Surface mu heavy chain signals down-regulation of the V(D)J-recombinase machinery in the absence of surrogate light chain components.

Early B cell development is characterized by stepwise, ordered rearrangement of the immunoglobulin (Ig) heavy (HC) and light (LC) chain genes. Only one of the two alleles of these genes is used to produce a receptor, a phenomenon referred to as allelic exclusion. It has been suggested that pre-B cell receptor (pre-BCR) signals are responsible for down-regulation of the VDJH-recombinase machinery (Rag1, Rag2, and terminal deoxynucleotidyl transferase [TdT]), thereby preventing further rearrangement on the second HC allele. Using a mouse model, we show that expression of an inducible muHC transgene in Rag2-/- pro-B cells induces down-regulation of the following: (a) TdT protein, (b) a transgenic green fluorescent protein reporter reflecting endogenous Rag2 expression, and (c) Rag1 primary transcripts. Similar effects were also observed in the absence of surrogate LC (SLC) components, but not in the absence of the signaling subunit Ig-alpha. Furthermore, in wild-type mice and in mice lacking either lambda5, VpreB1/2, or the entire SLC, the TdT protein is down-regulated in muHC+LC- pre-B cells. Surprisingly, muHC without LC is expressed on the surface of pro-/pre-B cells from lambda5-/-, VpreB1-/-VpreB2-/-, and SLC-/- mice. Thus, SLC or LC is not required for muHC cell surface expression and signaling in these cells. Therefore, these findings offer an explanation for the occurrence of HC allelic exclusion in mice lacking SLC components.

Selection of Ig mu heavy chains by complementarity-determining region 3 length and amino acid composition.

Although it is generally accepted that Ig heavy chains (HC) are selected at the pre-B cell receptor (pre-BCR) checkpoint, the characteristics of a functional HC and the role of pre-BCR assembly in their selection have remained elusive. We determined the characteristics of HCs that successfully passed the pre-BCR checkpoint by examining transcripts harboring V(H)81X and J(H)4 gene segments from J(H)(+/-) and lambda5(-/-)mice. V(H)81X-J(H)4-HC transcripts isolated from cells before or in the absence of pre-BCR assembly had no distinguishing complementarity-determining region 3 traits. In contrast, transcripts isolated subsequent to passage through the pre-BCR checkpoint had distinctive complementarity-determining regions 3 of nine amino acids in length (49%) and a histidine at position 1 (73%). Hence, our data define specific structural requirements for a functional HC, which is instrumental in shaping the diverse B cell repertoire.

Immunoglobulin mu heavy chains do not mediate tyrosine phosphorylation of Ig alpha from the ER-cis-Golgi.

Signals delivered by Ig receptors guide the development of functional B lymphocytes. For example, clonal expansion of early mu heavy chain ( mu HC)-positive pre-B cells requires the assembly of a signal-competent pre-B cell receptor complex (pre-BCR) consisting of a mu HC, a surrogate L chain, and the signal dimer Ig alpha beta. However, only a small fraction of the pre-BCR is transported to the cell surface, suggesting that pre-BCR signaling initiates already from an intracellular compartment, e.g., the endoplasmic reticulum (ER). The finding that differentiation of pre-B cells and allelic exclusion at the IgH locus take place in surrogate L chain-deficient mice further supports the presence of a mu HC-mediated intracellular signal pathway. To determine whether a signal-competent Ig complex can already be assembled in the ER, we analyzed the consequence of pervanadate on tyrosine phosphorylation of Ig alpha in J558L plasmacytoma and 38B9 pre-B cells transfected with either a transport-competent IgL chain-pairing or an ER-retained nonpairing micro HC. Flow cytometry, combined Western blot-immunoprecipitation-kinase assays, and confocal microscopy revealed that both the nonpairing and pairing mu HC assembled with the Ig alpha beta dimer; however, in contrast to a pairing mu HC, the nonpairing mu HC was retained in the ER-cis-Golgi compartment, and neither colocalized with the src kinase lyn nor induced tyrosine phosphorylation of Ig alpha after pervanadate treatment of cells. On the basis of these findings, we propose that a signal-competent Ig complex consisting of mu HC, Ig alpha beta, and associated kinases is assembled in a post-ER compartment, thereby supporting the idea that a pre-BCR must be transported to the cell surface to initiate pre-BCR signaling.

