Igor Kuzin

Expanded CD23 + /CD21 hi B Cells in Inflamed Lymph Nodes Are Associated with the Onset of Inflammatory-Erosive Arthritis in TNF-Transgenic Mice and Are Targets of Anti-CD20 Therapy

Anti-CD20 B cell depletion therapy (BCDT) is very effective for some patients with rheumatoid arthritis (RA); however the pathogenic role of B lymphocytes in RA and the primary targets of BCDT are unknown. The human TNF transgenic (hTNF-Tg) mouse model of RA displays a chronic, progressive disease that spreads from distal to proximal joints and is generally considered to be adaptive immune system independent. We have previously reported that knee arthritis in hTNF-Tg mice is accompanied by structural and functional changes of the adjoining popliteal lymph node (PLN), detectable by contrast-enhanced magnetic resonance imaging. To better understand these changes, in this paper we show that onset of knee synovitis and focal erosions are paralleled by PLN contraction and accumulation of large numbers of B cells in the lymphatic sinus spaces within the node. Flow cytometry from TNF-Tg mice 2, 4–5, and 8–12 mo old demonstrated that B cell accumulation in the PLN follows ankle arthritis, but commences before knee disease, and involves early expansion of CD21hi, CD23+, IgMhi, CD1d+, activation marker-negative, polyclonal B cells that are found to be specifically restricted to lymph nodes draining inflamed, arthritic joints. The same B cell population also accumulates in PLNs of K/BxN mice with autoantigen-dependent arthritis. Strikingly, we show that BCDT ameliorates hTNF-Tg disease and clears follicular and CD21hi, CD23+ B cells from the PLNs. On the basis of these findings, we propose a model whereby B cells contribute to arthritis in mice, and possibly RA, by directly affecting the structure, composition, and function of joint-draining lymph nodes.

Normal Isotype Switching in B Cells Lacking the Iμ Exon Splice Donor Site: Evidence for Multiple Iμ-Like Germline Transcripts

Ig class switch recombination (CSR) in activated B cells is preceded by the generation of “switch” transcripts from the heavy chain constant region (CH) genes targeted for rearrangement. Switch transcripts include a sterile “I” exon spliced onto the first CH exon. Targeted mutations disrupting the expression or splicing of I exons severely hamper CSR to all tested CH loci, except μ. However, all μ switch transcript mutations tested so far have left the Iμ exon splice donor site intact. To test the possibility that the residual CSR activity in Iμ mutants could be due to splicing of a truncated Iμ exon, we generated new mutants specifically lacking the Iμ splice donor site. Surprisingly, normal CSR was observed in the Iμ splice donor mutants even in the absence of detectable spliced Iμ transcripts. In a search for potential alternative sources of switch-like transcripts in the μ locus, we identified two novel exons which map just upstream of the Sμ region and splice onto the Cμ1 exon. Their expression is detectable from early B cell developmental stages, and, at least in hybridomas, it does not require the Eμ enhancer. These studies highlight a unique structure for the μ locus I exon region, with multiple nested switch transcript-like exons mapping upstream of Sμ. We propose that all of these transcripts directly contribute to μ class switching activity.

Resistance of cellular membrane antigens to solubilization with Triton X-100 as a marker of their association with lipid rafts—analysis by flow cytometry

Lipid rafts are specialized micro-domains of the plasma membrane enriched in glycosphingolipid and cholesterol that play important role in signal transduction, membrane trafficking, and cell adhesion. A distinct feature of lipid rafts is their resistance to solubilization with non-ionic detergent Triton X-100 (TX-100). In this study, we used flow cytometry to evaluate TX-100 resistance of 74 cell membrane molecules expressed on normal human peripheral blood lymphocytes (PBL), thymocytes, and 12 lymphoid cell lines. Resistance of membrane molecules to solubilization with TX-100 was determined by comparing the intensities of fluorescence of cells treated with TX-100 or left untreated. The majority of antigens analyzed were easily solubilized with TX-100 that resulted in decreased fluorescence intensity. However, a group of antigens showed TX-100 resistance in the range of 20-100%. These included all glycosylphosphatidylinositol (GPI)-anchored antigens under study, as well as some glycolipid and trans-membrane antigens. With the few exceptions, antigen resistance to solubilization with TX-100 was stable parameter, which did not depend on cell type in which it was analyzed. There was a good correspondence between the antigens showing resistance to solubilization with TX-100 as evaluated by our flow cytometry method, and the antigens that were previously demonstrated in detergent-resistant membranes using a more standard method of physical fractionation. Taken collectively, our data suggest that flow cytometry is a useful method for rapid evaluation of the possible association of a membrane antigen with lipid rafts.

