Clearance of Pneumocystis carinii in mice is dependent on B cells but not on P carinii-specific antibody

Both CD4(+) T cells and B cells are critical for defense against Pneumocystis carinii infection; however, the mechanism by which B cells mediate protection is unknown. We show that P. carinii-specific IgM is not sufficient to mediate clearance of P. carinii from the lungs since CD40-deficient mice produced normal levels of specific IgM, but were unable to clear the organisms. Using chimeric mice in which the B cells were deficient in CD40 (CD40KO chimeras) we found that clearance of P. carinii infection is delayed compared with wild-type controls. These CD40KO chimeric mice produced normal levels of P. carinii-specific IgM, but did not produce class-switched IgG or IgA. Similarly, clearance of P. carinii was delayed in mice deficient in FcgammaRI and III (FcgammaRKO), indicating that P. carinii-specific IgG partially mediates opsonization and clearance of P. carinii. Opsonization of organisms by complement did not compensate for the lack of specific IgG or FcgammaR, since C3-deficient and C3-depleted FcgammaRKO mice were still able to clear P. carinii. Finally, micro MT and CD40KO chimeric mice had reduced numbers of activated CD4(+) T cells in the lungs and lymph nodes compared with wild-type mice, suggesting that B cells are important for activation of T cells in response to P. carinii. Together these data indicate that P. carinii-specific IgG plays an important, but not critical, role in defense against P. carinii. Moreover, these data suggest that B cells also mediate host defense against P. carinii by facilitating CD4(+) T cell activation or expansion.     

Impaired antibody response to group B streptococcal type III capsular polysaccharide in C3- and complement receptor 2-deficient mice

Group B Streptococcus (GBS) is the foremost bacterial cause of serious neonatal infections. Protective immunity to GBS is mediated by specific Abs to the organism's capsular polysaccharide Ags. To examine the role of complement in the humoral immune response to type III GBS capsular polysaccharide (III-PS), mice deficient in C3 or in CD21/CD35 (i.e., complement receptors 1 and 2; CR1/CR2) were immunized with III-PS. Mice deficient in C3 or Cr2 had an impaired primary immune response to III-PS. The defective response was characterized by low IgM levels and the lack of an isotype switch from IgM to IgG Ab production. Compared with wild-type mice, C3- and Cr2-deficient mice exhibited decreased uptake of III-PS by follicular dendritic cells within the germinal centers and impaired localization of III-PS to the marginal zone B cells. Complement-dependent uptake of capsular polysaccharide by marginal zone B cells appears necessary for an effective immune response to III-PS. The normal immune response in wild-type mice may require localization of polysaccharide to marginal zone B cells with subsequent transfer of the Ag to follicular dendritic cells.     

Hijacked DNA repair proteins and unchained DNA polymerases

Somatic hypermutation of immunoglobulin (Ig) genes occurs at a frequency that is a million times greater than the mutation in other genes. Mutations occur in variable genes to increase antibody affinity, and in switch regions before constant genes to cause switching from IgM to IgG. Hypermutation is initiated in activated B cells when the activation-induced deaminase protein deaminates cytosine in DNA to uracil. Uracils can be processed by either a mutagenic pathway to produce mutations or a non-mutagenic pathway to remove mutations. In the mutagenic pathway, we first studied the role of mismatch repair proteins, MSH2, MSH3, MSH6, PMS2 and MLH1, since they would recognize mismatches. The MSH2–MSH6 heterodimer is involved in hypermutation by binding to U:G and other mismatches generated during repair synthesis, but the other proteins are not necessary. Second, we analysed the role of low-fidelity DNA polymerases η, ι and θ in synthesizing mutations, and conclude that polymerase η is the dominant participant by generating mutations at A:T base pairs. In the non-mutagenic pathway, we examined the role of the Cockayne syndrome B protein that interacts with other repair proteins. Mice deficient in this protein had normal hypermutation and class switch recombination, showing that it is not involved.     

