B-cells In Lymph Nodes Research Articles

The influence of chronic hyperhomocysteinemia on the structure and immune processes of the spleen in young rats

Homocysteine is a sulfur-containing amino acid that is an intermediate product of methionine metabolism. Entering the body with products of animal origin, methionine undergoes a number of biochemical transformations. Hyperhomocysteinemia has a negative effect on the body, causing damage to all organs and systems and leading to disruption of homeostasis. The study aims to study the characteristics of changes in the structure and immune processes of the spleen of young rats with chronic hyperhomocysteinemia. Experiments were performed on 22 white male rats. The animals were divided into control and experimental groups (11 individuals in each group) during the experiment. Chronic hyperhomocysteinemia was achieved by administering D, L-thiolactone homocysteine hydrochloride to experimental group animals at a dose of 200 mg/kg of body weight intragastrically in a 1 % starch gel solution once a day for eight weeks. After the end of the experimental simulation of chronic hyperhomocysteinemia, the animals were removed from the experiment by anaesthetising by decapitation and using thiopental anaesthesia. Histological preparations were studied using an SEO SСAN light microscope. Experimental modelling of chronic hyperhomocysteinemia in young rats was not accompanied by significant morphological changes in the stroma and red pulp of the spleen. T-cell periarterial sheaths and B-cell nodules in animals from the experimental group underwent changes. B-cell proliferation leads to growth within the white pulp, expansion of the marginal zones, which become more blurred and infiltration of these cells into the red pulp. Evaluation of the red pulp in the spleen of young rats from the experimental group showed a particular expansion in the venous sinuses, which may be a sign of reaction to the influence of homocysteine and slight swelling of these vessels. The number of T-cells in young animals in the periarterial cuffs is slightly reduced, which can be explained by the increasing activity of macrophages. However, T-cells also infiltrated the red pulp. Such penetration of white blood cells into the red pulp is accompanied by the expansion and “washing out” of the marginal zones of lymph nodes. Thus, the increased homocysteine level significantly potentiated proliferation and partially inhibited apoptosis in T-lymphocytes but did not directly affect dormant T-cells.

Examination of the exposome in an animal model: The impact of high fat diet and rat strain on local and systemic immune markers following occupational welding fume exposure

The goal of this study was to investigate the impact of multiple exposomal factors (genetics, lifestyle factors, environmental/occupational exposures) on pulmonary inflammation and corresponding alterations in local/systemic immune parameters. Accordingly, male Sprague-Dawley (SD) and Brown Norway (BN) rats were maintained on either regular (Reg) or high fat (HF) diets for 24wk. Welding fume (WF) exposure (inhalation) occurred between 7 and 12wk. Rats were euthanized at 7, 12, and 24wk to evaluate local and systemic immune markers corresponding to the baseline, exposure, and recovery phases of the study, respectively. At 7wk, HF-fed animals exhibited several immune alterations (blood leukocyte/neutrophil number, lymph node B-cell proportionality)—effects which were more pronounced in SD rats. Indices of lung injury/inflammation were elevated in all WF-exposed animals at 12wk; however, diet appeared to preferentially impact SD rats at this time point, as several inflammatory markers (lymph node cellularity, lung neutrophils) were further elevated in HF over Reg animals. Overall, SD rats exhibited the greatest capacity for recovery by 24wk. In BN rats, resolution of immune alterations was further compromised by HF diet, as many exposure-induced alterations in local/systemic immune markers were still evident in HF/WF animals at 24wk. Collectively, HF diet appeared to have a greater impact on global immune status and exposure-induced lung injury in SD rats, but a more pronounced effect on inflammation resolution in BN rats. These results illustrate the combined impact of genetic, lifestyle, and environmental factors in modulating immunological responsivity and emphasize the importance of the exposome in shaping biological responses.

Systemic toxicity induced by topical application of heptafluorobutyric acid (PFBA) in a murine model

Heptafluorobutyric acid (PFBA) is a synthetic chemical belonging to the per- and polyfluoroalkyl substances (PFAS) group that includes over 5000 chemicals incorporated into numerous products. PFBA is a short-chain PFAS (C4) labeled as a safer alternative to legacy PFAS which have been linked to numerous health effects. Despite the high potential for dermal exposure, occupationally and environmentally, dermal exposure studies are lacking. Using a murine model, this study analyzed serum chemistries, histology, immune phenotyping, and gene expression to evaluate the systemic toxicity of sub-chronic dermal PFBA 15-day (15% v/v or 375 mg/kg/dose) or 28-day (3.75–7.5% v/v or 93.8–187.5 mg/kg/dose) exposures. PFBA exposure produced significant increases in liver and kidney weights and altered serum chemistries (all exposure levels). Immune-cell phenotyping identified significant increases in draining lymph node B-cells (15%) and CD11b + cells (3.75–15%) and skin T-cells (3.75–15%) and neutrophils (7.5–15%). Histopathological and gene expression changes were observed in both the liver and skin after dermal PFBA exposure. The findings indicate PFBA induces liver toxicity and alterations of PPAR target genes, suggesting a role of a PPAR pathway. These results demonstrate that sustained dermal exposure to PFBA induces systemic effects and raise concerns of short-chain PFAS being promoted as safer alternatives.

