The receptor TLR9, recognizing unmethylated bacterial DNA (CpG), is expressed by B cells and plays a role in the maintenance of serological memory. Little is known about the response of B cells stimulated with CpG alone, without additional cytokines. In this study, we show for the first time the phenotypic modification, changes in gene expression, and functional events downstream to TLR9 stimulation in human B cell subsets. In addition, we demonstrate that upon CpG stimulation, IgM memory B cells differentiate into plasma cells producing IgM Abs directed against the capsular polysaccharides of Streptococcus pneumoniae. This novel finding proves that IgM memory is the B cell compartment responsible for the defense against encapsulated bacteria. We also show that cord blood transitional B cells, corresponding to new bone marrow emigrants, respond to CpG. Upon TLR9 engagement, they de novo express AID and Blimp-1, genes necessary for hypersomatic mutation, class-switch recombination, and plasma cell differentiation and produce Abs with anti-pneumococcal specificity. Transitional B cells, isolated from cord blood, have not been exposed to pneumococcus in vivo. In addition, it is known that Ag binding through the BCR causes apoptotic cell death at this stage of development. Therefore, the ability of transitional B cells to sense bacterial DNA through TLR9 represents a tool to rapidly build up the repertoire of natural Abs necessary for our first-line defense at birth.
A clonal population of B cells expressing a V H 1-69-encoded idiotype accumulates in hepatitis C virus (HCV) associated mixed cryoglobulinemia (MC). These cells are phenotypically heterogeneous, resembling either typical marginal zone (MZ) B cells (IgM +IgD IntroductionHepatitis C virus (HCV) is associated with a spectrum of extrahepatic manifestations, the best characterized of which is type II mixed cryoglobulinemia (MC) [1]. MC is a benign monoCorrespondence: Dr. Massimo Fiorilli e-mail: massimo.fiorilli@uniroma1.it clonal lymphoproliferative disorder of B cells producing rheumatoid factor IgM that, in turn, forms cryoprecipitable immune complexes leading to small vessel vasculitis in vivo [1]. In a large proportion of MC patients, the monoclonal rheumatoid factor bears an idiotype encoded by the V H 1-69 heavy chain variable gene [2,3]. Several lines of evidence suggest that HCV activates B cells via the cross-reactivity of the V H 1-69-encoded idiotype with the E2 glycoprotein of the viral envelope [4,5] The CD21 low B cells expanded in CVID, in HCV + MC, and in HIV-infected patients as well as those found in the tonsil show signs of previous activation and proliferation, fail to proliferate in response to B-cell stimuli and are unable to flux calcium upon BCR cross-linking, although they are in general poised to secrete high levels of immunoglobulins [11][12][13][15][16][17][18][19][20]. In addition, CD21 low B cells express a peculiar array of homing and inhibitory receptors, the latter including CD22, CD72, CD32b, CD85j, CD85d, Fc receptor-like 4 (FCRL4), and sialic acid binding Ig-like lectin 6 (Siglec-6) [11,[15][16][17][18][19][20]. The contribution of these inhibitory receptors, and particularly of FCRL4 and Siglec-6, to the dysfunction of CD21 low B cells is supported by the partial recovery of the proliferative capacity and of effector function upon silencing of these genes with siRNA [21].We recently suggested that the CD21 + MZ-like V H 1-69 + B cells of MC patients also fail to proliferate in response to TLR9 ligation [13]. Here, we characterized the responses of these cells to B-cell stimuli and investigated inhibitory mechanisms. We show that MZ-like V H 1-69 + B cells are functionally exhausted since they fail to respond to TLR and BCR ligands, although their proliferative defect can be overcome by co-stimulation of TLR9 and BCR in the presence of interleukin(IL)-2 and IL-10. In addition, MZ-like V H 1-69 + B cells display increased constitutive and decreased BCRinduced phosphorylation of extracellular signal regulated kinase (ERK); this pattern, however, was also observed in a subpopulation of MZ B cells from healthy individuals. Finally, although the CD21 + MZ-like V H 1-69 + B cells do not express the inhibitory receptors of CD21 low B cells, they strikingly overexpress Stra13, a transcriptional repressor that acts as a key negative regulator of activation and cell cycle progression in B cells [22][23][24]. Our results indicate that the V H 1-69 + MZ B cells activated by HCV undergo premat...
