B-cell Follicles Research Articles

Deficient CCR7 signaling promotes TH2 polarization and B‐cell activation in vivo

The chemokine receptor CCR7 has a central role in regulating homing and positioning of T cells and DCs to lymph nodes (LNs) and participates in T-cell development and activation. In this study, we addressed the role of CCR7 signaling in T(H) 2 polarization and B-cell activation. We provide evidence that the lack of CCR7 drives the capacity of naïve CD4(+) T cells to polarize toward T(H) 2 cells. This propensity contributes to a lymph node environment in CCR7-deficent mice characterized by increased expression of IL-4 and increased frequency of T(H) 2 cells. We show that elevated IL-4 levels lead to B-cell activation characterized by up-regulated expression of MHC class II, CD23 and CD86. Activated B cells are in turn highly efficient in presenting antigen to CD4(+) T cells and thus potentially contribute to the T(H) 2 microenvironment. Taken together, our results support the idea of a CCR7-dependent patterning of T(H) 2 responses, with absent CCR7 signaling favoring T(H) 2 polarization, dislocation of T helper cells into the B-cell follicles and, as a consequence, B-cell activation.

In this issue

In this issue

Cover Picture: Eur. J. Immunol. 12/11

The cover image shows immunohistological analysis of a spleen section from QMxB6 mice 96 hours after immunization with NP-Ficoll. IgD staining (brown) determines the B-cell follicles and NP-staining (blue) the antibody-producing cells; plasmablasts (dark blue regions) are present in the red pulp, and germinal centres (pale blue) can be seen in the follicles; the T zones (which lack B cells) remain unstained. The image is kindly provided by Jennifer Marshall and relates to the study Marshall et al. (pp. 3506–3512) in which it is shown that Ig class-switch recombination during extrafollicular responses occurs at the B blast rather than the plasmablast stage.

Epstein‐barr virus in myasthenia gravis thymus: A matter of debate

Epstein-Barr Virus in Myasthenia Gravis Thymus: A Matter of Debate Barbara Serafini, PhD, Paola Cavalcante, PhD, Pia Bernasconi, PhD, Francesca Aloisi, PhD, and Renato Mantegazza, MD In this issue, 2 papers report absence/very low levels of EpsteinBarr virus (EBV) in early-onset myasthenia gravis (EOMG) thymuses. In contrast, we recently showed active EBV infection in all myasthenic thymuses analyzed. These discrepancies are unlikely due to sampling from patients with different clinical features as all or most patients tested had EOMG. The possible effect of treatment is difficult to discuss as the patients analyzed by Kakalacheva and colleagues apparently did not require immunosuppression (or any other treatment) before thymectomy, even if performed up to 3 years after onset; they therefore probably represent selected patients characterized by a mild phenotype. As neither description of treatment, nor clear indication of the patients’ status is given in the report by Meyer and colleagues, where the cohort is apparently quite inhomogeneous, we cannot comment adequately on this study. Rather, the discrepancies could be explained by differences in tissue processing (we analyzed not only paraffin samples as did the 2 other groups, but also fixed frozen and snap frozen samples) and use of different procedures and tools. Both studies have used in situ hybridization and immunohistochemistry as well as automated immunostaining systems optimized for viral detection in EBV-associated tumors, but which may not be sensitive enough to detect EBV in nonmalignant pathological conditions. Thus, Meyer and colleagues report that only rare cells expressing BZLF1 are generally present in tonsil from infectious mononucleosis patients. In contrast, using a similarly processed tissue (kindly provided by Prof. G. Niedobitek) and the same monoclonal antibody (BZ-1, kind gift of Prof. J. Middeldorp) but different antigen-unmasking and detection procedures, we found numerous BZLF1þ cells outside tonsillar B-cell follicles (Fig), suggesting that our immunohistochemical protocol has greater sensitivity, allowing detection of BZLF1þ cells in myasthenic thymus (Fig). In accordance with our findings, Kakalacheva and colleagues see rare cells expressing EBV nuclear antigen 2 (EBNA2), which indicates highly dysregulated EBV infection. The finding of very low to undetectable levels of EBV DNA in MG thymuses analyzed by Kakalacheva and colleagues suggests that their real-time polymerase chain reaction (PCR) may be less sensitive than the PCR method we used. To solve these issues, a correct approach would be exchanging optimally processed thymic samples and finding an agreement on the best procedures to analyze EBV infection in thymus. Finally, the finding by Kakalacheva and colleagues that EBV-specific T-cell and B-cell responses are unchanged in patients with MG compared to controls should be interpreted with caution given the small number of samples analyzed. To clarify this issue, larger, longitudinal serological studies and analyses of CD8 T-cell responses to specific EBV lytic antigens using pentamer technology should be performed.

