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. 2007 Jun;117(6):1558-65.
doi: 10.1172/JCI27628. Epub 2007 May 17.

Mature B cells class switched to IgD are autoreactive in healthy individuals

Affiliations

Mature B cells class switched to IgD are autoreactive in healthy individuals

Kristi Koelsch et al. J Clin Invest. 2007 Jun.

Abstract

Determination of the origin and fate of autoreactive B cells is critical to understanding and treating autoimmune diseases. We report that, despite being derived from healthy people, antibodies from B cells that have class switched to IgD via genetic recombination (and thus become class switched to C delta [C delta-CS] cells) are highly reactive to self antigens. Over half of the antibodies from C delta-CS B cells bind autoantigens on human epithelioma cell line 2 (HEp-2) cells or antinuclear antigens, and a quarter bind double-stranded DNA; both groups of antibodies are frequently polyreactive. Intriguingly, some C delta-CS B cells have accumulated basic residues in the antibody variable regions that mediate anti-DNA reactivity via somatic hypermutation and selection, while other C delta-CS B cells are naturally autoreactive. Though the total percentage was appreciably less than for C delta-CS cells, a surprising 31% of IgG memory cell antibodies were somewhat autoreactive, and as expected, about 24% of naive cell antibodies were autoreactive. We interpret these findings to indicate either that autoreactive B cells can be induced to class switch to IgD or that autoreactive B cells that use IgD as the B cell receptor are not effectively deleted. Determination of the mechanism by which the majority of C delta-CS B cells are autoreactive may be important in understanding peripheral tolerance mechanisms and may provide insight into the enigmatic function of the IgD antibody.

