Follicular Mantle Zone Research Articles

Mantle Cell Lymphoma- A Rare Occurrence: Case Report

Mantle Cell Lymphoma (MCL) is a relatively uncommon yet distinct type of malignant lymphoma whose clinical and pathological characterization has been limited by the small numbers of cases published to date. It comprises 3-7% of all Hodgkin’s lymphoma cases, with a median diagnosis age of 68 years. Based on immunophenotypic and molecular data, MCL is a neoplasm composed of cells resembling those residing in follicular mantle zones. Although MCL usually presents as advanced disease some patients may present with more limited (stages I-I) disease. We present the case of a 41-year-old male who exhibited generalized lymphadenopathy and hepatosplenomegaly. Peripheral smear showed absolute lymphocytosis and the bone marrow biopsy and flow cytometry identified the condition as mantle cell lymphoma. The patient’s management required a multidisciplinary approach, incorporating haematological, molecular, and genetic evaluations. Treatment included hyper-CVAD chemotherapy. Proteasome inhibitors (Bortezomib), mTOR inhibitors (Temsirolimus), and immunomodulatory drugs (Lenalidomide) have recently been added to the treatment options in MCL.This case underscores the significance of comprehensive evaluation and customized therapeutic strategies and re looking an old diagnosis in managing complex haematological disorders.

Histopathological staging and differential diagnosis of marginal zone lymphoma of gastric mucosa-associated lymphoid tissue

The purpose of this study was to explore the histopathological staging and differential diagnosis of marginal zone lymphoma in gastric mucosa-associated lymphoid tissue (MALT lymphoma). We performed detailed histomorphology and immunohistochemistry investigations as well as genetic testing on endoscopic biopsy and endoscopic mucosal resection specimens from 18 patients with gastric MALT lymphoma. We found that gastric MALT lymphoma typically begins as a small, isolated area outside the lymphoid follicular mantle zone or proliferates in a multifocal, patchy manner, gradually spreads to the interfollicular zone, forming diffuse proliferation, invades the gastric mucosal glands, and infiltrates or proliferates into the center of peripheral reactive lymphoid follicles. Abnormally proliferating lymphocytes invade the surrounding lymphoid follicles, resulting in damage, atrophy, and disappearance of their normal follicles as well as of the gastric mucosa glands, forming diffuse proliferation. Redifferentiation and proliferation lead to the transformation of lymphocytes; that is, MALT transitions into highly invasive lymphoma. Based on our findings in this study, we propose the following five stages in the process of development and progression of gastric MALT lymphoma: the stage of cell proliferation outside the lymphoid follicular mantle zone; the stage of heterogeneous proliferative lymphoepithelial lesion; the stage of reactive lymphoid follicular implantation; the stage of lymphoid follicular clonal proliferation; and the stage of MALT transforming into highly invasive lymphoma. We examined the differential diagnosis of histopathological features at each stage. The clinicopathological staging of gastric MALT lymphoma can help clinicians provide accurate treatment and track malignant cell transformation, thus playing a significant role in controlling its development and progression.

CD40 Signaling Promotes CXCR5 Expression in B Cells via Noncanonical NF-κB Pathway Activation.

Chemokine receptor CXCR5-mediated control of B cell trafficking in the lymphoid tissues plays a central role in orchestrating the B cell function, which not only guides the colocalization of B cells with follicular helper T cells in the follicular mantle zone but also determines the position of germinal center dark and light zones. However, the mechanisms that regulate the expression of CXCR5 in B cells remain unclear. Here, we show that the expression level of CXCR5 in B cells was substantially reduced in vitro culture conditions, while being maintained in the presence of CD40 signals. Furthermore, CD40 signaling promotes CXCR5 expression in B cells at least partially through noncanonical NF-κB signaling pathway activation. However, other non-B cells also contributed to the optimal expression of CXCR5 in B cells through cell-cell contact and cytokine secretion. Our findings suggest that CD40 signaling-mediated activation of the noncanonical NF-κB pathway promotes the expression of CXCR5 in a B cell-intrinsic way to orchestrate the trafficking of B cells.

