Infection, Immunity and Inflammation

We examined the frequency of such compartmentalization, the cell types involved, the constraints exerted on the different variants, and the role of immunoglobulin-complexed variants. We screened the hypervariable region (HVR1) of HCV isolates from 14 HBsAg-and HIV-seronegative patients with chronic HCV infection. HCV RNA was amplified and cloned from plasma, the immunoglobulin G (IgG)-bound fraction, and total and sorted BMCs (CD19؉, CD8؉, CD4؉, and CD14؉ cells). Compartmentalization was estimated using a matrix correlation test. The ratio of nonsynonymous/synonymous substitutions (d N /d S ratio) was calculated for each compartment. HCV RNA was detected in 3/3 BMC, 11/11 CD19؉, 10/11 CD14؉, 4/11 CD8؉ and 0/11 CD4؉ cell samples.

In vitro drug susceptibility testing of clinical isolates plays an important role in the management of HIV. Similar analyses for HBV will become important as therapeutic options increase. Analyses of clinical HBV isolates have been difficult due to the limitations of available culture systems. Site-directed mutagenesis has been used to study point mutations in laboratory HBV strains, however, the validity of such analyses is limited since mutations are removed from their natural genetic context. Aims: To develop methods for efficiently phenotyping full-length clinical HBV isolates. To assess natural variation in the adefovir susceptibility of wildtype HBV isolates derived from multiple patients. Methods: Sera (n=8) were collected from chronic hepatitis B patients entering trials of adefovir dipivoxil. Full-length HBV genomes were amplified using the methods of Gunther et al. (J.Virol 1995, 69: 5437). A novel vector that allowed the insertion and expression of full-length HBV genomes under the control of a CMV promoter was developed. Intracellular replication and adefovir susceptibility of cloned isolates were assayed by transient transfection. Results: Full-length HBV clones were isolated from all patients. There was a marked variation in the levels of replication between clones, with approximately 70% producing detectable replicative intermediates. Among the evaluable isolates, adefovir susceptibility did not differ significantly (12 clones from 8 patients, mean IC.50=0.26&0.08 FM). Conclusions: A novel vector that enables the efficient phenotyping of fulllength clinical HBV isolates has been developed. Despite variations in genetic background and replication levels, adefovir demonstrated similar and potent activity against all clinical HBV isolates.

In vitro drug susceptibility testing of clinical isolates plays an important role in the management of HIV. Similar analyses for HBV will become important as therapeutic options increase. Analyses of clinical HBV isolates have been difficult due to the limitations of available culture systems. Site-directed mutagenesis has been used to study point mutations in laboratory HBV strains, however, the validity of such analyses is limited since mutations are removed from their natural genetic context. Aims: To develop methods for efficiently phenotyping full-length clinical HBV isolates. To assess natural variation in the adefovir susceptibility of wildtype HBV isolates derived from multiple patients. Methods: Sera (n=8) were collected from chronic hepatitis B patients entering trials of adefovir dipivoxil. Full-length HBV genomes were amplified using the methods of Gunther et al. (J.Virol 1995, 69: 5437). A novel vector that allowed the insertion and expression of full-length HBV genomes under the control of a CMV promoter was developed. Intracellular replication and adefovir susceptibility of cloned isolates were assayed by transient transfection. Results: Full-length HBV clones were isolated from all patients. There was a marked variation in the levels of replication between clones, with approximately 70% producing detectable replicative intermediates. Among the evaluable isolates, adefovir susceptibility did not differ significantly (12 clones from 8 patients, mean IC.50=0.26&0.08 FM). Conclusions: A novel vector that enables the efficient phenotyping of fulllength clinical HBV isolates has been developed. Despite variations in genetic background and replication levels, adefovir demonstrated similar and potent activity against all clinical HBV isolates.

