Activation and Induction of Antigen-Specific T Follicular Helper Cells Play a Critical Role in Live-Attenuated Influenza Vaccine-Induced Human Mucosal Anti-influenza Antibody Response.

There is increasing interest recently in developing intranasal vaccines against respiratory tract infections. The antibody response is critical for vaccine-induced protection, and T follicular helper cells (TFH) are considered important for mediating the antibody response. Most data supporting the role for TFH in the antibody response are from animal studies, and direct evidence from humans is limited, apart from the presence of TFH-like cells in blood. We studied the activation and induction of TFH and their role in the anti-influenza antibody response induced by a live-attenuated influenza vaccine (LAIV) in human nasopharynx-associated lymphoid tissue (NALT). TFH activation in adenotonsillar tissues was analyzed by flow cytometry, and anti-hemagglutinin (anti-HA) antibodies were examined following LAIV stimulation of tonsillar mononuclear cells (MNC). Induction of antigen-specific TFH by LAIV was studied by flow cytometry analysis of induced TFH and CD154 expression. LAIV induced TFH proliferation, which correlated with anti-HA antibody production, and TFH were shown to be critical for the antibody response. Induction of TFH from naive T cells by LAIV was shown in newly induced TFH expressing BCL6 and CD21, followed by the detection of anti-HA antibodies. Antigen specificity of LAIV-induced TFH was demonstrated by expression of the antigen-specific T cell activation marker CD154 upon challenge by H1N1 virus antigen or HA. LAIV-induced TFH differentiation was inhibited by BCL6, interleukin-21 (IL-21), ICOS, and CD40 signaling blocking, and that diminished anti-HA antibody production. In conclusion, we demonstrated the induction by LAIV of antigen-specific TFH in human NALT that provide critical support for the anti-influenza antibody response. Promoting antigen-specific TFH in NALT by use of intranasal vaccines may provide an effective vaccination strategy against respiratory infections in humans.IMPORTANCE Airway infections, such as influenza, are common in humans. Intranasal vaccination has been considered a biologically relevant and effective way of immunization against airway infection. The vaccine-induced antibody response is crucial for protection against infection. Recent data from animal studies suggest that one type of T cells, TFH, are important for the antibody response. However, data on whether TFH-mediated help for antibody production operates in humans are limited due to the lack of access to human immune tissue containing TFH In this study, we demonstrate the induction of TFH in human immune tissue, providing critical support for the anti-influenza antibody response, by use of an intranasal influenza vaccine. Our findings provide direct evidence that TFH play a critical role in vaccine-induced immunity in humans and suggest a novel strategy for promoting such cells by use of intranasal vaccines against respiratory infections.

Clinical and biological effects of an agonist anti-CD40 antibody: a Cancer Research UK phase I study.

PURPOSE: This phase I study aimed to establish the biologic effects and MTD of the agonistic IgG1 chimeric anti-CD40 antibody ChiLob7/4 in patients (pts) with a range of CD40-expressing solid tumors and diffuse large B-cell lymphoma, resistant to conventional therapy. Potential mechanisms of action for agonistic anti-CD40 include direct cytotoxic effects on tumor cells and conditioning of antigen-presenting cells. EXPERIMENTAL DESIGN: ChiLob7/4 was given by IV infusion weekly for 4 doses at a range from 0.5 to 240 mg/dose. Validated ELISAs were used to quantify ChiLob7/4 in serum and test for anti-chimeric MAb (HACA) responses. Pharmacodynamic assessments included quantitation of T-cell, natural killer-cell, and B-cell numbers and activation in blood by flow cytometry and a panel of cytokines in plasma by Luminex technology. Planned dose escalation was in cohorts of 3 patients until MTD or biologic effect, defined as reduction of peripheral blood CD19(+) B cells to 10% or less of baseline. RESULTS: Twenty-nine courses of treatment were given to 28 subjects. The MTD was 200 mg × 4, with dose-limiting toxicity of liver transaminase elevations at 240 mg. At 200 mg (range between 2.1 mg/kg and 3.3 mg/kg based on patient body weight), the trough level pretreatment was above 25 µg/mL. Grade 1-2 infusion reactions were seen above the dose of 16 mg, but could be prevented with single-dose corticosteroid premedication. HACA responses were seen after doses between 1.6 mg and 50 mg, but not above this. There were dose-dependent falls in blood B-cell numbers accompanied by reduced expression of CD21, and transient reductions in NK cell numbers with increased CD54 expression from 50 mg upward. MIP-1ß and IL12 plasma concentrations rose after doses above 16 mg. Fifteen of 29 treatments were accompanied by disease stabilization for a median 6 months, the longest for 37 months. CONCLUSIONS: ChiLob7/4 can activate B and NK cells at doses that can be administered safely, and should be tested in combination with other antibodies and chemotherapy agents.

