Ageing and obesity similarly impair antibody responses.

Ageing is characterized by increased low-grade chronic inflammation, which is a significant risk factor for morbidity and mortality of elderly individuals. Similar to ageing, obesity is considered to be an inflammatory predisposition associated with chronic activation of immune cells and consequent local and systemic inflammation. Both ageing and obesity are characterized by reduced innate and adaptive immune responses. This review focuses on B cells, how they may contribute, at least locally, to low-grade chronic inflammation in ageing and obesity and on the mechanisms involved.

Immune response to influenza A/H1N1 vaccine in inflammatory bowel disease patients treated with anti TNF-α agents: effects of combined therapy with immunosuppressants.

BACKGROUND AND AIMS: Our first objective was to evaluate the immune response to the adjuvanted 2009 A/H1N1 pandemic (pH1N1) vaccine in inflammatory bowel disease (IBD) patients treated with anti-TNF-α alone or combined with immunosuppressants (IS). Second and third aims were the safety of pH1N1 vaccine and the effects on IBD clinical activity. METHODS: 36 patients with Crohn's disease (CD) and 26 with ulcerative colitis (UC) and thirty-one healthy control (HC) subjects were enrolled. 47 patients were on anti TNF-α maintenance monotherapy and 15 on anti TNF-α combined with IS. Sera were collected at baseline (T0) and 4 weeks after the vaccination (T1) for antibody determination by hemagglutination inhibition (HAI). Disease activity was monitored at T0 and T1. RESULTS: Seroprotective titers (≥1:40) in patients were comparable to HC. Seroconvertion rate (≥4 fold increase in HAI titer) was lower than HC in IBD patients (p=0.009), either on anti TNF-α monotherapy (p=0.034) or combined with IS (p=0.011). Geometric mean titer (GMT) of antibodies at T1 was significantly lower in patients on combined therapy versus those on monotherapy (p=0.0017) and versus HC (p=0.011). The factor increase of GMT at T1 versus T0 was significantly lower in IBD patients versus HC (p=0.042), and in those on combined immunosuppression, both versus monotherapy (p=0.0048) and HC (p=0.0015). None of the patients experienced a disease flare. CONCLUSION: Our study has shown a suboptimal response to pH1N1 vaccine in IBD patients on therapy with anti TNF-α and IS compared to those on anti-TNF-α monotherapy and HC.

The reduced expression of surrogate light chains in B cell precursors from senescent BALB/c mice is associated with decreased E2A proteins.

Senescent mice exhibit decreased numbers of pre-B cells in the bone marrow. Herein, we show that the molecules, lambda5 and VpreB, which comprise the surrogate light chain component of the pre-B cell receptor, are reduced in pro-B/early pre-B cells derived in vitro from the bone marrow of 18-27 months old BALB/c mice after stimulation with IL-7. Both lambda5 and VpreB expression were decreased at the mRNA level as indicated by semi-quantitative RT-PCR; this suggests that the reduced surrogate light chains seen in senescent B cell precursors result from dysfunctional transcriptional regulation. The transcription of surrogate light chains is regulated, in part, by E2A (E47) gene products. Levels of E2A proteins, including E47, were decreased in senescent B cell precursors by up to 90%. Reduced E2A (E47) expression and subsequent reduced transcription of the surrogate light chain components lambda5 and VpreB may, in part, explain the diminished production of B lineage cells observed in senescence.

Transgenic human lambda 5 rescues the murine lambda 5 nullizygous phenotype.

The human lambda 5 (hu lambda 5) gene is the structural homologue of the murine lambda 5 (m lambda 5) gene and is transcriptionally active in pro-B and pre-B lymphocytes. The lambda 5 and VpreB polypeptides together with the Ig mu H chain and the signal-transducing subunits, Ig alpha and Ig beta, comprise the pre-B cell receptor. To further investigate the pro-B/pre-B-specific transcription regulation of hu lambda 5 in an in vivo model, we generated mouse lines that contain a 28-kb genomic fragment encompassing the entire hu lambda 5 gene. High levels of expression of the transgenic hu lambda 5 gene were detected in bone marrow pro-B and pre-B cells at the mRNA and protein levels, suggesting that the 28-kb transgene fragment contains all the transcriptional elements necessary for the stage-specific B progenitor expression of hu lambda 5. Flow cytometric and immunoprecipitation analyses of bone marrow cells and Abelson murine leukemia virus-transformed pre-B cell lines revealed the hu lambda 5 polypeptide on the cell surface and in association with mouse Ig mu and mouse VpreB. Finally, we found that the hu lambda 5 transgene is able to rescue the pre-B lymphocyte block when bred onto the m lambda 5-/- background. Therefore, we conclude that the hu lambda 5 polypeptide can biochemically and functionally substitute for m lambda 5 in vivo in pre-B lymphocyte differentiation and proliferation. These studies on the mouse and human pre-B cell receptor provide a model system to investigate some of the molecular requirements necessary for B cell development.

