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To investigate the diagnostic value of intraoperative cytology and rapid immunocytochemistry in primary central nervous system lymphoma. 254 cases of lymphoma and 82 cases of non-lymphoma were collected from 2010 to 2023. Frozen section(FS) was using alone in 44 cases during 2010-2014, FS and intraoperative cytology(IC) were using in 251 cases during 2015 to 2022. Rapid immunocytochemical(RICC, CD20, GFAP) were using with FS + IC in 41 cases during 2021 to 2023. Method One: According to the results of archives, statistic the diagnostic accuracy of lymphoma during three time periods. Method Two: All cases were randomly renumbered, 4 neuropathologists compared the accuracy of independent histology and that of combining cytology. The archives showed the diagnostic accuracy of FS in PCNSL was 77.27%, FS + IC was 86.06%, FS + IC + RICC was 92.68%. The retrospective study demonstrated the diagnostic accuracy of FS was 79.76%, FS + IC was 87.33% and FS + IC + RICC was 92.68%. The positive predictive value, negative predictive value, sensitivity, specificity and accuracy of CD20 were 100%, 76.92%, 90.32%, 100% and 92.68%, respectively. The results of the paired χ 2  test was no statistically significant difference (0.05 < P < 0.1) between FS + IC + RICC and immunohistochemical (IHC) diagnosis of paraffin sections. The integration of IC + RICC + FS diagnosis can significantly enhance the intraoperative diagnostic accuracy of PCNSL and rectify potential errors that may occurred when relying solely on FS diagnosis., (© 2024. The Author(s).)

Background: CD20 + T cells represent up to 5% of circulating T lymphocytes. These cells have been shown to produce higher levels of IL-17A and IFN-γ than those of CD20 -  T lymphocytes. Some reports described the role of CD20 + T cells in autoimmune disorders such as multiple sclerosis and rheumatoid arthritis possibly due to their ability to produce these inflammatory cytokines. This study is aimed at describing the behavior of CD20 + T lymphocytes in the most frequent autoimmune disorder, i.e., Hashimoto's thyroiditis (HT), presenting isolated or associated to further autoaggressive disorders in a frame of poly-autoimmunity., Methods: The study group encompasses 65 HT patients: 23 presenting in isolated form (IT) and 42 with an associated non-endocrine autoimmune disorder [16 with chronic atrophic gastritis (CAG), 15 with nonsegmental vitiligo (VIT), and 11 with celiac disease (CD)]. Twenty healthy donors act as control group (HD). Chronic use of interfering drugs, severe or chronic disorders, and pregnancy and lactation were used as exclusion criteria. Whole blood samples (100 µl) were stained with fluorescent-labeled antibodies (anti-CD45, anti-CD3, anti-CD19, anti-CD16, anti-CD56, anti-CD4, anti-CD8, anti-CD20). Red blood cells were then lysed by adding 1 ml of hypotonic buffer, and samples were acquired on a Flow Cytometer., Results: CD3 + CD8 + CD20 + T lymphocytes' percentages, were significantly higher in the whole group of autoimmune patients compared to healthy donors (p = 0.0145). Dividing HT patients based on the type of presentation of autoimmune thyroiditis, CAG group showed the highest percentage of these cells as compared to HD and CD (p = 0.0058). IT patients showed higher percentages of CD3 + CD8 + CD20 + cells than those of HD patients although not reaching statistical significance. However, dividing IT group based on thyroid function, hypothyroid patients showed higher CD8 + CD20 + cell percentages than those of HD and euthyroid patients (p = 0.0111). Moreover, in IT patients, these cells were negatively correlated with FT4 levels (p = 0.0171; r = -0.4921)., Conclusions: These preliminary findings indicate that CD8 + CD20 + T cells are activated in patients with autoimmune thyroiditis and may behave differently according to the presence of poly-autoimmunity and hypothyroidism., (© 2024. The Author(s).)

Introduction: Immune dysregulation plays a major role in cancer progression. The quantification of lymphocytic spatial inflammation may enable spatial system biology, improve understanding of therapeutic resistance, and contribute to prognostic imaging biomarkers., Methods: In this paper, we propose a knowledge-guided deep learning framework to measure the lymphocytic spatial architecture on human H&E tissue, where the fidelity of training labels is maximized through single-cell resolution image registration of H&E to IHC. We demonstrate that such an approach enables pixel-perfect ground-truth labeling of lymphocytes on H&E as measured by IHC. We then experimentally validate our technique in a genetically engineered, immune-compromised Rag2 mouse model, where Rag2 knockout mice lacking mature lymphocytes are used as a negative experimental control. Such experimental validation moves beyond the classical statistical testing of deep learning models and demonstrates feasibility of more rigorous validation strategies that integrate computational science and basic science., Results: Using our developed approach, we automatically annotated more than 111,000 human nuclei (45,611 CD3/CD20 positive lymphocytes) on H&E images to develop our model, which achieved an AUC of 0.78 and 0.71 on internal hold-out testing data and external testing on an independent dataset, respectively. As a measure of the global spatial architecture of the lymphocytic microenvironment, the average structural similarity between predicted lymphocytic density maps and ground truth lymphocytic density maps was 0.86 ± 0.06 on testing data. On experimental mouse model validation, we measured a lymphocytic density of 96.5 ± %1% in a Rag2 +/- control mouse, compared to an average of 16.2 ± %5% in Rag2 -/- immune knockout mice (p<0.0001, ANOVA-test)., Discussion: These results demonstrate that CD3/CD20 positive lymphocytes can be accurately detected and characterized on H&E by deep learning and generalized across species. Collectively, these data suggest that our understanding of complex biological systems may benefit from computationally-derived spatial analysis, as well as integration of computational science and basic science., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Li, Heirman, Rickard, Sotolongo, Castillo, Adanlawo, Everitt, Hodgin, Watts, Janowczyk, Mowery, Barisoni and Lafata.)