Mantle cell lymphoma: recent insights into pathogenesis, clinical variability, and new diagnostic markers

Mantle cell lymphoma (MCL; previously called centrocytic lymphoma or lymphocytic lymphoma of intermediate differentiation) is a distinct subtype of B-cell lymphoma, accounting for approximately 3%-10% of all lymphoma diagnoses. The name refers to the growth pattern in early disease presentation resembling the normal mantle zone that surrounds the germinal center of the B-cell follicle. The hallmark of MCL is the t(11;14)(q13;q32), resulting in aberrant expression of the CCND1 gene and expression of cyclin D1 in the tumor cells. Expression and genomic profiling of MCL have provided new insight into the pathogenesis and will be summarized in this review. Pitfalls in the differential diagnosis versus B-cell chronic lymphocytic leukemia, B-cell prolymphocytic leukemia, cyclin D1-positive diffuse large B-cell lymphoma, hairy cell leukemia, and plasma cell tumors will be discussed, including the usefulness new diagnostic markers SOX11 and CD200. In situ MCL, MCL with an indolent clinical course, and cyclin D1-negative MCL are other topics of this review.

Follicular helper T cells: implications in neoplastic hematopathology

A distinct subset of T helper cells, named follicular T helper cells (T(FH), has been recently described. T(FH) cells are characterized by their homing capacities in the germinal centers of B-cell follicles where they interact with B cells, supporting B-cell survival and antibody responses. T(FH) cells can be identified by the expression of several markers including the chemokine CXCL13, the costimulatory molecules PD1 and inducible costimulator, and the transcription factor BCL6. They appear to be relevant markers for the diagnosis of angioimmunoblastic T-cell lymphoma (AITL) and have helped to recognize subsets of peripheral T-cell lymphoma, not otherwise specified, with nodal or cutaneous presentation expressing T(FH) antigens that might be related to AITL. In B-cell neoplasms, T(FH) cells are present within the microenvironment of nodular lymphocyte-predominant Hodgkin lymphoma and follicular lymphoma, where they likely support the growth of neoplastic germinal center-derived B cells. Interestingly, the amount of PD1+ cells in the neoplastic follicles might have a favorable impact on the outcome of follicular lymphoma patients. Altogether, the availability of antibodies directed to T(FH)-associated molecules has important diagnostic and prognostic implications in hematopathology. In addition, T(FH) cells could represent interesting targets in T(FH)-derived lymphomas such as AITL, or in some B-cell neoplasms where they act as part of the tumor microenvironment.

Characterization of Apoptosis Pathways (Intrinsic and Extrinsic) in Lymphoid Tissues of Calves Inoculated with Non-cytopathic Bovine Viral Diarrhoea Virus Genotype-1

Previous studies have shown that activation of effector caspase-3 is associated with the apoptosis of lymphocytes occurring during infection with bovine viral diarrhoea virus (BVDV); however, the regulation of the apoptosis pathways that induce cell death via activation of effector caspase-3 has not yet been clarified. The aim of this study was to examine immunohistochemically the expression of cleaved caspase (CCasp)-8 (initiator caspase of the extrinsic pathway), CCasp9 (initiator caspase of the intrinsic pathway) and Bcl-2 (an anti-apoptotic marker) in gut-associated lymphoid tissue (GALT) of the ileum from calves inoculated with a non-cytopathic strain of BVDV genotype-1. CCasp8 had similar expression to that of CCasp3. In interfollicular T-cell areas there was moderate apoptosis and evidence of moderate activation of initiator caspase-8. In B-cell follicles there was marked lymphocyte apoptosis and evidence of intense caspase-8 activation, highlighting the potentially major role of the extrinsic pathway in lymphocyte apoptosis in the GALT during BVDV infection. Additionally, there was a significant decrease in the number of CCasp9(+) cells from the start of the experiment and this was linked to inactivation of caspase-9. Therefore, the intrinsic pathway may play only a minor role in the induction of lymphocyte apoptosis. Finally, the observed overexpression of Bcl-2 protein could play a major role in protecting lymphocytes in the T-cell areas against apoptosis, while low levels of Bcl-2 expression could be associated with the follicular lymphocyte apoptosis occurring during BVDV infection.