IL-21 and IFN\u03b1 therapy rescues terminally\xa0differentiated NK cells and limits SIV reservoir in ART-treated macaques

Unlike HIV infection, which progresses to AIDS absent suppressive anti-retroviral therapy, nonpathogenic infections in natural hosts, such African green monkeys, are characterized by a lack of gut microbial translocation and robust secondary lymphoid natural killer cell responses resulting in an absence of chronic inflammation and limited SIV dissemination in lymph node B-cell follicles. Here we report, using the pathogenic model of antiretroviral therapy-treated, SIV-infected rhesus macaques that sequential interleukin-21 and interferon alpha therapy generate terminally differentiated blood natural killer cells (NKG2a/clowCD16+) with potent human leukocyte antigen-E-restricted activity in response to SIV envelope peptides. This is in contrast to control macaques, where less differentiated, interferon gamma-producing natural killer cells predominate. The frequency and activity of terminally differentiated NKG2a/clowCD16+ natural killer cells correlates with a reduction of replication-competent SIV in lymph node during antiretroviral therapy and time to viral rebound following analytical treatment interruption. These data demonstrate that African green monkey-like natural killer cell differentiation profiles can be rescued in rhesus macaques to promote viral clearance in tissues.

Immunization with recombinant rabies virus expressing Interleukin-18 exhibits enhanced immunogenicity and protection in mice.

Several studies have shown that interleukin-18 (IL-18) plays an important role in both innate and adaptive immune responses. In this study, we investigated the pathogenicity and immunogenicity of recombinant rabies virus expressing IL-18 (rHEP-IL18). Experimental results showed that Institute of Cancer Research (ICR) mice that received a single intramuscular immunization with rHEP-IL18 elicited the highest titers of serum neutralizing antibodies and the strongest cell-mediated immune responses to prevent the development of rabies disease, compared with immunization with the parent virus HEP-Flury. Mice inoculated with rHEP-IL18 developed significantly higher IFN-γ responses, increased percentages of CD4+ and CD8+ T-lymphocytes compared to HEP-Flury. Flow cytometry results show that rHEP-IL18 recruited more activated T- and B-cells in lymph nodes or peripheral blood, which is beneficial for virus clearance in the early stages of infection. A higher percentage of mice immunized with rHEP-IL18 survived wild-type rabies virus (RABV) challenge, compared to HEP-Flury mice. Our results show that rHEP-IL18 is promising as a novel vaccine for RABV prevention and control.

Evaluation of the irritancy and hypersensitivity potential following topical application of didecyldimethylammonium chloride

Didecyldimethylammonium chloride (DDAC) is a dialkyl-quaternary ammonium compound that is used in numerous products for its bactericidal, virucidal and fungicidal properties. There have been clinical reports of immediate and delayed hypersensitivity reactions in exposed individuals; however, the sensitization potential of DDAC has not been thoroughly investigated. The purpose of these studies was to evaluate the irritancy and sensitization potential of DDAC following dermal exposure in a murine model. DDAC induced significant irritancy (0.5 and 1%), evaluated by ear swelling in female Balb/c mice. Initial evaluation of the sensitization potential was conducted using the local lymph node assay (LLNA) at concentrations ranging from 0.0625–1%. A concentration-dependent increase in lymphocyte proliferation was observed with a calculated EC3 value of 0.17%. Dermal exposure to DDAC did not induce increased production of IgE as evaluated by phenotypic analysis of draining lymph node B-cells (IgE + B220+) and measurement of total serum IgE levels. Additional phenotypic analyses revealed significant and dose-responsive increases in the absolute number of B-cells, CD4 + T-cells, CD8 + T-cells and dendritic cells in the draining lymph nodes, along with significant increases in the percentage of B-cells (0.25% and 1% DDAC) at Day 10 following 4 days of dermal exposure. There was also a significant and dose-responsive increase in the number of activated CD44 + CD4 + and CD8 + T-cells and CD86 + B-cells and dendritic cells following exposure to all concentrations of DDAC. These results demonstrate the potential for development of irritation and hypersensitivity responses to DDAC following dermal exposure and raise concerns about the use of this chemical and other quaternary ammonium compounds that may elicit similar effects.

Abstract 1277: Tumor-specific human monoclonal antibodies isolated from cancer patients that bind antigens expressed on the outer plasma membrane of cancer cells

