... 1893 Nicolaas A. BosoV, Cornelia G. MeeuwsenO, Elisabeth De Glopper-Van der Veero, Theodoor W... more ... 1893 Nicolaas A. BosoV, Cornelia G. MeeuwsenO, Elisabeth De Glopper-Van der Veero, Theodoor W. Van den AkkerO, Jiri Radl*, Kornelisje A. Zwaagstra. and Robbed BennerO ... It may be that the Ly-1 B cell lineage is involved in BMG. ...
Early in ontogeny B cells preferentially use VH gene families which are most adjacent to the gene... more Early in ontogeny B cells preferentially use VH gene families which are most adjacent to the genes coding for the constant part of the immunoglobulin molecule. In conventional adult mice, however, a random usage of VH gene families has been found. We investigated the role of exogenous antigenic stimulation on this normalization of VH gene usage by B cells. Therefore, we made use of adult germ-free BALB/c mice fed a chemically defined ultrafiltered antigen-free diet (GF-CD) and neonatal conventional BALB/c mice. Both the adult GF-CD and the newborn conventional mice represent situations with minimal exogenous antigenic stimulation. The results obtained with RNA dot blot hybridization with probes specific for the different VH gene families showed in hybridomas from adult GF-CD BALB/c mice a preferential usage of the CH-proximal VH gene family PC7183. In hybridomas from 5-day-old conventional BALB/c mice a less frequent usage of the 5558 VH gene family was found and an increased usage of the PC7183 VH gene family than what would be expected from random usage. Evidence is presented that the RNA giving a positive signal with the PC7183 probe represents functional messages for IgM production.
Insight into the maintenance of naive T cells is essential to understand defective immune respons... more Insight into the maintenance of naive T cells is essential to understand defective immune responses in the context of aging and other immune compromised states. In humans, naive CD4+ T cells, in contrast to CD8+ T cells, are remarkably well retained with aging. Here, we show that low-affinity TCR engagement is the main driving force behind the emergence and accumulation of naive-like CD4+ T cells with enhanced sensitivity to IL-2 in aged humans. In vitro, we show that these CD45RA + CD25 dim CD4 + T cells can develop from conventional naive CD25 À CD4+ T cells upon CD3 cross-linking alone, in the absence of costimulation, rather than via stimulation by the homeostatic cytokines IL-2, IL-7, or IL-15. In vivo, TCR engagement likely occurs in secondary lymphoid organs as these cells were detected in lymph nodes and spleen where they showed signs of recent activation. CD45RA + CD25 dim CD4+ T cells expressed a broad TCRVb repertoire and could readily differentiate into functional T helper cells. Strikingly, no expansion of CD45RA + CD25 dim CD8+ T cells was detected with aging, thereby implying that maintenance of naive CD4+ T cells is uniquely regulated. Our data provide novel insight into the homeostasis of naive T cells and may guide the development of therapies to preserve or restore immunity in the elderly.
Monoclonal gammopathy of undetermined significance (MGUS) arises from a clonal expansion of plasm... more Monoclonal gammopathy of undetermined significance (MGUS) arises from a clonal expansion of plasma cells in the bone marrow, secreting monoclonal (M) paraprotein. MGUS is associated with increased susceptibility to infections, which may reflect altered B-cell repertoire. To investigate this, we examined the IgM, IgG and IgA B-cell repertoire diversity in MGUS at baseline and after influenza vaccination (n=16) in comparison to healthy controls (HCs) (n=16). The CDR3 region of the immunoglobulin heavy chain variable (IGHV) region gene was amplified and B-cell spectratypes analyzed by high-resolution electrophoresis. Spectratype Gaussian distribution, kurtosis and skewness were quantified to measure repertoire shifts. Both HC and MGUS baseline spectratypes show inter-individual variability that is more pronounced in the IGHG and IGHA repertoires. Overall, baseline B cell repertoire is more altered in MGUS, with oligoclonality observed in 50% (p=0.01). Post-vaccination, significant diff...
Healthy aging requires an optimal balance between pro-inflammatory and anti-inflammatory immune r... more Healthy aging requires an optimal balance between pro-inflammatory and anti-inflammatory immune responses. Although CD4+ T cells play an essential role in many immune responses, few studies have directly assessed the effect of aging on the balance between effector T (T eff ) cells and regulatory T (T reg ) cells. Here, we determined if and how aging affects the ratio between T reg and T eff cells. Percentages of both naive T reg (nT reg ; CD45RA+CD25 int FOXP3 low ) and memory T reg (memT reg ; CD45RA-CD25 high FOXP3 high ) cells were determined by flow cytometry in peripheral blood samples of healthy individuals of various ages (20-84 years). Circulating Th1, Th2 and Th17 effector cells were identified by intracellular staining for IFN-γ, IL-4 and IL-17, respectively, upon in vitro stimulation with PMA and calcium ionophore. Whereas proportions of nT reg cells declined with age, memT reg cells increased. Both Th1 and Th2 cells were largely maintained in the circulation of aged humans, whereas Th17 cells were decreased. Similar to memT reg cells, the 3 T eff subsets resided primarily in the memory CD4+ T cell compartment. Overall, T reg /T eff ratios were increased in the memory CD4+ T cell compartment of aged individuals when compared to that of young individuals. Finally, the relative increase of memT reg cells in elderly individuals was associated with poor responses to influenza vaccination. Taken together, our findings imply that aging disturbs the balance between T reg cells and T eff cells.