Marginal zone B cells transport IgG3-immune complexes to splenic follicles.

Ag administered together with specific IgG3 induces a higher Ab response than Ag administered alone, an effect requiring the presence of complement receptors 1 and 2 (CR1/2). In this study, we have investigated the fate of Ag, the development of germinal centers (GCs), and the Ab response after i.v. administration of IgG3 anti-trinitrophenyl (TNP) in complex with OVA-TNP. After 2 h, OVA-TNP was detected on marginal zone (MZ) B cells, and a substantial amount of Ag was detected in splenic follicles and colocalized with follicular dendritic cells (FDCs). After 10 d, the percentage of GCs and the IgG responses were markedly higher than in mice immunized with uncomplexed OVA-TNP. The effects of IgG3 were dependent on CR1/2 known to be expressed on B cells and FDCs. Using bone marrow chimeric mice, we demonstrate that an optimal response to IgG3-Ag complexes requires that CR1/2 is expressed on both cell types. These data suggest that CR1/2(+) MZ B cells transport IgG3-Ag-C complexes from the MZ to the follicles, where they are captured by FDCs and induce GCs and IgG production. This pathway for initiating the transport of Ags into splenic follicles complements previously known B-cell dependent pathways where Ag is transported by 1) MZ B cells, binding large Ags-IgM-C complexes via CR1/2; 2) recirculating B cells, binding Ag via BCR; or 3) recirculating B cells, binding IgE-Ag complexes via the low-affinity receptor for IgE, CD23.

BAFF maintains T-cell survival by inducing OPN expression in B cells.

Dysregulation of T-cell survival and apoptosis is the common cause of autoimmune diseases such as multiple sclerosis (MS). However, the factors inducing imbalance of T-cell survival and apoptosis in MS remains unclear. Here, we show that the resistance to apoptosis was associated with high levels of B-cell activating factor (BAFF). Blockade of BAFF with TACI (transmembrane activator and calcium modulator and cyclophilin ligand interactor)-IgG significantly reduced T-cell survival in myelin oligodendroglia glycoprotein (MOG)-induced chronic experimental allergic encephalitis (EAE). Furthermore, BAFF induced anti-apoptotic molecule Bcl2 expression in T cells by up-regulating osteopontin (OPN) secretion from B cells. BAFF mainly induced OPN expression in splenic CD21(-)CD23(+) B cells via a NF-kB dependent signaling pathway. In addition, we found that BAFF and OPN levels were increased in MS patients similar to the results obtained from our mice research. The study suggests that BAFF regulates T-cell survival by inducing OPN secretion in B cells in autoimmune diseases.

Optimal germinal center B cell activation and T-dependent antibody responses require expression of the mouse complement receptor Cr1.

