Journal: Nature Communications

Article Title: CXCR4 induces memory formation over exhaustion in CAR-T cells to achieve durable leukemia targeting

doi: 10.1038/s41467-025-67745-x

Figure Lengend Snippet: a Differential gene expression analysis of AML blasts ( n = 235 samples from 155 patients) versus normal hematopoietic stem/progenitor cells (HSPCs; n = 44 samples from 14 donors). Genes encoding surface proteins are highlighted in pink. Validated AML CAR targets (black) and candidate targets identified in our screen (blue) are annotated. log₂ fold-changes and FDR-corrected p-values from DESeq2 (Wald-test) are reported. b Heatmap of surface molecule expression across 31 non-hematopoietic tissues (GTEx dataset; median TPM for n = 4 to 803 donors per tissue). Candidate targets with low off-tumor expression and canonical AML CAR-T targets are annotated. Novel targets discovered in our cohort and replicated in beatAML are labeled in blue. c Representative flow cytometry histogram of CD25 expression in CD25⁻ and CD25⁺ AML patient samples (pink = anti-CD25 antibody, gray = isotype control). CD25⁺ AML was defined as > 70% CD25⁺ blasts by flow cytometry ( n = 12/66 patients). d AML cohort ranked by CD25 mRNA expression (black dots, n = 155 patient samples). For a subset, CD25 surface levels were validated by flow cytometry (pink bars; n = 55 patient samples). Middle: Expression log₂ fold-change relative to HSPCs for candidate and select validated AML CAR-T targets. Bottom: Mutational landscape for individual samples. For some samples, multiple individual measurements were made for different rounds of xenotransplantation. e In vitro cytotoxicity of CAR-T cells assessed by co-culture with primary AML blasts. Effector CAR constructs include: ACTT and CD19 (negative controls); CD33 (positive control); CD25; and CD96. Sorted PDX BM-derived AML blasts for a representative CD25/CD96 dual-positive patient sample (P12) were used as target cells. Co-culture experiments were setup for multiple effector:target ratios and %AML reduction was evaluated at 24 h and 72 h. Experiments were replicated using CAR-T cells derived from n = 3 CB donors (rep1-3). Each data point corresponds to average of three technical repicates. Y-axis reports the percentage of hCD33+ cells compared to AML blasts alone. Error bars correspond to mean ± SD. The non-adjusted p-value reported for each CAR construct versus ACTT (two-tailed T-test). Source data are provided as a Source data file for panel(s) ( a , d , e ).

Article Snippet: After blocking with horse serum, slides were incubated with mouse anti-human TCF1 antibody (R&D systems, #2203) (1:200), anti-human CD33 (R&D systems, MAB11371) (1:100), anti-human and mouse CXCL12 (R&D systems, MAB350) (1:20), or anti-human CCR7 (Abcam, ab253187) (1:100) and then HRP-conjugated horse anti-rabbit or anti-mouse IgG antibody (ImmPRESS, MP-7500).

Techniques: Gene Expression, Expressing, Labeling, Flow Cytometry, Control, In Vitro, Co-Culture Assay, Construct, Positive Control, Derivative Assay, Two Tailed Test

Journal: Nature Communications

Article Title: CXCR4 induces memory formation over exhaustion in CAR-T cells to achieve durable leukemia targeting