A subset of patients with common variable immunodeficiency (CVID), group 1a of the Freiburg classification, is characterized by increased B cells expressing low levels of CD21 (CD21 low ), lymphoproliferation and autoimmunity. The CD21 low B cells have been shown to be profoundly anergic, and defects of BCR-mediated calcium signaling and of T cells have been described in CVID 1a. We found that also the classical naïve B cells from CVID 1a patients, but not from CVID non-1a patients, proliferated poorly. The B cells of CVID 1a patients had a reduced capacity to divide reminiscent of the proliferative arrest associated with replicative senescence. Thus, we investigated whether lymphocyte dysfunction in CVID 1a was related to telomere-dependent replicative senescence, and found that both the B and the T cells from CVID 1a patients had significantly shorter telomeres compared with B and T cells from CVID non-1a patients. Telomere lengths in B and T cells were significantly correlated, indicating that the rate of telomere attrition in lymphocytes is an individual characteristic of CVID patients. Our findings suggest that telomere-dependent replicative senescence contributes to the immune dysfunction of CVID 1a patients, and may provide an important clue for a better understanding of the pathogenesis of CVID.Key words: Anergy . IntroductionCommon variable immunodeficiency (CVID) is a heterogeneous disorder characterized by reduced antibody levels with low to normal numbers of circulating B cells [1]. Only a small proportion of CVID patients can be classified by the underlying genetic defect [2], and current classifications are mainly based on the B-cell phenotype [3,4]. A subset of CVID is hallmarked by the expansion of an unusual population of B cells with reduced expression of CD21 (CD21 low ); these patients are classified as CVID group 1a according to the Freiburg study [3]. Clinically, CVID 1a is characterized by a late onset, autoimmunity and benign polyclonal lymphoproliferation [3,4]. In addition, several T-cell abnormalities have been described in CVID 1a patients [5]. 854The CD21 low B cells of CVID patients express an array of inhibitory receptor and fail to proliferate in response to BCRdependent and -independent stimuli [6,7]. However, anergy is not limited to CD21 low B cells, since all mature B cells from CVID 1a patients fail to flux calcium upon BCR triggering [8]. CD21 low B cells are also increased in patients with HIV infection [9], systemic lupus erythematosus [10], or mixed cryoglobulinemia secondary to HCV infection [11,12], all conditions characterized by B-cell hyperactivation. Although the CD21 low B cells of patients with HIV infection or HCV-associated mixed cryoglobulinemia have mutated immunoglobulin genes [10][11][12], the CD21 low B cells of CVID 1a patients are unmutated and mostly autoreactive [6,7]. Similarly to those of patients with CVID, the CD21 low B cells of patients with HIV infection are profoundly anergic to B-cell stimuli [9].In this study, we observed that the B cells fr...
