B cells are anergic in transgenic mice that express IgM anti-DNA antibodies

B lymphocytes in individuals with systemic lupus erythematosus (SLE) secrete pathogenic auto antibodies to DNA which cause clinical nephritis. (NZB x NZW) Fl (BW) female mice also secrete pathogenic anti-DNA auto antibodies, and therefore are considered to be an animal model of SLE. The rearranged immunoglobulin (Ig) genes that encode an anti-DNA antibody from a diseased BW mouse have been cloned, and transgenic (Tg) mice have been created by microinjection of these constructs into fertilized eggs from normal mice. As we reported previously, when the construct contains the Cγ2a heavy chain constant (C_H) region, the mice spontaneously secrete anti-DNA IgG and they develop mild nephritis. This demonstrated that the Ig encoded by the transgene is pathogenic. In contrast, here we report that when the construct contains the same anti-DNA Ig variable (V) regions used previously, along with the Cμ region, the auto reactive B cells are rendered tolerant. Most B cells in the Tg mice express the μ transgene product on their surface, and rearrangement of endogenous light chain genes is partially suppressed. Furthermore, most hybridomas made from Tg B cells secrete IgM anti-DNA. Despite this, the Tg mice have reduced levels of total serum Ig and they do not secrete anti-DNA IgM either spontaneously or following immunization with DNA. We conclude that most B cells in the Tg mice have been rendered anergic. Anergy is however reversible in vitro; lipopolysaccharide stimulation of Tg B cells leads to the production of a significant amount of IgM anti-DNA antibody. The studies demonstrate that in this line of Tg mice on a normal mouse genetic background potentially pathogenic B cells that express a high-affinity Ig specific for a natural autoantigen are subject to tolerance by induction of anergy.     

Evidence for selective pressure in the appearance of monoclonal immunoglobulins during aging: Studies in M54 μ-transgenic mice

Serum monoclonal immunoglobulins (M-Ig) appear during aging but little is known about the immunological factors which lead to their development. We have investigated whether such M-Ig occur as a clonally random process or result from V-region-directed selective pressures. We have analyzed a μ-transgenic mouse strain in which over 95% of all splenic B cells express the transgenic μ chain. All endogenous repertoire and unchain diversity are generated from the 5% of the B cells which express endogenous μ chains. Not one of the M-Ig detected in these mice were of transgene origin alone; 11 of the 14 M-Ig did not express a μ- chain and none of the μ chain containing M-Ig expressed the transgene allotype alone. This observation suggests that the B cells giving rise to M-Ig are heavily selected from among the small number of B cells which express endogenous Ig. The selective factors that might act on the endogenous B cell pools are discussed.     

Immune mechanisms leading to abnormal B cell selection and activation in New Zealand Black mice

Polyclonal B cell activation is a hallmark of the immune dysregulation in New Zealand Black (NZB) mice. We have previously shown that the splenic B cell activation is associated with increased CD80 expression. Here we show that abnormal expansions of CD80-expressing GC, CD5(+), marginal zone (MZ) precursor and MZ B cells produce this increase. To investigate the role of BCR engagement in the generation and activation of these populations, a non-self-reactive Ig Tg was introduced onto the NZB background. NZB Ig-Tg mice lacked Tg CD5(+) and peanut agglutinin(+) B cells, confirming the role of endogenous Ag in their selection. Although the increased proportion of MZ B cells was retained in NZB Ig-Tg mice, CD80 expression on these cells was reduced as compared to non-Tg NZB mice, suggesting a role for BCR engagement with endogenous Ag in their activation. Examination of CD40L-knockout NZB mice showed no difference in the abnormal activation or selection of the B cell populations, with the exception of GC cells, as compared to wild-type NZB mice. Thus, polyclonal B cell activation in NZB mice does not require CD40 engagement, but results, in part, from dysregulated BCR-specific mechanisms.     

