Cytoplasmic Igα Serine/Threonines Fine-Tune Igα Tyrosine Phosphorylation and Limit Bone Marrow Plasma Cell Formation

Signals from the pre-BCR and the BCR critically shape B cell development, survival, activation, and Ab production. The signal-transducing element of the pre-BCR and the BCR is the associated Igα/β heterodimer (1, 2). The Igα and Igβ cytoplasmic domains each contain one ITAM, a motif shared by many receptors of the immune system (3–5). The dually phosphorylated Igα/β ITAMs rapidly amplify the signal by recruiting and activating Src homology 2 domain-containing Src family kinases and spleen tyrosine kinase (Syk) (3, 6). These protein tyrosine kinases phosphorylate neighboring ITAMs and downstream effectors (6, 7). Mechanisms ensuring signal termination include dephosphorylation of Igα by Src homology 2 domain phosphatase-1 recruited to the transmembrane adapter CD22 (6, 8).

Many ITAM-containing receptors contain evolutionarily conserved serines or threonines surrounding their ITAM tyrosines (4, 5). Some of these residues, including those in Igβ, CD3γ, CD3δ, and FcεRI, are phosphorylation sites (9–14). Igα contains two evolutionarily conserved serines in positions 191 and 197 and a threonine in position 203. These residues were found phosphorylated in B cell lines and in vitro (4, 9–11), and experiments in myeloma cell lines showed that Igα S191, 197, and T203 transiently decrease BCR-mediated Igα ITAM tyrosine phosphorylation (15). Moreover, a recent study suggested that at least S197 is a target for phosphorylation by Syk, thereby negatively regulating Syk and Src family kinase-mediated Igα ITAM tyrosine phosphorylation (16). We therefore speculated that Igα serine/threonines also antagonize the in vivo function of the Igα ITAM tyrosines. To test this hypothesis, we generated mice with targeted mutations of S191, 197, and T203.

Gene targeting was performed employing a strategy previously used in the laboratory (17, 18). Chimeric mice were generated through injection of targeted C57BL/6 embryonic stem cells into C57BL/6 albino blastocysts (19). Animal care and experiments were conducted according to protocols approved by the Animal Care and Use Committee of Harvard Medical School and the Immune Disease Institute. The mice were kept in a specific pathogen-free facility. Homozygous mutant mice and age-matched control mice of the C57BL/6 background (Charles River Laboratories) or littermates were analyzed at 8–14 wk unless indicated otherwise. Igα ITAM tyrosine mutant (Igα^FF) mice and Igβ ITAM tyrosine mutant (Igβ^AA) mice were previously described (17, 20). Embryonic stem cells with the Igα^SATV allele are available from the authors upon request.

Spleen, bone marrow, peritoneal lavage, Peyer’s patches (PPs), mesenteric, inguinal, and axillary lymph nodes, as well as cultured B cells were harvested into PBS containing 2% FCS (Invitrogen). Single-cell suspensions were stained with conjugated monoclonal and polyclonal Abs purchased from BD Biosciences, eBioscience, and Southern Biotechnology Associates or derived from hybridoma cell lines in the laboratory. IgG2c and IgG2b staining of germinal center (GC) B cells and LPS-stimulated blasts was performed with polyclonal biotinylated anti-IgG2a, recognizing both IgG2a and the homolog IgG2c present in C57BL/6 mice (21), and anti-IgG2b (Southern Biotechnology Associates) after IgG subclass specificity testing by ELISA and titration on LPS-stimulated B cell cultures displaying Ig class switching to IgGc and IgG2b. All samples were analyzed with a BD FACSCalibur (BD Biosciences) and FlowJo software (Tree Star).

