Regulation of inherently autoreactive VH4-34 B cells in the maintenance of human B cell tolerance

Abstract

The study of human B cell tolerance has been hampered by difficulties in identifying a sizable population of autoreactive B lymphocytes whose fate could be readily determined. Hypothesizing that B cells expressing intrinsically autoreactive antibodies encoded by the VH4-34 heavy chain gene (VH4-34 cells) represent such a population, we tracked VH4-34 cells in healthy individuals. Here, we show that naive VH4-34 cells are positively selected and mostly restricted to the follicular mantle zone. Subsequently, these cells are largely excluded from the germinal centers and underrepresented in the memory compartment. In healthy donors but not in patients with systemic lupus erythematosus (SLE), these cells are prevented from differentiating into antibody-producing plasma cells. This blockade can be overcome ex vivo using cultures of naive and memory VH4-34 cells in the presence of CD70, IL-2, and IL-10. VH4-34 cells may therefore represent an experimentally useful surrogate for autoantibody transgenes and should prove valuable in studying human B cell tolerance in a physiological, polyclonal environment. Our initial results suggest that both positive and negative selection processes participate in the maintenance of tolerance in autoreactive human B cells at multiple checkpoints throughout B cell differentiation and that at least some censoring mechanisms are faulty in SLE.

Figure 1

Representation of VH4-34 cells in human B cell subsets. (a) Distribution of total, VH4-34 (9G4) and VH3 (LJ26) B cells into naive (N), germinal center (GC), and memory (M) subsets in a representative tonsil. Histograms demonstrate the frequency of 9G4 and LJ26 cells in each subset. Bold lines represent VH4-34 and VH3 cells. Isotype antibody controls for 9G4 are shown as superimposed dashed lines. (b) Similar Vresults were obtained with peripheral blood B cells. (c) 9G4 and LJ26 cells express similar levels of sIg (determined with anti-κ/λ antibodies). (d) Analysis of the sIg expression by tonsil B cells. At least 90% of cells in each subset express detectable levels of sIg. (e) Tonsil B cells were permeabilized and analyzed for either surface or cytoplasmic expression of VH4-34. (f) PCR analysis of VH4-34 rearrangements. Samples are as follows: MW, 100 bp ladder; Ø DNA, negative control without input DNA; 1–3, total tonsil B cells; 4–6, naive B cells; 7–9, memory B cells; 10–12, VH4-34–expressing Ramos B cell lymphoma; 13–15, Jurkat T cells. The following amounts of DNA template were used: lanes 1, 4, 7, 10, and 13 (33 ng); lanes 2, 5, 8, 11, and 14 (100 ng); lanes 3, 6, 9, 12, and 15 (300 ng). Noticeable amplification of rearranged VH4-34 was detected in memory cells only after 35 PCR cycles with the highest amount of template DNA (300 ng). The amount of product obtained with memory cells was four to ten times lower than that obtained with naive B cells (average of four experiments). Calculations based on equivalent amounts of Ramos cells or Ramos DNA indicate that less than 2% of memory cells express VH4-34.

Figure 2

VH4-34 cells primarily reside within the follicular mantle zone. (a) Purified tonsil naive B cells were fractionated into Bm1 and Bm2 subsets based on their expression of surface IgD and CD23 (dot plot on the left) and stained with 9G4 or LJ26 (histograms on the right). A two- to fourfold expansion of VH4-34 cells from the Bm1 (virgin naive) to the Bm2 (ligand-selected naive) compartment was consistently demonstrated in all samples analyzed. (b) Immunohistochemical staining of adjacent tonsil sections was performed with antibodies against B cells (anti-CD20), VH4-34 cells (9G4), VH4 cells (LC1), or T-cells (anti-CD3). A conventional distribution of B cells and T cells was observed within the follicular mantle (FM), dark zone (DZ), and light zones (LZ) of typical germinal center structures. VH4-34 cells were concentrated in the FM and were virtually absent from the germinal center proper. In contrast, B cells expressing VH4 antibodies distinct from VH4-34 were readily visualized inside the germinal centers (LC1 staining).

Figure 3

Expression of VH4-34 antibodies in plasma cells from healthy donors and SLE patients. (a) Bone marrow plasma cells from healthy donors were enriched using a MACS CD138 column, and the cytoplasmic expression of VH4-34 or VH3 antibodies was detected with anti–light chain and VH-specific anti-Id antibodies 9G4, LJ26, or LC1 (data not shown). In contrast to VH3 and other VH4 antibodies, expression of VH4-34 was absent from normal plasma cells. (b) Identical results were obtained with tonsillar cells. (c) Plasma cells from the peripheral blood of active SLE patients express VH4-34 antibodies with significant frequency (two representative examples are shown). Further analysis revealed that more than 95% of VH4-34 antibodies were of the IgG isotype (data not shown). (d) Differentiation of VH4-34 B cells into plasma cells in vitro. Naive and memory tonsil B cells were sorted and independently cultured in the presence of CD70, IL-2, and IL-10 as described. The frequency of plasma cells was determined by flow cytometry before fractionation (left panel) and after culture (middle panels), and morphological confirmation was obtained by Giemsa staining (right panel). Significant numbers of plasma cells with a CD38^Hi/CD20^Lo phenotype were obtained with naive and memory cell cultures. (e) Plasma cells obtained in culture were analyzed for intracellular expression of VH4-34 antibodies using FITC-conjugated 9G4 and anti-κ/λ-RPE. VH4-34⁺ plasma cells were detected at a frequency consistent with the relative abundance of VH4-34 cells in the initial samples and therefore were significantly higher in the naive cell cultures than in the memory cell cultures.