Identification and characterization of a loss-of-function human MPYS variant.

MPYS, also known as STING and MITA, is an interferon (IFN)ß stimulator essential for host defense against RNA, DNA viruses and intracellular bacteria. MPYS also facilitates the adjuvant activity of DNA vaccines. Here, we report identification of a distinct human MPYS haplotype that contains three non-synonymous single nucleotide polymorphisms (SNPs), R71H-G230A-R293Q (thus, named the HAQ haplotype). We estimate, in two cohorts (1,074 individuals), that ∼3% of Americans are homozygous for this HAQ haplotype. HAQ MPYS exhibits a > 90% loss in the ability to stimulate IFNß production. Furthermore, fibroblasts and macrophage cells expressing HAQ are defective in Listeria monocytogenes infection-induced IFNß production. Lastly, we find that the loss of IFNß activity is due primarily to the R71H and R293Q SNPs in HAQ. We hypothesize that individuals carrying HAQ may exhibit heightened susceptibility to viral infection and respond poorly to DNA vaccines.

B cell activation. III. B cell plasma membrane depolarization and hyper-Ia antigen expression induced by receptor immunoglobulin cross-linking are coupled.

We report investigation of the relationship between ligand-induced B cell plasma membrane depolarization and increased expression of membrane-associated, I-A subregion encoded (mI-A) antigens. Results demonstrate that equal frequencies of B cells are stimulated to undergo membrane depolarization and to increase mI-A expression in response to mitogen, anti-Ig, and thymus-independent (TI) or thymus-dependent (TD) antigens. Further, a cause-and-effect relationship between these two events is suggested by results that demonstrate that inhibition of anti-Fab--induced depolarization by valinomycin also inhibits the subsequent increase in mI-A antigen expression and "passive" (non-ligand-mediated) depolarization of murine B cells by K+ results in hyper-mI-A antigen expression. Based upon these results we hypothesize that antigen-mediated receptor cross-linking results in signal transduction via membrane depolarization, which is resultant in increased mI-A antigen synthesis and cell surface expression. This increase in mI-A antigen density may render the B cell more receptive to subsequent interaction with I-region-restricted helper T cells.

B cell activation. I. Anti-immunoglobulin-induced receptor cross-linking results in a decrease in the plasma membrane potential of murine B lymphocytes.

We report analyses of the effect of anti-Fab antibodies on plasma membrane potential of mouse B lymphocytes. Results indicate that divalent fragments of anti-Fab antibodies mitogenic for B cells stimulate membrane depolarization detectable by cytofluorometric analysis of 3,3'-dipentyloxacarbocyanine iodide-stained cells. Depolarization is detectable within 5 min of exposure to ligand and maximal within 1 h of exposure when greater than or equal to 80% of splenic B cells exhibit decreased membrane potential. The ineffectiveness of monovalent Fab antibody fragments in inducing this event suggests that receptor immunoglobulin cross-linking is essential. Frequencies of cells induced to enter cell cycle, as assessed by acridine orange cell cycle analysis, are equal to those induced to depolarize by lipopolysaccharide plus dextran sulfate or anti-Fab, which suggests a relationship between these events. However, membrane depolarization is itself an insufficient signal to promote subsequent thymidine uptake, as evidenced by the fact that doses of anti-Fab that are suboptimal for thymidine uptake induce maximal depolarization. These results suggest that cross-linking of surface immunoglobulin on B cells may provide an initial signal for activation but is itself insufficient to drive B cell proliferation.

Ia-mediated signal transduction leads to proliferation of primed B lymphocytes.

One of the most controversial questions in immunology is the molecular basis by which Th lymphocytes deliver activating signals to quiescent B lymphocytes during T cell-dependent immune responses. Recent studies suggest that T cell-dependent activation of quiescent B lymphocytes may involve signaling mediated by direct T helper cell-B cell contact. Since B cell membrane-associated MHC-encoded class II molecules (Ia) must be recognized by Th lymphocytes for generation of T cell-dependent humoral immune responses, they are obvious candidates for receptors of this signal. Here we report that stimulation of quiescent murine B cells with IL-4 and antibodies against the B cell antigen receptor for 12-16 h primes cells to proliferate in response to immobilized mIa binding ligands. In the presence of additional lymphokines, these B cells differentiate to secrete Ig of IgM and IgG classes. These results suggest that Ia molecules are receptors for direct, T helper cell-B cell contact mediated signaling that results in B cell proliferation.

