Early preplasma cells define a tolerance checkpoint for autoreactive B cells

Abstract

Ab-secreting plasma cells (PCs) are the effectors of humoral immunity. In this study, we describe regulation of autoreactive B cells specific for the ribonucleoprotein Smith (Sm) at an early pre-PC stage. These cells are defined by the expression of the PC marker CD138 and normal levels of CD19 and B220. They are present at a high frequency in normal mouse spleen and bone marrow, are Ag dependent, and are located predominantly along the T cell-B cell border and near bridging channels. Anti-Sm pre-PCs also occur at a high frequency in nonautoimmune mice and show additional phenotypic characteristics of PC differentiation. However, while some of these pre-PCs are Ab-secreting cells, those specific for Sm are not, indicating regulation. Consistent with this, anti-Sm pre-PCs have a higher turnover rate and higher frequency of cell death than those that do not bind Sm. Regulation of anti-Sm pre-PCs occurs upstream of the transcriptional repressor, B lymphocyte-induced maturation protein-1, expression. Regulation at this stage is overcome in autoimmune MRL/lpr mice and is accompanied by an altered B lymphocyte stimulator receptor profile. These data reveal a new B cell tolerance checkpoint that is overcome in autoimmunity.

FIGURE 1

CD138^int cells are present at a high frequency in the spleens and BM of non-Tg and 2-12H mice. A, CD138 expression by splenic B cells. Top row, CD19 and CD138 staining of total splenic lymphocytes in non-Tg and 2-12H littermates as determined by forward scatter (FSC) and side scatter (SSC). The right histogram is a representative isotype (IgG2a,κ) control stain (1.19 ± 0.25% (n = 4) falling within the CD19⁺, CD138⁺ quadrant). Middle row, Shown are histograms for IgM and Sm staining of CD19⁺ B cells from non-Tg and 2-12H mice. The indicated gate is that for the Sm⁺ population analyzed in the bottom row. Bottom row, Shown is the CD138 expression of gated Sm⁺ and Sm⁻ B cells from 2-12H mice and total B cells from non-Tg mice. The percentage of CD19⁺ B cells that are CD138^int is provided. B, Frequency of CD19⁺CD138^int B cells among CD19⁺ B cells. Each symbol represents a single mouse and a horizontal line marks the mean. Absolute numbers of CD138^int B cells are 5.48 × 10⁶± 1.16 × 10⁶ (n = 8) for 2-12H anti-Sm, 8.19 × 10⁶± 1.77 × 10⁶ (n = 8) for 2-12H non-Sm, and 2.37 × 10⁷± 6.47 × 10⁶ (n = 6) for non-Tg mice, 4.91 × 10⁶± (Figure legend continues) 4.66 × 10⁵ (n = 4) for CD19^+/− mice, 2.7 × 10⁵± 1.63 × 10⁵ (n = 8) in MD4 mice, and 1.20 × 10⁶± 4.76 × 10⁵ (n = 11) in MD4 × ML5 mice. C, Activation marker expression by CD19⁺ CD138⁻ B cells (shaded) and CD19⁺ CD138^int (black line) from non-Tg and 2-12H mice. Representative histograms are shown. Values of p are given for the differences between the CD138^int and CD138⁻ cells. D, Sm binding by CD138^int B cells. Histograms are gated on CD138^int B cells from the indicated mice as illustrated in A (upper right quadrant of top row). The percentage of anti-Sm CD138^int B cells is provided. The average percentage for non-Tg and 2-12H mice is given in Table I. E, CD138 expression by BM B cells. Top row, Histograms are gated on CD19⁺ cells to identify the recirculating IgD⁺CD23⁺ B cells. Middle row, Sm binding by gated IgD⁺ CD23⁺ B cells. Bottom row, CD138 expression by the indicated B cell subsets. The percentage of gated B cells that are CD138⁺ is given. F, Frequency of CD138^int B cells among the recirculating IgD⁺CD23⁺ B cells of the BM. Each symbol represents a single mouse and a horizontal line marks the mean. G, CD19⁺ CD138^int B cells are absent in anti-HEL MD4 mice, but present in anti-HEL/HEL MD4 × ML5 mice. Frequency of CD138^int B cells is given as percent of CD19⁺ B cells. The FSC and SSC for CD138⁻ (shaded) and CD138^int (solid line) B cells are shown along with the p values for the differences in mean fluorescence intensities. The gating for CD138^int cells is indicated in the top histograms. The CD138⁻ B cells were all CD19⁺ cells that fell outside the CD138^int gate. Lower graph shows real-time PCR results for CD138 expression using sorted CD138⁻ and CD138^int B cells from MD4 and MD4 × ML5 mice.

