CD20 is dispensable for B-cell receptor signaling but is required for proper actin polymerization, adhesion and migration of malignant B cells.

Surface protein CD20 serves as the critical target of immunotherapy in various B-cell malignancies for decades, however its biological function and regulation remain largely elusive. Better understanding of CD20 function may help to design improved rational therapies to prevent development of resistance. Using CRISPR/Cas9 technique, we have abrogated CD20 expression in five different malignant B-cell lines. We show that CD20 deletion has no effect upon B-cell receptor signaling or calcium flux. Also B-cell survival and proliferation is unaffected in the absence of CD20. On the contrary, we found a strong defect in actin cytoskeleton polymerization and, consequently, defective cell adhesion and migration in response to homeostatic chemokines SDF1α, CCL19 and CCL21. Mechanistically, we could identify a reduction in chemokine-triggered PYK2 activation, a calcium-activated signaling protein involved in activation of MAP kinases and cytoskeleton regulation. These cellular defects in consequence result in a severely disturbed homing of B cells in vivo.

CD20 role in pathophysiology of Hodgkin's disease.

Hodgkin's lymphoma (HL) is a tumor comprising non-malignant and malignant B-cells. Classical HL expresses CD15+ and CD30+ antigens, and 20 to 40% of patients are CD20+. This antigen is a ligand free protein present in B lymphocyte cells and its function is not well known. Some studies suggest that expression of CD20 may play a major role in Hodgkin's disease pathophysiology and may affect the patients' treatment prognosis, as well as relapse and refractory response. In the past few years, development of monoclonal anti-CD20 antibodies changed drastically the treatment for non-Hodgkin lymphomas in which CD20 is expressed. HL treatment is essentially composed of radiotherapy and chemotherapy; however, monoclonal anti-CD20 antibodies applicability is not well delimitated due to lack of information about clinical outcomes with anti-CD20 monotherapy or combined drug therapy using a classic regimen, as well as about CD20 pathophysiology mechanisms in B-cells tumors. The objective of our review is to discuss CD20 function in Hodgkin's lymphoma development, its influence on disease evolution and outcomes, as well as its effects on therapeutics and patients' prognostic.

CD20 role in pathophysiology of Hodgkin's disease / O papel do CD20 na fisiopatologia da doença de Hodgkin

Summary Hodgkin's lymphoma (HL) is a tumor comprising non-malignant and malignant B-cells. Classical HL expresses CD15+ and CD30+ antigens, and 20 to 40% of patients are CD20+. This antigen is a ligand free protein present in B lymphocyte cells and its function is not well known. Some studies suggest that expression of CD20 may play a major role in Hodgkin's disease pathophysiology and may affect the patients' treatment prognosis, as well as relapse and refractory response. In the past few years, development of monoclonal anti-CD20 antibodies changed drastically the treatment for non-Hodgkin lymphomas in which CD20 is expressed. HL treatment is essentially composed of radiotherapy and chemotherapy; however, monoclonal anti-CD20 antibodies applicability is not well delimitated due to lack of information about clinical outcomes with anti-CD20 monotherapy or combined drug therapy using a classic regimen, as well as about CD20 pathophysiology mechanisms in B-cells tumors. The objective of our review is to discuss CD20 function in Hodgkin's lymphoma development, its influence on disease evolution and outcomes, as well as its effects on therapeutics and patients' prognostic.