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Figures

Figure 1
Figure 1. Phenotype of Cδ-CS B cells.
(A and B) Flow cytometry sorting gates used to isolate the various B cell types analyzed from blood or tonsils, including Cδ-CS GC cells (IgD+IgMCD38+), naive B cells (IgD+IgM+ and CD38 tonsils or CD27 blood), and memory B cells (IgDCD27+ from the “memory” box in B). PC, plasma cell. Analysis of somatic mutation frequency (C) and usage of the JH6 gene segment (D) demonstrate that all Cδ-CS B cell subsets and those expressed herein are similarly unique from IgG and IgM B cells. (C) Shown is the percentage of VH genes with the indicated number of mutations for each subset analyzed. (D) Use of various JH gene segments. Included are transcripts from 625 Cδ-CS GC cell variable genes from 11 donors (by donor, n = 44, 44, 57, 44, 62, 34, 225, 59, 16, 21, and 19), 78 Cδ-CS plasma cell variable genes from 1 donor, 124 Cδ-CS memory cell variable genes from 4 donors (by donor, n = 20, 20, 28, and 56), 620 IgG GC, memory, and plasma cell variable genes from 14 donors (by donor, n = 18, 28, 174, 40, 108, 37, 25, 21, 18, 22, 15, 24, 19, and 71), 681 IgM GC, memory, and plasma cell variable genes from 18 donors (by donor, n = 18, 91, 51, 158, 17, 10, 16, 48, 30, 19, 28, 11, 36, 29, 13, 22, 20, and 64), and 267 naive variable genes from 6 donors (by donor, n = 47, 30, 24, 15, 24, and 127).
Figure 2
Figure 2. Antibodies from Cδ-CS B cells are frequently autoreactive.
(A) Cδ-CS–derived antibodies are highly reactive to HEp-2 and ANA antigens compared with naive- and IgG-derived antibodies. Antibodies tested included 89 Cδ-CS antibodies (from 3 donors, n = 37, 18, and 34), 70 naive B cell antibodies (from 3 donors, n = 39, 14, and 17), and 64 antibodies from IgG memory cells (from 4 donors, n = 25, 5, 12, and 20). The frequency of HEp-2 reactive antibodies was determined by screening the antibodies by immunofluorescence using commercially prepared HEp-2 slides (Supplemental Figure 1), and the frequency of ANA reactive antibodies using commercial ANA immunosorbent assays (see Methods). Antibodies that bound HEp-2 slides more intensely than negative control serums provided by the manufacturer or that bound ANA more intensely than the mean ± SD of all naive cell antibodies were considered positive in the 2 assays. Results indicated that the incidence of autoreactivity was significantly higher in Cδ-CS–derived antibodies than in naive- or IgG-derived antibodies (χ2, P < 0.0001). (B) Frequency of HEp-2 and ANA reactivity by donor. Cδ-CS antibodies bound more often than IgG or naive cell–derived antibodies (Student’s t test, P < 0.05). (C) Although IgG cells were generally more reactive in the ANA assays than were naive cells, the reactivity was low intensity. In contrast, significantly more Cδ-CS antibodies bound to ANA with high intensity. Red lines indicate mean ANA binding absorbances. Dashed lines indicate the thresholds for positive scoring (lower dashed line indicates ANA+ that is the naive cell mean ± SD; upper dashed line indicates ANAhigh); the high absorbance level was achieved more commonly for Cδ-CS antibodies.
Figure 3
Figure 3. Binding to ssDNA, dsDNA, LPS, or insulin by the expressed antibodies was measured by ELISA.
The degree of binding to antigen-coated microtiter plates (absorbance [OD415]) was measured at antibody concentrations of 6.67 × 10–8, 1.67 × 10–8, 4.17 × 10–9, and 1.04 × 10–9 M (which is 1 μg/ml and three 4-fold serial dilutions, x axis). All ELISA include 3H9 (red lines with diamonds) and H241 (red lines with squares) monoclonal antibodies with high and medium anti-DNA or polyreactivity, respectively. The assays were normalized based on the absorbencies of the 3H9 antibody. Percentages indicate the number of antibodies scored as positive. A total of 78 antibodies from naive B cells isolated from 3 donors (by donor, n = 37, 27, and 14 antibodies) were compared with 100 antibodies from Cδ-CS B cells from 3 donors (by donor, n = 45, 34, and 21 antibodies) and 64 antibodies from IgG memory cells from 4 donors (by donor, n = 25, 12, 7, and 20 antibodies). Blue lines represent the approximate positive thresholds determined by calculating the mean ± 2 SD of the naive antibodies (see Results).
Figure 4
Figure 4. Variability between donors and frequency of polyreactivity.
(A) Mean ± SEM for donors for the various assays performed. (B) The percentage of antibodies from each group (Cδ-CS B cells, naive B cells, or IgG memory B cells) that bound 0–1, 2, 3, or all 4 antigens tested (ssDNA, dsDNA, LPS, and insulin). Polyreactivity is defined as the binding of more than 1 antigen. The yellow portion of the chart indicates the percentage that bound up to 1 antigen and so were not polyreactive. Statistical significance (χ2, P = 0.04) was reached for Cδ-CS versus naive B cell antibodies.
Figure 5
Figure 5. Cδ-CS can occur for cells that express germline (natural) autoreactive antibodies as well as those that have acquired autoreactivity via somatic mutations.
(A) Cδ-CS antibodies encoded by unmutated (germline) variable genes display levels of HEp-2 autoreactivity, DNA binding, and polyreactivity similar to those from somatically mutated variable genes. Shown are the percentages of antibodies from each subpopulation. (B) Binding curves of 1 μg/ml anti-DNA antibodies from Cδ-CS B cells to DNA were used to calculate absorbencies (blue dots). Comparison of these absorbencies with the frequency of amino acid replacements (red bars) showed that there is no correlation between the affinity for dsDNA and the accumulation of somatic mutations. The variable genes of clones 14 and 17 (asterisks) were reverted to their germline sequences to determine whether the somatic mutations might cause DNA binding. (C) DNA binding is lost when 2 anti-DNA Cδ-CS antibodies (clone 14, blue square; clone 17, blue circle) were expressed from variable genes reverted to the germline unmutated sequences (clone 14, black square; clone 17, black circle). Anti-DNA binding was evaluated by ELISA (absorbance [OD415]) relative to the control 3H9 monoclonal antibody with high affinity for DNA (red line). GL, germline.

References

    1. Nemazee D.A., Burki K. Clonal deletion of B lymphocytes in a transgenic mouse bearing anti-MHC class I antibody genes. Nature. 1989;337:562–566. - PubMed
    1. Hartley S.B., et al. Elimination from peripheral lymphoid tissues of self-reactive B lymphocytes recognizing membrane-bound antigens. Nature. 1991;353:765–769. - PubMed
    1. Gay D., Saunders T., Camper S., Weigert M. Receptor editing: an approach by autoreactive B cells to escape tolerance. J. Exp. Med. 1993;177:999–1008. - PMC - PubMed
    1. Tiegs S.L., Russell D.M., Nemazee D. Receptor editing in self-reactive bone marrow B cells. J. Exp. Med. 1993;177:1009–1020. - PMC - PubMed
    1. Nossal G.J. Cellular mechanisms of immunologic tolerance. Annu. Rev. Immunol. 1983;1:33–62. - PubMed

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