Journal: Nature immunology

Article Title: St3gal1 and βII-spectrin pathways control CAR-T cell migration to target tumor sites

doi: 10.1038/s41590-023-01498-x

Figure Lengend Snippet: A. Clinical protocol for CAR T-cell infusion (CTL019). B. Blood T cell counts from patients (n = 15). Red dashed line, the mean ; blue line, total blood lymphocyte counts. C & D. Visualization of early sequestration (0–1 hr) of hHER2 CAR-T cells after adoptive transfer with PET/CT (89Zr-DFO-labeled T cells) (C) or IV-MPM (red, dextran-labeled vessels; green, GFP-expressing CAR-T cells; blue, SHG) (D). IV-MPM images from the lung at the indicated times post-injection (D). Scale bars, 50 μm. E. Representative 3D imaging of CAR-T cell accumulation in cleared lung 4 and 72 hr after i.v. injection (red, blood vessels; green, CAR-T cells). Scale bars, 1 mm. F. The cell density curve and histogram of the distribution of T cells extravasated from the nearest blood vessel (attraction distance, AD). G. 3D image of GFP-expressing CAR-T cells and CD31-labeled vessels. H. The number of injected CAR-T cells in the lung, blood, and tumor at the indicated times. I. in vitro migration of hHER2-CAR T cell (red) to hHER2-B16-BFP tumor spheroid (blue) and . Scale bars, 100 μm. J. CAR-T cells from the lung 72 hr after i.v. injection (T72; CFSE) and newly in vitro activated CAR-T cells (T0; CTFR) were co-transferred (1:1 mix) and the fold change in the homing index was analyzed 24 hr after injection (n = 4, 13 mice per experiment). K. Volcano plot and signaling pathway from RNA-seq analysis in CAR-T cells (T72 vs. T0). L. Percentage of Ki67+ cells in vitro activated CAR-T cells (0 hr) or CAR-T cells isolated from the lung after i.v. injection (4 and 72 hr). M. Flow cytometry results of PD-1 and Tim-3 expression levels on naïve and CAR-T cells isolated from the lung at indicated times post-injection. N. The cytotoxicity of CAR-T cells (PBS, T72, and T0) against B16-hHER2. O. The proportion of CAR-T cells distributed across different organs post-injection. Representative image of 5 mice (D-E). Representative data from three experiments with n = 3 per group (L-N) n = 3 mice per group (F, O). Results are shown mean ± SEM (H, J, L, N). The data were analyzed by ordinary one-way ANOVA with Tukey’s multiple comparison posttest (H) or two-tailed Student’s t test (N).

Article Snippet: For B16-hHER2 stable cell line, B16F10 mouse melanoma cells (ATCC) were transfected with human HER2 pcDNA3.1 mammalian expression plasmid (Addgene #16257) using Lipofectamine 2000 according to the manufacturer’s protocol.

Techniques: Adoptive Transfer Assay, Positron Emission Tomography-Computed Tomography, Labeling, Expressing, Injection, Imaging, In Vitro, Migration, RNA Sequencing, Isolation, Flow Cytometry, Comparison, Two Tailed Test

Journal: Nature immunology

Article Title: St3gal1 and βII-spectrin pathways control CAR-T cell migration to target tumor sites

