Fig 2: STING deficiency in EC but not in T cells results in impaired TEM in response to TNF-α.(A) Cultured MHEC from WT and STING–/– mice were lysed and analyzed by immunoblot to evaluate STING expression and β-actin, used as a loading control. (B and C) Quantification of adhesion and %TEM of WT and STING–/– Th1 cells perfused across WT and STING–/– MHEC (for WT Th1 groups: n = 3 independent experiments with WT and STING–/– MHEC preparations and Th1 preparations, using duplicate or triplicate coverslips). (D) Representative images of WT and STING–/– Th1 cell adhesion on ICAM1- and VCAM1-coated coverslips following perfusion under flow conditions. Scale bar: 100 μm. (E and F) Quantification of Th1 adhesion on ICAM1 (n = 3 independent experiments, triplicate coverslips) and on VCAM1 (n = 3 independent experiments). (G and H) Representative flow cytometry histograms and quantification of VLA-4 (G) and of LFA-1 (H) from WT and STING–/– Th1 cells (n = 3 independent Th1 cell preparations). Data are shown as mean ± SEM. ***P < 0.001; 1-way ANOVA (B and C) and t test (E and H).

Fig 3: STING modulation of T cell TEM is independent of NF-κΒ–inducible adhesion molecules and dependent on the ISG CXCL10.(A and B) Immunoblot (A) and quantification from 3 independent experiments (B) of STING and β-actin expression in control and STING KD HUVEC monolayers using CRISPR. (C and D) CD3+ T cells isolated from human blood were perfused under flow conditions across WT and STING KD HUVEC stimulated for 4 h with TNF-α, and accumulation (C) and %TEM (D) were quantified. n = 8 control and n = 10 TNF-α coverslips from 3 independent experiments. (E and F) Representative histograms of surface PECAM-1, ICAM1, and VCAM1 per cell fluorescent intensity on HUVEC (E) and MHEC (F). (G) Quantification of CXCL10 in supernatants collected from WT and STING–/– MHEC at baseline and in response to 4-h TNF-α stimulation from n = 3 cell preparations. (H and I) Quantification of accumulation (H) and %TEM (I) of Th1 cells perfused across control and anti-CXCL10-treated MHEC from n = 3 independent experiments. Data are shown as mean ± SEM values. *P < 0.05, **P < 0.01 and ***P < 0.001; t test.

Fig 4: ICAM-1 is widely upregulated in brain capillaries in CD19-CAR T cell treated mice.(A) Representative immunofluorescence images showing capillary labeling with anti-laminin antibody (neuronal nuclei are also labeled) and ICAM-1 staining. These images were analyzed to obtain the data in B and C, which show scatter plots of ICAM-1 and VCAM-1 immunofluorescence brightness, as normalized to mock control. Each dot indicates one mouse. One-sample Wilcoxon test with theoretical median set at 100, n=6. The mock control values (n=4) are all 100% and are only shown for reference. (D) Representative top down view of the cortical microvasculature by in vivo two-photon imaging. ICAM-1 and VCAM-1 were labeled with intravascular fluorescent antibodies in mice treated with mock (top row) or CAR T cells (bottom row). c, capillary; v, venule. (E) Scatter plots showing upregulation of ICAM-1 and VCAM-1 in hBMECs after treatment with cytokines. The y-axis indicates the fold change in mean fluorescence intensity as measured by flow cytometry, comparing each data point to the 0 pg/mL vehicle-only control within the same experiment. The x-axis indicates the concentration of each cytokine that was added to the culture media for 24h. The yellow shading indicates the approximate concentrations of each cytokine found in the blood of healthy volunteers, the blue shading indicates the range of concentrations reported in CAR T cell patients, and green shows overlap of these ranges. N=3 independent experiments, one-way ANOVA with Dunnett’s multiple comparisons test, only comparisons with the 0 pg/mL vehicle-only control were performed. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. All others are P>0.05.

Fig 5: CAR T cells strongly upregulate affinity of VLA-4 to VCAM after infusion.(A) Schematic of experimental design. (B) Timeline of experimental design. (C-F) Scatter plots showing flow cytometric measurements of T cell affinity to ICAM-1 (C), surface expression of integrin αL (LFA-1, D), affinity to VCAM-1 (E), and surface expression of integrin α4 (VLA-4, F). The y-axis indicates the mean fluorescence intensity (MFI, arbitrary units) for each analyte, and the x-axis indicates time relative to CAR T cell infusion, IP = cryopreserved infusion product. Measurements are compared between mice treated with mock transduced T cells (blue triangles) and mice treated with CD19-CAR T cells (red circles). Each data point indicates one mouse, the box plots show the mean and the whiskers show the standard deviation. N=3 independent experiments, for a total of 3–6 mice per group. All comparisons are by unpaired 2-sample t test with Welch correction, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns P>0.05. (G, H) Scatter plots showing movement velocity in the open-field test after treatment with antibody to block function of integrin α4 (G) or integrin αL (H). Values are normalized to mock treated control. Each dot indicates one mouse. Data is summarized from 3 or more independent experiments. Statistics are by one-sample Wilcoxon test with theoretical median set at 100, n=7–8. The mock control values (n=7–8) are only shown for reference.