Metabolic Reprogramming Commits Differentiation of Human CD27+IgD+ B Cells to Plasmablasts or CD27-IgD- Cells.

B cells play a crucial role in the pathogenesis of autoimmune diseases, such as systemic lupus erythematosus (SLE). However, the relevance of the metabolic pathway in the differentiation of human B cell subsets remains unknown. In this article, we show that the combination of CpG/TLR9 and IFN-α markedly induced the differentiation of CD27+IgD+ unswitched memory B cells into CD27hiCD38hi plasmablasts. The response was accompanied by mammalian target of rapamycin complex 1 (mTORC1) activation and increased lactate production, indicating a shift to glycolysis. However, CpG alone induced the differentiation of unswitched memory B cells into CD27-IgD- memory B cells with high cytokine production, but such differentiation was suppressed by IFN-α. AMP-activated protein kinase activation enhanced the differentiation to CD27-IgD- B cells, but it attenuated mTORC1 activation and differentiation into plasmablasts. High mTORC1 activation was noted in CD19+ B cells of patients with SLE and correlated with plasmablast differentiation and disease activity. Taken together, differential metabolic reprogramming commits the differentiation of human unswitched memory B cells into plasmablasts (the combination of CpG and IFN-α amplifies mTORC1-glycolysis pathways) or CD27-IgD- memory B cells (CpG alone amplifies the AMP-activated protein kinase pathway). The former metabolic pathway may play a pivotal role in SLE.

Duplex Quantitative PCR Assay for Detection of Haemophilus influenzae That Distinguishes Fucose- and Protein D-Negative Strains.

We have developed a specific Haemophilus influenzae quantitative PCR (qPCR) that also identifies fucose-negative and protein D-negative strains. Analysis of 100 H. influenzae isolates, 28 Haemophilus haemolyticus isolates, and 14 other bacterial species revealed 100% sensitivity (95% confidence interval [CI], 96% to 100%) and 100% specificity (95% CI, 92% to 100%) for this assay. The evaluation of 80 clinical specimens demonstrated a strong correlation between semiquantitative culture and the qPCR (P < 0.001).

Bacterial exploitation of phosphorylcholine mimicry suppresses inflammation to promote airway infection.

Regulation of neutrophil activity is critical for immune evasion among extracellular pathogens, yet the mechanisms by which many bacteria disrupt phagocyte function remain unclear. Here, we have shown that the respiratory pathogen Streptococcus pneumoniae disables neutrophils by exploiting molecular mimicry to degrade platelet-activating factor (PAF), a host-derived inflammatory phospholipid. Using mass spectrometry and murine upper airway infection models, we demonstrated that phosphorylcholine (ChoP) moieties that are shared by PAF and the bacterial cell wall allow S. pneumoniae to leverage a ChoP-remodeling enzyme (Pce) to remove PAF from the airway. S. pneumoniae-mediated PAF deprivation impaired viability, activation, and bactericidal capacity among responding neutrophils. In the absence of Pce, neutrophils rapidly cleared S. pneumoniae from the airway and impeded invasive disease and transmission between mice. Abrogation of PAF signaling rendered Pce dispensable for S. pneumoniae persistence, reinforcing that this enzyme deprives neutrophils of essential PAF-mediated stimulation. Accordingly, exogenous activation of neutrophils overwhelmed Pce-mediated phagocyte disruption. Haemophilus influenzae also uses an enzyme, GlpQ, to hydrolyze ChoP and subvert PAF function, suggesting that mimicry-driven immune evasion is a common paradigm among respiratory pathogens. These results identify a mechanism by which shared molecular structures enable microbial enzymes to subvert host lipid signaling, suppress inflammation, and ensure bacterial persistence at the mucosa.

Defense genes missing from the flight division.