B-1a and B-1b cells exhibit distinct developmental requirements and have unique functional roles in innate and adaptive immunity to S. pneumoniae

B-1a and B-1b lymphocytes were found to exhibit specialized roles in providing immunity to Streptococcus pneumoniae and differ dramatically in their developmental requirements. Transgenic mice overexpressing CD19 (hCD19Tg) generated B-1a cells and natural antibodies that provided protection during infection, while CD19-deficient (CD19(-/-)) mice lacked B-1a cells, lacked natural antibodies, and were more susceptible to infection. By contrast, pneumococcal polysaccharide (PPS) immunization protected CD19(-/-) mice during lethal challenge, whereas hCD19Tg mice remained unprotected. This resulted from differences in the B-1b subset: the key population found to produce protective PPS-specific antibody in both wild-type and CD19(-/-) mice. Thus, CD19(-/-) mice generated B-1b cells and protective adaptive PPS-specific antibody responses, whereas hCD19Tg mice lacked B-1b cells and adaptive PPS-specific antibody responses. This reciprocal contribution of B-1a and B-1b subsets to innate and acquired immunity reveals an unexpected division of labor within the B-1 compartment that is normally balanced by their coordinated development.     

Peripheral T cell tolerance as a tumor escape mechanism: deletion of CD4+ T cells specific for a monoclonal immunoglobulin idiotype secreted by a plasmacytoma

Tumors could escape an immune attack by inducing peripheral T cell tolerance. To test this, T cell receptor (TCR)-transgenic mice were injected with plasmacytoma cells secreting a highly tumor-specific antigen, a monoclonal immunglobulin (Ig), for which the transgene-encoded TCR is specific. The TCR recognizes a third hypervariable region idiotypic (Id) peptide of the Ig, presented by a class II molecule on host antigen-presenting cells. The TCR-transgenic mice have previously been shown to be protected against an Id⁺ plasmacytoma challenge. In the present experiments, the protection was deliberately overwhelmed by subcutaneous injection of large numbers of plasmacytoma cells. Such tumor mice, chronically exposed to increasing amounts of monoclonal Ig, delete Id-specific CD4⁺ T cells in their peripheral lymphoid organs and in the tumor. The residual CD4⁺ cells express endogenous, rather than transgene-encoded TCR α chains. Peripheral deletion, functional T cell unresponsiveness, and thymocyte deletion are all first detected at the same serum concentration of monoclonal Ig, ∼50 μg/ml (0.3 μM), and become more and more profound as the tumor burden increases. The results suggest that peripheral T cell tolerance to Id could be a tumor escape mechanism in patients with B cell malignancies. In addition, the findings have implications for T cell tolerance to Ig V regions in normal individuals.     

PB76: a novel surface glycoprotein preferentially expressed on mouse pre-B cells and plasma cells detected by the monoclonal antibody G-52

A monoclonal antibody (mAb) G-5-2 was isolated which binds to transformed as well as normal cells of the B lineage but not to cells of the T cell, myeloid lineages nor to fibroblasts. mAb G-5-2 reacts with pre-B and plasma cell-transformed lines, and it preferentially recognizes normal pre-B cells from fetal liver and bone marrow as well as plasma cells from spleen of mice. G-5-2⁺ fetal liver cells isolated by cell sorter express mRNA for μ heavy chain Ig gene and generate in vitro antibody-producing cells when co-cultured with lipopolysaccharide and rat thymocyte filler cells. During development the frequency and staining intensity of G-5-2⁺ cells in fetal liver from normal mice increases from 1% G-5-2⁺ cells at day 14 to～7% positive cells at day 18 of gestation. Several strains or normal mice contain comparable numbers of G-5-2⁺ cells as well as B-220⁺ and BP-1⁺ B cell precursors in the fetal liver. Mice carrying the xid mutation have 3-4-fold less G-5-2⁺ as well as B-220⁺ and BP-1⁺ cells in the fetal liver, suggesting that the effects of the xid mutation may be manifested from early stages of B cell development. Fetal liver cells from mice carrying the scid mutation were found to contain normal numbers of G-5-2⁺ as well as B-220⁺ and BP-1⁺ pre-B cells. These results indicate that differentiation from progenitors to pre-B cells in scid mice may occur normally; the scid mutation would thus appear to affect the process of rearrangement and expression of the Ig genes in the developing pre-B cells. mAb G-5-2 precipitates a 76-kDa glycoprotein from surface-radiolabeled pre-B cells and plasma cells. Taken together, these results indicate that G-5-2 mAb recognizes a novel B cell lineage-specific surface molecule called PB76 which is preferentially expressed by pre-B cells and plasma cells.     