FIGURE 2

Immunofluorescence analysis of nonautoimmune and autoimmune mice. Spleens were sectioned and stained with anti-CD138 and anti-IgM, anti-CD3, anti-CD11b, and anti-CD11c to identify B cells, T cells, macrophages, and DCs, respectively. A, Non-Tg: A representative follicle at ×10 magnification is shown in panel 1. B cells are blue, CD138 is red, and macrophages/DCs are green. The locations and corresponding photos for the higher magnifications (×40) are indicated. Panels 2 and 4 are stained identically to A1, but in panels 3 and 5, T cells are blue and B cells are not shown. White carets identify examples of CD138⁺ B cells. Panels 2 and 3 show a clustering of CD138^int B cells near a bridging channel (BC). Note that the CD138⁺ B cells are more frequent near the PALS and bridging channel. Panels 4 and 5 show an area of the follicle away from the bridging channel in which CD138⁺ B cells are infrequent. B, 2-12H: Panels 1–3 are as described for the corresponding photos in A. Panels 4–6 are higher magnification (×64) of the indicated areas indicated in panel 2. Panels 4 and 6 show B cells (blue), but not T cells; panels 5 and 7 show T cells (blue), but not B cells. Panels 2–6 show an area near a bridging channel. Note the greater concentration of CD138⁺ B cells near the PALS and bridging channel. White carets identify representative CD138⁺ B cells. The yellow carrot shows an example of a T cell near a cluster of CD138⁺ B cells. C, MRL/lpr: Panels 1 and 2 show a representative follicle at ×10 magnification. Panel 1 shows B cells (blue), macrophages/DCs (green), and T cells (red). In this view, T cells have infiltrated into the marginal sinus. Panel 2 shows the same follicle with B cells (blue), macrophages/DCs (green), and CD138 (red). Note that many T cells are CD138⁺. Panels 3 and 4 show the MZ area (×40 magnification), and panels 5 and 6 show a follicle (×40 magnification). Panels 3 and 5 show B cells (blue) but not T cells; panels 4 and 6 show T cells (blue), but not B cells. D, 2-12H MRL/lpr. Same as C. Panels 3 and 4 show the follicle; panels 5 and 6 show the MZ.

FIGURE 3

A subset of CD138^int B cells are IC Ig-M^high and secrete Ab, but anti-Sm CD138^int B cells do not secrete Ab. A, Surface and IC IgM on non-Tg CD138⁻ and CD138^int B cells. Nonpermeabilized and isotype controls do not show IC IgM staining (top row). CD138⁻ and CD138^int B cells were gated as indicated (bottom row; left histogram) and the percentage of IC IgM⁺ B cells that are IC IgM^high determined (second row; middle and right histograms) and displayed in B. The bottom and top gates indicate the IC IgM^low and IC IgM^high populations, respectively. B, The frequency of IC IgM^high cells among CD138⁻ and CD138^int B cells from non-Tg and 2-12H mice. The horizontal line marks the mean frequency. C, The number of ASCs per 10⁶ sorted CD138⁻ or CD138^int B cells detected by ELIS-POT. IgM and IgG ELISPOT assays were used for analysis of non-Tg mice, and IgM and anti-Sm ELISPOT assays were used for analysis of 2-12H mice. The total number of ASCs per spleen is presented in Table II.