B cell-specific deficiency for Smad2 in vivo leads to defects in TGF-beta-directed IgA switching and changes in B cell fate

Smad2 is a member of the intracellular mediators that transduce signals from TGF-beta receptors and activin receptors. Targeted inactivation of Smad2 in mice leads to early lethality before gastrulation. It was shown previously that TGF-betaRII deficiency in vivo leads to defects in B cell homeostasis, Ag responsiveness, and IgA class switch recombination of B cells. To investigate the importance of Smad2-mediated signaling in B lymphocytes, we generated a B cell-specific inactivation of Smad2 in mice (bSmad2(-/-)). bSmad2(-/-) mice had normal B cell numbers in the spleen but showed a reduced population of marginal zone B cells. In contrast, B cells in Peyer's patches and peritoneal B-1a cells of bSmad2(-/-) mice were increased in numbers. bSmad2(-/-) mice showed a reduced number of surface-IgA(+) B cells and of IgA-secreting cells in Peyer's patches, decreased levels of IgA in serum, and, after immunization with a T cell-dependent Ag, a reduced IgA response. Class switch recombination to IgA was impaired in Smad2-deficient B cells, when stimulated in vitro with LPS in the presence of TGF-beta. The growth-inhibitory effects of TGF-beta in LPS-stimulated B cells were not affected in Smad2-deficient B cells. In summary, our data indicate a crucial role of Smad2 in mediating signals for the TGF-beta-directed class switch to IgA and the induction of IgA responses in vivo. Other B cell functions like growth-inhibitory signaling, which are known to be regulated by signals via the TGF-betaR, are not affected in Smad2-deficient B cells.     

Targeted deletion of the IgA constant region in mice leads to IgA deficiency with alterations in expression of other Ig isotypes

A murine model of IgA deficiency has been established by targeted deletion of the IgA switch and constant regions in embryonic stem cells. B cells from IgA-deficient mice were incapable of producing IgA in vitro in response to TGF-beta. IgA-deficient mice expressed higher levels of IgM and IgG in serum and gastrointestinal secretions and decreased levels of IgE in serum and pulmonary secretions. Expression of IgG subclasses was complex, with the most consistent finding being an increase in IgG2b and a decrease in IgG3 in serum and secretions. No detectable IgA Abs were observed following mucosal immunization against influenza; however, compared with those in wild-type mice, increased levels of IgM Abs were seen in both serum and secretions. Development of lymphoid tissues as well as T and B lymphocyte function appeared normal otherwise. Peyer's patches in IgA-deficient mice were well developed with prominent germinal centers despite the absence of IgA in these germinal centers or intestinal lamina propria. Lymphocytes from IgA-deficient mice responded to T and B cell mitogens comparable to those of wild-type mice, while T cells from IgA-deficient mice produced comparable levels of IFN-gamma and IL-4 mRNA and protein. In conclusion, mice with targeted deletion of the IgA switch and constant regions are completely deficient in IgA and exhibit altered expression of other Ig isotypes, notably IgM, IgG2b, IgG3, and IgE, but otherwise have normal lymphocyte development, proliferative responses, and cytokine production.     

Abrogation of lupus nephritis in activation-induced deaminase-deficient MRL/lpr mice

We generated MRL/lpr mice deficient in activation-induced deaminase (AID). Because AID is required for Ig hypermutation and class switch recombination, these mice lack hypermutated IgG Abs. Unlike their AID wild-type littermates, AID-deficient MRL/lpr mice not only lacked autoreactive IgG Abs but also experienced a dramatic increase in the levels of autoreactive IgM. This phenotype in AID-deficient mice translated into a significant reduction in glomerulonephritis, minimal mononuclear cell infiltration in the kidney, and a dramatic increase in survival to levels comparable to those previously reported for MRL/lpr mice completely lacking B cells and well below those of mice lacking secreted Abs. Therefore, this study wherein littermates with either high levels of autoreactive IgM or autoreactive IgG were directly examined proves that autoreactive IgM Abs alone are not sufficient to promote kidney disease in MRL/lpr mice. In addition, the substantial decrease in mortality combined with a dramatic increase in autoreactive IgM Abs in AID-deficient MRL/lpr mice suggest that autoreactive IgM Abs might not only fail to promote nephritis but may also provide a protective role in MRL/lpr mice. This novel mouse model containing high levels of autoreactive, unmutated IgM Abs will help delineate the contribution of autoreactive IgM to autoimmunity.     