Abstract Introduction: Cancer cells express novel antigens that reflect their transformed state and may engender an antibody response. We hypothesize that some of these auto-antibodies detect neo-antigens that are shared by diverse tumor types and that a subset of these are expressed on the outer plasma membrane (PM) of cancer cells, but not normal cells. Here, we describe two human mAbs, isolated from lymph node B-cells of breast cancer patients, that bind antigens expressed only on the surface of cancer cells. Methods: We have optimized a hybridoma method for cloning human IgG monoclonal antibodies (mAbs) by screening for those that bind to live, heterologous cancer cell lines. We use whole cell immunofluorescence binding assays with human cancer cell lines, detecting cell-reactive human IgG with the Operetta. We use B-cells from lymph nodes obtained at surgery from stage I, ER+PR+ breast cancer patients and Stage I or II lung cancer patients, as well as peripheral blood B-cells from lung cancer patients responsive to PD-1 inhibitors. We used cDNA library expression methods to identify the tumor antigens and characterized the tumor-specific activities of two mAbs using immunohistochemistry (IHC), gene transfection, flow cytometry, immunoblotting, and invasion assays. Results: Two of the mAbs isolated from lymph nodes of patients with breast cancer bind to antigens that are only expressed on the outer PM of cancer cells. The antigen recognized by mAb 9H2 is known to be expressed on the outer PM of metastatic cells, while the antigen recognized by mAb 2B9 is a cytoplasmic protein not previously recognized to exist on the outer PM. IHC demonstrates that the 9H2 antigen is expressed only rarely on normal cells, but is commonly on breast and colon cancers (3 of 4 samples tested for each tumor type). Binding was also seen on 2 of 4 small cell lung cancers samples and 1 of 3 pancreatic cancers. The existence of the 2B9 antigen on the outer PM was confirmed by study of 293T cells transiently transfected with MYC-tagged antigen expression constructs and analyzed by flow cytometry with the mAb 2B9 and an anti-myc mAb. Treatment of MDA-MB-231 cells with the mAb 2B9 substantially reduced invasion in the transwell migration assay. An additional panel of 12 mAbs exhibited internalizing activity and is being evaluated for efficacy as antibody-drug conjugates Conclusion: Our panel of mAbs from cancer patients supports the hypothesis that patients with malignancies make antibodies that bind novel cancer-specific antigens. Two mAbs bind antigens expressed only on the surface of malignant cells; one has not previously been detected on the outer PM. The ability of mAb 2B9 to inhibit breast cancer cell invasion suggests that the anti-tumor immune response may be functional. Studying the anti-cancer antibody repertoire may identify new targets for mAb-based cancer diagnostics and therapeutics. Citation Format: Baron Heimbach, Cezary Swider, Huiwu Zhao, Paul Simon, R. Katherine Alpaugh, Michael Walker, David Krag, David Knight, Tung Chan, Hossein Borghaei, Scott Dessain. Tumor-specific human monoclonal antibodies isolated from cancer patients that bind antigens expressed on the outer plasma membrane of cancer cells. [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 1277. doi:10.1158/1538-7445.AM2015-1277

Immunophenotypic features by multiparameter flow cytometry can help distinguish low grade B-cell lymphomas with plasmacytic differentiation from plasma cell proliferative disorders with an unrelated clonal B-cell process.

Highly sensitive flow cytometry studies may incidentally identify B cell clones when used to assess plasma cell clonality in bone marrows. Clinical history, which can help differentiate related clones (low grade B cell lymphoma with plasmacytic differentiation/LBCL-PD) from unrelated ones (plasma cell proliferative disorder (PCPD) with an unrelated B cell clone), is often unavailable in referred specimens. We sought to identify morphologic or phenotypic features that would help predict the significance of these clones in the absence of history. We included only cases with identical light chain B and plasma cell clones, as determined by 6-color flow cytometry with additional DNA ploidy analysis, in which the relationship between clones could be established by review of medical records. There were 26 cases; 18 were related (14 were Waldenstrom macroglobulinemia) and eight were unrelated (seven multiple myeloma). Features seen exclusively in LBCL-PD include CD19+/CD45+ clonal plasma cell phenotype (66·7%, P=0·0022) and morphologic features such as paratrabecular bone marrow involvement, increased mast cells, and plasma cells surrounding B-cell nodules. Aneuploidy was identified exclusively in PCPD cases (75%, P=0·000028). We conclude that CD19+/CD45+ clonal plasma cell phenotype and aneuploidy are useful in distinguishing related clones (LBCL-PD) from unrelated clones (PCPD).

CD75: A B‐cell marker which should not be forgotten in lymphocyte predominant Hodgkin lymphoma