Serum ELISAs were performed by coating plates with 4-hydroxy-3-nitrophenylacetyl (NP) (22) conjugated to BSA, polyclonal goat anti-mouse Igκ/λ (Southern Biotechnology Associates), denatured salmon sperm DNA (Sigma-Aldrich), or purified mouse glucose phosphate isomerase-GST fusion protein generated in the laboratory of D. Mathis and C. Benoist (Harvard Medical School, Boston, MA). Serum was added to 96-well plates at a starting dilution of 1:25 or 1:50, followed by 3.5- or 5-fold serial dilutions. Subsequently, serum Ig was detected with polyclonal biotinylated goat anti-mouse IgM, IgG1, IgG2b, IgG3, and IgA (Southern Biotechnology Associates), streptavidin-alkaline phosphatase conjugate (Roche), and chromogenic substrate 4-nitrophenyl phosphate (Sigma-Aldrich). Serum IgG2c was detected using cross-reactive polyclonal biotinylated goat anti-mouse IgG2a (Southern Biotechnology Associates) (21). Mice were immunized by i.p. injection of NP (41)-Ficoll (NP conjugated with aminoethylcarboxymethyl-Ficoll), and aluminum hydroxide-precipitated NP-chicken gammaglobulin (CGG) purchased from Biosearch Technologies. Laboratory-developed purified mAbs (S43-10, D3-13F1, B1-8μ, 18-1-16, and S24/63/63) and Abs purchased from Southern Biotechnology Associates (HOPC-1, A-1, 11E10, B10, 15H6, and S107) were used for the determination of the serum concentrations of NP-specific Abs and total Ig, respectively.

Numbers of Ab-forming cells were determined by culturing spleen and bone marrow cells on polyvinylidene fluoride membranes (Pall Corporation), previously coated with polyclonal anti-Igκ and anti-Igλ (Southern Biotechnology Associates), in six-well plates for 3 to 4 h. Membrane-bound Ig was subsequently detected with biotinylated polyclonal anti-IgM, IgG1, IgG2c, IgG2a cross-reactive with IgG2c (21), IgG2b, and IgG3 (Southern Biotechnology Associates), a streptavidin–HRP conjugate (Jackson ImmunoResearch Laboratories), ECL reagent (GE/Amersham Biosciences), and autoradiography film (Kodak).

Splenic B cells were MACS purified by CD43 depletion of spleen cells (Miltenyi Biotec), resulting in isolation of cellular fractions containing >95% CD19⁺ B cells. The cells were cultured in complete DMEM (Invitrogen), 10% FCS (Invitrogen) with polyclonal anti-IgM F(ab')2 fragments (Jackson Immunoresearch Laboratories), LPS from Escherichia coli O55:B5 (Sigma-Aldrich), or synthesized 20-mer of CpG DNA. B cells were labeled with CFSE (Molecular Probes) according to the manufacturer’s instructions prior to culturing for 3 d. Following harvest of the cultures, B cells were stained with dead cell exclusion marker TO-PRO-3 (Molecular Probes) and analyzed on an FACSCalibur (BD Biosciences). The percentage of cells that had undergone one or more cell division was calculated using the proliferation platform functions of FlowJo software (Tree Star).

Kidney sections were fixed in 10% formalin (Sigma-Aldrich) and stained with H&E or periodic acid-Schiff. Immunofluorescence staining of frozen kidney sections was performed with TRITC-conjugated goat anti-mouse IgG (Southern Biotechnology Associates) and FITC-conjugated rabbit anti-human C3d (DakoCytomation).

Purified splenic B cells in RPMI medium (Invitrogen) were stimulated with the indicated doses of polyclonal anti-IgM F(ab')2 fragments (Jackson Immunoresearch Laboratories) and whole-cell lysates prepared by immediate lysis in 1% Nonidet P-40 lysis buffer, 1 mM sodium orthovanadate, 5 mM sodium pyrophosphate, 10 mM sodium fluoride, 2 mM PMSF, and further protease inhibitors (Roche). A total of 20–40 μg protein per lane was subjected to an 8 or 10% SDS-PAGE (Hoefer) under reducing conditions and wet transferred (BioRad Transblot Cell) to polyvinylidene fluoride membranes (Millipore) according to the manufacturer’s instructions. Immunoblotting Abs raised against human p-Tyr⁵²⁵/⁵²⁶Syk, p-Thr²⁰²/Tyr²⁰⁴ERK, and ERK were obtained from Cell Signaling Technology, mAb 4G10 from Upstate, Abs specific for Syk (N19), B cell linker protein (H80) from Santa Cruz Biotechnology, and polyclonal rabbit Igα and Igβ from Y. M. Kim and H. Ploegh (Whitehead Institute for Biomedical Research, Cambridge, MA). Images shown were captured and quantified with a Fujifilm LAS-3000 imaging system (Fujifilm).

