Abstract
Adoptive transfer of genetically engineered chimeric antigen receptor (CAR) T cells is becoming a promising treatment option for hematological malignancies. However, T cell immunotherapies have mostly failed in individuals with solid tumors. Here, with a CRISPR–Cas9 pooled library, we performed an in vivo targeted loss-of-function screen and identified ST3 β-galactoside α-2,3-sialyltransferase 1 (ST3GAL1) as a negative regulator of the cancer-specific migration of CAR T cells. Analysis of glycosylated proteins revealed that CD18 is a major effector of ST3GAL1 in activated CD8+ T cells. ST3GAL1-mediated glycosylation induces the spontaneous nonspecific tissue sequestration of T cells by altering lymphocyte function-associated antigen-1 (LFA-1) endocytic recycling. Engineered CAR T cells with enhanced expression of βII-spectrin, a central LFA-1-associated cytoskeleton molecule, reversed ST3GAL1-mediated nonspecific T cell migration and reduced tumor growth in mice by improving tumor-specific homing of CAR T cells. These findings identify the ST3GAL1–βII-spectrin axis as a major cell-intrinsic program for cancer-targeting CAR T cell migration and as a promising strategy for effective T cell immunotherapy.
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Data availability
The Protein Data Bank accession code is 5E6U. Signaling pathway analysis was performed with Enrichr using the NCI-Nature 2016 database for pathway enrichment analysis (http://amp.pharm.mssm.edu/Enrichr). Sequencing datasets have been deposited in the Gene Expression Omnibus database under accession codes GSE227429 (CRISPR screen), GSE227509 (CD8+ T cell bulk RNA sequencing) and GSE227430 (CAR T cell bulk RNA sequencing). All other data are included in the article and Supplementary Information files. Source data are provided with this paper.
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Acknowledgements
We thank the High Content Image Core (University of Rochester) for help with fluorescence imaging and use of the Dragonfly spinning-disc confocal. We thank Y. Gao and P. Rock for their technical assistance on the manuscript. We especially thank M. Jin (Weill Cornell) for the anti-HER2 CAR DNA construct and for his help with micro-PET/CT imaging, L. Gan (Augusta University) for help with βII-spectrin transgenic mouse generation, R. Burack and J. Moore for human sample collections and data review, J. Ashton, D. Ghoneim and J. Malik for help with CRISPR transcriptomic analyses and depositing RNA-sequencing data and all members of the Kim Laboratory for their comments during the course of these studies and input during preparation of the manuscript. We obtained hHER2 transgenic mice from Genentech. This study was funded by National Institutes of Health grant AI102851 (M.K. and P.W.O.), National Institutes of Health grant AI147362 (M.K. and R.E.W.), National Institutes of Health grant T32AI007285 (A.M.A. and C.J.S.), National Science and Technology Council in Taiwan MOST 111-2314-B-182A-069 and MOST 111-2321-B-182-002 (T.-C.F.) and K-GRC GO! KRICT project BSF23-113 (K.-D.K.).
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Conceptualization: Y.H., H.-R.K., B.L.W. and M.K. methodology: Y.H., H.-R.K., B.L.W., W.S.S., M.K., J.R.L., A.M.A., C.J.S., K.L., R.K.M., K.-D.K., T.C., E.B.L., N.S.R., K.V., T.-C.F., A.L.-T.Y., P.W.O., R.E.W., C.-D.J., P.M.R. and S.A.G. investigation: Y.H., H.-R.K., B.L.W., W.S.S., J.R.L., A.M.A., C.J.S., K.L., K.-D.K., T.C., E.B.L., N.S.R., K.V. and T.-C.F. funding acquisition: A.M.A., P.W.O., M.K., R.E.W. and K.-D.K. supervision: M.K. writing, original draft: Y.H., H.-R.K., B.L.W. and M.K.
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Extended data
Extended Data Fig. 1 hHER2 CAR-T cell assays and in vivo distribution.