Delivery of B cell receptor-internalized antigen to endosomes and class II vesicles.

B cell receptor (BCR)-mediated antigen processing is a mechanism that allows class II-restricted presentation of specific antigen by B cells at relatively low antigen concentrations. Although BCR-mediated antigen processing and class II peptide loading may occur within one or more endocytic compartments, the functions of these compartments and their relationships to endosomes and lysosomes remain uncertain. In murine B cells, at least one population of class II- containing endocytic vesicles (i.e., CIIV) has been identified and demonstrated to be distinct both physically and functionally from endosomes and lysosomes. We now demonstrate the delivery of BCR-internalized antigen to CIIV within the time frame during which BCR-mediated antigen processing and formation of peptide-class II complexes occurs. Only a fraction of the BCR-internalized antigen was delivered to CIIV, with the majority of internalized antigen being delivered to lysosomes that are largely class II negative. The extensive colocalization of BCR-internalized antigen and newly synthesized class II molecules in CIIV suggests that CIIV may represent a specialized subcellular compartment for BCR-mediated antigen processing. Additionally, we have identified a putative CIIV-marker protein, immunologically related to the Igalpha subunit of the BCR, which further illustrates the unique nature of these endocytic vesicles.

Distinct signal thresholds for the unique antigen receptor-linked gene expression programs in mature and immature B cells.

Although it is well established that immature B lymphocytes are exquisitely sensitive to tolerance induction compared with their mature counterparts, the molecular basis for this difference is unknown. We demonstrate that signaling by B cell antigen receptors leads to distinct and mutually exclusive biologic responses in mature and immature B cells: upregulation of CD86, CD69, and MHC class II in mature cells and receptor editing in immature cells. These responses can be induced simply by elevation of intracellular free calcium levels, as occurs after receptor aggregation. Importantly, induction of immature B cell responses requires much smaller increases in intracellular free calcium than does induction of mature B cell responses. These differences in biologic response and sensitivity to intracellular free calcium likely contributes to selective elimination at the immature stage of even those B cells that express low affinity for self-antigens.

Definition of conditions that enable antigen-specific activation of the majority of isolated trinitrophenol-binding B cells.

In an effort to further elucidate the early cellular events in generation of antibody responses, we have determined the requirements for antigen-specific initiation of the G0 to G1 transition by isolated trinitrophenol (TNP) -binding B lymphocytes. TNP-binding cells were isolated from normal B6D2F1 splenocyte populations using hapten affinity fractionation on disulfide-bonded TNP-gelatin-coated plates. Populations prepared in this way are greater than or equal to 96% immunoglobulin positive and 70-95% antigen binding. Isolated cells were cultured for 48 h in the presence of a variety of TNP conjugates including TNP-Brucella abortus (Ba), TNP-Ficoll, TNP-sheep erythrocytes (SRBC), TNP-human gamma globulin (HGG), or TNP-ovalbumin (OVA) before being harvested and subjected to acridine orange cell cycle analysis. As many as 80% of cells were in cycle by 48 h in response to TNP-Ba, a thymus-independent (TI1 antigen. A smaller proportion (congruent to 40%) were in cycle in response to TNP-Ficoll, a TI2 antigen. Significant activation was not detected in cultures challenged with the thymus-dependent immunogens TNP-SRBC, TNP-HGG, and TNP-OVA. Addition of interleukin 1 (IL-1), IL-2, B cell growth factor, and/or T cell-replacing factor to cultures did not facilitate responses to these immunogens, suggesting a requirement for antigen-specific T cell help for entry into cell cycle induced by thymus dependent antigens. Activation by TNP-Ba was antigen specific and independent of accessory cells, occurring with equal efficiency in bulk and single-cell cultures. Activation by TNP-Ba was inhibitable by anti-Fab and anti-mu antibodies, but not by anti-delta antibodies. Results indicate that activation of TNP-binding cells to enter cell cycle by TNP-Ba is independent of accessory cells and requires interaction of antigen with cell surface IgM. Exposure to thymus-dependent TNP-immunogens plus nonspecific helper factors is insufficient to cause entry of TNP-binding cells into cycle.

Identification of the tyrosine phosphatase PTP1C as a B cell antigen receptor-associated protein involved in the regulation of B cell signaling.