Bone marrow cells inhibit the generation of autologous EBV-specific CTL.

Recently, we reported that human bone marrow cells (BMC) inhibited the proliferative (recall) response of lymphocytes to Epstein-Barr virus (EBV) and cytomegalovirus (CMV) protein antigens [12]. To clarify further the effect of BMC on the immune response to viral antigens, we obtained PBL from EBV IgG antibody positive kidney transplant recipients (R) and their living-related donors (LRD) 1 year after renal transplantation and generated EBV-specific CTL in vitro in the presence or absence of autologous BMC. The addition of freshly aspirated autologous iliac crest BMC from either R or LRD caused a significant inhibitory effect on the generation of EBV-specific CTL from CTL precursors, in contrast to the addition of autologous PBL used as controls (62.29 +/- 10.85% inhibition using BMC from the kidney transplant recipients; 74.47 +/- 15.21% inhibition using BMC from the living-related donors). This inhibitory effect was only exerted during the CTL generation phase; but not in the effector CTL killing phase. The expression of CD94, a component of the killer inhibitory receptor (KIR) on CD3(+) cells was elevated in the cultures with BMC, in contrast to the cultures without BMC. The BMC inhibitory effect was partially abrogated by pre-incubation of the CTL effectors with anti-CD94 monoclonal antibody, in contrast with its isotype control. In addition, supernatants obtained from the CTL generating cultures with BMC contained high levels of prostaglandin E(2) (PGE(2)), and EBV-specific CTL activity was inhibited by the addition of exogenous PGE(2) in the absence of BMC. The induction of CD40L cell surface expression by anti-CD3 was also decreased on the effector T cell population when BMC were added. There was a concomitant reduction in protein kinase C (PKC) activity. These studies demonstrate that BMC exert an inhibitory effect on T cell-mediated immunity to viral antigens in humans by regulating autologous effector T cell generation and early T cell activation signaling pathways.

Bone marrow cells promote TH2 polarization and inhibit virus-specific CTL generation.

This laboratory recently reported that human bone marrow cells (BMC) inhibit the generation of virus-specific CTL in culture. The culture supernatants contained increased levels of prostaglandin E(2) (PGE(2)) (shown to favor TH2 cell development) and also inhibited EBV-CTL effector cell development. In this study, we obtained PBL from Epstein-Barr virus (EBV) IgG antibody positive kidney transplant recipients (R) and their living-related donors (LRD) one year after renal transplantation. EBV-specific CTL were then generated in vitro by stimulating PBL with autologous EBV-transformed B cells (EBV-B) in the presence or absence of autologous BMC. The addition of BMC to the EBV-CTL generation cultures increased the intracellular expression in CD3+ cells of IL-4,-5,-6,-10, and -13. These CD3+ cells also expressed increased levels of the TH2 associated receptor CCR3. Inhibition was even observed by preparing EBV-CTL generating cultures in trans-wells that separated the autologous BMC from the PBL + EBV-B. It was then observed that CD3+ cells obtained after 7 days of culture in the presence of autologous BMC could be used as inhibitors of EBV-CTL generation. Protein Kinase A (PKA), a cAMP kinase that is involved in the upregulation of TH2 cytokine activity, was increased in EBV-CTL cultures by the presence of BMC. Additionally, IL-4-mediated signal transduction and activation of transcription (STAT-6) phosphorylation was slightly increased. These results show that the BMC inhibition is mediated by soluble factors (cytokines) and that cell-cell contact in this autologous system is not required, so that BMC (at least partially, via cytokine production) promote TH2 polarization in culture. Moreover, TH2 cells induced by culturing with autologous BMC directly inhibit EBV-CTL generation, and TH2 associated PKA, CCR3, and STAT-6 phosphorylation are enhanced by BMC.

Senescent BALB/c mice exhibit decreased expression of lambda5 surrogate light chains and reduced development within the pre-B cell compartment.

Although senescent BALB/c mice (approximately 2 years old) have reduced numbers of small pre-B cells, early pre-B cells (CD43+CD25+B220+) are present in comparable numbers within the bone marrow of both young (3-6-month-old) and senescent BALB/c mice. The transition of CD43+ pre-B cells to the CD43- pre-B cell compartments is dependent on proliferation and clonal maturation dictated by the pre-B cell receptor (mu/lambda5/VpreB). In vivo, senescent CD43+B220+ pro-B/early pre-B cells demonstrated reduction of lambda5 mRNA, by RT-PCR analysis, and of both surface and cytoplasmic lambda5 protein. Decreased lambda5 protein expression was also seen among pro-B/pre-B cells derived from senescent bone marrow after stimulation in vitro with IL-7. We propose that diminished expression of the lambda5 surrogate light chain results in decreased pre-B cell receptor formation and contributes to reduced recruitment of nascent CD43+ pre-B cells into the CD43- large and small pre-B cell compartments.