Influence of the nude and X-Iinked immune deficiency genes on expression of × and λ light chains

The relative amounts of Igx and Igλ₁ anti-2,4-dinitrophenyl antibodies were measured at various times after immunizing mice with prototype thymus-dependent (TD), thymus-independent type 1 (TI-1) and thymus-independent type 2 (TI-2) antigens. Similar amounts of Igλ₁ were produced after TD and TI-2 immunization and somewhat less was produced after a TI-1 stimulus. In contrast, Igx levels were much greater after TD than after TI-1 or TI-2 antigen. The amount of light chain isotype produced appeared to depend on the molecular form in which the hapten was presented, although possible adjuvant effects were not ruled out. Levels of Igx and Igλ present in nonimmune sera were measured in normal, xid and nude mice. The x/λ, ratio was higher in xid than in normal mice and the difference was demonstrated by F₁ analysis to be due to an X-linked gene. Conversely, the x/λ ratio was lower in nude than in normal mice. This was true for the CBA/Tufts (Igh^j), CBA. Igh^b and C57BL/10 strains. However, there were no detectable differences in the relative frequencies of surface Igx- and Igλ -bearing B cells in adult CBA/Tufts, CBA/N and nude mice. Hence, serum ratios may reflect differences at the level of B cell triggering. Two possible explanations for these differences are discussed. Igx and Igλ may be expressed on functionally distinct B cell subsets. (For instance Igλ -producing cells might be readily triggered by T1 antigens whereas Igx-producing cells are more dependent on T cell signals. Such functional subsets could be determined by light chain expression). Alternatively, cells producing Igx antibody are selected for because they have a higher affinity for antigen. If so, triggering of cells producing high affinity Igx or their subsequent selection is T cell-dependent.     

Development of nephritis but not sialadenitis in autoimmune-prone BAFF transgenic mice lacking marginal zone B cells

B cell-activating factor belonging to the TNF family (BAFF) is a B cell survival factor required for B cell maturation. BAFF transgenic (Tg) mice develop autoimmune disorders characterized by autoantibody production, which leads to nephritis and salivary gland destruction (sialadenitis), features reminiscent of systemic lupus erythematosus and Sj?gren's syndrome (SS), respectively. Disease in BAFF Tg mice correlates with the expansion of the marginal zone (MZ) B cell compartment and the abnormal presence of MZ-like B cells in the blood, LN and inflamed salivary glands, suggesting a role for these cells in BAFF-induced autoimmunity. Lymphotoxin-beta (LTbeta)-deficient mice show disrupted splenic architecture, lack MZ B cells and some peripheral LN, and are unable to mount T cell-dependent immune responses. BAFF Tg mice lacking LTbeta (LTbetaDelta-BTg) retained these defects, yet still developed nephritis associated with the presence of B-1 B cells in the kidneys. However, in contrast to old BAFF Tg mice, aging LTbetaDelta-BTg mice no longer developed sialadenitis. Thus, autoimmune disorders in BAFF Tg mice are possibly events coordinated by MZ and B-1 B cells at separate anatomical sites.     