Birds have a smaller repertoire of immune genes than mammals. In our efforts to study antiviral responses to influenza in avian hosts, we have noted key genes that appear to be missing. As a result, we speculate that birds have impaired detection of viruses and intracellular pathogens. Birds are missing TLR8, a detector for single-stranded RNA. Chickens also lack RIG-I, the intracellular detector for single-stranded viral RNA. Riplet, an activator for RIG-I, is also missing in chickens. IRF3, the nuclear activator of interferon-beta in the RIG-I pathway is missing in birds. Downstream of interferon (IFN) signaling, some of the antiviral effectors are missing, including ISG15, and ISG54 and ISG56 (IFITs). Birds have only three antibody isotypes and IgD is missing. Ducks, but not chickens, make an unusual truncated IgY antibody that is missing the Fc fragment. Chickens have an expanded family of LILR leukocyte receptor genes, called CHIR genes, with hundreds of members, including several that encode IgY Fc receptors. Intriguingly, LILR homologues appear to be missing in ducks, including these IgY Fc receptors. The truncated IgY in ducks, and the duplicated IgY receptor genes in chickens may both have resulted from selective pressure by a pathogen on IgY FcR interactions. Birds have a minimal MHC, and the TAP transport and presentation of peptides on MHC class I is constrained, limiting function. Perhaps removing some constraint, ducks appear to lack tapasin, a chaperone involved in loading peptides on MHC class I. Finally, the absence of lymphotoxin-alpha and beta may account for the observed lack of lymph nodes in birds. As illustrated by these examples, the picture that emerges is some impairment of immune response to viruses in birds, either a cause or consequence of the host-pathogen arms race and long evolutionary relationship of birds and RNA viruses.

The absence of immunoglobulin D B cell receptor-mediated signals promotes the production of autoantibodies and exacerbates glomerulonephritis in murine lupus.

Immunoglobulin (Ig)D is the major antigen receptor isotype co-expressed with IgM on the surface of most peripheral B cells in mice and humans. However, the biological role of IgD as B cell receptor (BCR) has remained unclear. Previous studies have indicated that IgD may play a role in B cell tolerance. To understand the role of IgD in B cell tolerance and autoimmunity, we have examined the development of autoimmune syndrome in lpr mice deficient for IgD. The present study showed that IgD deficiency did not alter lymphoproliferation and lymphocyte activation in lpr mice. The survival and proliferation of B cells were not affected by the absence of IgD, indicating that IgD BCR-mediated signals do not have an important role in negative selection of autoreactive B cell clones. Interestingly, compared to IgD-competent littermates, lpr mice with IgD deficiency had elevated autoantibody production, increased deposition of immune complex in the kidney and more severe nephritis. Accumulation of abnormal CD4(-) CD8(-) αß(+) T cells was accelerated in IgD(-/-) lpr mice compared to lpr mice. These results suggest that IgD BCR-mediated signals may be involved in the differentiation of autoreactive B cells into plasma cells and abnormal T cell expansion.

Cutting edge: Helminth infection induces IgE in the absence of mu- or delta-chain expression.

Infections with helminth parasites are associated with an IgE isotype switch and high serum IgE concentrations. IgE is rapidly bound by the high affinity IgE receptor (Fc epsilonRI), thereby sensitizing Fc epsilonRI-bearing basophils and mast cells for IgE-inducible effector functions such as IL-4 production. The development of Ab-secreting B cells is dependent on IgM and consequently, muMT mice, which lack surface IgM, are considered devoid of Abs. In this study we report the unexpected finding that C57BL/6 muMT mice generate robust IgE responses upon infection with three distinct helminth parasites, Heligmosomoides polygyrus, Trichuris muris, and Schistosoma mansoni. IgE is produced despite an apparent block in B cell development and licenses basophils for IgE-induced IL-4 production. Our findings reveal the existence of an evolutionarily conserved, IgM-independent pathway for the production of IgE upon infection with helminth parasites.

A new population of cells lacking expression of CD27 represents a notable component of the B cell memory compartment in systemic lupus erythematosus.