Follicular dendritic cells (FDCs) and complement receptor (Cr)1 and complement receptor (Cr)2 are important for the generation of humoral immunity. Cr1/2 expression on B cells and FDCs was shown to provide a secondary signal for B cell activation, to facilitate transport of Ag in immune follicles, and to enhance retention of immune complexes by FDCs. We show in this study that murine B cells predominantly express the Cr2 product from the Cr2 gene, whereas FDCs almost exclusively express the Cr1 isoform generated from the Cr2 gene. To define the specific role of Cr1, we created an animal that maintains normal cell-restricted expression of Cr2 but does not express Cr1. Cr1-deficient (Cr1KO) mice develop normal B1 and B2 immature and mature B cell subsets and have normal levels of naive serum Abs but altered levels of natural Abs. Immunization of the Cr1KO animal demonstrates deficient Ab responses to T-dependent, but not T-independent, Ags. Germinal centers from the immunized Cr1KO animal possess a deficiency in activated B cells, similar to that seen for animals lacking both Cr1 and Cr2 or C3. Finally, animals lacking only Cr1 respond similarly to wild-type animals to infections with Streptococcus pneumoniae, a pathogen to which animals lacking C3 or both Cr1 and Cr2 are particularly sensitive. Altogether, these data suggest that the production of Cr1, primarily by FDCs, is critical in the generation of appropriately activated B cells of the germinal center and the generation of mature Ab responses.

Systemic human CR2-targeted complement alternative pathway inhibitor ameliorates mouse laser-induced choroidal neovascularization.

PURPOSE: Genetic associations and the presence of complement components within pathological structures of age-related macular degeneration (AMD) have generated the hypothesis that AMD is caused by chronic local complement activation. Since the majority of activity in the common terminal pathway results from engagement of the amplification loop, the alternative pathway has been proposed as a logical therapeutic target. We recently generated a factor H (fH)-based complement inhibitor (CR2-fH) with the capacity to be "targeted" to sites of complement C3 activation. We asked whether the human therapeutic (TT30) is effective in a mouse model of AMD. METHODS: Choroidal neovascularization (CNV) was induced by argon laser photocoagulation of Bruch's membrane. Every other day, mice received intravenous injections of TT30 or vehicles, and after 6 days, the presence or absence of CNV and CNV-related changes were evaluated. Area of CNV, photoreceptor cell function, gene expression for complement components and cytokines, vascular endothelial growth factor (VEGF) protein levels, and TT30 bioavailability were determined. RESULTS: CNV development, which has previously been shown to require local complement activation, could be reduced by intravenous TT30 delivery. Specific inhibition of the alternative pathway not only reduced angiogenesis in CNV, but also ameliorated changes in several associated disease-related biomarkers, including diminished retinal function and molecular events known to be involved in AMD such as VEGF production. After intravenous injection, TT30 localized to CNV lesion sites in the retinal pigmented epithelium-choroid. CONCLUSION: Systemic administration of TT30 was found to reduce CNV pathology. These data may open new avenues for novel systemic AMD treatment strategies.

Expansion of functionally anergic CD21-/low marginal zone-like B cell clones in hepatitis C virus infection-related autoimmunity.

Homeostasis of peripheral B cell subsets is disturbed during chronic hepatitis C virus (HCV) infection, leading to the occurrence of autoimmunity and B cell lymphoproliferation. However, mechanisms by which HCV causes lymphoproliferation remain controversial. We report in this article on the elevated number of clonal CD21(-/low)IgM(+)CD27(+) marginal zone (MZ)-like B cells, which correlates with autoimmunity and lymphoproliferation in HCV patients. We found an increase in autoreactive BCRs using V(H)1-69 and V(H)4-34 genes in CD21(-/low) MZ B cells. CD21(-/low) MZ B cells showed impaired calcium-mediated signaling, did not upregulate activation markers, and did not proliferate in response to BCR triggering. CD21(-/low) MZ B cells also were prone to dying faster than their CD21(+) counterparts, suggesting that these B cells were anergic. CD21(-/low) MZ B cells, in contrast, remained responsive to TLR9 stimulation. Gene array analyses revealed the critical role of Early growth response 2 and Cbl-b in the induction of anergy. Therefore, HCV patients who display high frequencies of unresponsive CD21(-/low) MZ B cells are more susceptible to developing autoimmunity and/or lymphoproliferation. These cells remain in peripheral blood controlled by functional anergy instead of being eliminated, and chronic antigenic stimulation through TLR stimulation may create a favorable environment for breaking tolerance and activating these cells.