Genetic analysis of mouse embryonic stem cells bearing Msh3 and Msh2 single and compound mutations

We have previously described the use of homologous recombination and CRE-loxP-mediated marker recycling to generate mouse embryonic stem (ES) cell lines homozygous for mutations at the Msh3, Msh2, and both Msh3 and Msh2 loci (2). In this study, we describe the analysis of these ES cells with respect to processes known to be affected by DNA mismatch repair. ES cells homozygous for the Msh2 mutation displayed increased resistance to killing by the cytotoxic drug 6-thioguanine (6TG), indicating that the 6TG cytotoxic mechanism is mediated by Msh2. The mutation rate of the herpes simplex virus thymidine kinase 1 (HSV-tk1) gene was unchanged in Msh3-deficient ES cell lines but markedly elevated in Msh2-deficient and Msh3 Msh2 double-mutant cells. Notably, the HSV-tk1 mutation rate was 11-fold higher, on average, than that of the hypoxanthine-guanine phosphoribosyl transferase (Hprt) locus in Msh2-deficient cells. Sequence analysis of HSV-tk1 mutants from these cells indicated the presence of a frameshift hotspot within the HSV-tk1 coding region. Msh3-deficient cells displayed a modest (16-fold) elevation in the instability of a dinucleotide repeat, whereas Msh2-deficient and Msh2 Msh3 double-mutant cells displayed markedly increased levels of repeat instability. Targeting frequencies of nonisogenic vectors were elevated in Msh2-deficient ES cell lines, confirming the role of Msh2 in blocking recombination between diverged sequences (homeologous recombination) in mammalian cells. These results are consistent with accumulating data from other laboratories and support the current model of DNA mismatch repair in mammalian cells.     

IL-4 induces the specific rearrangement of gamma 1 genes on the expressed and unexpressed chromosomes of lipopolysaccharide-activated normal murine B cells

Small, resting, surface IgM+/surface IgD+ murine B cells undergo an Ig class switch to IgG1 or IgE after stimulation with LPS and T cell supernatants containing IL-4. To firmly establish the role of IL-4 in the directed switch recombination observed in IgG1-secreting cells, we have 1) used highly purified native IL-4 instead of T cell supernatants, 2) used resting B cells from F1 mice in which the active IgH allele was determined before culture, 3) taken advantage of the allelic differences in the restriction fragment lengths of mu, gamma 1, gamma 2b, and gamma 3 loci to determine the status of the CH genes on both the expressed and unexpressed chromosomes, and 4) used different restriction enzymes to distinguish between deletion and rearrangement of a given CH gene. Our results indicate that LPS alone induces rearrangement of the gamma 3 genes on both chromosomes whereas stimulation with LPS plus IL-4 results in deletion of gamma 3 genes and rearrangement of gamma 1 genes on both chromosomes. The studies definitively establish the role of IL-4 in directed switch recombination to the gamma 1 locus in LPS-stimulated murine B cells.     