The total number of spontaneously occurring ("background") IgM-, IgG-, and &A-secreting cells and... more The total number of spontaneously occurring ("background") IgM-, IgG-, and &A-secreting cells and the frequency of antigen-specific IgM-, IgG-, and IgA-secreting cells were determined in germ-free BALB/c mice fed a chemically defined ultrafiltered diet (GF-CD), in specific pathogen-free BALB/c mice fed an autoclaved natural ingredient diet (SPF-NI), and in conventional BALB/c mice fed nonautoclaved natural ingredients (CV-NI). This was done by means of the ELISA-plaque assay. The results did not show differences among the various groups of mice with regard to the total numbers of &@-secreting cells in the various lymphoid organs. Also the frequencies of IgM-secreting cells specific for DNP27-BSA and the anti-idiotypic monoclonal antibodies Ac38 and Acl46 did not differ significantly among GF-CD, SPF-NI, and CV-NI mice. GF-CD mice, however, did show substantially decreased numbers of IgG-and IgA-secreting cells in their lymphoid organs. Furthermore, there were striking differences in the frequencies of antigen-specific IgG-and IgA-secreting cells between GF-CD mice and the two other groups of mice. These results indicate that exogenous antigenic stimulation has a great effect on both the total numbers and the specificity repertoires of background IgG-and IgA-secreting cells. Such an influence could not be detected with regard to the background IgM-secreting cells. This suggests two distinct compartments of background Ig-secreting cells: a very stable, endogenously regulated compartment consisting mainly of IgM-secreting cells, and another compartment, consisting mainly of IgG-and &A-secreting cells, whose numbers and specificity repertoire appeared to be influenced by exogenous antigenic stimulation. Q 1988 Academic press, IX.
Antimicrobial peptides are important effectors of innate immunity throughout the plant and animal... more Antimicrobial peptides are important effectors of innate immunity throughout the plant and animal kingdoms. In the mammalian small intestine, Paneth cell α-defensins are antimicrobial peptides that contribute to host defense against enteric pathogens. To determine if α-defensins also govern intestinal microbial ecology, we analyzed the intestinal microbiota in mice expressing a human αdefensin (DEFA5) and in mice lacking an enzyme required for processing of murine α-defensins. We detected significant α-defensin-dependent changes in microbiota composition, but not in total bacterial numbers, in these complementary models. Furthermore, DEFA5-expressing mice had striking losses of Segmented Filamentous Bacteria and fewer interleukin 17-producing lamina propria T cells. These data ascribe a new homeostatic role for α-defensins in regulating the makeup of the commensal microbiota.
The vast majority of rodent splenic marginal zone (MZ)-B cells are naive IgM + cells. A small fra... more The vast majority of rodent splenic marginal zone (MZ)-B cells are naive IgM + cells. A small fraction of these MZ-B cells carry mutated V-genes, and represent IgM + memory MZ-B cells. Here we reveal further heterogeneity of B cells with a MZ-B cell phenotype, by providing evidence for the existence of class-switched memory MZ-B cells in the rat. In essence, we observed IGHV5 encoded C transcripts, among FACS-purified MZ-B cells, defined as HIS24 low HIS57 bright cells. Furthermore, we found that most IgG encoding transcripts are mutated. There is no significant difference in IGHV5 repertoire and subclass usage of these IgG encoding transcripts collected from B cells with a MZ-B cell phenotype and B cells with a follicular (FO) B cell phenotype. However, the IGHV5 genes encoding for IgG antibodies of MZ-B cells exhibited significantly fewer mutations, compared to those with a FO-B cell phenotype. In one rat we found a clonally related set of IgG encoding sequences, of which one was derived from the MZ-B cell fraction and the other from the FO-B cell fraction. We speculate that these two subpopulations of class-switched B cells are both descendants from naive FO-B cells and are generated in germinal centers. Class-switched memory cells with a MZ-B cell phenotype may provide the animal with a population of IgG memory cells that can respond rapidly to blood-borne pathogens.