Recent data implicating loss of PTP1C tyrosine phosphatase activity in the genesis of the multiple hemopoietic cell defects found in systemic autoimmune/immunodeficient motheaten (me) and viable motheaten (mev) mice suggest that PTP1C plays an important role in modulating intracellular signaling events regulating cell activation and differentiation. To begin elucidating the role for this cytosolic phosphatase in lymphoid cell signal transduction, we have examined early signaling events and mitogenic responses induced by B cell antigen receptor (BCR) ligation in me and mev splenic B cells and in CD5+ CH12 lymphoma cells, which represent the lymphoid population amplified in motheaten mice. Despite their lack of functional PTP1C, me and mev B cells proliferated normally in response to LPS. However, compared with wild-type B cells, cells from the mutant mice were hyperresponsive to normally submitogenic concentrations of F(ab')2 anti-Ig antibody, and they exhibited reduced susceptibility to the inhibitory effects of Fc gamma IIRB cross-linking on BCR-induced proliferation. Additional studies of unstimulated CH12 and wild-type splenic B cells revealed the constitutive association of PTP1C with the resting BCR complex, as evidenced by coprecipitation of PTP1C protein and phosphatase activity with BCR components and the depletion of BCR-associated tyrosine phosphatase activity by anti-PTP1C antibodies. These results suggest a role for PTP1C in regulating the tyrosine phosphorylation state of the resting BCR complex components, a hypothesis supported by the observation that PTP1C specifically induces dephosphorylation of a 35-kD BCR-associated protein likely representing Ig-alpha. In contrast, whereas membrane Ig cross-linking was associated with an increase in the tyrosine phosphorylation of PTP1C and an approximately 140-kD coprecipitated protein, PTP1C was no longer detected in the BCR complex after receptor engagement, suggesting that PTP1C dissociates from the activated receptor complex. Together these results suggest a critical role for PTP1C in modulating BCR signaling capacity, and they indicate that the PTP1C influence on B cell signaling is likely to be realized in both resting and activated cells.

Role of the Syk autophosphorylation site and SH2 domains in B cell antigen receptor signaling.

To explore the mechanism(s) by which the Syk protein tyrosine kinase participates in B cell antigen receptor (BCR) signaling, we have studied the function of various Syk mutants in B cells made Syk deficient by homologous recombination knockout. Both Syk SH2 domains were required for BCR-mediated Syk and phospholipase C (PLC)-gamma 2 phosphorylation, inositol 1,4,5-triphosphate release, and Ca2+ mobilization. A possible explanation for this requirement was provided by findings that recruitment of Syk to tyrosine-phosphorylated immunoglobulin (Ig) alpha and Ig beta requires both Syk SH2 domains. A Syk mutant in which the putative autophosphorylation site (Y518/Y519) of Syk was changed to phenylalanine was also defective in signal transduction; however, this mutation did not affect recruitment to the phosphorylated immunoreceptor family tyrosine-based activation motifs (ITAMs). These findings not only confirm that both SH2 domains are necessary for Syk binding to tyrosine-phosphorylated Ig alpha and Ig beta but indicate that this binding is necessary for Syk (Y518/519) phosphorylation after BCR ligation. This sequence of events is apparently required for coupling the BCR to most cellular protein tyrosine phosphorylation, to the phosphorylation and activation of PLC-gamma 2, and to Ca2+ mobilization.

Qualitative regulation of B cell antigen receptor signaling by CD19: selective requirement for PI3-kinase activation, inositol-1,4,5-trisphosphate production and Ca2+ mobilization.

Genetic ablation of the B cell surface glycoprotein CD19 severely impairs the humoral immune response. This requirement is thought to reflect a critical role of CD19 in signal transduction that occurs upon antigen C3dg coligation of antigen receptors with CD19 containing type 2 complement receptors (CR2). Here we show that CD19 plays a key accessory role in B cell antigen receptor signaling independent of CR2 coligation and define molecular circuitry by which this function is mediated. While CD19 is not required for antigen-mediated activation of receptor proximal tyrosines kinases, it is critical for activation of phosphatidylinositol 3-kinase (PI3-kinase). PI3-Kinase activation is dependent on phosphorylation of CD19 Y484 and Y515. Antigen-induced CD19-dependent PI3-kinase activation is required for normal phosphoinositide hydrolysis and Ca2+ mobilization responses. Thus, CD19 functions as a B cell antigen receptor accessory molecule that modifies antigen receptor signaling in a qualitative manner.

Differential susceptibility of neonatal and adult murine spleen cells to in vitro induction of B-cell tolerance.

