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

Journal: bioRxiv

Article Title: Borrelia Burgdorferi binds Serum Amyloid A and Modulates Subcutaneous Adipose Tissue Immune Signaling

doi: 10.64898/2026.01.29.702514

Figure Lengend Snippet: Single Cell RNA Sequencing (scRNA seq) (10X Genomics) of 31,953 adipose tissue stromal vascular fraction (SVF) cells (13,023 from Bb-infected tissue, 18,930 from uninfected tissue) (n=3 Uninfected control, n=2 Bb- infected tissue specimens). (A) Uniform Manifold Approximation and Projection (UMAP) plots indicate 7 unique cell types with a total of 10 clusters. (B) Bubble plot shows adipose stem/progenitor cells and monocyte/macrophage (Mono/Mø) clusters and their expressed immune focused pathways from MSigDB Hallmark gene sets. (C) Total SAA (SAA1+SAA2) gene expression across tissue cohorts. (D) SAA-related genes expressed per cluster showing average expression across cell type and percent of cells expressing each gene of interest. Genes upregulated during infection were plotted in red and genes downregulated after infection plotted in blue. The size of the bubble is based on the percentage of cells within that cell type cluster expression each gene. (E) Total SAA gene expression (SAA1+SAA2) across cell clusters within each tissue cohort. Abbreviations: ASPC: adipose stem/progenitor cells; LEC: lymphatic endothelial cells; Mono/Mø: monocytes/macrophages; SMC: smooth muscle cells.

Article Snippet: Spirochetes were pelleted at 5000 x g for 5 minutes, washed with 1x PBS, then resuspended in solution with SAA2 recombinant protein (hSAA2-His: MedChemExpress HY-P70985, Elabscience PKSH033532; mSAA2-His: antibodies online ABIN7088660) at 1μg/mL, 10μg/mL or 40μg/mL for 1h at room temperature.

Techniques: Single Cell, RNA Sequencing, Infection, Control, Gene Expression, Expressing

Journal: bioRxiv

Article Title: Borrelia Burgdorferi binds Serum Amyloid A and Modulates Subcutaneous Adipose Tissue Immune Signaling

doi: 10.64898/2026.01.29.702514

Figure Lengend Snippet: SAA2-bound spirochetes were stained for the presence of attached His-tagged protein (SAA2) detected by flow cytometry using an anti-His-tag fluorescent (PE/Cy5) conjugated antibody. (A) Histogram plots showing human SAA (hSAA2 and hSAA1), relative to non-stained controls (grey) and positive control protein (brown) known to bind Borrelia (Peptidoglycan Recognition Protein 1 (PGLYRP1) – Ref 39). The dashed line represents 40 µg/mL of hSAA2. The solid line represents 10µg/mL hSAA2. (B) Streptococcus pneumoniae incubated with 40µg/mL recombinant hSAA2 showing no increase in fluorescence compared to control. (C) Repeat binding assays showing histograms representing SAA2 binding multiple isolates of Bb sensu stricto (B31, CA8, HP19, CT-1, NT-1).

Article Snippet: Spirochetes were pelleted at 5000 x g for 5 minutes, washed with 1x PBS, then resuspended in solution with SAA2 recombinant protein (hSAA2-His: MedChemExpress HY-P70985, Elabscience PKSH033532; mSAA2-His: antibodies online ABIN7088660) at 1μg/mL, 10μg/mL or 40μg/mL for 1h at room temperature.

Techniques: Staining, Flow Cytometry, Positive Control, Incubation, Recombinant, Fluorescence, Control, Binding Assay

Journal: bioRxiv

Article Title: Borrelia Burgdorferi binds Serum Amyloid A and Modulates Subcutaneous Adipose Tissue Immune Signaling

doi: 10.64898/2026.01.29.702514

Figure Lengend Snippet: (A) Experimental workflow diagram of flow cytometry binding assay. Spirochetes cultured at 37°C for 24h were allowed to bind recombinant His-tagged mSAA2. Anti-His tag fluorescent (PE/Cy5) primary conjugated antibodies were bound to SAA-spirochete complexes to label protein attached to spirochetes. Spirochetes were washed and used for flow cytometry analysis compared to unstained spirochete complexes. (B) Histogram plots indicating murine SAA2 (mSAA2) binds SAA2 relative to staining control ( Bb + mSAA2) and positive control protein peptidoglycan recognition protein-1 (PGLYRP1 ref 39). (C) Binding of mSAA2 to Bb sensu stricto strains B31, CA8, HP19, CT-1, NT-1.

Article Snippet: Spirochetes were pelleted at 5000 x g for 5 minutes, washed with 1x PBS, then resuspended in solution with SAA2 recombinant protein (hSAA2-His: MedChemExpress HY-P70985, Elabscience PKSH033532; mSAA2-His: antibodies online ABIN7088660) at 1μg/mL, 10μg/mL or 40μg/mL for 1h at room temperature.

Techniques: Flow Cytometry, Binding Assay, Cell Culture, Recombinant, Staining, Control, Positive Control

Journal: bioRxiv

Article Title: Borrelia Burgdorferi binds Serum Amyloid A and Modulates Subcutaneous Adipose Tissue Immune Signaling

doi: 10.64898/2026.01.29.702514

Figure Lengend Snippet: (A) Workflow diagram of labeled spirochetes added to stained cells to be measured by flowcytometry. Spirochetes were allowed to bind SAA2 previously described and labeled with CFSE. Separately, differentiated THP-1 macrophage-like cells were labeled with membrane stain DiD. Labeled spirochetes were added to stained cells and harvested at 0.5-3.0h post incubation at 37°C. (B) Flow cytometry controls of CFSE-stained Bb N40 spirochetes (green) and DiD-labeled cells (red) showing detection capability of both positive- and negative-stained populations. (C) Contour plots of THP-1 cells and spirochetes incubated with either hSAA2, 5% normal human serum (NHS), and hPGLYRP1 (10µg) comparing levels of double positive populations over time (0.5, 1.0, 2.0, 3.0h in co-culture). Contour plots show percentages of cells in each quadrant in which triplicate plots overlayed. Each replicate value from quadrant 2 (top right) used for quantification .

Article Snippet: Spirochetes were pelleted at 5000 x g for 5 minutes, washed with 1x PBS, then resuspended in solution with SAA2 recombinant protein (hSAA2-His: MedChemExpress HY-P70985, Elabscience PKSH033532; mSAA2-His: antibodies online ABIN7088660) at 1μg/mL, 10μg/mL or 40μg/mL for 1h at room temperature.

Techniques: Labeling, Staining, Membrane, Incubation, Flow Cytometry, Co-Culture Assay