Anti‐CD20 monoclonal antibodies such as Rituximab, Ofatumumab, and Obinutuzumab are widely used to treat lymphomas and autoimmune diseases. They act by depleting B cells, mainly through Fc‐dependent ...effectors functions. Some patients develop resistance to treatment but the underlying mechanisms are poorly understood. Here, we performed a genome‐wide CRISPR/Cas9 screen to identify genes regulating the efficacy of anti‐CD20 antibodies. We used as a model the killing of RAJI B cells by Rituximab through complement‐dependent‐cytotoxicity (CDC). As expected, the screen identified MS4A1, encoding CD20, the target of Rituximab. Among other identified genes, the role of Interferon Regulatory Factor 8 (IRF8) was validated in two B‐cell lines. IRF8 KO also decreased the efficacy of antibody‐dependent cellular cytotoxicity and phagocytosis (ADCC and ADCP) induced by anti‐CD20 antibodies. We further show that IRF8 is necessary for efficient CD20 transcription. Levels of IRF8 and CD20 RNA or proteins correlated in normal B cells and in hundreds of malignant B cells. Therefore, IRF8 regulates CD20 expression and controls the depleting capacity of anti‐CD20 antibodies. Our results bring novel insights into the pathways underlying resistance to CD20‐targeting immunotherapies.
In B cells, IRF8 protein induces MS4A1 transcription which encodes CD20 protein. CD20 is recognized by anti‐CD20 antibodies such as Rituximab, Ofatumumab and Obinutuzumab. Complement‐dependent cytotoxicity (CDC), Antibody‐Dependent Cell Cytotoxicity (ADCC) and Antibody‐Dependent Cellular Phagocytosis (ADCP) are then induced. Knocking‐out of IRF8 reduces CD20 levels and sensitivity to anti‐CD20 antibodies.
CD8⁺ T cells are believed to play an important role in multiple sclerosis (MS), yet their role in MS pathogenesis remains poorly defined. Although myelin proteins are considered potential ...autoantigenic targets, prior studies of myelin-reactive CD8⁺ T cells in MS have relied on in vitro stimulation, thereby limiting accurate measurement of their ex vivo precursor frequencies and phenotypes. Peptide:MHC I tetramers were used to identify and validate 5 myelin CD8⁺ T cell epitopes, including 2 newly described determinants in humans. The validated tetramers were used to measure the ex vivo precursor frequencies and phenotypes of myelin-specific CD8⁺ T cells in the peripheral blood of untreated MS patients and HLA allele-matched healthy controls. In parallel, CD8⁺ T cell responses against immunodominant influenza epitopes were also measured. There were no differences in ex vivo frequencies of tetramer-positive myelin-specific CD8⁺ T cells between MS patients and control subjects. An increased proportion of myelin-specific CD8⁺ T cells in MS patients exhibited a memory phenotype and expressed CD20 compared to control subjects, while there were no phenotypic differences observed among influenza-specific CD8⁺ T cells. Longitudinal assessments were also measured in a subset of MS patients subsequently treated with anti-CD20 monoclonal antibody therapy. The proportion of memory and CD20⁺ CD8⁺ T cells specific for certain myelin but not influenza epitopes was significantly reduced following anti-CD20 treatment. This study, representing a characterization of unmanipulated myelin-reactive CD8⁺ T cells in MS, indicates these cells may be attractive targets in MS therapy.
A small proportion of multiple sclerosis (MS) patients develop new disease activity soon after starting anti-CD20 therapy. This activity does not recur with further dosing, possibly reflecting deeper ...depletion of CD20-expressing cells with repeat infusions. We assessed cellular immune profiles and their association with transient disease activity following anti-CD20 initiation as a window into relapsing disease biology. Peripheral blood mononuclear cells from independent discovery and validation cohorts of MS patients initiating ocrelizumab were assessed for phenotypic and functional profiles using multiparametric flow cytometry. Pretreatment CD20-expressing T cells, especially CD20
CD8
T cells with a highly inflammatory and central nervous system (CNS)-homing phenotype, were significantly inversely correlated with pretreatment MRI gadolinium-lesion counts, and also predictive of early disease activity observed after anti-CD20 initiation. Direct removal of pretreatment proinflammatory CD20
CD8
T cells had a greater contribution to treatment-associated changes in the CD8
T cell pool than was the case for CD4
T cells. Early disease activity following anti-CD20 initiation was not associated with reconstituting CD20
CD8
T cells, which were less proinflammatory compared with pretreatment. Similarly, this disease activity did not correlate with early reconstituting B cells, which were predominantly transitional CD19
CD24
CD38
with a more anti-inflammatory profile. We provide insights into the mode-of-action of anti-CD20 and highlight a potential role for CD20
CD8
T cells in MS relapse biology; their strong inverse correlation with both pretreatment and early posttreatment disease activity suggests that CD20-expressing CD8
T cells leaving the circulation (possibly to the CNS) play a particularly early role in the immune cascades involved in relapse development.