The relative susceptibility of neonatal and adult murine splenocytes to induction of B-cell tolerance was studied in vitro. Adult cells required approximately 1,000-fold more trinitrophenyl-human gamma globulin to be rendered tolerant than did cells from 9- to 12-day-old neonates. The potential effects of suppressor T cells were excluded by pretreating the cultured B cells with anti-Thy-1 and C' and the helper T cells with anti-Ly-2.2 and C'. The possible role of cell surface immunoglobulin isotypes in contributing to this observed difference is discussed.

B-cell tolerance. IV. Differential role of surface IgM and IgD in determining tolerance susceptibility of murine B cells.

During ontogeny IgD appears later than IgM on splenocytes of neonatal mice (1) and at a time when mice develop a markedly increased immune responsiveness (2). Based on these observations, it was suggested that IgD serves as a "triggering" isotype for induction of immune responses, whereas surface IgM functions as a tolerizing receptor (3). To test this hypothesis, the susceptibility of adult splenocytes (which are predominantly mu(+)delta(+)[4-6]) and neonatal splenocytes (which bear predominantly IgM [mup(+); 1, 4-6]) to tolerance induction were compared. The results indicate that neonatal splenic B cells responsive to thymus dependent (TD) antigens are exquisitely susceptible to tolerance induction compared with those from adult mice (7-9). However, cells from both adult and neonatal mice were highly susceptible to tolerance induction when thymus independent (TI) antigen was used as immunogen (8). These results suggest that the major precursor for the TD response is a mu(+)delta(+)-cell which appears late in ontogeny and is resistant to tolerance induction and that the mup(+)-cell is the major precursor for the TI response and is highly susceptible to tolerance induction. Other differences between responders for TI and TD antigens have been described previously (10-12). To test this concept, adult splenocytes were treated with papain under conditions in which IgD, but not five other surface molecules, was removed (13). Such treated splenocytes were shown to be markedly susceptible to tolerance induction, resembling TD responders from neonatal animals. This experiment was interpreted as indicating that IgD confers resistance to tolerance induction on mu(+)delta(+)-cells. To prove this interpretation, it is necessary to show that specific removal of IgD with anti-delta also results in increased susceptibility to tolerance induction and that treatment with anti-mu does not have a similar effect. In the present studies, we have removed surface IgM or IgD by antibody-induced capping and assessed the tolerance susceptibility of the treated cells. Our results demonstrate that removal of IgD, but no IgM, from TD responders increases their susceptibility to tolerance induction.

B-cell tolerance. II. Trinitrophenyl human gamma globulin-induced tolerance in adult and neonatal murine B cells responsive to thymus-dependent and independent forms of the same hapten.

Neonatal splenic B cells which are responsive to thymus-dependent antigens (TD) are exquisitely susceptible to induction of tolerance (1,2). This state of tolerance is not mediated by suppressor T cells and is not a result of suboptimal macrophage function (1 and footnote one). In adult mice, induction of B-cell tolerance is not achieved with doses of antigen 1,000-fold higher (1) than those required to produce the same degree of unresponsiveness in neonates. In contrast to these results, studies with T-independent (TI) antigens indicate that neonatal and adult splenic B cells are equally susceptible to tolerance induction (3,4). However, such studies have not ascertained whether the neonate is more resistant to tolerance induction or the adult is hypersusceptible, i.e., does the induction of tolerance in cells responsive to TI antigens resemble that of adult or neonatal cells responsive to TD antigens? The answer is pertinent to determining the relative maturity of the B cells which can be tolerized or respond to TI or TD antigens. We report here the direct comparison of tolerogen sensitivity of adult and neonatal TD and TI responses by inducing tolerance in vitro with trinitophenyl human gamma globulin (TNP(17)HgG) and assaying unresponsiveness with TD and TI forms of the TNP determinant.

B-cell tolerance. III. Effect of papain-mediated cleavage of cell surface IgD on tolerance susceptibility of murine B cells.

Under defined conditions, papain removes IgD from cells while leaving IgM, H-2, Ia, Lyb-2, and complement receptor intact. The effect of such treatment with papain on the induction of tolerance in murine splenic B cells was determined in an in vitro system. Treatment of the cells with papain has no effect on subsequent antibody responsiveness presumably because surface receptors regenerate before and during incubation with immunogen. Removal of increasing amounts of IgD results in increasing susceptibility of thymus-dependent responsive cells to tolerance induction. The tolerance susceptibility of thymus-independent responsive cells, which we have previously suggested are immature cells that bear only IgM, is unaffected by cleavage of IgD. If cells are incubated for 24 h after treatment with papain, cell surface IgD and tolerance resistance return. These results indicate that a surface molecule affects susceptibility of B cells to induction of tolerance and suggest that this molecule may be IgD.

