B-lymphocyte Compartment Research Articles

Vitamin A-dependent transcriptional activation of the nuclear factor of activated T cells c1 (NFATc1) is critical for the development and survival of B1 cells

B1 cells represent a distinct subset of B cells that produce most of the natural serum IgM and much of the gut IgA and function as an important component of early immune responses to pathogens. The development of B1 cells depends on the nuclear factor of activated T cells c1 (NFATc1), a transcription factor abundantly expressed by B1 cells but not by conventional B2 cells. However, the factors that regulate the expression of NFATc1 in B1 cells remain unknown. Here we show that a vitamin A-deficient diet results in reduction of NFATc1 expression in B1 cells and almost complete loss of the B1 cell compartment. As a consequence, vitamin A-deficient mice have reduced serum IgM and are unable to mount T cell-independent antibody responses against bacterial antigens. We demonstrate that injection of all-trans retinoic acid induces the expression of NFATc1, particularly from the constitutive P2 promoter, and leads to the increase of the B1 cells. Thus, the retinoic acid-dependent pathway is critical for regulating NFATc1 expression and for maintenance of the natural memory B cell compartment.

Expansion of Immature/Transitional B-Lymphocytes in Patients with Active Chronic Graft-Versus-Host Disease.

Background: Chronic graft-versus-host disease (cGVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (HSCT) and an important cause of non-relapse mortality. The diagnosis of cGVHD depends mainly on clinical signs and histopathological confirmation. CGVHD is known to impair immune reconstitution. Recently, we observed that percentages of both non-class-switched (CD19+/IgD+/CD27+) and class-switched memory B-lymphocytes(CD19+/IgD−/CD27+) were significantly lower in patients with active cGVHD when compared to patients never experiencing cGVHD (p=0.003 and p= 0.001). The ratio of immature/memory B-lymphocytes (CD21−/CD27+) was significantly higher in patients with active cGVHD when compared to patients never experiencing cGVHD (p=0.0046). In the current study we investigated the influence of cGVHD on the pattern of immune reconstitution of B-cell subpopulations within the first years after HSCT.Methods: Ninety-nine patients (median age 42 years, range 17–62 years) were analyzed 3 months to 12 years after HSCT. The total of 212 sampling events included 141 samples obtained during active cGVHD and 71 samples from time points without cGVHD. Evidence of cGVHD in at least one organ as defined by the NIH Consensus Development Project was required to assign the sampling event into the active cGVHD group. The series included 10 patients (3 without and 7 developing cGVHD) assessed serially in 3 months' intervals within the first years after HSCT. Peripheral blood leukocytes were analyzed by multiparameter flow cytometry after staining for CD19, staining for surface Ig and the B-lymphocyte memory marker CD27 as well as staining for CD21, which is absent on immature/transitional B-lymphocytes. The patients were scored for cGVHD activity according to the NIH Consensus Development Project criteria at every sampling event.Results: While the CD21−/CD27+ ratio as well as the percentage of immature/transitional B-lymphocytes decreased in the first years after HSCT in patients without cGVHD, these parameters remained significantly higher over the years in patients with active cGVHD (p=0.001). In logistic regression analysis a higher CD21−/CD27+ ratio and higher percentage of immature B-lymphocytes (CD21−) significantly correlated with active cGVHD. The odds ratio increased from 1.028 to 14.532 between the second and seventh year after HSCT. Until the end of the first year after HSCT serially sampled patients without cGVHD and the ones with cGVHD responding to immunosuppressive therapy presented a decrease of immature/transitional B-lymphocytes and increase of class-switched memory B cells whereas this pattern was disturbed in all serial patients with refractory cGVHD.Conclusions: Our study demonstrates that cGVHD is associated with an impairment of the reconstitution of the B-lymphocyte compartment leading to a long-lasting expansion of immature/transitional B-lymphocytes. Prospective studies with larger patient numbers analyzed serially are warranted for further assessment of the role of B-cell subpopulations in the pathogenesis of cGVHD and as potential biomarkers for cGVHD activity.