Treatment with TNFα blockers induces phenotypical and functional aberrations in peripheral B cells.

To dissect the mechanisms of anti-TNFα-induced autoimmunity we examined the phenotype and function of B cells from anti-TNFα-treated patients. Levels of Lyn, Syk, SHP-1, tyrosine 348 phospho-Syk (Y348-Syk) and tyrosine phosphorylated (P-Y) proteins were evaluated and B-cell-surface CD20, CD21 and CD5 were also assessed in 29 patients treated with TNF-α blockers. Following treatment, Lyn, but not Syk or SHP-1, significantly increased particularly in patients with spondyloarthropathies. Increased Lyn levels following treatment correlated with increased Lyn activity as evidenced by a 2.9-fold increase of Y348-Syk (a Lyn target). Peripheral B-cells from 56.3% of the patients displayed a tendency towards increased P-Y levels without any BCR-initiated activation during treatment. CD20, but not CD21, significantly increased in patients with rheumatoid arthritis. Circulating CD5+ B-cells were also significantly expanded during treatment. Our findings suggest that B cells in anti-TNFα-treated patients display functional and phenotypical aberrations that may enhance our understanding of TNF-α blocker-induced autoimmunity.

CR2+ marginal zone B cell production of pathogenic natural antibodies is C3 independent.

Intestinal ischemia-reperfusion (IR)-induced damage requires complement receptor 2 (CR2) for generation of the appropriate natural Ab repertoire. Pathogenic Abs recognize neoantigens on the ischemic tissue, activate complement, and induce intestinal damage. Because C3 cleavage products act as ligands for CR2, we hypothesized that CR2(hi) marginal zone B cells (MZBs) require C3 for generation of the pathogenic Abs. To explore the ability of splenic CR2(+) B cells to generate the damaging Ab repertoire, we adoptively transferred either MZBs or follicular B cells (FOBs) from C57BL/6 or Cr2(-/-) mice into Rag-1(-/-) mice. Adoptive transfer of wild type CR2(hi) MZBs but not CR2(lo) FOBs induced significant damage, C3 deposition, and inflammation in response to IR. In contrast, similarly treated Rag-1(-/-) mice reconstituted with either Cr2(-/-) MZB/B1 B cells (B1Bs) or FOBs lacked significant intestinal damage and displayed limited complement activation. To determine whether C3 cleavage products are critical in CR2-dependent Ab production, we evaluated the ability of the natural Ab repertoire of C3(-/-) mice to induce damage in response to IR. Infusion of C3(-/-) serum into Cr2(-/-) mice restored IR-induced tissue damage. Furthermore, Rag-1(-/-) mice sustained significant damage after infusion of Abs from C3(-/-) but not Cr2(-/-) mice. Finally, adoptive transfer of MZBs from C3(-/-) mice into Rag-1(-/-) mice resulted in significant tissue damage and inflammation. These data indicate that CR2 expression on MZBs is sufficient to induce the appropriate Abs required for IR-induced tissue damage and that C3 is not critical for generation of the pathogenic Abs.

Exosomes containing glycoprotein 350 released by EBV-transformed B cells selectively target B cells through CD21 and block EBV infection in vitro.

Exosomes are nano-sized membrane vesicles released from a wide variety of cells, formed in endosomes by inward budding of the endosomal limiting membrane. They have immune stimulatory-, inhibitory-, or tolerance-inducing effects, depending on their cellular origin, which is why they are investigated for use in vaccine and immune therapeutic strategies. In this study, we explored whether exosomes of different origins and functions can selectively target different immune cells in human peripheral blood. Flow cytometry, confocal laser scanning microscopy, and multispectral imaging flow cytometry (ImageStream) revealed that exosomes derived from human monocyte-derived dendritic cells and breast milk preferably associated with monocytes. In contrast, exosomes from an EBV-transformed B cell line (LCL1) preferentially targeted B cells. This was not observed for an EBV(-) B cell line (BJAB). Electron microscopy, size-distribution analysis (NanoSight), and a cord blood transformation assay excluded the presence of virions in our LCL1 exosome preparations. The interaction between LCL1-derived exosomes and peripheral blood B cells could be blocked efficiently by anti-CD21 or anti-gp350, indicating an interaction between CD21 on B cells and the EBV glycoprotein gp350 on exosomes. The targeting of LCL1-derived exosomes through gp350-CD21 interaction strongly inhibited EBV infection in B cells isolated from umbilical cord blood, suggesting a protective role for exosomes in regulating EBV infection. Our finding also suggests that exosome-based vaccines can be engineered for specific B cell targeting by inducing gp350 expression.

