Chimeric antigen receptor (CAR) T-cells targeting CD19 demonstrate remarkable efficacy in treating B-lineage acute lymphoblastic leukemia (BL-ALL), yet up to 39% of treated patients relapse with CD19(−) disease. We report that CD19(−) escape is associated with downregulation, but preservation, of targetable expression of CD20 and CD22. Accordingly, we reasoned that broadening the spectrum of CD19CAR T-cells to include both CD20 and CD22 would enable them to target CD19(−) escape BL-ALL while preserving their upfront efficacy. Supplementary informationThe online version of this article (https://

Purpose: Rituximab [chimeric anti-CD20 monoclonal antibody], alone or combined with chemotherapy, is used in the treatment of non-Hodgkin's lymphoma (NHL). Rituximab binds to CD20 and inhibits intracellular survival/growth pathways leading to chemo/ immunosensitization of tumor cells in vitro. The contribution of rituximab Fc-FcR interaction in signaling is not known. This study examined the role of Fc-FcR interactions in rituximab-induced signaling using rituximab (Fab') 2 fragments as well as rituximab devoid of the CH2 Fc-binding domain (CH2 -). Experimental Design: Rituximab (CH2 -) and rituximab (Fab') 2 were tested for their activity on B-NHL cell lines. Cell signaling and sensitization to chemotherapy and immunotherapy were examined. The in vitro studies were validated in mice bearing tumor xenografts. Results: Although the modified antibodies were defective in antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity functions, they retained all other biological activities such as inhibition of cell proliferation, induction of cell aggregation, and apoptosis induction. In addition, similar to rituximab, the modified antibodies inhibited the activity of cell survival/growth pathways and their associated transcription factors (e.g., NF-κB, YY1, SP-1), and signal transducers and activators of transcription 3 (STAT-3), and downregulated the expression of antiapoptotic gene products, such as Bcl-2/Bcl XL , which regulate drug resistance. The modified antibodies, similar to rituximab, sensitized resistant B-NHL cells to both CDDP and Fas ligandinduced apoptosis. Furthermore, treatment of nude mice bearing Raji tumor cell xenografts with the combination of rituximab (Fab') 2 or rituximab and CDDP resulted in similar and significant inhibition of tumor growth. Conclusion: These findings reveal that rituximab-mediated inhibition of intracellular signaling pathways and leading to chemo/immuno-sensitization of resistant B-NHL is Fc independent. (Clin Cancer Res 2009;15(21):6582-94) Rituximab (chimeric mouse anti-human CD20 monoclonal antibody) is a genetically engineered monoclonal antibody used for the treatment of patients with relapsed or refractory low-grade or follicular B-cell non-Hodgkin's lymphoma (NHL) and in patients with relapsed stage III/IV follicular lymphoma (1, 2). Rituximab is composed of murine Variable (V) and human IgG1 Constant (C) regions. Rituximab functions by binding to the CD20 antigen expressed on the surface of normal and malignant B cells. The overall response in patients is 50% when it is used as a single agent (3), and the response rate

Despite remarkable progress in the development and authorization of vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there is a need to validate vaccine platforms for broader application. The current intramuscular vaccines are designed to elicit systemic immunity without conferring mucosal immunity in the nasal compartment, which is the first barrier that SARS-CoV-2 virus breaches before dissemination to the lung. We report the development of an intranasal subunit vaccine that uses lyophilized spike protein and liposomal STING agonist as an adjuvant. This vaccine induces systemic neutralizing antibodies, IgA in the lung and nasal compartments, and T-cell responses in the lung of mice. Single-cell RNA sequencing confirmed the coordinated activation of T/B-cell responses in a germinal center-like manner within the nasal-associated lymphoid tissues, confirming its role as an inductive site to enable durable immunity. The ability to elicit immunity in the respiratory tract can prevent the establishment of infection in individuals and prevent disease transmission.Mucosal vaccination can stimulate both systemic and mucosal immunity and has the advantage of being a non-invasive procedure suitable for immunization of large populations. However, mucosal vaccination is

Transgenic coexpression of a class I–restricted tumor antigen–specific T cell receptor (TCR) and CD8αβ (TCR8) redirects antigen specificity of CD4+ T cells. Reinforcement of biophysical properties and early TCR signaling explain how redirected CD4+ T cells recognize target cells, but the transcriptional basis for their acquired antitumor function remains elusive. We, therefore, interrogated redirected human CD4+ and CD8+ T cells by single-cell RNA sequencing and characterized them experimentally in bulk and single-cell assays and a mouse xenograft model. TCR8 expression enhanced CD8+ T cell function and preserved less differentiated CD4+ and CD8+ T cells after tumor challenge. TCR8+CD4+ T cells were most potent by activating multiple transcriptional programs associated with enhanced antitumor function. We found sustained activation of cytotoxicity, costimulation, oxidative phosphorylation– and proliferation-related genes, and simultaneously reduced differentiation and exhaustion. Our study identifies molecular features of TCR8 expression that can guide the development of enhanced immunotherapies.

Natural killer (NK) cells are a highly heterogeneous population of innate lymphocytes that constitute our first line of defense against several types of tumors and microbial infections. Understanding the heterogeneity of these lymphocytes requires the ability to integrate their underlying phenotype with dynamic functional behaviors. We have developed and validated a single-cell methodology that integrates cellular phenotyping and dynamic cytokine secretion based on nanowell arrays and bead-based molecular biosensors. We demonstrate the robust passivation of the polydimethylsiloxane (PDMS)-based nanowells arrays with polyethylene glycol (PEG) and validated our assay by comparison to enzyme-linked immunospot (ELISPOT) assays. We used numerical simulations to optimize the molecular density of antibodies on the surface of the beads as a function of the capture efficiency of cytokines within an open-well system. Analysis of hundreds of individual human peripheral blood NK cells profiled ex vivo revealed that CD56dimCD16+ NK cells are immediate secretors of interferon gamma (IFN-γ) upon activation by phorbol 12-myristate 13-acetate (PMA) and ionomycin (< 3 h), and that there was no evidence of cooperation between NK cells leading to either synergistic activation or faster IFN-γ secretion. Furthermore, we observed that both the amount and rate of IFN-γ secretion from individual NK cells were donor-dependent. Collectively, these results establish our methodology as an investigational tool for combining phenotyping and real-time protein secretion of individual cells in a high-throughput manner.