Cluster of differentiation 20 (CD20) is a B cell membrane protein that is targeted by monoclonal antibodies for the treatment of malignancies and autoimmune disorders but whose structure and function ...are unknown. Rituximab (RTX) has been in clinical use for two decades, but how it activates complement to kill B cells remains poorly understood. We obtained a structure of CD20 in complex with RTX, revealing CD20 as a compact double-barrel dimer bound by two RTX antigen-binding fragments (Fabs), each of which engages a composite epitope and an extensive homotypic Fab:Fab interface. Our data suggest that RTX cross-links CD20 into circular assemblies and lead to a structural model for complement recruitment. Our results further highlight the potential relevance of homotypic Fab:Fab interactions in targeting oligomeric cell-surface markers.
Based on the known and emerging biology of autoimmune diseases and COVID‐19, it was hypothesised that whilst B‐cell depletion should not necessarily expose people to severe SARS‐CoV‐2‐related issues, ...it may inhibit or blunt the protective immunity following infection and vaccination. This is supported clinically, as the majority of SARS‐CoV‐2 infected, CD20‐depleted people with autoimmunity, have recovered. However, in CD‐20 treated people until naïve B‐cells repopulate, based on B‐cell repopulation‐kinetics and vaccination responses, from published rituximab, and unpublished ocrelizumab (NCT00676715, NCT02545868) trial data shown here suggests that it may be possible to undertake dose‐interruption to maintain inflammatory disease control, whilst allowing effective vaccination against SARS‐CoV‐29, if and when an effective vaccine is available.
Summary
Although most autoimmune diseases are considered to be CD4 T cell‐ or antibody‐mediated, many respond to CD20‐depleting antibodies that have limited influence on CD4 and plasma cells. This includes rituximab, oblinutuzumab and ofatumumab that are used in cancer, rheumatoid arthritis and off‐label in a large number of other autoimmunities and ocrelizumab in multiple sclerosis. Recently, the COVID‐19 pandemic created concerns about immunosuppression in autoimmunity, leading to cessation or a delay in immunotherapy treatments. However, based on the known and emerging biology of autoimmunity and COVID‐19, it was hypothesised that while B cell depletion should not necessarily expose people to severe SARS‐CoV‐2‐related issues, it may inhibit protective immunity following infection and vaccination. As such, drug‐induced B cell subset inhibition, that controls at least some autoimmunities, would not influence innate and CD8 T cell responses, which are central to SARS‐CoV‐2 elimination, nor the hypercoagulation and innate inflammation causing severe morbidity. This is supported clinically, as the majority of SARS‐CoV‐2‐infected, CD20‐depleted people with autoimmunity have recovered. However, protective neutralizing antibody and vaccination responses are predicted to be blunted until naive B cells repopulate, based on B cell repopulation kinetics and vaccination responses, from published rituximab and unpublished ocrelizumab (NCT00676715, NCT02545868) trial data, shown here. This suggests that it may be possible to undertake dose interruption to maintain inflammatory disease control, while allowing effective vaccination against SARS‐CoV‐29, if and when an effective vaccine is available.
B cell depletion via anti-CD20 antibodies is a highly effective treatment for multiple sclerosis (MS). However, little is known about the maturation/activation stage of the returning B cell ...population after treatment cessation and the wider effects on other immune cells. In the present study, 15 relapsing-remitting MS patients receiving 1,000 mg of rituximab were included. B, T, and myeloid cells were analyzed before anti-CD20 administration and in different time intervals thereafter over a period of 24 mo. In comparison to the phenotype before anti-CD20 treatment, the reappearing B cell pool revealed a less mature and more activated phenotype: 1) reappearing B cells were significantly enriched in transitional (before: 10.1 ± 1.9%, after: 58.8 ± 5.2%) and mature naive phenotypes (before: 45.5 ± 3.1%, after: 25.1 ± 3.5%); 2) the frequency of memory B cells was reduced (before: 36.7 ± 3.1%, after: 8.9 ± 1.7%); and 3) reappearing B cells showed an enhanced expression of activation markers CD25 (before: 2.1 ± 0.4%, after: 9.3 ± 2.1%) and CD69 (before: 5.9 ± 1.0%, after: 21.4 ± 3.0%), and expressed significantly higher levels of costimulatory CD40 and CD86. T cells showed 1) a persistent increase in naive (CD4⁺: before: 11.8 ± 1.3%, after: 18.4 ± 3.4%; CD8⁺: before: 12.5 ± 1.4%, after: 16.5 ± 2.3%) and 2) a decrease in terminally differentiated subsets (CD4⁺: before: 47.3 ± 3.2%, after: 34.4 ± 3.7%; CD8⁺: before: 53.7 ± 2.1%, after: 49.1 ± 2.7%).