Resumo O linfoma de Hodgkin (LH) é um tumor composto por células B não malignas e malignas. O LH clássico expressa antígenos CD15+ e CD30+, mas apenas cerca de 20 a 40% dos pacientes expressa também antígeno CD20. Este antígeno é uma proteína sem ligante presente nas células de linfócitos B cuja função não é bem conhecida. Alguns estudos sugerem que a expressão de CD20 pode ter um papel importante na fisiopatologia da doença de Hodgkin e pode afetar o prognóstico dos pacientes ao tratamento, bem como recaída e refratariedade. Nos últimos anos, o desenvolvimento de anticorpos monoclonais anti-CD20 mudou drasticamente o tratamento para linfomas não Hodgkin em que o CD20 é expresso. O tratamento do LH é composto essencialmente de radio e quimioterapia; no entanto, o espaço dos anticorpos monoclonais anti-CD20 não está bem delimitado em decorrência de: falta de informação sobre o desfecho clínico, seja na monoterapia com anti-CD20, seja na terapêutica combinada com o regime clássico; falta de informação sobre os mecanismos de fisiopatologia CD20 em tumores de células B. O objetivo desta revisão é discutir sobre a função do CD20 no desenvolvimento do linfoma de Hodgkin, sua influência na evolução da doença e os resultados, bem como os efeitos sobre terapêutica e prognóstico dos pacientes.

The CD20 homologue MS4A4 directs trafficking of KIT toward clathrin-independent endocytosis pathways and thus regulates receptor signaling and recycling.

MS4A family members differentially regulate the cell cycle, and aberrant, or loss of, expression of MS4A family proteins has been observed in colon and lung cancer. However, the precise functions of MS4A family proteins and their mechanistic interactions remain unsolved. Here we report that MS4A4 facilitates trafficking of the receptor tyrosine kinase KIT through endocytic recycling rather than degradation pathways by a mechanism that involves recruitment of KIT to caveolin-1-enriched microdomains. Silencing of MS4A4 in human mast cells altered ligand-induced KIT endocytosis pathways and reduced receptor recycling to the cell surface, thus promoting KIT signaling in the endosomes while reducing that in the plasma membrane, as exemplified by Akt and PLCγ1 phosphorylation, respectively. The altered endocytic trafficking of KIT also resulted in an increase in SCF-induced mast cell proliferation and migration, which may reflect altered signaling in these cells. Our data reveal a novel function for MS4A family proteins in regulating trafficking and signaling, which could have implications in both proliferative and immunological diseases.

Activated pancreatic stellate cells sequester CD8+ T cells to reduce their infiltration of the juxtatumoral compartment of pancreatic ductal adenocarcinoma.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) is characterized by a prominent desmoplastic microenvironment that contains many different immune cells. Activated pancreatic stellate cells (PSCs) contribute to the desmoplasia. We investigated whether distinct stromal compartments are differentially infiltrated by different types of immune cells. METHODS: We used tissue microarray analysis to compare immune cell infiltration of different pancreaticobiliary diseased tissues (PDAC, ampullary carcinoma, cholangiocarcinoma, mucinous cystic neoplasm, chronic inflammation, and chronic pancreatitis) and juxtatumoral stromal (<100 µm from tumor) and panstromal compartments. We investigated the association between immune infiltrate and patient survival times. We also analyzed T-cell migration and tumor infiltration in LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre (KPC) mice and the effects of all-trans retinoic acid (ATRA) on these processes. RESULTS: Juxtatumoral compartments in PDAC samples from 2 independent groups of patients contained increased numbers of myeloperoxidase(+) and CD68(+) cells compared with panstromal compartments. However, juxtatumoral compartments of PDACs contained fewer CD8(+), FoxP3(+), CD56(+), or CD20(+) cells than panstromal compartments, a distinction absent in ampullary carcinomas and cholangiocarcinomas. Patients with PDACs that had high densities of CD8(+) T cells in the juxtatumoral compartment had longer survival times than patients with lower densities. In KPC mice, administration of ATRA, which renders PSCs quiescent, increased numbers of CD8(+) T cells in juxtatumoral compartments. We found that activated PSCs express cytokines, chemokines, and adhesion molecules that regulate T-cell migration. In vitro migration assays showed that CD8(+) T cells, from patients with PDAC, had increased chemotaxis toward activated PSCs, which secrete CXCL12, compared with quiescent PSCs or tumor cells. These effects could be reversed by knockdown of CXCL12 or treatment of PSCs with ATRA. CONCLUSIONS: Based on studies of human PDAC samples and KPC mice, activated PSCs appear to reduce migration of CD8(+) T cells to juxtatumoral stromal compartments, preventing their access to cancer cells. Deregulated signaling by activated PSCs could prevent an effective antitumor immune response.