doi: 10.1038/s41467-025-67745-x

Figure Lengend Snippet: a AML samples ranked by normalized CD96 mRNA expression (black dots, n = 155 patients). CD96 surface expression (flow cytometry) shown for a subset (blue bars, n = 95 patients). Bottom heatmap shows mutational profiles per sample, as in Fig. b Representative flow cytometry histogram showing CD96 surface expression in CD96⁻ and CD96⁺ patient-derived AML samples, as in Fig. . Anti-CD96 antibody staining is shown in blue, isotype control in gray. 98 AML samples were analyzed; cases with >70% CD96⁺ leukemic blasts were defined as CD96⁺ (blue, n = 54), others as CD96⁻ (gray, n = 44). c Peripheral blood %AML chimerism pre-CAR-T injection and at endpoint in untreated ( n = 21), CXCR4(-) CD96 CAR-T-treated ( n = 23), and hCXCR4(+) CD96 CAR-T-treated ( n = 18) animals. Red dots indicate animals that achieved complete remission (0% AML chimerism in PB). d AML chimerism in peripheral blood, spleen, bone marrow, and liver of PDX mice as in ( c ), stratified by treatment. Group sizes by treatment: untreated ( n = 3, 9, 4, 5), CXCR4(-) CD96 CAR-T ( n = 6, 9, 5, 3), and hCXCR4(+) CD96 CAR-T ( n = 4, 6, 4, 4), PDX mice of Patients 12–15, respectively (See Table ). Statistical comparisons were made using unpaired two-sided Mann-Whitney U tests. The non-adjusted p-value is reported. For ( c , d ) Box and whisker plots: central lines show median, lower and upper hinges show first and third quartiles (the 25th and 75th percentiles) and whiskers extend up to 1.5 * IQR. e ) H&E and CD33 immunohistochemistry of bone marrow from Patient 13-derived PDX (untreated, CXCR4(-), or hCXCR4(+) CD96 CAR-T treated) (scale: 20 µm). Representative images selected from sections prepared from n = 3 animals per treatment group. Source data are provided as a Source data file for ( a , c , d ).

Article Snippet: After blocking with horse serum, slides were incubated with mouse anti-human TCF1 antibody (R&D systems, #2203) (1:200), anti-human CD33 (R&D systems, MAB11371) (1:100), anti-human and mouse CXCL12 (R&D systems, MAB350) (1:20), or anti-human CCR7 (Abcam, ab253187) (1:100) and then HRP-conjugated horse anti-rabbit or anti-mouse IgG antibody (ImmPRESS, MP-7500).

Techniques: Expressing, Flow Cytometry, Derivative Assay, Staining, Control, Injection, MANN-WHITNEY, Whisker Assay, Immunohistochemistry

Journal: Nature Communications

Article Title: CXCR4 induces memory formation over exhaustion in CAR-T cells to achieve durable leukemia targeting

doi: 10.1038/s41467-025-67745-x

Figure Lengend Snippet: a Schematic of study design. b Peripheral blood %AML chimerism pre-CAR-T treatment and at endpoint in untreated ( n = 22 animals), CD25-targeted CAR-T-treated ( n = 32 animals), and mCXCR4(⁺) CD25 CAR-T-treated ( n = 27 animals). c AML chimerism in PB, spleen, bone marrow, and liver of PDX mice, stratified by treatment. Group sizes by treatment: untreated ( n = 4, 9, 8, 15 animals), CXCR4( ⁻) CD25-targeted CAR-T ( n = 10, 10, 8, 7 animals), and mCXCR4( ⁺ ) CD25-targeted CAR-T ( n = 12, 8, 7, 8 animals), PDX mice of Patients 10-13, respectively. Statistical comparisons made using unpaired two-sided Mann-Whitney U tests. The non-adjusted p-value is reported. For b , c Box and whisker plots: central lines show median, lower and upper hinges show first and third quartiles (the 25th and 75th percentiles) and whiskers extend up to 1.5 * IQR. d Longitudinal kinetics of AML cells (hCD45⁺CD33⁺), CAR-T cells (hCD45⁺CD3⁺), and murine leukocytes (mCD45⁺) in nucleated cell fraction from PB (red cells were excluded by lysis pre-FACS). Lines and shaded regions indicate mean ± SD. The number of mice included at each time point is shown. e Kaplan-Meier survival curves for PDX animals stratified by treatment arm including CXCR4(-) CD25-targeted CAR-T ( n = 27 animals), mCXCR4(+) CD25-targeted CAR-T ( n = 32 animals), and mock/ACTT-treated ( n = 10 animals) groups. Individuals removed for paired analysis or time-controlled sampling were censored (see Table ). Shaded regions show 95% confidence intervals. f H&E and CD33 IHC staining of femur sections from PDX (P11). Representative images selected from sections prepared from n = 5 mice per treatment group (scale: 20 µm) g ) Laser scanning microscopy (LSM) imaging and 3D reconstruction of liver sections acquired from PDX (P10), harvested 10 days after injection with CXCR4(-) or mCXCR4(+) CD25-targeted CAR-T. IF staining was performed for CXCL12 (green), CD3 (red) and DAPI (blue). Images were acquired on a Zeiss Axio Observer 7. Z-stack 3D reconstruction was performed using ImarisViewer. Representative images selected from sections prepared from n = 3 mice per treatment group (scale: 30 µm). Source data are provided as a Source data file for ( b , c , d , e ). “Fig. 3a” created in BioRender. Liang, M. ( https://BioRender.com/5hfwv50 ) licensed under CC BY 4.0 .

