Fig 1: PA T cells upregulate CD69 in DC co-culture. (A) OT-II mice were i.v. injected with 0.1LD50 LM-OVA. dLNs (draining LNs) and spleen were harvested on stated days and CD69 expression on CD4 T cells as a percentage was determined by FACS. n = 5 mice per group, and total 55 mice in this experiment. Results are representative of three independent experiments (N = 3). N = 3 for independent repeats of the experiment. *P < 0.05, **P < 0.01, ***P < 0.001 (Unpaired Student’s t test). n (replicates of biological samples) and N (number of independent repeats of the experiments) designations, as well as statistical symbols are used henceforth. (B) Left: Representative staining of previously activated CD4 T cells (PA T) after resting 48 h, CD69 expression was compared with co-cultured with DC1940 cell-line or B6 BMDCs. Red line is positive control which stands for PA T co-cultured with DC1940 in the presence of 10 µg/mL OVA. Three replicates in each group (n = 3), results are representative of eight independent experiments (N = 8). Right: Pooled data from eight independent experiments are shown. Normalized CD69 mean fluorescence intensity (MFI) by the PA T group in multiple independently repeated experiments (N = 8) was analyzed for fold change of CD69 MFI. **P < 0.01, ****P < 0.0001 (Unpaired Student’s t test). (C) Similar to (B) except that naïve freshly magnetically isolated OT-II splenic CD4 cells were used in place of PA T. Three replicates in each group (n = 3), results are representative of three independent experiments (N = 3). (D) Similar to (B) except that B6 MEF and 3T3 cells were used in place of DCs. Three replicates in each group (n = 3), results are representative of four independent experiments (N = 4). (E) Magnetically isolated naïve CD4 T cells from B6 mice were activated in vitro with anti-CD3e and anti-CD28. Same experiment as in (B) was performed using B6 splenic CD11c+ cells and DC1940 as the stimulator. Three replicates in each group (n = 3), and results are representative of three independent experiments (N = 3). (F) Left: Similar to (D) except that MEF and 3T3 were in place of DCs. Three replicates in each group (n = 3), results are representative of three independent experiments (N = 3). Right: pooled data from three independent experiments are shown. Normalized CD69 mean fluorescence intensity (MFI) by the B6 activated CD4 group in multiple independently repeated experiments (N = 3) was analyzed for fold change of CD69 MFI. ns means no significant difference (Unpaired Student’s t test). Pooled data for the panels (C and D) from multiple experiments are shown in Fig. S3 as marked
Fig 2: The effect of sunitinib treatment on intratumoral MDSC, Tregs, CD8+ T cells and activation status of the CD8 T cell population. C57Bl6 mice were subcutaneously inoculated with TC-1 tumor cells (n = 3–6 mice/group). On day 15 after tumor inoculation, sunitinib treatment was started, daily, i.p., for a period of 9 consecutive days. Three increasing dosages of sunitinib were used: 20, 40 and 60 mg/kg body weight. Mice were sacrificed and tumors and spleens were harvested. The levels of (A) MDSCs (CD11b+Gr1+), (B) Tregs (CD4+FoxP3+), (C) CD8+ T cells (CD8+; black bars) and the activation status of CD8+ T cells (CD69+; white bars) were analyzed by immunostaining and multicolor fluorescence cytometry. Experiments were repeated twice. Shown here are averages and SD for each experimental group (*p < 0.05; **p < 0.01; ***p < 0.001).
Fig 3: Multiplex ISH/ICC development.Multiplex ISH/ICC assay for detection of 4 targets simultaneously on naïve and stimulated lymphocytes for 24 hours. (A) Detection of CD69 and Notch1 proteins and mRNAs. Channels are split below merged image to help visualization. (B) Multiplex negative control. Scrambled-sequence probes and isotype antibodies were used. (C) ISH-only control in which CD69 and Notch1 targeted probe were used in combination with species-specific antibody isotype controls for both target ICC. (D) ICC-only control in which scrambled-sequence probes were used in combination with specific anti-CD69 and anti-Notch1 antibodies for ICC. White color in the images corresponds to actual overlapping signals from different probes and/or antibodies: in fact to avoid bleed-through of fluorescence emission, we combined sequential scanning to ensure no signal bleed-through between channels. Scale bar is equal to 10 µm.
Fig 4: PA T encounter with DCs results in limited activation involving upregulation ofIl2andBcl2. (A) PA T cells were co-cultured with DC1940 for 24 h, then CD4+ populations were gated into CD69+ and CD69- cells by FACS. The expression of CD103, CXCR5, CD62L, CCR9, CCR7, CD127 and CD83 between CD69+ and CD69- cells were compared. Three replicates in each group (n = 3), and results are representative of three independent experiments (N = 3). (B) CD69+ and CD69- cells cultured in complete RPMI for 24 h without additional treatment, then the cytokines released to the supernatant were measured by ELISA. Pooled data from three independent experiments are shown. (C) Gene expression of Il7r, Il2 and Bcl2 was determined by QPCR in PA T cells or in CD69 positive or negative populations purified by FACS sorting. Three replicates in each group (n = 3), and results are representative of three independent experiments (N = 3). GAPDH was used for normalization
Fig 5: Therapeutic immunization and sunitinib enhance intratumoral and intrasplenic levels of total CD8+ T cells, but not MDSC and Tregs. Mice were s.c. inoculated with TC-1 tumor cells (n = 3 mice/group). On day 14 after tumor inoculation mice were vaccinated once, i.m., with 5 × 106 SFVeE6,7 particles. One day later, sunitinib treatment of 20 and 40 mg/kg body weight was started, i.p., for a period of 9 consecutive days. The levels of (A) MDSCs (Gr1+CD11b+), (B) Tregs (CD4+FoxP3+), (C) activated total CD8+ T cells (CD8+CD69+), and (D) activated E7-antigen specific CD8+ T cells (CD8+E7+CD69+) in tumors and spleens were analyzed by immunostaining and fluorescence cytometry on day 24. Experiments were repeated twice. Shown here are averages and SD for each experimental group (*p < 0.05).
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