Fig 2: Activation and proliferation of human B cells are enhanced by HP06T07. Human PBMCs (5 × 105) cultured with or without HP06T07 (red), ODN 2395 (green), and negative control GC (black) at different concentrations (0.01, 0.03, 0.1, 0.3, 1, and 3 μM) for 24 h. Cells were collected, and the dead cells were excluded using an aqua dead cell stain kit. Cells were then stained with CD3, CD19, CD80, and CD86. Representative plots and histograms of CD80 (A) and CD86 (C) expression analyzed using flow cytometry (gated on CD3- CD19+ B cells). (B, D) Expression of CD80 and CD86 on CD3− CD19+ B cells. (E–H) Human PBMCs (5 × 105) incorporated with CFSE and cultured with or without HP06T07, ODN 2395, and the negative control GC at different concentrations (0.01, 0.03, 0.1, 0.3, 1, and 3 μM) for 3 days. CD19+ B cell (E, F) and CD3+ T cell (G, H) proliferation was measured by decreasing the CFSE content. All data are presented as means ± SEM of three technical replicates from two donors per group. Statistical significance of differences between HP06T07 and ODN 2395 groups were determined (*P < 0.05, **P < 0.01, and ****P < 0.0001).

Fig 3: U-IRI promotes late immune cell accumulation.Wild-type mice were subjected to 27 min of ischemia/reperfusion injury (IRI) with contralateral nephrectomy (IRI/CL-NX) or unilateral IRI (U-IRI) and sacrificed on day 1, 7, 14, and 30 after injury. The injured kidneys and normal control kidneys (defined as day 0) were analyzed. a Quantitative RT-PCR analysis for Adgre1 (F4/80), Itgax, Ly6g, Cd3e, Cd4, and Cd8a was performed on whole-kidney RNA. Data are presented as mean ± SD. n = 10 kidneys/time point/model. Two-way ANOVA is summarized in Supplementary Table 1. *p < 0.05 (p = 0.0449), ****p < 0.0001 at the indicated time points (by Bonferroni multiple comparison). b Contralateral (CL), U-IRI and IRI/CL-NX kidney sections on day 14 after IRI were immunostained with F4/80, CD11c, Ly6G, CD3ε, CD4, and CD8α. Images that are representative of those taken from 10 kidneys/model are shown at 40x magnification. Scale bars, 50 µm. c F4/80-, CD11c-, Ly6G-, CD3ε-, CD4-, and CD8α-positive areas as in b were quantified. Data were presented as mean ± SD. n = 10 kidneys/group. Two-way ANOVA is summarized in Supplementary Table 3. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 in the indicated subgroup analyses by Tukey multiple comparison.

Fig 4: Dual depletion of T cells and neutrophils attenuates kidney tubule atrophy.WT mice were treated as described in Methods with either PBS or a combination of antibodies (Ab)-against Thy1.2 and Ly6G beginning 5 days after unilateral ischemia/reperfusion injury (U-IRI) and sacrificed on day 30. a The IRI kidney sections were immunostained with CD3ε and Ly6G. Nine kidneys/group were sectioned and stained, and representative images are shown. Scale bars, 25 µm. b CD3ε-, CD8α-, and Ly6G-positive areas were quantified using Image J. Data are presented as mean ± SD. n = 9 kidney sections/group. ****p < 0.0001 by unpaired two-tailed t-test. c Quantitative RT-PCR analysis for Cd3e, Cd4, Cd8a, and Ly6g was performed on whole-kidney RNA on day 30 after U-IRI ± dual T-cell/neutrophil depletion. Data are presented as mean ± SD. n = 9 kidneys/group. ***p < 0.001, ****p < 0.0001 by unpaired two-tailed t-test. d Kidney-to-body weight ratios on day 30 following U-IRI ± dual T-cell/neutrophil depletion. Data are presented as mean ± SD. n = 9 kidneys/group. **p < 0.01 by unpaired two-tailed t-test. e Kidney sections from day 30 after U-IRI ± dual T-cell/neutrophil depletion were immunostained with lotus tetragonolobus lectin (LTL, dark gray). Nine kidneys were sectioned and stained and representative images are shown. Scale bars, 1 mm. f LTL-positive area was quantified from the entire section (left panel) and as a percentage of the section (right panel). Data are presented as mean ± SD. n = 9 kidneys/group. ***p < 0.001 by unpaired two-tailed t-test. g Quantitative RT-PCR analysis for Lrp2 and Slc34a1 was performed on whole-kidney RNA from U-IRI mice ± dual T-cell/neutrophil depletion. Data are presented as mean ± SD. n = 9 kidneys/group. *p < 0.05 by unpaired two-tailed t-test.

Fig 5: PMA and ionomycin activated immune cells isolated from BALB/c (splenocytes) can infiltrate breast cancer spheroids.a1 No significant difference in the number of CD3+ cells in splenocytes between inactivated and activated splenocytes. n = 6 a2 Activated splenocytes express higher levels of the CD69 activation maker in comparison to the inactivated splenocytes. Statistical significance was calculated using a one-way ANOVA, Kruskal–Wallis test, n = 9 biologically independent animals. Data represents mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. b Representative images showing phase contrast as well as confocal microscopic images of spheroids with b1 inactivated splenocytes, b2 activated for 3 hr, and b3 activated for 24 hr. In the confocal images, green represents breast cancer spheroids and red represents splenocytes. The scale bar in the phase-contrast microscopic images represents 250 µm.