Cutting edge: signaling and cell surface expression of a mu H chain in the absence of lambda 5: a paradigm revisited.

Pre-B cell receptor (pre-BCR) signals are essential for pro-B cells to mature efficiently into pre-B cells. The pre-BCR is an Ig-like transmembrane complex that is assembled from two mu H chains (mu HC) and two surrogate L chains consisting of the non-covalently associated polypeptides VpreB and lambda5. In lambda5(-/-) mice, pro-B cell maturation is impaired, but not completely blocked, implying that a mu HC induces differentiation signals in the absence of lambda5. Using a mouse model, in which transgenic mu HC expression can be controlled by tetracycline, we show that in the absence of lambda5, the transgenic mu HC promotes in vivo differentiation of pro-B cells, induces IL-7-dependent cell growth, and is expressed on the surface of pre-B cells. Our findings not only show that an incomplete pre-BCR can initiate signals, but also challenge the paradigm that an IgHC must associate with an IgLC or a SLC to gain transport and signaling competency.

Cytoplasmic micro heavy chain confers sensitivity to dexamethasone-induced apoptosis in early B-lineage acute lymphoblastic leukemia.

Most childhood acute lymphoblastic leukemia (ALL) arises from early B-lineage cells,and response to steroid treatment is critical to successful ALL therapy. To investigate the effect of the pre-B cell receptor (pre-BCR) complex on the response of leukemic cells to steroids, cytoplasmic micro protein (cyto mu) was transfected into cyto mu-, steroid-resistant early B cell lines. The presence of cyto mu and the assembled pre-BCR complex conferred sensitivity to dexamethasone-induced apoptosis. Both intrinsic and extrinsic apoptosis pathways are involved in this cell death. However, if the transfected cyto micro protein is unable to assemble the pre-BCR complex, the cells remain resistant to dexamethasone. These findings suggest a role for the pre-BCR complex in the response of ALL cells to treatment and provide insight into the mechanism of steroid response in the treatment of pre-B ALL.

An Ig mu-heavy chain transgene inhibits systemic lupus erythematosus immunopathology in autoimmune (NZB x NZW)F1 mice.

Intrinsic defects in the B lymphoid lineage are involved in predisposition for systemic lupus erythematosus in (NZB x NZW)F(1) (NZB/W) mice. In addition, a contribution of CD4(+) T cells has been shown to be crucial for the development of fatal glomerulonephritis. To further dissect the role of B and T cells in lupus immunopathology we used Ig mu-heavy chain (muHC) transgenic (Tg) NZB/W mice that we recently established to study mechanisms of B cell tolerance. The Tg NZB/W mice have a very restricted B cell repertoire and only a very minor population of B cells having endogenously rearranged muHC Ig loci are able to undergo isotype switch. Here we analyzed the influence of the restricted B cell repertoire on the development of IgG anti-DNA antibodies and glomerulonephritis as well as the hyperactivation of T(h) cells. IgG anti-DNA antibodies developed delayed but consistently in the Tg NZB/W mice, suggesting that a strong selective mechanism for the development of these autoantibodies is operative. Despite significant autoantibody titers in Tg NZB/W mice, very little immune deposits in the glomeruli and no evidence for renal inflammation were found. The Tg mice have a significantly prolonged survival time and most of the Tg mice lived much longer than 1 year. Interestingly, the generalized T cell activation that normally correlates and coincides with the progression of the disease in NZB/W mice is strongly reduced in older Tg animals. The absence of IgG3 anti-DNA antibodies and the strong reduction of IgG2a anti-DNA antibodies in the Tg mice suggests that particularly the activation of T(h)1 cells is inhibited. This result shows that a significant restriction in the B cell repertoire prevents hyperactivation of T(h) cells and supports the model that T cell hyperactivation in NZB/W mice is secondary to specific interactions with a subpopulation of presumably autoreactive B lymphocytes.

The level of expression of mu heavy chain modifies the composition of peripheral B cell subpopulations.