Background: In patients with untreated CD20-positive diffuse large B-cell lymphoma (DLBCL), a phase 3 trial was carried out to evaluate the efficacy and safety of zuberitamab plus CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone; Hi-CHOP) versus rituximab plus CHOP (R-CHOP) treatment regimens., Methods: In a 2:1 ratio, eligible patients were assigned randomly to receive treatment of six cycles of either 375 mg/m 2 zuberitamab or rituximab together with conventional CHOP chemotherapy. The objective response rate (ORR) at C6D50 served as the primary endpoint, and a non-inferiority margin of 10% was established. The secondary endpoints included the complete response (CR) rate at C6D50, duration of response (DOR), progression-free survival (PFS) and event-free survival (EFS) judged by blinded-independent review committee (BIRC), overall survival (OS) and safety outcomes., Results: Of the 487 randomized patients, 423 patients including 287 in the Hi-CHOP and 136 in the R-CHOP groups completed the C6D50 assessment. For the full analysis set (FAS) and per-protocol set (PPS), BIRC-assessed ORR at C6D50 for the Hi-CHOP and R-CHOP groups were 83.5% versus 81.4% and 95.3% versus 93.7%, respectively. The non-inferiority was confirmed as the lower limit of the two-sided 95% CI for the intergroup differences of -5.2% and -3.3%; both were >-10% in the FAS and PPS. The BIRC-assessed CR rate of Hi-CHOP was significantly higher in PPS (85.7% vs 77.3%, p=0.038), but comparable in FAS (75.2% vs 67.9%, p=0.092). After a median follow-up of 29.6 months, patients in the Hi-CHOP group had a slight advantage with regard to the DOR (HR 0.74, p=0.173), PFS (HR 0.67, p=0.057), EFS (HR 0.90, p=0.517) and OS (HR 0.60, p=0.059). Patients with the germinal-center B cell-like subtype who received Hi-CHOP exhibited statistically significant improvements in ORR (p=0.034) and CR rate (p=0.038) at C6D50, EFS (p=0.046) and OS (p=0.014). Treatment-emergent adverse event occurrence rates were comparable across groups (all p>0.05). Infusion-related responses occurred more often in the Hi-CHOP group (32.1% vs 19.9%, p=0.006), all of grade 1-3 severity., Conclusions: Zuberitamab (375 mg/m 2 ) plus CHOP was non-inferior to R-CHOP regarding ORR but exhibited a higher CR rate and was well tolerated in CD20-positive, previously untreated Chinese patients with DLBCL., Trial Registration Number: Chinese Clinical Trial Registry, ChiCTR2000040602, retrospectively registered., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Abstract: Although CD20 expression is typically scarce in mycosis fungoides (MF), it is more commonly associated with T-cell lymphomas. Nevertheless, isolated instances of CD20-positive MF have been documented infrequently. Here, we present a unique case of CD20-positive MF in a 30-year-old man who manifested with a hypopigmented patch on the anterior chest. Histopathological examination revealed epidermotropic infiltrates of small- to medium-sized lymphocytes with hyperchromatic and cerebriform nuclei aligned along the basal and low-mid layers of the epidermis. Immunophenotypic analysis demonstrated neoplastic T cells expressing CD4+, CD8+, and CD3+ with the loss of CD7. Intriguingly, a notable subset of the neoplastic T cells exhibited CD20 expression. This case contributes to the sparse literature on CD20-positive MF and underscores its diagnostic and clinical ramifications. The role of B cells has been more thoroughly characterized in T-cell lymphomas other than MF. However, its significance in MF remains unclear due to the scarcity of reported cases. Some hypotheses propose that the B cells' expression might indicate immune dysregulation or complex interactions within the tumor microenvironment. Another perspective suggests it could signify a progression of the disease towards a more aggressive lymphoma phenotype. Further investigation and documentation of similar cases is imperative to elucidate the clinical features, prognosis, and optimal therapeutic strategies. The long-term prognosis and outcomes in patients with hypopigmented MF and CD20 positivity remain ambiguous, underscoring the necessity for continued research and scrutiny of analogous cases., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Purpose: Despite chimeric antigen receptor (CAR) T-cell therapy has achieved great advances in recent year, approximately 50% of relapsed/refractory B cell acute lymphoblastic leukemia (r/r B-ALL) patients treated with CAR-T experience relapse 6 months post CAR-T treatment. CD20 express on 30 to 50% of B-ALL, which makes CD20 Monoclonal Antibody as one of the potential therapy strategies to decrease the tumor burden and improve the efficacy of CAR-T therapy. Adding Rituximab to chemotherapy protocol had been demonstrated to improve the outcome for CD20-positive ALL. However, rare study explored the influence of Rituximab combined with CAR-T therapy., Methods: We retrospectively analyzed 20 r/r B-ALL patients who received CAR-T therapy, all of whom had failed multiple lines of therapy. Before CAR-T infusion, we administered Rituximab to 10 patients with high CD20 expression at a dose of 375 mg/m 2 for 1 day. Meanwhile, we selected 10 patients with the comparable features who underwent CAR-T treatment without Rituximab in the same period as the control group. In vitro, the surface molecule expression and killing of CAR-T post Rituximab-treated B-ALL cells co-incubated with CAR-T cells were detected by flow cytometry., Results: The median follow-up of Rituximab and Control groups were 29.27 and 9.83 months. We found that adding Rituximab may confer a favorable prognosis compared with Control group. The 2-year overall survival (OS) and leukemia-free survival (LFS) rates both were longer in the Rituximab group (90% vs. 26.7%, p = 0.0342; 41.7% vs. 25%, p = 0.308). In vitro, we observed that Rituximab-treated tumour cells are more sensitive to CAR-T killing and a broad range of cytokines and chemokines were produced when Rituximab-treated Nalm-6 cells co-cultured with 19-22CAR-T cells, such as interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and interleukin-2 (IL-2). To investigate whether Rituximab has an effect on CAR-T persistence, we stimulated CAR-T cells repeatedly in vitro with Rituximab-treated Nalm-6 to evaluate the changes in CAR-T surface exhaustion molecules at different times. We found that the expression of exhaustion molecules (LAG-3, PD-1, TIM-3) on CAR-T cells were significantly lower in the Rituximab group than in the Control group., Conclusion: Rituximab combined with CAR-T therapy is effective for improving the long-term prognosis of B-ALL patients who have failed multiple lines of therapy. In vitro, we observed that rituximab potentially improves CAR-T efficacy by sensitizing ALL to CART-mediated cytotoxicity and reducing CAR-T exhaustion., (© 2024. The Author(s).)

T cells play critical roles in adipose tissue (AT) inflammation. The role of CD20 + T cell in AT dysfunction and their contributing to insulin resistance (IR) and type 2 diabetes progression, is not known. The aim was to characterize CD20 + T cells in omental (OAT), subcutaneous (SAT) and peripheral blood (PB) from subjects with obesity (OB, n = 42), by flow cytometry. Eight subjects were evaluated before (T1) and 12 months post (T2) bariatric/metabolic surgery (BMS). PB from subjects without obesity (nOB, n = 12) was also collected. Higher percentage of CD20 + T cells was observed in OAT, compared to PB or SAT, in OB-T1. CD20 expression by PB CD4 + T cells was inversely correlated with adiposity markers, while follicular-like CD20 + T cells were positively correlated with impaired glucose tolerance (increased HbA1c). Notably, among OB-T1, IR establishment was marked by a lower percentage and absolute number of PB CD20 + T cells, compared nOB. Obesity was associated with higher percentage of activated CD20 + T cells; however, OAT-infiltrated CD20 + T cells from OB-T1 with diabetes displayed the lowest activation. CD20 + T cells infiltrating OAT from OB-T1 displayed a phenotype towards IFN-γ-producing Th1 and Tc1 cells. After BMS, the percentage of PB CD4 + CD20 + T cells increased, with reduced Th1 and increased Th17 phenotype. Whereas in OAT the percentage of CD20 + T cells with Th1/17 and Tc1/17 phenotypes increased. Interestingly, OAT from OB pre/post BMS maintained higher frequency of effector memory CD20 + T cells. In conclusion, CD20 + T cells may play a prominent role in obesity-related AT inflammation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Graves' disease (GD), an autoimmune thyroid disease, is one of the main autoimmune diseases in the general population. It is known that the pathophysiology of this disease may be related to immunological mechanisms dysregulation. These mechanisms can be influenced by GD therapies, such as iodide or antithyroid drugs (ATD)., Objective: Verify relation between clinical, biochemical and treatment modalities used prior to surgery and histopathological characteristics observed in total thyroidectomy products from patients previously diagnosed with Graves' disease. Furthermore, these data were related to composition of lymphocytic infiltrate in terms of proportions of lymphocytes CD4 + , CD8 + , CD25 + and CD20 + . We aim to contribute to the understanding of the evolution pattern of GD, whose pathophysiology is not yet completely understood., Methods: Cross-sectional study assessing thyroidectomy products for the presence of lymphocytic infiltrate, as well as the proportion and intensity of CD4 + , CD8 + , CD25 + and CD20 + markers. We selected 50 patients who underwent total or partial thyroidectomy in a tertiary service between 1996 and 2013 due to GD with histopathological confirmation. The control group (non-autoimmune disease group) consisted of 12 patients with histopathological data compatible with normal perilesional thyroid parenchyma. The intensity of lymphocytic infiltrate and immunohistochemical expression of the markers CD4 + (helper T lymphocytes), CD8 + (cytotoxic T lymphocytes), CD25 + (regulatory T lymphocytes) and CD20 + (B lymphocytes) were retrospectively evaluated and relationship with ultrasound, laboratory and clinical data was assessed., Results: No differences were found in intensity, presence of lymphoid follicles, and expression of CD4+/CD8+/CD25+ in patients with GD who did or did not use ATD or iodide. In the group that did not use ATD, a higher proportion of CD20 + expression was found. The GD group was associated with hyperplastic epithelium and the control group was associated with simple epithelium. There was no difference in ultrasound thyroid volume between the groups. In GD patients with mild lymphocytic infiltrate, higher free thyroxin (FT4) levels were observed than those in patients with no infiltrate or moderate infiltrate., Conclusion: We found a lower proportion of intrathyroidal CD20 + B lymphocytes in patients under use of methimazole. However, no difference was observed in intrathyroidal lymphocyte subpopulations related to the short-term use of iodide. The understanding of thyroid autoimmunity, as well as identifying points of pharmacological modulation, are very important for advancement and improvement in treatments for these diseases., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Denise Engelbrecht Zantut Wittmann reports equipment, drugs, or supplies and statistical analysis were provided by State University of Campinas. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Classical Hodgkin Lymphoma (CHL) is a rare malignant neoplasm of the lymphatic system. While CHL typically responds well to conventional treatments, some cases may experience relapse to other subtypes, with the development of secondary peripheral T-cell lymphoma (PTCL) being relatively uncommon. Herein, we report a rare case of nodal T follicular helper cell lymphomas,nos (nTFHL-NOS) secondary to CHL, accompanied by aberrant CD20 expression and clonal rearrangements of T-cell receptor (TCR) and immunoglobulin (IG). A 74-year-old male, was diagnosed with CHL, leaning toward the mixed cell type, 6 years ago. He received six cycles of the Adriamycin, Bleomycin, Vinblastine, Dacarbazine (ABVD) regimen, achieving complete clinical remission. The patient was admitted to our hospital due to the appearance of multiple skin nodules 66 months later. Histopathological analysis revealed nTFHL-NOS, with aberrant CD20 expression and clonal rearrangements of TCR and IG. The patient underwent two cycles of chemotherapy with brentuximab vedotin and the Gemcitabine-Oxaliplatin (G-mox) regimen, resulting in a reduction of the skin lesions to 2 cm × 1 cm. We discuss this rare case and review related literature., (© 2024. The Author(s) under exclusive licence to The Japanese Society for Clinical Molecular Morphology.)