Histopathology, parasite density and cell phenotypes of the popliteal lymph node in canine visceral leishmaniasis

While enlargement of popliteal lymph nodes (LN) is frequently described in canine visceral leishmaniasis (CVL), there are few histopathologic studies of lymph nodes during this chronic immunopathological condition. Besides a detailed histopathologic analysis, we have characterized the parasite load and major immunophenotypic features of the LN in Leishmania ( Leishmania) chagasi-infected dogs. Our major histopathological findings highlight that hypertrophy/hyperplasia of LN cortical and medullary zones was the principal characteristic observed in asymptomatic dogs (AD), whereas atrophy of LN cortical zone was predominant in symptomatic animals (SD). The LN parasite density detected by anti- Leishmania immunohistochemical assay or expressed as Leishman Donovan Units was also highly correlated with the skin parasitism, the most reliable parameter to decode the clinical status of CVL. The major LN immunophenotypic changes during ongoing CVL were an increased frequency of T-lymphocytes, particularly CD8 + T-cells, up-regulation of MHC-II expression by lymphocytes and decreased levels of CD21 + B-cells. Our findings further demonstrated that changes in the LN B-lymphocyte compartment exhibited a negative correlation with the skin parasite load. Conversely, we also showed evidence for a positive association between skin parasitism and LN T-cell-mediated immunity, suggesting that T-cells, especially CD8 + lymphocytes, may have a Type-2 immunological profile in this lymphoid tissue in response to CVL.

B-cell function in CNS inflammatory demyelinating disease: a complexity of roles and a wealth of possibilities

Multiple sclerosis is a heterogeneous disease associated with a rupture of immunological tolerance toward CNS antigens in both T- and B-lymphocyte compartments. The association of human leukocyte antigen genes with higher susceptibility to multiple sclerosis, and the capacity of immune-modulating drugs that block leukocyte transit into the CNS (e.g., natalizumab) to limit pathogenesis confirms that immune cells play important roles in disease progression. An immune therapy that depletes B cells selectively (rituximab) is in clinical trials for multiple sclerosis. Experiments performed in the multiple sclerosis animal model of experimental autoimmune encephalomyelitis have highlighted multiple possible roles for B cells, ranging from being crucial for pathogenesis to being necessary for recovery from clinical disease. Thus, we may expect that patients in whom B cells are key players in the pathogenic process should benefit from rituximab, whereas those in whom B cells are involved predominantly in regulation may show disease exacerbation.

Hoxb3 deficiency impairs B lymphopoiesis in mouse bone marrow

Hox genes are involved in hematopoietic lineage commitment and differentiation. In this study, we investigated the roles of Hoxb3 in hematopoiesis by examining the phenotypes of a Hoxb3 knockout mutant mouse line. Despite previous reports describing the apparently normal phenotype of these mutant mice, we found that by 6 months of age, Hoxb3(-/-) mice began to exhibit significantly impaired B lymphopoiesis in the bone marrow (BM). The cellularity was reduced by 30% in mutant BM compared to age- and sex-matched heterozygous and wild-type controls. The population size of B220(+)CD43(+) progenitor B cells showed a twofold reduction while that of B220(+)CD43(-)IgM(-) precursor B cells was decreased fivefold. Sorting-purified Hoxb3(-/-) progenitor B cells displayed significantly reduced proliferative response to IL-7 in culture, consistent with our findings of reduced IL-7 receptor expression in Hoxb3(-/-) progenitor B cells. However, the peripheral B cell pool in the spleen of Hoxb3(-/-) mice was maintained with a similar size as in wild-type littermates. Analysis of T-cell development in the thymus and B1 cell compartment in the peritoneal cavity showed no significant changes. Thus, our findings suggest that the Hoxb3 gene plays an essential role in regulating B lymphopoiesis in the BM of adult mice.

BAFF-R Receptor Also Functions in the Nucleus of Normal and Neoplastic Human B Lymphocytes.

