Fig 2: 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 3: 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 4: CD38 CAR construct and expression on primary T cells. (A, B) The construction of two CD38 CARs. The CD38-ScFv sequences were selected from two different CD38 antibody sequences (CAR21 and CAR22) with the CD8 hinge and transmembrane region, the CD28 cytoplasmic costimulatory region and the CD3ζ signaling domain. (C) The copy numbers of supernatant viral vectors produced by the PG13 cell line were detected by RT-qPCR. (D) The transduction efficiency of CD38 CAR in primary T cells from different donors (n= 5). The transduction efficiency was defined as the percentage of CD3-positive and myc-positive cells among total live cells. (E) The vector copy number of CD38 CAR integration per primary T cell was detected by RT-qPCR. (F) The expression of CD38 on the surface of human primary T cells transduced with the CD38 CAR was detected by flow cytometry.

Fig 5: 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).