Human memory B cells comprise isotype-switched and nonswitched cells with both subsets displaying somatic hypermutation. In addition to somatic hypermutation, CD27 expression has also been considered a universal memory B cell marker. We describe a new population of memory B cells containing isotype-switched (IgG and IgA) and IgM-only cells and lacking expression of CD27 and IgD. These cells are present in peripheral blood and tonsils of healthy subjects and display a degree of hypermutation comparable to CD27+ nonswitched memory cells. As conventional memory cells, they proliferate in response to CpG DNA and fail to extrude rhodamine. In contrast to other recently described CD27-negative (CD27neg) memory B cells, they lack expression of FcRH4 and recirculate in the peripheral blood. Although CD27neg memory cells are relatively scarce in healthy subjects, they are substantially increased in systemic lupus erythematosus (SLE) patients in whom they frequently represent a large fraction of all memory B cells. Yet, their frequency is normal in patients with rheumatoid arthritis or chronic hepatitis C. In SLE, an increased frequency of CD27neg memory cells is significantly associated with higher disease activity index, a history of nephritis, and disease-specific autoantibodies (anti-dsDNA, anti-Smith (Sm), anti-ribonucleoprotein (RNP), and 9G4). These findings enhance our understanding of the B cell diversification pathways and provide mechanistic insight into the immunopathogenesis of SLE.

Immunoglobulin deficiencies and susceptibility to infection among homozygotes and heterozygotes for C2 deficiency.

About 25% of C2-deficient homozygotes have increased susceptibility to severe bacterial infections. C2-deficient homozygotes had significantly lower serum levels of IgG2, IgG4, IgD, and Factor B, significantly higher levels of IgA and IgG3 and levels of IgG1 and IgM similar to controls. Type 1 (28 bp deletion in C2 exon 6 on the [HLA-B18, S042, DR2] haplotype or its fragments) and type II (non-type I) C2-deficient patients with increased susceptibility to bacterial infection had significantly lower mean levels of IgG4 (p < 0.04) and IgA (p < 0.01) than those without infections (who had a higher than normal mean IgA level) but similar mean levels of other immunoglobulins and Factor B. Of 13 C2-deficient homozygotes with infections, 85% had IgG4 deficiency, compared with 64% of 25 without infections. IgD deficiency was equally extraordinarily common among infection-prone (50%) and noninfection-prone (70%) homozygous type I C2-deficient patients. IgD deficiency was also common (35%) among 31 type I C2-deficient heterozygotes (with normal or type II haplotypes), but was not found in 5 type II C2-deficient heterozygotes or 1 homozygote. Thus, C2 deficiency itself is associated with many abnormalities in serum immunoglobulin levels, some of which, such as in IgG4 and IgA, may contribute to increased susceptibility to infection. In contrast, IgD deficiency appears not to contribute to increased infections and appears to be a dominant trait determined by a gene or genes on the extended major histocompatibility complex (MHC) haplotype [HLA-B 18, S042, DR2] (but probably not on type II C2-deficient haplotypes) similar to those previously identified on [HLA-B8, SC01, DR3] and [HLA-B18, F1C30, DR3].

Cells expressing the Epstein-Barr virus growth program are present in and restricted to the naive B-cell subset of healthy tonsils.

In this paper we demonstrate, for the first time, that Epstein-Barr virus (EBV)-infected cells expressing the lymphoblastoid growth program are present in healthy carriers of the virus. Previously we observed that latently infected naive B cells are present in tonsils only when viral replication is detected, suggesting that these may represent newly infected B cells. We have tested this idea by performing a reverse transcription-PCR analysis for the expression of latent genes (EBNA2 and the EBNA3s) that are characteristically expressed only by newly infected cells expressing the growth latency program. EBNA2 expression is regularly detected in purified naive (IgD(+)) tonsillar B cells (13 of 16 tonsils tested) but was never found in the IgD(-) population (0 of 16). More detailed analysis revealed that the mRNAs for the latent genes EBNA1 (3 of 3 tonsils tested), EBNA3a (3 of 5), EBNA3b (3 of 5), EBNA3c (3 of 5), LMP1 (6 of 6), and LMP2 (5 of 6) were also present in the IgD(+) population, but the EBNA1Q-K transcript, characteristic of nonlymphoblastoid forms of latency, was never detected (0 of 6). Finally, we demonstrate that the latently infected naive (IgD(+)) cells express CD80 (B7.1), a marker characteristically expressed on activated naive lymphoblasts but absent from resting naive B cells. The infected naive (IgD(+)) population in the tonsil therefore has the viral and cellular phenotype of a B-cell directly infected with EBV-an activated lymphoblast expressing the growth program.