Progressive Transformation of Germinal CentersA Mimicker of Lymphoma

Progressive transformation of germinal centers (PTGC) is an uncommon variant of non-specific reactive follicular hyperplasia. PTGC is a clinically asymptomatic condition characterised by persistent lymphadenopathy. PTGC may occur before, concurrently with or after the onset of nodular lymphocyte predominant Hodgkin’s lymphoma (NLPHL). It may also be confused for follicular lymphoma and mantle zone lymphoma. We report an interesting case of PTGC in a 23 year female with swelling in the left side of neck of one year duration. Histology revealed PTGC, which was confirmed by immunohistochemistry which showed CD3+, CD20+ in appropriate B and T cell region and negative for CD15, CD30 and bcl-2. As PTGC mimics lymphoma, it should be followed up carefully.

The role of the Chaperonin containing t-complex polypeptide 1, subunit 8 (CCT8) in B-cell non-Hodgkin's lymphoma

The chaperonin containing t-complex polypeptide 1 (CCT) is known to mediate folding of proteins. CCT, subunit 8 (CCT8), is the θ subunit of CCT complex chaperonin. CCT8 has been reported to be dysregulated in several tumor tissues. In this study, we investigated the role of CCT8 in B-cell non-Hodgkin’s lymphoma (NHL). Clinically, the expression levels of CCT8 in reactive lymphoid hyperplasia (RLH) and B-cell NHL specimens were investigated using immunohistochemical analysis. We found that CCT8 was highly expressed in proliferating germinal center cells compared with the quiescent cells of the follicular mantle zone. Furthermore, CCT8 was highly expressed in progressive lymphomas than in indolent lymphomas. Kaplan-Meier curve showed that high expression of CCT8 was significantly associated with shorter overall survival in patients with diffuse large B-cell lymphoma. Moreover, we demonstrated that CCT8 could promote the proliferation of B-cell NHL cells. In addition, we found that CCT8 could accelerate the G1/S transition in B-cell NHL. Finally, we demonstrated that overexpression of CCT8 could reverse cell adhesion-mediated drug resistance (CAM-DR) phenotype. Our study may shed new insights into the important role of CCT8 in cancer development.

Abstract 1287: Characterization of B cells infiltrating human breast cancer

Abstract Recent advances in tumor immunology have shown that an important link exists between tumor infiltrating lymphocytes (TIL) and patient outcome. In human breast cancer (BC), more extensive lymphocyte infiltration is associated with a better prognosis and also predicts responses to pre-operative chemotherapy. TIL are principally composed of T cells; however, approximately 20% of BC patients have a significant B cell infiltration, which can be diffuse and/or aggregated in stroma-located tertiary lymphoid structures (TLS). B cell infiltration in human BC has been associated with better survival and a longer disease-free interval, although the pro-/anti-tumor activities of B cells remain under debate. We undertook this study to more fully characterize the B cell infiltrate in human BC and the role humoral immunity potentially plays in generating effective anti-tumor immune responses. Flow cytometric analysis of fresh breast tissue homogenates detected an increased density of infiltrating B cells in all untreated primary BC (n = 223) compared with normal (mammary reductions; n = 53) and non-tumor non-adjacent (NANT; n = 198) breast tissues. This increase is not observed in BC tissue from metastatic and recurrent BC nor neoadjuvant-treated primary tumors. Potential correlations between infiltrating B cell density and clinicopathological parameters were examined, revealing that B cell density was associated with Ki67, ER and PgR status but not with age, tumor size, nodal status, grade, vascular invasion, HER-2 status, stage, histotype, subtype or in situ lesions. B cell infiltration was also correlated with the number of infiltrating CD4+ and CD8+ T cells and CD14+ monocytes. All stages of B cell differentiation were detected in the infiltrate; however, memory B cells significantly increased in BC compared to normal breast tissues. An increase in centroblasts and centrocytes, the germinal center B cells, was associated with a TFH cell presence (our data previously demonstrating that TFH cells predict good clinical outcomes). Furthermore, the presence of centroblasts-centrocytes and TFH cells was positively correlated with antibody-secreting cells, suggesting local humoral immune responses are generated in TLS. This hypothesis is supported by the increased immunoglobulin secretion detected in primary supernatants from tumors compared with normal breast tissues. Embedded BC tissues, examined by confocal microscopy, confirm that infiltrating B cells are principally located in TLS, which are composed of marginal, follicular mantle and germinal center zones surrounded by T cells. These data show that B cell infiltration is correlated with the overall extent of TIL and their presence likely functions to generate a humoral immune response adjacent to the tumor bed. B cells in extensively infiltrated tumors potentially play other important functional roles in generating effective anti-tumor immune responses, an aspect currently under investigation. Citation Format: Soizic Garaud, Laurence Buisseret, Céline Naveaux, Anaïs Boisson, Jean-Nicolas Lodewyckx, Hugues Duvillier, Ligia Craciun, Denis Larsimont, Karen Willard-Gallo. Characterization of B cells infiltrating human breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1287. doi:10.1158/1538-7445.AM2015-1287

