PMC

Cells were gated first on singlets, and then on CD20⁺ B lymphocytes. All CD20⁺ B cells were further analyzed based on their CD21 and CD27 surface molecule expression. Statistically significant differences between DP CD21⁺CD27⁺ and SP CD21⁺ B cell subsets are shown. Indicates significant differences between SP CD27⁺ B cells and other B cell subsets for the specified tissue.

Cells were gated first on singlets, and then on CD20⁺ B lymphocytes. All CD20⁺ B cells were further analyzed based on their CD21 and CD27 surface molecule expression. Statistically significant differences between DP CD21⁺CD27⁺ and SP CD21⁺ B cell subsets are shown. Indicates significant differences between SP CD27⁺ B cells and other B cell subsets for the specified tissue.

Singlets were gated first to eliminate doublets and finally gating was performed on CD20⁺ B lymphocytes. All CD20⁺ B cells were further analyzed based on their CD21 and CD27 surface molecule expression. The percentage of BrdU⁺ cells is indicated in the top box of each panel. Note that DP CD21⁺CD27⁺ B cells had higher BrdU⁺ proliferating cells than other B cell subsets.

Singlets were gated first to eliminate doublets and finally gating was performed on CD20⁺ B lymphocytes. All CD20⁺ B cells were further analyzed based on their CD21 and CD27 surface molecule expression. BrdU was injected intraperitoneally and tissues were collected 24 hrs after inoculation. Increased proliferation of DP CD21⁺CD27⁺ B cell subsets compared to other B cells subsets were observed in all healthy normal rhesus macaques. *Indicates significant differences between BrdU expression of DP CD21⁺CD27⁺ B cells and other B cell subsets.

(A) A representative dot and contour plots showing distribution of CD27 and CD21 phenotypes on CD20⁺ B cells from a normal uninfected healthy rhesus macaque (BB01). Singlets were gated first to eliminate doublets and finally gating was performed on CD20⁺ B lymphocytes. All CD20⁺ B cells were further analyzed based on their CD21 and CD27 surface molecule expression. Each quadrant shows percentages of specified populations. (B) Mean frequencies (± standard deviation) of single positive (SP) CD27⁺, SP CD21⁺, double positive (DP) CD21⁺CD27⁺ and double negative CD21⁻CD27⁻ B cell subsets are shown as bars for different tissues from uninfected rhesus macaques. Note that in lymphoid tissues including lymph node, bronchoalveolar lavage, spleen, tonsil and jejunum LPL there were increased percentages of DP CD21⁺CD27⁺ B cells compared to peripheral blood and BM B lymphocytes. Statistical significant differences between each group of cells are shown.

(A). All 4 subpopulations (CD21⁻CD27⁺, CD21⁺CD27⁺, CD21⁺CD27⁻, and CD21⁻CD27⁻) of CD3⁻CD20⁺ B cells were sorted from peripheral blood mononuclear cells (PBMC) and stimulated in the presence of pokeweed mitogen, SAC, and CpG-2006 for 6 days. Unstimulated (filled histogram) and stimulated (open histogram) sorted B cell cultures are shown as histogram for expression of anti-CD20, anti-CD138, and anti-CD86. There was significant upregulation of CD86, downregulation of CD20 in all B cell subsets, and upregulation of CD138 expression in DP CD21⁺CD27⁺ B cells. This experiment was repeated independently 3 times and data shown is the mean value of the representative plot. (B) Supernatants collected from B cell subsets (CD21⁻CD27⁺, CD21⁺CD27⁺, CD21⁺CD27⁻, and CD21⁻CD27⁻), CD3⁺ T cells and total PBMCs following 6 days stimulation. Sandwich ELISA was performed to determine the level of secretory IgG in culture supernatant. Mean frequency (± standard error) of IgG production is shown for all B cell subsets, T cells and total PBMCs. Increased levels of IgG production were detected in DP CD21⁺CD27⁺ B cells following stimulation compared to other B cell subsets. Indicates significant differences between IgG production of DP CD21⁺CD27⁺ B cells and other B cell subsets.