Age related variation in expression of CD21 and CD32 on bovine lymphocytes: a cross-sectional study.

It is difficult to induce active immune responses in neonatal calves, partly due to limited functional ability of the immune system and partly due to immune inhibitory effects of maternal antibodies. CD21 (complement receptor 2), an activating receptor, and CD32 (Fc gamma receptor II), an inhibitory receptor, can both be expressed by mature B lymphocytes. Studies of the signalling pathways regulating B cell activation suggest that these receptors have mutually antagonistic effects, that may determine immune responsiveness in the first months of life. In a cross-sectional study, blood was collected from 41 Holstein calves, 1-90 days of age, and 12 mature cows. The absolute number of CD21 and CD32 positive cells increased from birth until 90 days of age, with CD21+ cells showing a greater relative increase compared to CD32+ cells. Approximately 89% of CD21+ cells also expressed CD32. In calves, CD32+ cells consisted of two distinct populations, characterized to be CD14+CD32+IgM(-) (44%) and CD14(-)CD32+IgM+ (53%) cells, consistent with monocytes and B cells respectively. Mean fluorescence intensity (MFI) for CD21 did not change appreciably up to 90 days of age, and mean values were slightly lower than for adults. MFI for CD32 on CD21+ lymphocytes increased slightly over the first 3 months of life, but values were lower than for adults. Over 92% of calf membrane immunoglobulin M+ (mIgM) B cells expressed CD21, however only 60-80% of CD21+ cells were mIgM positive, regardless of age. Although the CD21+IgM(-) cells were not characterized further, it was evident that CD21 is not a suitable surrogate marker for B lymphocytes in calves. Most importantly this study showed that from birth, the majority of circulating B cells express both CD21 and CD32. This suggests that these cells are subject to activating and inhibiting influences mediated by these receptors from birth. Expression of CD21 does not appear to be a limiting factor in neonatal B cell responses.

Increased B cell deletion and significantly reduced auto-antibody titre due to premature expression of human complement receptor 2 (CR2, CD21).

The involvement of complement receptor 2 (CR2) in B cell tolerance and autoimmune disease has been revealed over the past decade or so. Our previous studies have established that mice prematurely expressing human CR2 under the control of a lambda light chain promoter (in particular the hCR2(high) line) have a marked deficit in their immune response to various antigens and fail to develop collagen-induced arthritis. This phenotype appears to be the result of irreversible changes in B cell signalling pathways and suggested that hCR2 expressing mice are protected from developing autoimmune disease. To test this hypothesis, we examined the ability of the hCR2 to block the development of spontaneous autoimmune disease on the C57BL/6j-Fas(lpr/)Fas(lpr) (B6(lpr)) background. We found that expression of hCR2 on the B6(lpr) background resulted in a significant reduction in levels of anti-nuclear antibodies (ANA) generated as mice aged but the levels of ANA were still higher than those found in age matched C57BL/6j (B6) mice. B cells from hCR2(high) mice were found to display a higher baseline level of apoptosis, whether analysed ex vivo or after in vitro culture, than their B6 counterparts and this was apparently linked to both surface IgM expression by the B cells and C3 levels in the mice. Our data also provides evidence that B cell survival in the presence of hCR2 is heavily modified by the background strain of the mouse. Overall, we have demonstrated that mice expressing hCR2 on their B cells during bone marrow development display a higher degree of apoptosis which may lead to a deletion of autoreactive B cells and be protective against the development of autoimmune disease.