Article Snippet: After blocking with horse serum, slides were incubated with mouse anti-human TCF1 antibody (R&D systems, #2203) (1:200), anti-human CD33 (R&D systems, MAB11371) (1:100), anti-human and mouse CXCL12 (R&D systems, MAB350) (1:20), or anti-human CCR7 (Abcam, ab253187) (1:100) and then HRP-conjugated horse anti-rabbit or anti-mouse IgG antibody (ImmPRESS, MP-7500).

Techniques: MANN-WHITNEY, Whisker Assay, Lysis, Sampling, Immunohistochemistry, Laser-Scanning Microscopy, Imaging, Injection, Staining

Journal: bioRxiv

Article Title: Chimeric Antigen Receptors Transmit Piconewton Forces that are Coupled with T Cell Function

doi: 10.1101/2025.10.23.684052

Figure Lengend Snippet: A. Proposed mechanism of action for dasatinib in CAR T cells which leads to Lck inhibition. B . Representative RICM and tension images of CAR T cells treated with escalating doses of dasatinib. C and D. Plots quantifying CAR T cell forces and cell spread area as a function of dasatinib treatment. N ≥ 162 from 5 independent transductions. ***= 0.0001, ****<0.0001. E . Plot showing cytotoxicity as a function of dose-dependent dasatinib CAR T cell treatment. Data is averaged from two technical replicates using the same batch of CAR T cells. F . Relationship between CAR T cell tension and cytotoxicity showing correlation. G . Schematic showing three additional engineered CARs with 4-1BB costimulatory domain, ITAM mutants lacking key tyrosine residues, and finally CAR lacking a cytoplasmic domain along with representative RICM and 8 pN locked tension (Cy3B) images. H . Plot of single cell tension levels for each CAR T cell construct tested. N ≥ 162 from 5 independent transductions. One-way ANOVA. ***= 0.0001 , ****<0.0001. I . Representative RICM and 8 pN tension images showing the tension and spread area of cells on CD19 and CD33 antigens. J. Quantification of cellular tension levels from n≥90 cells/condition from 3 separate transductions. Each color indicates a biological replicate from a unique transduction using the same human donor. The larger data points indicate the mean of each replicate while the small data points represent the single cell measurements. N ≥ 106 cells/condition from 3 independent transductions. Student’s Two-Tailed Paired T Test, ****<0.0001.

Article Snippet: Biotinylated CD19 protein (Cat#CD9-H82E9-25ug), biotinylated CD33 protein (Cat#CD3-H82E7-25ug), and AlexaFluor 647 Anti-FMC63 Ab (Cat#FM3-AM534-25tests) were purchased from AcroBioSystems (Newark, DE).

Techniques: Inhibition, Construct, Transduction, Two Tailed Test