Surface Antigen CD20 Research Articles

Single-cell transcriptome analysis reveals CD34 as a marker of human sinoatrial node pacemaker cardiomyocytes

The sinoatrial node regulates the heart rate throughout life. Failure of this primary pacemaker results in life-threatening, slow heart rhythm. Despite its critical function, the cellular and molecular composition of the human sinoatrial node is not resolved. Particularly, no cell surface marker to identify and isolate sinoatrial node pacemaker cells has been reported. Here we use single-nuclei/cell RNA sequencing of fetal and human pluripotent stem cell-derived sinoatrial node cells to reveal that they consist of three subtypes of pacemaker cells: Core Pacemaker, Sinus Venosus, and Transitional Cells. Our study identifies a host of sinoatrial node pacemaker markers including MYH11, BMP4, and the cell surface antigen CD34. We demonstrate that sorting for CD34+ cells from stem cell differentiation cultures enriches for sinoatrial node cells exhibiting a functional pacemaker phenotype. This sinoatrial node pacemaker cell surface marker is highly valuable for stem cell-based disease modeling, drug discovery, cell replacement therapies, and the targeted delivery of therapeutics to sinoatrial node cells in vivo using antibody-drug conjugates.

The pro-differentiating capability of a flavonoid-rich extract of Citrus bergamia juice prompts autophagic death in THP-1 cells

Acute myeloid leukemia (AML) is a hematologic neoplasm, characterized by a blockage of differentiation and an unconstrained proliferation of immature myeloid cells. Recently, the survival of leukemia patients has increased thanks to the use of differentiating agents, though these may cause serious side effects. Hence, the search for safer differentiating compounds is necessary. Our aim was to assess the pro-differentiating effects of a flavonoid-rich extract of bergamot juice (BJe) in human monocytic leukemia THP-1 cells, an in vitro AML model. For the first time, we showed that treatment with BJe induced differentiation of THP-1 cells, changes in cell morphology and increased expression of differentiation-associated surface antigens CD68, CD11b and CD14. Moreover, BJe enhanced protein levels of autophagy-associated markers, such as Beclin-1 and LC3, as well as induced the phosphorylation of the MAPKs JNK, ERK and p38, hence suggesting a potential mechanism underlying its antiproliferative effects. Indeed, parallel experiments highlighted that BJe was able to hamper THP-1 cell growth. In conclusion, our study suggests that BJe induces the differentiation of THP-1 cells and reduces their proliferation, highlighting its potential in differentiation therapy of AML.

Disulfiram Inhibits Opsonin-Independent Phagocytosis and Migration of Human Long-Lived In Vitro Cultured Phagocytes from Multiple Inflammatory Diseases.

Disulfiram (DSF), an anti-alcoholism medicine, exerts treatment effects in patients suffering from persistent Borreliosis and also exhibits anti-cancer effects through its copper chelating derivatives and induction of oxidative stress in mitochondria. Since chronic/persistent borreliosis is characterized by increased amounts of pro-inflammatory macrophages, this study investigated opsonin-independent phagocytosis, migration, and surface marker expression of in vivo activated and in vitro cultured human monocyte-derived phagocytes (macrophages and dendritic cells) with and without DSF treatment. Phagocytosis of non-opsonized Dynabeads® M-450 and migration of macrophages and dendritic cells were monitored using live cell analyzer Juli™ Br for 24 h, imaging every 3.5 min. To simultaneously monitor phagocyte function, results were analyzed by a newly developed software based on the differential phase contrast images of cells before and after ingestion of Dynabeads. DSF decreased the phagocytic capacities exhibited by in vitro enriched and long-lived phagocytes. Although no chemotactic gradient was applied to the test system, vigorous spontaneous migration was observed. We therefore set up an algorithm to monitor and quantify both phagocytosis and migration simultaneously. DSF not only reduced phagocytosis in a majority of these long-lived phagocytes but also impaired their migration. Despite these selective effects by DSF, we found that DSF reduced the expression densities of surface antigens CD45 and CD14 in all of our long-lived phagocytes. In cells with a high metabolic activity and high mitochondrial contents, DSF led to cell death corresponding to mitochondrial oxidative stress, whereas metabolically inactive phagocytes survived our DSF treatment protocol. In conclusion, DSF affects the viability of metabolically active phagocytes by inducing mitochondrial stress and secondly attenuates phagocytosis and migration in some long-lived phagocytes.

Isatuximab-Based Regimens As Salvage Therapy for Patients with Multiple Myeloma: Sardinian, Multicenter, Retrospective Clinical Practice Experience with 27 Cases Outside of Controlled Clinical Trials