Dendritic cell sarcomas/tumours of the breast: report of two cases.

Extranodal follicular dendritic cell sarcoma/tumours (FDCS/Ts) and interdigitating dendritic cell sarcoma/tumours (IDCS/Ts) are rare neoplasms. We present two cases of FDCS/T and IDCS/T of the breast. The FDCS/T case (case 1) presented in a 31-year-old woman and the IDCS/T case (case 2) in a 67-year-old woman who both showed a firm lump in the left breast. The FDCS/T lesion superficially appeared as an anaplastic carcinoma and the IDCS/T was reminiscent of a spindle cell sarcomatoid carcinoma. Nevertheless both lesions were negative for keratins while case 1 displayed neoplastic cells strongly positive for CD21, vimentin and focally for CD68 and S-100 protein. The tumour cells of case 2 were positive for S-100, CD68 and CD45. In breast, an unusual keratin negative tumour composed predominantly of spindle cells arranged in fascicles, storiform pattern or whorls with a lymphoid rich stroma should raise suspicion for FDCS/Ts or IDCS/Ts. The distinction from malignant tumours with similar features is discussed.

Laser-capture microdissection of oropharyngeal epithelium indicates restriction of Epstein-Barr virus receptor/CD21 mRNA to tonsil epithelial cells.

BACKGROUND: Epstein-Barr virus colonizes the oropharynx of a majority of individuals. It infects B lymphocytes and epithelial cells and can contribute to the development of both lymphoid and epithelial tumors. The virus uses CD21 for attachment to B cells which constitutively express the protein. Infection of epithelial cells in vitro is also more efficient if CD21 is available. However, its potential contribution to infection in vivo has been difficult to evaluate as discrepant results with antibodies have made it difficult to determine which, if any, epithelial cells in the oropharynx express CD21. METHODS: To reevaluate CD21 expression by an alternative method, epithelial cells were isolated by laser-capture microdissection from formalin-fixed sections of tissues from various parts of the oropharynx and mRNA was amplified with primers specific for the exons of CD21 which code for the Epstein-Barr virus binding site. RESULTS: CD21 mRNA was expressed in tonsil epithelium, but not in epithelium from buccal mucosa, uvula, soft palate or tongue. CONCLUSIONS: CD21 does not contribute to infection of most normal epithelial tissues in the oropharynx, but may contribute to infection of epithelial cells in the tonsil, where virus has been demonstrated in healthy carriers.

CXCL13 expression and follicular dendritic cells in relation to B-cell infiltration in periodontal disease tissues.

BACKGROUND AND OBJECTIVE: B lymphocyte is the dominant infiltrating cell type in periodontitis lesions. CXCL13, produced by follicular dendritic cells, endothelial cells and fibroblasts, is crucial for B-cell trafficking. An association between chronic inflammation and lymphoid organogenesis has been reported in infection and in autoimmune responses, in which T-cell/B-cell follicles with a follicular dendritic cell network are formed. The aim of this study was to examine CXCL13 expression and follicular dendritic cell distribution in relation to B-cell infiltration in chronic inflammatory periodontal lesions. MATERIAL AND METHODS: Fifty-eight gingival tissue biopsies from patients with periodontitis and 25 samples from subjects with gingivitis were analyzed. Gene expression for CXCL13 and for the CD21 long isoform was analyzed using the reverse transcription-polymerase chain reaction. Immunohistochemical analysis was performed using antibodies to CXCL13, CXCR5, follicular dendritic cells, CD3 and CD19 on serial cryostat sections. RESULTS: mRNA for CXCL13 was expressed in both periodontitis and gingivitis tissues. The number of CXCL13+ cells was significantly higher in periodontitis than in gingivitis in connective tissues subjacent to the pocket epithelium and positively correlated with the number of CD19+ cells. CXCL13+ cells were distributed in B-cell-dominant areas both with and without follicular dendritic cells. Although obvious reticular networks of follicular dendritic cells were not found in periodontitis and gingivitis, the accumulation of follicular dendritic cells in B-cell-dominant areas in periodontitis was observed in some patients. CONCLUSION: These findings suggested that CXCL13 and follicular dendritic cells were involved in B-cell recruitment to, and B-cell distribution in, chronic inflammatory periodontal lesions.