We examined the frequency of such compartmentalization, the cell types involved, the constraints exerted on the different variants, and the role of immunoglobulin-complexed variants. We screened the hypervariable region (HVR1) of HCV isolates from 14 HBsAg-and HIV-seronegative patients with chronic HCV infection. HCV RNA was amplified and cloned from plasma, the immunoglobulin G (IgG)-bound fraction, and total and sorted BMCs (CD19؉, CD8؉, CD4؉, and CD14؉ cells). Compartmentalization was estimated using a matrix correlation test. The ratio of nonsynonymous/synonymous substitutions (d N /d S ratio) was calculated for each compartment. HCV RNA was detected in 3/3 BMC, 11/11 CD19؉, 10/11 CD14؉, 4/11 CD8؉ and 0/11 CD4؉ cell samples.

B-cell-activating factor of the TNF family, (BAFF), and a proliferation-inducing ligand (APRIL) regulate B-lymphocyte survival and activation. We report that BAFF, but not APRIL, increased the chemotactic response of primary human B cells to CCL21, CXCL12, and CXCL13. The BAFF-induced increase in B-cell chemotaxis was totally abolished by blockade of BAFF-R and was strongly dependent on the activation of PI3K/AKT, NF-kappaB, and p38MAPK pathways. BAFF had similar effects on the chemotaxis of naive and memory B cells in response to CCL21 but increased more strongly that of memory B cells to CXCL13 than that of naive B cells. Our findings indicate a previously unreported role for the BAFF/BAFF-R pair in mature B-cell chemotaxis. The synergy between CXCL13 and BAFF produced by stromal cells and follicular dendritic cells may have important implications for B-cell homeostasis, the development of normal B-cell areas, and for the formation of germinal center-like follicles that may be observed in various autoimmune diseases.

The signal transducer and activator of transcription 3 (STAT3) transcription factor pathway plays an important role in many biological phenomena. STAT3 transcription is triggered by cytokine-associated signals. Here, we use isolated human B cells to analyse the role of STAT3 in interleukin (IL)-10 induced terminal B cell differentiation and in immunoglobulin (Ig)A production as a characteristic readout of IL-10 signalling. We identified optimal conditions for inducing in-vitro IgA production by purified blood naive B cells using IL-10 and soluble CD40L. We show that soluble CD40L consistently induces the phosphorylation of nuclear factor (NF)-kB p65 but not of STAT3, while IL-10 induces the phosphorylation of STAT3 but not of NF-kB p65. Interestingly, while soluble CD40L and IL-10 were synergistic in driving the terminal maturation of B cells into IgA-producing plasma cells, they did not co-operate earlier in the pathway with regard to the transcription factors NF-kB p65 or STAT3. Blocking either NF-kB p65 or STAT3 profoundly altered the production of IgA and mRNA for activation-induced cytidine deaminase (AID), an enzyme strictly necessary for Ig heavy chain recombination. Finally, the STAT3 pathway was directly activated by IL-10, while IL-6, the main cytokine otherwise known for activating the STAT3 pathway, did not appear to be involved in IL-10-induced-STAT3 activation. Our results suggest that STAT3 and NF-kB pathways co-operate in IgA production, with soluble CD40L rapidly activating the NF-kB pathway, probably rendering STAT3 probably more reactive to IL-10 signalling. This novel role for STAT3 in B cell development reveals a potential therapeutic or vaccine target for eliciting IgA humoral responses at mucosal interfaces.