Stromal cells directly mediate the re-establishment of the lymph node compartments after transplantation by CXCR5 or CCL19/21 signalling

Lymph nodes (LN) are highly organized and have characteristic compartments. Destruction of these compartments leads to an inability to fulfil their immunological function. However, it is not yet clearly understood which mechanisms are involved in the development and maintenance of this organization. After transplantation of LN into the mesentery, the LN regenerate to fully functional LN. In this study, the question was addressed, how stromal cells in the B-cell follicles (follicular dendritic cells), which were identified by CD21/CD35, and stromal cells in the T-cell area (gp38+ cells) are involved via chemokine signalling. The gp38+ cells and CD21/CD35+ cells were detected in the transplanted LN (EGFP, plt/plt and CXCR5(-/-) mice) over a period of 8 weeks to analyse their competence to reconstruct the compartmental organization. The presence of gp38+ cells was stable during regeneration and these cells reconstructed the T-cell area within 4 weeks. After transplantation of plt/plt LN CCL19/CCL21 expression was observed leading to partial restoration of the T-cell area. In contrast, there were changes in the presence and morphology of CD21/CD35+ cells within the B-cell area during reconstruction, which was dependent on the presence of B cells and CXCL13/CXCR5 signalling. Hence, CD21/CD35+ cells and gp38+ cells are involved in the establishment of the compartmental organization of lymph nodes but using different ways to recruit lymphocytes via chemokine signalling.

Multiple sclerosis: BAFF and CXCL13 in cerebrospinal fluid

Background: There is increasing evidence of B-cell involvement in the pathogenesis of multiple sclerosis (MS). B-cell activating factor (BAFF) has an essential role in B-cell homeostasis. The chemokine CXCL13 has an important role in the formation and maintenance of B-cell follicles. Objective: To measure BAFF and CXCL13 levels in the cerebrospinal fluid (CSF) of patients with MS compared to patients with other neurological diseases. Methods: Cytokine/chemokine levels were measured by an enzyme-linked immunosorbent assay (ELISA). Results: In MS patients, BAFF levels were highest in patients with secondary progressive disease, and were higher during relapse in patients with relapsing–remitting and secondary progressive disease. CXCL13 levels were also higher during relapse. There was a positive correlation between CXCL13 and the IgG index, and an inverse correlation between BAFF and the IgG index. The implications of this finding are discussed. Conclusion: During relapse, we found various positive correlations between BAFF, CXCL13 and the cytokines IL-6 and IL-10. These findings show that molecules that are essential for B-cell recruitment, survival, maturation and function may be working in concert to affect B-cell homeostasis in MS and contribute to the pathophysiology of the disease.

Dysregulated Generation of Follicular Helper T Cells in the Spleen Triggers Fatal Autoimmune Hepatitis in Mice

To clarify mechanisms involved in the development of autoimmune hepatitis (AIH), we recently developed a mouse model of spontaneous AIH by inducing a concurrent loss of Foxp3(+) regulatory T cells and programmed cell death 1 (PD-1)-mediated signaling. Fatal AIH in these mice was characterized by severe T-cell infiltration and huge production of antinuclear antibodies (Abs). This study aims to identify induction sites, responsible T-cell subsets, and key molecules for induction of AIH. To develop the mouse model of AIH, neonatal thymectomy (NTx) was performed on PD-1-deficient (PD-1(-/-)) mice. We then conducted neonatal splenectomy or in vivo administration of Abs to cytokines, chemokines, or cell-surface molecules. In NTx-PD-1(-/-) mice, either neonatal splenectomy or in vivo CD4(+) T-cell depletion suppressed CD4(+) and CD8(+) T-cell infiltration in the liver. In the induction phase of AIH, splenic CD4(+) T cells were localized in B-cell follicles with huge germinal centers and showed the Bcl6(+) inducible costimulator (ICOS)(+) interleukin (IL)-21(+) IL-21 receptor (IL-21R)(+) follicular helper T (T(FH)) cell phenotype. Blocking Abs to ICOS or IL-21 suppressed T(FH)-cell generation and induction of AIH. In addition, IL-21 produced by T(FH) cells drove CD8(+) T-cell activation. Splenic T(FH) cells and CD8(+) T cells expressed CCR6, and CCL20 expression was elevated in the liver. Administration of anti-CCL20 suppressed migration of these T cells to the liver and induction of AIH. Dysregulated T(FH) cells in the spleen are responsible for the induction of fatal AIH, and CCR6-CCL20 axis-dependent migration of splenic T cells is crucial to induce AIH in NTx-PD-1(-/-) mice.