Introduction: Isatuximab (Isa), a humanized monoclonal antibody directed against the surface antigen CD38, has been available in Italy since October 2021 for the treatment of relapsed or refractory Multiple Myeloma (RRMM) in combination with Pomalidomide (P) and Dexamethasone (d) (IsaPd) after two lines of therapy with lenalidomide and a proteosome inhibitor, and from April 2022 in combination with carfilzomib (K) after one line of treatment. Patients eligible for these therapies must not be refractory to Daratumumab. Here, we report preliminary efficacy and safety data for Isatuximab in a Sardinian study of RRMM patients treated with IsaPd and IsaKda as salvage therapy outside of controlled clinical trials. The primary objective of this study was to evaluate the toxicity profile and quality of response to Isatuximab-based regimens administered as salvage therapy in a real-world setting. Methods: The 2 cohorts included 27 patients with RRMM from 4 Sardinian centers who received at least one cycle of IsaPd (16 patients) or IsaKd (11 patients) as salvage therapy between January 2022 and March 2023. Patients received Isa 10 mg/kg IV weekly for the first 4 weeks (wks), then every 2 weeks in combination with Pomalidomide and Dexamethasone (4mg PO days 1-21; 40mg [20mg at > 75 years] PO or IV weekly) every 28 days for the IsaPd regimen. The IsaKd regimen consisted of administration of 10 mg/kg Isatuximab intravenously once weekly in the first cycle, then once every 2 weeks in subsequent cycles. Both arms received Carfilzomib at a dose of 20 mg/m2 on days 1 and then 56 mg/m2 on days 8, 9, 15, and 16 of the first cycle, then 56 mg/m2 on days 1, 2, 8, 9, 15, and 16 in subsequent cycles, while 20 mg of Dexamethasone was administered on days 1, 2, 8, 9, 15, 16, 22, and 23 of each cycle. Results: The median age of the 16 IsaPd-treated patients was 67.5 years (range 48-89 years) and 61 years (range 49-84 years) for the IsaKd population; 44% and 64% were male, respectively (Table 1). The median number of prior therapies was 2 (range 2-4) for the IsaPd and 1 (range 1-4) for the IsaKd cohort; 11 (68.75%) and 9 (82%) received prior autologous stem cell transplantation (ASCT), respectively. Four patients in both populations started treatment because of biochemical relapse, and no refractory patients were recorded in the IsaKd cohort. Three patients (2 in the IsaPd population and 1 in the IsaKd cohort) had creatinine clearance ≤30 mL/min. Among the IsaKd-cohort, 5 (46%) had R-ISS 2 stage and three had a 17p deletion. At the last data collection, 13 cases in the IsaPD population were evaluable for response. The median number of courses administered to date was 4 (range, 1-14). The overall response rate (ORR) was 69%, with 2 stringent complete remissions (sCR) (15%) and 1 very good partial remissions (VGPRs) (7.7%). The median time to first response was 1 month, while the median time to best response was 2 months. In the IsaKd population, 10 patients were evaluable with an overall response rate (ORR) of 100%, with 1 complete remission (sCR) (10%), 1 complete remission (10%), and 5 very good partial remissions (VGPRs) (50%). A median time to first response of 1 month was also recorded in this population, whereas the median time to best response was 3 months. Renal insufficiency and age > 70 years had no significant impact on the likelihood of achieving a response in either population.In the IsaPd cohort, after a median follow-up of 6 months (range 1-14 months), 9 patients discontinued treatment, 7 due to disease progression, 1 due to toxicity, and 1 due to patient choice) Six patients died (5 patients from progressive disease and 1 from secondary acute myeloid leukemia). Common grade 3 or 4 adverse events were fatigue (57%), thrombocytopenia (35%), neutropenia (57%), lymphocytopenia (15.9%), anemia (29%), and infection (21%). Infusion reactions occurred in only 2 patients (12.5%) and were grade 1 or 2 without treatment discontinuation. In the IsaKd population, the median follow-up time was 4 months (range 1-8 months). 1 patient discontinued treatment and died due to disease progression. Grade 3 or 4 adverse events were fatigue (18%), thrombocytopenia (18%), neutropenia (18%), anemia (36%), and infection (63%) No infusion reactions occurred. Conclusions: Despite the limited number of cases, our preliminary real-world data confirm that Isa-based treatment is an effective and safe therapy for RRMM patients, broadly consistent with results from controlled clinical trials.

A Single Bout of Exercise Enhances the Efficacy of Rituximab Against Autologous Chronic Lymphocytic Leukaemia B Cells Ex Vivo By Transiently Increasing Natural Killer Cell Frequency in Blood, and Simultaneously Mobilises CD5 +CD19 +CD20 + B Cells into Blood

