Complement receptor 2/CD21- human naive B cells contain mostly autoreactive unresponsive clones

Abstract

Complement receptor 2-negative (CR2/CD21(-)) B cells have been found enriched in patients with autoimmune diseases and in common variable immunodeficiency (CVID) patients who are prone to autoimmunity. However, the physiology of CD21(-/lo) B cells remains poorly characterized. We found that some rheumatoid arthritis (RA) patients also display an increased frequency of CD21(-/lo) B cells in their blood. A majority of CD21(-/lo) B cells from RA and CVID patients expressed germline autoreactive antibodies, which recognized nuclear and cytoplasmic structures. In addition, these B cells were unable to induce calcium flux, become activated, or proliferate in response to B-cell receptor and/or CD40 triggering, suggesting that these autoreactive B cells may be anergic. Moreover, gene array analyses of CD21(-/lo) B cells revealed molecules specifically expressed in these B cells and that are likely to induce their unresponsive stage. Thus, CD21(-/lo) B cells contain mostly autoreactive unresponsive clones, which express a specific set of molecules that may represent new biomarkers to identify anergic B cells in humans.

Figure 1

CVID and RA patients may display an increased frequency of CD21^−/lo B cells in their blood. (A) CVID group Ia patients were previously defined as patients displaying a frequency of CD21^−/lo naive B cells > 20% of total B cells. Each diamond represents a person and (B) dot plots show CD19 and CD21 expression on CD27⁻-gated peripheral B cells from representative CVID group Ia patients. (C) Increased CD21^−/lo B-cell frequency in RA patients. Dot plots show CD19 and CD21 expression on CD27-depleted peripheral B cells from a representative healthy donor (HD10) and RA patients. (D) The frequencies of CD21^−/lo naive B cells are compared between healthy donors and RA patients. Each diamond represents a person and statistically significant differences are indicated.

Figure 2

CD21^−/lo naive B cells express autoreactive BCRs. (A) A majority of CD21^−/lo B cells from RA and CVID patients express IgM and IgD. Dot plots show IgM and IgD expression gated on CD19⁺CD21⁺(left) and CD19⁺CD21^−/lo (right) CD27-depleted B cells. (B) Most CD21^−/lo naive B cells express autoreactive antibodies. Antibodies cloned from single CD21⁺CD27⁻CD10⁻ and CD21^−/loCD27⁻CD10⁻ B cells from 2 healthy donors (HD10 and 11), 3 RA, and 3 CVID patients were tested by ELISA for reactivity against HEp-2 lysates. Dotted lines show ED38-positive control. Horizontal lines show cutoff OD₄₀₅ for positive reactivity. For each B-cell population, the frequency of HEp-2–reactive and non–HEp-2–reactive clones is summarized in pie charts, with the number of antibodies tested indicated in the centers. The frequency of HEp-2–reactive B cells was higher in CD21^−/lo than in CD21⁺ B cells in all persons. (C) CD21^−/lo B cells express highly autoreactive antibodies. The frequency of highly HEp-2–reactive antibodies, arbitrarily defined by ELISA OD₄₀₅ > 2, expressed by CD21⁺ and CD21^−/lo B cells from HD, RA, and CVID patients is represented. A large fraction of CD21^−/lo B cells from most persons was highly HEp-2 reactive. P values calculated using a paired t test are indicated when significant. (D) A majority of highly autoreactive antibodies from CD21^−/lo B cells express kappa light chains. The frequency of highly HEp-2–reactive CD21^−/lo B cells from HD, RA, and CVID patients is represented subdivided among kappa and lambda clones. Many highly HEp-2–reactive CD21^−/lo B cells express kappa light chains, suggesting that receptor editing using lambda light chains was not induced to silence these clones.