Combinatorial chemotherapeutic efficacy in non-Hodgkin lymphoma can be predicted by a signaling model of CD20 pharmacodynamics.

Combination chemotherapy represents the standard-of-care for non-Hodgkin lymphoma. However, the development of new therapeutic regimens is empirical and this approach cannot be used prospectively to identify novel or optimal drug combinations. Quantitative system pharmacodynamic models could promote the discovery and development of combination regimens based upon first principles. In this study, we developed a mathematical model that integrates temporal patterns of drug exposure, receptor occupancy, and signal transduction to predict the effects of the CD20 agonist rituximab in combination with rhApo2L/TNF-related apoptosis inducing ligand or fenretinide, a cytotoxic retinoid, upon growth kinetics in non-Hodgkin lymphoma xenografts. The model recapitulated major regulatory mechanisms, including target-mediated disposition of rituximab, modulation of proapoptotic intracellular signaling induced by CD20 occupancy, and the relative efficacy of death receptor isoforms. The multiscale model coupled tumor responses to individual anticancer agents with their mechanisms of action in vivo, and the changes in Bcl-xL and Fas induced by CD20 occupancy were linked to explain the synergy of these drugs. Tumor growth profiles predicted by the model agreed with cell and xenograft data, capturing the apparent pharmacologic synergy of these agents with fidelity. Together, our findings provide a mechanism-based platform for exploring new regimens with CD20 agonists.

Molecular mechanisms of the antitumor effects of anti-CD20 antibodies.

Anti-CD20 monoclonal antibodies (mAbs) have become the mainstay in the treatment of non-Hodgkin's lymphomas and have shown significant activity in patients with B-cell chronic lymphocytic leukemia. Antitumor action of these antibodies results from triggering of indirect effector mechanisms of the immune system that include activation of complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), or phagocytosis. Moreover, some studies indicate direct influence of anti-CD20 mAbs on tumor cells that leads to induction of various types of cell death. Despite the wealth of data on the mechanisms of cytotoxicity that accumulated over the last two decades their relative contribution to the therapeutic outcome is still difficult to predict in individual patients. Elucidation of molecular mechanisms of anti-CD20 mAbs action is necessary to deliver their maximal activity in rationally designed combinations with other therapeutic approaches and to design next generation anti-CD20 mAb with improved ability to eliminate tumor cells.

CD20 as a target for therapeutic type I and II monoclonal antibodies.

The last decade has seen the monoclonal antibody (mAb), rituximab, transform clinical management of many non-Hodgkin lymphomas and more recently provide new opportunities for controlling autoimmune conditions, such as rheumatoid arthritis. Although not yet fully determined, the explanation for this success appears to lie with the inherent properties of its target, CD20, which allow rituximab to recruit potent cytotoxic effectors with unusual efficiency. In this review we detail the properties of CD20 that make it such an effective therapeutic target and describe how different mAbs change the membrane distribution and internalization of CD20 and have distinct modes of cytotoxic activity.

CD20 deficiency in humans results in impaired T cell-independent antibody responses.

CD20 was the first B cell differentiation antigen identified, and CD20-specific mAbs are commonly used for the treatment of B cell malignancies and autoantibody-mediated autoimmune diseases. Despite this the role of CD20 in human B cell physiology has remained elusive. We describe here a juvenile patient with CD20 deficiency due to a homozygous mutation in a splice junction of the CD20 gene (also known as MS4A1) that results in "cryptic" splicing and nonfunctional mRNA species. Analysis of this patient has led us to conclude that CD20 has a central role in the generation of T cell-independent (TI) antibody responses. Key evidence to support this conclusion was provided by the observation that although antigen-independent B cells developed normally in the absence of CD20 expression, antibody formation, particularly after vaccination with TI antigens, was strongly impaired in the patient. Consistent with this, TI antipolysaccharide B cell responses were severely impeded in CD20-deficient mice. Our study therefore identifies what we believe to be a novel type of humoral immunodeficiency caused by CD20 deficiency and characterized by normal development of antigen-independent B cells, along with a reduced capacity to mount proper antibody responses.