Administration of interleukin -5 or -10 activates peritoneal B-1 cells and induces autoimmune hemolytic anemia in anti-erythrocyte autoantibody-transgenic mice

Activation mechanisms of B-1 (Ly-1 B) cells have been suggested to be different from those of conventional B cells. To assess the role of various interleukins (IL) in the activation of B-1 cells, we injected IL-4, IL-5 or IL-10 into nonanemic anti-red blood cells (RBC) autoantibody-transgenic mice, in which conventional B cells are clonally deleted but peritoneal B-1 cells persist without secreting Ig. Intraperitoneal or intramuscular injection of IL-5 or IL-10, but not IL-4, increased the number of antibody-producing peritoneal B-1 cells by four- to five-fold, resulting in increased anti-RBC serum autoantibody and induction of hemolytic anemia. These results suggest that IL-5 or IL-10 may play an important role in the terminal differentiation of B-1 cells into antibody-producing cells in vivo.     

A murine Ig light chain transgene reveals IGKV3 gene contributions to anti-collagen types IV and II specificities

A subset of autoimmune diseases result from autoantibodies targeting epitopes on matrix collagen. The most extensively studied are anti-glomerular basement membrane glomerulonephritis (or its systemic counterpart Goodpasture’s disease) that destroys kidneys and lungs, and rheumatoid arthritis that leads to disabling arthritis. Autoantibodies in these disorders bind evolutionarily conserved conformational epitopes on the noncollagenous domain 1 (NC1) of the alpha3 chain of type IV [alpha3(IV)NC1] collagen in glomerular and alveolar basement membranes, and on native or citrullinated type II collagen (CII) in joint cartilage, respectively. The genetic origins of pathogenic anti-collagen B cells in these diseases is unknown, but observations from murine models raise the possibility that they overlap despite distinct in vivo immunopathologies. Monoclonal autoantibodies isolated from mice immunized with alpha3(IV)NC1 collagen or CII show a biased use of Ig light chains (LC) encoded by genes of the IGKV3 subgroup (previously Vk21 family), paired with diverse Ig heavy chains. To further explore this relationship and determine if a single murine IGKV3 LC independently predisposes to both anti-collagen responses, we generated a novel transgenic (Tg) C57BL/6 mouse that expresses a productively rearranged IGKV3-encoded LC, termed mLCV3-Tg, in conjunction with endogenously rearranged Ig heavy chains. Tg mice are also genetically deficient in endogenous kappa chains to permit tracking of the mLCV3 transgene. We show that mLCV3-Tg mice are susceptible to humoral autoimmunity against both collagen chains. Anti-alpha3(IV)NC1 collagen, but not anti-CII, mLCV3-encoded Ig are detected in serum of unmanipulated Tg mice, while Toll-like receptor ligands induce secretion of mLCV3-Tg autoantibodies of both collagen specificities from splenocytes ex vivo. This indicates developmental survival of mLCV3-Tg B cells reactive with each antigen, and is consistent with production of the two anti-collagen autoIg from distinct B cell populations. Reduced B cell numbers, low serum Ig kappa levels, low cell surface Ig kappa density, and abundant endogenous lambda chain expression suggest that subsets of IGKV3-encoded B cells are regulated in vivo by mechanisms that include deletion, anergy, and LC editing. These results support the notion that murine IGKV3 LCs contribute structural fitness to antigen binding sites that support diverse anti-collagen autoimmune responses, that these responses are regulated in vivo, and that these cells can nonetheless readily escape immune regulation.     

ACh receptor protein drives primary and memory autoantibody responses in chimeric human-SCID mice

The native antigen that drives the T-helper cells regulating production of muscle acetylcholine receptor (AChR) autoantibodies is unknown. Human T cell lines activated by autoantigens in vitro are of unproven relevance to B cell help. Here we report the functional interaction and unprecedented longevity of AChR-specific human T and B lymphocytes residing in SCID mice. Lymphoid cells from myasthenia gravis (MG) patients and healthy subjects were injected ip. Recombinant human AChR-alpha1-subunit-1-210 was injected after day 75. Human AChR-specific Ig was produced rapidly in MG-SCID mice challenged once. Only 1 of 32 control hu-SCID mice produced AChR-specific Ig. This required multiple immunizations, was initially cross-reactive with Torpedo AChR, and had a slow course. Thus, memory T and B lymphocytes specific for human AChR-alpha1-subunit are readily demonstrable in MG patients, interact to produce autoantibody of the same restricted specificity found in the donor's serum, and are long-lived without exogenous autoantigen challenge. In healthy subjects, AChR-specific lymphocytes are infrequent and exhibit naive response characteristics, including apparent affinity maturation of Ig specificity.     