Chronic Lymphocytic Leukaemia (CLL) is characterised by the proliferation and accumulation of clonal B cells (B-CLL cells) and is often treated with anti-CD20 monoclonal antibody immunotherapies, including rituximab. One of the mechanism-of-action of rituximab is antibody-dependent cellular cytotoxicity (ADCC) which occurs when natural killer (NK) cells detect rituximab bound to CD20 + target cells. Rituximab often fails to induce stringent disease eradication, due in part to the diffuse distribution of clonal cells across multiple lymphoid and non-lymphoid tissues where NK cell frequency can be low. It is well established that an individual bout of aerobic exercise induces a transient relocation of lymphocytes - including NK cells and B cells - into peripheral blood. We hypothesised that this exercise-induced lymphocytosis could be harnessed to enhance the efficacy of rituximab in CLL by relocating both target and effector cells together with rituximab into blood. In this pilot study n = 20 treatment naïve patients with CLL (mean ± SD: age = 62 ± 10 years; height = 174.0 ± 7.5 cm; body mass = 83.3 ± 16.8 kg; body fat = 31.7 ± 9.8 %; blood leukocytes = 30.70 ± 22.21 ×10 9/L; anaerobic threshold = 14.1 ± 2.9 mL.kg -1.min -1) participated. Participants cycled at a moderate intensity (15% above their anaerobic threshold) for ~30-minutes, with blood samples collected pre-, post-, and 1-hour post-exercise. Given the importance of CD16 + NK cells in evoking ADCC, we enumerated NK cell subsets in blood samples collected pre, post-, and 1-hour post-exercise by flow cytometry. As expected, exercise induced a preferential increase of CD56 +CD16 + (+255%, p < 0.001) and mature, cytotoxic CD56 +CD57 +CD16 + NK cells (+322%, p < 0.001) pre- to post-exercise. Next, using immunomagnetic negative separation, NK cells and primary B-CLL cells were isolated from blood pre- and post-exercise and incubated together with heat inactivated foetal calf serum, with or without the presence of rituximab to determine specific lysis using a calcein-release assay. Rituximab mediated cell lysis increased by +129% following exercise ( p < 0.001), with no change in antibody independent NK cell lysis of B-CLL cells - independent of rituximab - following exercise ( p = 0.25). Blocking CD16 on NK cells - vital for ADCC - blunted the effects of exercise on B-CLL cell lysis ( p = 0.84). In a subset of patients ( n = 9) we also explored the effects of autologous time-point matched plasma (instead of heat inactivated foetal calf serum) on rituximab mediated ADCC, which evoked a +92% increase in lysis pre- to post-exercise ( p = 0.038). Collectively, our results suggest that augmented efficacy of rituximab mediated ADCC was driven by an increase in CD16 + NK cells. We posited that the enhancement to rituximab mediated ADCC would have greater, clinically relevant implications if there was also a concomitant exercise-induced mobilisation of B-CLL cells expressing CD20 into blood from different body tissues. Flow cytometry revealed a +63% increase in CD5 +CD19 +CD20 + B-CLL cells ( p = 0.002) in blood after exercise. Further analyses revealed that CD5 +CD19 +CD20 + B-CLL cells with a phenotype consistent with recent egress from lymphoid tissue (CD5 brightCXCR4 dim; 70%, p = 0.004) and B-CLL cells with a propensity to migrate to peripheral tissues (CD5 dimCXCR4 bright; 67%, p = 0.002) were mobilised, with no change to overall CD20 surface antigen density ( p = 1.0) - determined by median fluorescence intensity. Furthermore, exercise evoked a +69% ( p = 0.022) increase in CD5 +CD19 +CD20 + B-CLL cells expressing CD49d, which is considered one of the strongest predictors of CLL prognosis. Taken together, these data demonstrate that exercise increased the frequency of CD20 + B-CLL cells with lymphoid origins and prognostic relevance into the blood, therefore rendering them susceptible to rituximab mediated ADCC. Our results show that individual bouts of moderate intensity aerobic exercise temporarily increased the number of cytotoxic CD16 + NK cells, and CD20 + B-CLL cells in blood. Additionally, our ex vivo investigations demonstrated enhanced rituximab mediated ADCC following exercise. Thus, exercise could be explored as a means of improving clinical responses in patients receiving rituximab, and/or other anti-CD20 monoclonal antibodies such as, Obinutuzumab.

TP53 Deficiency in AML Confers Resistance to CAR T-Cells That Can be Overcome By Targeting the Cholesterol or Wnt Pathways

Introduction TP53-mutant acute myeloid leukemia (AML) and myelodysplastic neoplasms (MDS) are distinct clinicogenomic entities characterized by chemotherapy resistance and high relapse rates after allogeneic stem cell transplantation, and thus, represent an unmet clinical need. Chimeric antigen receptor (CAR) T-cell therapy might be a promising therapeutic option for TP53-mutant AML/MDS. However, the AML-intrinsic determinants of response to T-cell-based approaches in AML, in particular the impact of TP53-deficiency, are largely unknown. In the present study, we addressed the biologically and clinically highly relevant question as to whether, and, if so, how TP53 deficiency in AML cells might confer resistance to CAR T-cell therapy. Results We took advantage of recently developed, CRISPR/Cas9-engineered TP53 isogenic AML cell lines harboring TP53 null, missense, or wildtype alleles to exclude other potential genetic confounders of therapy resistance. Flow cytometry-based co-culture assays using these isogenic cell lines together with CAR T-cells targeting the common AML surface antigens CD33, CD371, CD123, and CD117, showed that AML cells harboring TP53 null or missense mutations were relatively resistant to CAR T-cells in vitro. CAR T-cells engaging TP53-deficient leukemia cells proliferated less, upregulated exhaustion markers, displayed increased trogocytosis, and were ultimately unable to control AML cell outgrowth. Live-cell imaging revealed a longer engagement time of CAR T-cells with TP53-deficient AML cells before killing occurred as judged by propidium iodide influx. Immunodeficient mice xenografted with TP53-deficient AML cells and treated with CAR T-cells exhibited significantly shortened survival compared to TP53-wildtype AML controls. Transcriptional profiling revealed that the cholesterol pathway was upregulated in TP53-deficient AML cells under CAR T-cell attack. Simultaneously, CAR T-cells engaging TP53-deficient AML demonstrated a downregulated Wnt pathway, in particular the master regulators of T-cell fate TCF7 and EOMES. Rational pharmacological targeting of either of these pathways, blocking by Simvastatin and activation by BIO-acetoxime, respectively, rescued TP53-deficient AML cell sensitivity to CAR T-cell-mediated killing in vitro. Discussion and Outlook Using a combination of in vitro co-incubation assays, live-cell imaging, gene expression profiling and a therapeutic in vivo model we could demonstrate that TP53 deficiency in AML cells confers relative resistance to CAR T-cell therapy and elucidate some of the underlying molecular mechanisms in leukemia target cells and CAR T-cells. We thus propose a model, in which the intrinsic apoptotic defect in TP53-deficient AML/MDS cells results in a longer duration of the cellular interaction between CAR T-cells and TP53-deficient AML/MDS cells. This longer temporal interaction eventually leads to reduced CAR T-cell proliferation and enhanced CAR T-cell exhaustion with an overall decrease in AML cell killing (figure 1). Furthermore, we identified the cholesterol pathway as a potential therapeutic vulnerability of TP53-deficient AML cells engaged by CAR T-cells, and the Wnt pathway as a promising avenue to enhance the efficacy of CAR T-cell therapy in TP53-deficient AML/MDS. Our data suggest that the combination of CAR T-cell therapies with pharmacological co-interventions - as exemplified in this study - may be a preferable strategy towards more efficacious and tolerable cellular therapies for patients with TP53-mutant myeloid neoplasms.