Splenic Marginal Zone Lymphoma in 5 Dogs (2001–2008)

Splenic marginal zone lymphomas (MZL) in dogs arise from the marginal zone of B-cell follicles and can progress slowly. To describe clinical features, treatment, and outcome of dogs with splenic MZL. Five dogs with naturally occurring MZL. Clinical, laboratory, and follow-up data were retrospectively reviewed. Diagnosis was based on clinical, histopathological, and immunophenotypic features. All dogs had stage IV disease; among them, 2 were symptomatic (substage "b") because of splenic rupture. Four dogs underwent splenectomy and adjuvant doxorubicin, and 1 dog underwent surgery only. Three out of the 4 dogs treated with surgery and chemotherapy died of causes unrelated to lymphoma, after 760, 939, and 1,825 days, whereas the remaining dog was alive and in complete remission after 445 days. The dog not receiving any adjuvant treatment had recurrence of the tumor after 180 days. Splenic MZL appears indolent and can benefit from splenectomy, with or without systemic chemotherapy.

Cellular source and molecular form of TNF specify its distinct functions in organization of secondary lymphoid organs

AbstractSecondary lymphoid organs provide a unique microenvironment for generation of immune responses. Using a cell type–specific conditional knockout approach, we have dissected contributions of tumor necrosis factor (TNF) produced by B cells (B-TNF) or T cells (T-TNF) to the genesis and homeostatic organization of secondary lymphoid organs. In spleen, lymph nodes and Peyer patches, the cellular source of TNF, and its molecular form (soluble versus membrane-bound) appeared distinct. In spleen, in addition to major B-TNF signal, a complementary T-TNF signal contributed to the microstructure. In contrast, B-TNF predominantly controlled the development of follicular dendritic cells and B-cell follicles in Peyer patches. In lymph nodes, cooperation between TNF expressed by B and T cells was necessary for the maintenance of microarchitecture and for generation of an efficient humoral immune response. Unexpectedly, soluble but not membrane TNF expressed by B cells was essential for the organization of the secondary lymphoid organs. Thus, the maintenance of each type of secondary lymphoid organ is orchestrated by distinct contributions of membrane-bound and soluble TNF produced by B and T lymphocytes.

Permissive environment for B‐cell maturation in myositis muscle in the absence of B‐cell follicles

Myositis muscle contains antigen-matured B-cells and plasma cells. Myositis muscle biopsy specimens were examined for nodular collections of T-cells, B-cells, myeloid dendritic cells, plasma cells, and follicular dendritic cells. Immunoglobulin and B-cell-activating factor (BAFF) transcripts were quantitated. Laser-capture microdissection was used to isolate single plasma cells, and their immunoglobulin transcripts were sequenced. Dense inflammatory infiltrates contained histological elements of ectopic lymphoid tissue but not B-cell follicles. Immunoglobulin transcript sequence analysis demonstrated spatially distributed, clonally related B-cells and plasma cells, suggesting local maturation of B-cells into plasma cells in myositis muscle. Regions of dense cellular infiltrates in myositis muscle are sometimes areas of B-cell maturation into antibody-producing plasma cells. An atypical lymphoid histology, lacking concentrated collections of germinal-center-like B-cell follicles, is capable of antigen-stimulated clonal maturation of antibody-producing plasma cells.