Objective. Blood platelets represent a link between hemostasis, inflammation, and tissue repair. Their role in immune responses and inflammation mainly involves many molecules, among which Toll-like receptor, major histocompatibility complex class I, CD40 and CD154/CD40 ligand (CD40L). As platelets are the major purveyor of soluble CD40L (sCD40L), we sought to determine their involvement in CD40/CD40L-dependent immune responses and to understand the interactions between platelets and peripheral B lymphocytes. Materials and Methods. We examined the capacity of platelets to bind nonstimulated B cells, and phenotypic changes by flow cytometry and confocal scanning laser microscopy. Modulation of cytokines/chemokines and total levels of immunoglobulin (Ig) A, IgG, IgM, and IgG subclasses in supernatants of coculture, platelets, and B lymphocytes was performed by sandwich enzyme-linked immunosorbent assay and differential production of cytokine mRNA as determined by reverse transcriptase polymerase chain reaction. Results. In coculture, platelets and B lymphocytes were mutually activated, as demonstrated by the increased expression of platelet CD62p and B-cell CD86. Platelet/B-cell interactions were accompanied by changes in membrane expression of CD40 and CD40L by both platelets and B lymphocytes. IL12p70 and IL8 gene transcription were significantly reduced, which was attributable to B cells. Conversely, there was a significant, platelet-dependent reduction of sCD40L and RANTES mRNA expression. After a 3-day incubation with platelets, differentiated B cells increased their in vitro production of IgG1, IgG2, and IgG3, but not IgG4, IgA, or IgM. Conclusion. These data emphasize the potentially important role of platelets in the adaptive immune response. Platelets have an immunoregulatory role that might be applied clinically in multitransfused patients (e.g., hematopoietic stem cell transplantation). Ó

Protein A (SpA) of Staphylococcus aureus is known to target the paratope of immunoglobulins expressing V(H)3 genes, and to delete marginal zone B-cells and B-1a in vivo. Here, we have discovered that SpA endows S. aureus with the potential to subvert B-cell trafficking in the host. We found that SpA, whose Fc binding site has been inactivated, binds essentially to naïve B-cells and induces a long-lasting decrease in CXCR4 expression and in B-cell chemotaxis to CXCL12. Competition experiments indicated that SpA does not interfere with binding of CXCR4 ligands and does not directly bind to CXCR4. This conclusion is strongly supported by the inability of SpA to modulate clathrin-mediated CXCR4 internalization, which contrasts with the potent effect of anti-IgM antibodies. Microscopy and biochemical experiments confirmed that SpA binds to the surface IgM/IgD complex and induces its clathrin-dependent internalization. Concomitantly, the SpA-induced signaling leads to PKC-dependent CXCR4 ...

B-cell expression of certain Toll-like receptors (TLRs) is important in linking innate and adaptive immune responses in normal and pathological conditions. The expression of TLR9 plays a role in the recognition of conserved pathogen motifs in a manner that is dependent on B-cell localization, deduced from B-cell phenotype. The nature of TLR9 function is unclear. A first step in unravelling the function of this pattern recognition receptor is to discover the precise nature of the cell types that express TLR9. This study used three-colour flow cytometry to characterize the B lymphocytes from human peripheral blood mononuclear cells (PBMCs) that express TLR9 on the surface. We sorted TLR9-positive B and non-B cells from the PBMC population and detected TLR9 expression on naïve and memory B cells. Moreover, we identified two discrete subpopulations of B cells: CD19 + CD27 ) CD23 + cells and CD19 + CD27 high CD80 + cells. These subpopulations expressed high levels of membrane TLR9 and exhibited a strong in vitro response to binding a relevant CpG motif by secreting high levels of interleukin-6 (compared to controls). Our finding that this pattern recognition receptor is expressed on a variety of cell subsets adds to the current understanding of the functional complexity of B-cell membrane TLR9.

