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In breast cancer, humoral immune responses may contribute to clinical outcomes, especially in more immunogenic subtypes. Here, we investigated B lymphocyte subsets, immunoglobulin expression, and clonal features in breast tumors, focusing on aggressive triple-negative breast cancers (TNBC). In samples from patients with TNBC and healthy volunteers, circulating and tumor-infiltrating B lymphocytes (TIL-B) were evaluated. CD20 + CD27 + IgD - isotype-switched B lymphocytes were increased in tumors, compared with matched blood. TIL-B frequently formed stromal clusters with T lymphocytes and engaged in bidirectional functional cross-talk, consistent with gene signatures associated with lymphoid assembly, costimulation, cytokine-cytokine receptor interactions, cytotoxic T-cell activation, and T-cell-dependent B-cell activation. TIL-B-upregulated B-cell receptor (BCR) pathway molecules FOS and JUN, germinal center chemokine regulator RGS1, activation marker CD69, and TNFα signal transduction via NFκB, suggesting BCR-immune complex formation. Expression of genes associated with B lymphocyte recruitment and lymphoid assembly, including CXCL13, CXCR4, and DC-LAMP, was elevated in TNBC compared with other subtypes and normal breast. TIL-B-rich tumors showed expansion of IgG but not IgA isotypes, and IgG isotype switching positively associated with survival outcomes in TNBC. Clonal expansion was biased toward IgG, showing expansive clonal families with specific variable region gene combinations and narrow repertoires. Stronger positive selection pressure was present in the complementarity determining regions of IgG compared with their clonally related IgA in tumor samples. Overall, class-switched B lymphocyte lineage traits were conspicuous in TNBC, associated with improved clinical outcomes, and conferred IgG-biased, clonally expanded, and likely antigen-driven humoral responses. SIGNIFICANCE: Tumor-infiltrating B lymphocytes assemble in clusters, undergoing B-cell receptor-driven activation, proliferation, and isotype switching. Clonally expanded, IgG isotype-biased humoral immunity associates with favorable prognosis primarily in triple-negative breast cancers., (©2021 The Authors; Published by the American Association for Cancer Research.)

Objective To construct the eukaryotic expression vector of CD20 and overexpress soluble CD20 proteins in HEK293T cells. Methods The total RNA from human peripheral blood mononuclear cells (PBMCs) was used to amplify the coding sequence of CD20 by reverse transcription PCR, and then the CD20 gene was cloned into the expression vector pCMV3-C-His. After PCR and sequencing validation, the recombinant CD20 protein was transiently expressed in HEK293T cells and detected by ELISA and Western blot analysis. Moreover, the expression conditions were optimized to improve the expression level of CD20. Results The coding sequence of CD20 was successfully cloned into eukaryotic expression vector pCMV3-C-His. After 72 hours of transfection, the expression of CD20 was detected both in intracellular and supernatant by Western blot analysis. The passage number of HEK293T cells was related to the expression level of CD20 in supernatant. Conclusion The coding sequence of CD20 was successfully cloned into the eukaryotic expression vector, and CD20 was over expressed in HEK293T cells.

Although the CD20-targeted monoclonal antibody rituximab (RTX) has revolutionized the therapeutic landscape for B-cell malignancy, relapsed and refractory disease due to RTX resistance continue to constitute major challenges, illustrating the need for better therapies. Here, we apply drug-free macromolecular therapeutics (DFMT) that amplifies CD20 cross-linking to enhance apoptosis in RTX-resistant cells. Bispecific engager (anti-CD20 Fab' conjugated with oligonucleotide1) pretargets CD20 and the deletion of Fc-region minimizes its premature endocytosis in resistant cells that rapidly internalize and consume CD20/RTX complexes. Second-step delivery of multivalent polymeric effector (linear copolymer conjugated with multiple copies of complementary oligonucleotide 2) simultaneously hybridizes multiple CD20-bound engagers and strengthens CD20 ligation. Moreover, the restoration of CD20 expression by the pretreatment of cells with a polymer-gemcitabine conjugate, a CD20 expression enhancer, unleashes the full potential of DFMT in the CD20-deficient resistant cells. Hence, amplification of CD20 cross-linking is achieved by (1) the enhancement of surface CD20 accessibility, (2) the increase in CD20 expression, and (3) multimeric CD20 binding, which ultimately translates into the amplified activation of a wide range of innate apoptotic responses. We demonstrated that the altered molecular signaling pathway that originally results in RTX resistance could be circumvented and compensated by other DFMT-augmented pathways. Of note, our preliminary data provide proof-of-concept that CD20 cross-linking amplification emerges as an important strategy for overcoming RTX resistance.