Characterization of CD3+/CD20+ canine large-cell lymphoma.

Immunophenotyping of canine large-cell lymphoma (LCL) for B-cell and T-cell surface antigens is commonly performed to better predict the clinical outcome. Expression of surface antigen CD3 is associated with T-cell malignancies; surface antigen CD20 is expressed on B cells. However, a small subset of canine LCLs expresses both CD3 and CD20 (CD3+/CD20+); this form of lymphoma remains poorly defined at the molecular level. In a retrospective study, we aimed to better characterize immunophenotypic properties and antigen receptor clonality of CD3+/CD20+ LCL. We selected formalin-fixed, paraffin-embedded tissues from 10 cases of CD3+/CD20+ LCL and breed-matched controls of peripheral large T-cell lymphoma (PTCL) and diffuse large B-cell lymphoma (DLBCL). Using PCR for antigen receptor rearrangement (PARR), we identified monoclonal T-cell receptor gamma (TCRγ) rearrangements in all CD3+/CD20+ cases. Three of 10 cases had monoclonal rearrangements in the immunoglobulin heavy chain (IgH), supportive of cross-lineage rearrangement. There was no significant difference in the frequency of antigen receptor rearrangement between CD3+/CD20+ and PTCL cases. In comparison with DLBCL, CD3+/CD20+ LCL had TCRγ rearrangement more frequently and IgH rearrangement less frequently, respectively. Immunolabeling of the B-cell marker PAX5 occurred less frequently in all CD3+/CD20+ LCL cases compared to the DLBCL controls. Immunolabeling for BCL-2 was robust, regardless of immunophenotype. Nuclear Ki67 positivity was variable in CD3+/CD20+ cases, indicating a heterogeneity in proliferation. Overall, cases of canine CD3+/CD20+ LCL had properties similar to PTCL, suggesting a similar histogenesis of these 2 subsets.

Identifying Suitable Targets for Alzheimer's Disease and Other Eight Common Neurological Disorders Using the Human Plasma Proteome: A Mendelian Randomization Study.

Neurological disorders, such as Alzheimer's disease (AD), comprise a major cause of health-related disabilities in human. However, biomarkers towards pathogenesis or novel targets are still limited. To identify the causality between plasma proteins and the risk of AD and other eight common neurological diseases using a Mendelian randomization (MR) study. Exposure data were obtained from a genome-wide association study (GWAS) of 2,994 plasma proteins in 3,301 healthy adults, and outcome datasets included GWAS summary statistics of nine neurological disorders. Inverse variance-weighted MR method as the primary analysis was used to estimate causal effects. Higher genetically proxied plasma myeloid cell surface antigen CD33 level was found to be associated with increased risk of AD (odds ratio [OR] 1.079, 95% confidence interval [CI] 1.047-1.112, p = 8.39×10-7). We also discovered the causality between genetically proxied elevated prolactin and higher risk of epilepsy (OR = 1.068, 95% CI = 1.034-1.102; p = 5.46×10-5). Negative associations were identified between cyclin-dependent kinase 8 and ischemic stroke (OR = 0.927, 95% CI = 0.896-0.959, p = 9.32×10-6), between neuralized E3 ubiquitin-protein ligase 1 and migraine (OR = 0.914, 95% CI = 0.878-0.952, p = 1.48×10-5), and between Fc receptor-like protein 4 and multiple sclerosis (MS) (OR = 0.929, 95% CI = 0.897-0.963, p = 4.27×10-5). The findings identified MR-level protein-disease associations for AD, epilepsy, ischemic stroke, migraine, and MS.

Role of CD14 in human disease.