Anti-CD20 antibody rituximab in the treatment of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic, autoimmune reaction-driven systemic inflammatory disease that affects joints and several other organs. Although anti-TNF therapy and combination therapy with traditional anti-rheumatic drugs have improved the treatment of RA, still quite a significant proportion of patients do not reach adequate anti-rheumatic response. The understanding of the pathogenesis of RA has developed markedly during the last two decades, and this has brought up new targets for anti-rheumatic therapy. B cells have been found to have a pivotal role in the development of arthritis both in experimental models and in humans. Rituximab, an anti-CD20 antibody that depletes B cells, has been introduced in the treatment of RA, and it has proven to be safe and efficacious in RA. This review gives an overview on the mechanism of action of rituximab in RA and summarizes the published clinical data of rituximab in the treatment of RA.

Engaging the lysosomal compartment to combat B cell malignancies.

The combination of rituximab, a type I anti-CD20 mAb, with conventional chemotherapy has significantly improved the outcome of patients with B cell malignancies. Regardless of this success, many patients still relapse with therapy-resistant disease, highlighting the need for the development of mAbs with higher capacity to induce programmed cell death. The so-called type II anti-CD20 mAbs (e.g., tositumomab) that trigger caspase-independent B cell lymphoma cell death in vitro and show superior efficacy as compared with rituximab in eradicating target cells in mouse models are emerging as the next generation of therapeutic anti-CD20 mAbs. In this issue of the JCI, Ivanov and colleagues identify the lysosomal compartment as a target for type II mAbs (see the related article beginning on page 2143). These data encourage the further clinical development of type II mAbs as well as other lysosome-targeting drugs in the treatment of B cell malignancies.

Spotlight on anti-CD20.

Immune modulators, such as interferon beta and glatiramer acetate, have focused on T-cells as the primary therapeutic target, although both drugs affect other cell types as well. There has been a renewed interest in the potential roles of both antibody-dependent and antibody-independent B-cell responses in multiple sclerosis (MS) and its animal model. Accumulating data suggest that the contribution of B-cells and their secreted products to central nervous system (CNS) inflammatory diseases may relate to the abilities of B-cells to (1) differentiate into plasmocytes that produce antibodies, (2) function as antigen-presenting cells, contributing to Tcell activation, (3) produce effector cytokines that may modulate the local immune environment, (4) harbour the Epstein Barr virus in a chronically activated state, and (5) play a role in formation and maintenance of new lymphoid foci within the CNS. Understanding the biological and clinical impact of selective B-cell interventions such as rituximab (Rituxan, Genentech Inc., San Francisco, CA, USA) becomes of particular interest. This review will present the rational for B-cell based therapies in MS and related diseases and preliminary data suggesting a therapeutic benefit in MS and related diseases. In addition, other therapies aiming at CD20 will be reviewed.

[B cell depletion therapy using anti-CD20 antibodies in rheumatoid arthritis].

Some patients with rheumatoid arthritis (RA) suffer from disease that is refractory to both conventional therapy and newer biological agents such as tumor necrosis factor (TNF) inhibitors. In recent years, there has been growing interest in, and enhanced understanding of, the contribution of B cells to the immunopathogenesis of RA. Rituximab (RTX), a chimeric monoclonal antibody against CD20 that effectively depletes B cells in peripheral blood, has been licensed for the treatment of malignant lymphoma for almost 10 years. Efficacy of rituximab in RA has already been demonstrated in randomized control trials, and US Food and Drug Administration has approved the combination of RTX with methotrexate for use in patients with RA, who have had an inadequate response to one or more TNF antagonist therapies. The long-term efficacy, especially about joint damage, and long-term safety need to be further investigated.

What signals are generated by anti-CD20 antibody therapy?

