B-cell tolerance defects in the B6.Aec1/2 mouse model of Sjögren's syndrome

Abstract

Purpose: Primary Sjögren's syndrome (SjS) is an autoimmune disorder characterized by lymphocytic infiltration of the salivary and lacrimal glands, B-cell clonal expansions and an increased risk of lymphoma. In order to understand the role of B cells in this disorder, the antibody repertoire and B-cell maturation were studied in a mouse model of SjS called B6.Aec1/2.

Methods: B6.Aec1/2 serum was analyzed for antibodies by ELISA and immunoprecipitation, B-cell development by flow cytometry, and antibody gene rearrangements by CDR3 spectratyping and quantitative PCR. In order to test the functional consequences of the observed defects, B6.Aec1/2 mice were crossed with anti-dsDNA antibody heavy chain knock-in mice (B6.56R).

Results: B6.Aec1/2 mice exhibit B-cell clonal expansions, have altered serum immunoglobulin levels and spontaneously produce multireactive autoantibodies. B6.Aec1/2 mice also have decreased numbers of bone marrow pre-B cells and decreased frequencies of kappa light chain gene deletion. These findings suggest that B6.Aec1/2 mice have a defective early B-cell tolerance checkpoint. B6.56R.Aec1/2 mice unexpectedly had lower anti-dsDNA antibody levels than B6.56R mice and less salivary gland infiltration than B6.Aec1/2 mice.

Conclusions: These data suggest that the early tolerance checkpoint defect in B6.Aec1/2 mice is not sufficient to promulgate disease in mice with pre-formed autoantibodies, such as B6.56R. Rather, B6.Aec1/2 mice may require a diverse B-cell repertoire for efficient T-B-cell collaboration and disease propagation. These findings imply that therapies aimed at reducing B-cell diversity or T-B interactions may be helpful in treating SjS.

Fig. 1. IgM, IgA and IgG antibody levels and anti-dsDNA antibodies

Sera were obtained from B6 (n=23 females), B6.Aec1/2 (n =19 females) mice that ranged in age from 2 mo to 24 mo. Average OD values (of duplicate readings) for individual mice are indicated (grey symbols indicate B6, black symbols indicate B6.Aec1/2). a. Serum IgM, IgA and IgG levels. b. Serum anti-dsDNA IgM, anti-dsDNA IgA and anti-dsDNA IgG antibody levels. Vertical lines represent the confidence interval for one SD. The central horizontal line indicates the arithmetic mean. Asterisks indicate p < 0.05 by two-tailed Mann-Whitney test. All ELISA measurements shown were performed in duplicate in the same assay at the same dilution (see materials and methods) on the same day. The OD₄₀₅ measurements were subtracted from the background value (medium alone). These conditions apply to all mouse serum ELISA studies unless specified otherwise.

Fig. 2. Spontaneous hybridoma panel analysis

a. 70 hybridomas were generated from a 20-mo-old female B6.Aec1/2 spleen without any stimulation. Among them, 41 expressed IgM, 5 expressed IgG and 1 expressed IgA. Three clones expressed light chain but not IgM, IgG or IgA heavy chains. Twenty clones did not secrete any identifiable heavy chain or light chain (NS = non-secretors). b. Kappa light chain sequencing of clonally related hybridomas. Upper and lower panels represent two expanded clones with the same Vκ, Jκ and CDR3 length. The germline Vκ and Jκ sequences are shown in the top row for comparison. Horizontal dashes indicate that the nucleotide sequence of the clone is identical to the germline sequence. Sequence changes that result in amino acid replacements are indicated in bold underlined font. Silent mutations are given in regular font. Gaps in the sequence are indicated by angled hatch marks. The sequences in the gap are identical to the germline sequence (not shown). Nucleotide positions are numbered according to their positions in the germline sequences, starting with the Vκ framework 1 sequence. In the upper group of clonally related hybridomas, two of the three hybridomas (indicated by 2×) have an identical Vκ-23-Jκ5 sequence, which has 3 mutations compared to the germline sequences. The third hybridoma (this sequence was recovered only once, 1×) has two mutations compared to the germline sequence. In the lower group of clonally related hybridomas, two of the hybridomas had sequences that were identical to the germline sequence and the third hybridoma had one nucleotide substitution resulting in a silent mutation in Jκ5. FR3 = framework region 3. IF = in-frame rearrangement (productive). OF = out-of-frame rearrangement (non-productive). c. CDR3 spectratyping of splenocytes from the same B6.Aec1/2 mouse used for hybridoma analysis. J606.1-J_H2 rearrangements were shown from three independent PCRs. Shared peaks in all three independent amplifications (putative clonal expansions) are indicated by the asterisks. d. CDR3 spectratyping of splenocytes from a 23-month-old female B6 mouse. MOPC21-J_H2 rearrangements are shown from three independent PCRs.