We used Listeria monocytogenes, a gram-positive, facultative intracellular bacterium, to study th... more We used Listeria monocytogenes, a gram-positive, facultative intracellular bacterium, to study the gut mucosal immune responses following oral infection. We employed a germfree (GF) mouse model to try to accentuate the development of a humoral mucosal immune response in the gut, and we used oral colonization with one of the mutants, actA-negative (DeltaactA) L. monocytogenes, to restrict infection largely to the gut. The DeltaactA mutant was able to colonize the intestinal mucosa of formerly GF mice for long periods of time without causing disease while eliciting secretory immunoglobulin A (IgA) responses, as evidenced by gut tissue fragment culture assays. Flow cytometric analyses and immunohistochemical methods showed the development of only minimal germinal center reactions (GCR) in Peyer's patches and more robust GCR in mesenteric lymph nodes. Pronounced increases in total (natural) IgA production occurred in gut tissues by day 7 and were maintained for up to 90 days. Levels of specific IgA were modest in gut tissues on day 14, increased until day 76, and stabilized at day 90. We also observed a significant rise in serum IgA and IgG1 levels following oral infection by listeriae. Upon colonization, the organisms mainly infected the intestines and intestinal lumen, and we only sporadically observed few colony-forming bacteria in the liver and spleen. We observed a marked rise in IgA-secreting cells, including listeria-specific IgA antibody-secreting cells, in the lamina propria of the small intestine by enzyme-linked immunospot assays. To ascertain whether some of the IgA was specific for listeriae, we performed Western blot analysis to test the reactivity of IgA from fragment cultures to antigens in sonicates of L. monocytogenes. We detected IgA binding to antigenic proteins with molecular masses of 96, 60, 40, and 14 kDa in the Listeria sonicates.
We have mapped and annotated the variable region of the immunoglobulin heavy (IGH) gene locus of ... more We have mapped and annotated the variable region of the immunoglobulin heavy (IGH) gene locus of the Brown Norway (BN) rat (assembly V3.4; Rat Genomic Sequence Consortium). In addition to known variable region genes, we found 12 novel previously unidentified functional IGHV genes and 1 novel functional IGHD gene. In total, the variable region of the rat IGH locus is composed of at least 353 unique IGHV genes, 21 IGHD genes, and 5 IGHJ genes, of which 131, 14, and 4 are potentially functional genes, respectively. Of all species studied so far, the rat seems to have the highest number of functional IGHV genes in the genome. Rat IGHV genes can be classified into 13 IGHV families based on nucleotide sequence identity. The variable region of the BN rat spans a total length of approximately 4.9 Mb and is organized in a typical translocon organization. Like the mouse, members of the various IGHV gene families are more or less grouped together on the genome, albeit some members of IGHV gene families are found intermingled with each other. In the rat, the largest IGHV gene families are IGHV1, IGHV2, and IGHV5. The overall conclusion is that the genomic organization of the variable region of the rat IGH locus is strikingly similar to that of the mouse, illustrating the close evolutionary relationship between these two species.
This study investigates the influence of exogenous antigenic stimulation on the serum immunoglobu... more This study investigates the influence of exogenous antigenic stimulation on the serum immunoglobulin levels and the levels of circulating natural antibodies against carbohydrate antigens. Thus, BALB/c mice, raised in a germ-free environment and fed a chemically defined, ultrafiltered diet (GF-CD), were employed.These mice had normal serum IgM levels, but IgG and IgA levels were approximately 5% of conventionally reared littermates. The concentrations of all four IgG isotypes were equally low. The variable part of the heavy chains of naturally occurring BALB/c antibodies against a number of carbohydrate antigens, including 3-fucosyllactosamine (3-FL), levan and dextran, are encoded by V~4 4 1 , and these antibodies express cross-reactive idiotopes recognized by the monoclonal antibodies 6C4 and 6B1. Antibodies against levan and dextran were lower in GF-CD than in conventional mice, but levels of anti-3FL antibodies, and 6C4 and 6B1 idiotopes, were comparable to those in conventional animals. Peptidoglycan polysaccharide complexes (PPC) are carbohydrate antigens of bacterial origin, like levan and galactan. Naturally occurring antibodies against PPC were found in the serum of conventional mice, but were severely reduced in GF-CD mice.The results indicate that most naturally occurring antibodies against carbohydrate antigens of bacterial origin found in conventional mice are caused by exogenous stimulation.
BB BioBreeding DB Diabetes prone DR Diabetes resistant FOXP3 Forkhead box P3 HC Hydrolysed casein... more BB BioBreeding DB Diabetes prone DR Diabetes resistant FOXP3 Forkhead box P3 HC Hydrolysed casein To the Editor: Diabetes-prone (DP)-BioBreeding (BB) rats show reduced natural regulatory T cell (nTreg; CD4 + / CD25 + /forkhead box P3 [FOXP3] + ) levels and function, and spontaneously develop type 1 diabetes from 70 days of age [1, 2]. Adoptive transfers of nTregs from diabetesresistant (DR)-BB rats to DP-BB rats prevents diabetes in DP-BB rats [3, 4]. Environmental factors such as diet are critical triggers for the development of type 1 diabetes [5].