Distinct structural compartmentalization of the signal transducing functions of major histocompatibility complex class II (Ia) molecules.

Class II major histocompatibility complex encoded proteins (MHC class II or Ia molecules) are principal plasma membrane proteins involved in activation of both B and T cells during antigen-driven immune responses. Recent data indicate that class II molecules are more than simply recognition elements that provide a ligand for the T cell antigen receptor. Changes in B cell physiology that follow class II binding are now recognized as being required not only for the induction of T cell activation, but also for B cell activation and proliferation. It is interesting to note that class II molecules appear to transduce signals via two distinct mechanisms depending upon the differentiative state of the B cell on which they are expressed. While one of these pathways, involving cAMP generation and protein kinase C localization in the cytoskeletal/nuclear compartment, is seen in resting B cells, the second is seen in primed B cells and involves tyrosine kinase activation, inositol lipid hydrolysis, and Ca2+ mobilization. Use of this pathway is correlated with ability of class II to transduce signals leading to B cell proliferation. To begin to address the molecular basis of this unique, activation-dependent, differential coupling of class II to signaling pathways, we conducted mutational analysis of class II structural requirements for signal transduction. Here we report that the cytoplasmic (Cy) domains of I-Ak class II molecules are not required for either receptor-mediated activation of protein tyrosine phosphorylation or Ca2+ mobilization. This is in contrast to the requirement of the Cy domain of beta chain of class II for the alternate signaling pathway and efficient antigen presentation to autoreactive T cell lines. Disparate distribution of functional motifs within the MHC class II molecules may reflect use of distinct receptor associated effector molecules to sustain different modes of signal transduction in various class II-expressing cells.

Ligand-independent signaling functions for the B lymphocyte antigen receptor and their role in positive selection during B lymphopoiesis.

Signal transduction through the B cell antigen receptor (BCR) is determined by a balance of positive and negative regulators. This balance is shifted by aggregation that results from binding to extracellular ligand. Aggregation of the BCR is necessary for eliciting negative selection or activation by BCR-expressing B cells. However, ligand-independent signaling through intermediate and mature forms of the BCR has been postulated to regulate B cell development and peripheral homeostasis. To address the importance of ligand-independent BCR signaling functions and their regulation during B cell development, we have designed a model that allows us to isolate the basal signaling functions of immunoglobulin (Ig)alpha/Igbeta-containing BCR complexes from those that are dependent upon ligand-mediated aggregation. In vivo, we find that basal signaling is sufficient to facilitate pro-B --> pre-B cell transition and to generate immature/mature peripheral B cells. The ability to generate basal signals and to drive developmental progression were both dependent on plasma membrane association of Igalpha/Igbeta complexes and intact immunoregulatory tyrosine activation motifs (ITAM), thereby establishing a correlation between these processes. We believe that these studies are the first to directly demonstrate biologically relevant basal signaling through the BCR where the ability to interact with both conventional as well as nonconventional extracellular ligands is eliminated.

Interleukin-induced increase in Ia expression by normal mouse B cells.

The constitutive culture supernatant (SN) of the macrophage tumor line P388D1 (P388 SN) and the concanavalin A (Con A)-induced culture supernatant of the T cell hybridoma FS6-14.13 (FS6 Con A SN) were shown to contain nonspecific factors capable of inducing increased Ia expression by normal resting B cells in a dose-dependent manner. In six consecutive experiments the relative increase in Ia expression induced by P388 SN was 4.9 +/- 0.9, with FS6 Con A SN 10.7 +/- 1.5, and with a combination of both preparations 13.0 +/- 1.7. This increase in Ia expression was observed to occur in virtually all the B cells, reaching maximum levels within 24 h of culture. The interleukin-induced increase in B cell Ia expression occurred in the absence of ancillary signals provided by ligand-receptor Ig cross-linking and despite the fact that virtually all the control B cells, cultured in the absence of factors, remained in G0. These results suggest that functional receptors for at least some interleukins are expressed on normal resting B cells and their effects can be manifest in the absence of additional activating signals. The increased Ia expression induced by the nonspecific factor preparations was shown to be correlated with enhanced antigen-presenting capacity by the B cells to T cell hybridomas. The nature of the interleukins responsible for these effects remains to be definitively determined, however, the activity of FS6 Con A SN was shown to correlate with B cell growth factor activity and increased B cell Ia expression was not observed using interleukin 2 (IL-2) or interferon-gamma, prepared by recombinant DNA technology.

