtained using Student's t-test or Two-Way ANOVA with Tukey's multiple comparisons test. ND: Not done.