Randy T. Fischer

Patterns of microRNA expression characterize stages of human B-cell differentiation

Mature B-cell differentiation provides an important mechanism for the acquisition of adaptive immunity. Malignancies derived from mature B cells constitute the majority of leukemias and lymphomas. These malignancies often maintain the characteristics of the normal B cells that they are derived from, a feature that is frequently used in their diagnosis. The role of microRNAs in mature B cells is largely unknown. Through concomitant microRNA and mRNA profiling, we demonstrate a potential regulatory role for microRNAs at every stage of the mature B-cell differentiation process. In addition, we have experimentally identified a direct role for the microRNA regulation of key transcription factors in B-cell differentiation: LMO2 and PRDM1 (Blimp1). We also profiled the microRNA of B-cell tumors derived from diffuse large B-cell lymphoma, Burkitt lymphoma, and chronic lymphocytic leukemia. We found that, in contrast to many other malignancies, common B-cell malignancies do not down-regulate microRNA expression. Although these tumors could be distinguished from each other with use of microRNA expression, each tumor type maintained the expression of the lineage-specific microRNAs. Expression of these lineage-specific microRNAs could correctly predict the lineage of B-cell malignancies in more than 95% of the cases. Thus, our data demonstrate that microRNAs may be important in maintaining the mature B-cell phenotype in normal and malignant B cells.

IL-21 and BAFF/BLyS Synergize in Stimulating Plasma Cell Differentiation from a Unique Population of Human Splenic Memory B Cells

Both constitutive Ig secretion by long-lived plasma cells (PC) and the recurrent differentiation of memory (mem) B cells into PC contribute to the maintenance of serologic mem. However, the relative contribution of each is unknown. In this study, we describe a novel population of human postswitched mem B cells that rapidly differentiate into PC and thus contribute to serologic mem. These IgG+ B cells reside in the region of human spleen analogous to the murine marginal zone and have not previously been examined. These cells are highly responsive to IL-21 in the context of CD40 stimulation. Uniquely, IgG+ marginal zone analog B cells are exquisitely sensitive to the combination of IL-21 and B cell-activating factor belonging to the TNF family (BAFF/BLyS) that synergize in the absence of further costimulation to induce up-regulation of B lymphocyte-induced maturation protein-1 and drive PC differentiation. Other cytokine combinations are not active in this regard. This is the first demonstration that this unique population of mem B cells can respond specifically and exclusively to IL-21 and BAFF/BLyS by differentiating into IgG-secreting PC, and thus contributing to serologic mem in an Ag-independent manner.

Identification and Characterization of a Human CD5+ Pre-Naive B Cell Population

We have identified a distinct pre-naive B cell population circulating in human peripheral blood that exhibits an intermediate phenotype between transitional and naive B cells. Like human transitional B cells, these cells express CD5 but have intermediate densities of CD38, CD10, CD9, and the ABCB1 transporter compared with transitional and naive B cells. These pre-naive B cells account for a majority of circulating human CD5+ B cells. Importantly, CD5+ pre-naive B cells could be induced to differentiate into cells with a naive phenotype in vitro. CD5+ pre-naive B cells show only partial responses to BCR stimulation and CD40 ligation and undergo more spontaneous apoptosis and cell death than do naive B cells, whereas BAFF/BLyS (B cell-activating factor belonging to the TNF family) did not enhance their survival compared with naive B cells. In contrast, CD5+ pre-naive B cells carry out certain functions comparable to naive B cells, including the capacity to differentiate into plasma cells and the ability to function as APCs. Notably, an increased proportion of CD5+ pre-naive B cells were found in peripheral blood of patients with systemic lupus erythematosus. These results have identified a unique intermediate in human naive B cell development within the peripheral blood and derangements of its homeostasis in patients with systemic lupus erythematosus.

Impact of anti-interleukin-6 receptor blockade on circulating T and B cell subsets in patients with systemic lupus erythematosus

Background Circulating plasmablasts/plasma cells and activated B and T cells are increased in systemic lupus erythematosus (SLE). Interleukin (IL)-6 induces differentiation of B cells into antibody-forming cells and of T cells into effector cells. Objective To examine the hypothesis that blocking IL-6 would reverse some of the immune abnormalities present in SLE. Methods Fifteen patients with SLE with mild-to moderate disease activity were treated with biweekly infusions of tocilizumab, a humanised anti-IL-6 receptor monoclonal antibody for 12 weeks. Lymphocyte subsets (analysed by flow cytometry) and serum immunoglobulin levels were compared at baseline and at weeks 6 and 12. Results Tocilizumab decreased activated T and B cells, the frequency of CD27highCD38highIgD− plasmablasts/plasma cells and IgD−CD27+ post-switched memory B cells as well as IgG+ memory B cell, whereas it increased the frequency of IgD+CD27− antigen-inexperienced B cells. Among antigen-inexperienced IgD+CD27− B cells, CD38low mature naïve B cells increased significantly and CD38IntermediateCD5+ pre-naïve B cells showed a decreasing trend, whereas CD38highCD5+ transitional type 1 B cells did not change. Most of the changes occurred in patients who had abnormal values at baseline. IgG, IgA, IgG1 and IgG3 serum levels decreased albeit within the normal range. The frequency of CD4+CD45RA+CCR7+ naïve T cells increased. Conclusions In vivo blockade of the IL-6 receptor decreases lymphocyte activation and restores B and T cell homoeostasis by either blocking differentiation and/or trafficking in patients with SLE and leads to normalisation of the abnormal B and T cell subsets seen at baseline.