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 and after CUBIC clearing.
Extended Data Fig. 2 hHER2 CAR-T cell distribution.
A. 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 or MHCII, green; CAR-T cell). 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 ± SEM, 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 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-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.
Extended Data Fig. 3 In vivo T cell CRISPR screen.
a. Volcano plot shows comparison of differentially expressed genes in naïve vs. in vitro activated CD8 T cells. Red denotes genes increased and blue denotes genes decreased in T cells. P values were adjusted using the Benjamini–Hochberg method. b. Heatmap of RNA-sequencing data from naïve vs. in vitro activated CD8 T cell samples (n = 3). Heatmap represents top 1,316 genes chosen from highest 0.25% p-value summary. c. Retroviral construct used for sgRNA delivery. d. A schematic of the experimental design showing timeline of T cell stimulation, transduction, enrichment, and flow analysis. T cells were purified by FACS sorting at day 7 and i.v injected into recipient tumor bearing mice. e. Scatter plot of the enrichment of candidates (n = 5 sgRNAs per gene) showing that sgRNAs targeting essential genes (left) were significantly reduced in the samples after two representative in vivo screens, but not the non-targeting guides (right). f. Genes with positive beta score (Tmigratory) and negative beta score (TSequestered) (>2 standard deviations from the mean (red)) are highlighted in yellow and blue, respectively.
Extended Data Fig. 4 Identification of CD18.
a. The spectrum of one of the CD18 peptides obtained by nanospray-ion trap tandem mass spectrometry. b. The deconvoluted MS/MS spectrum of tryptic peptide NVTR with 3 HexNAc. M: the parent ion of NVTR-HexNAc(3) at m/z 549.760 Da. M-18: loss one H2O in NVTR-HexNA(3). a2: the observed m/z 186.1218 Da matched to the a2 ion. y1: the observed m/z 175.1261 Da matched to the y1 ion. c. The crystal structure of LFA-1 headpiece (5E6U.pbd). The region encircled by dots from the left view is enlarged (right), representing the O-glycosylation on Thr256 in βI-domain.
Extended Data Fig. 5 Activated T cell migration in vitro.
a. Recruitment of CAR-T cells into dLN was blocked by LFA-1-blocking Ab and PTx treatments. Percentages of total transferred CD8 + T cells are shown. b & c. Activated CD8+ T cell migration on ICAM-1-coated plates ± CXCL12 (B) or CXCL10 (C) with varying concentrations as indicated. Data were collected from 3 independent experiments (n = 3, 11–20 individual cells per mouse). Data represent mean ± SEM. Statistical analyses were performed using one-way ANOVA with Bonferroni post-test. d. Representative Transwell assay of in vitro activated CD8+ T cell migration in response CXCL12 (2 µg/ml). Data were collected from 3 independent experiments (1 × 106 cells/well). Data represent mean ± SEM. Statistical analyses were performed using two-sided, unpaired Student’s t test. e. Schematic depicting the flow chamber assay. f & g. Activated CD8+ T cell migration on ICAM-1 coated plates with flow. Cells were treated with PTx (6 h) (F) and/or gallein (30 min) (G) ± CXCL12 (G) where indicated. Cells were allowed to settle for 10 minutes prior to 1 dyne/cm2 of fresh media being run across the surface of the plate. Data were collected from 3 independent experiments (n = 3, 25–45 individual cells per mouse). Data represent mean ± SEM. Statistical analyses were performed using one-way ANOVA with Bonferroni post-test. h. Activated CD8+ T cell migration on bEND.3 coated plates. bEND.3 cells were allowed to grow to a monolayer for at least 12 h prior to imaging. CD8+ were treated with PTx (6 h) where indicated. T cells were allowed to settle for 10 minutes prior to 1 dyne/cm2 of fresh media being run across the surface of the plate. Data were collected from 3 independent experiments (n = 3, 20–39 individual cells per mouse). Data represent mean ± SEM. Statistical analyses were performed using two-sided, unpaired Student’s t test.