To the Editor The derivation of lymphocyte predominant (LP) cells of nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) from germinal center (GC) B cells is supported by the following features: the expression of the BCL6 gene product and CD40 by LP cells 1, 2; the occurrence of numerous CD4+/PD1 T cells surrounding the LP cells, as seen in normal GCs and progressively transformed GCs (PTGCs); the presence of a follicular dendritic cell (FDC) meshwork (CD21+) within the tumor nodules 3; and the global gene expression profile 3. On these grounds, immunophenotyping by immunohistochemical analysis using B-cell markers for detecting LP cells is generally considered the golden standard in the diagnosis of NLPHL 3. LP cells are positive for CD20, CD79a, CD75, BCL6, Pax5, and OCT2. To evaluate the impact of B-cell markers in detecting LP cells of NLPHL, we analyzed the histopathologic and immunophenotypic patterns of NLPHL from 42 patients (total of 52 samples). These cases were obtained from our files at Aviano Centro di Riferimento Oncologico (CRO) during the period from 1986 to 2005. Immunohistochemical studies were performed on paraffin-embedded sections from formalin-fixed samples and Bouin-fixed samples. Informed written consent was obtained from the patients, and tissue collection was approved by Aviano CRO Institutional Review Board. Immunohistochemical stains were performed with a panel of monoclonal and polyclonal antibodies, listed in Supporting Information, using an automated immunostainer (Ventana Medical Systems, Tucson, AZ) according to the company's protocols. Serial sections were used for assessment of antigen coexpression. The diagnosis of NLPHL was based on the established WHO criteria 3. Typical NLPHL patterns were recognized in 31 patients, while histopathologic variants were found in the remaining 11 patients. The typical NLPHL patterns, including pattern A and pattern B according to Fan et al. 4, consisted of a predominantly nodular growth with non-neoplastic B-cells and LP cells located within the B-cell nodules. The LP cells were rarely found outside the nodules. Conversely, histopathologic NLPHL variants were characterized by the presence of LP tumor cells outside the B-cell nodules. LP cells were positive for all the B cell markers tested in all cases. CD75, also known as CDw75, a marker of mature B cells of GC derivation, was useful in detecting LP cells, especially those admixed with reactive B cells and FDCs in nodules of typical NLPHL patterns (31 out of 42) (Fig. 1). LP cells expressed CD75 strongly, while small reactive B cells with a mantle cell phenotype in the nodules showed weak cytoplasmic positivity in the Golgi area but no membranous staining. The consequence was that in B-cell nodules of typical NLPHL patterns CD75 selectively highlighted LP cells through their cytoplasmic and membranous staining (Fig. 1). CD20 was expressed in LP cells and in the small reactive B cells in the background with an equal intensity of staining. CD20 could be difficult to interpret in B-cell nodules of typical NLPHL patterns, while in areas with B-cell depletion of histopathologic variants of NLPHL the CD20 stained LP cells were detected easier. LP cells stained for CD75 were more numerous than those stained for CD20. The OCT2 and PAX5, B-cell transcription factors were consistently coexpressed. However, their expression was even worse than CD20 in detecting LP cells because these markers preferentially stained small reactive B cells. Our results lead us to conclude that CD75 is superior to other B-cell markers in detecting LP cells, especially in typical NLPHL patterns. Therefore, CD75 should not be forgotten in the immunohistochemical panel for detecting LP cells. Recognizing histopathologic NLPHL patterns is clinically relevant because of the adverse prognostic impact displayed by the histopathologic NLPHL variants as demonstrated by the German Hodgkin Study Group 5 and also because of the favorable prognostic impact displayed by the typical NLPHL patterns as recently reported by us in the American Journal of Haematology 6. Antonino Carbone1* Annunziata Gloghini2 1Department of Pathology, Centro di Riferimento Oncologico Aviano, Istituto Nazionale Tumori, IRCCS, I-33081 Aviano, Italy; 2Department of Diagnostic Pathology and Laboratory Medicine, Fondazione IRCCS, Istituto Nazionale Tumori, I-20133 Milano, Italy Additional Supporting Information may be found in the online version of this article. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Subcutaneous versus Intravenous Administration of Rituximab: Pharmacokinetics, CD20 Target Coverage and B-Cell Depletion in Cynomolgus Monkeys

The CD20-specific monoclonal antibody rituximab (MabThera®, Rituxan®) is widely used as the backbone of treatment for patients with hematologic disorders. Intravenous administration of rituximab is associated with infusion times of 4–6 hours, and can be associated with infusion-related reactions. Subcutaneous administration of rituximab may reduce this and facilitate administration without infusion-related reactions. We sought to determine the feasibility of achieving equivalent efficacy (measured by endogenous B-cell depletion) and long-term durability of CD20 target coverage for subcutaneously administered rituximab compared with intravenous dosing. In these preclinical studies, male cynomolgus monkeys were treated with either intravenous rituximab or novel subcutaneous formulation of rituximab containing human recombinant DNA-derived hyaluronidase enzyme. Peripheral blood samples were analyzed for serum rituximab concentrations, peripheral B-cell depletion, and CD20 target coverage, including subset analysis according to CD21+ status. Distal lymph node B-cell depletion and CD20 target coverage were also measured. Initial peak serum concentrations of rituximab were significantly higher following intravenous administration than subcutaneous. However, the mean serum rituximab trough concentrations were comparable at 2 and 7 days post-first dose and 9 and 14 days post-second dose. Efficacy of B-cell depletion in both peripheral blood and distal lymph nodes was comparable for both methods. In lymph nodes, 9 days after the second dose with subcutaneous and intravenous rituximab, B-cell levels were decreased by 57% and 42% respectively. Similarly, levels of peripheral blood B cells were depleted by >94% for both subcutaneous and intravenous dosing at all time points. Long-term recovery of free unbound surface CD20 levels was similar, and the duration of B-cell depletion was equally sustained over 2 months for both methods. These results demonstrate that, despite initial peak serum drug level differences, subcutaneous rituximab has similar durability, pharmacodynamics, and efficacy compared with intravenous rituximab.

MRas- knock-out mouse: B-cell phenotype and reduced macrophage infiltration in atherosclerotic plaques at the aortic root

Background: Recently, we identified by genome-wide association study the MRas gene locus on human chromosome 3q22 to be associated with coronary artery disease (CAD) (rs9818870; p=7.44x10-13). M-Ras is a member of the Ras superfamily of small GTPases, which act as molecular switches in diverse cellular functions and thereby regulate a variety of biological processes. M-Ras has been implicated in the regulation of TNFα-stimulated LFA-1 activation and integrin-mediated leukocyte adhesion downstream of various inflammatory cytokines. Aim: To further study the pathomechanism underlying the association between MRas and CAD in a knockout mouse model. Methods: We crossbred the MRas-KO mice onto the ApoE-KO background and fed these mice with western diet. In parallel, we performed adhesion and migration assays with macrophages, monocytes, B- and T-cells to study the influence of MRas on adhesion and migration as a potential pathomechanism of atherosclerosis. Results: The MRas/ApoE-KO mouse showed no difference in plaque size at the aortic root in comparison to the ApoE-KO mouse after 10 weeks of high fat diet, but there were significant less macrophages in the plaque (n=19/10/15; ∼10%; p=0.002). In addition, the ratio between macrophage and collagen in the plaque at the aortic root was significant higher in the MRas/ApoE-KO than in the ApoE-KO under high fat diet (n=19/9/15; p=0.002). Furthermore, we observed a 15% reduction of CD19+ B-cells in lymphnodes in MRas-KO mice (p<0.05), but not in bone marrow, spleen and blood. Moreover, we noticed a 20% reduction of leukocyte adhesion in in vitro assays with an endothelial cell line (p<0.05). Again, especially B-cell adhesion was reduced in MRas-KO mice in comparison to WT mice. In vitro migration assays show also a reduced migration of B-cells towards the chemokine CxCl12 (p<0.01). Conclusion: Our data suggests that MRas-KO has no effect on plaque formation at the aortic root but on the plaque stability by less macrophage infiltration. This could be linked to the reduction of adhesion and migration by B-cells.

