Fig 1: Modelling the in vivo GBM tumor niche in a ‘GBM-a-on-Chip’ microphysiological system.(A) A schematic diagram illustrating a microfluidics-based GBM-on-a-Chip model to investigate ? the interactions of immune cell (CD8+ T-cells) with brain microvessels, ? tumor-associated macrophages (TAMs) and ? GBM tumor cells in an engineered 3D brain-mimicking ECM. (B) A schematic illustrating the procedures of cell preparation in the microphysiological system. Biomimetic TAMs (CD68+CD163+) were prepared by differentiating monocyte-like U937 cells with 5 nM of PMA for 24 hr, followed by treatments of conditioned-media of GBM cells for 3 days. Simultaneously, fresh allogeneic CD8+ T-cells were isolated from PBMCs and activated and expanded for 3 days with IL-2. (C) Representative confocal immunofluorescence images showing a 3D brain microvessel lumen (yellow) in contact with CD8+ T-cells (green) and GBM (PN, GBML20) tumor cells (red). Scale bar is 50 µm. (D) Representative time-lapsed images showing a single CD8+ T-cell extravasating through brain microvessels (yellow, 0–1 hr), infiltrating through ECM (1–4 hr), and interacting with GBM tumor cells (red, 4–6 hr). Scale bar is 50 µm. (E) Quantified CD8+ T-cell migration speed at different time points of infiltration, indicating the relatively maximum migration speed after extravasation and before contacting with GBM cells. (F) Representative immunofluorescence images showing the distinct counts of allogeneic CD8+ T-cell infiltrate in the PN (GBML20), CL (GBML08) and MES (GBML91) GBM subtypes in GBM-on-a-Chip after 3 days’ culture. Note that CD8+ T-cells (green) were in contact with brain microvessels (yellow), TAMs (blue) and GBM tumor cells (red). Scale bar is 50 µm. (G) Quantified results showing more infiltrated allogeneic CD8+ T-cells in the PN GBM as compared to the CL and MES GBMs. (H) Migration trajectories of infiltrated CD8+ T-cell (n > 20) for 2 hr in different GBM subtypes. (I) Quantified migration speed of infiltrated CD8+ T-cell, showing faster migration speed in the PN GBM as compared to the CL and MES GBMs at the observation window. Note that the speed range (0–6 µm/min) represents different infiltration stages of different T-cells. (J) Quantified GBM cell apoptosis ratio with the presence or absence of IL-2-activated allogeneic CD8+ T-cell in different GBM niches based on caspase-3/7 activation. Error bars represent ± standard error of the mean (s.e.m.). p-Values were calculated using the Student’s paired sample t-test. *, p<0.05.
Fig 2: Oxidized-desialylated LDL inhibits LAK cell cytotoxicity in vitro. A) Activated and expanded LAK cells were cultured in serum free X-VIVO 10 media in a 24 well plate with 0.1 µg/ml IL-2 in the absence or presence of native LDL, oxidized only LDL, desialylated only LDL, or oxidized-desialylated LDL at 50 µg/ml for 72 hours. Then LAK cells were washed three times with X-VIVO 10 serum free media to remove residual external LDL and incubated in a 4-hr killing assay with K562 cells at a 10:1 effector to target ratio. Percent cytotoxicity was determined by flow cytometry. B) Quantification of K562 cell death. **indicates a statically significant difference between native LDL (control) and oxidized-desialylated LDL treated LAK cells, and oxidized only LDL vs oxidized-desialylated LDL treated LAK cells (p<0.0001).*Indicates significant difference between native LDL and desialylated only LDL, and desialylated only LDL vs oxidized-desialylated (p <0.001) treated LAK cells, n = 5 per group. ns indicates not statistically significant. Statistical significance determined using two-way ANOVA with Tukey posthoc test. Error bars represent standard deviation.