Splenic cells were labeled with Indo-1 (Molecular Probes), and BCR-mediated calcium flux of B cells resuspended at 2 × 10⁶ cells/ml RPMI 1640/2% FCS was assayed by analysis of the 405 nm/485 nm emission ratio after excitation with a 350-nm UV laser on an FACSVantage flow cytometer (BD Biosciences). Data were analyzed with the FlowJo analysis program (Tree Star).

Averages, geometric means, SD, logarithmic SD, and medians were calculated as indicated. The p values were determined by applying the two-tailed Student t test for unpaired samples to data sets.

The Igα cytoplasmic domain contains serines at position 191 and 197 in close proximity of ITAM tyrosine 193 and a threonine at position 203 adjacent to non-ITAM tyrosine 204 (Fig. 1A). We generated mice with targeted mutations in the Igα encoding mb-1 gene that resulted in replacement of serines 191 and 197 with alanine and threonine 203 with valine (Fig. 1B, Supplemental Fig. 1A–C). The mutant allele was termed Igα^SATV, and mice homozygous for this allele were analyzed. Immunoblotting of anti-IgM–stimulated splenic B cells from Igα^SATV/SATV mice showed enhanced Igα tyrosine phosphorylation. Specifically, Igα tyrosine phosphorylation normalized to total Igα expression was transiently increased by 10–30% in B cells from Igα^SATV/SATV mice compared with controls, whereas total Igα expression was slightly decreased when normalized to Igβ and ERK (Fig. 1C and data not shown). These results suggest that Igα serine/threonines modulate BCR-induced Igα tyrosine phosphorylation in primary splenic B cells as predicted by previous results (15, 16).

Hypothesizing that the Igα serine/threonines antagonize the in vivo Igα ITAM tyrosine function, we speculated that Igα ^SATV/SATV mice might display features opposite to those of Igα ITAM tyrosine (Igα^FF/FF) mutant mice. Although Igα^SATV/SATV mice did not show phenotypes opposite to those observed in Igα^FF/FF mice, the mutant mice had some features that were opposite to those observed in Igα^FF/FF mice also carrying a heterozygous or homozygous Igβ ITAM tyrosine mutation (Igβ^AA). Thus, the percentage of pre-B cells among lymphocytes was increased by approximately half, with the greatest increase in the percentage of cells detected at the small pre-B cell stage (68%) (Fig. 2A, Supplemental Fig. 2A) and without differences in bone marrow cellularity (Igα ^SATV/SATV: 20.4 ± 10.7 × 10⁶ cells/femur; controls: 16.1 ± 7.5 × 10⁶ cells/femur in 7–12-wk-old mice; n = 18, p = 0.159). Similarly, immature B cell fractions in the bone marrow and transitional B cells in the spleen were expanded by approximately half in Igα ^SATV/SATV mice compared with controls (Fig. 2B, 2C, Supplemental Fig. 2B), whereas total B cell numbers in the spleen were normal (Supplemental Fig. 2C). In contrast, Igα^FF and Igβ^AA compound mutants, but not Igα^FF/FF mice or Igβ^AA/AA, showed impaired pre-B cell and later stage B cell development (Supplemental Fig. 2D) (17, 20). These results suggest that Igα serine/threonines negatively regulate the size of the pre-, immature, and transitional B cell compartments. These findings are in line with a previous study suggesting that Igα serine/threonines antagonize the in vivo function of Igα/β ITAM tyrosine signaling at the pre-B cell stage as indicated by increased pre-BCR–mediated calcium responses in pre-B cells expressing serine and threonine mutant Igα (16).

The fraction of marginal zone B cells in Igα^SATV/SATV mice was reduced by 25–30% compared with controls (Fig. 2D, Supplemental Fig. 2E). The size of the mature B cell compartment was also slightly reduced (Fig. 2C). The size of the peritoneal B1 cell compartment and the ratio of Igκ- to Igλ-expressing cells in the spleen and bone marrow were normal (Supplemental Fig. 2F, 2G). Following immunization with the TD Ag NP-CGG, Igα^SATV/SATV mice had normal NP-specific IgM and IgG1 responses compared with controls (Supplemental Fig. 2H). The anti-IgM–induced calcium response, as well as surface expression of BCR components on mature B cells from Igα^SATV/SATV mice, was largely unchanged (Supplemental Fig. 3A, 3B). Further, no major differences in BCR-induced in vitro B cell responses were detected, as suggested by largely normal anti-IgM–mediated Syk and ERK phosphorylation, CD69 expression, and proliferation of splenic B cells from the mutant animals (Supplemental Fig. 3C, 3D). These results suggest that Igα serine/threonines modulate marginal zone B cell numbers, but do not play a critical role in regulating Igλ usage, surface BCR expression, B1 cell development, and Ag-specific IgG1 responses.