In the peripheral blood (PB) of multiple myeloma (MM) patients, clonotypic B cells are present th... more In the peripheral blood (PB) of multiple myeloma (MM) patients, clonotypic B cells are present that express the identical V(D)J rearrangements as the malignant plasma cells in the bone marrow. In the present study, the proliferative capacity of clonotypic B cells from MM patients (n ¼ 10) and the ability to differentiate in vitro was determined using the CD40-culturing system. For six patients, the presence of clonotypic B cells expressing variant immunoglobulin (Ig) isotypes was assessed by Ig isotypespecific allele-specific oligonucleotide reverse transcription polymerase chain reaction (ASO-RT-PCR) after culturing with CD40L and interleukin 4 (IL-4). In three out of six patients, clonotypic B cells expressing variant isotypes were detected both before and after culturing. The ability of clonotypic B cells to undergo B-cell differentiation was studied by abrogating CD40 signalling accompanied by IL-10 and
Primary plasma cell leukaemia (PCL) is a rare plasma cell malignancy, which is related to multipl... more Primary plasma cell leukaemia (PCL) is a rare plasma cell malignancy, which is related to multiple myeloma (MM) and is characterized by a poor prognosis. In a previous study we demonstrated that PCL plasma cells display a high expression of CD27, in contrast to MM plasma cells. The present study was set out to assess the functional properties of CD27 expressed on PCL plasma cells by triggering with its ligand CD70. Using CD27-expressing purified plasma cells from a PCL patient we demonstrated that CD27-triggering modestly inhibited spontaneous and dexamethasone-induced apoptosis. In vitro stimulation and Western blotting showed that activation of p38 and extracellular-regulated kinase 1/2 (ERK1/2) mitogen-activated protein kinases (MAPK) was associated with CD27-mediated signal transduction. Specific inhibition of p38 and ERK1/2 MAPK abolished the anti-apoptotic effects of CD27-triggering. Interestingly, simultaneous inhibition of p38 and ERK1/2 strongly sensitized PCL cells for dexamethasone-induced apoptosis. Finally, in dexamethasone-treated PCL cells, CD27-triggering was associated with persistent DNA-binding activity of activator protein 1 (AP-1) but not of nuclear factor-jB. These findings suggest that, in primary PCL, specific antiapoptotic pathways exist that might provide novel therapeutic targets.
Next to conventional B cells (or B-2 cells), peritoneal B-1 cells have been shown to contribute s... more Next to conventional B cells (or B-2 cells), peritoneal B-1 cells have been shown to contribute significantly to the production of IgA-secreting plasma cells in the gut. Evidence for this was mainly based on studies comprising manipulated animals, including lethally X-irradiated and transgenic mice. To examine the ability of peritoneal B-1 cells from untreated mice to switch actively to IgA in vivo, we performed RT-PCR analysis on FACS-sorted peritoneal B-cell subsets from untreated BALB/c mice in order to examine the presence of germline Ca mRNA and mature Ca mRNA transcripts. Germline Ca and mature Ca transcripts were readily detectable in peritoneal B-1 cells (defined as IgMbright/IgDOU), but not, or very little, in peritoneal B-2 cells (defined as IgMaul/IgDbrigrt). Moreover, by subdividing the B-l-cell population in CD5 B-la cells and CD5-B-lb cells, it was shown that in vivo expression of germline Ca and mature Ca transcripts was largely restricted to the B-lb-cell lineage. These results indicate that peritoneal B-1 cells indeed are capable to switch to IgA under normal physiological conditions and hereby further support the view that B-1 cells contribute significantly to the mucosal IgA response, albeit this function appears to be restricted to the B-lb-cell subset.
Early in ontogeny B cells preferentially use VH gene families which are most adjacent to the gene... more Early in ontogeny B cells preferentially use VH gene families which are most adjacent to the genes coding for the constant part of the immunoglobulin molecule. In conventional adult mice, however, a random usage of VH gene families has been found. We investigated the role of exogenous antigenic stimulation on this normalization of VH gene usage by B cells. Therefore, we made use of adult germ-free BALB/c mice fed a chemically defined ultrafiltered antigen-free diet (GF-CD) and neonatal conventional BALB/c mice. Both the adult GF-CD and the newborn conventional mice represent situations with minimal exogenous antigenic stimulation. The results obtained with RNA dot blot hybridization with probes specific for the different VH gene families showed in hybridomas from adult GF-CD BALB/c mice a preferential usage of the CH-proximal VH gene family PC7183. In hybridomas from 5-day-old conventional BALB/c mice a less frequent usage of the 5558 VH gene family was found and an increased usage of the PC7183 VH gene family than what would be expected from random usage. Evidence is presented that the RNA giving a positive signal with the PC7183 probe represents functional messages for IgM production.
Monoclonal gammopathy of undetermined significance (MGUS) arises from a clonal expansion of plasm... more Monoclonal gammopathy of undetermined significance (MGUS) arises from a clonal expansion of plasma cells in the bone marrow, secreting monoclonal (M) paraprotein. MGUS is associated with increased susceptibility to infections, which may reflect altered B-cell repertoire. To investigate this, we examined the IgM, IgG and IgA B-cell repertoire diversity in MGUS at baseline and after influenza vaccination (n=16) in comparison to healthy controls (HCs) (n=16). The CDR3 region of the immunoglobulin heavy chain variable (IGHV) region gene was amplified and B-cell spectratypes analyzed by high-resolution electrophoresis. Spectratype Gaussian distribution, kurtosis and skewness were quantified to measure repertoire shifts. Both HC and MGUS baseline spectratypes show inter-individual variability that is more pronounced in the IGHG and IGHA repertoires. Overall, baseline B cell repertoire is more altered in MGUS, with oligoclonality observed in 50% (p=0.01). Post-vaccination, significant diff...