The cell surface antigen CD14 is primarily understood to act as a co-receptor for toll-like receptors (TLRs) to activate innate immunity responses to pathogens and tissue injury in macrophages and monocytes. However, roles for CD14 are increasingly being uncovered in disease responses in epithelial and endothelial cells. Consistent with these broader functions, CD14 expression is altered in a variety of non-immune cell types in response to a several of disease states. Moreover, soluble CD14 activated by factors from both pathogens and tissue damage may initiate signalling in a variety of non-immune cells. This review examined the current understanding CD14 in innate immunity as well as its potential functions in nonimmune cells and associated human diseases.

Study on in vitro differentiation of human adenoid-derived mesenchymal stem cells into olfactory sensory neurons

Objective: To investigate the feasibility of isolation and culture of human adenoid-derived mesenchymal stem cells (aMSCs) in vitro, and to observe the differentiation of aMSCs into olfactory sensory neurons. Methods: Adenoid tissues surgically removed from children with adenoid hypertrophy in the Second Xiangya Hospital of Central South University from September to November of 2020 were collected. The adenoid tissues were digested and isolated by trypsin and then cultured with adhesion method. The expressions of cell surface antigens CD45, CD73 and CD90 on aMSCs of P5 generation were tested by flow cytometry, and the ability of osteogenic and adipogenic induction were used to identify cell differentiation ability. Then, aMSCs were induced into differentiation by retinoic acid (RA), sonic hedgehog (SHH), basic fibroblast growth factor (bFGF), RA+SHH, RA+bFGF, SHH+bFGF and RA+SHH+bFGF, respectively. The morphology of differentiated cells was observed under inverted microscope. The expression of β-tubulin 3, which was the specific marker of sensory neuron, the expressions of growth associated protein-43 (GAP43) and olfactory maker protein (OMP), which were the specific markers of olfactory sensory neuron, were detected by immunofluorescence antibody assay. The expression intensities were compared by Chi-square test of four-grid table data. Results: aMSCs were successively isolated and cultured from human adenoid tissues. P0 cells generation had good adhesion and proliferation performance. P2 cells were basically purified. P5 cells expressed CD73 and CD90 with the purity of 99.3% and 99.75% respectively, without CD45 expression. P5 cells had a good ability of osteogenic differentiation and adipogenic differentiation. Neuron-like morphology and expression of β-tubulin 3 were found in differentiated cells after induced by RA, SHH, or bFGF, respectively. An induction of expression of GAP43 was found in differentiated cells of bFGF+SHH group and RA+SHH+bFGF group, without expression of OMP of each group. The intensity of GAP43 expression of RA+SHH+bFGF group was stronger than that of bFGF+SHH group (χ2=17.48, P<0.005). Conclusions: aMSCs can be cultured from human adenoid tissues, with the stably passaged and good differentiation ability. As a new population of mesenchymal stem cells, aMSCs have the neuroregenerative properties and could differentiate into immature olfactory sensory neurons under the induction of RA+SHH+bFGF in vitro.

Effect of Dichloromethane Extraction Phase of Patrinia Scabiosaefolia Fisch. Stem on Proliferation and Differentiation of K562 Cells

To explore the effect of dichloromethane extraction phase of ethanol extract from stem of Patrinia scabiosaefolia Fisch.(DPSS) on proliferation and differentiation of K562 cells and its related mechanism. MTT assay was used to detect the effects of DPSS at 0, 25, 50, 100 and 200 μg/ml on the proliferation of K562 cells at 24, 48 and 72 hours. Flow cytometry was used to analyze the changes of cell cycle and apoptosis at 24 and 48 hours. Wright-Giemsa staining was used to observe the morphological changes of K562 cells. The cell surface antigens CD33 and CD11b were detected by flow cytometry. The proliferation of K562 cells treated with different concentrations of DPSS was inhibited in a time-dose dependent manner (r=-0.96). Cell cycle analysis showed that with the increase of DPSS concentration, cells in G2/M phase increased (r=0.88), and cells were blocked in G2/M phase. Flow cytometry results showed that with the apoptosis rate of K562 cells was the highest when treated with 200 μg/ml DPSS for 48 h. Morphological observation showed that the K562 cell body increased, the amount of cytoplasm increased, the ratio of nucleus to cytoplasm decreased, and the nuclear chromatin was rough after DPSS treatment. Cell differentiation antigen, CD33 and CD11b, were positively expressed after treated with DPSS. DPSS can induce apoptosis through cell cycle arrest, inhibit the proliferation of K562 cells, and induce K562 cells to differentiate into monocytes, which has a potential anti-leukemia effect.

CAR T-Cell Immunotherapy Treating T-ALL: Challenges and Opportunities.

T-cell acute lymphoblastic leukemia (T-ALL), a form of T-cell malignancy, is a typically aggressive hematological malignancy with high rates of disease relapse and a poor prognosis. Current guidelines do not recommend any specific treatments for these patients, and only allogeneic stem cell transplant, which is associated with potential risks and toxicities, is a curative therapy. Recent clinical trials showed that immunotherapies, including monoclonal antibodies, checkpoint inhibitors, and CAR T therapies, are successful in treating hematologic malignancies. CAR T cells, which specifically target the B-cell surface antigen CD19, have demonstrated remarkable efficacy in the treatment of B-cell acute leukemia, and some progress has been made in the treatment of other hematologic malignancies. However, the development of CAR T-cell immunotherapy targeting T-cell malignancies appears more challenging due to the potential risks of fratricide, T-cell aplasia, immunosuppression, and product contamination. In this review, we discuss the current status of and challenges related to CAR T-cell immunotherapy for T-ALL and review potential strategies to overcome these limitations.