The 14.1 surrogate light chain promoter has lineage- and stage-restricted activity.

The 14.1 surrogate light chain protein is expressed on human pre-B lymphocytes in association with Vpre-B and the mu Ig heavy chain to form the pre-B receptor. The 14.1 chain has also been called the lambda (lambda)-like (LL) protein and is homologous to murine lambda5. The 14.1(IGLL1) gene is expressed in a lineage- and stage-restricted manner. To understand the molecular mechanism of the 14.1 gene tissue- and stage-specific expression, we analyzed the 5'-flanking region and characterized the promoter for this gene. In this report, we identify two DNase I-hypersensitive sites located at 2.6 kilobases (HSS 1) and 0.2 kilobases (HSS 1) upstream of 14.1 exon 1. These hypersensitive sites are present in pre-B lymphocyte cell lines, but absent in mature B and T cell lines. We have used RNase protection analysis to localize the 5' major transcriptional start site and rapid amplification of 5' cDNA ends to identify multiple start sites within the TATA-less, GC-rich 14.1 promoter. The region encompassing HSS 2 was analyzed for promoter activity. Transfection of cell lines with a series of truncated segments of the 5' flanking region linked to the luciferase reporter gene revealed that the 14.1 promoter is lineage- and stage-specific, and we localized this activity to positions +150 to +227 relative to the 5' major transcriptional start site.

The human lambda immunoglobulin enhancer is controlled by both positive elements and developmentally regulated negative elements.

We have recently reported the localization of the first transcriptional enhancer in the human lambda (lambda) immunoglobulin light chain locus. Enhancer activity was contained on a 1.2 kb SstI fragment, with partial activity retained on a core 111 bp PstI-SstI fragment. This enhancer is located 11.7 kb downstream of C lambda 7, the most 3' lambda constant region gene. Using a chloramphenicol acetyl transferase (CAT) assay system, we have now determined the boundaries of the complete enhancer and find it is two- to four-fold as active as the core fragment in both pre-B and B cell lines. Interestingly, a larger fragment, containing the complete enhancer as well as 5' and 3' flanking sequences has four- to eight-fold reduced activity when tested in pre-B cell lines, but full activity in B cell lines. This suggests the presence of developmentally regulated negative elements flanking the human lambda enhancer which prevent or reduce its activity at a developmentally incorrect time. By using in vivo footprinting we have begun to examine the protein interactions within this enhancer in a more physiologically relevant manner and have identified motifs which are shared with the murine lambda enhancers, as well as motifs unique to the human lambda enhancer.

Identification and localization of a developmental stage-specific promoter activity from the murine lambda 5 gene.

The lambda 5 protein is expressed in pre-B cells in association with VpreB and mu-heavy chains, and is critical for differentiation to the B cell stage. Pre-B cell-specific expression of the lambda 5 and VpreB genes is regulated at the level of transcription initiation. In this report, we have identified several DNase l-hypersensitive sites 2.5- to 6.0-kb downstream of the lambda 5 gene, which are present in the pre-B cell line 70Z/3, but not in the myeloma cell line j558L. These sites, however, were shown to have no transcriptional enhancer activity as measured by transient transfection. Enhancer activity was identified within a 361-bp fragment (-296 to +65, where +1 is the major 5' transcription initiation site) upstream of the mouse lambda 5 gene. This activity is orientation and position independent, and is also tissue and differentiation stage specific (active in pre-B but not B and T cells). Deletion constructs indicate that three adjacent areas (-210 to -169, -153 to -64, and -64 to -22) are all necessary for enhancer activity. Pre-B cell-specific promoter activity was shown to reside within the -219 to +109 fragment. Basal promoter activity resides within the -64 to +109 fragment, but is not tissue specific or stage specific. A negative element within the -101 to -64 region is active in all lymphoid cell lines tested and therefore cannot by itself be responsible for the tissue and stage specificity. The data indicate that the elements responsible for the enhancer activity (-210 to -22) are part of the lambda 5 gene promoter and likely confer the tissue and stage specificity via positive elements within the -210 to -22 region.

Physical location of the human immunoglobulin lambda-like genes, 14.1, 16.1, and 16.2.