doi: 10.1038/s41590-023-01498-x

Figure Lengend Snippet: A. Left: Deep 3D imaging (1 mm thick) of CAR-T cell accumulation in the cleared mouse lung after 72 hr after i.v. injection (red; CD11c (top) or MHCII (bottom), green; CAR-T cell (GFP)). Right: Graph shows colocalization of red signal versus GFP (CAR-T cells). Pearson coefficient was generated as Green (CAR-T cell) /Red (anti-CD11c or anti-MHCII). Data are presented as mean ± SEM; n = 3 mice/group. B. Flow cytometric analysis of OT-I T cells in the lung, blood, and tumor after i.v. injection (4, 24, 48, and 72 hr; mean ± s.e.m., n = 3 mice per group). C. PCA of differentially expressed genes in CAR-T cells (T72 vs. T0). D. Gating strategy for detection of hHER2-CAR-T cells from organs (lungs) E. Expression of Ki67+ in CAR-T cells isolated from the lung after i.v. injection. F. Relative expression of IFN-γ and TNF-α in CAR-T cells isolated from lung at indicated times after i.v. injection. Data are presented as mean ± SEM; n = 3 mice/group. G. Representative flow cytometry results show cell surface PD-1 and Tim-3 expression levels in OT-I naïve T cells, freshly activated in vitro (0 hr), or isolated from the lung after 72 hr post-injection. H. hHER2-CAR-T cells isolated from the lung or dLN after 72 hr i.v. injection were cocultured with B16-hHER2 for 24 hr. Dead B16 cells were stained with 7-amino-actinomycin D (7-AAD). The data reflect 3 independent experiments (the mean ± SEM, n = 3). The data were analyzed by two-tailed Student’s t test (*P = 0.002). I. Representative flow cytometry results showing cell surface PD-1 and Tim-3 expression levels in naïve and CAR-T cells isolated from the lung at the indicated times post-injection.

Article Snippet: For B16-hHER2 stable cell line, B16F10 mouse melanoma cells (ATCC) were transfected with human HER2 pcDNA3.1 mammalian expression plasmid (Addgene #16257) using Lipofectamine 2000 according to the manufacturer’s protocol.

Techniques: Imaging, Injection, Generated, Expressing, Isolation, Flow Cytometry, In Vitro, Staining, Two Tailed Test

Journal: Nature immunology

Article Title: St3gal1 and βII-spectrin pathways control CAR-T cell migration to target tumor sites

doi: 10.1038/s41590-023-01498-x

Figure Lengend Snippet: A. Gating strategy for detection of hHER2-CAR-T cells. A median of > 90% of mouse CD8 T cells transduced with the hHER2-CAR retrovirus were positive for the transgene as judged by flow cytometry analysis. B. B16F10 cells were transfected with mammalian human HER2 construct with lipofectamine. Cells were grown in the presence of G418 and sorted twice to generate single cell clones. C. CAR-T-mediated B16-HER2 cell killing assay. Apoptotic cells were stained for Annexin V and analyzed by flow cytometry. D. Whole mouse lung before (left) and after (right) CUBIC clearing.

Article Snippet: For B16-hHER2 stable cell line, B16F10 mouse melanoma cells (ATCC) were transfected with human HER2 pcDNA3.1 mammalian expression plasmid (Addgene #16257) using Lipofectamine 2000 according to the manufacturer’s protocol.

Techniques: In Vivo, Transduction, Flow Cytometry, Transfection, Construct, Clone Assay, Staining

Journal: Nature immunology

Article Title: St3gal1 and βII-spectrin pathways control CAR-T cell migration to target tumor sites

doi: 10.1038/s41590-023-01498-x

Figure Lengend Snippet: A. Schematic for the development of a pooled in vivo CRISPR screening platform in mouse CD8 T cells (Created with BioRender.com) B. Rank-ordered norm z-score of the beta values for all three OT-I T cell distribution screens in B16-OVA bearing mice (yellow, migratory genes; blue, sequestered genes). The top three sequestered and the top five migratory genes are indicated. C. Genes rank based on the difference in beta scores across sequestered and migratory conditions. D. Sectored scatter plot of gene-level beta values from Tsequestered vs. Tmigratory cell populations isolated from B16-OVA bearing mice. Significant migratory genes and sequestered gene signatures (FDR < 5%) are colored yellow and blue, respectively. Ellipse (gray dashed lines), 5% FDR threshold delineated by normal ellipse fit to differential scores. E. qPCR of St3gal1 in mouse and human CD8 T cells (naïve vs. activated) as compared to three averaged housekeeping genes (n = 3–11). F. Expression levels of St3gal1 in activated CD8 T cells (from Days 0 to 5). Loading control: β-actin. G. qPCR of Spc24 in mouse and human CD8 T cells (naïve vs. activated) as compared to three averaged housekeeping genes (n = 3–4). H. Expression levels of Spc24 in activated CD8 T cells (from Days 0 to 5). Loading control: β-actin. I. Expression levels of Spc24 and St3gal1 in Cas-9 hHER2 CAR-T cells with sgSpc24 and sgSt3gal1, respectively. Loading control: β-actin. J. The cytotoxicity of CAR-T cells (PBS, sgSpc24 cells, and sgSt3gal1 cells) against B16-hHER2 (n = 3). K. The distribution of CAR-T cells with (WT; CTFR)/without (KO; CFSE) Spc24 or St3gal1 24 hr after injection (n = 3). The fold change in the homing index, as determined by (KOsample/WTsample)/(KOinput/WTinput). Results are shown mean ± SEM (E, G, J-K). The data reflect three independent experiments (J, K) The data were analyzed by two-tailed Student’s t test (E, G).