Abstract 1669: Characterization of B-cells infiltrating human breast cancer and their presence in peritumoral tertiary lymphoid structures

Abstract Recent advances in tumor immunology show an important link exists between tumor infiltrating lymphocytes (TIL) and patient outcome. In human breast cancer (BC) more extensive lymphocyte infiltration is associated with a better prognosis and can also predict responses to pre-operative chemotherapy. Tertiary lymphoid structures (TLS) detected at the tumor periphery have been correlated with a good prognosis in lung and colorectal cancer. Our recent work was the first to demonstrate that TLS are present in BC, located adjacent to the tumor bed in extensively infiltrated tumors. TLS organization is similar to a lymph node, with a CD3+ T-cell zone adjacent to a CD20+ B-cell follicle where germinal centers containing CD23+ follicular dendritic cells, Bcl6+ TFH cells and Ki67+ cells are found. Our laboratory recently discovered that TLS-associated CD4+ Tfh cells are present in extensively infiltrated tumors and showed they signal a good prognosis. This important presence of Tfh-containing TLS adjacent to the tumor bed suggests that B-cells may also play a role in generating effective anti-tumor immune responses. We therefore undertook to fully characterize the B-cells infiltrating human BC. Flow cytometric analysis of fresh breast tumor homogenates detected an increase in CD45+ leukocytes in all BC subtypes compared with normal and non-tumor non-adjacent (NANT) breast tissue irrespective of lymphocyte infiltration levels. The infiltrate was dominated by CD3+ T-cells (65-86% of CD45+) in normal and malignant tissues. In contrast, B-cells were elevated in tumors compared to normal and NANT tissue, particularly in extensively infiltrated high proliferative BC subtypes. This increase was paralleled by a relative decrease in CD8+ T-cells. CD45/CD19/CD38/IgD labeling revealed that approximately 50% of the infiltrating B-cells are memory cells in contrast to normal tissues, which contain less than 15%. Centroblasts and centrocytes, which are follicular B cells, were significantly increased in extensively infiltrated tumors in association with Tfh cells. We assessed immunoglobulin isotypes in primary supernatants from breast tissue homogenates, detecting decreased IgA and increased IgG and IgM per mm3 in the tumor. Patient sera, particularly from high proliferative subtypes, had elevated IgG4 compared with low proliferative subtypes and healthy donors. Immunofluorescent analysis of embedded tissues show that TLS contain a marginal zone region(CD20+CD27+IgD-), a follicular mantle zone (CD20+IgD+) and a germinal center zone (CD20+Ki67+CD35+CD21+PD-1+). Tumor-associated TLS are surrounded by T-cells (CD4+ and CD8+). These data reveal that when B-cells are present in breast tumors they are principally associated with organized TLS and may therefore play an important functional role in comprehensive anti-tumor immune responses, an aspect currently under further investigation. Citation Format: Soizic Garaud, Laurence Buisseret, Chunyan Gu, Edoardo Migliori, Jean-Nicolas Lodewyckx, Hugues Duvillier, Ligia Craciun, Denis Larsimont, Karen Willard-Gallo. Characterization of B-cells infiltrating human breast cancer and their presence in peritumoral tertiary lymphoid structures. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1669. doi:10.1158/1538-7445.AM2014-1669