CD20 is the first-line diagnostic marker of B-cells, which serves as the target of the therapeutic monoclonal antibodies in B-cell lymphomas and leukemias. Recently, aberrant CD20 expression has been described in a small series of papillary thyroid carcinomas (PTCs). We aimed to evaluate CD20 immunoexpression and to perform clinicopathologic correlation in a large set of thyroid tumors, including a cohort of high-grade thyroid cancer. A total of 625 cases of thyroid tumor comprised tissue microarrays of 538 PTCs and 47 follicular adenomas, and whole-slide sections of 40 aggressive thyroid carcinomas (10 radioiodine-refractory PTCs and 8 poorly differentiated, 5 anaplastic, and 17 medullary thyroid carcinomas) were immunostained with anti-CD20 monoclonal antibody. BRAF V600E mutation was tested by direct sequencing in 478 cancers. Our study found that a small subset of PTCs (<10%, mainly of classic variant) exhibited aberrant membranous expression of CD20. These tumors displayed less aggressive histological features and had a lower prevalence of BRAF V600E mutation. We also discovered that CD20 expression was maintained in 6%-20% of aggressive thyroid cancers but not observed in follicular adenomas. All CD20-positive tumor cells were negative for CD79a and PAX5. Aberrant expression of CD20 by thyroid cancer cells may present a diagnostic pitfall in cytologic evaluation of thyroid and cervical masses. Residual expression of CD20 in aggressive cancers may offer promise for translational implications, which merits further experimental investigation., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) is a rare lymphoma entity with an incidence of 0.1 to 0.2/100 000/y. Compared with the more common subtypes of classical Hodgkin lymphoma, NLPHL is characterized by distinct pathological and clinical features. Histologically, the disease-defining lymphocyte predominant cells consistently express CD20 but lack CD30. Clinically, NLPHL mostly has a rather indolent course, and patients usually are diagnosed in early stages. The prognosis of early-stage NLPHL is excellent, with progression-free survival and overall survival rates exceeding 90% after involved-field radiotherapy (IF-RT) alone (stage IA) or combined modality treatment consisting of a brief chemotherapy with 2 cycles of ABVD (doxorubicin, bleomycin, vinblastine, dacarbazine) chemotherapy followed by IF-RT (early stages other than stage IA). In contrast, patients with advanced disease at diagnosis tend to relapse either with NLPHL histology or with histological transformation into aggressive B-cell non-Hodgkin lymphoma despite more aggressive first-line treatment with 6 to 8 cycles of multiagent chemotherapy. However, even NLPHL patients with multiple relapses successfully respond to salvage therapy in many cases. Salvage therapies range from single-agent anti-CD20 antibody treatment to high-dose chemotherapy followed by autologous stem cell transplantation. Treatment at disease recurrence should be chosen on the basis of various factors, including histology at relapse, time to relapse, extent of disease at relapse, and prior treatment. Because death among NLPHL patients is more often caused by therapy-related late effects than lymphoma-related complications, optimizing the risk-benefit ratio of treatment by decreasing toxicity whenever possible is the major goal of clinical research in this disease., Competing Interests: Conflict-of-interest disclosure: The authors declare no competing financial interests., (© 2016 by The American Society of Hematology. All rights reserved.)

Background: The appropriate number of chemotherapy cycles for limited stage diffuse large B-cell lymphoma (DLBCL) patients without gross residual lesions after complete resection, has not been specifically questioned. We performed a multicenter, single-arm, phase 2 study to investigate the feasibility of 3 cycles of abbreviated R-CHOP chemotherapy in low-risk patients with completely resected localized CD20+ DLBCL., Results: Between December 2010 and May 2013, we recruited 23 patients. One was excluded due to ineligibility, and hence, 22 were included in the final analysis. The primary sites comprised the intestine (n = 15), cervical lymph nodes (n = 4), stomach (n = 1), tonsil (n = 1), and spleen (n = 1). All patients successfully completed the 3 cycles of planned R-CHOP chemotherapy. Over a median follow-up of 39.5 months (95% confidence interval, 29.9-47.1 months), both the estimated 2-year disease-free survival and overall survival rates was 95% confidence interval, 85.9-104.1%. Only one patient with an international prognostic index of 2 experienced relapse and died. The most common grade 3 or 4 toxicity condition included neutropenia (n = 8, 36.4%). Three patients experienced grade 3 febrile neutropenia, but no grade 3 or 4 non-hematologic toxicity was observed., Materials and Methods: DLBCL patients without residual lesions after resection were enrolled and R-CHOP chemotherapy was repeated at 3-week-intervals over 3 cycles. The primary endpoint was 2-year disease-free survival., Conclusions: Three cycles of abbreviated R-CHOP immunochemotherapy is feasible for completely resected low risk localized DLBCL.