Identifying Phenotypically and Clinically Distinct Entities within the Spectrum of Marginal Zone Lymphoma and Lymphoplasmacytic Lymphoma/Waldenstrom Macroglobulinemia

Abstract 2652The mature B-cell neoplasms marginal zone lymphoma (MZL), lymphoplasmacytic lymphoma (LPL) and Waldenstrom macroglobulinemia (WM) display considerable overlap in clinic presentation, morphology, immunophenotype and genetic features. There is known to be poor reproducibility in the classification of these disorders and often only minimal diagnostic material is available. Multiparameter flow cytometry is a valuable tool for diagnosis and monitoring of B-cell disorders that can be applied to low cellularity samples or when the neoplastic cells represent a minority of total leucocytes. However, even when using large numbers of markers it is not possible to discriminate between MZL, LPL and WM and the aberrant expression of certain markers appears to occur with similar frequency in the different disease categories. The aim of this study was to determine whether there are phenotypically distinct entities within the spectrum of marginal zone and lymphoplasmacytic lymphomas, and to correlate these entities with clinical features.Samples from 146 people with a diagnosis of MZL, LPL or WM (122 bone marrow, 13 peripheral blood, 10 tissue biopsies), 8 with hairy cell leukemia, 10 reactive lymph node biopsies and 10 normal bone marrow samples were analysed using a panel of 39 markers. A minimum of 50,000 cells were acquired and analysed using a BD FACSCanto II with Diva software. Median fluorescence intensities were recorded for neoplastic CD19+ B-cell populations as well as for germinal centre (GC) and non-GC lymph node B-cells, B-progenitors, mature B-cells and plasma cell populations. Unsupervised cluster analysis was performed using dChip software. Clinical features and outcome data were collected and recorded centrally (www.hmrn.org.uk).Cluster analysis identified 5 entities with significant differences in their immunophenotypic profile. Neoplastic cells in all the MZL/LPL/WM groups typically expressed CD31, CD39 and CD49d with lack of CD10, CD23 and weaker expression of LAIR1 (CD305), CXCR5 (CD185) and CD22 compared to normal B-cells. CD5 and CD43 were expressed in a subset of cases but co-expression occurred in <1% of cases. The groups are characterised as follows: (1) clustered with normal mature B-cells, characterised by weaker CD20/CD95 and stronger CXCR5 expression relative to other MZL/LPL/WM cases, shorter survival, anemia and lymphadenopathy, typically requiring therapeutic intervention at diagnosis; (2) clustered with HCL, characterised by CD25 and CD11c expression and a higher degree of extranodal involvement; (3) did not cluster with other B-cell types, characterised by very strong CD79b and surface IgM expression often with anemia requiring supportive care; (4) characterised by strong CD24 expression with relatively good clinical course; and (5) clustered with normal bone marrow plasma cells, characterised by weak CD20, CD24 and IgM expression and also with better clinical features.Group2 year survivalAnemia (Hb <10.5g/dL)Lympho-cytosis (>4 × 10^9/L)Raised B2M (>2.7mg/L)Lymph-adenopathy (≥2 nodes)Extranodal involvementTherapeutic intervention at diagnosis115/24 (63%)10/19 (53%)9/19 (47%)8/10 (80%)12/19 (63%)4/19 (21%)14/19 (74%)228/37 (76%)8/25 (32%)11/25 (44%)6/11 (55%)9/23 (39%)10/23 (43%)9/25 (36%)325/31 (81%)14/24 (58%)5/23 (22%)11/18 (61%)4/19 (21%)5/19 (26%)13/24 (54%)422/27 (81%)5/22 (23%)5/22 (23%)10/17 (59%)5/20 (25%)4/20 (20%)8/22 (36%)522/26 (85%)5/16 (31%)5/16 (31%)6/8 (75%)8/14 (57%)5/14 (36%)5/16 (31%)The results indicate that the current diagnostic categories of MZL and LPL/WM have a great degree of overlap and comprise a number of phenotypically distinct entities which also appear to have different clinical features and outcomes. Enhancing the classification is likely to facilitate diagnosis, monitoring and identification of the underlying features of these B-cell malignancies. Disclosures:Rawstron:BD Biosciences: Patents & Royalties.