We read with great interest the article by Charles et al 1 describing anergy of V H 1-69 ϩ CD21 low B cells from patients with hepatitis C virus (HCV)-associated mixed cryoglobulinemia (MC). Anergy of these cells was made evident by decreased calcium mobilization and low production of IgM. These authors reported that the V H 1-69 ϩ B cells with a CD21 high phenotype were not anergic, but data from our laboratory challenge this conclusion. It is worth noting that in Charles et al's study, calcium mobilization in different subsets of B cells was very variable, and that the analyses could be biased by contaminating V H 1-69 Ϫ CD21 high B cells because G6 ϩ cells were not gated in those experiments. Furthermore, IgM production does not appear to be a reliable marker of anergy in CD21 low B cells, because the CD21 low B cells of patients with common variable immunodeficiency or with HIV infection have defects of proliferative responses although they produce significant amounts of IgM. 2,3 Unfortunately, proliferation studies were not reported in Charles et al's article.We investigated the functional properties of V H 1-69 ϩ B cells from several patients with HCV-associated MC. As with Charles's study, we exploited the G6 antibody 4 (provided by R. Jefferis, University of Birmingham, Birmingham, United Kingdom) to identify these cells. Phenotypic characteristics suggest that the V H 1-69 ϩ CD21 high (IgM ϩ CD27 ϩ ) marginal zone (MZ)-like B cells are the precursors of their CD21 low (CD11c ϩ ) counterparts, considering that some of them express low-level CD11c ( Figure 1A,C). The proliferative responses of V H 1-69 ϩ B cells were analyzed by the CFSE dilution flow cytometric method 5 ; the strategy of analysis is illustrated in Figure 1B and D. We found that both the CD21 low ( Figure 1B) and the CD21 high ( Figure 1D) V H 1-69 ϩ B cells failed to proliferate in response to the stimulation of Toll-like receptor (TLR) 9 with CpG; similar findings were obtained with the TLR7 ligand R848 (not shown). Furthermore, V H 1-69 ϩ B cells failed to differentiate to CD20 low/neg plasmablasts ( Figure 1B,D). The data shown in Figure 1D are representative of the findings in 3 HCV ϩ MC patients with highly enriched (Ͼ 75% of total B cells) CD21 high V H 1-69 ϩ B cells.Clonal B cells of HCV ϩ MC patients provide a unique model for investigating the pathophysiology of human MZ B cells. Murine MZ B cells expand massively on stimulation by bloodborne microbial antigens 6 and, similarly, the antigenic pressure of HCV appears to result in the robust clonal expansion of V H 1-69 ϩ MZ B cells in MC. 7 We provide evidence that these cells undergo premature proliferative anergy, which takes place when the V H 1-69 ϩ B cells still retain a CD21 high MZ-like phenotype and have not yet shifted to a fully "exhausted" 3 CD21 low phenotype. Interestingly, microarray gene expression profiling studies 8 showed that murine MZ B cells stimulated in vivo by Streptococcus pneumoniae rapidly up-regulate Stra13, a negative regulator of B-cell proliferatio...
IntroductionThe use of anti-retroviral therapy (ART) has dramatically reduced HIV-1 associated morbidity and mortality. However, HIV-1 infected individuals have increased rates of morbidity and mortality compared to the non-HIV-1 infected population and this appears to be related to end-organ diseases collectively referred to as Serious Non-AIDS Events (SNAEs). Circulating miRNAs are reported as promising biomarkers for a number of human disease conditions including those that constitute SNAEs. Our study sought to investigate the potential of selected miRNAs in predicting mortality in HIV-1 infected ART treated individuals.Materials and MethodsA set of miRNAs was chosen based on published associations with human disease conditions that constitute SNAEs. This case: control study compared 126 cases (individuals who died whilst on therapy), and 247 matched controls (individuals who remained alive). Cases and controls were ART treated participants of two pivotal HIV-1 trials. The relative abundance of each miRNA in serum was measured, by RTqPCR. Associations with mortality (all-cause, cardiovascular and malignancy) were assessed by logistic regression analysis. Correlations between miRNAs and CD4+ T cell count, hs-CRP, IL-6 and D-dimer were also assessed.ResultsNone of the selected miRNAs was associated with all-cause, cardiovascular or malignancy mortality. The levels of three miRNAs (miRs -21, -122 and -200a) correlated with IL-6 while miR-21 also correlated with D-dimer. Additionally, the abundance of miRs -31, -150 and -223, correlated with baseline CD4+ T cell count while the same three miRNAs plus miR-145 correlated with nadir CD4+ T cell count.DiscussionNo associations with mortality were found with any circulating miRNA studied. These results cast doubt onto the effectiveness of circulating miRNA as early predictors of mortality or the major underlying diseases that contribute to mortality in participants treated for HIV-1 infection.
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