... 1893 Nicolaas A. BosoV, Cornelia G. MeeuwsenO, Elisabeth De Glopper-Van der Veero, Theodoor W... more ... 1893 Nicolaas A. BosoV, Cornelia G. MeeuwsenO, Elisabeth De Glopper-Van der Veero, Theodoor W. Van den AkkerO, Jiri Radl*, Kornelisje A. Zwaagstra. and Robbed BennerO ... It may be that the Ly-1 B cell lineage is involved in BMG. ...
Early in ontogeny B cells preferentially use VH gene families which are most adjacent to the gene... more Early in ontogeny B cells preferentially use VH gene families which are most adjacent to the genes coding for the constant part of the immunoglobulin molecule. In conventional adult mice, however, a random usage of VH gene families has been found. We investigated the role of exogenous antigenic stimulation on this normalization of VH gene usage by B cells. Therefore, we made use of adult germ-free BALB/c mice fed a chemically defined ultrafiltered antigen-free diet (GF-CD) and neonatal conventional BALB/c mice. Both the adult GF-CD and the newborn conventional mice represent situations with minimal exogenous antigenic stimulation. The results obtained with RNA dot blot hybridization with probes specific for the different VH gene families showed in hybridomas from adult GF-CD BALB/c mice a preferential usage of the CH-proximal VH gene family PC7183. In hybridomas from 5-day-old conventional BALB/c mice a less frequent usage of the 5558 VH gene family was found and an increased usage of the PC7183 VH gene family than what would be expected from random usage. Evidence is presented that the RNA giving a positive signal with the PC7183 probe represents functional messages for IgM production.
Insight into the maintenance of naive T cells is essential to understand defective immune respons... more Insight into the maintenance of naive T cells is essential to understand defective immune responses in the context of aging and other immune compromised states. In humans, naive CD4+ T cells, in contrast to CD8+ T cells, are remarkably well retained with aging. Here, we show that low-affinity TCR engagement is the main driving force behind the emergence and accumulation of naive-like CD4+ T cells with enhanced sensitivity to IL-2 in aged humans. In vitro, we show that these CD45RA + CD25 dim CD4 + T cells can develop from conventional naive CD25 À CD4+ T cells upon CD3 cross-linking alone, in the absence of costimulation, rather than via stimulation by the homeostatic cytokines IL-2, IL-7, or IL-15. In vivo, TCR engagement likely occurs in secondary lymphoid organs as these cells were detected in lymph nodes and spleen where they showed signs of recent activation. CD45RA + CD25 dim CD4+ T cells expressed a broad TCRVb repertoire and could readily differentiate into functional T helper cells. Strikingly, no expansion of CD45RA + CD25 dim CD8+ T cells was detected with aging, thereby implying that maintenance of naive CD4+ T cells is uniquely regulated. Our data provide novel insight into the homeostasis of naive T cells and may guide the development of therapies to preserve or restore immunity in the elderly.
Monoclonal gammopathy of undetermined significance (MGUS) arises from a clonal expansion of plasm... more Monoclonal gammopathy of undetermined significance (MGUS) arises from a clonal expansion of plasma cells in the bone marrow, secreting monoclonal (M) paraprotein. MGUS is associated with increased susceptibility to infections, which may reflect altered B-cell repertoire. To investigate this, we examined the IgM, IgG and IgA B-cell repertoire diversity in MGUS at baseline and after influenza vaccination (n=16) in comparison to healthy controls (HCs) (n=16). The CDR3 region of the immunoglobulin heavy chain variable (IGHV) region gene was amplified and B-cell spectratypes analyzed by high-resolution electrophoresis. Spectratype Gaussian distribution, kurtosis and skewness were quantified to measure repertoire shifts. Both HC and MGUS baseline spectratypes show inter-individual variability that is more pronounced in the IGHG and IGHA repertoires. Overall, baseline B cell repertoire is more altered in MGUS, with oligoclonality observed in 50% (p=0.01). Post-vaccination, significant diff...
Healthy aging requires an optimal balance between pro-inflammatory and anti-inflammatory immune r... more Healthy aging requires an optimal balance between pro-inflammatory and anti-inflammatory immune responses. Although CD4+ T cells play an essential role in many immune responses, few studies have directly assessed the effect of aging on the balance between effector T (T eff ) cells and regulatory T (T reg ) cells. Here, we determined if and how aging affects the ratio between T reg and T eff cells. Percentages of both naive T reg (nT reg ; CD45RA+CD25 int FOXP3 low ) and memory T reg (memT reg ; CD45RA-CD25 high FOXP3 high ) cells were determined by flow cytometry in peripheral blood samples of healthy individuals of various ages (20-84 years). Circulating Th1, Th2 and Th17 effector cells were identified by intracellular staining for IFN-γ, IL-4 and IL-17, respectively, upon in vitro stimulation with PMA and calcium ionophore. Whereas proportions of nT reg cells declined with age, memT reg cells increased. Both Th1 and Th2 cells were largely maintained in the circulation of aged humans, whereas Th17 cells were decreased. Similar to memT reg cells, the 3 T eff subsets resided primarily in the memory CD4+ T cell compartment. Overall, T reg /T eff ratios were increased in the memory CD4+ T cell compartment of aged individuals when compared to that of young individuals. Finally, the relative increase of memT reg cells in elderly individuals was associated with poor responses to influenza vaccination. Taken together, our findings imply that aging disturbs the balance between T reg cells and T eff cells.