Multiple sclerosis (MS) is a disease of the central nervous system (CNS) characterized by inflammation and autoimmune responses. This review explores the participation of T cells, particularly certain CD3 + CD20 + T cells, in the clinical manifestations of MS and highlights their presence in diagnosed patients. These T cells show aberrant expression of CD20, normally considered a B-cell marker. In this review, relevant journal articles available in PubMed and CINAHL were identified by employing diverse search terms, such as MS, CD3 + CD20 + T cells, the incidence and significance of CD3 + CD20 + T cells in MS patients, and the impact of rituximab treatment. The search was limited to articles published in the ten-year period from 2014 to 2024. The results of this review suggest that most scholars agree on the presence of CD3 + CD20 + T cells in cerebrospinal fluid. Emerging concepts relate to the fundamental role of CD20-expressing T cells in determining the target and efficacy of MS therapeutics and the presence of T cells in the cerebrospinal fluid of MS patients. The results clearly show that CD20 + T cells indicate disease chronicity and high disease activity.

Mycosis fungoides (MF) represents the most common type of primary cutaneous T-cell lymphoma. Recognition of MF variants with divergent immunophenotypes is important for accurate diagnosis and appropriate management, as they can be confused with other lymphoma subtypes. We present a case of a 49-year-old male previously diagnosed with a cutaneous lymphoproliferative disorder with an unusual NK/T-cell phenotype. He presented with a 10-year history of pelvic girdle rash involving the right hip and upper thigh. The lesions were characterized as atrophic patches concentrated in sun-protected areas and involving 10% of the body surface area. Shave biopsies revealed an atypical epidermotropic infiltrate composed of hyperchromatic small to medium-sized lymphocytes with perinuclear halos and "tagging" along the dermal-epidermal junction. The immunophenotype was unusual in that the neoplastic lymphocytes showed complete loss of pan T-cell antigens along with expression of CD56, cytotoxic markers, and weak CD20. All other B-cell markers were negative. The combination of clinical findings, in addition to the histopathologic and immunophenotypic profile, were diagnostic of null T-cell phenotype MF with aberrant expression of CD56 and CD20. Null T-cell phenotype MF is very uncommon, can be diagnostically challenging, and can mislead the diagnosis of aggressive lymphoma subtypes., (© 2024 The Authors. Journal of Cutaneous Pathology published by John Wiley & Sons Ltd.)

The CD20 antigen is a key target for several diseases including lymphoma and autoimmune diseases. For over 20 years, several monoclonal antibodies were developed to treat CD20-related disorders. As many therapeutic proteins, their clinical use is however limited due to their nature with a costly biotechnological procedure and side effects such as the production of anti-drug neutralizing antibodies. Nucleic acid aptamers have some advantages over mAbs and are currently investigated for clinical use. We herein report the selection of DNA aptamer by using a peptide-based CE-SELEX (Capillary Electrophoresis-Systematic Evolution of Ligands by Exponential Enrichment) method. It was demonstrated that these aptamers bind specifically a CD20-expressing human cell line, with K d estimated from isothermal titration calorimetry experiments in the micromolar range. This study demonstrates that the CE-SELEX is suitable as alternative method to the conventional Cell-SELEX to discover new cell-targeting compounds., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Recently, new and advanced techniques have been adopted to design and produce nanobodies, which are used in diagnostic and immunotherapy treatments. Traditionally, nanobodies are prepared from camelid immune libraries that require animal treatments. However, such approaches require large library sizes and complicated selection procedures. The current study has employed CDR grafting and site-directed mutagenesis techniques to create genetically engineered nanobodies against the tumor marker CD20 (anti-CD20 nanobodies) used in leukemia treatment., Methods and Results: In this study, we utilized the swapping method to graft CDRs from the VH Rituximab antibody to VHH CDRs. We aimed to enhance the binding affinity of the nanobodies by substituting the amino acids (Y101R-Y102R-Y107R) in the VHH-CDR3. To assess the binding capacity of the mutated nanobodies, we conducted an ELISA test. Moreover, through flow cytometry analysis, we compared the fluorescence intensity of the grafted CD20 and mutant nanobodies with that of the commercially available human anti-CD20 in Raji cells. The results showed a significant difference in the fluorescence intensity of the grafted nanobodies and mutant nanobodies when compared to the commercially available human anti-CD20., Conclusion: The approach we followed in this study makes it possible to create multiple anti-CD20 nanobodies with varying affinities without the need for extensive selection efforts. Additionally, our research has demonstrated that computational tools are highly reliable in designing functional nanobodies., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Rituximab (RTX) plus chemotherapy (R-CHOP) applied as a first-line therapy for lymphoma leads to a relapse in approximately 40% of the patients. Therefore, novel approaches to treat aggressive lymphomas are being intensively investigated. Several RTX-resistant (RR) cell lines have been established as surrogate models to study resistance to R-CHOP. Our study reveals that RR cells are characterized by a major downregulation of CD37, a molecule currently explored as a target for immunotherapy. Using CD20 knockout (KO) cell lines, we demonstrate that CD20 and CD37 form a complex, and hypothesize that the presence of CD20 stabilizes CD37 in the cell membrane. Consequently, we observe a diminished cytotoxicity of anti-CD37 monoclonal antibody (mAb) in complement-dependent cytotoxicity in both RR and CD20 KO cells that can be partially restored upon lysosome inhibition. On the other hand, the internalization rate of anti-CD37 mAb in CD20 KO cells is increased when compared to controls, suggesting unhampered efficacy of antibody drug conjugates (ADCs). Importantly, even a major downregulation in CD37 levels does not hamper the efficacy of CD37-directed chimeric antigen receptor (CAR) T cells. In summary, we present here a novel mechanism of CD37 regulation with further implications for the use of anti-CD37 immunotherapies., Competing Interests: No potential conflict of interest was reported by the author(s)., (© 2024 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Mosunetuzumab (Mosun) is a CD20xCD3 T-cell engaging bispecific antibody that redirects T cells to eliminate malignant B cells. The approved step-up dose regimen of 1/2/60/30 mg IV is designed to mitigate cytokine release syndrome (CRS) and maximize efficacy in early cycles. A population pharmacokinetic (popPK) model was developed from 439 patients with relapsed/refractory B-Cell Non-Hodgkin lymphoma receiving Mosun IV monotherapy, including fixed dosing (0.05-2.8 mg IV every 3 weeks (q3w)) and Cycle 1 step-up dosing groups (0.4/1/2.8-1/2/60/30 mg IV q3w). Prior to Mosun treatment, ~50% of patients had residual levels of anti-CD20 drugs (e.g., rituximab or obinutuzumab) from prior treatment. CD20 receptor binding dynamics and rituximab/obinutuzumab PK were incorporated into the model to calculate the Mosun CD20 receptor occupancy percentage (RO%) over time. A two-compartment model with time-dependent clearance (CL) best described the data. The typical patient had an initial CL of 1.08 L/day, transitioning to a steady-state CL of 0.584 L/day. Statistically relevant covariates on PK parameters included body weight, albumin, sex, tumor burden, and baseline anti-CD20 drug concentration; no covariate was found to have a clinically relevant impact on exposure at the approved dose. Mosun CD20 RO% was highly variable, attributed to the large variability in residual baseline anti-CD20 drug concentration (median = 10 μg/mL). The 60 mg loading doses increased Mosun CD20 RO% in Cycle 1, providing efficacious exposures in the presence of the competing anti-CD20 drugs. PopPK model simulations, investigating Mosun dose delays, informed treatment resumption protocols to ensure CRS mitigation., (© 2024 The Author(s). Clinical and Translational Science published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