The biologic properties of leukemias arising from BCR/ABL-mediated transformation vary as a function of developmental origin and activity of the p19ARF gene

Recent reports have shown that upon expression of appropriate oncogenes, both stem cells and more differentiated progenitor populations can serve as leukemia-initiating cells. These studies suggest that oncogenic mutations subvert normal development and induce reacquisition of stem-like features. However, no study has described how specific mutations influence the ability of differentiating cell subsets to serve as leukemia-initiating cells and if varying such cellular origins confers a functional difference. We have examined the role of the tumor suppressor gene p19(ARF) in a murine model of acute lymphoblastic leukemia and found that loss of p19(ARF) changes the spectrum of cells capable of tumor initiation. With intact p19(ARF), only hematopoietic stem cells (HSCs) can be directly transformed by BCR/ABL expression. In a p19(ARF)-null genetic background expression of the BCR/ABL fusion protein renders functionally defined HSCs, common lymphoid progenitors (CLP), and precursor B-lymphocytes competent to generate leukemia stem cells. Furthermore, we show that leukemias arising from p19(ARF)-null HSC versus pro-B cells differ biologically, including relative response to drug insult. Our observations elucidate a unique mechanism by which heterogeneity arises in tumor populations harboring identical genetic lesions and show that activity of p19(ARF) profoundly influences the nature of tumor-initiating cells during BCR/ABL-mediated leukemogenesis.

CD23 + CD21 high CD1d high B Cells in Inflamed Lymph Nodes Are a Locally Differentiated Population with Increased Antigen Capture and Activation Potential

CD23+CD21highCD1dhigh B cells in inflamed nodes (Bin cells) accumulate in the lymph nodes (LNs) draining inflamed joints of the TNF-α–transgenic mouse model of rheumatoid arthritis and are primarily involved in the significant histological and functional LN alterations that accompany disease exacerbation in this strain. In this study, we investigate the origin and function of Bin cells. We show that adoptively transferred GFP+ sorted mature follicular B (FoB) cells home preferentially to inflamed LNs of TNF-α–transgenic mice where they rapidly differentiate into Bin cells, with a close correlation with the endogenous Bin fraction. Bin cells are also induced in wild-type LNs after immunization with T-dependent Ags and display a germinal center phenotype at higher rates compared with FoB cells. Furthermore, we show that Bin cells can capture and process Ag-immune complexes in a CD21-dependent manner more efficiently than can FoB cells, and they express greater levels of MHC class II and costimulatory Ags CD80 and CD86. We propose that Bin cells are a previously unrecognized inflammation-induced B cell population with increased Ag capture and activation potential, which may facilitate normal immune responses but may contribute to autoimmunity when chronic inflammation causes their accumulation and persistence in affected LNs.

Chronic exposure to tumor necrosis factor in vivo induces hyperalgesia, upregulates sodium channel gene expression and alters the cellular electrophysiology of dorsal root ganglion neurons

The goal of these studies was to investigate the links between chronic exposure to the pro-inflammatory cytokine tumor necrosis factor (TNF), hyperalgesia and the excitability of dorsal root ganglion (DRG) sensory neurons. We employed transgenic mice that constitutively express TNF (TNFtg mice), a well-established model of chronic systemic inflammation. At 6 months of age, TNFtg mice demonstrated increased sensitivity to both mechanical and thermal heat stimulation relative to aged-matched wild-type controls. These increases in stimulus-evoked behaviors are consistent with nociceptor sensitization to normal physiological stimulation. The mechanisms underlying nociceptor sensitization were investigated using single-cell analysis to quantitatively compare gene expression in small-diameter (<30μm) DRG neurons. This analysis revealed the upregulation of mRNA encoding for tetrodotoxin-resistant (TTX-R) sodium (Na+) channels (Nav1.8, Nav1.9), Na+ channel β subunits (β1-β3), TNF receptor 1 (TNFR1) and p38α mitogen-activated protein kinase in neurons of TNFtg mice. Whole-cell electrophysiology demonstrated a corresponding increase in TTX-R Na+ current density, hyperpolarizing shifts in activation and steady-state inactivation, and slower recovery from inactivation in the TNFtg neurons. Increased overlap of activation and inactivation in the TNFtg neurons produces inward Na+ currents at voltages near the resting membrane potential of sensory neurons (i.e. window currents). The combination of increased Na+ current amplitude, hyperpolarized shifts in Na+ channel activation and increased window current predicts a reduction in the action potential threshold and increased firing of small-diameter DRG neurons. Together, these data suggest that increases in the expression of Nav1.8 channels, regulatory β1 subunits and TNFR1 contribute to increased nociceptor excitability and hyperalgesia in the TNFtg mice.