The human immunoglobulin lambda-like (IGLL) genes, which are homologous to the human immunoglobulin lambda (IGL) light chain genes, are expressed only in pre-B cells and are involved in B cell development. Three IGLL genes, 14.1, 16.1, and 16.2 are present in humans as opposed to one, lambda 5 (Igll), found in the mouse. To precisely map the location of the human IGLL genes in relation to each other and to the human IGL gene locus, at 22q11.1-2, a somatic cell hybrid panel and pulsed field gel electrophoresis (PFGE) were used. Hybridization with a lambda-like gene-specific DNA probe to somatic cell hybrids revealed that these genes reside on 22q11.2 between the breakpoint cluster region (BCR) and the Ewing sarcoma breakpoint at 22q12 and that gene 16.1 was located distal to genes 14.1 and 16.2. Gene 14.1 was found by PFGE to be proximal to 16.2 by at least 30 kilobases (kb). A 210 kb Not I fragment containing genes 14.1 and 16.2 is adjacent to a 400 kb Not I fragment containing the BCR locus, which is just distal to the IGL-C (IGL constant region) genes. We have determined that the IGLL genes 14.1 and 16.2 are approximately 670 kb and 690 to 830 kb distal, respectively, to the 3'-most IGL-C gene in the IGL gene locus, IGL-C7. We thus show the first physical linkage of the IGL and the IGLL genes, 14.1 and 16.2. We discuss the relevance of methylation patterns and CpG islands to expression, and the evolutionary significance of the IGLL gene duplications. Consistent with the GenBank nomenclature, these human IGLL genes will be referred to as IGLL1 (14.1), IGLL2 (16.2), and IGLL3 (16.1), reflecting their position on chromosome 22, as established by this report.

Identification and localization of an enhancer for the human lambda L chain Ig gene complex.

A strong transcriptional enhancer was identified for the human lambda L chain Ig gene complex. Enhancer activity was measured by activation of the chloramphenicol acetyl transferase (CAT) gene in a transient assay using both mouse and human B lymphoid cell lines. The smallest fragment identified with enhancer activity was 111 bp, which resides 11.7 kb downstream (3') of C lambda 7, a constant region gene we have recently isolated and identified as functional in the human population. Enhancer activity is orientation independent, tissue specific (active in all B cell lines tested and not in a T cell line), and independent of NF kappa B, similar to the mouse lambda enhancers recently reported. The human lambda enhancer is active in both mouse and human B cell lines; interestingly, the mouse lambda enhancers are active in mouse lines but not in a human B cell line. DNA sequence comparison of the mouse and human lambda enhancers indicates a higher degree of homology (average of 72.5%) within the 111-bp enhancer core region identified here than for the remaining flanking sequence compared (average of 42%). This discovery of an enhancer in the human lambda locus (HuE lambda), which is clearly distinct from that of the H and L chain loci, will help to determine the mechanism for the ordered expression and rearrangement of these gene complexes in B cell ontogeny. The presence of only one enhancer in the human C lambda complex 3' of all the C genes suggests that the evolutionary duplication of the L locus differs from that seen in mouse; in mouse the duplication unit was JCJC-enhancer, whereas the human JC lambda s duplicated without the enhancer.

Nuclear disintegration of target cells by killer B lymphocytes from tumor-bearing mice.

Normal murine B lymphocytes are not known to be effectors of the Fc receptor-mediated, antibody-dependent cellular cytotoxicity (ADCC). In contrast, we report here that highly purified splenic B cells from mammary tumor-bearing mice develop the potential of lysing antibody-coated target cells. These lymphocytes are characterized by being G-10 nonadherent, nylon wool adherent, sIg+, FcR+, Thy 1.2-, asialo GM1-, and the immunoglobulin heavy-chain genes of both chromosomes are rearranged. The lytic reaction is characterized by a noninterdigitating binding and by the appearance of endocytotic vesicles in the target cells. Nuclear disintegration occurs 18 h after initial effector-target cell conjugate formation. At such time, only minor cytoplasmic membrane alterations are evident. The emergence of killer B cells in tumor-bearing hosts indicates that all lymphoreticular cell types bearing Fc receptors are capable of mediating ADCC.

Intergenic exchange maintains identity between two human lambda light chain immunoglobulin gene intron sequences.

Evidence for gene conversion or unequal double crossover in the human lambda light chain immunoglobulin locus is presented. The high level of J2C2-J3C3 intron cross-hybridization, the identity of the J lambda and J lambda 3 coding and intron sequences, the presence of multiple base differences between the C lambda 2 and C lambda 3 coding regions, and the presence of both the unconverted and converted alleles in the normal gene pool, suggest that a recombinational event has resulted in the conversion of the J lambda 2 coding and intron sequences to those of J lambda 3 and its flanking sequences. Intergenic exchanges, such as the one described here, may provide a mechanism to maintain sequence homogeneity and functionality among the duplicated members of the human lambda gene family.