The spleen and bone marrow of the model mice and healthy recipient control mice
were harvested at the 6th or 16th week after BMT. Single cell suspensions from
spleens and bone marrows were labeled with anti-CD3 PE, -CD4 FITC, -CD8 AF700
(BD Pharmingen, San Diego, CA, USA) for T cells, anti-CD19 FITC (BD) for B
cells, anti-CXCR3 BV421 and anti-CD80 FITC for immune molecular examination,
anti-CD4 FITC (BD) and anti-Foxp3 PE (eBioscience, San Diego, CA, USA) for
regulatory T cells (Treg), anti-IFN-γ PE and -IL-17A PE-cy7 (BD) for Th1/Th17
subsets, respectively. Before detecting the secretion of IFN-γ and IL-17A in T
cells, cell suspensions were activated with anti-mouse CD3/CD28 microbeads for 3
hours. The spleen mononuclear cells of mice transplanted with A20 lymphoma cells
were stained with anti-B220 PE and -H-2Kb BV421 (eBioscience) to
examine the ratio of tumor cells. Activated human PBMCs with anti- human
CD3/CD28 beads were stained with anti-human CD3 FITC and -CD69 PE for T cell
activation analysis or with anti-human CD4 PE-cy7, -T-bet PE, -Foxp3 AF647,
-GATA3 APC and -ROR?t PE (eBioscience) for T cell differentiation analysis after
incubation with SHR0302 (1 μM) for 24 h. Fixable viability dye or DAPI (BD) were
used to distinguish live cells from dead cells.
were harvested at the 6th or 16th week after BMT. Single cell suspensions from
spleens and bone marrows were labeled with anti-CD3 PE, -CD4 FITC, -CD8 AF700
(BD Pharmingen, San Diego, CA, USA) for T cells, anti-CD19 FITC (BD) for B
cells, anti-CXCR3 BV421 and anti-CD80 FITC for immune molecular examination,
anti-CD4 FITC (BD) and anti-Foxp3 PE (eBioscience, San Diego, CA, USA) for
regulatory T cells (Treg), anti-IFN-γ PE and -IL-17A PE-cy7 (BD) for Th1/Th17
subsets, respectively. Before detecting the secretion of IFN-γ and IL-17A in T
cells, cell suspensions were activated with anti-mouse CD3/CD28 microbeads for 3
hours. The spleen mononuclear cells of mice transplanted with A20 lymphoma cells
were stained with anti-B220 PE and -H-2Kb BV421 (eBioscience) to
examine the ratio of tumor cells. Activated human PBMCs with anti- human
CD3/CD28 beads were stained with anti-human CD3 FITC and -CD69 PE for T cell
activation analysis or with anti-human CD4 PE-cy7, -T-bet PE, -Foxp3 AF647,
-GATA3 APC and -ROR?t PE (eBioscience) for T cell differentiation analysis after
incubation with SHR0302 (1 μM) for 24 h. Fixable viability dye or DAPI (BD) were
used to distinguish live cells from dead cells.