Detection of Epstein–Barr virus and B-cell follicles in the multiple sclerosis brain: what you find depends on how and where you look

Sir, The research article by Willis et al . (2009) and the letter to the editor by Peferoen et al . (2010) challenge our earlier finding that infection with Epstein–Barr virus is a common feature in the multiple sclerosis brain, provided that autopsy samples containing immune infiltrates are analysed (Serafini et al ., 2007). Both groups also fail to detect meningeal B-cell follicles, the structures we first found in a proportion of patients with secondary progressive multiple sclerosis (Serafini et al ., 2004; Magliozzi et al ., 2007; Howell et al ., 2009) and then identified as main, though not unique, intracerebral sites of Epstein–Barr virus persistence and reactivation (Serafini et al ., 2007). Based on their inability to find follicle-like structures in 94 multiple sclerosis brain specimens collected by the Netherlands Brain Bank and in a small number of specimens obtained from the UK Multiple Sclerosis Tissue Bank, Peferoen et al . (2010) ‘question the importance and significance of ectopic B cell follicles as a pathogenic feature of multiple sclerosis’, a scenario we proposed by showing that these structures are associated with more extensive cortical pathology and aggressive clinical course (Magliozzi et al ., 2007; Howell et al ., 2009). Inability to detect meningeal B-cell follicles in brain tissue of the Dutch Multiple Sclerosis cohort was recently reported also by Kooi et al . (2009) and Torkildsen et al . (2010). Given the mounting serological evidence (Ascherio and Munger, 2007; Farrell et al ., 2009; Sundstrom et al ., 2009; Levin et al ., 2010; Lunemann et al ., 2010) and immunological (Jilek et al ., 2008; Lunemann and Munz, 2009; Pender, 2009; Jaquiery et al ., 2010) of an association between Epstein–Barr virus and multiple sclerosis, understanding …

Characterization of ex vivo healthy human axillary lymph nodes with high resolution 7 Tesla MRI

ObjectiveTo characterize ex vivo healthy human axillary lymph nodes on 7 Tesla MRI and to correlate the findings with pathological analysis as a first step towards non-invasive staging of breast cancer patients in the future.MethodsFour axillary lymph node dissection (ALND) specimens from 2 autopsy patients, who had no cancer, were examined on a clinical 7 Tesla MRI system. For morphological analysis a 3D T1-weighted fat-suppressed fast-field-echo [isotropic resolution 180 μm] was acquired. For quantitative analyses 2D T1-, 3D T2-, T2*- and diffusion-weighted images were acquired. The ALNDs were mapped and stained for precise correlation of MRI to pathology. Nodes were sliced in 3 μm sections, Haematoxylin & Eosin stained, and examined by an experienced pathologist.ResultsMRI detected all 45 nodes and 6 additional nodes that were not detected at pathological analysis. B-cell follicles, efferent- and afferent lymph vessels and blood vessels were identified. Mean T1, T2, T2*, ADC values (± standard deviation) were 944 ± 113 ms, 32 ± 2 ms, 16 ± 2 ms, 0.39 ± 0.09·10−3 mm2/s, respectively.Conclusions7 Tesla MRI of ex vivo human axillary lymph nodes correlated well with pathology. MRI detected all nodes present in the specimens and allowed visualization of fine structural detail. Pathology-correlated quantitative MRI data are presented.

Meningeal T cells associate with diffuse axonal loss in multiple sclerosis spinal cords

A link between diffuse axonal loss and diffuse inflammation has been established in the brain of patients with progressive multiple sclerosis (MS). In the present paper, we sought to determine whether such a link could be similarly demonstrated in the spinal cord of patients with progressive MS. A neuropathological quantitative assessment of inflammation and axonal loss was performed in the cervical spinal cord of 18 patients with progressive MS and 5 control subjects. As previously reported, we found a mean 25% decrease of axonal density in the normal-appearing white matter (NAWM) of MS versus control spinal cords. T-cell perivascular infiltrates were rare, but a robust diffuse inflammation was observed in both the normal-appearing parenchyma and the meninges. The extent of diffuse axonal loss in the NAWM correlated with both the density of major histocompatibility complex (MHC) class II(+) microglia in the NAWM and, surprisingly, the density of CD3(+) T cells in the meninges. Interestingly, close interactions between T cells and MHC class II(+) macrophages were observed in the meninges of spinal cords from MS patients. Recent studies assigned a major role to meningeal B-cell follicles in the pathophysiology of secondary progressive MS. The present work also emphasizes the link between meningeal inflammation and parenchymal lesions and points to a specific role exerted by both meningeal T cells and activated microglia in diffuse axonal loss in the spinal cord.