Immunotherapy with rituximab (a chimeric anti-CD20 monoclonal antibody), alone or in combination with chemotherapy, has improved the treatment outcome of patients with non-Hodgkin's lymphoma (NHL), but the in vivo mechanisms by which rituximab exerts its effects have not been elucidated. The mechanisms underlying resistance are not known. In addition to the proposed actions mediated by rituximab (such as complement-dependent cytotoxicity, antibody-dependent cellular cytotoxicity, and apoptosis), rituximab may signal the tumor cells and inhibit constitutively activated survival signaling pathways (Raf-1-MEK1/2-ERK1/2, p38 MAPK, NF-kappaB, and Akt), resulting in inhibition of cell growth and of selectively anti-apoptotic gene products such as Bcl-2 and Bcl-(xL). The inhibition of these anti-apoptotic gene products by rituximab sensitizes drug-resistant tumor cells to apoptosis induced by a variety of cytotoxic chemotherapeutic drugs. Also, rituximab sensitizes NHL cells to apoptosis resulting from upregulation of death receptors, implicating a novel in vivo role of host involvement in rituximab-mediated effects. We have developed rituximab-resistant clones that do not respond to rituximab-mediated cell signaling. The clones exhibited hyperactivated cell survival pathways and overexpression of anti-apoptotic gene products and could not be chemosensitized by rituximab. Inhibitors of the survival signaling pathways reverse drug resistance in both wildtype cells and resistant clones. These findings identify several novel intracellular pathways modifyed by rituximab that sensitize NHL cells to both chemotherapy and immunotherapy, as well as several therapeutic targets whose modifications reverse resistance. These findings have clinical relevance for both prognosis and novel treatment strategies for patients with NHL.

Enhanced expression of CD20 in human tumor B cells is controlled through ERK-dependent mechanisms.

Rituximab, a chimeric Ab directed against CD20, induces apoptosis in targeted cells. Although the majority of B cell malignancies express the CD20 Ag, only approximately 50% of patients will respond to single-agent rituximab. The available data suggest that a decreased CD20 expression could account for the lack of response observed in some patients treated with rituximab. Despite the potential critical role of CD20 in the biology of B cell malignancies, the mechanisms controlling its expression are poorly understood. We evaluated the effect of the immune modulator agent bryostatin-1 on the expression of CD20 in non-Hodgkin's lymphoma cells. Using the B cell lines, DB and RAMOS, as well as tumor cells derived from a chronic lymphocytic leukemia patient, we demonstrated that bryostatin-1 enhanced the expression of both CD20 mRNA and protein. The enhanced expression of CD20 was associated with increased transcriptional activity of the CD20 gene, whereas the stability of CD20 mRNA was not affected. The effect of bryostatin-1 on CD20 expression in non-Hodgkin's lymphoma cells was mediated through the MAPK kinase/ERK signal transduction pathway and involved protein kinase C, but was independent of p38 MAPK and was insensitive to dexamethasone. Cells pretreated with bryostatin-1 were more susceptible to the proapoptotic effect of anti-CD20 Ab. Overall, these data demonstrate for the first time that ERK phosphorylation is required for the up-regulated expression of CD20 on B cell malignancies. The findings also suggest that bryostatin-1 and rituximab could be a valuable combined therapy for B cell malignancies.

Cellular and molecular signal transduction pathways modulated by rituximab (rituxan, anti-CD20 mAb) in non-Hodgkin's lymphoma: implications in chemosensitization and therapeutic intervention.