Extended Data Fig. 6 TIRFM measurements of LFA-1 distribution.
a. Schematic of TIRFM measurements. Molecules near the surface fluoresce more brightly than molecules between microvilli and those farther from the substrate. b. Human CD8 T cells labeled for LFA-1 spreading on glass, BSA, or ICAM-1 coated substrate. Contrast and brightness have been adjusted for visibility, but the original gray values are indicated in the scale bars to the right of each image. All images in the same row are at the same magnification. Scale bars, 5 μm. Representative images from > five independent experiments are shown.
Extended Data Fig. 7 βII-spectrin expression in T cells.
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 TNF 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) 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.
Supplementary information
Supplementary Table 1
MS data.
Supplementary Table 2
LC–MS/MS analysis data.
Supplementary Table 3
CD19 CAR T cell participant information.
Supplementary Video 1
In vitro CAR T cell killing assay. WT CD8+ T cells or hHER2 CAR T cells were co-incubated with B16-hHER2 cells (green) in the presence of propidium iodide (red); scale bar, 20 μm.
Supplementary Video 2
In vivo CAR T cell migration. hHER2 CAR T cells (green) were intravenously injected by the tail vein of the B12-hHER2-bearing mouse, and intravital imaging was simultaneously performed to visualize T cells in the lung, LNs and tumor. The blood vessels were labeled with anti-CD31 (red). Time is marked as min:s; scale bars, 50 μm (parts 1, 3 and 4) and 20 μm (part 2).
Supplementary Video 3
CAR T cell accumulation. hHER2 CAR T cells (green) were intravenously injected by the tail vein of the B12-hHER2-bearing mouse, and intravital imaging was performed 48 and 72 h after infusion to visualize T cells in the lung. The blood vessels were labeled with anti-CD31 (red). Time is marked as min:s; scale bar, 50 μm.
Supplementary Video 4
CAR T cell accumulation. Deep 3D imaging (1-mm thick) of CAR T cell accumulation in a cleared mouse lung 72 h after intravenous injection (red, blood vessels (CD31); green, CAR T cells (GFP)).
Supplementary Video 5
In vitro CAR T cell migration to a tumor spheroid. Long-term time-lapse imaging (9 h) of hHER2 CAR T cells (red) embedded in 3D Matrigel around an hHER2-B16 (blue) spheroid ± PTx pretreatment; scale bars, 100 µm. Time is represented in h:min.
Supplementary Video 6
In vitro CD8+ T cell migration. Migration of naive and activated (day 5) mouse CD8+ T cells on ICAM-1 ± CXCL12-coated plates. Time is marked as min:s; scale bar, 50 μm.
Supplementary Video 7
In vitro CD8+ T cell migration. Migration of activated (day 5) mouse CD8+ T cells on ICAM-1-coated plates with (1 dyn cm–2) and without (0 dyn cm–2) flow; scale bar, 50 μm.
Supplementary Video 8
In vitro CD8+ T cell migration with βII-spectrin expression. Activated CD8+ T cells were transfected with GFP only (mock) or βII-spectrin cDNA and migrated on ICAM-1 ± CXCL12-coated plates. Time is marked as min:s; scale bar, 20 μm.
Supplementary Video 9
LFA-1-mediated CD8+ T cell spreading. TIRF microscopy images of activated CD11a–mYFP CD8+ T cell adhesion on ICAM-1 ± CXCL12-coated plates with and without βII-spectrin expression. LFA-1 intensity (YFP) is shown on a pseudocolor scale (from low (blue) to high (red)). Each frame is 15 s; scale bar, 10 μm.
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Hong, Y., Walling, B.L., Kim, HR. et al. ST3GAL1 and βII-spectrin pathways control CAR T cell migration to target tumors. Nat Immunol 24, 1007–1019 (2023). https://doi.org/10.1038/s41590-023-01498-x
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DOI: https://doi.org/10.1038/s41590-023-01498-x
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