Primary central nervous system (PCNS) extranodal NK/T-cell lymphoma, nasal type (ENKTCL), is an exceedingly rare tumor. To the best of our knowledge, only 27 cases and only one reported aberrant CD20 expression have been documented in the literature. Here we present a second case of PCNS ENKTCL with aberrant CD20 expression in a 43-year-old immunocompetent Chinese female. The patient presented with tremors, weakness in the right upper limb, and a slow reaction. Magnetic resonance imaging revealed multiple brain lesions. A histological examination revealed a diffuse distribution of intermediate-sized pleomorphic lymphocytes with angiocentric growth. The tumor cells expressed CD2, CD3, CD56, T-cell intracellular antigen-1, granzyme B, and Epstein-Barr virus-encoded RNAs (EBERs), with additional partial and weak CD20 and CD30 expression. Despite a confirmatory pathological diagnosis, the patient refused treatment and was discharged, ultimately dying from the disease. In the literature review, the clinical, immunohistochemical, EBERs, treatment, and prognostic features of PCNS ENKTCL were summarized. Although PCNS ENKTCT is extremely rare, it does occur and should always be included in differential diagnoses. CD20 expression should be evaluated routinely with relevant markers. The accumulation of cases is crucial for developing an effective treatment strategy for this rare and aggressive malignancy., (© 2023 Japanese Society of Neuropathology.)

In order to explore a new mode for the diagnosis of angioimmunoblastic T-cell lymphoma (AITL), 31 cases of AITL and 28 cases of peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) were used as the study subjects. Identifying T follicular helper (TFH) cells with CD4, CD10, Bcl-6, and PD-1, identifying proliferative B cells with CD20 and EZH2, identifying proliferative follicular dendritic cells (FDCs) with CD21 and CD23, and analyzing the value of TFH/B/FDC proliferation and immunolocalization in the diagnosis of AITL. (1) Outside the inherent lymphoid follicles, simultaneous proliferation of TFH/B/FDC (a new diagnostic mode) were observed in AITL [83.87%; 26/31], with their immunolocalizations in the same site [83.87%; 26/31], while this phenomenon was not observed in 28 cases of PTCL-NOS (P<0.05). (2) The sensitivity and specificity of using this new mode to diagnose AITL were both high (83.87%, 100%), which was superior to CD2 (100%, 0%), CD3 (100%, 0%), CD4 (100%, 32.14%), CD5 (100%, 25%), CD10 (61.9%, 100%), Bcl-6 (42.86%, 100%), PD-1 (83.87%, 96.43%), and its Youden Index (0.84) was the highest. The areas under the curve (AUC) of CD10, Bcl-6, PD-1, and new mode to diagnosis AITL were 0.81, 0.71, 0.90, and 0.92, respectively, while the new mode had the highest AUC. The simultaneous proliferation of TFH/B/FDC cells outside the inherent lymphoid follicles can be used to assist in the diagnosis of AITL, and the simultaneous spatiotemporal proliferation of TFH/B/FDC cells is a specific immunomorphology of AITL.

Chimeric antigen receptor (CAR) T cell therapies have demonstrated immense clinical success for B cell and plasma cell malignancies. We tested their impact on the viral reservoir in a macaque model of HIV persistence, comparing the functions of CD20 CAR T cells between animals infected with simian/human immunodeficiency virus (SHIV) and uninfected controls. We focused on the potential of this approach to disrupt B cell follicles (BCFs), exposing infected cells for immune clearance. In SHIV-infected animals, CAR T cells were highly functional, with rapid expansion and trafficking to tissue-associated viral sanctuaries, including BCFs and gut-associated lymphoid tissue (GALT). CD20 CAR T cells potently ablated BCFs and depleted lymph-node-associated follicular helper T (T FH ) cells, with complete restoration of BCF architecture and T FH cells following CAR T cell contraction. BCF ablation decreased the splenic SHIV reservoir but was insufficient for effective reductions in systemic viral reservoirs. Although associated with moderate hematologic toxicity, CD20 CAR T cells were well tolerated in SHIV-infected and control animals, supporting the feasibility of this therapy in people living with HIV with underlying B cell malignancies. Our findings highlight the unique ability of CD20 CAR T cells to safely and reversibly unmask T FH cells within BCF sanctuaries, informing future combinatorial HIV cure strategies designed to augment antiviral efficacy., Competing Interests: Declaration of interests The authors declare the following competing interests: H.-P.K. is or was a consultant to and has or had ownership interests in Rocket Pharmaceuticals, Homology Medicines, Vor Biopharma, and Ensoma, Inc., (Copyright © 2024 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Background: A bidirectional promoter-driven chimeric antigen receptor (CAR) cassette provides the simultaneous expression of two CARs, which significantly enhances dual antigen-targeted CAR T-cell therapy., Methods: We developed a second-generation CAR directing CD19 and CD20 antigens, incorporating them in a head-to-head orientation from a bidirectional promoter using a single Sleeping Beauty transposon system. The efficacy of bidirectional promoter-driven dual CD19 and CD20 CAR T cells was determined in vitro against cell lines expressing either, or both, CD19 and CD20 antigens. In vivo antitumor activity was tested in Raji lymphoma-bearing immunodeficient NOD-scid IL2Rgamma null (NSG) mice., Results: Of all tested promoters, the bidirectional EF-1 α promoter optimally expressed transcripts from both sense (CD19-CAR) and antisense (GFP.CD20-CAR) directions. Superior cytotoxicity, cytokine production and antigen-specific activation were observed in vitro in the bidirectional EF-1 α promoter-driven CD19/CD20 CAR T cells. In contrast, a unidirectional construct driven by the EF-1 α promoter, but using self-cleaving peptide-linked CD19 and CD20 CARs, showed inferior expression and in vitro function. Treatment of mice bearing advanced Raji lymphomas with bidirectional EF-1 α promoter-driven CD19/CD20 CAR T cells effectively controlled tumor growth and extended the survival of mice compared with group treated with single antigen targeted CAR T cells., Conclusion: The use of bidirectional promoters in a single vector offers advantages of size and robust CAR expression with the potential to expand use in other forms of gene therapies like CAR T cells., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

The mammalian olfactory system detects and discriminates between millions of odorants to elicit appropriate behavioral responses. While much has been learned about how olfactory sensory neurons detect odorants and signal their presence, how specific innate, unlearned behaviors are initiated in response to ethologically relevant odors remains poorly understood. Here, we show that the 4-transmembrane protein CD20, also known as MS4A1, is expressed in a previously uncharacterized subpopulation of olfactory sensory neurons in the main olfactory epithelium of the murine nasal cavity and functions as a mammalian olfactory receptor that recognizes compounds produced by mouse predators. While wildtype mice avoid these predator odorants, mice genetically deleted of CD20 do not appropriately respond. Together, this work reveals a CD20-mediated odor-sensing mechanism in the mammalian olfactory system that triggers innate behaviors critical for organismal survival., (© 2024. The Author(s).)