The B cell receptor (BCR) has a decisive role in transducing signals required for the development of B cells and their survival in the periphery. However, the processes that initiate these signals remain unclear and concepts of constitutive and ligand-dependent signaling have been proposed. Using a mu-transgenic mouse model, we have analyzed the impact of high surface IgM expression on the composition of the splenic B cell population. kappa-deficient mice homozygous for the H3-mu transgene have B cells with a higher BCR surface density than H3 heterozygous mice. This higher BCR expression is associated with an increase in the percentage and the total number of splenic B cells. In addition, an important proportion of CD23(-)CD21(+) marginal zone (MZ) B cells can be observed in H3 homozygous mice. However, these modifications operate in the absence of impairment of the positive selection process of the H3-mu/lambda1 combination over the H3-mu/lambda2 + 3 ones. These results suggest that (i) a constitutive BCR signaling directly correlated with BCR surface density is responsible for the efficient B cell colonization of the periphery with an accumulation of B cells in the MZ and (ii) a ligand-dependent BCR signal is responsible for the clonotype composition of the mature B cell repertoire.

Interleukin-4 transgenic mice develop glomerulosclerosis independent of immunoglobulin deposition.

Mice with constitutive transgenic (tg) expression of IL-4 develop autoimmune-type disorders resembling human lupus nephritis. The kidneys show progressive glomerulosclerosis with immunoglobulin (Ig) and complement deposition. This study investigated the roles of renal IL-4 expression and glomerular Ig deposition in the pathogenesis of glomerulosclerosis in IL-4 tg mice. Treatment of these mice with IL-4 neutralizing antibody prevented renal disease. IL-4 tg mice treated with methylprednisolone (MP) showed increased mesangial collagen deposition with only trace amounts of glomerular Ig. To analyze the relevance of Ig deposition in the development of the renal lesions, IL-4 tg mice were cross-bred with mu chain-deficient mice (muMT-/-), which are unable to produce Ig. IL-4 tg/muMT-/- mice developed progressive glomerulosclerosis with mesangial accumulation of collagen types I, IV and V despite complete absence of glomerular Ig deposits. Renal IL-4 expression was observed in both anti-IL-4- and MP-treated IL-4 tg mice as well as in IL-4 tg/muMT-/- mice. No statistical difference in the number of glomerular T cells and macrophages between any of the groups was evident. Our data demonstrate that in this model glomerulosclerosis can develop independently of and prior to Ig deposition, and suggest that the initial accumulation of glomerular extracellular matrix is due to renal IL-4 expression. Our results point to a novel mechanism for the development of glomerulosclerosis which may have implications for human disease.

Inhibition of intracellular transport of B cell antigen receptor complexes by Kaposi's sarcoma-associated herpesvirus K1.

The B cell antigen receptor (BCR) is a large complex that consists of a disulfide-linked tetramer of two transmembrane heavy (mu) chains and two light (lambda or kappa) chains in association with a heterodimer of Igalpha and Igbeta. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a transforming protein called K1, which has structural and functional similarity to Igalpha and Igbeta. We demonstrate that K1 downregulates the expression of BCR complexes on the surface. The NH(2)-terminal region of K1 specifically interacts with the mu chains of BCR complexes, and this interaction retains BCR complexes in the endoplasmic reticulum, preventing their intracellular transport to the cell surface. Thus, KSHV K1 resembles Igalpha and Igbeta in its ability to induce signaling and to interact with mu chains of the BCR. However, unlike Igalpha and Igbeta, which interact with mu chains to direct BCR complexes to the cell surface, K1 interacts with mu chains to block the intracellular transport of BCR complexes to the cell surface. These results demonstrate a unique feature of the K1 transforming protein, which may confer virus-infected cells with a long-term survival advantage.

Investigation of anti-WI-1 adhesin antibody-mediated protection in experimental pulmonary blastomycosis.

Infection with Blastomyces dermatitidis elicits strong antibody responses to the surface adhesin WI-1. The antibodies are directed chiefly against the adhesive domain, a 25-amino-acid repeat. Tandem-repeat-specific monoclonal antibodies (mAbs) were studied for their opsonic activity in vitro and their capacity to adoptively transfer protection in murine experimental blastomycosis. mAbs to WI-1 enhanced binding and entry of B. dermatitidis yeasts into J774. 16 cells but did not enhance killing or growth inhibition of the yeast. Passive transfer of 8 mAbs to WI-1 into 3 different inbred strains of mice also did not improve the course of experimental infection and sometimes worsened it. mu-deficient mice were more resistant to experimental blastomycosis than were intact littermates, and passive transfer of the mAbs into these mice did not protect them against experimental infection. Thus, antibody to WI-1 does not appear to improve the outcome of murine blastomycosis and may enhance the infection.