B-lymphocyte stimulator (BLyS) is a relatively newly described tumor necrosis factor (TNF) superfamily cytokine involved in cell survival and proliferation in normal and neoplastic B cells, particularly in the aggressive B cell non-Hodgkin's lymphomas (NHL-B). BLyS binds to three receptors: BAFF-R (or BR3), BCMA and TACI. However, recent studies have shown that BLyS regulates B cell survival and proliferation predominately through BAFF-R. Mice with mutant BAFF-R show significant decrease in the peripheral blood B-lymphocyte compartment, similar to the phenotype of BLyS knockout mice. BLyS/BAFF-R signaling functions through activation of the critical transcription factor NF-kB pathways, especially the NF-kB2 pathway, which mainly involves the p52/ rel-B complex.Our previous studies have shown that BLyS is constitutively expressed in aggressive NHL-B cells leading to increased survival and proliferation of the malignant B cells. In this study, we found by western blotting and confocal microscopy that BAFF-R, the major B cell associated cell membrane receptor for BLyS, was also present in the B cell nucleus, in addition to its location in plasma membrane and cytoplasm in both normal peripheral blood B lymphocytes and NHL-B cells. Nuclear presence can be increased by anti-IgM and soluble CD154 treatment in normal peripheral blood B lymphocytes, and is constitutively expressed in aggressive NHL-B. Inhibition of BLyS expression by specific BLyS siRNA decreases nuclear BAFF-R level and survival in LBCL cells. Immunostaining experiments show that in the cytoplasm of normal and neoplastic B cells, BAFF-R interacts with the improtin a and b which are members of the classic karypherin pathway. A candidate nuclear localization sequence (NLS) was also identified in the BAFF-R protein sequence, and mutation of this putative NLS can block BAFF-R entering nucleus, which is consistent with our hypothesis that BAFF-R undergoes nuclear translocation. To further investigate the functional role of BAFF-R in nucleus, we performed confocal immunostaining analysis and co-immunoprecipitation, that shows that BAFF-R co-localizes with some NF-kB family members such as c-rel in the B cell nucleus. We also found that nuclear BAFF-R/c-rel complex can bind to the NF-kB binding site on the promoters of NF-kB target genes such as BLyS, CD154, Bcl-xL and Bfl-1 /A1 by chromatin immnoprecipitation (ChIP) assay. Luciferase reporter assays show that BAFF-R has transactivation activity on these NF-kB target genes. Furthermore, NLS mutant BAFF-R decreases NF-kB target gene promoter activity, compared to wild type BAFF-R, and NHL-B cell proliferation. These findings indicates that in addition to activating NF-kB pathways at the plasma membrane, BAFF-R may also promote survival and proliferation of both normal and NHL-B cell in the nucleus by directly regulating transcriptional activity of key NF-kB target genes and may functions as a transcriptional co- factor with NF-kB.

Control of the B1-cell compartment by cross talk between the C5a receptor and toll-like receptor 2

Control of the B1-cell compartment by cross talk between the C5a receptor and toll-like receptor 2

Pre-B cell receptor expression is necessary for thymic stromal lymphopoietin responsiveness in the bone marrow but not in the liver environment.

IL-7 and thymic stromal lymphopoietin (TSLP) are two major cytokines controlling murine B cell development. IL-7 has been studied extensively, but only recently has it become possible to unravel the role of TSLP in detail. We studied the biological activities of TSLP in B cell development at distinct ages in the mouse. On the one hand, TSLP is able to give rise to a measurable B1 cell compartment derived from fetal liver pro-B cells, although, as is the case for B2 cells, it does not play a prevalent role in the development of this subset. On the other hand, TSLP drives the proliferation of pro-B cells from the fetal and neonatal liver, but in the bone marrow environment, B cell precursors require pre-B cell receptor expression for TSLP responsiveness.

Regeneration of natural antibody repertoire after massive ablation of lymphoid system: robust selection mechanisms preserve antigen binding specificities.

