Journal: Nature Communications

Article Title: Enhancing CAR- and TCR-mediated targeting of cancer via an immune synapse-stabilizing receptor

doi: 10.1038/s41467-025-65897-4

Figure Lengend Snippet: A A schematic showing interaction between CLL1.CAR and CD38.SSR. CAR and SSR configurations are denoted on the right B Representative flow plots showing co-expression of CAR and SSRs on day7 post co-transduction (TD). SSR expression was measured by detection of a surrogate maker truncated NGFR. C Residual tumor counts after a 3-day culture of CD38+ Molm13 (AML) and CCRF-CEM (T-ALL) with non-transduced T cells (NT) or SSR T cells at an E:T ratio 1:4. Data represent 3 donors. P values were determined using one way ANOVA with Tukey’s correction for multiple comparisons. Bar graphs show mean+S.E.M. (* P < 0.05, ** P < 0.01, *** P < 0.001. ns, non-significant). D Histograms showing CLL1 and CD38 levels in indicated cell lines. E Residual tumor counts and T-cell expansion after a 3-day co-culture of Molm13 and CCRF-CEM with CAR T cells at a E:T ratio 1:4. Data represent 6 donors from 3 independent experiments. P values were determined using one way ANOVA with Tukey’s correction for multiple comparisons. Bar graphs show mean + S.E.M. (* P < 0.05, ** P < 0.01, *** P < 0.001. ns, non-significant). F Histograms showing CLL1 and CD38 levels in pre-sorted THP1 and CLL1-low THP1 post-sorting. G Residual tumor counts and fold T-cell expansion after 3 day culture at a E:T ratio 1:4. Data represent 6 donors from 2 independent experiments. P values were determined using one way ANOVA with Tukey’s correction for multiple comparisons. Bar graphs show mean + S.E.M. (*P < 0.05. ns, non-significant). H CD38 expression in the parental and knock-out line of Molm13. I Residual tumor counts and fold T-cell expansion after 3 day culture at a E:T ratio 1:4. Data represent 4 donors from 2 independent experiments. P values were determined using one way ANOVA with Tukey’s correction for multiple comparisons. Bar graphs show mean + S.E.M. (* P < 0.05, ** P < 0.01, *** P < 0.001. ns, non-significant). (* P < 0.05, ** P < 0.01. ns, non-significant).

Article Snippet: To investigate LAT downstream signaling, 1 × 10 6 CAR.SSR T cells were loaded into a well of a 24-well plate pre-coated with 0.3 μg anti-CLL1 CAR antibody and 1 μg recombinant human CD38 (Sino Biological, Cat: 10818) for 30 min on ice, followed by stimulation at 37 °C in a water bath for indicated time-points.

Techniques: Expressing, Transduction, Co-Culture Assay, Knock-Out

Journal: Nature Communications

Article Title: Enhancing CAR- and TCR-mediated targeting of cancer via an immune synapse-stabilizing receptor

doi: 10.1038/s41467-025-65897-4

Figure Lengend Snippet: A Schematic illustration (left) and representative images (right) of calcium influx (green) in T cells (blue) at pre-contact or post-contact with Molm13 (red) over the indicated time course. B Calcium (Ca++) flux in T cells interacting with Molm13 over the indicated timepoints, and the mean area under the curve (AUC) for the initial 30 min post-contact. Each dot represents a single cell. Data include single cells (42 cells (NT), 34 cells (CAR), 30 cells (CAR + ΔSSR), 30 cells (CAR + SSR)) combined from 3 independent donors and experiments. P values were determined using one way ANOVA with Tukey’s correction for multiple comparisons. Dot plots show mean + S.E.M. (* P < 0.05, ** P < 0.01, ns, non-significant). C Western blots showing the detection of phospho-proteins and GAPDH harvested from CAR or CAR.SSR T cells stimulated with plate-coated anti-CLL1 CAR antibody and recombinant CD38 at indicated time points. Data were repeated in three independent experiments using 3 donors. MW, molecular weight. D Densitometry analysis of pPLCγ1, pERK1/2 and pNF-KB normalized to GAPDH. E Schematic of an SSR construct containing a point mutation Y132F in the LAT endo-domain, and flow plots (bottom) showing the co-expression of CAR and SSR or SSR with Y132F on day 6 post transduction. F Residual tumor counts (left) and T-cell expansion normalized to day 0 (right) in a 3 day co-culture with Molm13. Data represent two independent experiments with 4 donors. P values were determined using one way ANOVA with Tukey’s correction for multiple comparisons. Bar graphs show mean + S.E.M. (* P < 0.05, ns, non-significant).

Article Snippet: To investigate LAT downstream signaling, 1 × 10 6 CAR.SSR T cells were loaded into a well of a 24-well plate pre-coated with 0.3 μg anti-CLL1 CAR antibody and 1 μg recombinant human CD38 (Sino Biological, Cat: 10818) for 30 min on ice, followed by stimulation at 37 °C in a water bath for indicated time-points.