Cluster of differentiation 20 (CD20) is a nonglycosylated, multispanning transmembrane protein specifically integrated by B lymphocytes. Similar to CD20, another four-pass transmembrane protein, claudin 18.2, has attracted attention as an emerging therapeutic target for cancer. However, their poor solubility and toxic nature often hinder downstream applications, such as antibody drug development. Therefore, developing a cost-effective method for producing drug targets with multiple membrane-spanning domains is crucial. In this study, a high yield of recombinant CD20 was achieved through an E. coli-based in vitro coupled transcription-translation system. Surface plasmon resonance results showed that rituximab (an antileukemia drug) has nanomolar affinity with the CD20 protein, which aligns with published results. Notably, a previously hard-to-express claudin 18.2 recombinant protein was successfully expressed in the same reaction system by replacing its membrane-spanning domains with the transmembrane domains of CD20. The folding of the extracellular domain of the chimeric protein was verified using a commercial anti-claudin 18 antibody. This study provides a novel concept for promoting the expression of four-pass transmembrane proteins and lays the foundation for the large-scale industrial production of membrane-associated drug targets, similar to claudin 18.2., (Copyright © 2023 Elsevier Inc. All rights reserved.)

One reason for a lack of response to rituximab as well as infusion-related anaphylactic adverse events is the development of antidrug Abs to rituximab. Besides rituximab, a number of other therapeutic Abs targeting CD20 are nowadays available as alternatives. In this study, we investigated the potential cross-reactivity of (human) anti-rituximab Abs to three other anti-CD20 mAbs: ofatumumab, obinutuzumab, and ocrelizumab. In 25 cases of anti-rituximab Abs, cross-reactivity was examined using both direct binding assays and inhibition immunoassays. Although no cross-reactivity was observed to ofatumumab or obinutuzumab, 8 of 25 samples also showed reactivity toward ocrelizumab in at least one of the two assays. Furthermore, in three cases of anti-ocrelizumab Abs, cross-reactivity to rituximab was observed in an inhibition immunoassay, albeit not in a direct binding assay. Our results suggest that obinutuzumab or ofatumumab are safe anti-CD20 alternatives in case of the presence of anti-rituximab Abs. It is advisable to proceed cautiously if switching from rituximab to ocrelizumab (or vice versa) is considered in case these alternatives may not be available., (Copyright © 2024 by The American Association of Immunologists, Inc.)

Elimination of the binding of immunoglobulin Fc to Fc gamma receptors is highly desirable for the avoidance of unwanted inflammatory responses to therapeutic antibodies and fusion proteins. Many different approaches have been used in the clinic, but they have not been systematically compared. We have now produced a matched set of anti-CD20 antibodies with different Fc subclasses and variants and compared their activity for binding to C1q, Fc-gamma receptors and in cell-based assays. Most of the variants still have significant levels of activity in one or more of these assays and many of them have impaired temperature stability compared with the corresponding wild-type antibody.

Although most cancer drugs modulate the activities of cellular pathways by changing posttranslational modifications (PTMs), little is known regarding the extent and the time- and dose-response characteristics of drug-regulated PTMs. In this work, we introduce a proteomic assay called decryptM that quantifies drug-PTM modulation for thousands of PTMs in cells to shed light on target engagement and drug mechanism of action. Examples range from detecting DNA damage by chemotherapeutics, to identifying drug-specific PTM signatures of kinase inhibitors, to demonstrating that rituximab kills CD20-positive B cells by overactivating B cell receptor signaling. DecryptM profiling of 31 cancer drugs in 13 cell lines demonstrates the broad applicability of the approach. The resulting 1.8 million dose-response curves are provided as an interactive molecular resource in ProteomicsDB.

Several monoclonal antibodies targeting the CD20 have been produced and Ofatumumab is a case in point. Although whole antibodies target cancer cells effectively, their applications are restricted in some ways. Single-chain fragment variable antibodies, rather than employing the entire structure of antibodies, have proven a practical approach for creating completely functional antigen-binding fragments. In current research, the DNA coding sequence of V L and V H of the wild and mutant forms of ofatumumab were joined with a flexible linker ( GGGGS ) 3 separately. Using the E. coli BL21 (DE3) expression system, the V L -linker- V H genes were cloned into the pET-28 a (+), and the associated recombinant proteins were produced. Purified and refolded scFvs (scFv-C and scFv- V 3 ) represented a concentration of around 0.7 mg/ml from 1 L of initial E. coli culture with a molecular weight of about 27 kDa. Affinity measurement disclosed anti-CD20 scFv- V 3 possesses a higher affinity constant compared to anti-CD20 scFv- C . The recombinant scFvs exclusively attach to Raji cells but not to Jurkat cells, according to a cell-ELISA analysis. The MTT test signified anti-CD20 scFvs could affect cell viability in Raji cells but had no impact on Jurkat cells and also, Raji cells viability was affected more significantly by anti-CD20 scFv- V 3 .

Introduction: Although CD20 is classically a B cell marker, in the last three decades, dim expression has been noted on a subset of T cells as well that has been independently verified by a number of groups. Our understanding of these cells and their function is not well established., Methods: A thorough review of original articles on CD20 + T cells was undertaken of Pubmed by using combination of phrases including "CD20 + ", "CD20-positive" and "T cells". Articles in English were considered, and there was no time restriction., Results: CD20 + T cells express the standard T cell markers and, in comparison to CD20¯ T cells, appear to express greater inflammatory cytokines and markers of effector function. Although the ontogeny of these cells is still being established, the current theory is that CD20 may be acquired by trogocytosis from B cells. CD20 + T cells may be found in healthy controls and in a wide range of pathologies including autoimmune diseases, haematological and non-haematological malignancies and human immunodeficiency virus (HIV) infections. One of the best studied diseases where these cells are found is multiple sclerosis (MS) where a number of therapeutic interventions, including anti-CD20 depletion, have been shown to effectively deplete these cells., Conclusion: This review summarises the latest understanding of CD20 + T cells, their presence in various diseases, their putative function and how they may be an ongoing target of CD20-depleting agents. Unfortunately, our understanding of these cells is still at its infancy and ongoing study in a wider range of pathologies is required., (© 2022. Crown.)

Cluster of differentiation 20 (CD20) is an integral membrane protein expressed mainly on different developmental stages of B lymphocytes and rarely on T lymphocytes, and it functions as a link to B cell antigen receptor (BCR) and immune microenvironment via regulating calcium ion influx, cell cycle progression and interaction between isotypic BCRs and their co-receptors. Diverse therapeutic monoclonal antibodies (mAbs) targeting CD20 are generated and grouped into two types based on the ability to redistribute CD20 into lipid rafts, which results in huge differences in response. Currently, multiple anti-CD20 mAbs have been approved as drugs for neurological and neuromuscular diseases with promising clinical efficacy. This review aims to summarize the potential mechanisms, development and current evidence for anti-CD20 therapy in neurological and neuromuscular diseases., (Copyright © 2022 Elsevier Inc. All rights reserved.)

