Identification of functional human splenic memory B cells by expression of CD148 and CD27

Abstract

Memory B cells isolated from human tonsils are characterized by an activated cell surface phenotype, localization to mucosal epithelium, expression of somatically mutated immunoglobulin (Ig) variable (V) region genes, and a preferential differentiation into plasma cells in vitro. In spleens of both humans and rodents, a subset of memory B cells is believed to reside in the marginal zone of the white pulp. Similar to tonsil-derived memory B cells, splenic marginal zone B cells can be distinguished from naive follicular B cells by a distinct cell surface phenotype and by the presence of somatic mutations in their Ig V region genes. Although differences exist between human naive and memory B cells, no cell surface molecules have been identified that positively identify all memory B cells. In this study, we have examined the expression of the receptor-type protein tyrosine phosphatase CD148 on human B cells. CD148(+) B cells present in human spleen exhibited characteristics typical of memory B cells. These included an activated phenotype, localization to the marginal zone, the expression of somatically mutated Ig V region genes, and the preferential differentiation into plasma cells. In contrast, CD148(-) B cells appeared to be naive B cells due to localization to the mantle zone, the expression of surface antigens typical of unstimulated B cells, and the expression of unmutated Ig V region genes. Interestingly, CD148(+) B cells also coexpressed CD27, whereas CD148(-) B cells were CD27(-). These results identify CD148 and CD27 as markers which positively identify memory B cells present in human spleen. Thus, assessing expression of these molecules may be a convenient way to monitor the development of memory B cell responses in immunocompromised individuals or in vaccine trials.

Figure 1

CD148⁺ and CD148⁻ human B cells represent phenotypically distinct subpopulations of B cells. (A) MNC from human spleen were incubated with PE-labeled anti-CD20 mAb and either FITC-labeled anti-CD148 mAb (bold histogram) or FITC-labeled isotype control IgG₁ mAb (thin histogram). The FITC fluorescence of CD20⁺ B cells was assessed by flow cytometric analysis. The values represent the mean ± SD of CD148⁺ B cells from nine different donor spleens. (B) MNC from human spleen were incubated with FITC- or PE-labeled anti-CD148, PerCp-labeled anti-CD20, and one of the following PE- or FITC-labeled mAbs: anti-IgM, anti-IgD, anti-IgA, anti-CD21, anti-CD23, anti-CD27, anti-CD39, anti-CD80, anti-CD86, or anti-CD95. CD148⁺CD20⁺ (bold histogram) and CD148⁻CD20⁺ (thin histogram) cells were gated, and the fluorescence and the light scattering characteristics of each population were assessed by flow cytometric analysis. The fluorescence of CD148⁺CD20⁺ and CD148⁻CD20⁺ cells incubated with an isotype control mAb was within the first log of the histogram plot. These results are representative of six different spleen samples.

Figure 1

CD148⁺ and CD148⁻ human B cells represent phenotypically distinct subpopulations of B cells. (A) MNC from human spleen were incubated with PE-labeled anti-CD20 mAb and either FITC-labeled anti-CD148 mAb (bold histogram) or FITC-labeled isotype control IgG₁ mAb (thin histogram). The FITC fluorescence of CD20⁺ B cells was assessed by flow cytometric analysis. The values represent the mean ± SD of CD148⁺ B cells from nine different donor spleens. (B) MNC from human spleen were incubated with FITC- or PE-labeled anti-CD148, PerCp-labeled anti-CD20, and one of the following PE- or FITC-labeled mAbs: anti-IgM, anti-IgD, anti-IgA, anti-CD21, anti-CD23, anti-CD27, anti-CD39, anti-CD80, anti-CD86, or anti-CD95. CD148⁺CD20⁺ (bold histogram) and CD148⁻CD20⁺ (thin histogram) cells were gated, and the fluorescence and the light scattering characteristics of each population were assessed by flow cytometric analysis. The fluorescence of CD148⁺CD20⁺ and CD148⁻CD20⁺ cells incubated with an isotype control mAb was within the first log of the histogram plot. These results are representative of six different spleen samples.

Figure 2

Morphological differences between sort-purified human CD148⁺ and CD148⁻ splenic B cells. Human splenic B cells were incubated with FITC-labeled anti-CD148 and PE-labeled anti-CD20 and sorted into CD20⁺ B cell populations that were either CD148⁻ or CD148⁺. After sort-purification, the (A) CD148⁻ B cells and (B) CD148⁺ B cells were cytocentrifuged onto glass slides and analyzed by Giemsa staining.

Figure 3

Localization of CD148⁺CD27⁺ and CD148⁻CD27⁻ B cells in follicles of human spleen. Serial tissue sections of human spleen were incubated with anti-IgD antiserum alone (blue; A and D), anti-IgD antiserum (blue) and anti-CD27 mAb (red; B and E), or anti-IgD antiserum (blue) and anti-IgM mAb (red; C). The anti-IgD polyclonal antibody was visualized after the addition of alkaline phosphatase–conjugated anti–goat Ig and phosphatase-specific substrate. The anti-CD27 or anti-IgM mAb was visualized by an anti–mouse Ig-specific Vectastain kit. Original magnification of A, B, and C: ×40; of D and E: ×100. The follicular mantle zone (FM), marginal zone (MZ), germinal center (G or GC), and T cell zones (T) are indicated.