Fig 3: Oxidized-desialylated LDL downregulates cytotoxicity receptor CD56 and upregulates the CD3 receptor. Activated and expanded LAK cells were cultured in serum free X-VIVO 10 media in a V-bottom 96 well plate with IL-2 in the absence or presence of native LDL or oxidized-desialylated LDL at 50 µg/ml for 72 hours. Then LAK cells were washed three times with X-VIVO 10 serum free media, reconstituted in PBS, and labeled with anti-CD56 antibodies, as well as with the dead staining dye SytoxBlue. A) Live cells were gated and plotted against CD3 and CD56. B) Oxidized-desialylated LDL decreased the number of CD3-CD56+ cells. C) The number of NKT cells (CD3+ CD56+) also decreased significantly. D) The number of CD3 positive cells increased significantly upon oxidized-desialylated LDL treatment of LAK cells. n = 5 per group. Error bars represent standard deviation. ** indicates statically significant differences (p<0.0001), determined using one-way ANOVA with Tukey posthoc test.
Fig 4: Ex vivo reactivation of spike-specific CD4+ T Cells reveals durable and functional immune memory in SARS-CoV-2-recovered individuals.a) Representative flow cytometry plots 20 hours after Vehicle control or Spike-stimulation of PBMCs from HC and CoV2+ individuals demonstrating T cell upregulation of CD40L and ICOS on CD45RA-CD4+ T cells. b) Enumeration of total CD40L+ICOS+ and c) CXCR5+CD40L+ICOS+ (cTfh) per 1e6 CD4+ T Cells and paired CoV2+ data from Visit 1 and Visit 2 represented as frequency of spike minus vehicle. d) Representative flow cytometry plots and e) number of CD69+ICOS+ CD4+ T Cells producing intracellular cytokines and number producing cytokine after incubation with spike minus number after incubation with vehicle. f) Relative distribution of effector cytokine production in memory T Cell compartments (CCR6+/- cTfh and non-cTfh) following ex vivo stimulation for 20 hrs; (IFN-y; blue) (IL-2; red) (IL-17A; yellow) from (d). g) Antigen-specific T cell proliferation of sorted CD4+ naive or memory T cells in control and CoV2+ PBMCs. Proliferation following 5-6 day co-culture with SARS-CoV-2 spike protein-pulsed autologous monocytes. h) Antigen-specific expansion represented as frequency of spike minus vehicle, CXCR3+CPDlow responding cells. i) Representative flow cytometry plots and j) quantification of spike-specific CD8+ T Cells in control and Cov2+ PBMCs stimulated with SARS-CoV-2 spike protein. a-h) Significance was determined by Kruskal-Wallis test correcting for multiple comparisons using FDR two-stage method. Adjusted p values are reported. i-j) Significance was determined by two-tailed, non-parametric Mann-Whitney tests. a-j) Data represented as mean and SD; Each symbol represents one donor. a-f, i-j) n=7 HN, n=14 HC, n=14 CoV2+(2 experiments). g-h) n=3 V1 HC, n=4 V2 HC, n=3 V1 CoV2+, n=4 V2 CoV2+ (2 experiments).
Fig 5: Enhanced uptake of oxidized-desialylated LDL by LAK cells. LAK cells were cultured in serum free X-VIVO 10 media in a V-bottom 96 well plate with IL-2 in the absence or presence of native pHrodo Green Conjugate LDL or oxidized-desialylated pHrodo Green Conjugate LDL at 10 µg/ml for 1, 2, 8, 16, 32, and 72 hours. The percentage of LDL positive cells was measured by flow cytometry. A) Qualitative flow data showing uptake of oxidized-desialylated LDL and native LDL, which shows the differences in uptake between the two forms of LDL. FSCH stands for forward scatter cell signal height which facilitates selection of single cells. B) Time course of native LDL and oxidized-desialylated LDL uptake by LAK cells. * Indicates statically significant differences between native LDL (control) and oxidized-desialylated LDL treated LAK cells (p<0.001). Statistical significance determined using multiple t-tests (one per group) and corrected for multiple comparisons using the Holm-Sidak method. n = 3 per time point. Error bars represent standard deviation. For some points, errors bars are shorter than the symbol, and error bars are not shown.
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