Epstein-Barr Virus Latent Infection and BAFF Expression in B Cells in the Multiple Sclerosis Brain: Implications for Viral Persistence and Intrathecal B-Cell Activation

A cardinal feature of multiple sclerosis (MS) is the persistent intrathecal synthesis of antibodies. Our previous finding that a large fraction of B cells infiltrating the MS brain are infected with Epstein-Barr virus (EBV) raises the possibility that this virus, because of its ability to establish a latent infection in B cells and interfere with their differentiation, contributes to B-cell dysregulation in MS. The aim of this study was to gain further insight into EBV latency programs and their relationship to B-cell activation in the MS brain. Immunohistochemical analysis of postmortem MS brain samples harboring large EBV deposits revealed that most B cells in white matter lesions, meninges, and ectopic B-cell follicles are CD27+ antigen-experienced cells and coexpress latent membrane protein 1 and latent membrane protein 2A, 2 EBV-encoded proteins that provide survival and maturation signals to B cells. By combining laser-capture microdissection with preamplification reverse transcription-polymerase chain reaction techniques, EBV latency transcripts (latent membrane protein 2A, EBV nuclear antigen 1) were detected in all MS brain samples analyzed. We also found that B cell-activating factor of the tumor necrosis factor family is expressed in EBV-infected B cells in acute MS lesions and ectopic B-cell follicles. These findings support a role for EBV infection in B-cell activation in the MS brain and suggest that B cell-activating factor of the tumor necrosis factor family produced by EBV-infected B cells may contribute to this process resulting in viral persistence and, possibly, disruption of B-cell tolerance.

Global lymphoid tissue remodeling during a viral infection is orchestrated by a B cell–lymphotoxin-dependent pathway

AbstractAdaptive immune responses are characterized by substantial restructuring of secondary lymphoid organs. The molecular and cellular factors responsible for virus-induced lymphoid remodeling are not well known to date. Here we applied optical projection tomography, a mesoscopic imaging technique, for a global analysis of the entire 3-dimensional structure of mouse peripheral lymph nodes (PLNs), focusing on B-cell areas and high endothelial venule (HEV) networks. Structural homeostasis of PLNs was characterized by a strict correlation between total PLN volume, B-cell volume, B-cell follicle number, and HEV length. After infection with lymphocytic choriomeningitis virus, we observed a substantial, lymphotoxin (LT) β-receptor–dependent reorganization of the PLN microarchitecture, in which an initial B-cell influx was followed by 3-fold increases in PLN volume and HEV network length on day 8 after infection. Adoptive transfer experiments revealed that virus-induced PLN and HEV network remodeling required LTα1β2-expressing B cells, whereas the inhibition of vascular endothelial growth factor-A signaling pathways had no significant effect on PLN expansion. In summary, lymphocytic choriomeningitis virus-induced PLN growth depends on a vascular endothelial growth factor-A–independent, LT- and B cell–dependent morphogenic pathway, as revealed by an in-depth mesoscopic analysis of the global PLN structure.

Visualizing influenza virus capture in the lymph node following vaccination

Visualizing influenza virus capture in the lymph node following vaccination

Multiple checkpoints keep follicular helper T cells under control to prevent autoimmunity

Follicular helper T (Tfh) cells select mutated B cells in germinal centres, which can then differentiate into long-lived high affinity memory B cells and plasma cells. Tfh cells are regulated by a unique molecular programme orchestrated by the transcriptional repressor Bcl6. This transcription factor turns down expression of multiple genes, including transcriptional regulators of other T helper lineages and a vast amount of microRNAs. This enables Tfh cells to express a suite of chemokine receptors, stimulatory ligands and cytokines that enable migration into B-cell follicles, and provision of effective help to B cells. Not surprisingly, dysregulation of this powerful helper subset can lead to a range of autoantibody-mediated diseases; indeed, aberrant accumulation of Tfh cells has been linked with systemic lupus erythematosus, Sjogren's disease and autoimmune arthritis. Here we dissect multiple checkpoints that operate throughout Tfh cell development and maturation to maintain immunological tolerance while mounting robust and long-lasting antibody responses.