Pathology Quiz Case 1

A 70-YEAR-OLD MAN PRESENTED WITH A REcurrent right parotid mass. Five months earlier, he had presented at an outside hospital with a 4.3 3.2 2.6-cm level 2 mass in the right side of his neck. Two months after the excision, the mass recurred. Magnetic resonance imaging of the neck showed a 6 5-cm ill-defined mass of low signal intensity in superficial and deep parotid lobes, with infiltration of the surrounding soft tissues (Figure1). A chest radiograph revealed multifocal opacities. Positron emission tomography–computed tomography showed diffusehypermetabolic lymphadenopathy throughout thebody and hypermetabolic activity in the right parotid gland. An excisional biopsy specimen of a level 2 lymph node on the right side demonstrated follicular hyperplasia, increased immunoblasts, germinal centers, and mantle zones, with no evidence of lymphoma. The patient underwent a right total parotidectomy with facial nerve preservation. Pathologic examination demonstrated marked periductal and interlobular sclerosis with prominent collagen bands, lymphoid hyperplasia with germinal centers, numerous small lymphocytes, and plasma cells (Figure 2 and Figure 3). Immunohistochemical analysis revealed markedly increased IgG4 cells (Figure 4), with more than 80 IgG4-positive plasma cells per high-power field and an IgG4-IgG ratio of 10:1.Pathologic examination showed no evidence of lymphoma. Serologic tests demonstrated elevated levels of IgG (2940 mg/dL [reference range, 690-1660 mg/dL]) and IgG4 (1030 mg/dL [reference range, 4-86]) (to convert IgG and IgG4 levels to grams per liter, multiply by 0.01). Prednisone and azathioprine therapy was initiated, and the patient showed rapid improvement of symptoms and resolution of pulmonary opacities on computed tomograms of the chest. The level of IgG4 decreased to 19.4 mg/dL after 1 month and was then successfully maintained within normal limits, with no disease recurrence at the 9-month follow-up visit. What is your diagnosis?

항암화학요법에 실패한 Mantle 세포 림프종의 수술적 시도 1예

Mantle cell lymphoma (MCL) describes B-cell non-Hodgkin’s lymphomas that are derived from cells of the follicular mantle zone. MCL usually begins with lymph node enlargement and can easily spread to extra nodal organs such as the marrow, liver and gastrointestinal tracts. At times it begins in a lymphocyte outside a lymph node, such as in the gastrointestinal tract. MCL is treated the same way as other aggressive B-cell lymphomas with multidrug therapy, such as ESHAP (Etoposide, Methylprednisone, Cytarabine, Cisplatin). Most mantle cell lymphoma patients treated with treatment combination chemotherapy. But, it has tendency to recur after chemotherapy and like our case, planning of chemotherapy after total surgical resection of the neck mass that could be a new way of treatment. We report this case of surgical treatment on a recurrent cervical mass after 6cycles of chemotherapy. (J Clinical Otolaryngol 2009;20:106-109)

Status of the Immunoglobulin Heavy Chain and Light Chain Genes in Chronic Lymphocytic Leukemia and Related Disorders