Figure 4

Nucleotide sequence of Ig V_H5 genes isolated from CD148⁻ and CD148⁺ splenic B cells. Nucleotide sequences of V_H5 Ig genes cloned from sort-purified CD148⁻ and CD148⁺ human splenic B cells are shown. The germline V_H5 (V_H251) sequence was derived from Tomlinson et al. (reference 40). For clarity, only sequences homologous to V_H251 are shown. Each dash represents identity with the germ-line sequence; nucleotide differences are indicated. The FR and CDRs are indicated.

Figure 4

Nucleotide sequence of Ig V_H5 genes isolated from CD148⁻ and CD148⁺ splenic B cells. Nucleotide sequences of V_H5 Ig genes cloned from sort-purified CD148⁻ and CD148⁺ human splenic B cells are shown. The germline V_H5 (V_H251) sequence was derived from Tomlinson et al. (reference 40). For clarity, only sequences homologous to V_H251 are shown. Each dash represents identity with the germ-line sequence; nucleotide differences are indicated. The FR and CDRs are indicated.

Figure 4

Nucleotide sequence of Ig V_H5 genes isolated from CD148⁻ and CD148⁺ splenic B cells. Nucleotide sequences of V_H5 Ig genes cloned from sort-purified CD148⁻ and CD148⁺ human splenic B cells are shown. The germline V_H5 (V_H251) sequence was derived from Tomlinson et al. (reference 40). For clarity, only sequences homologous to V_H251 are shown. Each dash represents identity with the germ-line sequence; nucleotide differences are indicated. The FR and CDRs are indicated.

Figure 5

Nucleotide sequence of Ig V_H6 genes isolated from CD148⁻ and CD148⁺ splenic B cells. Nucleotide sequences of V_H6 Ig V genes cloned from sort-purified CD148⁻ and CD148⁺ human splenic B cells are shown. The germline V_H6 (V_HVI) sequence was derived from Tomlinson et al. (reference 40). Each dash represents identity with the germ-line sequence; nucleotide differences are indicated. The FR and CDRs are indicated.

Figure 5

Nucleotide sequence of Ig V_H6 genes isolated from CD148⁻ and CD148⁺ splenic B cells. Nucleotide sequences of V_H6 Ig V genes cloned from sort-purified CD148⁻ and CD148⁺ human splenic B cells are shown. The germline V_H6 (V_HVI) sequence was derived from Tomlinson et al. (reference 40). Each dash represents identity with the germ-line sequence; nucleotide differences are indicated. The FR and CDRs are indicated.

Figure 5

Nucleotide sequence of Ig V_H6 genes isolated from CD148⁻ and CD148⁺ splenic B cells. Nucleotide sequences of V_H6 Ig V genes cloned from sort-purified CD148⁻ and CD148⁺ human splenic B cells are shown. The germline V_H6 (V_HVI) sequence was derived from Tomlinson et al. (reference 40). Each dash represents identity with the germ-line sequence; nucleotide differences are indicated. The FR and CDRs are indicated.

Figure 6

Frequency of mutations in Ig V genes isolated from CD148⁻ and CD148⁺ splenic B cells. The frequency of somatic mutations in (a) V_H5 or (b) V_H6 genes isolated from CD148⁻ (white bars) and CD148⁺ B cells (black bars) was calculated as the number of nucleotide changes detected per base pairs sequenced. Each value represents the mean rate of mutation for either the entire V_H5 or V_H6 gene (V_H), or for the different regions (FR, CDR) of the Ig V gene for all of the V_H5 and V_H6 clones analyzed.

Figure 7

CD148⁺ B cells differentiate into plasma cells in vitro. Sort-purified (a) CD148⁺ and (b) CD148⁻ B cells were cultured for 3 d with anti-CD40 mAb (15 μg/ml), IL-2 (100 U/ml), and IL-10 (100 U/ml), and then recultured for an additional 3 d with IL-2 and IL-10. Plasma cell differentiation was then assessed by flow cytometric analysis after immunofluorescent staining with FITC–anti-CD38 mAb (x axis) and PE–anti-CD20 mAb (y axis). The values represent the percentage of cells in each quadrant of the dot plot. Similar results were obtained in a second experiment.

Figure 8

CD148⁻ and CD148⁺ B cells differ in their ability to secrete Igs. Sort-purified CD148⁺ (gray bars) and CD148⁻ B cells (white bars) were cultured with (a) anti-CD40 mAb (15 μg/ml) and IL-4 (400 U/ml), (b) anti-CD40 mAb and IL-2 (100 U/ ml) and IL-10 (100 U/ml), or (c and d) SAC (0.01%) plus IL-2 with or without IL-10 or anti-CD40 mAb. The amounts of IgG, IgE, IgG₄, IgA, and IgM secreted were determined by isotype-specific immunoassays after 12 d. Each value represents the mean ± SEM of four to six replicates. The results shown are representative of data obtained from between 5 and 11 independent experiments.

Figure 8

CD148⁻ and CD148⁺ B cells differ in their ability to secrete Igs. Sort-purified CD148⁺ (gray bars) and CD148⁻ B cells (white bars) were cultured with (a) anti-CD40 mAb (15 μg/ml) and IL-4 (400 U/ml), (b) anti-CD40 mAb and IL-2 (100 U/ ml) and IL-10 (100 U/ml), or (c and d) SAC (0.01%) plus IL-2 with or without IL-10 or anti-CD40 mAb. The amounts of IgG, IgE, IgG₄, IgA, and IgM secreted were determined by isotype-specific immunoassays after 12 d. Each value represents the mean ± SEM of four to six replicates. The results shown are representative of data obtained from between 5 and 11 independent experiments.