American Society of Hematology

Figure 3.

$Figure 3. ELISpot analysis of anti-Sm ASCs in 2-12H, 2-12H/Faslpr, and 2-12HMRL/Faslpr mice. (A) Spleen cells were stained for expression of CD19, CD138, and Sm. The CD138 expression of CD19+, Sm+ cells is shown. Boxes indicate the CD138int and CD138hi populations. The percent of each population is provided. Data are representative of 3 or more mice of each strain. The percentages of CD138int B cells are as follows: 17% ± 2.4% for 2-12H, 14% ± 3.1% for 2-12H/Faslpr, 7.0% ± 1.1% for 2-12H MRL, and 7.7% ± 0.50% for 2-12H MRL/Faslpr. The percentages of CD138hi B cells are as follows: 0.13% ± 0.07% for 2-12H, 0.09% ± 0.01% for 2-12H/Faslpr, 1.4% ± 0.46% for 2-12H MRL, and 1.9% ± 0.66% for 2-12H MRL/Faslpr. (B) Size, IgM expression, and CXCR4 expression are shown for anti-Sm CD138int and CD138hi B cells from mice of the indicated strains. The gates are illustrated in panel A. Data are representative of 3 or more mice. (C) Spleen cells were stained for CD19, CD138, CD21, and CD23. The gating scheme used to sort the FO, MZ, and CD138+ cells is indicated using 2-12H spleen cells. The top histogram shows the identification of CD19+, CD138+ cells sorted for ELISpot analysis. The CD19+ CD138– cells were further fractionated by CD21 and CD23 expression to identify FO and MZ B cells as shown. (D) The number of small and large anti-Sm ELISpots was determined for each of the indicated cell populations. The definition of large and small ELISpots is given in “Materials and methods.” The average size of small ELISpots characteristic of MZ and FO B cells is 2 × 10–3 ± 4 × 10–4 mm2, and the average size of the large ELISpots characteristic of CD138+ pre-PCs is 1.7 × 10–2 ± 4 × 10–4 mm2. Examples of the small FO/MZ-like spots and large CD138+ pre-PC ELISpots are shown. The number of FO anti-Sm ELISpots per 105 cells in 2-12H and 2-12H/Faslpr mice is 23.3 ± 2.36 and 3.51 ± 2.48, respectively (P < .001). The number of MZ anti-Sm ELISpots per 105 cells in 2-12H and 2-12H/Faslpr mice is 183 ± 19.2 and 22.5 ± 10.9, respectively (P = .002). The number of CD138+ pre-PC anti-Sm ELISpots per 105 cells from 2-12H, 2-12H/Faslpr, and 2-12H MRL/Faslpr mice is 26.0 ± 5.6, 1540 ± 135, and 7600 ± 265, respectively (P < .01 for all comparisons). ND indicates none detected.$

ELISpot analysis of anti-Sm ASCs in 2-12H, 2-12H/Fas^lpr, and 2-12HMRL/Fas^lpr mice. (A) Spleen cells were stained for expression of CD19, CD138, and Sm. The CD138 expression of CD19⁺, Sm⁺ cells is shown. Boxes indicate the CD138^int and CD138^hi populations. The percent of each population is provided. Data are representative of 3 or more mice of each strain. The percentages of CD138^int B cells are as follows: 17% ± 2.4% for 2-12H, 14% ± 3.1% for 2-12H/Fas^lpr, 7.0% ± 1.1% for 2-12H MRL, and 7.7% ± 0.50% for 2-12H MRL/Fas^lpr. The percentages of CD138^hi B cells are as follows: 0.13% ± 0.07% for 2-12H, 0.09% ± 0.01% for 2-12H/Fas^lpr, 1.4% ± 0.46% for 2-12H MRL, and 1.9% ± 0.66% for 2-12H MRL/Fas^lpr. (B) Size, IgM expression, and CXCR4 expression are shown for anti-Sm CD138^int and CD138^hi B cells from mice of the indicated strains. The gates are illustrated in panel A. Data are representative of 3 or more mice. (C) Spleen cells were stained for CD19, CD138, CD21, and CD23. The gating scheme used to sort the FO, MZ, and CD138⁺ cells is indicated using 2-12H spleen cells. The top histogram shows the identification of CD19⁺, CD138⁺ cells sorted for ELISpot analysis. The CD19⁺ CD138^– cells were further fractionated by CD21 and CD23 expression to identify FO and MZ B cells as shown. (D) The number of small and large anti-Sm ELISpots was determined for each of the indicated cell populations. The definition of large and small ELISpots is given in “Materials and methods.” The average size of small ELISpots characteristic of MZ and FO B cells is 2 × 10^–3 ± 4 × 10^–4 mm², and the average size of the large ELISpots characteristic of CD138⁺ pre-PCs is 1.7 × 10^–2 ± 4 × 10^–4 mm². Examples of the small FO/MZ-like spots and large CD138⁺ pre-PC ELISpots are shown. The number of FO anti-Sm ELISpots per 10⁵ cells in 2-12H and 2-12H/Fas^lpr mice is 23.3 ± 2.36 and 3.51 ± 2.48, respectively (P < .001). The number of MZ anti-Sm ELISpots per 10⁵ cells in 2-12H and 2-12H/Fas^lpr mice is 183 ± 19.2 and 22.5 ± 10.9, respectively (P = .002). The number of CD138⁺ pre-PC anti-Sm ELISpots per 10⁵ cells from 2-12H, 2-12H/Fas^lpr, and 2-12H MRL/Fas^lpr mice is 26.0 ± 5.6, 1540 ± 135, and 7600 ± 265, respectively (P < .01 for all comparisons). ND indicates none detected.

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