Fig 2: IL-27 help B cells for HBsAg-specific and total antibody secretion. (A) FACS-purified TFH cells were first primed with HBsAg for 3 h, washed and then cultured with autologous memory and naïve B cells in the presence and absence IL-21 and IL-27 neutralizing antibody for 5 days. Supernatant was harvested and IgG, IgM and IgA were quantified by ELISA (CHB: n=5, HC-vacc: n=5). (B-E) Representative ELISpot image indicate HBsAg-specific as well as total IgG, IgM and IgA secreting cells in HC-vacc and CHB patients after 5 days culture in the presence of rIL-21, rIL-27, R848+IL-2 alone and in different combinations. No stimulation was given in controls. Bar graphs shows cumulative data of six subjects in each group and indicate mean of the no. of antigen specific as well as total antibody producing cell spot. P values were determined by Kruskal-Wallis test (ANOVA) with Dunn’s post hoc test for multiple comparisons or paired t test. Bars represent mean with standard deviation. Data comparisons were made between controls (without any stimulation) of HC-vacc and CHB with different stimulations and p values are flagged with *. Comparisons between HC-vacc and CHB were also analyzed and p values are flagged with ♦. *,♦ indicates p<0.05, **,♦♦ p<0.01 and ***,♦♦♦ p<0.001. (F) Levels of plasma antibodies were determined by ELISA and significance was calculated by non-parametric, 2-tailed Mann-Whitney U test.

Fig 3: 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 4: 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.

Fig 5: 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.