Autoreactive B Cells Escape Clonal Deletion by Expressing Multiple Antigen Receptors

IgH and L chain transgenes encoding a phosphocholine (PC)-specific Ig receptor were introduced into recombinase-activating gene (Rag-2−/−) knockout mice. The PC-specific B cells that developed behaved like known autoreactive lymphocytes. They were 1) developmentally arrested in the bone marrow, 2) unable to secrete Ab, 3) able to escape clonal deletion and develop into B1 B cells in the peritoneal cavity, and 4) rescued by overexpression of bcl-2. A second IgL chain was genetically introduced into Rag-2−/− knockout mice expressing the autoreactive PC-specific Ig receptor. These dual L chain-expressing mice had B cells in peripheral lymphoid organs that coexpressed both anti-PC Ab as well as Ab employing the second available L chain that does not generate an autoreactive PC-specific receptor. Coexpression of the additional Ig molecules rescued the autoreactive anti-PC B cells and relieved the functional anergy of the anti-PC-specific B cells, as demonstrated by detection of circulating autoreactive anti-PC-Abs. We call this novel mechanism by which autoreactive B cells can persist by compromising allelic exclusion receptor dilution. Rescue of autoreactive PC-specific B cells would be beneficial to the host because these Abs are vital for protection against pathogens such as Streptococcus pneumoniae.

Flow cytometric measurement of fluorescence (Förster) resonance energy transfer from cyan fluorescent protein to yellow fluorescent protein using single-laser excitation at 458 nm.

Use of distinct green fluorescent protein (GFP) variants permits the study of protein–protein interactions and colocalization in viable transfected cells by fluorescence (Förster) resonance energy transfer (FRET). Flow cytometry is a sensitive method to detect FRET. However, the typical dual‐laser methods used in flow cytometric FRET assays are not generally applicable because they require a specialized krypton ultraviolet (UV) laser. The purpose of this work was to develop a flow cytometric method to detect FRET between cyan fluorescent protein (CFP; donor) and yellow fluorescent protein (YFP; acceptor) by using the 458‐nm excitation from a single tunable argon‐ion laser.

Flow cytometric assessment of the signaling status of human B lymphocytes from normal and autoimmune individuals

Abnormalities in lymphocyte signaling cascades are thought to play an important role in the development of autoimmune disease. However, the large amount of cellular material needed for standard biochemical assessment of signaling status has made it difficult to evaluate putative abnormalities completely using primary lymphocytes. The development of technology to employ intracellular staining and flow cytometry to assess the signaling status of individual cells has now made it possible to delineate the perturbations that are present in lymphocytes from patients with autoimmune disease. As an example, human B cells from the Ramos B cell line and the periphery of systemic lupus erythematosus (SLE) patients or normal nonautoimmune controls were assessed for activation of the NF-κB and mitogen activated protein kinase (MAPK) signaling cascades by intracellular multiparameter flow cytometric analysis and biochemical Western blotting. In combination with fluorochrome conjugated antibodies specific for surface proteins that define B cell subsets, antibodies that recognize activated, or phosphorylated inhibitors of κB (IκB) as well as the extracellular regulated kinase (ERK), jun N-terminal kinase (JNK) or p38 MAPKs were used to stain fixed and permeabilized human B cells and analyze them flow cytometrically. Examination of the known signaling pathways following engagement of CD40 on human B cells confirmed that intracellular flow cytometry and Western blotting equivalently assay CD154-induced phosphorylation and degradation of IκB proteins as well as phosphorylation of the MAPKs ERK, JNK and p38. In addition, B cells from the periphery of SLE patients had a more activated status immediately ex vivo as assessed by intracellular flow cytometric analysis of phosphorylated ERK, JNK and p38 when compared with B cells from the periphery of normal, nonautoimmune individuals. Together, these results indicate that multiparameter intracellular flow cytometric analysis of signaling pathways, such as the NF-κB and MAPK cascades, can be used routinely to assess the activation status of a small number of cells and thus delineate abnormalities in signaling molecules expressed in primary lymphocytes from patients with autoimmune disease.

IL-21 and BAFF/BLyS synergize in stimulating plasma cell differentiation from human marginal zone B cells as well as from circulating peripheral blood B cells from autoimmune patients

IL-21 promotes plasma cell (PC) differentiation while BAFF promotes B-cell survival. Here, we report that IL-21 synergizes with BAFF to elicit BLIMP-1 induction, PC differentiation and IgG production from a novel population of human splenic memory B cells. These human marginal zone analogue B cells are exquisitely sensitive to IL-21 and BAFF in the absence of further co-stimulation. The ability of IgG+ marginal zone analogue to respond specifically and exclusively to IL-21 and BAFF demonstrates that they are uniquely poised to respond to antigen-independent signals and differentiate into IgG-producing PC, thereby replenishing serologic memory. Importantly, peripheral blood B cells from a portion of patients with systemic lupus erythematosus and rheumatoid arthritis were highly responsive to stimulation with IL-21 and BAFF. These data suggest that IL-21 and BAFF may be capable of inducing PC differentiation from memory B cells with autoreactive specificities and thereby contribute to autoimmunity.