Humoral autoimmunity in mice overexpressing B cell surface CD19: vital role for MHC class II

B6 mice bearing a transgene (Tg) for human CD19 (hCD19) harbor increased numbers of splenic Ig-secreting cells (IgSC), increased serum levels of total Ig and autoantibodies, but decreased numbers of splenic B cells. To assess the influence of MHC class II (MHCII) to this phenotype, MHCII-deficient CD19-Tg mice were generated. Compared to MHCII-sufficient CD19-Tg mice, splenic IgSC numbers and serum Ig and autoantibody levels were markedly diminished, and the decrease in splenic B cell numbers was aggravated. Remarkably, genetic reconstitution of these MHCII-deficient mice with a human DQ8 Tg resulted in a hierarchical restorative pattern. Restoration of splenic B cell numbers was complete; restoration of numbers of splenic IgSC and of serum Ig levels was partial; and restoration of circulating autoantibody levels was virtually non-existent. Thus, MHCII expression has a profound effect on B cells which can be uncoupled from global Ig and autoantibody production in the hCD19-Tg model. This raises the possibility that MHCII affects B cells in a manner that, at least in part, is independent of helper T cell function.     

Involvement of CD28 cosignalling in the T cell-mediated suppression of the IgG antibody response against the TI-2 antigen alpha(1-->3) dextran.

The humoral immune response against alpha(1-->3) dextran (Dex) in BALB/c mice is characterized by the formation of predominantly IgM antibodies bearing the J558 idiotype. IgG antibodies do not appear in euthymic mice. In athymic animals, however, the response proceeds to a vigorous IgG production. In euthymic mice formation of IgG is suppressed by J558 idiotype specific regulatory T cells recognizing in association with I-Ed and in cognate T/B interaction the V(H) CDR3 derived peptide of the J558 idiotype. Only B-2 lymphocytes produce IgG whereas B-1 cells do not participate in the production of this Ig class. Using novel synthetic all alpha(1-->3)-D-gluco configured tetrasaccharide the Dex-specific B cells can for the first time be analyzed in FACS. In experiments using this newly designed low molecular Dex no signs of B cell apoptosis can be found. This demonstrates a true silencing of persisting Bgamma memory cells as previously suggested by adoptive transfer experiments. In this suppression a further involvement of CD28 and B7-1 interaction can be demonstrated which delivers a necessary costimulatory suppression signal in addition to the cognate TCR/peptide-I-Ed interaction between J558 specific T cells and J558 idiotype bearing B cells.     

Isolation of peritoneal precursors of B-1 cells in the adult mouse

Two weeks of daily peritoneopheresis of adult mice result in the selective depletion of B-1 cells, followed by the appearance of a population of B220⁺IgM^?lymphocytes in the peritoneal cavity. These cells share with bone marrow (BM) pre-B cells expression of λ5, V_preB, and RAG-1 genes and a higher fraction of unrearranged V to DJ heavy (H) chain immunoglobulin (Ig) gene segments, when compared with mature B lymphocytes. Upon transfer to SCID recipients, sorted peritoneal B220⁺IgM^? cells fail to colonize the BM, repopulate very few B cells in the spleen, but entirely reconstitute the B-1 cell compartment in the peritoneal and pleuropericardial cavities. In contrast, parallel transfers of sorted BM B220⁺IgM^? cells result in reconstitution of the BM and spleen B lineage cell compartments, but in no coelomic B cell repopulation. Both types of pre-B cells reconstitute splenic plasma cells of donor origin, but with markedly distinct efficiencies: the ratio of IgM-plasma cell/B cell numbers in the spleens of peritoneal pre-B cell recipients is more than 500-fold higher than that of recipients reconstituted by BM pre-B cells. We take these data to indicate that (1) differentiative commitment to the B-1 cell population occurs before selection events on mature cells; (2) B-1 precursors exist or may be locally produced in the adult mouse; (3) there is a lineage-related differential ability of mature B cells to undergo terminal differentiation to high-rate Ig secretion.     