NK cells are promising cellular therapeutics against hematological and solid malignancies. Immunogenetic studies have identified that various activating killer cell Ig-like receptors (KIRs) are associated with cancer outcomes. Specifically, KIR2DS2 has been associated with reduced incidence of relapse following transplant in hematological malignancies and improved outcomes in solid tumors, but the mechanism remains obscure. Therefore, we investigated how KIR2DS2 expression impacts NK cell function. Using a novel flow cytometry panel, we show that human NK cells with high KIR2DS2 expression have enhanced spontaneous activation against malignant B cell lines, liver cancer cell lines, and primary chronic lymphocytic leukemia cells. Surface expression of CD16 was increased on KIR2DS2 high NK cells, and, accordingly, KIR2DS2 high NK cells had increased activation against lymphoma cells coated with the clinically relevant anti-CD20 Abs rituximab and obinutuzumab. Bulk RNA sequencing revealed that KIR2DS2 high NK cells have upregulation of NK-mediated cytotoxicity, translation, and FCGR gene pathways. We developed a novel single-cell RNA-sequencing technique to identify KIR2DS2 + NK cells, and this confirmed that KIR2DS2 is associated with enhanced NK cell-mediated cytotoxicity. This study provides evidence that KIR2DS2 marks a population of NK cells primed for anticancer activity and indicates that KIR2DS2 is an attractive target for NK-based therapeutic strategies., (Copyright © 2022 The Authors.)

Objective: T cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT) is a novel immunosuppressive molecule. This study aimed to investigate the expression of TIGIT on B cells and the function of TIGIT + CD20 + B cells in gastric cancer (GC)., Methods: Tumor tissue paraffin-embedded sections and clinicopathological data from 194 patients with GC were collected. Dual immunohistochemistry was performed to detect the expression of TIGIT on B cells. Multiplex immunofluorescence was used to initially explore the relationship between TIGIT + CD20 + B cells and the exhaustion of CD8 + T cells., Results: In GC, TIGIT + CD20 + B cells were observed in intratumor, peritumor, and tertiary lymphoid structures (TLS). Patients with GC having high peritumoral TIGIT + CD20 + B cells infiltration had inferior clinical outcomes and could benefit from adjuvant chemotherapy (ACT). In GC tissues, PD-1 + CD8 + T cells were more closer to TIGIT + CD20 + B cells than to TIGIT - CD20 + B cells., Conclusions: Peritumoral TIGIT + CD20 + B cells infiltration was an independent prognostic predictor for patients with GC and a potential biomarker for ACT selection. TIGIT + CD20 + B cells might affect the exhaustion of CD8 + T cells in GC., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background and Objectives: Rituximab is an anti-CD20 monoclonal antibody approved in several diseases, including chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), rheumatoid arthritis (RA), and anti-neutrophil cytoplasmic antibody-associated vasculitis (AAV). The influence of underlying disease on rituximab pharmacokinetics has never been investigated for several cancer and non-cancer diseases simultaneously. This study aimed at assessing this influence using an integrated semi-mechanistic model accounting for target-mediated elimination of rituximab., Methods: Rituximab concentration-time data from five studies previously published in patients with CLL, DLBCL, FL, RA, and AAV were described using a two-compartment model with irreversible binding of rituximab to its target antigen. Both underlying disease and target antigen measurements were assessed as covariates., Results: Central volume of distribution was [95% confidence interval] 1.7-fold [1.6-1.9] higher in DLBCL than in RA, FL, and CLL, and it was 1.8-fold [1.6-2.1] higher in RA, FL, and CLL than in AAV. First-order elimination rate constants were 1.8-fold [1.7-2.0] and 1.3-fold [1.2-1.5] higher in RA, DLBCL, and FL than in CLL and AAV, respectively. Baseline latent antigen level (L 0 ) was 54-fold [30-94], 20-fold [11-36], and 29-fold [14-64] higher in CLL, DLBCL, and FL, respectively, than in RA and AAV. In lymphoma, L 0 increased with baseline total metabolic tumor volume (p = 6.10 -7 ). In CLL, the second-order target-mediated elimination rate constant (k deg ) increased with baseline CD20 count on circulating B cells (CD20 cir , p = 0.0081)., Conclusions: Our results show for the first time that rituximab pharmacokinetics is strongly influenced by underlying disease and disease activity. Notably, neoplasms are associated with higher antigen amounts that result in decreased exposure to rituximab compared to inflammatory diseases. Our model might be used to estimate unbound target amounts in upcoming studies., (© 2021. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Chimeric antigen receptor (CAR) T cell therapies have resulted in profound clinical responses in the treatment of CD19-positive hematological malignancies, but a significant proportion of patients do not respond or relapse eventually. As an alternative to CAR T cells, T cells can be engineered to express a tumor-targeting T cell receptor (TCR). Due to HLA restriction of TCRs, CARs have emerged as a preferred treatment moiety when targeting surface antigens, despite the fact that functional differences between engineered TCR (eTCR) T and CAR T cells remain ill-defined. Here, we compared the activity of CAR T cells versus engineered TCR T cells in targeting the B cell malignancy-associated antigen CD20 as a function of antigen exposure. We found CAR T cells to be more potent effector cells, producing higher levels of cytokines and killing more efficiently than eTCR T cells in a short time frame. However, we revealed that the increase of antigen exposure significantly impaired CAR T cell expansion, a phenotype defined by high expression of coinhibitory molecules and effector differentiation. In contrast, eTCR T cells expanded better than CAR T cells under high antigenic pressure, with lower expression of coinhibitory molecules and maintenance of an early differentiation phenotype, and comparable clearance of tumor cells., Competing Interests: The authors report no conflict of interest., (© 2022 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Background: Despite therapeutic advances, Non-Hodgkin lymphoma (NHL) relapses can occur. The development of radioimmunotherapy (RIT) with α-emitters is an attractive alternative. In this study, we investigated the potential of α-RIT in conjunction with 212 Pb-rituximab for the treatment of NHL., Methods: EL4-hCD20-Luc cells (mouse lymphoma cell line) were used for in vitro and in vivo studies. Biodistribution and efficacy studies were performed on C57BL/6 mice injected intravenously with 25 × 10 3 cells., Results: 212 Pb-rituximab (0.925-7.4 kBq/mL) inhibit proliferation of EL4-hCD20-Luc cells in vitro. Biodistribution of 203/212 Pb-rituximab in mice showed a significant tumour uptake and suggested that the liver, spleen, and kidneys were the organs at risk. For efficacy studies, mice were treated at either 11 days (early stage) or 20-30 days after injection of tumour cells (late stage). Treatment with 277.5 kBq 212 Pb-rituximab significantly prolonged survival. Even at an advanced tumour stage, significant tumour regression occurred, with an increase in the median survival time to 28 days, compared with 9 days in the controls., Conclusions: These results show the efficacy of 212 Pb-rituximab in a murine syngeneic lymphoma model, in terms of significant tumour regression and increased survival, thereby highlighting the potency of α-RIT for the treatment of NHL., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: Small extracellular vesicles (sEVs) including exosomes, carrying the CD20, could be involved in immunotherapy resistance in diffuse large B cell lymphoma (DLBCL). We have reported endogenous brain-derived neurotrophic factor/TrkB (tropomyosin-related kinase B) survival axis in DLBCL. Here, we performed a comparative study of sEV production by germinal centre B cell (GCB) and activated B cell (ABC)-DLBCL cell lines, and analysed TrkB activation on this process., Methods: GCB (SUDHL4 and SUDHL6) and ABC (OCI-LY3, OCI-LY10 and U2932) cell lines were used. sEVs were characterised using nanoparticle tracking analysis technology and western blot. CD20 content was also analysed by enzyme-linked immunoassay, and complement-dependent cytotoxicity of rituximab was investigated. 7,8-Dihydroxyflavone (7,8-DHF) was used as a TrkB agonist. In vivo role of sEVs was evaluated in a xenograft model., Results: sEVs production varied significantly between DLBCL cells, independently of subtype. CD20 level was consistent with that of parental cells. Higher CD20 expression was found in sEVs after TrkB activation, with a trend in increasing their concentration. sEVs determined in vitro and in vivo protection from rituximab, which seemed CD20 level-dependent; the protection was enhanced when sEVs were produced by 7,8-DHF-treated cells., Conclusions: DLBCL-derived sEVs have the differential capacity to interfere with immunotherapy, which could be enhanced by growth factors like neurotrophins. Evaluating the sEV CD20 level could be useful for disease monitoring., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: We present a unique case of primary breast CD20-positive extranodal NK/T cell lymphoma with stomach involvement in a young Chinese female patient., Case Presentation: The patient presented with a mass in her right breast that rapidly increased in size over approximately 2 months. Upper gastrointestinal endoscopy showed a giant serpentine ulcer in the stomach. Biopsy was performed, and microscopic inspection revealed that the fibrous tissue was diffusely involved by medium to large abnormal lymphocytes. The cytoplasm was low to moderate. The tumor cells had irregular nuclei and inconspicuous nucleoli. The lymphoid cells were strongly immunoreactive to CD20, CD3, CD4, CD56, TIA-1, EBER, and Ki-67 (90%). Epstein-Barr virus genomes were also found in tumor cells by in situ hybridization. A whole-body positron emission tomography (PET)-CT scan revealed intense FDG uptake in the right breast and greater curvature of the stomach. Monoclonal rearrangements of the T cell receptor (TCR-γ) and immunoglobulin heavy chain (IgH) were identified by genetic analysis. Whole-genome next-generation sequencing was performed, and up to 12 gene mutations, including a frameshift mutation in exon 4 of the BCOR (G97Rfs*87; 44.3%) gene and a base substitution mutation (Q61H 17.6%) in exon 3 of the KRAS gene, were detected. Kyoto Encyclopedia of Genes and Genomes pathway analyses were performed using the database for annotation, visualization, and integrated discovery, which showed that rare primary breast CD20-positive extranodal NK/T cell lymphoma had a unique genetic background compared with diffuse large B cell lymphoma and extranodal NK/T cell lymphoma without CD20 expression. The patient received four cycles of the modified SMILE regimen. The second whole-body PET-CT scan revealed that the right breast mass was significantly smaller than before; additionally, FDG uptake in the stomach wall disappeared., Conclusions: Systemic examination, extensive immunohistochemistry, and molecular profiling are essential for an accurate diagnosis. More similar cases are required to clarify the biological pathways and even the potential molecular mechanisms of rare lymphomas, which may help direct further treatment., (© 2021. The Author(s).)