The total number of spontaneously occurring ("background") IgM-, IgG-, and &A-secreting cells and... more The total number of spontaneously occurring ("background") IgM-, IgG-, and &A-secreting cells and the frequency of antigen-specific IgM-, IgG-, and IgA-secreting cells were determined in germ-free BALB/c mice fed a chemically defined ultrafiltered diet (GF-CD), in specific pathogen-free BALB/c mice fed an autoclaved natural ingredient diet (SPF-NI), and in conventional BALB/c mice fed nonautoclaved natural ingredients (CV-NI). This was done by means of the ELISA-plaque assay. The results did not show differences among the various groups of mice with regard to the total numbers of &@-secreting cells in the various lymphoid organs. Also the frequencies of IgM-secreting cells specific for DNP27-BSA and the anti-idiotypic monoclonal antibodies Ac38 and Acl46 did not differ significantly among GF-CD, SPF-NI, and CV-NI mice. GF-CD mice, however, did show substantially decreased numbers of IgG-and IgA-secreting cells in their lymphoid organs. Furthermore, there were striking differences in the frequencies of antigen-specific IgG-and IgA-secreting cells between GF-CD mice and the two other groups of mice. These results indicate that exogenous antigenic stimulation has a great effect on both the total numbers and the specificity repertoires of background IgG-and IgA-secreting cells. Such an influence could not be detected with regard to the background IgM-secreting cells. This suggests two distinct compartments of background Ig-secreting cells: a very stable, endogenously regulated compartment consisting mainly of IgM-secreting cells, and another compartment, consisting mainly of IgG-and &A-secreting cells, whose numbers and specificity repertoire appeared to be influenced by exogenous antigenic stimulation. Q 1988 Academic press, IX.
Antimicrobial peptides are important effectors of innate immunity throughout the plant and animal... more Antimicrobial peptides are important effectors of innate immunity throughout the plant and animal kingdoms. In the mammalian small intestine, Paneth cell α-defensins are antimicrobial peptides that contribute to host defense against enteric pathogens. To determine if α-defensins also govern intestinal microbial ecology, we analyzed the intestinal microbiota in mice expressing a human αdefensin (DEFA5) and in mice lacking an enzyme required for processing of murine α-defensins. We detected significant α-defensin-dependent changes in microbiota composition, but not in total bacterial numbers, in these complementary models. Furthermore, DEFA5-expressing mice had striking losses of Segmented Filamentous Bacteria and fewer interleukin 17-producing lamina propria T cells. These data ascribe a new homeostatic role for α-defensins in regulating the makeup of the commensal microbiota.
The vast majority of rodent splenic marginal zone (MZ)-B cells are naive IgM + cells. A small fra... more The vast majority of rodent splenic marginal zone (MZ)-B cells are naive IgM + cells. A small fraction of these MZ-B cells carry mutated V-genes, and represent IgM + memory MZ-B cells. Here we reveal further heterogeneity of B cells with a MZ-B cell phenotype, by providing evidence for the existence of class-switched memory MZ-B cells in the rat. In essence, we observed IGHV5 encoded C transcripts, among FACS-purified MZ-B cells, defined as HIS24 low HIS57 bright cells. Furthermore, we found that most IgG encoding transcripts are mutated. There is no significant difference in IGHV5 repertoire and subclass usage of these IgG encoding transcripts collected from B cells with a MZ-B cell phenotype and B cells with a follicular (FO) B cell phenotype. However, the IGHV5 genes encoding for IgG antibodies of MZ-B cells exhibited significantly fewer mutations, compared to those with a FO-B cell phenotype. In one rat we found a clonally related set of IgG encoding sequences, of which one was derived from the MZ-B cell fraction and the other from the FO-B cell fraction. We speculate that these two subpopulations of class-switched B cells are both descendants from naive FO-B cells and are generated in germinal centers. Class-switched memory cells with a MZ-B cell phenotype may provide the animal with a population of IgG memory cells that can respond rapidly to blood-borne pathogens.