The incidence of chronic lymphocytic leukemia (CLL) is low in Asian countries including Japan, while it is the most common type of leukemia in western countries. It has been evident that the immunoglobulin heavy chain variable region (IGHV) gene mutation status can predict the prognosis in CLL; unmutated IGHV genes correlate with a worse prognosis than mutated genes. Over-representation of selected IGHV genes is noted in western CLL, in particular IGHV1-69, IGHV4-34, IGHV3-7, and IGHV3-21. Although their relative frequencies vary between cohorts, the most frequent gene in western countries is IGHV1-69, which is found in about 10–20% of all CLL patients. Several studies have shown very unusual Ig characteristics in CLL: highly restricted IGHV gene usage and very similar antigen-binding sequences (stereotyped antigen receptors), suggesting a role for antigen selection during the development and maintenance of the disease. For the purpose of clarifying the characteristics of CLL in the Japanese population, we analyzed both IGHV and Ig light chain (κ-chain, IGK and λ-chain, IGL) genes in 81 CLL cases and compared the findings with cases of 52 leukemic chronic lymphoproliferative disorders (CLPD) including 6 hairy cell leukemia (HCL), 1 prolymphocytic leukemia (PLL), 31 indolent lymphoma in leukemic phase (15 mantle cell lymphoma (MCL), 7 follicular lymphoma (FL), 5 splenic marzinal zone lymphoma (SMZL), and 4 lymphoplasmacytic lymphoma (LPL)) and 14 cases that could not be classified further. Of the 81 Japanese CLL patients, 17 (21.3%) had unmutated IGHV, and 63 (78.7%) had mutated IGHV. The number of CLL with mutated IGHV was at a higher frequency compared to previous reports from western countries. It may be partly explained by the fact that the commonly unmutated IGHV1-69 type was rare (1.2%), but the commonly mutated IGHV4-34 type was frequent (27.2%) in the Japanese CLL patients. We previously reported that IGHV1-69 CLL is rare in Japan (1/44), which is confirmed by the present study of newly diagnosed cases (0/37). Moreover, only 1 of 65 CLL patients was reported to use IGHV1-69 in China. These findings suggest that IGHV1-69 is extremely rare in Asia. Similar to reports from Scandinavian countries, IGHV3-21 cases showed biased λ-chain expression (5/6), but were not associated with overuse of IGLV3-21 (V2-14) in our cohort. Recently, studies of B-cell antigen receptors (BCRs) in patients with CLL identified that subsets of cases expressed almost identical BCRs. We also found a pair of CLL patients who had the same IGHV4-39, IGHD6-13, IGHJ5 (heavy chain) and IGKV1-39 (O12), IGKJ1 or 2 segment with remarkably similar H and L CDR3 sequences. The use of IGHV, IGKV and IGLV was significant different when compared between CLL and leukemic CLPD. IGHV4-34, which was the most preferentially used in CLL patients (21/81, 26.0%), was used rarely in CLPD patients (4/52, 7.7%, p = 0.007). Of the 4 CLPD patients with IGHV4-34, 3 were MCL (CD5+) and 1 was unclassified CLPD (CD5 −). As leukemic cells of all CLL cases were CD5+, only 1 of the 25 patients using IGHV4-34 had CD5 negative cells. In normal B-cell development, naive IGHV4-34 B-cells are positively selected and mostly restricted to the follicular mantle zone but these cells are largely excluded from the germinal centers. This mechanism may be relevant to IGHV4-34 usage being underrepresented in CLPD other than MCL, which mainly consisted of GC- or post-GC-derived lymphomas/leukemias. In CLPD patients, only 1 patient with SMZL used the IGHV1-69 gene. Interestingly, IGHV1-69 was associated with IGHD5-24, IGHJ3, IGKV3-20 and IGLKJ1, which have been previously identified to comprise one of the stereotypical BCR gene subsets in patients with CLL. In the CLL patients, IGKV3-11 (L6) and IGLV3-21 (V2-14) were the most frequent IGKV (7/43) and IGLV (11/35), respectively. However, in the CLPD patients, IGKV3-11 and IGLV3-21 were used by none (0/26, p = 0.03) and only 1 MCL patient (1/22, p = 0.002), respectively. To date little data has been obtained on CLL in Japan and other Asian countries, where the susceptibility to CLL is very low. Thus, it is important to investigate genetic and environmental differences between Asian and western countries to identify risk factors that give rise to this disease. In addition, a comparison of the disease features of CLL with other lymphoproliferative disorders will further elucidate the clinical and pathogenetic characteristics of CLL.

Molecular cloning and expression analysis of pig CD79α

The CD79α (immunoglobulin α, Igα), a part of B cell receptor (BCR) complex, forms a heterodimer with CD79β (Igβ) and plays an important role in the B cell signaling. In this study, we have cloned pig Cd79a cDNA using RT-PCR and determined the complete cDNA sequence of pig Cd79a. Pig Cd79a cDNA contains an open reading frame (672 bp) encoding 223 amino acids. The putative amino acid identity of pig CD79α with those of human, cattle and mouse are 70.4, 81.4, and 67.7%, respectively. Alignment of the CD79α amino acid sequence with those of mammalian species showed that the extracellular domain is the most divergent, whereas transmembrane region and cytoplasmic tail including immunoreceptor tyrosine-based activation motif (ITAM) are largely conserved. Pig Cd79a mRNA was detected mainly in lymphoid tissues by RT-PCR. The highest level of Cd79a mRNA expression was observed in mesenteric lymph node and spleen. Relatively low level of Cd79a mRNA expression was observed in lung, thymus and small intestine. The lowest level of Cd79a mRNA expression was observed in large intestine. Flow cytometry analyses demonstrated that human CD79α antibody recognizes a CD79α in pig B cells. Further, immunohistochemistry analysis using human CD79α antibody on pig spleen was revealed that CD79α is strongly expressed in the follicular mantle zone rather than in the germinal center. Future study will be focused on defining the functional role of CD79α during the course of pig infectious diseases and the formation of neoplasm.