A BENEFICIAL EFFECT OF LONG-TERM TREATMENT OF RITUXIMAB IN INTERSTITIAL LUNG DISEASE ASSOCIATED WITH SYSTEMIC SCLEROSIS

A BENEFICIAL EFFECT OF LONG-TERM TREATMENT OF RITUXIMAB IN INTERSTITIAL LUNG DISEASE ASSOCIATED WITH SYSTEMIC SCLEROSIS

Post-Cardiac Arrest Syndrome Is Not Associated With an Early Bacterial Translocation.

The aim of this study was to investigate bacterial translocation and its possible role in the development of post-resuscitation inflammatory response following Cardio-Pulmonary Resuscitation (CPR) after cardiac arrest. Munich female swine were employed for a model of cardiac arrest via application of electrical current. After 7 min, CPR was initiated, and animals were either successfully return to spontaneous circulation (ROSC) within 40 min or not (no-ROSC). At the end of experimental period and prior to sacrifice, samples from the intestine, mesenteric lymph nodes (MLN), liver and portal vein blood were obtained. Evaluation of inflammation and gut permeability was performed; MLN, liver and portal vein samples were analyzed for 16 s rRNA detection and cytokine mRNA expression. A decreased expression of the tight junction protein Occludin, with higher levels of inflammation, greater epithelial disintegration, ulceration, loss of crypts and villi height were found in the intestines of the ROSC swine in comparison to no-ROSC. The macrophage surface antigen CD-14 staining was relatively more intense in the ROSC than in no-ROSC. Higher levels of TNF-α mRNA expression were present in the liver of the ROSC group. Finally, despite the inflammatory response and the gut mucosal alterations in ROSC group, no bacterial translocation was detected in liver, MLN and portal vein. We show that resuscitation from cardiac arrest induces inflammatory response and intestinal permeability in swine 4h after resuscitation, but not a bacterial translocation. Bacterial translocation is not an early phase phenomenon but probably part of the pathophysiologic sequelae.

Monoclonal antibody-based therapies for Waldenström's macroglobulinemia

Monoclonal antibodies have established an important role in the treatment armamentarium of hematological malignancies, including Waldenström's macroglobulinemia. Rituximab, an anti-CD20 monoclonal antibody, is established as standard therapy for this unique low grade lymphoma, due to its effectiveness and safety as monotherapy, in combination with chemotherapy or other targeted therapies in WM. Newer monoclonal antibodies, targeting CD20 or other surface antigens, have shown to be effective in patients with WM. In the current review we attempt to provide an overview of the mechanisms of action of monoclonal antibodies and discuss clinical evidence that support their use in WM and their therapeutic potential.

Alemtuzumab clearance, lymphocyte count, and T‐cell chimerism after hematopoietic stem cell transplant in sickle cell disease

AbstractStudy ObjectiveAlemtuzumab is a monoclonal antibody that targets the cell surface antigen CD52 on lymphocytes. Although it is used for the treatment of hematologic malignancies, such as chronic lymphocytic leukemia, and incorporated into many hematopoietic stem cell transplant (HSCT) conditioning regimens, few studies have evaluated the pharmacology of alemtuzumab in adult patients with sickle cell disease (SCD). We therefore examined the pharmacokinetics (PK) and pharmacodynamics (PD) of alemtuzumab in adults with SCD who received a matched related donor HSCT to determine if the clearance of alemtuzumab affects transplant outcomes.DesignPK and PD analysis of patient data from a single‐center clinical trial.SettingClinical research center.PatientsTwenty‐two adult patients with SCD who received one of two nonmyeloablative allogeneic HSCT regimens: alemtuzumab and total body irradiation (Alem‐TBI) or pentostatin, cyclophosphamide, alemtuzumab, and total body irradiation (Pento‐Cy‐Alem‐TBI).Measurements and Main ResultsAlemtuzumab serum concentrations, absolute lymphocyte counts, T‐cell (CD3), and myeloid (CD14/15) chimerism were collected at distinct time points and analyzed. A semi‐mechanistic PK population model was built to understand inter‐individual differences in pharmacology. Alemtuzumab was detectable up to 28 days post‐HSCT. The mean alemtuzumab level 7 days after transplant for patients on Alem‐TBI was 818 ng/ml, significantly lower than the mean level of 1502 ng/ml for patients on Pento‐Cy‐Alem‐TBI (p &lt; 0.001), but this difference decreased as time progressed. The clearance of alemtuzumab was linear, and the half‐life was longer in the Pento‐Cy‐Alem‐TBI group (average half‐life = 61.1 h) compared to the Alem‐TBI group (average half‐life = 44.1 h) (p &lt; 0.001). The CD3 chimerism at 2 and 4 months after transplant positively correlated with alemtuzumab levels collected on day 14 after transplant (R2 = 0.40 and p = 0.004 at 2 months, R2 = 0.36 and p = 0.005 at 4 months), but this significance was lost by 6 months after HSCT. No correlation was seen between alemtuzumab levels and CD14/15 chimerism.ConclusionBetween 2 and 4 months after transplant, higher alemtuzumab levels measured 14 days after transplant correlated with patients having better engraftment, suggesting more lymphodepletion may be needed to reduce graft failure in these two non‐myeloablative matched related donor HSCT regimens.