An explanation for the defect in secretion of IgM Mott cells and their predominant occurrence in the Ly-1 B cell compartment.

Mott cells are a variant form of plasma cell in which the immunoglobulin (Ig), rather than being secreted, accumulates in rough endoplasmic reticulum-derived vesicles called Russell bodies. We have examined the molecular cause of this defect and the in vivo origin of IgM Mott cells. Our examination of the Ig variable region gene sequences of two IgM Mott hybridomas derived from C.B-20 Ly-1 B cells showed all to be germ line. In a series of mix and match transfection experiments, the Mott phenotype was only reconstituted when the original Mott specificity was expressed as an IgM, suggesting that both the specificity and the isotype were critical to the formation of Russell bodies. Based on our finding that Russell body formation was dependent on the Ig isotype being IgM, we suggest that the Mott phenotype is apparent only after differentiation of B cells into plasma cells and that probably the major cause of the IgM Mott phenotype is low-affinity interaction of the Mott Ig with some as yet unknown intracellular component(s) being stabilized by the intrinsic high avidity of the pentameric secreted form of IgM. Consistent with this proposal was the finding that after in vitro lipopolysaccharide (LPS) stimulation of sorted Ly-1 B cells derived from C.B-20 mice, Mott cells represented up to 5% of the IgM plasma cells in the culture. LPS stimulation of conventional B cells also induced the appearance of IgM Mott cells, but at the much reduced level of 0.1%, suggesting that the major, if not the only, source of Mott cells in vivo is the Ly-1 B cell population. A possible causal relationship between the elevated frequency of Mott cells in the Ly-1 B cell-derived LPS blasts and the repertoire selection inherent in the development of these B cells is discussed.     

Effects of IgM Allotype Suppression on Serum IgM Levels,B-1 and B-2 Cells,and Antibody Responses ir Allotype Heterozygous F1 Mice

IgM allotype heterozygous F1 mice were independently suppressed for Igh6a or Igh6b to evaluate the contribution of B-1 and B-2 cells to natural serum IgM levels and Ab responses. B-2 B cells expressing IgM of the suppressed allotype were evident in the spleens of suppressed mice 4 to 6 weeks after cessation of the suppression regimen, whereas B-1 B cells of the suppressed allotype were undetectable for up to 9 months. Although serum IgM of the suppressed allotype was initially depleted in mice suppressed for either allotype, by 7 months of age, there were detectable levels of IgM of the suppressed allotype in the serum; however, the levels were significantly below that found in nonsuppressed mice. When mice were immunized with either the T-independent or T-dependent form of phosphorylcholine, those suppressed for either allotype, and consequently depleted of B-1 B cells of that allotype, did not respond with phosphorylcholine-specific IgM of the suppressed allotype. In contrast, when mice were immunized with α1-3 dextran, the Igh6a allotype-suppressed mice were able to produce dextran-specific IgM of that allotype. These results show that allotype-bearing B-1 cells of both allotypes can be effectively suppressed by this suppression protocol and this produces long-lasting effects on B-1 cell levels and serum IgM of the suppressed allotype. These observations reflect the derivation of the majority of B-1 cells from fetal-neonatal precursors, which cannot be replaced by newly emerging B-2 cells of adult origin. Their ablation by antibody treatment results in permanent alterations to the adult B-cell repertoire.     

The effect of an immunoglobulin mu transgene on B cell maturation.