In this study, we show that IFNa increases the chemotaxis of human B cells to CCL20, CCL21 and CXCL12 in a dose-and time-dependent manner. The effect was maximal with 2000 IU ml ÿ1 IFNa. It peaked at 24 h and decreased thereafter. At 24 h, IFNa had increased B-cell chemotaxis to CCL20 by 20 6 6.2% (n 5 9, P < 0.002), to CCL21 by 20 6 8.5% (n 5 14, P < 0.0001) and to CXCL12 by 16.3 6 4.2% (n 5 12, P < 0.003) without changing CCR6, CCR7 or CXCR4 expression. IFNa enhanced the migration of memory B cells to CCL20, CCL21 and CXCL12 2.6-fold more strongly than that of naive B cells. The triggering of chemokine receptors by their ligands resulted in the activation of phosphatidylinositide-3 kinase (PI3K)/protein kinase B (PKB), inhibitory NF-jB (IjBa) RhoA and extracellular signalregulated protein kinase 1/2 (ERK1/2). All these effectors except ERK1/2 are crucial for B-cell chemotaxis. IFNa modulated the requirements for B-cell chemotaxis, which became dependent on ERK1/2, more dependent on PI3K, RhoA and nuclear factor-jB but less dependent on Gbc and phospholipase C activation. IFNa also decreased ligand-induced chemokine receptor internalization in a manner dependent on PI3K/AKT and RhoA but not on IjBa and ERK1/2. Our data characterize chemokine receptor signaling in human B cells and clarify the relevance of downstream pathways in B-cell chemotaxis and chemokine receptor internalization. They also suggest that non-class I PI3K are involved in B-cell chemotaxis.

Tumor cells from eight adult patients with T-cell chronic malignancies were investigated with a series of mono- clonal antibodies recognizing T-cell differentiation anti- gens. This series allowed definition of discrete subpopula- tions of mature T cells with functional specialization. All six patients with S#{233}zary syndrome and one patient with T-chronic lymphocytic leukemia had cells with the same C ERTAIN STUDIES have shown that malignant cells in S#{233}zary syndrome (55) and rare cases of chronic lymphocytic leukemia (CLL) are T cells.&#x27;5 In other studies it has been shown in a few cases that these cells have some functional properties of mature T cells.68

The tumor necrosis factor (TNF) family member B cell activating factor (BAFF) binds B cells and enhances B cell receptor-triggered proliferation. We find that B cell maturation antigen (BCMA), a predicted member of the TNF receptor family expressed primarily in mature B cells, is a receptor for BAFF. Although BCMA was previously localized to the Golgi apparatus, BCMA was found to be expressed on the surface of transfected cells and tonsillar B cells. A soluble form of BCMA, which inhibited the binding of BAFF to a B cell line, induced a dramatic decrease in the number of peripheral B cells when administered in vivo. Moreover, culturing splenic cells in the presence of BAFF increased survival of a percentage of the B cells. These results are consistent with a role for BAFF in maintaining homeostasis of the B cell population.

We analyzed the modulation of human B cell chemotaxis by the gp120 proteins of various HIV-1 strains. X4 and X4/R5 gp120 inhibited B cell chemotaxis toward CXCL12, CCL20, and CCL21 by 40 -50%, whereas R5 gp120 decreased inhibition by 20%. This gp120-induced inhibition was strictly dependent on CXCR4 or CCR5 and lipid rafts but not on CD4 or V H 3-expressing BCR. Inhibition did not impair the expression or ligand-induced internalization of CCR6 and CCR7. Our data suggest that gp120/ CXCR4 and gp120/CCR5 interactions lead to the cross-desensitization of CCR6 and CCR7 because gp120 does not bind CCR6 and CCR7. Unlike CXCL12, gp120 did not induce the activation of phospholipase C␤3 and PI3K downstream from CXCR4, whereas p38 MAPK activation was observed. Similar results were obtained if gp120-treated cells were triggered by CCL21 and CCL20. Our results are consistent with a blockade restricted to signaling pathways using phosphatidylinositol-4,5-bisphosphate as a substrate. X4 and X4/R5 gp120 induced the cleavage of CD62 ligand by a mechanism dependent on matrix metalloproteinase 1 and 3, CD4, CXCR4, G␣ i , and p38 MAPK, whereas R5 gp120 did not. X4 and X4/R5 gp120 also induced the relocalization of cytoplasmic CD95 to the membrane and a 23% increase in CD95-mediated apoptosis. No such effects were observed with R5 gp120. The gp120-induced decrease in B cell chemotaxis and CD62 ligand expression, and increase in CD95-mediated B cell apoptosis probably have major deleterious effects on B cell responsiveness during HIV infection and in vaccination trials. The Journal of Immunology, 2005, 175: 302-310.