Comparison of B Cell Depletion Mediated by Intravenous (IV) Rituxan® and Rituxan Given Subcutaneous (SC) In Cynomolgus Monkeys

AbstractAbstract 3980Rituximab (MabThera)® is a chimeric mouse/human monoclonal antibody recognizing the B cell specific trans-membrane protein CD20. Rituximab's CD20 specific binding facilitates the prolonged depletion of both malignant and endogenous CD20+ B cells. Rituximab is currently FDA approved for monotherapy and combinatorial therapies in the treatment of multiple forms of Non Hodgkin's Lymphoma (NHL), Chronic Lymphocytic Leukemia (CLL) and Diffuse Large B cell Lymphomas (DLBCL), as well as for the treatment of moderate to severe rheumatoid arthritis patients who are refractory to TNF antagonists (in combination with methotrexate, MTX). As with most large protein based biologics, the proto-typical route of administration of rituximab is by intravenous (IV) infusion. Following delivery to the circulation, rituximab distributes into both primary and secondary lymphoid tissues throughout the body. This distribution is essential to full target coverage of CD20+ cells and therefore critical for the efficacy in targeting those cells for depletion. For other monoclonal antibodies in other clinical settings, the conversion from IV to SC administration has resulted in an improved tolerability with less infusion-related reactions, shorter administration times, an increased patient-convenience and an improved cost-effectiveness. These advantages are anticipated for the SC administration of rituximab as well.Here we describe the results of an IV/SC pharmacokinetic/pharmacodynamic (PKPD) cynomolgus monkey study comparing the traditional IV infusion of rituximab to a novel SC dosing route of rituximab administration using an SC formulation of rituximab containing recombinant human hyaluronidase (rHuPH20). rHuPH20 transiently degrades the local interstitial matrix component, hyaluronan, at the SC injection site, acting as a permeation enhancer and allowing SC administration of high volumes. Cynomolgus monkeys were treated twice, one week apart, with the rituximab SC formulation or with IV rituximab (2 × 10 mg/kg IV or SC). CD20 target coverage and depth of distal lymph node B-cell depletion was measured 7 days later, while the prolonged target coverage and duration of peripheral blood B-cell depletion was evaluated over an extended two month period. The results indicate similar rituximab trough concentrations in serum as well as similar B-cell depletion efficacy in both peripheral blood and distal secondary lymphoid tissue after IV and SC dosing. In conclusion, the results of the study suggest that the SC versus IV dosing routes do not influence the non-clinical efficacy in this B cell depletion model given that similar trough concentrations of rituximab are reached with both administration routes. Disclosures:Del Nagro:Genentech/Roche: Employment. Mao:Roche: Employment. Dabbagh:Roche: Consultancy, Employment. Birnboeck:Roche: Consultancy, Employment. Richter:Roche: Employment.

Distribution of immune cells in head and neck cancer: CD8+ T-cells and CD20+B-cells in metastatic lymph nodes are associated with favourable outcome in patients with oro- and hypopharyngeal carcinoma

BackgroundTumour infiltrating lymphocytes (TIL) are generally considered to represent a host immune response directed against tumour antigens. TIL are also increasingly recognised as possible prognostic parameters. However, the effects observed are variable indicating that results cannot be extrapolated from type of tumour to another. Moreover, it has been suggested that primary solid tumours may be ignored by the immune system and that a meaningful immune response is only mounted in regional lymph nodes.MethodsWe have examined the local distribution of immune cells in tumour-related compartments in head and neck squamous cell carcinomas (HNSCC). In a second step, the prognostic impact of these cells on disease-free survival (DFS) was analysed. A total of 198 tissue cores from 33 patients were evaluated using tissue mircroarray technique and immunohistochemistry. Tumour-infiltrating immune cells were identified using antibodies specific for CD3, CD8, GranzymeB, FoxP3, CD20 and CD68 and quantified using an image analysis system.ResultsWe demonstrate a relative expansion of FoxP3+ regulatory T-cells (Treg) and of cytotoxic T-cells among tumour infitrating T-cells. We also show that intratumoural CD20+ B-cells are significantly more frequent in metastatic deposits than in primary tumours. Furthermore, we observed a reduced number of peritumoural CD8+ T-cells in metastatic lymph nodes as compared to univolved regional nodes suggesting a local down-modulation of cellular immunity. All other immune cells did not show significant alterations in distribution. We did not observe an association of tumour infiltrating immune cells at the primary site with outcome. However, increased numbers of intraepithelial CD8+ TIL in metastatic tumours as well as large numbers of peritumoural B-cells in lymph node metastases were associated with favourable outcome. Unexpectedly, no effect on patient outcome was observed for Treg in any compartment.ConclusionOur results suggest that alterations in lymphocyte distribution in regional lymph nodes rather than at the primary tumour site may be relevant for patient prognosis. Moreover, we demonstrate that in addition to cellular immunity humoral immune responses may be clinically relevant in anti-tumour immunity.

A human monoclonal autoantibody to breast cancer identifies the PDZ domain containing protein GIPC1 as a novel breast cancer-associated antigen

BackgroundWe have been studying the native autoimmune response to cancer through the isolation of human monoclonal antibodies that are cancer specific from cancer patients. To facilitate this work we previously developed a fusion partner cell line for human lymphocytes, MFP-2, that fuses efficiently with both human lymph node lymphocytes and peripheral blood lymphocytes. Using this unique trioma fusion partner cell line we isolated a panel of autologous human monoclonal antibodies, from both peripheral blood and lymph node lymphocytes, which are representative of the native repertoire of anti-cancer specific antibodies from breast cancer patients.MethodsThe current study employs immunocytochemistry, immunohistochemistry, Western blot analysis as well as Northern blots, Scatchard binding studies and finally SEREX analysis for target antigen identification.ResultsBy application of an expression cloning technique known as SEREX, we determined that the target antigen for two monoclonal antibodies, 27.B1 and 27.F7, derived from lymph node B-cells of a breast cancer patient, is the PDZ domain-containing protein known as GIPC1. This protein is highly expressed not only in cultured human breast cancer cells, but also in primary and metastatic tumor tissues and its overexpression appears to be cancer cell specific. Confocal microscopy revealed cell membrane and cytoplasmic localization of the target protein, which is consistent with previous studies of this protein.ConclusionWe have determined that GIPC1 is a novel breast cancer-associated immunogenic antigen that is overexpressed in breast cancer. Its role, however, in the initiation and/or progression of breast cancer remains unclear and needs further clarification.