Induction of CD4(+) T-cell-independent immunoglobulin responses by inactivated influenza virus

Through cognate interaction between antigen-specific B-cell and CD4(+) alphabeta T cells, the CD4(+) alphabeta T cells secrete cytokines that initiate immunoglobulin (Ig) class switching from IgM to IgG. In this study, we show that formalin-inactivated influenza PR8 virus induces virus-specific IgM and IgG responses in the absence of CD4(+) T cells and that all four subclasses of IgG are produced. The immunized CD4-deficient mice were also found to be completely protected against lethal infection with live, pathogenic influenza virus. The ability of CD4(+) T-cell-deficient mice to generate these IgG responses was not found to be impaired when these mice were depleted of CD8(+) T cells with an anti-CD8 monoclonal antibody. In contrast, alphabeta T-cell-deficient mice (TCRbeta(-/-)) were not found to produce significant amounts of IgG upon immunization with formalin-inactivated PR8 virus. These results suggest that CD4(-) CD8(-) double-negative alphabeta T cells are playing a role in regulating Ig class switching in the absence of CD4(+) T cells.     

Differentiation of B lineage cells from liver of neonatal mice: generation of immunoglobulin isotype diversity in vitro

The development and differentiation of B cells expressing different immunoglobulin (Ig) isotypes was studied in cultures of murine neonatal liver cells. Before culture, 5 to 15% of the liver cells were mu + pre-B cells; 1 to 3% had surface IgM and less than 0.1% had slgG. During 4 days in culture the number of pre-B cells declined, whereas the number of IgM B cells increased greater than 20-fold; IgG B cells also increased in number. Of the four subclasses, IgG3+ and IgG2b+ cells predominated, each representing 3 to 10% of the total B cells at day 4. IgG1+ and IgG2a+ cells were present in lower numbers, representing 1 to 5% and 0.3 to 2.5% of B cells, respectively. Most IgG+ cells also expressed sIgM. Only a minority (less than 10%) of the sIgM+ cells were sIgD+, and most sIgG+ cells were sIgD-. Few T cells were present in these cultures (less than 0.5% in newborn liver), and sIgG+ cells were generated in normal frequencies in cultures of cells from nude mice. The numbers of B cells expressing each IgG subclass were similar in cultures from athymic nu/nu mice, nu/+ heterozygous littermates, and normal BALB/c mice. Plasmablasts and plasma cells appeared over a 14-day culture interval, and these expressed cytoplasmic IgM, IgG3, IgG1, IgG2b, IgG2a, and IgA. Measurable amounts of the first four isotypes were detected in the culture supernatants by radioimmunoassay. These results indicate that neonatal B cells can undergo isotype switching in the absence of T cell help, and that the expression of sIgD may not be a prerequisite for cells to switch Ig isotypes.     

Role of the mismatch repair gene, Msh6, in suppressing genome instability and radiation-induced mutations

Mismatch repair (MMR) is critical for preserving genomic integrity. Failure of this system can accelerate somatic mutation and increase the risk of developing cancer. MSH6, in complex with MSH2, is the MMR protein that mediates DNA repair through the recognition of 1- and 2-bp mismatches. To evaluate the effects of MSH6 deficiency on genomic stability we compared the frequency of in vivo loss of heterozygosity (LOH) between MSH6-proficient and deficient, 129S2xC57BL/6 F1 hybrid mice that were heterozygous for our reporter gene Aprt. We recovered mutant cells that had functionally lost APRT protein activity and categorized the spectrum of mutations responsible for the LOH events. We also measured the mutant frequency at the X-linked gene, Hprt, as a second reporter for point mutation. In Msh6-/-Aprt+/- mice, mutation frequency at Aprt was elevated in both T cells and fibroblasts by 2.5-fold and 5.7-fold, respectively, over Msh6+/+Aprt+/- littermate controls. While a modest increase in mitotic recombination (MR) was observed in MSH6-deficient fibroblasts compared to wild type controls, point mutation was the predominant mechanism leading to APRT deficiency in both cell types. Base substitution, consisting of multiple types of transitions, accounted for all of the point mutations identified within the Aprt coding region. We also assessed the role of MSH6 in preventing mutations caused by a common environmental mutagen, ionizing radiation (IR). In Msh6-/-Aprt+/- mice, 4Gy of X-irradiation induced a significant increase in point mutations at both Aprt and Hprt in T cells, but not in fibroblasts. These findings indicate that MutS alpha reduces spontaneous and IR-induced mutation in a cell type-dependant manner.     