In mu 17.2.25-transgenic (M54) mice the absolute number of surface IgM (sIgM) B cells in lymphoid organs is drastically reduced compared to normal C57BL/6 mice and a high frequency of B cells express the immunoglobulin (Ig) encoded by the transgene rather than endogenous Ig on the surface. To determine the effect of a mu transgene on B cell development, adoptive cell transfers were performed using mu transgenic (M54) bone marrow and fetal liver cells. The data presented support the following conclusions: (a) adult transgenic bone marrow contains functional B cell precursors able to mature and repopulate the spleen and peritoneum of recipient mice. The relative frequency of transgene (sIgMa) and endogenous (sIgMb) surface sIgM-positive B cells reconstituted by transgenic bone marrow in allotype-matched C57BL/6 recipients is the same as in the M54 donors; (b) serum analysis indicates that transgenic bone marrow donor cells can reconstitute B cells in congenic and severe combined immunodeficient (SCID) recipient mice; (c) transgenic fetal liver cells are not a richer source of precursors for B cells expressing endogeneous Ig; (d) in transgenic mice sIgM+ B cells are not restricted to the CD5+ phenotype, however, the relative frequency of sIgMb B cells that are CD5+ is higher in transgenic than normal mice; and (e) bone marrow cells from adult normal and transgenic mice are able to generate CD5+ B lymphocytes in the spleen and peritoneum of allotype-congenic and neonatal SCID recipient mice. The results indicate that the presence of a complete mu heavy chain transgene does not result in a selective developmental block of "conventional" bone marrow-derived pre-B and B cells.     

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.     

Strong antigenic selection shaping the immunoglobulin heavy chain repertoire of B-1a lymphocytes in lambda 2(315) transgenic mice

The B lymphocyte compartment of the recombinant mouse line L2 consists predominantly of CD5(+) B-1 cells that exclusively express the transgenic (Tg) lambda 2 L chain of the plasmacytoma MOPC315. Using such Tg mice as a simplified model to study positive selection processes, we show that restriction to a single L chain results in a strongly oligoclonal IgH chain repertoire in fetal and neonatal liver-derived B cells, as well as in peritoneal CD5(+) B-1 lymphocytes from adult mice. In contrast, in vitro differentiated fetal liver Pro/PreB-I cells from L2 embryos show a clear increase of complementarity determining region three (CDR3) diversity. These data suggest that the antibody repertoire of B-1a cells is strongly selected by antigen during fetal as well as adult peritoneal phase.     

Distinct and overlapping roles of CXCR5 and CCR7 in B-1 cell homing and early immunity against bacterial pathogens

CXC chemokine receptor (CXCR)5 and CC chemokine receptor (CCR)7 are the major chemokine receptors required for B cell homing and microenvironmental localization during antigen-independent and -dependent B cell differentiation. Here, we show markedly decreased B-1 B cell numbers in the peritoneal cavity of CXCR5-/- and CXCR5-/-CCR7-/- double-deficient mice paralleled by reduced antigen-induced phosphorylcholine-specific immunoglobulin (Ig)M responses after intraperitoneal (i.p.) administration of streptococcal antigen. CCR7-/- mice also revealed a partial reduction in peritoneal B-1 cell numbers combined with a reduced humoral response to i.p. injected bacterial antigen. However, opposite roles of CXCR5 and CCR7 were observed when the frequency of peritoneal B-2 cells was analyzed. CXCR5-/- mice almost completely lacked B-2 cells, whereas CCR7 deficiency engendered an increase in peritoneal B-2 cells. In addition, CCR7-/- mice had enhanced, splenic IgM+ plasma cell responses, whereas the extrafollicular B cell response of the CXCR5-/- mice was not significantly altered compared with wild-type controls. Thus, the two chemokine receptors exert divergent forces at multiple levels of the innate immune response. CXCR5 plays a dominant role in peritoneal B-1 B cell homing and body cavity immunity, but both chemokine receptors are needed for a proportional peritoneal B-2 cell homing and balanced development of an early splenic B cell response.