The [HLA-B18, F1C30, DR3] conserved extended haplotype carries a susceptibility gene for IgD deficiency.

We showed previously that the conserved extended MHC haplotype [HLA-B8, SCO1, DR3] carries recessive susceptibility genes for IgA and IgG4 deficiency and dominant genes for IgD and IgG3 deficiency. [HLA-B18, F1C30, DR3] has similar class II and III regions to [HLA-B8, SC01, DR3] and is common in the Basques. We therefore studied serum immunoglobulin concentrations in Basque homozygotes, heterozygotes, and noncarriers of (FIC30, DRB1*0301, DRB3*02, DQA1*0501. DQB1*0201) (F1C30, DR3). As shown by others, no subjects were deficient in IgA, IgM, or IgG subclasses. In contrast, 29% of homozygotes and three of seven double heterozygotes with (SC01, DRB1*0301, DRB3*0101, DQA1*0501, DQB1*0201) (presumed homozygotes for IgD deficiency susceptibility genes) were IgD deficient. Thus, 32% of presumed homozygotes were IgD deficient compared with 1.6% of noncarriers. Of haplotype heterozygotes, 25% were IgD deficient. The high frequency of IgD deficiency in both homozygotes and heterozygotes for (F1C30, DR3) suggests a partially penetrant dominant susceptibility gene for IgD deficiency on [HLA-B18, F1C30, DR3].

Absence of IgD-CD27(+) memory B cell population in X-linked hyper-IgM syndrome.

The present study analyzed peripheral blood B cell populations separated by IgD and CD27 expression in six males with X-linked hyper-IgM syndrome (XHIM). Costimulation of mononuclear cells from most of the patients induced no to low levels of class switching from IgM to IgG and IgA with Staphylococcus aureus Cowan strain (SAC) plus IL-2 or anti-CD40 mAb (anti-CD40) plus IL-10. Measurable levels of IgE were secreted in some of the patients after stimulation with anti-CD40 plus IL-4. Costimulation with SAC plus IL-2 plus anti-CD40 plus IL-10 yielded secretion of significant levels of IgG in addition to IgM, but not IgA. The most striking finding was that peripheral blood B cells from all of the six patients were composed of only IgD+ CD27(-) and IgD+ CD27(+) B cells; IgD- CD27(+) memory B cells were greatly decreased. IgD+ CD27(+) B cells from an XHIM patient produced IgM predominantly. Our data indicate that the low response of IgG production in XHIM patients is due to reduced numbers of IgD- CD27(+) memory B cells. However, the IgG production can be induced by stimulation of immunoglobulin receptors and CD40 in cooperation with such cytokines as IL-2 and IL-10 in vitro.

The swine Ig heavy chain locus has a single JH and no identifiable IgD.

Sequence analysis of 84 V region cDNAs expressed with IgM, IgG and IgA from both adult and newborn swine suggested that their JH segments had been derived from the same germline JH. Only a single hybridizing JH segment could be identified in genomic DNA, in a JH-C mu cosmid and in a Sacl fragment of the cosmid extending 5 kb 5' of the EnhH. The single germline JH segment mapped 6 kb 5' to C mu. This JH had a sequence identical to 40 of 42 JH segments expressed in a newborn piglet and 25 of 42 expressed by adult swine. None of the 19 JH segments which varied from the germline sequence were identical to each other and half of the nucleotide changes were silent. No cosmid DNA hybridizing with heterologous probes for C delta could be found within 20 kb 3' of C mu and C delta could not be cloned from genomic or cDNA libraries. A conserved IgD fragment could be amplified from human, mouse and rat genomic DNA but not from rabbit, swine or cattle. We hypothesize that heavy chain organization and constituency in homeothermic vertebrates is correlated with the site of secondary antibody repertoire development and the mechanism(s) used.

Mouse anti-mouse IgD monoclonal antibodies generated in IgD-deficient mice.

