Fig. 4. CD11b+Gr1+CD84hi cells exhibit potent capacity for T cell suppression and increased ROS production.
(A) Overview of FACS approach using two different tissues (spleen and primary tumor) from WT and PyMT were subjected to T cell activation, ROS formation and qPCR assays. (B-C) Splenic CD11b+Gr1+CD84hi cells from tumor-bearing mice suppress T cell proliferation. Histogram overly (B) and quantitative bar charts (C) showing CD4/CD8 T cell proliferation measured by FACS in control samples with T cells only (black), T cells activated by CD3/CD28 (blue), activated T cells plus CD11b+Gr1+ cells from control spleens (orange), activated T cells plus CD11b+Gr1+CD84−/lo cells (purple) and activated T cells plus CD11b+Gr1+CD84hi (red) from spleen of tumor-bearing mice. (C) Statistical analysis unpaired t-test (Mean ± SEM of n = 3) *P< 0.05. (D-E) T cell suppression analysis using CD11b+Gr1+CD84hi and CD84−/lo cells isolated from primary tumors. Histogram overly (D) and quantitative bar charts (E) showing CD4/CD8 T cell proliferation measured by FACS in control samples T cells (black), T cells activated by CD3/CD28 (blue), activated T cells plus CD11b+Gr1+CD84−/lo cells (purple) and activated T cells plus CD11b+Gr1+CD84hi (red) from tumor of tumor-bearing mice. (E) Statistical analysis unpaired t-test (Mean ± SEM of n = 3) *P< 0.05. (F-G) CD11b+Gr1+CD84hi cells from tumor-bearing mice show increased ROS formation compared to CD11b+Gr1+CD84−/lo; PMA-treated cells were used as positive control. ROS was measured by FACS using H2DCFDA. (E) Statistical analysis of ROS assay unpaired t-test (Mean ± SEM of n = 3) *P< 0.05.