Differential expression of CD22 (Lyb8) on murine B cells

Abstract

Previous studies have established the distribution, biochemistry and functional attributes of human CD22, a B cell-restricted glycoprotein. Recently, molecular cloning of the murine CD22 equivalent revealed this molecule to be the same as the previously described Lyb8 alloantigen. Using the anti-Lyb8 mAb Cy34.1.2, the present report documents the expression patterns of CD22 within the murine B cell compartment. The results demonstrate that in the bone marrow, murine CD22 is absent on the surface of pro-B cells, pre-B cells and newly emerging IgM+ B cells. CD22 is present at a low density on immature IgMhi B cells and fully expressed on mature recirculating B cells. In the periphery, murine CD22 is expressed at mature levels on all B cell subsets including follicular, marginal zone, B1 and switched B cells. Further studies showed CD22 to be retained on activated murine B cells for extended periods. Finally, in combination with CD23 and heat stable antigen, CD22 can be used to delineate the immature splenic B cells, and distinguish them from follicular and marginal zone cells. Together, the results demonstrate murine CD22 to be a useful pan marker for all mature B cell subsets.

Fig. 1

CD22 is expressed on mature B cells in the bone marrow. Bone marrow cells were spun through Fico-Lite-LM and stained with anti-CD22, and either anti-B220, BP-1, anti-lgD or anti-CD23. The contour plots are derived from the small light scatter gated cells One of three experiments

Fig. 2

CD22 is restricted to the lgM⁺ B cell fraction in the bone marrow. Bone marrow cells were spun through Fico-Lite-LM and stained with FITC–B220, Texas Red–anti-IgM and biotin–anti-CD22 plus PE–avidin. The histograms in the right side of the figure illustrate the expression of CD22 on the pro- and pre- (A), immature (B), and mature (C) B cell populations The gates for each of these populations are shown in the left hand panel The plots are derived from the small light scatter gated cells The isotype control for CD22 staining is shown in (C). Mean normalized fluorescence intensity values demonstrated that populations A and B expressed 8.3 ± 1 5% (SD) and 12.5 ± 2 8% CD22 levels respectively when compared with population C [(100%) average raw scale value of 5 1 ± 0 9] One of eight experiments

Fig. 3

Surface expression of CD22 first appears on lgM^hi immature B cells Bone marrow cells were spun through Fico-Lite-LM and stained with FITC–B220, Texas Red–anti-IgM and biotin–anti-CD22 plus PE–avidm The histogram overlays in the bottom panel demonstrate the staining pattern of CD22 on the IgM¹⁰ emerging B cells (A) and the lgM^hi immature B cells (B) The gates for each of these populations are shown in the upper panel The plots are derived from the small light scatter gated cells Mean normalized fluorescence intensity values demonstrated that population A expressed 66 2 ± 4 7% (SD) when compared with population B [(100%) average raw scale value of 0.8 ± 0 3]. One of eight experiments.

Fig. 4

CD22 and IgD are differentially expressed on splenic B cell populations Spleen cells were spun through Fico-Lite-LM and stained with Cyanine 5 18–anti-B220, PE–anti-CD23, FITC–anti-HSA and either biotin–anti-CD22 or biotin–anti-lgD plus Texas Red–avidn The upper two contour plots show the gating strategies for the total B220⁺ compartment (left panel) and CD23 and HSA defined B cell populations (right panel) The lower left panels compare the CD22 and IgD expression levels on follicular (CD23⁺) and marginal zone (CD23⁻, HSA⁺⁺) B cells The lower right panels compare the CD22 and IgD expression levels on marginal zone and immature (CD23⁻, HSA⁺⁺⁺) B cells The isotype control is shown for comparison in the CD22 and IgD histograms. Mean normalized fluorescence intensity values demonstrated that populations B and C expressed 98 6 ± 3 4% (SD) and 57 7 ± 0 8% CD22 levels respectively when compared with population A [(100%) average raw scale value of 3 5 ± 0 9]. One of six experiments.

Fig. 5

Comparison of CD23 and HSA defined splenic B cell populations in BALB/c and C3H mice. Spleen cells from two strains of mice were spun through Fico-Lite-LM and stained with Cyanine 518–anti-B220, PE–anti-CD23 and FITC-anti-HSA The CD23 and HSA contours are derived from the B220⁺ gated cells. One of five experiments

Fig. 6

Splenic CD23⁻, HSA⁺⁺⁺ B cells are eliminated in IL-7 deprived mice. BALB/c mice were treated with anti-IL-7 (M25) or control (Flag-M1) antibody for 4 weeks Subsequent to treatment, spleen cells were spun through Fico-Lite-LM and stained with Cyanine 5.18–anti-B220, PE–anti-CD23, FITC–anti-HSA and biotin–anti-CD22 plus Texas Red–avidin The upper panels illustrate the CD23- and HSA-defined B220⁺ B cell subsets from control treated and IL-7 deprived mice. The lower panel shows the CD22 expression patterns on the CD23⁻ gated subset from each mouse. One of six experiments.

Fig. 7

Peritoneal B1 cells display mature levels of CD22. Peritoneal lavage cells were spun through Fico-Lite-LM and stained with PE–anti-CD5, Texas Red–anti-IgM and biotin–anti-CD22 plus FITC–avidin The level of CD22 expression is shown for the B1a (CD5⁺, lgM⁺) and B1b (CD5⁻, lgM⁺) subsets Each histogram also contains the isotype control for comparison. One of three experiments

Fig. 8

CD22 expression is maintained on activated B cells T cell-depleted spleen cells were incubated for 4 days with 40 µg/ml of lipopolysaccharide with IL-4 (1000 U/ml) or recombinant CD40 ligand (trimeric form) plus IL-4 At the end of the culture period, cells were harvested, washed and stained with FITC–anti-IgM and biotin–anti-CD22 plus Cyanine 5 18–avidin The CD22 histograms are derived from the lgM⁺ gated cells The isotype control for each group is shown for comparison One of three experiments.

Fig. 9

CD22 is expressed on lgA⁺ Peyer’s patch B cells Cells from multiple Peyer's patches were pooled, spun through Fico-Lite-LM, and stained with FITC-anti-IgA, Texas Red-anti-IgM and biotin–anti–CD22 plus PE-avidin The histogram overlays represent the expression levels of CD22 on the resident lgA⁺ and lgM⁺ B cells. The isotype control is shown for comparison