Figure 3

CD21^−/lo B cells contain antinuclear and anticytoplasmic clones. The frequencies of antinuclear (A) and anticytoplasmic (C) clones in CD21⁺ and CD21^−/lo B cells from HD, RA, and CVID patients are shown. P values calculated using a paired t test are indicated when significant. (B) ANAs expressed by CD21^−/lo B cells showed a diversity of nucleolar (RA01 21⁻ κ181, RA01 21⁻ κ137, RA14 21⁻ κ15, and RA14 21⁻ κ83), nucleolar and speckled (RA19 21⁻ κ127), and speckled (CVID 21⁻ κ12) antinuclear staining patterns. (D) Anticytoplasmic CD21^−/lo B cells can react with well-defined intracellular structures, which include mitochondria (RA01 21⁻ κ140, and CVID321 21⁻ κ82), the Golgi (RA14 21⁻ κ18), and cytoplasmic fibers (RA19 21⁻ κ61, RA14 21⁻ κ62, and RA01 21⁻ κ157 that is likely to recognize actin).

Figure 4

CD21^−/lo naive B cells fail to properly respond to BCR triggering. CD21^−/lo naive B cells from (A) RA01 and (B) CVID321 patients fail to up-regulate CD69 and CD25 expression after stimulation with F(ab′2) anti-IgM, recombinant human CD40L, and TLR9 agonist CpG. Total naive B cells containing both CD21^−/lo and CD21⁺ B cells were plated and dot plots show CD69 and CD25 expression after 2 days according to gated CD21 expression. (C) BCR triggering in CD21^−/lo naive B cells does not properly increase [Ca²⁺]_i. [Ca²⁺]_i was assessed by flow cytometry in CD21^−/lo and CD21⁺ naive B cells from a healthy donor (HD) and RA01 and CVID321 patients. Both B-cell populations were loaded with Fluo-3 and expressed similar levels of surface IgM expression when experiments were performed. Arrow indicates when F(ab′)2 anti-IgM was added. Data from each group of persons are representative of 2-3 independent experiments.

Figure 5

CD21^−/lo naive B cells do not proliferate after BCR and CD40 cotriggering and are prone to cell death. (A) Total naive B cells from healthy donors (HD), and RA and CVID patients containing both CD21^−/lo and CD21⁺ B cells were labeled with CFSE and stimulated with F(ab′2) anti-IgM and/or recombinant human CD40L. Dot plots show CD21 expression according to CFSE dilution after 3 days. CFSE dilution in CD21⁺ but not in CD21^−/lo B cells stimulated with F(ab′2) anti-IgM and recombinant human CD40L reveals that some CD21⁺ B cells underwent up to 2 divisions, whereas CD21^−/lo B cells did not divide. (B) Increased frequency of annexin V⁺ propidium iodide–negative (PI⁻) early apoptotic, and annexin V⁺ PI⁺ late apoptotic and dead cells in freshly isolated CD21^−/lo naive B cells from RA and CVID patients compared with their CD21⁺ B-cell counterparts. (C) BCR, CD40, and/or TLR9 triggering do not prevent the apoptosis and cell death of CD21^−/lo naive B cells. Total naive B cells containing both CD21^−/lo and CD21⁺ B cells from RA01 or CVID321 patients were plated, and annexin V and PI staining was assessed after 12 hours. BCR, CD40, and TLR9 stimulation prevents late apoptosis and cell death in CD21⁺ but not in CD21^−/lo B cells.

Figure 6

Gene array comparisons of CD21^−/lo and CD21⁺ naive B cells from RA and CVID patients using the Affymetrix Human Genome U133 Plus 2.0 Array. (A) Transcripts differentially expressed by CD21^−/lo and CD21⁺ naive B cells from 2 RA and 3 CVID group Ia patients are shown. (B) Selected transcripts well known to B-cell biology or of potential interest are presented. Up- and down-regulated transcripts are indicated in red and green, respectively. The magnitude of expression is depicted by the color bar.

Figure 7

CD21^−/lo B cells from RA and CVID patients display a phenotype reflecting gene array profiling data. Overlays for the indicated markers were obtained when CD27-depleted B cells isolated from the blood of patient RA01 were gated on either CD19⁺CD21^−/lo (bold line) or CD19⁺CD21⁺ (dashed line) B cells. The similar phenotypes of CD21^−/lo B cells from CVID patients and healthy donors are shown in supplemental Figures 7 and 8.