Mutations in Igalpha (CD79a) result in a complete block in B-cell development.

Mutations in Btk, mu heavy chain, or the surrogate light chain account for 85-90% of patients with early onset hypogammaglobulinemia and absent B cells. The nature of the defect in the remaining patients is unknown. We screened 25 such patients for mutations in genes encoding components of the pre-B-cell receptor (pre-BCR) complex. A 2-year-old girl was found to have a homozygous splice defect in Igalpha, a transmembrane protein that forms part of the Igalpha/Igbeta signal-transduction module of the pre-BCR. Studies in mice suggest that the Igbeta component of the pre-BCR influences V-DJ rearrangement before cell-surface expression of mu heavy chain. To determine whether Igalpha plays a similar role, we compared B-cell development in an Igalpha-deficient patient with that seen in a mu heavy chain-deficient patient. By immunofluorescence, both patients had a complete block in B-cell development at the pro-B to pre-B transition; both patients also had an equivalent number and diversity of rearranged V-DJ sequences. These results indicate that mutations in Igalpha can be a cause of agammaglobulinemia. Furthermore, they suggest that Igalpha does not play a critical role in B-cell development until it is expressed, along with mu heavy chain, as part of the pre-BCR.

The C(H)1 and transmembrane domains of mu in the context of a gamma2b transgene do not suffice to promote B cell maturation.

Mice carrying a gamma2b transgene have been shown previously to be deficient in B cell development. In particular, a developmental block exists at the pre-B cell stage. The few B cells that develop all express endogenous micro heavy chains. The phenotype suggests that gamma2b exerts a strong feedback inhibition on endogenous Ig gene rearrangement, but, unlike micro, cannot support further B cell development. In this study we have created hybrid transgenes between gamma2b and micro. Transgenic mice with a C(H)1 domain of micro, or both a C(H)1 and transmembrane/cytoplasmic domain of micro replacing the respective domains of a gamma2b transgene, have the same B cell defect as gamma2b transgenic mice. Interestingly, the severity of the defect is correlated with the level of expression of the transgene, suggesting that the degree of feedback inhibition of Ig gene rearrangement depends on the level and timing of Ig production. Crossing the gamma2b/micro transgenes into a Bcl-x(L) transgenic line allows immature gamma2b B cells to survive, but not to develop to maturity. Therefore, the missing function of micro is not simply an anti-apoptotic effect.

The Ig heavy chain intronic enhancer core region is necessary and sufficient to promote efficient class switch recombination.

The intronic IgH enhancer E(mu), which consists of the core enhancer (cE(mu) flanked by 5' and 3' matrix attachment regions (MAR), has been implicated in the control of IgH locus recombination and transcription. Both cE(mu) and the MAR are required to enhance transcription of an IgH transgene. To elucidate the regulatory functions of cE(mu) versus its associated MAR in IgH class switch recombination (CSR), we have assayed ES cell lines which have targeted deletions of these elements, both individually and in combination, by the Rag-2-deficient blastocyst complementation method. Mutant B cells from chimeric mice were activated in culture and the influence of the mutations on CSR was assessed by analysis of B cell hybridomas. We find that the cE(mu) is necessary and sufficient for providing the functions of E(mu) required for efficient CSR at the IgH locus. However, the 5' and 3' MAR sequences, as well as the known I(mu) transcription start sites and the bulk of I(mu) coding sequences, were dispensable for the process.

Biogenesis and function of IgM: the role of the conserved mu-chain tailpiece glycans.

The tailpiece of secretory Ig-mu-chains (mu(s)tp) is highly conserved throughout evolution: in particular, a carboxy-terminal cysteine residue (Cys575) and a glycan linked to Asn563 are found in all species sequenced so far. Here we show that the mu(s)tp oligosaccharide moieties are important for the binding of J-chains and for the process of IgM polymerization. In the absence of the mu(s)tp glycans, pentamers cannot be assembled and polymers containing six or more subunits are secreted. Despite their increased valency, these molecules have a lower association rate with antigen than wild-type polymers. Unexpectedly, the C-terminal oligosaccharides also affect kinetic parameters on unpolymerized subunits. Thus, monomers lacking the C-terminal sugars because of either site-directed mutagenesis or selective enzymatic deglycosylation with endoglycosidase H, have a lower k(on) for the antigen. Taken together, our results indicate that the C-terminal mu-chain glycans can shape the structure of mu(s2)L2 subunits and their further assembly into polymers.