Anti-immunoglobulin M activates nuclear calcium/calmodulin-dependent protein kinase II in human B lymphocytes.

We and others have previously shown that the nuclear protein, Ets-1, is phosphorylated in a calcium-dependent manner after ligation of immunoglobulin (Ig) M on B lymphocytes. As this phosphorylation was independent of protein kinase C activity, we tested whether a calcium/calmodulin-dependent protein kinase (CaM kinase) might phosphorylate the Ets-1 protein after elevation of intracellular free calcium concentrations. The dephosphorylated form of Ets-1 has been shown to bind to chromatin, suggesting that the operative kinase should be detectable in the nucleus. We prepared nuclear extracts from two human B cell lines in which increased intracellular free calcium levels correlated with increased phosphorylation of the Ets-1 protein. Activity of the CaM kinases was determined using a synthetic peptide substrate both in the absence and presence of an inhibitor specific for the CaM kinase family, KN-62. Stimulation of cells with anti-IgM led to increased activity of a nuclear kinase that could phosphorylate the peptide, and this activity was reduced by 10 microM KN-62. Kinase activity was reduced in lysates preadsorbed using an antibody specific for CaM kinase II. Two-dimensional phosphopeptide maps of the Ets-1 protein from cells incubated with ionomycin or anti-IgM contained two unique phosphopeptides that were absent in untreated cells. Incubation of isolated Ets-1 protein with purified CaM kinase II produced phosphorylation of peptides that migrated identically to those found in cells incubated with either anti-IgM or ionomycin. These data suggest a model of signal transduction by the antigen receptor on B lymphocytes in which increased intracellular free calcium can rapidly activate nuclear CaM kinase II, potentially resulting in phosphorylation and regulation of DNA-binding proteins.

Antigens varying in affinity for the B cell receptor induce differential B lymphocyte responses.

The B cell receptor (BCR) triggers a variety of biological responses that differ depending upon the properties of the antigen. A panel of M13 phage-displayed peptide ligands with varying affinity for the 3-83 antibody was generated to explore the role of antigen-BCR affinity in cell activation studies using primary 3-83 transgenic mouse B cells. Multiple parameters of activation were measured. T cell-independent B cell proliferation, antibody secretion, induction of germline immunoglobulin gamma1 transcripts, and B cell production of interleukin (IL) 2 and interferon gamma responses were better correlated with antigen-BCR affinity than with receptor occupancy. In contrast, other responses, such as upregulation of major histocompatibility complex class II and B7.2 (CD86), secretion of IL-6, and B cell proliferation in the context of CD40 signaling were only weakly dependent on antigen affinity. Biochemical analysis revealed that at saturating ligand concentrations the ability of phage to stimulate some early signaling responses, such as Ca++ mobilization and tyrosine phosphorylation of syk or Igalpha, was highly affinity dependent, whereas the ability to stimulate Lyn phosphorylation was less so. These data suggest that the BCR is capable of differential signaling. The possibility that differential BCR signaling by antigen determines whether an antibody response will be T independent or dependent is discussed.

Interference with immunoglobulin (Ig)alpha immunoreceptor tyrosine-based activation motif (ITAM) phosphorylation modulates or blocks B cell development, depending on the availability of an Igbeta cytoplasmic tail.

To determine the function of immunoglobulin (Ig)alpha immunoreceptor tyrosine-based activation motif (ITAM) phosphorylation, we generated mice in which Igalpha ITAM tyrosines were replaced by phenylalanines (Igalpha(FF/FF)). Igalpha(FF/FF) mice had a specific reduction of B1 and marginal zone B cells, whereas B2 cell development appeared to be normal, except that lambda1 light chain usage was increased. The mutants responded less efficiently to T cell-dependent antigens, whereas T cell-independent responses were unaffected. Upon B cell receptor ligation, the cells exhibited heightened calcium flux, weaker Lyn and Syk tyrosine phosphorylation, and phosphorylation of Igalpha non-ITAM tyrosines. Strikingly, when the Igalpha ITAM mutation was combined with a truncation of Igbeta, B cell development was completely blocked at the pro-B cell stage, indicating a crucial role of ITAM phosphorylation in B cell development.