Journal: Nature Communications

Article Title: Split-design approach enhances the therapeutic efficacy of ligand-based CAR-T cells against multiple B-cell malignancies

doi: 10.1038/s41467-024-54150-z

Figure Lengend Snippet: a Schematic representations of ligand-based conventional and split-design CAR approaches. The extracellular domains of BAFF and APRIL were used as target moieties to generate conventional CAR-T cells, referred to as APRIL CAR and BAFF CAR, respectively. b , d Representative images of cell‒cell conjugates captured at 100× oil objective magnification using a laser scanning confocal microscope (Nikon, A1R). APRIL or 9E10-IgG4m (pre-incubated with Myc-APRIL) CAR-T cells were co-cultured with RPMI8226-GFP cells ( b ), while BAFF or 9E10-IgG4m (pre-incubated with Myc-BAFF) CAR-T cells were co-cultured with IM9-GFP cells ( d ). Fluorescent labels included Hoechst (blue), anti-PKC-θ (red), and GFP (green) and a merged view of all stains. Scale bar = 10 μm. c , e Statistical analysis of the mean fluorescence intensity of PKC-θ at the IS in panels b and d, respectively. In panel c, sample sizes: APRIL CAR, n = 37; 9E10-IgG4m, n = 39. In panel e, BAFF CAR, n = 34; 9E10-IgG4m, n = 44. All n values represent individual cells. P values were determined by paired two-tailed t -tests. f , g Cytotoxicity assays of conventional and split-design CAR-T cells against the indicated target cells at various E:T ratios for 24 h in triplicate. h , i Inflammatory cytokine release assay. Conventional CAR-T cells or sCAR-T cells along with 1 nM corresponding switches were co-cultured with the specific target cells for 24 h at an E:T ratio of 1:1 in triplicate. Two-way ANOVA multiple comparisons in Dunnett correction were used to assess significance. j , l Schematic representations of ligand-based split-design CAR and FDA-approved CAR, referred to as BCMA CAR ( j ) and CD19 CAR ( l ), respectively. k , m Cytotoxicity assays of FDA-approved CAR-T cells and split-design CAR-T cells against the indicated target cells at various E:T ratios for 24 h in triplicate. Data in this figure are representative of three independent experiments. Error bars represent mean ± SD. NS indicates not significant. Source data are provided in the Source Data file.

Article Snippet: For western blot, anti-human CD19 antibody (Boster, BM4935) and anti-human CD22 antibody (Boster, BM4178) were used to verify the KO efficiency .

Techniques: Microscopy, Incubation, Cell Culture, Fluorescence, Two Tailed Test, Release Assay

Journal: Nature Communications

Article Title: Split-design approach enhances the therapeutic efficacy of ligand-based CAR-T cells against multiple B-cell malignancies

doi: 10.1038/s41467-024-54150-z

Figure Lengend Snippet: a Timeline of in vivo experiments. Consistent results were obtained in two independent experiments ( n = 5 mice). b Representative bioluminescence images of mice subjected to different treatments. Colors represent the luminescence intensity (red, highest; blue, lowest). c , d Quantification of the average radiance (p/s/cm /sr) of the luminescence, related to APRIL- ( c ) and BAFF-( d )-based CAR-T-cell therapy. Two-way ANOVA multiple comparisons in Dunnett correction were used to assess significance. e Evaluation of serum inflammatory cytokine release by ELISA 24 h after CAR-T-cell infusion. One-way ANOVA multiple comparisons in Tukey correction were used to assess significance. f , g Survival curves of the mice subjected to the indicated treatments. Survival curves were compared using the log-rank (Mantel‒Cox) test. h Timeline of in vivo experiments. Consistent results were obtained in two independent experiments ( n = 5 mice). i Representative bioluminescence images of mice subjected to different treatments. Colors represent the luminescence intensity (red, highest; blue, lowest). j Quantification of the average radiance (p/s/cm /sr) of the luminescence. Two-way ANOVA multiple comparisons in Dunnett correction were used to assess significance, comparing 9E10-IgG4m CAR-T (with Myc-BAFF) and CD19/CD22 CAR-T. k Evaluation of serum inflammatory cytokine release by ELISA 24 h after CAR-T-cell infusion. One-way ANOVA multiple comparisons in Dunnett correction were used to assess significance. l Assessment of the presence of persistent human CD3 + (hCD3 + ) T cells in peripheral blood by flow cytometry over a 3-week follow-up period. Two-way ANOVA multiple comparisons in Dunnett correction were used to assess significance, comparing 9E10-IgG4m CAR-T (with Myc-BAFF) with BAFF-CAR-T at each time point. m Survival curves of mice subjected to the indicated treatments, compared using the log-rank (Mantel‒Cox) test. All n represents biological replicates from different mice. Data in this figure are representative of one of two independent experiments. Error bars represent mean ± SEM. NS indicates not significant. Source data are provided in the Source Data file.