The clinical application of rituximab (chimeric mouse anti-human CD20 mAb, Rituxan, IDEC-C2B8), alone and/or combined with chemotherapy, has significantly ameliorated the treatment outcome of patients with relapsed and refractory low-grade or follicular non-Hodgkin's lymphoma (NHL). The exact in vivo mechanisms of action of rituximab are not fully understood, although antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and apoptosis have been suggested. We have proposed that modifications of the cellular signaling pathways by rituximab may be crucial for its clinical response. The B-cell restricted cell surface phosphoprotein CD20 is involved in many cellular signaling events including proliferation, activation, differentiation, and apoptosis upon crosslinking. Monomeric rituximab chemosensitizes drug-resistant NHL cells via selective downregulation of antiapoptotic factors through the type II mitochondrial apoptotic pathway. Several signaling pathways are affected by rituximab which are implicated in the underlying molecular mechanisms of chemosensitization. ARL (acquired immunodeficiency syndrome (AIDS)-related lymphoma) and non-ARL cell lines have been examined as in vitro model systems. In ARL, rituximab diminishes the activity of the p38MAPK signaling pathway resulting in inhibition of the interleukin (IL)-10/IL-10R autocrine/paracrine cytokine autoregulatory loop leading to the inhibition of constitutive STAT-3 activity and subsequent downregulation of Bcl-2 expression leading to chemosensitization. Rituximab upregulates Raf-1 kinase inhibitor protein (RKIP) expression in non-ARL cells. Through physical association with Raf-1 and nuclear factor kappaB (NF-kappa B)-inducing kinase (NIK), RKIP negatively regulates two major survival pathways, namely, the extracellular signal-regulated kinase1/2 (ERK1/2) and the NF-kappa B pathways, respectively. Downmodulation of the ERK1/2 and NF-kappa B pathways inhibits the transcriptional activity of AP-1 and NF-kappa B transcription factors, respectively, both of which lead to the downregulation of Bcl-(xL) (Bcl-2 related gene (long alternatively spliced variant of Bcl-x gene)) transcription and expression and sensitization to drug-induced apoptosis. Bcl-(xL)-overexpressing cells corroborated the pivotal role of Bcl-(xL) in chemosensitization. The specificity of rituximab-mediated signaling and functional effects were corroborated by the use of specific pharmacological inhibitors. Many patients do not respond and/or relapse and the mechanisms of unresponsiveness are unknown. Rituximab-resistant B-NHL clones were generated to investigate the acquired resistance to rituximab-mediated signaling, and chemosensitization. Resistant clones display different phenotypic, genetic and functional properties compared to wild-type cells. This review summarizes the data highlighting a novel role of rituximab as a signal-inducing antibody and as a chemosensitizing agent through negative regulation of major survival pathways. Studies presented herein also reveal several intracellular targets modified by rituximab, which can be exploited for therapeutic and prognostic purposes in the treatment of patients with rituximab- and drug-refractory NHL.

The role of B cell-mediated T cell costimulation in the efficacy of the T cell retargeting bispecific antibody BIS20x3.

In this study, we investigated the role of the naturally occurring B cell-mediated T cell costimulation in the antitumor efficacy of the bispecific Ab BIS20x3. BIS20x3 has a dual specificity for both CD20 and CD3 and has previously been shown to effectively direct the lytic potential of cytolytic T cells toward malignant, CD20(+) B cells. BIS20x3 instigated T cell-B cell interaction caused a dose-dependent activation of T cells that was 30 times stronger when compared with T cell activation induced by monovalent anti-CD3 Abs. The activation of T cells by BIS20x3 and B cells appeared functional and resulted in the rapid induction of high lytic potential in freshly isolated peripheral T cells. BIS20x3-mediated T cell-B cell interaction resulted in a significant up-regulation of ICAM-1 on B cells and the activation of T cells was found to be dependent on the interaction of ICAM-1 with LFA-1 and trans-activation by the NF-kappaB pathway. Also, the lytic potential of freshly isolated T cells activated via BIS20x3 appeared to be dependent on NF-kappaB signaling in the target B cells. Interestingly, the costimulatory signaling effects described in this study appeared specifically related to the targeting against CD20 because targeting against CD19, by a CD3xCD19-directed bispecific Ab, was significantly less effective in inducing T cell activation and T cell-mediated B cell lysis. Together these results demonstrate that the malignant B cells actively contribute to their own demise upon CD20-directed bispecific Ab-mediated T cell targeting.