Techniques: Western Blot, Recombinant, Molecular Weight, Construct, Mutagenesis, Expressing, Transduction, Co-Culture Assay

Journal: Nature Communications

Article Title: Enhancing CAR- and TCR-mediated targeting of cancer via an immune synapse-stabilizing receptor

doi: 10.1038/s41467-025-65897-4

Figure Lengend Snippet: A Representative flow plots showing expression of CD38 and CLL1 in PBMCs including CD14+ monocytes, CD3 + T cells, CD3-CD56 + NK cells and CD19 + B cells. B Residual PBMCs after a 24-h co-culture with autologous T cells at a 1:4 E:T ratio. Data represent 4 donors. P values were determined using one way ANOVA with Tukey’s correction for multiple comparisons. Bar graphs show mean + S.E.M. (** P < 0.01. ns, non-significant). C Quantification of a burst forming unit-erythroid (BFU-E) and colony forming unit-granulocyte/macrophage (CFU-GM) from hematopoietic stem cells/progenitors (HSPC) after 5-h co-culture of indicated CAR T cells with CD34+ cord blood cells at a E:T ratio 10:1, and expanded for 12 days in semi-solid methylcellulose. Data represent 4 donors. P values were determined using one way ANOVA with Tukey’s correction for multiple comparisons. Bar graphs show mean + S.E.M. (* P < 0.05, ** P < 0.01, *** P < 0.001. ns, non-significant). D Flow histograms show surface CLL1 and CD38 levels on AML blasts (CD33 + /CD34 + ) from the PBMCs collected from 2 patients (Pt1 and Pt2). E The residual AML blasts were detected at 24 h or 72 h after co-culture with CLL1 CAR T cells at a 1:1 E:T ratio. Data represent 3 independent donors. P values were determined using one way ANOVA with Tukey’s correction for multiple comparisons, or two-tailed, paired Student’s t test for CAR vs CAR + SSR in residual AML blasts from Pt2 at 72 h. Bar graphs show mean + S.E.M. (* P < 0.05, ** P < 0.01, *** P < 0.001. ns, non-significant).

Article Snippet: To investigate LAT downstream signaling, 1 × 10 6 CAR.SSR T cells were loaded into a well of a 24-well plate pre-coated with 0.3 μg anti-CLL1 CAR antibody and 1 μg recombinant human CD38 (Sino Biological, Cat: 10818) for 30 min on ice, followed by stimulation at 37 °C in a water bath for indicated time-points.

Techniques: Expressing, Co-Culture Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Enhancing CAR- and TCR-mediated targeting of cancer via an immune synapse-stabilizing receptor

doi: 10.1038/s41467-025-65897-4

Figure Lengend Snippet: A A schematic showing enhancement of cytotoxicity of survivin-specific TCR (Sur-TCR) by a CD38.SSR. B Representative flow plots showing co-expression of Sur-TCR and SSR on day 7 post-transduction (TD), and ( C ) expansion of TCR T cells derived from 3 HLA-A2- and 3 HLA-A2+ donors on day 10. Data represent 6 donors from two independent experiments. P values were determined using one way ANOVA with Tukey’s correction for multiple comparisons. Bar graphs show mean + S.E.M. (ns, non-significant). D Histograms showing intracellular survivin, surface HLA-A2 and CD38 levels in leukemia cell lines BV173, THP1 and TALL1. E Residual tumor counts after a 3-day co-culture with Sur-TCR T cells at a 1:4 E:T ratio. Data represent 6 donors from two independent experiments in co-culture with BV173 and THP1, and 3 donors from one independent experiment in co-culture with TALL1. P values were determined using one way ANOVA with Tukey’s correction for multiple comparisons. Bar graphs show mean + S.E.M. (* P < 0.05, ** P < 0.01, *** P < 0.001. ns, non-significant). Percentages of ( F ) granzyme B/CD107a double positive T cells and ( G ) cytokine positive T cells after 4-h co-culture with indicated leukemia cells at 1:1 E:T ratio. Data represent 3 donors. P values were determined using one way ANOVA with Tukey’s correction for multiple comparisons. Bar graphs show mean + S.E.M. (* P < 0.05, ** P < 0.01, *** P < 0.001. ns, non-significant). H Schematic timeline of the mouse experiment. I Leukemia progression was monitored by bioluminescence. J Tumor burden AUC between day 0 and day 26. The data represent 5 mice in NT and 4 mice in for TCR, TCR.ΔSSR and TCR.SSR. P values were determined using one-way ANOVA with Dunnet’s correction for multiple comparisons compared to NT. Bar graphs show mean + S.E.M. (* P = 0.0394). K Overall mouse survival in the THP1 model (NT, n = 5, TCR, n = 5, TCR.ΔSSR, n = 5, TCR.SSR, n = 4). Statistical significance was determined by a log rank test. (* P = 0.0214).

Article Snippet: To investigate LAT downstream signaling, 1 × 10 6 CAR.SSR T cells were loaded into a well of a 24-well plate pre-coated with 0.3 μg anti-CLL1 CAR antibody and 1 μg recombinant human CD38 (Sino Biological, Cat: 10818) for 30 min on ice, followed by stimulation at 37 °C in a water bath for indicated time-points.

Techniques: Expressing, Transduction, Derivative Assay, Co-Culture Assay