Role of mu heavy chain in B cell development. I. Blocked B cell maturation but complete allelic exclusion in the absence of Ig alpha/beta.

There is good evidence for a signaling role played by Ig heavy chain in the developmental transition through the pre-B cell stage. We have previously described signal-capable or signal-incapable mutants of mu heavy chain in which a signaling defect is caused by failure to associate with the Ig alpha/beta heterodimer. To further characterize the role of Ig heavy chain-mediated signaling in vivo, as well as in B cell development and allelic exclusion, we have created transgenic mice in which the B cells express these signal-capable and signal-incapable mutant mu chains. Failure of mu to signal via Ig alpha/beta results in a block in B cell development in mice expressing the signal-incapable mu. A small number of B cells in these animals do escape the developmental block and are expressed in the spleen and the periphery as B220+ transgenic IgM+ cells. These cells respond to LPS by proliferating but show no response to T-independent-specific Ag. In contrast, B cells expressing the signal-capable B cell receptor show a strong signaling response to Ag-specific stimulus. There is no Ig alpha seen in association with signal-deficient IgM. Thus, the B cell receptor complex is not assembled, and no signal can be delivered. Despite the block in developmental signaling, allelic exclusion is complete. There is no detectable coexpression of transgenic IgM and endogenous murine IgM, nor is there rearrangement of the endogenous heavy chain genes. This suggests that differing signaling mechanisms are responsible for the developmental transition and allelic exclusion and thus allows for separate examination of these signaling mechanisms.

Roles of calnexin and Ig-alpha beta interactions with membrane Igs in the surface expression of the B cell antigen receptor of the IgM and IgD classes.

Membrane Igs (mIgs) of all five classes are associated with Ig-alpha beta dimers on the B cell surface. While mIgM requires the presence of these two associated molecules for its surface expression, mIgD does not. To study the structural basis for this differential Ig-alpha beta dependence, we created mutant mIgM and mIgD molecules (chimeras and those with reciprocal point mutations in their transmembrane sequences) and identified two amino acid residues in the transmembrane region of the mIg heavy chains responsible for this transport difference. Without Ig-alpha beta, mIgM and mutant mIgD molecules remained endoglycosidase H sensitive, consistent with endoplasmic reticulum (ER) localization. The molecular chaperone calnexin has previously been implicated in retaining unassembled mIgs in the ER. However, we found that inhibition of the association between calnexin and newly synthesized mIgs by castanospermine treatment did not lead to surface expression of normally retained mIgs. Therefore, calnexin cannot be the only retention molecule. Furthermore, for both wildtype and mutant mIgDs, association with calnexin was rather transient, thereby ruling out calnexin being a major ER retention molecule for mIgDs. Our study with castanospermine also showed that calnexin is required for wildtype mIgD surface expression only if Ig-alpha beta is absent, while the latter alone can function to promote mIg folding, assembly, and transport. Further study using our system will help to identify novel molecules and characterize their involvement in the control of mIg transport.

Gamma 2b provides only some of the signals normally given via mu in B cell development.

B cell development is a complex process involving interactions between B cell precursors, stroma, and known and unknown ligands and cytokines. In order to more fully understand the requirements for Ig in that development we have created transgenic mice that carry a gamma 2b transgene and express it early in B cell development. Previously it was believed that these B cells arrested in their development prior to the pro- to pre-B cell transition. We show here that in conventional gamma 2b mice, B cell development actually arrests later, at the pre-B cell stage. This shows for the first time that a constant region different from mu can allow signaling through the pre-B cell receptor, but cannot promote complete development. The pro- and pre-B cells in the conventional gamma 2b transgenics are not fully functional since they cannot grow in IL-7 without stromal cells. This is a novel phenotype, separating development from stroma independence. The few, mature B cells that do develop in these mice express both mu and gamma 2b simultaneously, and are CD5+. Expression of a Bcl-2 transgene allows survival of gamma 2b transgenic immature B cells, but does not promote full maturation, indicating that normally mu provides both an anti-apoptotic signal and a differentiation signal. One line of gamma 2b mice, the C line, does not have this phenotype. B cell development is accelerated in this unconventional line, and the developing B cells have a very different phenotype from both normal mice and conventional gamma 2b mouse lines, but are very similar to mu transgenics. Mature B cells are largely CD5-, gamma 2b-only expressing. This unique phenotype is apparently due to the activation in B cell precursors of a gene at the insertion site of the transgene, circumventing the need for mu. Comparison of conventional gamma 2b transgenics with the C line and mu transgenics reveals the multiple signals required throughout B cell development.