FIGURE 4

Anti-Sm CD138^int B cells are ASCs in autoimmune mice. A, CD19⁺CD138^int and CD19⁺CD138^high B cells are present in non-Tg MRL/lpr mice and as anti-Sm and non-Sm B cells in 2-12H MRL/lpr mice. T cells from MRL/lpr mice express CD138 and have been excluded from the histograms of the top row by anti-CD3 staining and gating on the CD3-negative cells. The histograms of the middle row are gated on CD19⁺ lymphocytes, and the bottom row on the indicated Sm-binding or non-Sm-binding populations. B, Frequency of CD19⁺CD138^int and CD19^lowCD138^high B cells as a percentage of CD19⁺ B cells. Each symbol represents a single mouse, and the horizontal line marks the mean frequency. The number of CD19⁺CD138^int B cells is as follows: 9.48 × 10⁶± 3.55 × 10⁶ for non-Tg MRL/lpr (frequency: 6.8 ± 2.5% of CD19⁺ B cells); 2.12 × 10⁶± 1.49 × 10⁶ for 2-12H MRL/lpr anti-Sm (frequency: 3.0 ± 2.1% of CD19⁺ B cells); and 2.67 × 10⁶± 1.31 × 10⁶ for 2-12H MRL/lpr non-Sm (frequency: 3.8 ± 1.8% of CD19⁺ B cells). The number of CD19^lowCD138^high cells are as follows: 1.46 × 10⁶± 8.46 × 10⁵ for non-Tg MRL/lpr (frequency: 1.04 ± 0.174% of CD19⁺ B cells); 1.48 × 10⁵± 1.20 × 10⁵ for 2-12H MRL/lpr anti-Sm (frequency: 0.66 ± 0.130% of CD19⁺ B cells); and 4.27 × 10⁵± 3.52 × 10⁵ for 2-12H MRL/lpr non-Sm (frequency: 0.900 ± 0.180% of CD19⁺ B cells). The number and frequency of CD138^int from 2-12H nonautoimmune mice is given in Fig. 1. The number of anti-Sm CD138^high B cells in 2-12H mice is 4.24 × 10⁴± 8.33 × 10³ (frequency: 0.127 ± 0.0249% of CD19⁺ B cells). The number of CD138^high B cells in nonautoimmune non-Tg mice is 2.92 × 10⁵± 5.78 × 10⁴ (frequency: 0.23 ± 0.047% of CD19⁺ B cells) (using gates (Figure legend continues) identical to those used for 2-12H mice). The frequency of anti-Sm CD138^high B cells in 2-12H and 2-12H MRL/lpr mice differs significantly (p = 0.0084). C, Sm binding by CD138^int B cells. Histograms are gated on CD138^int B cells from the indicated mice as illustrated in A (upper right quadrant of top row). The percentage of anti-Sm CD138^int B cells is provided. The average percentage for non-Tg and 2-12H mice is given in Table I. D, FSC, SSC, and activation marker expression for CD19⁺CD138⁻ (shaded), CD19⁺CD138^int (thin black line), and CD19^lowCD138^high (thick black line) B cells from 2-12H Tg MRL/lpr mice. Representative histograms are shown. Anti-IgM levels on T cells are shown as a negative control for IgM expression in the first panel. The p values for the differences between CD138⁻ and CD138^int (top value) and between CD138^int and CD138^high (bottom value) are given. E, The frequency of IC IgM^high cells among CD138⁻, CD138^int, and CD138^high B cells from non-Tg and 2-12H Tg MRL/lpr mice. The horizontal line marks the mean frequency. The p values for the relevant comparisons are shown. F, ASCs among 10⁶ sorted CD138⁻, CD138^int, and CD138^high B cells from non-Tg MRL/lpr and 2-12H MRL/lpr mice. *, Statistical significance from CD138⁻ B cells (p < 0.05). The ELISPOT assays were as described for Fig. 1. Note that the scale is different from that for Fig. 3C.

FIGURE 5

Anti-Sm CD138^int B cells have a high turnover rate and a high frequency are undergoing apoptosis. A, Seven-day BrdU incorporation by CD138⁻ (□) and CD138^int (■) B cells from non-Tg, 2-12H, and 2-12H MRL/lpr mice (n = 4). Error bars indicate SD. On the right are representative histograms to illustrate the gatings used to measure the frequency CD138⁻ and CD138^int populations that have incorporated BrdU. B, Cell cycle analysis of CD138^int B cells based on propidium iodide (PI) incorporation. The frequency of PI⁺ cells in the G₂/S gate is shown for each population. Representative of two independent experiments using cells pooled from three mice. C, Detection of early apoptosis using VAD-FMK staining of CD138⁻ (□) and CD138^int (■) B cells in non-Tg, 2-12H, and 2-12H MRL/lpr mice (n = 6). Error bars indicate SD. On the right are representative histograms illustrating the gates for VAD-FMK and CD138 staining used to generate the frequency of apoptotic cells. Values of p are for the comparison between the indicated CD138^int populations.

FIGURE 6

Blimp-1 mRNA is up-regulated in CD138^int B cells that secrete Ab. Shown are Blimp-1 mRNA levels from sorted CD138⁻ (□) and CD138^int (■) B cells from non-Tg mice and sorted anti-Sm CD138⁻ and CD138^int B cells from 2-12H and 2-12H MRL/lpr mice. The right panel shows Blimp-1 mRNA levels in CD138^int (■) and CD138^high () B cells in 2-12H MRL/lpr. In the lower graph are relative Blimp-1 levels from sorted CD138⁻ and CD138^int B cells from MD4 and MD4 × ML5 mice. Data are representative of two independent experiments using pooled mice. Values of p are shown for the indicated comparisons.

FIGURE 7

Differential expression of BAFF-R, TACI, and BCMA on anti-Sm and non-Tg CD138^int B cells. BAFF-R, TACI, and BCMA mRNA levels in CD138⁻ (□) and CD138^int (■) non-Tg B cells and anti-Sm B cells from 2-12H and 2-12H MRL/lpr mice. Data are representative of two independent experiments using pooled mice. Values of p are shown for the indicated comparisons.