Article Snippet: For western blot, anti-human CD19 antibody (Boster, BM4935) and anti-human CD22 antibody (Boster, BM4178) were used to verify the KO efficiency .

Techniques: In Vivo, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: Nature Communications

Article Title: Split-design approach enhances the therapeutic efficacy of ligand-based CAR-T cells against multiple B-cell malignancies

doi: 10.1038/s41467-024-54150-z

Figure Lengend Snippet: a Timeline of in vivo experiments. Consistent results were obtained in two independent experiments ( n = 5 mice). b Representative bioluminescence images of mice subjected to different treatments. Colors represent the luminescence intensity (red, highest; blue, lowest). c Evaluation of serum inflammatory cytokine release by ELISA 24 h after CAR-T-cell infusion. One-way ANOVA multiple comparisons in Dunnett correction were used to assess significance. d Quantification of the average radiance (p/s/cm 2 /sr) of the luminescence. Two-way ANOVA multiple comparisons in Sidak correction were used to assess significance, comparing 9E10-IgG4m CAR-T (with Myc-APRIL) with BCMA CAR-T. e Survival curves of mice subjected to the indicated treatments, compared using the log-rank (Mantel‒Cox) test. f Timeline of the in vivo experiments. Consistent results were obtained in two independent experiments ( n = 5 mice). g Representative bioluminescence images of mice subjected to different treatments. Colors represent the luminescence intensity (red, highest; blue, lowest). h Evaluation of serum inflammatory cytokine release by ELISA 24 hours after CAR-T-cell infusion. One-way ANOVA multiple comparisons in Dunnett correction were used to assess significance. i Quantification of the average radiance (p/s/cm 2 /sr) of the luminescence. Two-way ANOVA multiple comparisons in Dunnett correction were used to assess significance, comparing 9E10-IgG4m CAR-T (with Myc-BAFF) with CD19 CAR-T. j Assessment of the presence of tumor cells (GFP + CD19 + or GFP + CD19 - ) in peripheral blood by flow cytometry on the 18th day of the experiment. One-way ANOVA multiple comparisons in Dunnett correction were used to assess significance. k Survival curves of the mice subjected to the indicated treatments, compared using the log-rank (Mantel‒Cox) test. All n represents biological replicates with different mice. Data are in this figure representative of one of two independent experiments. Error bars represent mean ± SEM. NS indicates not significant. Source data are provided in the Source Data file.

Article Snippet: For western blot, anti-human CD19 antibody (Boster, BM4935) and anti-human CD22 antibody (Boster, BM4178) were used to verify the KO efficiency .

Techniques: In Vivo, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: Science immunology

Article Title: Persistent STAT5 activation reprograms the epigenetic landscape in CD4 + T cells to drive polyfunctionality and antitumor immunity