We used Listeria monocytogenes, a gram-positive, facultative intracellular bacterium, to study th... more We used Listeria monocytogenes, a gram-positive, facultative intracellular bacterium, to study the gut mucosal immune responses following oral infection. We employed a germfree (GF) mouse model to try to accentuate the development of a humoral mucosal immune response in the gut, and we used oral colonization with one of the mutants, actA-negative (DeltaactA) L. monocytogenes, to restrict infection largely to the gut. The DeltaactA mutant was able to colonize the intestinal mucosa of formerly GF mice for long periods of time without causing disease while eliciting secretory immunoglobulin A (IgA) responses, as evidenced by gut tissue fragment culture assays. Flow cytometric analyses and immunohistochemical methods showed the development of only minimal germinal center reactions (GCR) in Peyer's patches and more robust GCR in mesenteric lymph nodes. Pronounced increases in total (natural) IgA production occurred in gut tissues by day 7 and were maintained for up to 90 days. Levels of specific IgA were modest in gut tissues on day 14, increased until day 76, and stabilized at day 90. We also observed a significant rise in serum IgA and IgG1 levels following oral infection by listeriae. Upon colonization, the organisms mainly infected the intestines and intestinal lumen, and we only sporadically observed few colony-forming bacteria in the liver and spleen. We observed a marked rise in IgA-secreting cells, including listeria-specific IgA antibody-secreting cells, in the lamina propria of the small intestine by enzyme-linked immunospot assays. To ascertain whether some of the IgA was specific for listeriae, we performed Western blot analysis to test the reactivity of IgA from fragment cultures to antigens in sonicates of L. monocytogenes. We detected IgA binding to antigenic proteins with molecular masses of 96, 60, 40, and 14 kDa in the Listeria sonicates.
We have mapped and annotated the variable region of the immunoglobulin heavy (IGH) gene locus of ... more We have mapped and annotated the variable region of the immunoglobulin heavy (IGH) gene locus of the Brown Norway (BN) rat (assembly V3.4; Rat Genomic Sequence Consortium). In addition to known variable region genes, we found 12 novel previously unidentified functional IGHV genes and 1 novel functional IGHD gene. In total, the variable region of the rat IGH locus is composed of at least 353 unique IGHV genes, 21 IGHD genes, and 5 IGHJ genes, of which 131, 14, and 4 are potentially functional genes, respectively. Of all species studied so far, the rat seems to have the highest number of functional IGHV genes in the genome. Rat IGHV genes can be classified into 13 IGHV families based on nucleotide sequence identity. The variable region of the BN rat spans a total length of approximately 4.9 Mb and is organized in a typical translocon organization. Like the mouse, members of the various IGHV gene families are more or less grouped together on the genome, albeit some members of IGHV gene families are found intermingled with each other. In the rat, the largest IGHV gene families are IGHV1, IGHV2, and IGHV5. The overall conclusion is that the genomic organization of the variable region of the rat IGH locus is strikingly similar to that of the mouse, illustrating the close evolutionary relationship between these two species.
This study investigates the influence of exogenous antigenic stimulation on the serum immunoglobu... more This study investigates the influence of exogenous antigenic stimulation on the serum immunoglobulin levels and the levels of circulating natural antibodies against carbohydrate antigens. Thus, BALB/c mice, raised in a germ-free environment and fed a chemically defined, ultrafiltered diet (GF-CD), were employed.These mice had normal serum IgM levels, but IgG and IgA levels were approximately 5% of conventionally reared littermates. The concentrations of all four IgG isotypes were equally low. The variable part of the heavy chains of naturally occurring BALB/c antibodies against a number of carbohydrate antigens, including 3-fucosyllactosamine (3-FL), levan and dextran, are encoded by V~4 4 1 , and these antibodies express cross-reactive idiotopes recognized by the monoclonal antibodies 6C4 and 6B1. Antibodies against levan and dextran were lower in GF-CD than in conventional mice, but levels of anti-3FL antibodies, and 6C4 and 6B1 idiotopes, were comparable to those in conventional animals. Peptidoglycan polysaccharide complexes (PPC) are carbohydrate antigens of bacterial origin, like levan and galactan. Naturally occurring antibodies against PPC were found in the serum of conventional mice, but were severely reduced in GF-CD mice.The results indicate that most naturally occurring antibodies against carbohydrate antigens of bacterial origin found in conventional mice are caused by exogenous stimulation.
BB BioBreeding DB Diabetes prone DR Diabetes resistant FOXP3 Forkhead box P3 HC Hydrolysed casein... more BB BioBreeding DB Diabetes prone DR Diabetes resistant FOXP3 Forkhead box P3 HC Hydrolysed casein To the Editor: Diabetes-prone (DP)-BioBreeding (BB) rats show reduced natural regulatory T cell (nTreg; CD4 + / CD25 + /forkhead box P3 [FOXP3] + ) levels and function, and spontaneously develop type 1 diabetes from 70 days of age [1, 2]. Adoptive transfers of nTregs from diabetesresistant (DR)-BB rats to DP-BB rats prevents diabetes in DP-BB rats [3, 4]. Environmental factors such as diet are critical triggers for the development of type 1 diabetes [5].