Arsenite exposure inhibits the erythroid differentiation of human hematopoietic progenitor CD34+\u2009cells and causes decreased levels of hemoglobin

Arsenic exposure poses numerous threats to human health. Our previous work in mice has shown that arsenic causes anemia by inhibiting erythropoiesis. However, the impacts of arsenic exposure on human erythropoiesis remain largely unclear. We report here that low-dose arsenic exposure inhibits the erythroid differentiation of human hematopoietic progenitor cells (HPCs). The impacts of arsenic (in the form of arsenite; As3+) on red blood cell (RBC) development was evaluated using a long-term culture of normal human bone marrow CD34+-HPCs stimulated in vitro to undergo erythropoiesis. Over the time course studied, we analyzed the expression of the cell surface antigens CD34, CD71 and CD235a, which are markers commonly used to monitor the progression of HPCs through the stages of erythropoiesis. Simultaneously, we measured hemoglobin content, which is an important criterion used clinically for diagnosing anemia. As compared to control, low-dose As3+ exposure (100 nM and 500 nM) inhibited the expansion of CD34+-HPCs over the time course investigated; decreased the number of committed erythroid progenitors (BFU-E and CFU-E) and erythroblast differentiation in the subsequent stages; and caused a reduction of hemoglobin content. These findings demonstrate that low-dose arsenic exposure impairs human erythropoiesis, likely by combined effects on various stages of RBC formation.

Quantitative Assessment of the Evolution of Therapeutic Target Antigen Expression Level in Diffuse Large B-Cell Lymphoma in Response to Treatment

Introduction:Approximately one-third of patients (pts.) with diffuse large B-cell lymphoma (DLBCL) develop relapsed or refractory (R/R) disease after frontline rituximab-(R) based chemo immunotherapy. Variability in expression of the B-cell surface antigen CD20, and possibly CD19, is thought to be an important mechanism of treatment failure, but there is vast heterogeneity in the reported incidence, with most reports utilizing Immunohistochemical (IHC) staining which is non-quantitative in nature. New therapeutics including CAR T-cells, antibodies (Ab) (tafasitimab) and Ab-drug conjugates (loncastuximab teserine) target CD19, while several bispecific engager Abs. in development target CD20. Understanding the evolution of target antigen expression in R/R DLBCL can provide information regarding selection and sequencing of therapies. We sought to quantitate the change in surface expression of CD19 and CD20 in R/R DLBCL after R-based chemo immunotherapy relative to pretreatment levels, to provide insight into the relative stability of these targets.Methods:Pts. diagnosed with DLBCL after January 2014, who were R/R to frontline R-based chemo immunotherapy (R-CHOP or R-EPOCH) were retrospectively identified. Pt. and disease related clinical variables at diagnosis (Dx) and first R/R were recorded. Data from IHC were obtained, as were archival flow cytometry assays performed on tumor biopsies at Dx and in the R/R setting. Using FCS Express® software, neoplastic cells were gated, and fluorescence intensity (FI) of CD19 and CD20 expression were reported (using fluorochromes APC-A for CD19 and PECy7 for CD20, respectively). Multivariate linear mixed model was used to compare median and geometric mean FI of CD19 and CD20 between Dx and R/R, while adjusting for other clinical variables.Results:A total of 51 flow cytometry assays (26 Dx and 25 R/R) were analyzed for 33 pts. Median age at Dx was 64 (range, 41-76) yrs., 24 pts. (73%) were male and 29 (88%) had IPI ≥ 2. 11 (33%) pts. had a prior indolent lymphoma. Cell of origin at Dx was GCB in 16 (49%) and non-GCB in 12 (36%) pts., while two had a double-hit rearrangement at diagnosis. Treatment was R-CHOP in 27 cases (82%) and R-EPOCH in 6 (18%). Median time to R/R was 10.4 months.There was a significant reduction in median FI of CD20 from Dx [median: 40,610 (range: 167 - 259,962)] to R/R [median: 11,596 (range: 63 - 79,592)], representing a mean reduction of 63% at R/R relative to Dx (P= 0.01; 95% CI: 20-73%). Similar change was observed in geometric mean FI of CD20, which was reduced 65% at R/R relative to Dx (P< 0.01; 95%CI: 31-82%). In comparison, on IHC, CD20 was reported to be negative at R/R relative to Dx in only one of 16 pts. for whom IHC results were available. Median and geometric mean FI of CD19 at R/R were 38% and 20% lower compared to Dx, respectively, but these differences were not statistically significant (P= 0.08 and 0.39, respectively) (Table). When examining the relative change in FI at R/R in individual cases, compared to the mean FI of all Dx cases, we observed that 21 out 25 R/R cases (84%) had reduction of CD20 whereas only 14/25 (56%) had reduction of CD19. Interestingly, 7/25 (28%) R/R cases had an increase in CD19 expression by >80% (Figure).When adjusting for clinical variables such as age, sex, presence of B symptoms, bone marrow (BM) involvement, PS and prior indolent lymphoma, the change in CD20 median FI from Dx to R/R maintained statistical significance (p=0.01). Reduction in CD20 geometric mean FI from Dx to R/R was significantly associated with age >60 years (p=0.04), BM involvement (p <0.01) and >1 site of extra nodal involvement (p= 0.03) at Dx.Conclusions:Quantitative assessment by flow cytometry revealed a significant decline in expression of CD20 at R/R compared to Dx in the majority of patients with DLBCL treated with R-based chemo immunotherapy. CD19 expression was unchanged in most R/R cases but was found to be dramatically upregulated in a subset of R/R cases. Given the role of CD19 mediated pathways in B-cell NHL and its association with PI3K pro-survival signaling, these data merit further exploration as a potential mechanism of treatment resistance. These findings also highlight the importance for repeat tissue biopsy at the time of suspected R/R DLBCL, with focus on therapeutic target expression, as it may influence treatment decisions or enrollment in clinical trials exploring efficacy of newer agents. [Display omitted] DisclosuresCaimi: TG Therapeutics: Honoraria; Seattle Genetics: Consultancy; Amgen Therapeutics.: Consultancy; XaTek: Patents & Royalties: Royalties from patents (wife); Verastem: Consultancy; Genentech: Research Funding; Kite Pharmaceuticals: Consultancy; ADC Theraputics: Consultancy, Research Funding. Hill: Celgene (BMS): Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Kite, a Gilead Company: Consultancy, Honoraria, Other: Travel Support, Research Funding; Karyopharm: Consultancy, Honoraria, Research Funding; AstraZenica: Consultancy, Honoraria; Epizyme: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Gentenech: Consultancy, Honoraria, Research Funding; Incyte/Morphysis: Consultancy, Honoraria, Research Funding; Beigene: Consultancy, Honoraria, Research Funding.