doi: 10.1126/sciimmunol.aba5962

Figure Lengend Snippet: The schema depicts the timeline of the experimental procedures. A20WT tumor cells were subcutaneously inoculated to mice. When tumor sizes reached 170 mm2, mice were randomly grouped and received either no treatment (No Tx), CTX only, CTX + control CD19CAR T cells, or CTX + CASTAT5 CD19CAR T cells. (A) Expression of CAR and CASTAT5 in transduced T cells. Total T cells purified from CD45.1 mice were transduced to express CD19CAR alone or co-express CD19CAR and CASTAT5. Representative dot plots show the levels of CD19CAR and CASTAT5 in transduced T cells. CD19CAR was measured by anti-Fab stain and CASTAT5 was evaluated by Thy1.1 expression. (B) Tumor growth curves of each mouse under each condition are shown. The numbers indicate the number of tumor-free mice among treated mice at the end point. (C) Relapsed tumors did not lose CD19 expression. Mice treated with CD19CAR T cells had initial tumor regression followed by relapse. Relapsed tumors were resected and processed into single cell suspension for evaluation of CD19 expression by flow-cytometry. Tumors from untreated mice were used for comparison. Representative dot plots shown represent the co-stain of CD19 and B220. (D) Frequencies of host B cells in blood. At the indicated time points, tail blood was collected and the presence of host B cells in blood was evaluated by CD19 and B220 co-stain. (E) Detection of CD19CAR viral vector in bone marrow by PCR. 30 days after T cell transfer, a cohort of mice were killed and bone marrow aspirates were collected. The presence of CD19CAR T cells was evaluated by PCR detection of the CD19CAR viral vector. (F) Mice cured by CTX+CASTAT5 CD19CAR T cells were resistant to A20WT re-challenge. Live A20WT tumor cells were subcutaneously injected to cured mice and a group of naive mice used as controls. Tumor sizes in mice 3 weeks after tumor inoculation are shown.

Article Snippet: CD26-CD19 cells were generated by transfecting cells with a vector encoding murine CD19 (pCMV3-mCD19, SinoBiological Inc.) using Lipofectamine 2000.

Techniques: Expressing, Purification, Staining, Flow Cytometry, Plasmid Preparation, Injection

Journal: Science immunology

Article Title: Persistent STAT5 activation reprograms the epigenetic landscape in CD4 + T cells to drive polyfunctionality and antitumor immunity

doi: 10.1126/sciimmunol.aba5962

Figure Lengend Snippet: (A) Viral transduction efficiency in CD4+ and CD8+ T cells. CD4+ and CD8+ T cells were separately purified from CD45.1 mice. These cells were transduced to express CD19CAR alone or co-express CD19CAR and CASTAT5. Representative dot plots show the levels of CD19CAR and CASTAT5 in transduced T cells. CD19CAR was measured by anti-Fab stain and CASTAT5 was evaluated by Thy1.1 expression. The numbers in plots indicate the percentage of cells in the corresponding quadrant. (B) Experimental setup and treatment conditions. The schema depicts the experimental procedures. The different combinations of engineered CD4+ and CD8+ CD19CAR T cells used for adoptive transfer are outlined in the chart. (C) The frequencies of donor CD8+ T cells in recipient mice during the course of CD19CAR T cell therapy. At the indicated time points, tail blood was collected from mice and subjected to flow cytometry to detect the donor CD8+ T cells (CD45.1+CD8+). The frequencies of donor CD8+ T cells were plotted against time. The frequencies of host B cells (CD19+B220+) in blood were also evaluated by flow cytometry and summarized in (D). (E) Tumor growth curves of mice in each group. The numbers indicate the number of tumor-free mice among treated mice at the end point of experiment. (F and G) Phenotypic characterization of donor CD4+ and CD8+ CD19CAR T cells in selected groups. Ten days after T cell transfer, splenocytes isolated from a cohort of mice from the specified groups (3 mice/group) were subject to flow cytometry analysis. Splenocytes from groups V and VI were stimulated with PMA/ionomycin or A20 tumor cells for ICS to examine the cytokine profiles of control CD4+ and CASTAT5 CD4+ CD19CAR T cells (F). Numbers in representative dot plots indicate percentage of cells in the corresponding quadrant. Donor CD8+ T cells from groups I, IV and VI, which expressed CD19CAR alone, or co-expressed CASTAT5 and CD19CAR in the absence or presence of CASTAT5 CD4+ CD19CAR T cells, respectively, were examined for expression of the specified markers. The frequencies of cells positive for each of the markers and the mean fluorescence intensity (MFI) of these markers are summarized in bar graphs shown as mean ± s.d. of five samples per group. (G). n.s., not significant; *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: CD26-CD19 cells were generated by transfecting cells with a vector encoding murine CD19 (pCMV3-mCD19, SinoBiological Inc.) using Lipofectamine 2000.

Techniques: Transduction, Purification, Staining, Expressing, Adoptive Transfer Assay, Flow Cytometry, Isolation, Fluorescence