Unlike Igα^FF/FF mice, which showed normal IgG2c and IgG2b concentrations in the serum (17), serum concentrations of IgG2c and IgG2b in unimmunized, 8–10-wk-old Igα^SATV/SATV mice were 4-fold increased compared with controls, with no such increase seen in the case of IgM, IgG1, IgG3, and IgA (Fig. 3A and data not shown). These findings contrasted decreased NP-specific IgG2c and IgG2b responses to NP-Ficoll, whereas NP-specific IgM and IgG3 responses were only slightly reduced or normal (Fig. 3B, Supplemental Fig. 4A). Further, NP-specific IgG2c and IgG2b responses to NP-CGG were normal in the mutant mice (Fig. 3C). However, unimmunized, 8–10-wk-old Igα^SATV/SATV mice displayed low concentrations of IgG2c and IgG2b Abs recognizing NP, ssDNA, and the metabolic enzyme glucose phosphate isomerase (GPI), whereas such Abs were below or at the detection limit in controls (Fig. 3D). Anti-ssDNA, GPI, and NP IgM serum concentrations were normal in Igα^SATV/SATV mice, whereas IgG1 and IgG3 with these specificities were undetectable in both wild-type and mutant mice (Supplemental Fig. 4B). Autoantibody production in the human and mouse can be associated with spontaneous germinal center (GC) formation, the development of autoimmune disorder including glomerulonephritis, and high autoantibody titers as the disease progresses. No evidence of spontaneous GCs in lymph nodes, spleen, and bone marrow or enhanced GC formation in the PPs and mesenteric lymph nodes was found in 8–12-wk-old mutant mice (Supplemental Fig. 4C). Nine-month-old Igα^SATV/SATV mice did not show evidence of kidney inflammation or IgG or complement deposition in the kidneys compared with age-matched controls (Supplemental Fig. 4D). Serum ssDNA and GPI IgG2c and IgG2b were readily detectable in 9-mo-old wild-type as well as the mutant mice, but differences between mutants and controls, which were seen in 8–10-wk-old mice, had largely disappeared (Supplemental Fig. 4E). These results suggest that Igα serine/threonines limit the production of serum total IgG2c and IgG2b as well as serum NP, ssDNA, and GPI-specific IgG2c and IgG2b in unimmunized, 8–10-wk-old mice. The results further suggest that Igα serine/threonines are required for an efficient IgG2c and IgG2b response to a T cell-independent type II (TI-II) Ag, but do not play a critical role in regulating the T cell-dependent IgG2c and IgG2b response.

Increased serum Ig concentrations may be the result of an expanded bone marrow plasma cell compartment, as plasma cells residing in the bone marrow are a major source of serum Ig (23, 24). Indeed, quantification of Ab- secreting cells in the bone marrow revealed that the number of cells secreting IgG2c and IgGb were increased 4- to 5-fold in Igα^SATV/SATV mice compared with controls, whereas the number of cells secreting IgM, IgG1, or IgG3 were normal (Fig. 4A). B cell stimulation through the BCR and other receptors results in B cell activation, proliferation, Ig class switching, and differentiation to plasmablasts found in secondary lymphoid organs such as the spleen, some of which go on to become long-lived resting plasma cells homing to the bone marrow (23, 24). No gross enlargement of the proportion of Igα^SATV/SATV B cells switching to IgG2c and IgG2b in response to LPS in vitro, nor any major changes in the percentage of IgG2c- and IgG2b-positive cells in GCs of PPs and mesenteric lymph nodes of the mutant mice were detected (Fig. 4B, 4C). Further, the number of splenic cells secreting IgG2c and IgG2b as well as LPS-induced IgG2c and IgG2b secretion in vitro were not increased (Fig. 4C, 4D). These findings suggest that Igα serine/threonines critically control formation of IgG2c- and IgG2b-secreting cells in the bone marrow and serum IgG2c and IgG2b concentrations, but not Ig class switching to or production of cells secreting these isotypes in the spleen.

