Arrested B lymphopoiesis and persistence of activated B cells in adult interleukin 7(-/)- mice

Abstract

Interleukin 7 is a crucial factor for the development of murine T and B lymphocytes. We now report that, in the absence of interleukin 7, B lymphocyte production takes place exclusively during fetal and perinatal life, ceasing after 7 wk of age. In peripheral organs, however, the pool of B lymphocytes is stable throughout adult life and consists only of cells that belong to the B1 and marginal zone (MZ) compartments. This is accompanied by a 50-fold increase in the frequency of immunoglobulin (Ig)M- and IgG-secreting cells, and the concentration of serum immunoglobulins is increased three- to fivefold. Both the MZ phenotype and the increase in serum IgM are T cell independent. These findings reveal a previously undescribed pathway of B lymphopoiesis that is active in early life and is interleukin 7 independent. This pathway generates B1 cells and a normal sized MZ B lymphocyte compartment.

Figure 1

Adult IL-7^−/− mice do not accumulate B cells in the periphery. Spleen cell suspension from C57BL/6 and IL-7^−/− mice were prepared at the indicated ages and the number of live B220⁺IgM⁺ B cells was determined. The figure shows the logarithmic mean and standard deviations of three to five animals per time point.

Figure 2

Bone marrow B lymphopoiesis is arrested in adult IL-7^−/− mice. (a) Expression of B220, IgM, and CD19 by wild-type (left) and IL-7^−/− (right) bone marrow cells. Dot plots show B220/IgM stainings and the B220⁺IgM⁻ gate used for histogram analysis. Histograms show the percentage of CD19⁺ cells in the B220⁺IgM⁻ population. Numbers represent percentage of live lymphoid cells in the bone marrow. The average number of cells recovered from the femurs of wild-type and IL-7^−/− were the following: wild-type 6.10⁶ (± 2.3), 25 × 10⁶ (± 7.0), and 30.8 × 10⁶ (± 4.5); IL-7^−/− 4.8 × 10⁶ (± 0.5), 12.6 × 10⁶ (± 4.5), and 19.4 × 10⁶ (± 6.3), at 1, 4, and 7 wk of age, respectively. Results are representative of at least three mice for each age. (b) Expression of CD24 (HSA) and BP-1 among B220⁺IgM⁻ (left histogram) and B220⁺ (right histogram) cells in the bone marrow of wild-type (open) and IL-7^−/− (filled) mice.

Figure 2

Bone marrow B lymphopoiesis is arrested in adult IL-7^−/− mice. (a) Expression of B220, IgM, and CD19 by wild-type (left) and IL-7^−/− (right) bone marrow cells. Dot plots show B220/IgM stainings and the B220⁺IgM⁻ gate used for histogram analysis. Histograms show the percentage of CD19⁺ cells in the B220⁺IgM⁻ population. Numbers represent percentage of live lymphoid cells in the bone marrow. The average number of cells recovered from the femurs of wild-type and IL-7^−/− were the following: wild-type 6.10⁶ (± 2.3), 25 × 10⁶ (± 7.0), and 30.8 × 10⁶ (± 4.5); IL-7^−/− 4.8 × 10⁶ (± 0.5), 12.6 × 10⁶ (± 4.5), and 19.4 × 10⁶ (± 6.3), at 1, 4, and 7 wk of age, respectively. Results are representative of at least three mice for each age. (b) Expression of CD24 (HSA) and BP-1 among B220⁺IgM⁻ (left histogram) and B220⁺ (right histogram) cells in the bone marrow of wild-type (open) and IL-7^−/− (filled) mice.

Figure 3

B1 lymphocytes are present in normal numbers. (a) Percentage of Mac-1⁺ and Mac-1⁻ B cells (IgM⁺) in the peritoneal cavity at 8 wk of age. Plots were gated to exclude dead cells, peritoneal macrophages, and granulocytes. (b) CD5 expression on gated peritoneal B1 cells (filled line) and on IgM⁻ cells (open line) from C57BL/6 (left panel) or IL-7^−/− (right panel) mice. (c) CD23 and CD43 expression by gated IgM⁺ cells in the spleen of C57BL/6 (left panel) or IL-7^−/− (right panel) mice. The numbers in the panels represent the percentage of CD23^lowCD43⁺ cells.

Figure 4

IL-7^−/− mice B lymphocytes are predominantly activated cells. Expression of activation markers by splenic B cells of IL-7^−/− and wild-type mice at 10 wk of age. Dot plots on the left show the B220⁺IgM⁺ gate used for histogram analysis in wild-type (top) and IL-7^−/− (bottom) cells. Histograms on the right show from top to bottom the size of B220⁺IgM⁺ cells of wild-type (open) and IL-7^−/− (filled) mice, measured by FSC, and the expression of CD21, CD23, and IgD. Splenocytes were gated for live lymphoid cells, and the results are representative of more than 20 adult animals analyzed.

Figure 5

B lymphocytes in IL-7^−/− mice are predominantly activated in the absence of thymus-derived cells. Expression of B cell activation markers in splenic B lymphocytes from IL-7^−/−, nu/nu and C57BL/6J nu/nu mice at 7 wk of age. The histograms show, from top to bottom, the size of B220⁺IgM⁺ cells of C57BL/6J nu/nu (open), and IL-7^−/−, nu/nu (filled) mice, measured by FSC, and the expression of CD21, CD23, and IgD. Splenocytes were gated for live B cells as in Fig. 4. The result is representative of two adult animals analyzed.

Figure 6

Antibody production and serum Ig levels. (a) Frequency of IgM and IgG plasma cells in the spleen, and of IgM plasma cells in the bone marrow, as determined by ELISA-spot assay in young (6–9 wk) and old (16 wk) mice. Results are displayed as number of antibody-secreting cells per 10⁵ B220⁺IgM⁺-positive cells for wild-type (open symbols) and IL-7^−/− (closed symbols) mice. (b) IgM, IgG, and IgA serum antibody titers from 6-wk-old IL-7^−/− (black bars) and wild-type (white bars) mice. Results show the mean and standard deviation (n = 6).