Impaired naive and memory B-cell responsiveness to TLR9 stimulation in human immunodeficiency virus infection.

Toll-like receptor 9 (TLR9) agonists such as unmethylated bacterial CpG DNAs activate B lymphocytes directly, potentially influencing their function and homeostasis. To assess B-cell responsiveness to TLR9 agonists in human immunodeficiency virus (HIV) disease, we examined the ability of naive and memory B cells to proliferation and to increase surface expression of CD80 in response to CpG oligonucleotides (ODN). CpG ODN induced expression of CD80 similarly in B cells from HIV-infected persons and from healthy controls. In contrast, proliferation responses to CpG ODN were markedly impaired in both naive and memory B-cell subsets from HIV-infected persons. Naive B-cell proliferation defects were related to plasma HIV RNA and, among memory B cells, to the frequencies of CD21-negative cells. Importantly, TLR9 mRNA levels were significantly diminished in freshly prepared naive B cells and especially so in memory B cells from HIV-positive viremic donors, suggesting a possible underlying mechanism for the observed functional impairments. Dose-response studies indicated that optimal induction of CD80 expression was achieved with much lower concentrations of CpG ODN than optimal induction of proliferation. We propose that the relatively low threshold of activation that is required for CD80 induction by CpG ODN might explain the preservation of this response in B cells from HIV-infected persons despite diminished TLR9 expression. Impaired responsiveness to TLR9 agonists may contribute to defects in humoral immunity in HIV infection.

Characterization of a late transitional B cell population highly sensitive to BAFF-mediated homeostatic proliferation.

We have characterized a distinct, late transitional B cell subset, CD21(int) transitional 2 (T2) B cells. In contrast to early transitional B cells, CD21(int) T2 B cells exhibit augmented responses to a range of potential microenvironmental stimuli. Adoptive transfer studies demonstrate that this subset is an immediate precursor of both follicular mature and marginal zone (MZ) B cells. In vivo, a large percentage of CD21(int) T2 B cells has entered the cell cycle, and the cycling subpopulation exhibits further augmentation in mitogenic responses and B cell-activating factor of the TNF family (BAFF) receptor expression. Consistent with these features, CD21(int) T2 cells exhibit preferential responses to BAFF-facilitated homeostatic signals in vivo. In addition, we demonstrate that M167 B cell receptor (BCR) idiotypic-specific B cells are first selected within the cycling CD21(int) T2 population, ultimately leading to preferential enrichment of these cells within the MZ B cell compartment. These data, in association with the coordinate role for BAFF and microenvironmental cues in determining the mature BCR repertoire, imply that this subset functions as a unique selection point in peripheral B cell development.

Epstein-Barr virus (EBV) infection in B-cell non-Hodgkin's lymphomas in children: virus latency and its correlation with CD21 and CD23 molecules.

Epstein Barr virus (EBV) infection of human B lymphocytes in vitro results in immortalisation of the cells and augmented membranous expression of numerous B-cell activation molecules, including CD23. Other studies demonstrated that only those B lymphocytes which carry the surface CD21 (EBV receptor) become transformation-competent. Inspired by the relatively unclear relations between expression of EBV and those of CD21 and CD23 in in vivo conditions we have decided to define correlations between tissue markers of EBV and of CD21 and CD23 molecules in B-cell non-Hodgkin's lymphomas (NHLs) in children. The studies were performed on an archival tissue material originating from children with B-cell NHLs (n=26) using immunocytochemical techniques, in situ hybridisation, and PCR. Our studies confirmed the latent phase of EBV infection in all of the EBV-positive patients. Viral proteins as well as viral RNAs (EBERs) was found both in the cytoplasm, in cell nuclei and in cell membranes of mainly the transformed lymphocytes B. Expression of the latent proteins (EBNA2 and LMP1) and that of EBERs in B-cell NHLs was significantly higher as compared to children with nonneoplastic lesions. The studies demonstrated reciprocally positive correlations between expressions of CD21 and CD23 in our children, but no correlation could be demonstrated between expression of EBV tissue markers and that of CD21 and/or CD23. Positive correlation was confirmed between expression of EBNA2 and LMP1 as well as between expression of the two proteins and EBERs in B-cell NHLs. Our studies have shown mainly latency III pattern of EBV. We have also demonstrated a novel form of EBV latency with no EBERs expression. The high detectability of EBV-positive cases both in the group of B-cell NHLs (77%), and in the group with non-neoplastic lesions (64%) suggested that only more pronounced tissue expression of EBV markers in B-cell NHLs as compared to the non-neoplastic material may point to a potential role of EBV in pathogenesis of lymphoma in this group of population in our country.