The Igα cytoplasmic domain contains two serines and one threonine in addition to the well-characterized ITAM and non-ITAM tyrosine phosphorylation sites (3, 4, 17, 18). In line with previous studies (15, 16), the present analysis of mice with targeted mutations of the cytoplasmic Igα serine/threonines suggests that Igα serine and threonine phosphorylation dampens Igα tyrosine phosphorylation in anti-IgM–stimulated splenic B cells. One might thus expect that the Igα serine/threonines antagonize the in vivo function of the Igα ITAM tyrosines as well. The abnormalities observed in early B cell development of Igα^SATV/SATV mice were indeed compatible with the idea that Igα serine/threonines negatively regulate BCR signaling. The decreased marginal zone B cell numbers and TI-II responses in the mutants, although not opposing the phenotype of Igα ITAM tyrosine-deficient mice, which also had decreased marginal zone B cell numbers and normal TI-II responses (17), support this notion as well, given that mice deficient in negative regulators such as CD22 also exhibit impaired marginal zone B cell development and TI-II responses (8). CD22-deficient mice and Igα^SATV/SATV mice further resembled each other in regard to production of low concentrations of autoantibodies, although autoreactive IgG accumulates only at older age in CD22-deficient mice (25), whereas differences to wild-type were found only in 8–10-wk-old but not aged Igα^SATV/SATV mice. However, any of these changes in Igα^SATV/SATV mice were of modest extent. Furthermore, no abnormalities in B1 cell development, Igλ usage, and the T cell-dependent IgG1 response were observed in the mutants, whereas Igα^FF/FF mice exhibit a phenotype in these respects (17).

In contrast, 8–10-wk-old Igα^SATV/SATV mice displayed strikingly elevated total serum IgG2c and IgG2b concentrations with a corresponding increase in IgG2c- and IgG2b-secreting bone marrow plasma cells, but normal in vitro Ig class switching to these isotypes, no accumulation of surface IgG2b- and IgG2c-positive GC cells in spontaneous GCs, and normal numbers of IgG2c- and IgG2b-secreting cells in the spleen, a major site of plasmablast production (26). Because Igα is not expressed at the plasma cell stage (27), it appears that Igα serine/threonines promote selection of cells into the bone marrow plasma cell compartment rather than survival of plasma cells. It is also possible that Igα serine/threonines limit the formation of bone marrow plasma cells by controlling the numbers of IgG2c- and IgG2b-expressing memory cells, which we did not analyze due to technical limitations. How can these observations be explained? Given the similarities between the IgM and the IgG BCR in structure and use of Igα for signal transduction (28), it is conceivable that the Igα^SATV/SATV mutation increases Igα tyrosine phosphorylation not only in IgM but also in IgG2c- and IgG2b-expressing cells and thereby enhances selection of the latter cells into the Igα-negative bone marrow plasma cell pool. Support for this idea comes from observations that strong Ag–BCR interactions, shown to correlate with increased Igα tyrosine phosphorylation, promote plasma cell differentiation in vitro, in T cell-independent, and in extrafollicular T cell-dependent responses (22, 30–32). The restriction of the phenotype to the IgG2c and IgG2b isotypes in Igα^SATV/SATV mice could reflect an as yet undefined difference in signal transduction through BCRs of the various IgG isotypes. In this context, the presence of low levels of self-reactive Abs of these isotypes in the mutant mice may suggest a link to the activation of cells expressing such specificities, given the similar isotype distribution of autoantibodies in mouse models of autoimmunity (33–35).

We thank J. Xia, S. Willms, and K. Jenssen for help with ELISAs, D. Ghitza, V. Smith, L. Du, C. Aristoff, A. Monti, A. Tetreault, C. Xiao, and A. Pellerin for mouse work and blastocyst injection, M. Nussenzweig, Y.M. Kim, H. Ploegh, D. Mathis, and C. Benoist for reagents, and all Rajewsky laboratory members and P. Schur for discussion and advice.

The authors have no financial conflicts of interest.