Monoclonal Abs against CD20 reduce the number of relapses in multiple sclerosis (MS); commonly this effect is solely attributed to depletion of B cells. Recently, however, a subset of CD3(+)CD20(+) T cells has been described that is also targeted by the anti-CD20 mAb rituximab. Because the existence of cells coexpressing CD3 and CD20 is controversial and features of this subpopulation are poorly understood, we studied this issue in detail. In this study, we confirm that 3-5% of circulating human T cells display CD20 on their surface and transcribe both CD3 and CD20. We report that these CD3(+)CD20(+) T cells pervade thymus, bone marrow, and secondary lymphatic organs. They are found in the cerebrospinal fluid even in the absence of inflammation; in the cerebrospinal fluid of MS patients they occur at a frequency similar to B cells. Phenotypically, these T cells are enriched in CD8(+) and CD45RO(+) memory cells and in CCR7(-) cells. Functionally, they show a higher frequency of IL-4-, IL-17-, IFN-γ-, and TNF-α-producing cells compared with T cells lacking CD20. CD20-expressing T cells respond variably to immunomodulatory treatments given to MS patients: they are reduced by fingolimod, alemtuzumab, and dimethyl fumarate, whereas natalizumab disproportionally increases them in the blood. After depletion by rituximab, they show earlier and higher repopulation than CD20(+) B cells. Taken together, human CD3(+)CD20(+) T cells pervade lymphatic organs and the cerebrospinal fluid, have a strong ability to produce different cytokines, and respond to MS disease modifying drugs., (Copyright © 2016 by The American Association of Immunologists, Inc.)

CD20 is a differentiation related cell surface phosphoprotein that is expressed during early pre-B cell stages until plasma cell differentiation, and is a suitable molecular target for B-cell malignancies by monoclonal antibodies such as rituximab, ofatumumab, obinutuzumab and others. CD20 expression is confirmed in most B-cell malignancies; however, the protein expression level varies in each patient, even in de novo tumors, and down-modulation of CD20 expression after chemoimmunotherapy with rituximab, resulting in rituximab resistance, has been recognized in the clinical setting. Recent reports suggest that genetic and epigenetic mechanisms are correlated with aberrantly low CD20 expression in de novo tumors and relapsed/refractory disease after using rituximab. Furthermore, some targeting drugs, such as lenalidomide, bortezomib and ibrutinib, directly or indirectly affect CD20 protein expression. CD20-negative phenotypically-changed DLBCL after rituximab use tends to show an aggressive clinical course and poor outcome with resistance to not only rituximab, but also conventional salvage chemo-regimens. Understanding of the mechanisms of CD20-negative phenotype may contribute to establish strategies for overcoming chemo-refractory B-cell malignancies. In this manuscript, recent progress of research on molecular and clinical features of CD20 protein and CD20-negative B-cell malignancies was reviewed.

Background: Mantle cell lymphoma (MCL), a B-cell lymphoma, pursues a relatively aggressive course, is resistant to long-term remission, and is associated with a poor prognosis. There is a pressing need for innovative treatment approaches against MCL. One such approach is targeted delivery of cytotoxic drugs to MCL cells., Materials and Methods: In the current investigation, we pursued a strategy to employ retinoid-based or curcumin-based nanoscale delivery particles, called nanodisks (NDs), for targeted drug delivery to MCL cells (Granta), and human follicular lymphoma (HF-1) cells. The cells were incubated with NDs made of CD20 single-chain variable antibody fragment (scFv)/apolipoprotein A-1 fusion protein, and loaded with either all-trans retinoic acid (ATRA) or curcumin, and cell apoptosis was measured using flow cytometry., Results and Conclusion: At 10 μM, curcumin-ND induced cell death more effectively than ATRA-ND. Combination of curcumin-ND and ATRA-ND significantly enhanced the biological activity of these drugs against lymphoma cells compared to individual treatments., (Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Purpose: Obinutuzumab (GA101), a novel glycoengineered type II anti-CD20 monoclonal antibody, demonstrated responses in single-arm studies of patients with relapsed/refractory non-Hodgkin lymphoma. This is the first prospective, randomized study comparing safety and efficacy of obinutuzumab with rituximab in relapsed indolent lymphoma. The primary end point of this study was the overall response rate (ORR) in patients with follicular lymphoma after induction and safety in patients with indolent lymphoma., Patients and Methods: A total of 175 patients with relapsed CD20(+) indolent lymphoma requiring therapy and with previous response to a rituximab-containing regimen were randomly assigned (1:1) to four once-per-week infusions of either obinutuzumab (1,000 mg) or rituximab (375 mg/m(2)). Patients without evidence of disease progression after induction therapy received obinutuzumab or rituximab maintenance therapy every 2 months for up to 2 years., Results: Among patients with follicular lymphoma (n = 149), ORR seemed higher for obinutuzumab than rituximab (44.6% v 33.3%; P = .08). This observation was also demonstrated by a blinded independent review panel that measured a higher ORR for obinutuzumab (44.6% v 26.7%; P = .01). However, this difference did not translate into an improvement in progression-free survival. No new safety signals were observed for obinutuzumab, and the incidence of adverse events was balanced between arms, with the exception of infusion-related reactions and cough, which were higher in the obinutuzumab arm., Conclusion: Obinutuzumab demonstrated a higher ORR without appreciable differences in safety compared with rituximab. However, the clinical benefit of obinutuzumab in this setting remains unclear and should be evaluated within phase III trials., Competing Interests: Authors' disclosures of potential conflicts of interest are found in the article online at www.jco.org. Author contributions are found at the end of this article., (© 2015 by American Society of Clinical Oncology.)