Abstract: The prognostic implications of the immunophenotype of the tumor-infiltrating lymphocytes (TILs) in primary cutaneous melanoma are well known. In recent years, the study of this immunophenotype has also resulted in immunotherapeutic consequences. The aims of this study were to characterize the subpopulations of TILs in primary cutaneous melanoma, in cases with and without metastasis, as well as the neovascularization associated with the primary neoplasm, and its influence on the development of metastasis. To this end, the immunophenotype of TILs and the neovascularization of 80 patients with primary cutaneous melanoma (40 each with metastatic and non-metastatic melanoma) were analyzed by immunohistochemistry for CD3, CD4, CD8, FOXP3, PD-1, CD31, and D2-40 antibodies. We found that higher frequencies of TILs with brisk pattern, and CD4+, CD8+, and CD20+ cells in TILs, and a lower frequency of CD31+ vessels were histopathological features associated with better prognosis in primary cutaneous melanoma. Our results support the notion that the immunohistochemical study of TILs and neovascularization in primary cutaneous melanoma may be helpful tools for identifying patients at increased risk of metastasis development., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Competing Interests: Declaration of Competing Interest ECS has no conflicts of interest to report. JS received honoraria for lectures, assembly of teaching material and participation in advisory boards from Alexion, Biogen, BMS/Celgene, Merck, Novartis, Sanofi/Genzyme and Roche.

Plants are promising drug-production platforms with high economic efficiency, stability, and convenience in mass production. However, studies comparing the equivalency between the original antibodies and those produced in plants are limited. Amino acid sequences that constitute the Fab region of an antibody are diverse, and the post-transcriptional modifications that occur according to these sequences in animals and plants are also highly variable. In this study, rituximab, a blockbuster antibody drug used in the treatment of non-Hodgkin's lymphoma, was produced in Nicotiana benthamiana leaves and Arabidopsis thaliana callus, and was compared to the original rituximab produced in CHO cells. Interestingly, the epitope recognition and antigen-binding abilities of rituximab from N. benthamiana leaves were almost lost. In the case of rituximab produced in A. thaliana callus, the specific binding ability and CD20 capping activity were maintained, but the binding affinity was less than 50% of that of original rituximab from CHO cells. These results suggest that different plant species exhibit different binding affinities. Accordingly, in addition to the differences in PTMs between mammals and plants, the differences between the species must also be considered in the process of producing antibodies in plants., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Epstein-Barr virus (EBV) is an oncogenic herpesvirus implicated in the pathogenesis of several malignant and non-malignant conditions. However, a number of fundamental aspects about the biology of EBV and the mechanism(s) by which this virus induces pathology remain unknown. One major obstacle has been the lack of a suitable animal model for EBV infection. In this study, using our recently established rabbit model of EBV infection, we examined the early events following primary EBV infection. We show that, both immunocompetent and immunosuppressed animals were readily susceptible to EBV infection. However, immunosuppressed animals showed marked splenomegaly and widespread infection. Following EBV infection, the virus primarily targeted naïve IgM + , CD20 + , CD21 + and CD79a + B cells. Infected cells expressed varying sets of viral latent/lytic gene products. Notably, co-expression of latent and lytic proteins in the same cell was not observed. Infected cells in type 0/1 latency (EBERs + ), were small and proliferating (Ki67 + ). By contrast, cells in type 2/3 latency (LMP1 + ), were large, non-proliferating (Ki-67 - ) and p53 + . Although infected B-cells were widely present in splenic follicles, they did not express germinal center marker, BCL-6. Taken together, this study shows for the first time, some of the early events following primary EBV infection., (© 2021. The Author(s).)

The presence of T cells that are dimly positive for the B cell marker CD20 is well-established in autoimmunity and correlates with disease severity in various diseases. Further, we previously identified that the level of CD20-positive T cells was three-fourfold elevated in ascites fluid of ovarian carcinoma patients, together suggesting a role in both autoimmunity and cancer. In this respect, treatment of autoimmune patients with the CD20-targeting antibody Rituximab has also been shown to target and deplete CD20-positive T cells, previously identified as IFN-gamma producing, low proliferative, CD8 cytotoxic T cells with an effector memory (EM) differentiation state. However, the exact phenotype and relevance of CD20-positive T cells remains unclear. Here, we set out to identify the transcriptomic profile of CD20-positive T cells using RNA sequencing. Further, to gain insight into potential functional properties of CD20 expression in T cells, CD20 was ectopically expressed on healthy human T cells and phenotypic, functional, migratory and adhesive properties were determined in vitro and in vivo. Together, these assays revealed a reduced transmigration and an enhanced adhesive profile combined with an enhanced activation status for CD20-positive T cells., (© 2021. The Author(s).)