Intrinsic B cell hypo-responsiveness in mice prematurely expressing human CR2/CD21 during B cell development.

We previously reported that human CR2 (hCR2) prematurely expressed under a murine Vlambda2 promoter/Vlambda2-4 enhancer minigene during the CD43+ CD25- late pro-B cell stage of development results in peripheral B cells with impaired responses to immunization with T-dependent antigens. Herein, we show that hCR2 transgenic (Tg) mice also demonstrate a severe defect in T-independent antigen responses and are substantially protected from clinical arthritis, synovitis and cartilage/bone destruction in a collagen-induced arthritis model. This outcome is found despite the apparently normal development of autoreactive T cells with equivalent cytokine and proliferative responses to antigen when compared to non-Tg control mice. These data suggest the presence of an intrinsic B cell defect in the hCR2 Tg mice. We also show that an hCR2-dependent Ca2+ influx can be generated in both developing and mature Tg B cells, but with different rates of decay as compared to control wild-type (WT) mice. In addition, although analysis of tyrosine-phosphorylated proteins in WT and Tg B cells following B cell receptor (BCR)-induced activation revealed the presence of distinctly different phosphorylation patterns, no differences were identified in several candidate protein targets. Overall, these data suggest that premature hCR2 expression and the consequences thereof during B cell development intrinsically alters the way mature B cells develop and subsequently respond to antigen through the BCR signaling complex.

Brain edema after intracerebral hemorrhage in rats: the role of inflammation.

BACKGROUND: Intracerebral hemorrhage (ICH) results in secondary brain edema and injury that may lead to death and disability. ICH also causes inflammation. It is unclear whether inflammation contributes to brain edema and neuron injury or functions in repairing the brain tissue. AIMS: To understand the effect of inflammation in ICH, we have carried out an investigation on the various aspects and the dynamic changes of inflammation. SETTINGS AND DESIGN: An ICH model was generated by injecting 50 microl autologous tail artery blood stereotactically into the right caudate nucleus of 30 rats, which were randomly divided into five ICH groups. Similarly, five Sham control groups were generated by inserting the needle to the right caudate nucleus of rats. MATERIALS AND METHODS: Rat behavior was evaluated over the time course (6 h, 24 h, 48 h, 72 h and 7 d) in each group. The rats were then killed by administering an overdose of pentobarbital. Following the euthanasia, the brain water content, neuronal loss, glia proliferation, inflammatory infiltration and brain morphology of the rats were measured. Additionally, the expression of TNF-alpha, IL-6, ICAM-1, VEGF, NF-kappaB, C3 and CR2 was analyzed by immunohistochemistry. STATISTICAL ANALYSIS: The data were analyzed by student's t test. RESULTS: Rat brain water content increased progressively over the time course and reached its peak at 48 h followed ICH. The maximum of inflammatory infiltrate (especially neutrophils) and immunopositive cells of TNF-alpha, IL-6 and NF-kappaB, were at 48 h. The expression of C3 and CR2 reached their peaks at 48-72 h, while the expression ICAM-1 and VEGF were at maximum at 72 h followed ICH. CONCLUSIONS: The results suggested that the inflammatory cytokines, complement system and VEGF may have a function in the development of the brain edema and neuron injury followed ICH.