Control of the sizes and contents of precursor B cell repertoires in bone marrow.

Ordered rearrangements of immunoglobulin (Ig) gene loci, first as DH to JH, then as VH to DHJH, and finally as VL to JL segment-specific recombinations occur 'in-frame' and 'out-of-frame'. 'In-frame' rearrangements lead to the expression of truncated DHJH-microC proteins and to microH chains. These H chain proteins have two major effects on precursor B cells. They suppress (as DJC mu proteins) or enhance (as full microH chain) the proliferation of precursor cells at the point where these precursors express these proteins. At the same time, they signal allelic exclusion of the microH chain alleles, so that VH to DHJH rearrangement at the second allele is suppressed. Regulation of precursor B cell proliferation and H chain allelic exclusion is mediated by a pre-B cell receptor that is composed of the microH chains and a surrogate L chain. This surrogate L chain is made up of two proteins encoded by the Vpre-B and lambda 5 genes that are expressed only at the early precursor cell stages just before and when H chain genes are first expressed. They are not found in later B cell development, when L chains are expressed, nor in any other cell of the body tested so far. The physiological roles of surrogate L chain and of the pre-B receptor have been clarified by generating mutant mice in which the lambda 5 gene has been inactivated by targeted disruption. Molecular mechanisms and cellular developments, by which the pre-B receptor controls proliferation and allelic exclusion, are discussed.

Constitutive Bcl-2 expression during immunoglobulin heavy chain-promoted B cell differentiation expands novel precursor B cells.

To test for effects on B cell differentiation, we introduced immunoglobulin mu heavy chain (HC) and Bcl-2 transgenes, separately or together, into recombination-activating gene 2 (RAG-2)-deficient mice. Transgenic Bcl-2 expression led to increased numbers of RAG-deficient pro-B cells, but did not promote their further differentiation. Expression of the mu HC transgene promoted the differentiation of RAG-deficient pro-B cells into pre-B cells that also expressed certain differentiation markers characteristic of even more mature B cell stages. However, the extent of the mu HC-dependent differentiation effects was greatly enhanced by coexpression of the transgenic Bcl-2 gene, and a subset of pre-B cells from both HC and HC, Bcl-2-transgenic RAG-2-deficient animals expressed surface mu HCs that were functional as judged by cross-linking experiments. These experiments demonstrate that the pro-B to pre-B transition in vivo cannot be effected by the expression of Bcl-2 alone, and that nontransformed immature B-lineage cells are competent to receive signals through a surface mu complex.

Atypical disease after Bordetella pertussis respiratory infection of mice with targeted disruptions of interferon-gamma receptor or immunoglobulin mu chain genes.

Using a murine respiratory challenge model we have previously demonstrated a role for Th1 cells in natural immunity against Bordetella pertussis, but could not rule out a role for antibody. Here we have demonstrated that B. pertussis respiratory infection of mice with targeted disruptions of the genes for the IFN-gamma receptor resulted in an atypical disseminated disease which was lethal in a proportion of animals, and was characterized by pyogranulomatous inflammation and postnecrotic scarring in the livers, mesenteric lymph nodes and kidneys. Viable virulent bacteria were detected in the blood and livers of diseased animals. An examination of the course of infection in the lung of IFN-gamma receptor-deficient, IL-4-deficient and wild-type mice demonstrated that lack of functional IFN-gamma or IL-4, cytokines that are considered to play major roles in regulating the development of Th1 and Th2 cells, respectively, did not affect the kinetics of bacterial elimination from the lung. In contrast, B cell-deficient mice developed a persistent infection and failed to clear the bacteria after aerosol inoculation. These findings demonstrate an absolute requirement for B cells or their products in the resolution of a primary infection with B. pertussis, but also define a critical role for IFN-gamma in containing bacteria to the mucosal site of infection.