Extracellular signal-regulated protein kinase 2 is required for efficient generation of B cells bearing antigen-specific immunoglobulin G

Activation of extracellular signal-regulated protein kinase (ERK) has been implicated in proliferation as well as differentiation in a wide variety of cell types. Using B-cell-specific gene-targeted mice, we report here that in T-cell-dependent immune responses, ERK2 is required to generate efficient immunoglobulin G (IgG) production. In its absence, the proportion of antigen-specific surface IgG1-bearing cells and the subsequent number of IgG1 antibody-secreting cells were decreased, despite apparently unimpaired class switch recombination. Notably, this defect was countered by overexpression of the antiapoptotic factor Bcl-2. Together, our results suggest that ERK2 plays a key role in efficient generation of antigen-specific IgG-bearing B cells by promoting their survival.     

Cannabinoid Receptor 2 (CB2) Plays a Role in the Generation of Germinal Center and Memory B Cells,but Not in the Production of Antigen-Specific IgG and IgM,in Response to T-dependent Antigens

The cannabinoid receptor 2 (CB2) has been reported to modulate B cell functions including migration, proliferation and isotype class switching. Since these processes are required for the generation of the germinal center (GC) and antigen-specific plasma and memory cells following immunization with a T-dependent antigen, CB2 has the capacity to alter the quality and magnitude of T-dependent immune responses. To address this question, we immunized WT and CB2^−/− mice with the T-dependent antigen 4-hydroxy-3-nitrophenylacetyl (NP)-chicken-gamma-globulin (CGG) and measured GC B cell formation and the generation of antigen-specific B cells and serum immunoglobulin (Ig). While there was a significant reduction in the number of splenic GC B cells in CB2^−/− mice early in the response there was no detectable difference in the number of NP-specific IgM and IgG₁ plasma cells. There was also no difference in NP-specific IgM and class switched IgG₁ in the serum. In addition, we found no defect in the homing of plasma cells to the bone marrow (BM) and affinity maturation, although memory B cell cells in the spleen were reduced in CB2^−/− mice. CB2-deficient mice also generated similar levels of antigen-specific IgM and IgG in the serum as WT following immunization with sheep red blood cells (sRBC). This study demonstrates that although CB2 plays a role in promoting GC and memory B cell formation/maintenance in the spleen, it is dispensable on all immune cell types required for the generation of antigen-specific IgM and IgG in T-dependent immune responses.     

ALKBH1 Is Dispensable for Abasic Site Cleavage during Base Excision Repair and Class Switch Recombination

Potential roles of the abasic site lyase activity associated with AlkB homolog 1 (ALKBH1) were assessed by studies focusing on the two cellular processes that create abasic sites as intermediates: base excision repair and class switch recombination. Alkbh1^−/− pups (lacking exon 3) were born at a lower than expected frequency from heterozygous parents, suggesting a reduced survival rate and non-Mendelian inheritance, and they exhibited a gender bias in favor of males (70% males and 30% females). To study ALKBH1’s potential involvement in DNA repair, fibroblasts were isolated from Alkbh1^−/− mice, spontaneously immortalized and tested for resistance to DNA damaging agents. Alkbh1^−/− and isogenic cells expressing hALKBH1 showed no difference in survival to the DNA damaging agents methyl-methionine sulfate or H₂O₂. This result indicates that ALKBH1 does not play a major role in the base excision repair pathway. To assess ALKBH1’s role in class switch recombination, splenic B cells were isolated from Alkbh1^−/− and Alkbh1^+/+ mice and subjected to switching from IgM to IgG1. No differences were found in IgG1 switching, suggesting that Alkbh1 is not involved in class switch recombination of the immunoglobulin heavy chain during B lymphocyte activation.