Characteristic appearances of the bone marrow in T‐cell large granular lymphocyte leukaemia

To augment the limited literature on bone marrow (BM) appearances in T-cell large granular lymphocyte (LGL) leukaemia and to identify a histological signature to aid in diagnosis of this condition. A descriptive analysis of the histology of the BM in T-cell LGL leukaemia was performed (n = 38). Antibodies against CD3, CD4, CD5, CD8, CD16, CD56, CD57 and CD20 or CD79a were employed. Antibodies against CD68 (macrophages) and CD34 (sinusoids) were also included. BM was normocellular or hypercellular in the majority of cases, with interstitial lymphoid infiltration in 97%. Lymphoid nodules were present in 55% and intrasinusoidal permeation in 58%. Apoptotic figures and haemosiderin deposition were common. All cases showed trilinear haematopoiesis with normal or increased megakaryopoiesis and erythropoiesis, but normal/reduced myelopoiesis. Reticulin was increased (Grade II-III). Immunohistochemistry revealed interstitial infiltration in all cases and helped to identify lymphoid nodules in two-thirds of cases. Preferential localization of CD8+ T lymphocytes to the interstitium and CD4+ T lymphocytes to the periphery of CD20+ B-cell nodules was seen in almost 90% of cases. Nodules with non-clonal B-cell centres surrounded by CD4+ cells, with interstitial CD8+ cells, are a characteristic finding in T-cell LGL leukaemia and may represent a histological signature for this condition.

The Presence of B-Cell Nodules Does Not Necessarily Portend a Less Favorable Outcome to Therapy in Patients With Acute Cellular Rejection of a Renal Allograft

The presence of B-cell nodules in kidney biopsies of patients undergoing acute renal allograft rejection has been reported to be associated with glucocorticoid resistance and a high risk of graft failure. In an attempt to corroborate this observation, biopsies of renal transplants that evidenced Banff grade I A acute rejection were examined for the presence of B- or T-cell nodules, the detection of which was correlated with the therapeutic response. Biopsies from 14 consecutive renal transplant recipients with a diagnosis of acute cellular rejection were examined for the presence of T (CD3-positive) or B (CD20-positive) cells by immunohistochemistry. All patients were biopsied because of a rise in serum creatinine. No biopsy showed evidence of acute humoral rejection. Immunofluorescence microscopy was negative for C4d deposition in peritubular capillaries. There were no neutrophils in the peritubular or glomerular capillaries. Five patients had T-cell nodules; four had B-cell nodules; three had both T- and B-cell nodules; two had no nodules. All biopsies contained CD3-positive cells in the tubules and in the interstitium. In all but one of the patients, episodes of acute rejection were treated with steroids (one received thymoglobulin). Furthermore two patients received mycophenolate mofetil and one, sirolimus. There were no significant differences among the groups in either the initial creatinine or the creatinine after therapy. The presence of B-cell nodules in renal allograft biopsies of patients experiencing acute cellular rejection did not portend a less favorable outcome.

Bcl-2 Antagonist ApoGossypol (NSC736630) Displays Single-Agent Activity in Bcl-2 Transgenic Mice and Has Superior Efficacy and Pharmacology with Less Toxicity Compared to Gossypol (NSC19048).