Rituximab is a monoclonal antibody against CD20. Rituximab combined with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) chemotherapy, termed R-CHOP, have improved the overall survival of patients with B-cell lymphoma in comparison with that of CHOP therapy. However, as with other molecularly-targeted therapies, resistance to rituximab could emerge sooner or later after rituximab administration. A number of mechanisms for rituximab resistance have been proposed, including downregulation of CD20 protein expression. Differential diagnosis of B-cell proliferation with reduced or lost CD20 expression includes not only B-cell lymphomas with CD20 downregulation, but also other tumorous and non-tumorous lesions. These include precursor B-cell neoplasms such as B acute lymphoblastic leukemia/lymphoblastic lymphoma (B-ALL/LBL) and hematogones, a normal precursor B-cell proliferation during regeneration of hematopoiesis, typically observed following bone marrow suppression by chemotherapy. It is important to distinguish these possibilities because distinct therapies are required for each. In this paper, we report a case where bone marrow infiltration of follicular lymphoma histopathologically mimicked hematogones or B-ALL/LBL when CD20 expression was downregulated in follicular lymphoma after R-CHOP therapy.

Understanding how viruses subvert host immunity and persist is essential for developing strategies to eliminate infection. T cell exhaustion during chronic viral infection is well described, but effects on antibody-mediated effector activity are unclear. Herein, we show that increased amounts of immune complexes generated in mice persistently infected with lymphocytic choriomeningitis virus (LCMV) suppressed multiple Fcγ-receptor (FcγR) functions. The high amounts of immune complexes suppressed antibody-mediated cell depletion, therapeutic antibody-killing of LCMV infected cells and human CD20-expressing tumors, as well as reduced immune complex-mediated cross-presentation to T cells. Suppression of FcγR activity was not due to inhibitory FcγRs or high concentrations of free antibody, and proper FcγR functions were restored when persistently infected mice specifically lacked immune complexes. Thus, we identify a mechanism of immunosuppression during viral persistence with implications for understanding effective antibody activity aimed at pathogen control., (Copyright © 2015 Elsevier Inc. All rights reserved.)

T cell dysfunction is well documented during chronic viral infections but little is known about functional abnormalities in humoral immunity. Here we report that mice persistently infected with lymphocytic choriomeningitis virus (LCMV) exhibit a severe defect in Fcγ-receptor (FcγR)-mediated antibody effector functions. Using transgenic mice expressing human CD20, we found that chronic LCMV infection impaired the depletion of B cells with rituximab, an anti-CD20 antibody widely used for the treatment of B cell lymphomas. In addition, FcγR-dependent activation of dendritic cells by agonistic anti-CD40 antibody was compromised in chronically infected mice. These defects were due to viral antigen-antibody complexes and not the chronic infection per se, because FcγR-mediated effector functions were normal in persistently infected mice that lacked LCMV-specific antibodies. Our findings have implications for the therapeutic use of antibodies and suggest that high levels of pre-existing immune complexes could limit the effectiveness of antibody therapy in humans., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Ponesimod, a novel selective sphingosine-1-phosphate 1 receptor modulator in the development for the treatment of autoimmune diseases, dose-dependently reduced lymphocyte counts in peripheral blood of healthy subjects. It rapidly and transiently reduced the number of circulating T and B cells, but not natural killer cells. T lymphocyte subsets exhibited differential sensitivities with a maximum decrease from baseline ranging from 67% to 89% following high doses. Naïve T cells were more sensitive than memory T cells. CD4(+) T cells were more sensitive than CD8(+) T cells or CD4(+)CD25(+) T regulatory cells. The differential effects on specialized T cell subsets may contribute to the immunomodulatory activity of ponesimod. The therapeutic potential of ponesimod has been recently shown in phase II studies of chronic plaque psoriasis and relapsing-remitting multiple sclerosis. Our data suggest that lymphocyte sequestration underlies the therapeutic potential of ponesimod in multiple autoimmune and chronic inflammatory diseases.