A compact marker/suicide gene that utilizes established clinical-grade reagents and pharmaceuticals would be of considerable practical utility to T-cell cancer gene therapy. Marker genes enable ...measurement of transduction and allow selection of transduced cells, whereas suicide genes allow selective deletion of administered T cells in the face of toxicity. We have created a highly compact marker/suicide gene for T cells combining target epitopes from both CD34 and CD20 antigens (RQR8). This construct allows selection with the clinically approved CliniMACS CD34 system (Miltenyi). Further, the construct binds the widely used pharmaceutical antibody rituximab, resulting in selective deletion of transgene-expressing cells. We have tested the functionality of RQR8 in vitro and in vivo as well as in combination with T-cell engineering components. We predict that RQR8 will make T-cell gene therapy both safer and cheaper.
•Marker genes enable detection and selection of T cells, whereas suicide genes enable selective destruction of T cells in case of toxicity.•RQR8 is a 136-amino-acid epitope-based marker/suicide gene that enables clinical selection, cell tracking, and deletion in case of toxicity.
Fluorescence microscopy, with its molecular specificity, is one of the major characterization methods used in the life sciences to understand complex biological systems. Super-resolution approaches
...can achieve resolution in cells in the range of 15 to 20 nm, but interactions between individual biomolecules occur at length scales below 10 nm and characterization of intramolecular structure requires Ångström resolution. State-of-the-art super-resolution implementations
have demonstrated spatial resolutions down to 5 nm and localization precisions of 1 nm under certain in vitro conditions. However, such resolutions do not directly translate to experiments in cells, and Ångström resolution has not been demonstrated to date. Here we introdue a DNA-barcoding method, resolution enhancement by sequential imaging (RESI), that improves the resolution of fluorescence microscopy down to the Ångström scale using off-the-shelf fluorescence microscopy hardware and reagents. By sequentially imaging sparse target subsets at moderate spatial resolutions of >15 nm, we demonstrate that single-protein resolution can be achieved for biomolecules in whole intact cells. Furthermore, we experimentally resolve the DNA backbone distance of single bases in DNA origami with Ångström resolution. We use our method in a proof-of-principle demonstration to map the molecular arrangement of the immunotherapy target CD20 in situ in untreated and drug-treated cells, which opens possibilities for assessing the molecular mechanisms of targeted immunotherapy. These observations demonstrate that, by enabling intramolecular imaging under ambient conditions in whole intact cells, RESI closes the gap between super-resolution microscopy and structural biology studies and thus delivers information key to understanding complex biological systems.
Anti-CD20 therapies have established efficacy in the treatment of immune-mediated neurological and non-neurological diseases. Rituximab, one of the first B-cell-directed therapies, is relatively ...inexpensive compared to newer anti-CD20 molecules, is available in many countries, and has been used off-label in pediatric patients with neuroimmune conditions. The objective of this paper is to describe the experience with rituximab in pediatric multiple sclerosis and other inflammatory immune-mediated disorders of the central nervous system (CNS), and to define a protocol for its use in clinical practice, in particular addressing doses, interval of administration, duration of treatment, and tests to perform at baseline and during follow-up.
Abstract We conducted a trial testing a CD20-specific CAR coupled with CD137 and the CD3ζ moiety in patients with chemotherapy refractory advanced diffuse large B cell lymphomas (DLBCL). Seven ...patients were enrolled. One of the two patients with no bulky tumor obtained a 14-month durable and ongoing complete remission by cell infusion only, and another attained a 6-month tumor regression. Four of five patients with bulky tumor burden were evaluable for clinical efficacy, three of which attained 3- to 6-month tumor regression. Delayed toxicities related to cell infusion are directly correlated to tumor burden and tumor-harboring sites, and mainly included cytokine release symptoms, tumor lysis symptoms, massive hemorrhage of the alimentary tract and aggressive intrapulmonary inflammation surrounding extranodal lesions. These results show firstly that anti-CD20 CART cells can cause prolonged tumor regression in combination with debulking conditioning regimens for advanced DLBCL. This study is registered at www.clinicaltrials.gov as NCT01735604.
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