Fig. 3. Bone marrow B-cell subsets and light chain gene rearrangements in B6 and B6.Aec1/2 mice

a. Percentages of bone marrow (% BM) B-cell subsets (Hardy fractions D, E and F as defined in the Methods) of B6 (n=9) and B6.Aec1/2 (n=11) mice. Subsets are defined as the fraction of CD19+ BM lymphocytes multiplied by 100%. Asterisks indicate fractions that differ significantly, p<0.05 by two-tailed Mann-Whitney test. b. Absolute B-cell numbers of BM fractions of B6.Aec1/2 (n=3) and B6 (n=5) mice. Cell counts were obtained by multiplying the number of viable B220+ BM lymphocytes in each mouse by the percentages of cells in each fraction. Asterisks indicate fractions that differ significantly, p<0.05 by two-tailed Mann-Whitney test. c. RS rearrangement frequencies in Fr. D bone marrow B cells from B6 and B6.Aec1/2 mice. The RS frequency is plotted as fold difference relative to kappa+ splenocytes from B6 mice. The average RS frequency is significantly lower in B6.Aec1/2 mice (p<0.05 by two-tailed Mann-Whitney test.) In all panels vertical lines represent the confidence interval for one SD. The central horizontal line indicates the arithmetic mean.

Fig. 4. Comparison of B-cell development and receptor editing in B6.56R and B6.56R.Aec1/2 mice

a. The percentages of bone marrow B cells that comprise fractions D, E and F (as defined in Methods) are shown for B6.56R (n=8) and B6.56R.Aec1/2 mice (n=14). b. RS rearrangement frequency in bone marrow fraction D cells from B6 (n = 14, 2–6 mo), B6.Aec1/2 (n=4, 2–14 mo), B6.56R (n=7, 2–6 mo), B6.Aec1/2.56R (n=3, 2–6 mo), and NOD (n=6, 2–6 mo). All PCRs were performed in duplicate. Data are depicted as fold difference relative to the RS level in B6 splenic B220+κ+ B cells. c. Percentages of IgM^b B cells in spenocytes of B6.56R (n=8) and B6.56R.Aec1/2 (n=7).

Fig. 5. Lymphocytic infiltration in the salivary glands of B6.Aec1/2 and B6.56R.Aec1/2 mice

a. Lymphocytic infiltrates in the submandibular glands of a female (left panel) and male (right panel) B6.Aec1/2 mouse. Sixteen B6.Aec1/2 mice were evaluated and 14 of them had infiltrates. b. Confocal images of salivary glands in female B6.Aec1/2 mice (n=2, age 6 months) reveal the presence of B cells and CD4 T cells. Green: anti-B220-Alexa488; Blue: anti-CD4-Alexa 647; Grey: phase-contrast image. c. Salivary gland focus score in young (<12 mo) and aged (>12 mo) B6.Aec1/2 (filled circles, young, n=11; old, n=5 ) and B6.56R.Aec1/2 (filled squares, young, n=14; old, n=3) mice. d. Focus scores of lymphocytic infiltrates in salivary glands of B6 (n=7, n=6 females), B6.Aec (n=16, n=10 females), B6.56R (n=8, n=5 females) and B6.56R.Aec1/2 (n=17, n=7 females). Female ages range from 5- to 25-mo-old, and males range from 6- to 12-mo-old. Asterisks indicate p <0.05 by two-tailed Mann-Whitney test. In panels c and d, vertical lines represent the confidence interval for one SD. The central horizontal line indicates the arithmetic mean.