The production of high-affinity monoclonal antibodies (mABs) is generally restricted to antigens recognised as foreign by the immune system. Here we report the generation of mouse mABs specific for mouse IgD. Mice rendered IgD-deficient by gene targeting and consequently immunologically fully responsive to mouse IgD, were used to elicit a humoral response against mouse IgD. Hybridomas producing mABs of high affinity were isolated and clones specific for non-allotypic determinants on the Fc or Fab portion of mouse IgD were obtained. The data show that mice lacking a protein of interest due to targeted gene inactivation can be utilised for the production of high-affinity mABs specific for that mouse protein and should also facilitate the generation of mABs specific for proteins highly conserved between species.

Oligomeric IgD augments and monomeric IgD inhibits the generation of IgG memory antibody responses in normal, but not in IgD-deficient, mice.

Dimeric or aggregated IgD causes augmentation of primary and secondary Ab responses in mice when injected a few days before or together with the primary dose of Ag. This effect is mediated by Th cells and seems to be linked to the up-regulation of receptors for IgD on CD4+ T cells. IgD-R cross-linking is needed for receptor up-regulation. Here we show that addition of monomeric IgD to dimeric or aggregated IgD blocks IgD-R up-regulation on T cells in vitro and in vivo, as well as their immunoaugmenting effect in vivo. More importantly, monomeric IgD injected 6 to 24 h before a primary Ag injection also inhibits 1) the up-regulation of IgD-R on T cells induced by Ag injection alone, and 2) the generation of IgG memory, as shown in the response to a second dose of Ag injected on day 10. These results suggest that IgD-R on T cells contribute to the T-B cell interaction involved in the priming for a secondary response. The augmenting effect of oligomeric IgD and the inhibiting effect of monomeric IgD on secondary Ab responses are not observed in IgD-/- (IgD-deficient) mice, although injection of oligomeric IgD leads to IgD-R up-regulation on T cells in these mice. These results indicate that IgD presented in the form of immune complexes, most likely on the surface of B cells, is a prerequisite for the immunoaugmenting effects exerted by IgD-R+ T cells. Thus, IgD is the only physiologic ligand for IgD-R on T cells.

Immunoglobulin D-deficient mice can mount normal immune responses to thymus-independent and -dependent antigens.

To examine the in vivo function of IgD we generated mice deficient for IgD by gene targeting. The IgD-mice show a reduced B-cell compartment with 30-50% less B cells in the spleen and lymph nodes but show a normal pre-B-cell compartment. The surface-IgD- B cells express two to three times more surface IgM than B cells of control animals. Serum concentrations of the immunoglobulin isotypes of IgD- mice are almost normal, indicating that surface-IgD expression is not necessary for class switching of B cells. Immunization experiments showed that IgD- mice could respond well to thymus-dependent and -independent antigens. After immunization normal germinal centers developed in the IgD- mice. These data suggest that IgD is not necessary for the induction of immune responses but may be important in homeostasis of cells in the B-cell compartment.

Immunoglobulin D (IgD)-deficient mice reveal an auxiliary receptor function for IgD in antigen-mediated recruitment of B cells.

To assess the role of immunoglobulin D (IgD) in vivo we generated IgD-deficient mice by gene targeting and studied B cell development and function in the absence of IgD expression. In the mutant animals, conventional and CD5-positive (B1) B cells are present in normal numbers, and the expression of the surface markers CD22 and CD23 in the compartment of conventional B cells indicates acquisition of a mature phenotype. As in wild-type animals, most of the peripheral B cells are resting cells. The IgD-deficient mice respond well to T cell-independent and -dependent antigens. However, in heterozygous mutant animals, B cells expressing the wild type IgH locus are overrepresented in the peripheral B cell pool, and T cell-dependent IgG1 responses are further dominated by B cells expressing the wild-type allele. Similarly, in homozygous mutant (IgD-deficient) animals, affinity maturation is delayed in the early primary response compared to control animals, although the mutants are capable of generating high affinity B cell memory. Thus, rather than being involved in major regulatory processes as had been suggested, IgD seems to function as an antigen receptor optimized for efficient recruitment of B cells into antigen-driven responses. The IgD-mediated acceleration of affinity maturation in the early phase of the T cell-dependent primary response may confer to the animal a critical advantage in the defense against pathogens.