Acute lymphoblastic leukemia cells that survive combination chemotherapy in vivo remain sensitive to allogeneic immune effects

Allogeneic hematopoietic stem cell transplantation is often performed for patients with acute lymphoblastic leukemia (ALL) whose disease has relapsed after chemotherapy treatment. However, graft versus leukemia (GVL) effects in ALL are generally weak and the mechanisms of this weakness are unknown. These studies tested the hypothesis that ALL cells that have survived conventional chemotherapy in vivo acquire relative resistance to the allogeneic GVL effect. C57BL/6 mice were injected with murine pre-B ALL lines driven by human mutations and then were treated with combination chemotherapy. ALL cells surviving therapy were analysed in vitro and in vivo for acquisition of resistance to chemotherapy, radiation, cytolytic T cells, NK cells, LAK cells and cytokines. In vivo drug treatment did lead to leukemia population with more rapid proliferation and also decreased sensitivity to vincristine, doxorubicin and radiation. However, drug treatment did not produce ALL populations that were less sensitive to GVL effects in vitro or in vivo.

Long-term immunologically competent human peripheral lymphoid tissue cultures in a 3D bioreactor.

Peripheral lymphoid organs (PLOs), the primary sites of development of adaptive immune responses, display a complex structural organization reflecting separation of cellular subsets (e.g., T and B lymphocytes) and functional compartments which is critical for immune function. The generation of in vitro culture systems capable of recapitulating salient features of PLOs for experimental, biotechnological, and clinical applications would be highly desirable, but has been hampered so far by the complexity of these systems. We have previously developed a three‐dimensional bioreactor system for long‐term, functional culture of human bone marrow cells on macroporous microspheres in a packed‐bed bioreactor with frequent medium change. Here we adapt the same system for culture of human primary cells from PLOs (tonsil) in the absence of specific exogenous growth factors or activators. Cells in this system displayed higher viability over several weeks, and maintain population diversity and cell surface markers largely comparable to primary cells. Light microscopy showed cells organizing in large diverse clusters within the scaffold pores and presence of B cell‐enriched areas. Strikingly, these cultures generated a significant number of antibody‐producing B cells when challenged with a panel of diverse antigens, as expected from a lymphoid tissue. Thus the three‐dimensional tonsil bioreactor culture system may serve as a useful model of PLOs by recapitulating their structural organization and function ex vivo. Biotechnol. Bioeng. 2011; 108:1430–1440.

Brief Report: Treatment of Tumor Necrosis Factor–Transgenic Mice With Anti–Tumor Necrosis Factor Restores Lymphatic Contractions, Repairs Lymphatic Vessels, and May Increase Monocyte/Macrophage Egress

Recent studies have demonstrated that there is an inverse relationship between lymphatic egress and inflammatory arthritis in affected joints. As a model, tumor necrosis factor (TNF)–transgenic mice develop advanced arthritis following draining lymph node (LN) collapse, and loss of lymphatic contractions downstream of inflamed joints. It is unknown if these lymphatic deficits are reversible. This study was undertaken to test the hypothesis that anti‐TNF therapy reduces advanced erosive inflammatory arthritis, associated with restoration of lymphatic contractions, repair of damaged lymphatic vessels, and evidence of increased monocyte egress.

Requirement for Enhancer Specificity in Immunoglobulin Heavy Chain Locus Regulation

The intronic Eμ enhancer has been implicated in IgH locus transcription, VDJ recombination, class switch recombination, and somatic hypermutation. How Eμ controls these diverse mechanisms is still largely unclear, but transcriptional enhancer activity is thought to play a central role. In this study we compare the phenotype of mice lacking the Eμ element (ΔEμ) with that of mice in which Eμ was replaced with the ubiquitous SV40 transcriptional enhancer (SV40eR mutation) and show that SV40e cannot functionally complement Eμ loss in pro-B cells. Surprisingly, in fact, the SV40eR mutation yields a more profound defect than ΔEμ, with an almost complete block in μ0 germline transcription in pro-B cells. This active transcriptional suppression caused by enhancer replacement appears to be specific to the early stages of B cell development, as mature SV40eR B cells express μ0 transcripts at higher levels than ΔEμ mice and undergo complete DNA demethylation at the IgH locus. These results indicate an unexpectedly stringent, developmentally restricted requirement for enhancer specificity in regulating IgH function during the early phases of B cell differentiation, consistent with the view that coordination of multiple independent regulatory mechanisms and elements is essential for locus activation and VDJ recombination.