Abstract: Erythema nodosum leprosum (ENL) occurs as an immunological complication of multibacillary leprosy (MBL). The pathogenesis of ENL is long considered to be a T-cell-mediated process. The role of B cells and plasma cells in ENL is not well described in the literature. Therefore, we investigated the B-cell and plasma cell infiltrates in the skin biopsies of biopsy-proven cases of ENL by immunohistochemistry and image morphometry and compared the result with paucibacillary leprosy and MBL. Moreover, we sought a correlation of the B-cell and plasma cell infiltrates with different clinical, hematological, histopathological, and bacteriological parameters as well as the T-cell subsets in the skin biopsies. Our study highlighted a significant reduction in the number of B cells from paucibacillary leprosy to MBL to ENL, although there was no significant variation in the plasma cell infiltrate. The plasma cell infiltrate correlated with absolute neutrophilia in the blood and the presence of eosinophils in the ENL lesions. Both B cells and plasma cells positively correlated with CD4-positive T-helper cells and the CD8-positive cytotoxic T cells. Besides, the B cells also correlated positively with the CD3-positive pan T cells in the biopsy and negatively correlated with the T-regulatory:T-cell ratio. Our results suggested the role of B cells and plasma cells even at the tissue level in the pathobiogenesis of ENL., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Pretransplant desensitization with rituximab has been applied to preformed donor-specific anti-human leukocyte antigen antibody (DSA)-positive recipients for elimination of preformed DSA. We investigated the impact of pretransplant desensitization with rituximab on anti-donor T cell responses in DSA-positive transplant recipients. To monitor the patients' immune status, mixed lymphocyte reaction (MLR) assays were performed before and after desensitization with rituximab. Two weeks after rituximab administration, the stimulation index (SI) of anti-donor CD4 + T cells was significantly higher in the DSA-positive recipients than in the DSA-negative recipients. To investigate the mechanisms of anti-donor hyper responses of CD4 + T cells after B cell depletion, highly sensitized mice models were injected with anti-CD20 mAb to eliminate B cells. Consistent with clinical observations, the SI values of anti-donor CD4 + T cells were significantly increased after anti-CD20 mAb injection in the sensitized mice models. Adding B cells isolated from untreated sensitized mice to MLR significantly inhibited the enhancement of anti-donor CD4 + T cell response. The depletion of the CD5 + B cell subset, which exclusively included IL-10-positive cells, from the additive B cells abrogated such inhibitory effects. These findings demonstrate that IL-10 + CD5 + B cells suppress the excessive response of anti-donor CD4 + T cells responses in sensitized recipients., (© 2021. The Author(s).)

Diffuse large B cell lymphoma (DLBCL) heterogeneity promotes recurrence and anti-CD20-based therapeutic resistance. Previous studies have shown that downregulation of MS4A1/CD20 expression after chemoimmunotherapy with rituximab leads to rituximab resistance. However, the mechanisms of CD20 loss remain unknown. We identified that pyruvate dehydrogenase kinase 4 (PDK4) is markedly elevated in DLBCL cells derived from both patients and cell lines with R-CHOP (rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone) resistance. We found that overexpression of PDK4 in DLBCL cells resulted in cell proliferation and resistance to rituximab in vitro and in vivo. Furthermore, loss of PDK4 expression or treatment with the PDK4 inhibitor dichloroacetate was able to significantly increase rituximab-induced cell apoptosis in DLBCL cells. Further studies suggested PDK4 mediates a metabolic shift, in that the main energy source was changed from oxidative phosphorylation to glycolysis, and the metabolic changes could play an important role in rituximab resistance. Importantly, by knocking down or overexpressing PDK4 in DLBCL cells, we showed that PDK4 has a negative regulation effect on MS4A1/CD20 expression. Collectively, this is the first study showing that targeting PDK4 has the potential to overcome rituximab resistance in DLBCL., (© 2021 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Anti-CD20 therapies have demonstrated considerable efficacy in the treatment of relapsing multiple sclerosis, constituting a high-efficacy treatment approach for reducing relapse risk and mitigating disability progression. These therapies have been shown to strongly deplete circulating B cells and small subsets of CD3+ CD4 and CD8 T cells that express low levels of CD20. While the clinical profiles of the various anti-CD20 monoclonal antibodies used in treating multiple sclerosis are well-described in the literature, greater understanding of the implications of their distinct molecular and pharmacological attributes is needed. In this review, we focus on four anti-CD20 monoclonal antibodies-rituximab, ocrelizumab, ofatumumab, and ublituximab-that are currently used, approved, or in late-stage clinical development for the treatment of multiple sclerosis. We provide clinical perspectives on the potential implications of differences in molecular structures, target epitopes, dosing regimens, mechanisms and impact on B-cell depletion and reconstitution, immunogenicity, administration-related reactions, and infection risks., (© 2021. The Author(s).)

Chronic active Epstein-Barr virus (CAEBV) infection is a rare disease with a high mortality rate. Our study aimed to summarize the clinicopathological characteristics of CAEBV infection in adults and improve knowledge of the disease. Data for 19 adult patients with CAEBV confirmed at our hospital from January 2010 to December 2019 were collected retrospectively. There were 14 males and 5 females, and the median age was 33 years (range 14-83). The main clinical manifestations included recurrent fever (84.2%, 16/19), splenomegaly (89.5%, 17/19), hepatomegaly (73.6%, 14/19), lymphadenopathy (42.1%, 8/19), abnormal liver function (78.9%, 15/19), hemopenia (94.7%, 18/19), and hemophagocytosis (52.6%, 10/19). A total of 22 specimens were collected from 19 patients for histopathology. Most of the biopsy specimens showed lymphocyte infiltration. Immunohistochemical staining and EBV-encoded small RNA (EBER) in situ hybridization were performed for 14 of the 22 samples. CD3 and CD20 staining were positive, with more CD3-positive cells than CD20-positive cells (100%, 14/14), and EBER in situ hybridization was positive in most cases (85.7%, 12/14). More than half of TCR gene rearrangement tests showed monoclonal rearrangement (66.6%, 4/6). Mortality was high, with most CAEBV patients dying during the period from diagnosis to the end of follow-up (12/19, 63%); the median survival time was only 20.75 months. Based on limited data, we consider that CAEBV is a disease with different ages of onset and is a complex and heterogeneous syndrome with features of both immunodeficiency and malignant neoplasms. Furthermore, the prognosis of adult-onset CAEBV appears to be very poor.

Objective: Our aim was to compare transcriptome and phenotype profiles of CD4+ T cells and CD19+ B cells in patients with Takayasu arteritis (TAK), patients with giant cell arteritis (GCA), and healthy donors., Methods: Gene expression analyses, flow cytometry immunophenotyping, T cell receptor (TCR) gene sequencing, and functional assessments of cells from peripheral blood and arterial lesions from TAK patients, GCA patients, and healthy donors were performed., Results: Among the most significantly dysregulated genes in CD4+ T cells of TAK patients compared to GCA patients (n = 720 genes) and in CD4+ T cells of TAK patients compared to healthy donors (n = 1,447 genes), we identified a follicular helper T (Tfh) cell signature, which included CXCR5, CCR6, and CCL20 genes, that was transcriptionally up-regulated in TAK patients. Phenotypically, there was an increase in CD4+CXCR5+CCR6+CXCR3- Tfh17 cells in TAK patients that was associated with a significant enrichment of CD19+ B cell activation. Functionally, Tfh cells helped B cells to proliferate, differentiate into memory cells, and secrete IgG antibodies. Maturation of B cells was inhibited by JAK inhibitors. Locally, in areas of arterial inflammation, we found a higher proportion of tertiary lymphoid structures comprised CD4+, CXCR5+, programmed death 1+, and CD20+ cells in TAK patients compared to GCA patients. CD4+CXCR5+ T cells in the aortas of TAK patients had an oligoclonal α/β TCR repertoire., Conclusion: We established the presence of a specific Tfh cell signature in both circulating and aorta-infiltrating CD4+ T cells from TAK patients. The cooperation of Tfh cells and B cells might be critical in the occurrence of vascular inflammation in patients with TAK., (© 2021, American College of Rheumatology.)