Natural Abs (NAbs) are Igs present in the serum and body fluids of healthy vertebrate animals, without any previous intentional immunization. NAbs often exhibit autoreactivity but also play an essential role in immunity, being a first line of defense against infectious microorganisms. We have previously analyzed the natural serum IgM Ab repertoire of normal mice, characterizing their reactivity with hundreds/thousands of self Ags; a significant similarity among different individuals was observed, and it was found that many reactivities of NAbs stably kept during adulthood were established early in life, implicating that period as a critical time window in the physiology of NAb repertoire selection. In the work reported here, experiments were conducted to address the role of normal lymphocyte ontogeny to the formation and stability of adult NAb repertoire. The massive destruction of the lymphoid system was promoted in adult mice with gamma-irradiation, and regeneration of hemopoietic tissues was granted by bone marrow or fetal liver inoculum. NAb repertoire regeneration was followed for 60 days after gamma-irradiation in bone marrow or fetal liver chimeric animals. The analysis of serum IgM reactivity with hundreds/thousands of self Ags showed that the NAb repertoire regenerated most of its original format after massive destruction of lymphoid compartments, characterizing autoreactive repertoire selection as a robust biological process. The data also show that regeneration of the NAb repertoire occurred similarly in fetal liver and bone marrow chimeras, although the latter animals poorly reconstituted their CD5(+) B1 cell compartment, suggesting that B1 cells are not essential for natural Ab regeneration.

Interleukin-10 modulates the sensitivity of peritoneal B lymphocytes to chemokines with opposite effects on stromal cell–derived factor-1 and B-lymphocyte chemoattractant

AbstractInterleukin-10 (IL-10) is constitutively produced by peritoneal B1a lymphocytes, and stromal cell–derived factor-1 (SDF-1) by mesothelial cells. Independent studies have shown that both IL-10 and SDF-1 are involved in the persistence of the peritoneal B-lymphocyte compartment. This study shows that IL-10 and SDF-1 act in synergy on peritoneal B lymphocytes. Indeed, autocrine production of IL-10 was absolutely required for all effects of SDF-1 on these cells, including increased proliferation, survival, and chemotaxis. Moreover, adding IL-10 to peritoneal B lymphocytes increased the effects of SDF-1. Neither IL-5, IL-6, nor IL-9 affected the response of peritoneal B lymphocytes to SDF-1. IL-10 was chemokinetic for peritoneal B lymphocytes, increasing their random mobility. It also potentiated the SDF-1–induced reorganization of the cytoskeleton without affecting CXCR4 gene expression by peritoneal B lymphocytes. Despite its chemokinetic properties, IL-10 abolished the migration of peritoneal B lymphocytes in response to B-lymphocyte chemoattractant (BLC), a chemokine targeting B lymphocytes to lymphoid organ follicles. The ability of B1a lymphocytes to produce IL-10 constitutively, combined with the opposite effects of this cytokine on the responses to SDF-1 and BLC, may account for the selective accumulation of B1 lymphocytes in body cavities.

The CD95 system and the death of a lymphocyte

Since the discovery of the CD95 receptor in 1989 and its ligand in 1993 as major initiators of apoptosis, extensive progress has been made in understanding their functions in programmed cell death. This review will summarize the cellular, molecular and biochemical requirements that play an important role in the CD95 system. Of importance in understanding the biology of CD95 are naturally occurring mutations in the mouse, thelprandgldmutations, in which the expression of either the receptor or its ligand is disturbed. This review will discuss the roles of the CD95 receptor/ligand system in negative selection of thymocytes and in the peripheral T- and B-lymphocyte compartment. Emphasis is put on its role in cell-mediated cytotoxicity, transplant rejection and on its pathophysiological role in organ damage. Finally, recent data are presented to give an overview of CD95-associated signalling proteins.

Memory cell generation ablated by soluble protein antigen by means of effects on T- and B-lymphocyte compartments.