Differential expression of oestrogen receptors in human secondary lymphoid tissues

Many autoimmune diseases including rheumatoid arthritis (RA), Sjögren's syndrome (SS) and systemic lupus erythematosus (SLE) occur much more frequently in women than in men. There is much evidence that oestrogen is the major cause of this gender difference. Interestingly, oestrogen relieves the symptoms of RA and SS but it exacerbates SLE. This contradictory effect of oestrogen on autoimmune diseases is not well understood. Most of the effects of oestrogen are mediated by two receptors: oestrogen receptor alpha and beta (ERalpha and ERbeta). To determine whether these contradictory effects of oestrogen relate to the involvement of distinct effects of the two ERs, we investigated expression of ERalpha and ERbeta in human secondary lymphoid tissues. We observed that, in tonsils, ERbeta is expressed in lymphocytes of germinal centres (GC) and the follicular mantle zone as well as in granulocytes, while ERalpha is expressed only in activated germinal centres but not in the follicular zone. ERbeta is the predominant ER in human leucocytes from peripheral blood, spleen and in leucocytes infiltrating cancers in both males and females. In addition, in different human lymphoma cell lines including Hodgkin lymphoma, Burkitt lymphoma, and multiple myeloma, ERbeta is abundant while ERalpha is not detectable. Our results indicate that ERbeta is the predominant type of ER in mature lymphocytes. We suggest that ERalpha and ERbeta have distinct roles in secondary lymphoid tissues and that further studies with ERbeta-specific agonists will help to elucidate the role of ERbeta in these tissues.

Autoimmune glomerulonephritis with spontaneous formation of splenic germinal centers in mice lacking the estrogen receptor alpha gene.

In mice, ovariectomy accelerates the progression of the end-stage renal disease glomerulosclerosis. In women, the incidence of this disease increases after menopause, and estrogen alters its progression. Polymorphisms in the human estrogen receptor alpha (ERalpha) gene have been suggested to constitute a genetic predisposition for lupus nephritis. Here we show that by 1 year of age, mice lacking ERalpha (ERalpha(-/-)) but not those lacking ERbeta (ERbeta(-/-)) exhibit immune complex-type glomerulonephritis, proteinuria, and destruction of tubular cells with severe infiltration of B lymphocytes in the kidney and the presence of anti-DNA antibodies in serum. No gender difference occurred in the incidence or severity of these symptoms. However, in female but not in male ERalpha(-/-) mice there were elevated serum levels of IgG3. Other prominent features of these mice were (i) spontaneous formation of germinal centers in the spleen in the absence of antigen challenge and (ii) infiltration of plasma cells in the kidney and plasmacytosis in the spleen. Immunohistochemistry indicated a selective expression of ERalpha protein in the germinal centers but not in the follicular mantle zone of murine spleens and human tonsils. Our results indicate that ERalpha has indispensable functions in the kidney and in germinal centers, and that defective ERalpha signaling results in glomerulonephritis.

Ectopic germinal center formation in rheumatoid synovitis.

Synovial inflammation in rheumatoid arthritis is closely related to the formation of ectopic lymphoid microstructures. In synovial tissue from some patients, one finds seemingly diffuse infiltrates; in others, T cells and B cells cluster in aggregates with interdigitating dendritic cells (DCs) but no follicular DCs (FDCs). In a third group, T cell/B cell follicles with germinal center (GC) reactions are generated. Within a given patient, aggregates and GCs are mutually exclusive and stable over time. Because antigen storage capacity, lymphoid density, and three-dimensional topography of GCs optimize immune responses, synovial GCs should play a crucial role in the breakdown of self-tolerance. We have identified factors critical for ectopic GCs, thereby transforming the synovial inflammatory process. Tissues with GCs produced 10- to 20-fold higher amounts of the chemokines CXCL13 and CCL21. CXCL13 derived from three sources, endothelial cells, synovial fibroblasts, and FDC networks. The level of CXCL13 transcripts strongly predicted GCs; however, some tissues had high levels of CXCL13 but lacked GCs. Tissue expression of LT-beta emerged as a second key factor. LT-beta protein was detected on follicular center and mantle zone B cells. Multivariate regression analysis identified CXCL13 and LT-beta as the only cytokines predicting GCs. Remarkably, LT-alpha did not contribute independently. The contribution of B cells to ectopic lymphoid organogenesis was not limited to LT-beta production. Rather, synovial tissue B cells were critical in regulating T cell activation. In adoptive transfer experiments in human synovium-SCID mouse chimeras, activation of synovium-derived CD4 T cells was strictly dependent on T cell/B cell follicles. Depletion of synovial tissue B cells abrogated T cell function, and non-B cell antigen-presenting cells could not maintain T cell stimulation. Unexpectedly, GC function in the rheumatoid lesion was also dependent on CD8 T cells. The majority of T cell receptors derived from CD8 T cells were shared between distinct GCs. Depletion of CD8 T cells disrupted synovial GCs, FDC networks disappeared, and transcription of LT-beta, IgG, and Igkappa declined. Follicle-sustaining CD8 T cells were located at the edge of or within the mantle zone. Cell-cell communication in the mantle zone, including CD8 T cells, appears to be critical for ectopic GC formation in rheumatoid synovitis.