Altered expression of Bcl-2-family proteins plays a central role in apoptosis dysregulation in cancer and leukemia, promoting malignant cell expansion and contributing to chemoresistance. In this study, we compared the toxicity, pharmacology, and efficacy in mice of Gossypol (NSC19048) and ApoGossypol (NSC736630), a semi-synthetic analog of natural product Gossypol, in which two reactive aldehydes were eliminated from the compound (Becattini, et al Chem & Biol 11(3):389-95, 2004 Mar.). Gossypol and ApoGossypol mimic endogenous BH3 peptide-containing antagonists of anti-apoptotic Bcl-2-family proteins, competing with the BH3 peptide binding sites on Bcl-2, Bcl-XL, Mcl-1, Bcl-W, and Bcl-B, but not Bfl-1, with IC 50 of 0.5-2 M (Zhai, et al. Cell Death & Differ 13:1419-21, 2006). These compounds thus represent broad-spectrum antagonists of anti-apoptotic Bcl-2-family proteins. ApoGossypol displayed biostability comparable to Gossypol in plasma from mice, rats, dogs, and humans. Liver microsome analysis demonstrated no metabolites of either ApoGossypol or Gossypol, though both compounds underwent some glucuronidation. Apogossypol appeared more stable in human and mouse liver microsomal preparations than gossypol. Pharmacokinetic studies of i.v. and orally delivered Gossypol and ApoGossypol in mice showed that ApoGossypol has improved AUC (50–75% higher) due principally to reduced clearance rate. Both Gossypol and ApoGossypol exhibited similar oral bioavailability (10–13%) and have similar Cmax (10-20 M). Toxicity studies of i.p. and orally administered compounds showed that ApoGossypol is far less toxic than Gossypol. Daily dosing studies showed that mice tolerate doses of ApoGossypol 2–4-times higher than Gossypol. Hepatotoxicity and gastrointestinal toxicity (intestinal edema and ileus) represented the major adverse activities of these compounds, with ApoGossypol far less toxic than Gossypol. Efficacy of Gossypol and ApoGossypol was compared in a transgenic mouse model of Bcl-2-driven disease, where a human Bcl-2 gene from a t(14;18)-containing lymphoma drives continuous Bcl-2 expression in B-cells (Katsumata, et al. Proc Natl Acad Sci 89: 11376; 1992). These mice develop splenomegally and have expanded populations of mature non-cycling B-cells in lymph nodes, resembling low-grade follicular lymphoma in humans. In vitro, Bcl-2-expressing B-cells were more sensitive to ApoGossypol than Gossypol, with LD50 values of 3–5 M and 7.5–10 M, respectively. In vivo, using the maximum tolerated dose of Gossypol (0.06 mmoles/kg) for sequential daily dosing (daily × 5 days × 3 weeks), ApoGossypol displayed superior activity to Gossypol in terms of reducing splenomegally and reducing B-cell counts in spleen. At higher does (0.12–0.24 mmole/kg), ApoGossypol showed still greater in vivo activity, producing larger reductions in splenomegally and in B-cell counts in spleen. Taken together, these studies indicate that ApoGossypol is superior to parent compound Gossypol with respect to pharmacology, toxicology, and efficacy, suggesting that further development of this compound for cancer therapy is warranted. (Supported by NCI-RAID and NCI contract funds N01-CM-07110 and N01-CM-52205, and by a grant to the CLL Research Consortium P01-CA081534).

Rituximab in refractory antineutrophil cytoplasmic antibody-associated vasculitis: what is the current evidence?

Antineutrophil cytoplasmic antibody (ANCA)associated vasculitis (AAV) is recognized as a chronic, relapsing and potentially fatal disease. Cyclophosphamide and steroids remain the mainstay of treatment of AAV [1]. Unfortunately, in up to 10% of patients, disease remains refractory despite conventional therapy [1]. In addition, conventional therapy has some serious side effects and hence limits its long-term use. For these refractory patients and those intolerant of conventional therapy, there are limited therapeutic options. B-lymphocytes have been implicated in the pathogenesis of AAV, by being the precursors of plasma cells (PCs), which produce ANCA [2] and in the formation of granuloma in Wegener’s granulomatosis (WG) [3]. Apart from the important role in antibody production, B-cells can also affect other aspects of the immune regulation such as immune response regulation, antigen presentation and cytokine production. This has been recognized in autoimmune diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus, where the disease process is B-cell dependent and antibody independent [4,5]. Rituximab (RIT) is a chimeric monoclonal anti-CD20 antibody, which depletes B-lymphocytes, and has been proposed as salvage therapy for refractory disease. RIT consists of human constant regions linked to murine variable domains [6]. The murine Fab domain of RIT binds the CD20 antigen, which is a transmembrane protein located on the surface of mature B-cells. The CD20 antigen is involved in regulation of the transmembrane calcium conductance and cell-cycle progression during human B-cell activation [7]. B-cell activation may differentiate into either shortlived (in the absence of T-cell help) or long-lived PCs that are regulated by ligation of CD40 provided by helper T-cells [8] (Figure 1). RIT acts through antibody-mediated apoptosis, antibody-dependent cellularcytotoxicity and complement-mediated toxicity [9]. RIT induces 98% depletion of peripheral blood B-cells, but only 40–70% of lymph node B-cells are depleted [2]. Long-lived PCs do not express CD20 and this may explain why RIT-induced depletion of CD20þ B-cells in some cases does not decrease either total serum IgG levels or ANCA levels [2,10]. The rationale behind the use of RIT in AAV is based on the following three assumptions.

Depletion of B Cells by a Humanized Anti-CD20 Antibody PRO70769 in Macaca Fascicularis

PRO70769 is a humanized IgG1 monoclonal antibody against the CD20 molecule that is present on normal and malignant B cells. PRO70769 is being evaluated for treatment of B-cell-mediated diseases and is in a phase 1 trial for rheumatoid arthritis. As part of the preclinical toxicology evaluation, B-cell depletion profiles and safety of PRO70769 were assessed in cynomolgus monkeys. Animals were administered drug (IV) on days 1 and 15 with 10, 50, or 100 mg/kg PRO70769 and killed 2 weeks after the second dose and after a 3-month recovery period. In a parallel study, animals were not necropsied but instead were retreated with a second cycle of PRO70769 administered under an identical regimen. PRO70769 suppressed B cells in the blood to undetectable levels and significantly reduced B cells in lymphoid tissues. Splenic B cells were depleted to a greater extent compared with lymph node B cells. A second cycle of treatment resulted in a greater extent of depletion in lymphoid tissues compared with the depletion observed after one cycle of treatment; however, residual B cells in lymphoid tissues were still detectable, even at the highest dose. The rate of B-cell recovery in peripheral blood appeared similar between one and two cycles of treatment. Upon depletion there was a change in the profile of lymph node B-cell subsets. After recovery, B-cell subsets were reconstituted to normal levels. Depletion of CD20-expressing cells and lymphoid follicular atrophy were the only treatment-related effects.