Article Snippet: For B16-hHER2 stable cell line, B16F10 mouse melanoma cells (ATCC) were transfected with human HER2 pcDNA3.1 mammalian expression plasmid (Addgene #16257) using Lipofectamine 2000 according to the manufacturer’s protocol.

Techniques: In Vivo, CRISPR, Isolation, Expressing, Control, Injection, Two Tailed Test

Journal: Nature immunology

Article Title: St3gal1 and βII-spectrin pathways control CAR-T cell migration to target tumor sites

doi: 10.1038/s41590-023-01498-x

Figure Lengend Snippet: A & B. MAL-II binding to CD8 T cells (A) and St3gal1 KO CAR-T cells (B). C. Immunoprecipitation of MAL-II-binding proteins in CD8 T cells. D. Western blot of the activated CD8 T cell lysates after enzyme treatments. In the graph, each bar represents the calculated molecular weight of protein bands. E. Immunoprecipitation of MAL-II binding CD18 in activated CD8 T cells. F. Recruitment of CAR-T cells into lung tissue (n = 3 mice per group). G. The numbers of WT or LFA-1 (CD11a)-KO CAR-T cells 24 hr after CAR-T cells injection (n = 3). H. In vitro CD8 T cell migration on ICAM-1-coated plates. (n = 3, 13–38 individual cells per mouse). I. In vitro activated CD8 T cell migration on ICAM-1-coated plates ± CXCL12. (n = 3, 10–57 individual cells per mouse). J. LFA-1 FRET assay with CD11a-mYFP and CD18-mCFP. Scale bars, 5 μm. (n = 3, 20–40 individual cells per condition). K. The density of microvilli of CD8 T cells by SEM (n = 6–8). Scale bars, 1 μm. L. The ratio of the TIRFM signal from the LFA-1 accumulation of CD8 T cells at sites of contact with the surface (n=3). M. Changes in the endocytosis of LFA-1 in CD8 T cells after treatment with α2,3-neuraminidase (n = 3–4). N. Time course of surface and intracellular LFA-1 expression. O & P. Changes in the endocytosis (O) and cell surface expression (P) of LFA-1 in CAR-T cells. (n = 3). Q & R. The migration of activated CD8 T cells pretreated with Exo1 (Q) or transfected with Rab13 siRNA (R) (n =3, 20–68 individual cells per mouse). S. The cytotoxicity of CAR-T cells against B16-hHER2 (n = 3). T & U. The percentage of hHER2 CAR-T cells in peripheral tissue sites (T) and tumors (U) 24 hours after adoptive transfer (n=3 mice per group). Results are shown mean fluorescence intensities (MFI) ± SEM (A-B) and mean ± SEM (C, F-M, O-P). All data and representative images reflect three independent experiments. Statistical analyses were performed using one-way ANOVA with Bonferroni posttest (A-C, H-I, Q-R), two-sided Mann-Whitney test (F, T-U), two-sided, unpaired Student’s t test (G, J) or two-tailed Student’s t test (K-M, O-P). NS; not significant.

Article Snippet: For B16-hHER2 stable cell line, B16F10 mouse melanoma cells (ATCC) were transfected with human HER2 pcDNA3.1 mammalian expression plasmid (Addgene #16257) using Lipofectamine 2000 according to the manufacturer’s protocol.