CD22 as a target of passive immunotherapy

CD22 is a 135-kd B-cell restricted sialoglycoprotein present in the cytoplasm of virtually all B-lineage cells but expressed on the B-cell surface only at mature stages of differentiation. In humans, the vast majority of IgM +IgD + B cells express cell-surface CD22, while in lymphoid tissues CD22 expression is high in follicular mantle and marginal zone B cells and weak in germinal center B cells. In B-cell malignancies, CD22 expression ranges from 60% to 80% depending on the histological type and on the assays used. The function of the CD22 molecule is uncertain, although recent studies have suggested roles for the molecule both as a component of the B-cell activation complex and as an adhesion molecule. CD22-deficient mice have a reduced number of mature B cells in the bone marrow and circulation; the B cells have a shorter lifespan and enhanced apoptosis, thus indicating a key role of this antigen in B-cell development/survival. After binding with its natural ligand(s) or antibodies, CD22 is rapidly internalized; this provides a potent costimulatory signal in primary B-cell and proapoptotic signals in neoplastic B cells. Preclinically CD22 has been shown to be an effective target for immunotherapy of B-cell malignancies using either “naked” or toxin-labeled or radiolabeled monoclonal antibodies. Clinical trials in patients with non-Hodgkin's lymphoma (NHL) (both indolent and aggressive disease) are now ongoing with a humanized naked anti-CD22 antibody (epratuzumab, Amgen Inc, thousand Oaks, CA and Immunomedics Inc, Morris Plains, NJ) used as single agent or in combination with other monclonal antibodies (ie, rituximab) and/or chemotherapy. Preliminary data from these studies showed these approaches to be effective and well-tolerated. Semin Oncol 30:253-257. © 2003 Elsevier Inc. All rights reserved.

Reduced expression of cyclin-dependent kinase inhibitor p27Kip1 in feline lymphoma.

Expression of p27Kip1 was identified in feline lymphoid tissues by immunohistochemistry. In normal lymphoid tissues, p27Kip1 was detected as a distinct nuclear stain in lymphocytes of the follicular mantle zone and interfollicular small lymphocytes, whereas activated lymphoblasts in the germinal center were negative. Lymphoid hyperplasia was similarly immunolabeled but with an expanded mantle zone and marginal zone of p27Kip1-reactive lymphocytes. Both T- and B-cell lymphomas lacked p27Kip1 immunolabel and were determined to be proliferative based on immunohistochemical detection of the Ki-67 antigen. Scattered p27Kip1-immunolabeled lymphocytes were detected throughout the lamina propria of most specimens characterized as lymphoplasmacytic enteritis. The results of this study suggest that the antiproliferative effect of the cell cycle regulator p27Kip1 is abrogated in feline lymphoma, presumably allowing cells to bypass the G1-S checkpoint of the cell cycle.

Regulation of inherently autoreactive VH4-34 B cells in the maintenance of human B cell tolerance

The study of human B cell tolerance has been hampered by difficulties in identifying a sizable population of autoreactive B lymphocytes whose fate could be readily determined. Hypothesizing that B cells expressing intrinsically autoreactive antibodies encoded by the VH4-34 heavy chain gene (VH4-34 cells) represent such a population, we tracked VH4-34 cells in healthy individuals. Here, we show that naive VH4-34 cells are positively selected and mostly restricted to the follicular mantle zone. Subsequently, these cells are largely excluded from the germinal centers and underrepresented in the memory compartment. In healthy donors but not in patients with systemic lupus erythematosus (SLE), these cells are prevented from differentiating into antibody-producing plasma cells. This blockade can be overcome ex vivo using cultures of naive and memory VH4-34 cells in the presence of CD70, IL-2, and IL-10. VH4-34 cells may therefore represent an experimentally useful surrogate for autoantibody transgenes and should prove valuable in studying human B cell tolerance in a physiological, polyclonal environment. Our initial results suggest that both positive and negative selection processes participate in the maintenance of tolerance in autoreactive human B cells at multiple checkpoints throughout B cell differentiation and that at least some censoring mechanisms are faulty in SLE.

Regulation of inherently autoreactive VH4-34 B cells in the maintenance of human B cell tolerance.