S1965 B Adaptive: A Case of Rituximab-Associated Colitis and SARS-CoV-2 Vaccination

Introduction: Rituximab (RTX) is a monoclonal antibody against the CD20 surface antigen on B-Cell lymphocytes and is used in various hematologic and autoimmune conditions. Our case describes a patient on RTX therapy with pancolitis that coincided with SARS-CoV-2 vaccination (Moderna-mRNA1273). We attempt to explore a potential correlation of mRNA vaccination platforms, RTX therapy and new onset colitis. Case description/methods: A 49 year old male presented with 6 weeks of watery, non-bloody diarrhea, right upper quadrant pain, >10 pound weight loss and fever. History was significant for stage IVa follicular lymphoma in remission on RTX maintenance therapy, splenectomy and SARS-CoV-2 vaccination (Moderna mRNA-1273) 2 days prior to onset of diarrhea. Infectious work up was negative and CT imaging demonstrated diffuse colonic wall thickening with new interval adjacent pancolonic fat stranding. Initial colonoscopy demonstrated diffuse scattered clean based ulcers between 1-8 mm in size throughout the colon and terminal ileum. Biopsies revealed active chronic ileitis with eosinophils, occasional crypt abscess and crypt destruction negative for HSV, CMV or signs of recurrent lymphoma. He was then treated with oral budesonide, bentyl and loperamide but returned 6 weeks later without any relief in abdominal pain or diarrhea. Repeat colonoscopy demonstrated circumferential hyperemia/edema with apthous ulcers throughout the colon. We plan to treat as a Crohn's patient with corticosteroid induction and trial Vedolizumab for maintenance of remission. Discussion: The novel use of mRNA vaccines has been noted to be game changing in its production time as well as its effectiveness at preventing COVID-19 illness. The mechanism of immunity is proposed to be a combination of generation of neutralizing antibodies and T-helper cell activation. To our knowledge, mRNA-1273 has not directly caused colitis. Literature also describes occurrences of fulminant colitis in patients on RTX therapy and is believed to be secondary to dysregulation caused by depletion of CD20+ B cells and upregulation of autoreactive T-cells. While RTX induced colitis may arise de novo, there may be a correlation between the immunogenicity of mRNA vaccines like mRNA-1273 and the induction of colitis in patients while on immunosuppressants. This case demonstrates the increased need for further evaluation of the pathogenesis of RTX induced colitis as well as the immunogenic response seen in COVID-19 vaccination platforms.

Circulating miR-221/222 reduces CD4+ T cells by inhibiting CD4 expression in colorectal cancer.

Many patients with cancers have low levels of CD4+ in their peripheral blood. However, the molecular mechanism is still unclear. Here, we found that the blood levels of miR-221 and miR-222 were dramatically increased in patients with colorectal cancer (CRC), and both circulating miR-211 and miR-222 served as sensitive diagnostic markers with an area under the curve of 0.8790 and 0.9148, respectively. Transfection of either miR-221 or miR-222 resulted in the reduction of the surface CD4 antigen level but not the surface CD8 antigen level. The luciferase reporter assay showed that miR-221/222 directly regulated CD4 expression in human primary T cells. These data showed that miR-221/222 levels were upregulated in the blood of patients with CRC and that the expression of CD4 in human primary T cells was inhibited by miR-221/222. These findings provide a novel strategy for modulating the number of CD4+ T cells in the blood and further adjusting the microenvironment suitable for immunotherapy.