Techniques: Binding Assay, Immunoprecipitation, Western Blot, Molecular Weight, Injection, In Vitro, Migration, Expressing, Transfection, Adoptive Transfer Assay, Fluorescence, MANN-WHITNEY, Two Tailed Test

Journal: Nature immunology

Article Title: St3gal1 and βII-spectrin pathways control CAR-T cell migration to target tumor sites

doi: 10.1038/s41590-023-01498-x

Figure Lengend Snippet: A. LFA-1 immunoprecipitates obtained from human T cells bound on ICAM-1 (IC-1)- or poly-L lysine (PLL)-coated cover glasses. LFA-1-associated proteins were identified by silver staining and mass spectrometry. B. Expression levels of αII-spectrin, βII-spectrin, and MyH9 in activated T cells (from Days 0 to 5). Loading control: β-actin. C. A confocal FRAP experiment with activated CD11a-mYFP CD8+ T cells (n = 3). Scale bars, 1 μm. D. & E. Representative TIRFM images of activated CD11a-mYFP CD8+ T cell adhesion on ICAM-1-coated plates (D) and the area and spreading speed calculated from zone including all TIRF signals for each cell (E). Scale bar, 20 μm. Each dot represents one cell (n = 3). Bar = mean. F. Naïve and activated CD8 T cell migration on ICAM-1- or ICAM-1+CXCL12-coated surfaces. (n = 3. 21–42 individual cells per mouse). G. Schematic illustrating the regulation of LFA-1 endocytic recycling via βII-spectrin or St3gal1 expression (Created with BioRender.com). H. βII-spectrin (GFP+) expression in T cells isolated from GFP-ROSAβII-spectrin mice (TβII-spectrin) after in vitro treatment with TAT-Cre recombinase. I. Cross sections image in the middle of hHER2-CAR T cell (WT and TβII-spectrin)-endothelial cell (bEND.3 cells) contacts by transmission EM. Black arrows, LFA-1 stainings at the cell-cell contacts. Scale bars, 2 μm and 0.1 μm. J. The ratio of the TIRFM signal from the LFA-1 accumulation of hHER2-CAR T cells (WT and TβII-spectrin) at sites of contact with the ICAM-1-coated surface (n = 3). K. Flow cytometry analysis of MAL-II binding to naïve and activated CD8 T cells (WT, TβII-spectrin, and TβII-spectrin + St3gal1-shRNA-treated cells) (n = 3). Representative images or data were collected from three independent experiments (A-B, F, H) or two independent experiments (I). Results are shown the mean ± SEM (C, F) and mean fluorescence intensities (MFI) ± SEM (K). Statistical analyses were performed by ordinary two-way ANOVA (C), two-sided, unpaired Student’s t test (E-F, J, K).

Article Snippet: For B16-hHER2 stable cell line, B16F10 mouse melanoma cells (ATCC) were transfected with human HER2 pcDNA3.1 mammalian expression plasmid (Addgene #16257) using Lipofectamine 2000 according to the manufacturer’s protocol.

Techniques: Silver Staining, Mass Spectrometry, Expressing, Control, Migration, Isolation, In Vitro, Transmission Assay, Flow Cytometry, Binding Assay, shRNA, Fluorescence

Journal: Nature immunology

Article Title: St3gal1 and βII-spectrin pathways control CAR-T cell migration to target tumor sites