The study of human B cell tolerance has been hampered by difficulties in identifying a sizable population of autoreactive B lymphocytes whose fate could be readily determined. Hypothesizing that B cells expressing intrinsically autoreactive antibodies encoded by the VH4-34 heavy chain gene (VH4-34 cells) represent such a population, we tracked VH4-34 cells in healthy individuals. Here, we show that naive VH4-34 cells are positively selected and mostly restricted to the follicular mantle zone. Subsequently, these cells are largely excluded from the germinal centers and underrepresented in the memory compartment. In healthy donors but not in patients with systemic lupus erythematosus (SLE), these cells are prevented from differentiating into antibody-producing plasma cells. This blockade can be overcome ex vivo using cultures of naive and memory VH4-34 cells in the presence of CD70, IL-2, and IL-10. VH4-34 cells may therefore represent an experimentally useful surrogate for autoantibody transgenes and should prove valuable in studying human B cell tolerance in a physiological, polyclonal environment. Our initial results suggest that both positive and negative selection processes participate in the maintenance of tolerance in autoreactive human B cells at multiple checkpoints throughout B cell differentiation and that at least some censoring mechanisms are faulty in SLE.

The Perifollicular and Marginal Zones of the Human Splenic White Pulp: Do Fibroblasts Guide Lymphocyte Immigration?

We investigate the white pulp compartments of 73 human spleens and demonstrate that there are several microanatomical peculiarities in man that do not occur in rats or mice. Humans lack a marginal sinus separating the marginal zone (MZ) from the follicles or the follicular mantle zone. The MZ is divided into an inner and an outer compartment by a special type of fibroblasts. An additional compartment, termed the perifollicular zone, is present between the follicular MZ and the red pulp. The perifollicular zone contains sheathed capillaries and blood-filled spaces without endothelial lining. In the perifollicular zone, in the outer MZ, and in the T cell zone fibroblasts of an unusual phenotype occur. These cells stain for the adhesion molecules MAdCAM-1, VCAM-1 (CD106), and VAP-1; the Thy-1 (CD90) molecule; smooth muscle alpha-actin and smooth muscle myosin; cytokeratin 18; and thrombomodulin (CD141). They are, however, negative for the peripheral node addressin, the cutaneous lymphocyte antigen, CD34, PECAM-1 (CD31), and P- and E-selectin (CD62P and CD62E). In the MZ the fibroblasts are often tightly associated with CD4-positive T lymphocytes, whereas CD8-positive cells are almost absent. Our findings lead to the hypothesis, that recirculating CD4-positive T lymphocytes enter the human splenic white pulp from the open circulation of the perifollicular zone without crossing an endothelium. Specialized fibroblasts may attract these T cells and guide them into the periarteriolar T cell area.

Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) is associated with a naïve B-cell phenotype in human tonsils.

In B cells, signaling through the B-cell antigen receptor (BCR) is negatively modulated by the co-ligation of immunoglobulin (Ig)-immunoreceptor tyrosine-based inhibitory motif (ITIM)-bearing molecules such as FcgammaRIIB1, B-cell transmembrane protein CD72, paired immunoglobulin-like receptor PIR-B, leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1), Ig-like transcript ILT2, biliary glycoprotein BGP-1 and B-cell co-receptor CD22. The co-expression of multiple Ig-ITIM receptors may provide B cells with different mechanisms of regulating inhibitory pathways at different stages of differentiation. In this study, we have examined the expression of a newly defined Ig-ITIM receptor, PECAM-1 (CD31) on human B-cells. Human tonsillar B cells were purified using negative selection by depleting T cells with a combination of monoclonal antibodies and magnetic bead separation. Following purification, the pattern of PECAM-1 expression was analyzed in B-cell subpopulations using two- and three-colour fluorescence. To complement this work, PECAM-1 localization in the context of distinct areas of human tonsil was defined by immunohistochemical analysis of tonsil sections. Finally to investigate somatic mutation, Ig variable (V) region genes belonging to the nonpolymorphic VH6 family were amplified by polymerase chain reaction (PCR), subcloned and sequenced from sort-purified CD19+ PECAM-1+ and CD19+ PECAM-1- B cells. Our results demonstrate that PECAM-1 is associated with an unstimulated resting B-cell phenotype, localization to the follicular mantle and marginal zones of human tonsil and expression of unmutated Ig V region genes. These studies suggest that PECAM-1 appears on the cell surface at the naive B-cell stage and is lost as B cells differentiate into memory cells, indicating that PECAM-1 is primarily involved in naive or immature B-cell function.