Adult C57BL/6 mice were injected with 100 micrograms of soluble, freshly deaggregated human serum albumin (HSA) to produce partial immunologic tolerance. Uninjected normal control (N) mice contain only approximately 100 B cells in their spleens with the capacity to (i) be activated in vitro into clonal proliferation by Escherichia coli lipopolysaccharide plus interleukins 2, 4, and 5, (ii) form IgG1 as well as IgM antibody, and (iii) display specificity for HSA when only IgG1 is allowed to score in an enzyme-linked immunosorbent assay (ELISA). Such N mice generate approximately 50,000 clonable anti-HSA IgG1 antibody-forming cell precursors in their spleens after T-dependent immunization with HSA absorbed onto alum and given with Bordetella pertussis adjuvant. Mice preinjected with soluble HSA (TOL) generate far fewer anti-HSA IgG1 antibody-forming cell precursors, termed anti-HSA memory cells. Splenocytes were transferred from N or TOL mice into lethally irradiated syngeneic recipients together with syngeneic bone marrow. Whereas N splenocytes generated plentiful memory cells within 2 weeks in antigenically challenged recipients, TOL splenocytes did not. Work with Ly-5 congenic mice ruled out memory cell generation from either the host or the bone marrow inoculum within this limited time. N T cells plus TOL B cells showed consistently lowered memory cell generation. TOL T cells plus N B cells showed an even greater lowering of adoptive memory cell generation. Thus the lowered response capacity of TOL mice resided in the T- and B-cell compartments. Attempts to show a suppressor component within the T-cell population were inconclusive, but a profound defect in capacity to respond to HSA in vitro was exhibited by the CD4+ T cells of TOL mice. B lymphocytes were harvested from T-dependently immunized mice 5 days after challenge, incubated with soluble HSA for 18 hr, and then adoptively transferred together with N T cells. The recently activated B cells were not rendered tolerant by this manipulation. The results argue for a major T-cell component in the process whereby soluble protein antigens ablate affinity maturation and memory cell generation.

Immunologic tolerance within the B-lymphocyte compartment: an adult tolerance model.

Immunologic tolerance within the B-lymphocyte compartment: an adult tolerance model.

Idiotype-specific regulation might contribute to specific unresponsiveness in dextran-primed mice

Priming of adult responder mice with optimal immunogenic doses of dextran α,1–6 results in reduced antibody responses to secondary antigenic challenge. We have now quantified dextran-specific clonal precursors in the large and small B-lymphocyte compartments within several days or several months after priming with either dextran or antiidiotypic antibodies directed to a dominant idiotype of C57BL/6 mice which accounts for more than 50 % of the antibody response. The results show that 《secondary unresponsiveness》 correlates with idiotype-directed depletion of the appropriate specificities from the immunocompetent resting B-cell pool.

Prognostic significance of splenic follicle size in splenectomized idiopathic thrombocytopenic purpura patients.

The prognostic significance of splenic follicle (B-lymphocyte compartment) size was studied in 62 patients splenectomized for idiopathic thrombocytopenic purpura (ITP). Patients with hyperplasia of splenic follicles (mean follicle diameter greater than 500 micron) were more likely to relapse or to develop additional autoimmune disorders than patients without hyperplastic splenic follicles (mean follicle diameter less than 500 micron) (p less than 0.01). The enlargement of splenic follicles had a positive predictive value of 27% and a negative predictive value of 100% for a poor outcome of splenectomy. Thus, the histological examination of spleens surgically removed for ITP seems to be an appropriate method to obtain the first indication of the possible long-term effect of splenectomy almost immediately after the operation.

Distinct patterns of expression of different protein kinase C mRNAs in rat tissues

The identification of multiple protein kinase C (PKC) cDNA sequences from rat, bovine, and human tissues has led to the discovery of a family of PKC genes. Using probes complementary to three cDNA sequences from rat brain, designated PKC-I, -II, and -III, the distribution of PKC transcripts in rat tissues was studied by in situ hybridization histochemistry. In brain, PKC-II and PKC-III transcripts colocalized except in the brain stem and spinal cord, where some cells appeared to contain only PKC-III message. PKC-I mRNA had a different, but partially overlapping, pattern of expression. In spleen, PKC-II and PKC-III, but not PKC-I, transcripts were concentrated in the marginal zone of white pulp, the major B-lymphocyte compartment of rat spleen. The distinct distributions of rat PKC mRNAs suggest that different members of the PKC family have specialized functions.