doi: 10.1038/s41590-023-01498-x

Figure Lengend Snippet: A. Generation of GFP-ROSAβII-spectrin mouse model (Rosa26tm(CAG-LSL-Sptbn1-IRES-GFP)). Mouse Sptbn1 cDNA was inserted into the CAG-STOP-GFP-Rosa targeting vector, CTV, between a floxed Stop cassette and the internal ribosome entry site (IRES) followed by the enhanced Green Fluorescent Protein gene (eGFP). Transcription is under control of the CAG promoter. The targeting vector contained Rosa26 homology arms (1 kb 5′ and 3.8 kb 3′), so that the entire loxP-stop-loxP-Tmc2-IRES-GFP transcriptional cassette was inserted into the first intron of Rosa26 gene on chromosome 6. B & C. Flow cytometry analysis of IFNγ and TNFα expression in hHER2-CAR transfected T cells from GFP-ROSAβII-spectrin mouse (TβII-spectrin) after treated with PBS or Tat-Cre Recombinase. Cells were co-cultured with B16-HER2 cells. Data represent mean ± SEM. n = 9. D. CAR-mediated B16-HER2 cell killing assay with TβII-spectrin cells after treated with PBS or Tat-Cre Recombinase. Cell death was stained for NucSpot. Data represent mean ± SEM. n = 9. E. Activated CD4+ T cell (OT-II) migration on ICAM-1 coated plates ± CXCL12. Cells were treated with PTx (6 hr) or Gallein (30 min) where indicated. Data were collected from 2 independent experiments (n = 2, 17–34 individual cells per mouse). Data represent mean ± SEM Statistical analyses were performed using one-way ANOVA with Bonferroni post-test. *P = 0.007. F. The pie charts depict the proportion of CD4 T cells distributed in the tumour, blood, LN/spleen, or lung/liver 72 h post-injection. G. Expression levels of βII-spectrin and St3gal1 in human CD4 and CD8 T cells (before and after activation). Loading control: β-actin. Representative western blot images from three independent experiments are shown. H. Expression levels of βII-spectrin in human CD8 memory T cells (CD8+CD45RO+CD45RA–CD56–CD57–). Loading control: β-actin. Representative western blot images from three independent experiments are shown.

Article Snippet: For B16-hHER2 stable cell line, B16F10 mouse melanoma cells (ATCC) were transfected with human HER2 pcDNA3.1 mammalian expression plasmid (Addgene #16257) using Lipofectamine 2000 according to the manufacturer’s protocol.

Techniques: Expressing, Plasmid Preparation, Control, Flow Cytometry, Transfection, Cell Culture, Staining, Migration, Injection, Activation Assay, Western Blot

Journal: Nature immunology

Article Title: St3gal1 and βII-spectrin pathways control CAR-T cell migration to target tumor sites

doi: 10.1038/s41590-023-01498-x

Figure Lengend Snippet: A. Schematic showing the assessment of tissue homing of TβII-spectrin (green) and Tcontrol (red) cells transfected with hHER2-CAR in hHER2 Tg mice bearing B16-hHER2 tumors. B. TβII-spectrin (green) and Tcontrol (red) hHER2-CAR were cotransferred (1:1 mix) and the fold change in the homing index was analyzed at each time point (n = 5 mice per condition). C. Represented deep 3D imaging (1-mm thick) of CAR-T cell accumulation in a cleared mouse lung 72 hr after i.v. injection (blue; blood vessels [CD31], green; TβII-spectrin, red; Tcontrol). Scale bars, 100 μm. D. Tumor growth and final tumor size from hHER2 Tg mice bearing B16-hHER2 tumors infused with TβII-spectrin and Tcontrol cells transfected with hHER2-CAR (n = 10 mice per group). E. Mouse survival curves up to 50 d after tumor inoculations. F. Frequency of CAR-T cells over total lymphocytes in B16-hHER2 tumours, lung, or dLN as measured by flow cytometry on day 22. respectively (n = 4). G. βII-spectrin western blot analysis of CAR-T cell infusion products obtained from 23 BCL patients who showed a complete recovery (CR), partial recovery (PR), or progressive disease (PD). Representative male and female samples from each patient group are shown. H. Quantification of total βII-spectrin protein expression in CAR-T cell products from patients with CR, PR, or PD as determined by western blotting. I. Correlation between βII-spectrin expression in CAR-T cell products and patient neurotoxic effects and cytokine release syndrome. Pearson correlation calculations were performed for linear correlations. J. Luminex assay of the proinflammatory cytokines IFN-γ, IL-6 and IL-1β in patient serum before (Day −5) and after (Day 2) CAR-T cell infusion. The data represent the mean ± SEM (B, D, F, H). Statistical analyses were performed by ordinary two-way ANOVA (D), two-sided, unpaired Student’s t test (B, D, H) or two-sided log-rank Mantel–Cox test (E). Whiskers represent maximum and minimum values (D, F, H).

Article Snippet: For B16-hHER2 stable cell line, B16F10 mouse melanoma cells (ATCC) were transfected with human HER2 pcDNA3.1 mammalian expression plasmid (Addgene #16257) using Lipofectamine 2000 according to the manufacturer’s protocol.

Techniques: Transfection, Imaging, Injection, Flow Cytometry, Western Blot, Expressing, Luminex