Contribution of alphabeta and gammadelta T cells to the generation of primary immunoglobulin G-driven autoimmune response in immunoglobulin- mu-deficient/lpr mice

Abstract

Class switch recombination (CSR) is a T-cell-dependent mechanism regulating isotype switching in activated mature B cells. Recently we showed that T-cell-independent CSRs occur spontaneously during B lymphopoiesis, but such cells are negatively selected by Fas signalling. In immunoglobulin mu-deficient mice, lack of Fas rescues isotype-switched B cells, resulting in generation of an autoimmune primary immunoglobulin G (IgG) repertoire in muMT/lpr mice. In the present study, we studied the role of alphabeta and gammadelta T cells in regulating this primary gammaH-driven repertoire. We found that a lack of alphabeta T cells significantly inhibited IgG production and autoimmunity in muMT/lpr mice, whereas a lack of gammadelta T cells resulted in augmented IgG production and autoimmunity. Also, a lack of T cells in muMT mice rescued isotype-switched B cells and serum IgG, probably owing to the lack of available FasL. We suggest that although CSRs in B-cell lymphopoiesis are T-cell independent, alphabeta T cells are important in the expansion of isotype-switched B-cell precursors and in promoting gammaH-driven autoimmunity, whereas gammadelta T cells regulate these cells.

Figure 1

Production of serum immunoglobulin G (IgG) in µMT/lpr mice lacking αβ, γδ, or both, T-cell subsets. Serum samples from 3–5-month-old mice, of the indicated genetic background, were collected and analysed by enzyme-linked immunosorbent assay (ELISA) to determine serum concentrations of total IgG. Concentrations were determined using an appropriate IgG standard curve, and results are expressed in µg/ml for individual mice and as group means. Each group contained six mice. *P-value of < 0·05 between µMT/lpr and µMT/lpr γδ^–/–, or between µMT/lpr αβ/γδ^–/– and µMT/lpr TCR-αβ^–/– mice.

Figure 2

Frequencies of antigen-forming cells (AFCs) in µMT/lpr mice deficient in αβ, γδ, or both, T cell subsets. Spleen cells from the indicated mice, at 3–5 months of age, were analysed in an enzyme-linked immunosorbent spot-forming cell assay (ELISPOT) to determine AFC frequency. (a) Spleen cells, in serial dilutions, were placed on filters (10⁴−10⁷ cells/filter). Spots were counted on each filter (when possible, depending on the density of spots at each dilution), and the frequencies of IgG-producing AFCs were calculated and expressed as number of AFCs per 10⁶ spleen cells. Results are expressed as mean ± standard error of the mean (SEM) of at least five mice in each group. **P-value of < 0·1 relative to µMT/lpr mice, *P-value of < 0·05 relative to µMT/lpr αβ^–/–. (b) A representative ELISPOT membrane cultured with 10⁶ spleen cells from the indicated mice.

Figure 3

Elevated levels of anti-chromatin immunoglobulin G (IgG) in γδ T-cell-deficient µMT/lpr mice. Serum samples from 3–5-month-old mice from the indicated genetic background were analysed by enzyme-linked immunosorbent assay (ELISA) for anti-chromatin IgG, as described in the Materials and methods. Antibody concentrations were determined using the anti-double-stranded DNA (dsDNA) monoclonal antibody (mAb), 3H9, at known concentrations, and are expressed in µg/ml for individual mice and group means. Results are expressed as mean ± standard error of the mean (SEM) for at least five mice in each group. *P-value of < 0·05 relative to µMT/lpr.

Figure 4

Tissue reactivity in γδ T-cell-deficient µMT/lpr mice. Serum samples from 3–5-month-old normal and µΜT/lpr TCR-δ^–/– mice were tested in a tissue array for tissue reactivity, as described in the Materials and methods. Shown are representative blot arrays (out of five in each group) obtained for a normal (top) and two different (3 months old) µΜT/lpr/TCR-δ^–/– serum samples (middle and bottom). Serum samples were diluted 1 : 3000 and adjusted to contain ≈ 1 µg/ml total IgG. Tissue-specific bands, identified by µΜT/lpr sera, are indicated by arrows. Tissues used are as follows: lane 1, brain; lane 2, heart; lane 3, skin; lane 4, kidney; lane 5, pancreas; lane 6, spleen; lane 7, muscle; lane 8, lung; lane 9, liver.

Figure 5

Detection of serum immunoglobulin G (IgG) and antigen-forming cells (AFCs) in µMT mice deficient for different T-cell subsets. Serum samples from 3–5-month-old T-cell-sufficient µMT mice, and from µMT mice deficient in αβ, γδ, or both, T-cell subsets, were analyzed for IgG production. (a) The concentrations of serum IgG were determined by enzyme-linked immunosorbent assay (ELISA), using an appropriate IgG standard curve. The results are expressed in µg/ml for individual mice, and as group means. Each group contained at least four mice. *P-value of < 0·2 relative to µMT/lpr αβ^–/–. (b) Detection of the γH chain, by Western blotting, in serum samples that were found to contain IgG by ELISA screening. A total of 8 µl of serum samples from the indicated mice were used for detection. A representative blot is shown. (c) AFC frequencies in spleens of the indicated mice were determined by enzyme-linked immunosorbent spot-forming cell assay (ELISPOT). The results represent the mean ± standard error of the mean (SEM) of at least four mice in each group. *P-value of < 0·1 relative to µMT/lpr αβ^–/–.