In the peripheral blood (PB) of multiple myeloma (MM) patients, clonotypic B cells are present th... more In the peripheral blood (PB) of multiple myeloma (MM) patients, clonotypic B cells are present that express the identical V(D)J rearrangements as the malignant plasma cells in the bone marrow. In the present study, the proliferative capacity of clonotypic B cells from MM patients (n ¼ 10) and the ability to differentiate in vitro was determined using the CD40-culturing system. For six patients, the presence of clonotypic B cells expressing variant immunoglobulin (Ig) isotypes was assessed by Ig isotypespecific allele-specific oligonucleotide reverse transcription polymerase chain reaction (ASO-RT-PCR) after culturing with CD40L and interleukin 4 (IL-4). In three out of six patients, clonotypic B cells expressing variant isotypes were detected both before and after culturing. The ability of clonotypic B cells to undergo B-cell differentiation was studied by abrogating CD40 signalling accompanied by IL-10 and
Primary plasma cell leukaemia (PCL) is a rare plasma cell malignancy, which is related to multipl... more Primary plasma cell leukaemia (PCL) is a rare plasma cell malignancy, which is related to multiple myeloma (MM) and is characterized by a poor prognosis. In a previous study we demonstrated that PCL plasma cells display a high expression of CD27, in contrast to MM plasma cells. The present study was set out to assess the functional properties of CD27 expressed on PCL plasma cells by triggering with its ligand CD70. Using CD27-expressing purified plasma cells from a PCL patient we demonstrated that CD27-triggering modestly inhibited spontaneous and dexamethasone-induced apoptosis. In vitro stimulation and Western blotting showed that activation of p38 and extracellular-regulated kinase 1/2 (ERK1/2) mitogen-activated protein kinases (MAPK) was associated with CD27-mediated signal transduction. Specific inhibition of p38 and ERK1/2 MAPK abolished the anti-apoptotic effects of CD27-triggering. Interestingly, simultaneous inhibition of p38 and ERK1/2 strongly sensitized PCL cells for dexamethasone-induced apoptosis. Finally, in dexamethasone-treated PCL cells, CD27-triggering was associated with persistent DNA-binding activity of activator protein 1 (AP-1) but not of nuclear factor-jB. These findings suggest that, in primary PCL, specific antiapoptotic pathways exist that might provide novel therapeutic targets.
Next to conventional B cells (or B-2 cells), peritoneal B-1 cells have been shown to contribute s... more Next to conventional B cells (or B-2 cells), peritoneal B-1 cells have been shown to contribute significantly to the production of IgA-secreting plasma cells in the gut. Evidence for this was mainly based on studies comprising manipulated animals, including lethally X-irradiated and transgenic mice. To examine the ability of peritoneal B-1 cells from untreated mice to switch actively to IgA in vivo, we performed RT-PCR analysis on FACS-sorted peritoneal B-cell subsets from untreated BALB/c mice in order to examine the presence of germline Ca mRNA and mature Ca mRNA transcripts. Germline Ca and mature Ca transcripts were readily detectable in peritoneal B-1 cells (defined as IgMbright/IgDOU), but not, or very little, in peritoneal B-2 cells (defined as IgMaul/IgDbrigrt). Moreover, by subdividing the B-l-cell population in CD5 B-la cells and CD5-B-lb cells, it was shown that in vivo expression of germline Ca and mature Ca transcripts was largely restricted to the B-lb-cell lineage. These results indicate that peritoneal B-1 cells indeed are capable to switch to IgA under normal physiological conditions and hereby further support the view that B-1 cells contribute significantly to the mucosal IgA response, albeit this function appears to be restricted to the B-lb-cell subset.
Early in ontogeny B cells preferentially use VH gene families which are most adjacent to the gene... more Early in ontogeny B cells preferentially use VH gene families which are most adjacent to the genes coding for the constant part of the immunoglobulin molecule. In conventional adult mice, however, a random usage of VH gene families has been found. We investigated the role of exogenous antigenic stimulation on this normalization of VH gene usage by B cells. Therefore, we made use of adult germ-free BALB/c mice fed a chemically defined ultrafiltered antigen-free diet (GF-CD) and neonatal conventional BALB/c mice. Both the adult GF-CD and the newborn conventional mice represent situations with minimal exogenous antigenic stimulation. The results obtained with RNA dot blot hybridization with probes specific for the different VH gene families showed in hybridomas from adult GF-CD BALB/c mice a preferential usage of the CH-proximal VH gene family PC7183. In hybridomas from 5-day-old conventional BALB/c mice a less frequent usage of the 5558 VH gene family was found and an increased usage of the PC7183 VH gene family than what would be expected from random usage. Evidence is presented that the RNA giving a positive signal with the PC7183 probe represents functional messages for IgM production.
Monoclonal gammopathy of undetermined significance (MGUS) arises from a clonal expansion of plasm... more Monoclonal gammopathy of undetermined significance (MGUS) arises from a clonal expansion of plasma cells in the bone marrow, secreting monoclonal (M) paraprotein. MGUS is associated with increased susceptibility to infections, which may reflect altered B-cell repertoire. To investigate this, we examined the IgM, IgG and IgA B-cell repertoire diversity in MGUS at baseline and after influenza vaccination (n=16) in comparison to healthy controls (HCs) (n=16). The CDR3 region of the immunoglobulin heavy chain variable (IGHV) region gene was amplified and B-cell spectratypes analyzed by high-resolution electrophoresis. Spectratype Gaussian distribution, kurtosis and skewness were quantified to measure repertoire shifts. Both HC and MGUS baseline spectratypes show inter-individual variability that is more pronounced in the IGHG and IGHA repertoires. Overall, baseline B cell repertoire is more altered in MGUS, with oligoclonality observed in 50% (p=0.01). Post-vaccination, significant diff...
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Papers by Nicolaas Bos