Acquirement of resistance to rituximab has been observed in lymphoma patients. To define mechanisms associated with rituximab resistance, we developed various rituximab-resistant cell lines (RRCL) and studied changes in CD20 expression/structure, lipid raft domain (LRD) reorganization, calcium mobilization, antibody-dependent cellular cytotoxicity, and complement-mediated cytotoxicity (CMC) between parental and RRCL. Significant changes in surface CD20 antigen expression were shown in RRCL. Decreased calcium mobilization and redistribution of CD20 into LRD were found in RRCL. Western blotting identified a unique 35 kDa protein band in RRCL, which was not seen in parental cells and was secondary to an increase in surface and cytoplasmic expression of IgM light chains. CD20 gene expression was decreased in RRCL. In vitro exposure to PS341 increased CD20 expression in RRCL and minimally improved the sensitivity to rituximab-associated CMC. Our data strongly suggest that the acquisition of rituximab resistance is associated with global gene and protein down-regulation of the CD20 antigen affecting LRD organization and downstream signaling. CD20 expression seems to be regulated at the pretranscriptional and posttranscriptional levels. Proteasome inhibition partially reversed rituximab resistance, suggesting the existence of additional mediators of rituximab resistance. Future research is geared to identify drugs and/or biological agents that are effective against RRCL.

Primary and secondary lymphoid organs are heavily innervated by the autonomic nervous system. Norepinephrine, the primary neurotransmitter secreted by post-ganglionic sympathetic neurons, binds to and activates β-adrenergic receptors expressed on the surface of immune cells and regulates the functions of these cells. While it is known that both activated and memory CD8⁺ T-cells primarily express the β2-adrenergic receptor (β2-AR) and that signaling through this receptor can inhibit CD8⁺ T-cell effector function, the mechanism(s) underlying this suppression is not understood. Under normal activation conditions, T-cells increase glucose uptake and undergo metabolic reprogramming. In this study, we show that treatment of murine CD8⁺ T-cells with the pan β-AR agonist isoproterenol (ISO) was associated with a reduced expression of glucose transporter 1 following activation, as well as decreased glucose uptake and glycolysis compared to CD8⁺ T-cells activated in the absence of ISO. The effect of ISO was specifically dependent upon β2-AR, since it was not seen in adrb2^−/− CD8⁺ T-cells and was blocked by the β-AR antagonist propranolol. In addition, we found that mitochondrial function in CD8⁺ T-cells was also impaired by β2-AR signaling. This study demonstrates that one mechanism by which β2-AR signaling can inhibit CD8⁺ T-cell activation is by suppressing the required metabolic reprogramming events which accompany activation of these immune cells and thus reveals a new mechanism by which adrenergic stress can suppress the effector activity of immune cells.

T cell engineering strategies have emerged as successful immunotherapeutic approaches for the treatment of human cancer. Chimeric Antigen Receptor T (CAR-T) cell therapy represents a prominent synthetic biology approach to re-direct the specificity of a patient’s autologous T cells toward a desired tumor antigen. CAR-T therapy is currently FDA approved for the treatment of hematological malignancies, including subsets of B cell lymphoma, acute lymphoblastic leukemia (ALL) and multiple myeloma. Mechanistically, CAR-mediated recognition of a tumor antigen results in propagation of T cell activation signals, including a co-stimulatory signal, resulting in CAR-T cell activation, proliferation, evasion of apoptosis, and acquisition of effector functions. The importance of including a co-stimulatory domain in CARs was recognized following limited success of early iteration CAR-T cell designs lacking co-stimulation. Today, all CAR-T cells in clinical use contain either a CD28 or 4-1BB co-stimulatory domain. Preclinical investigations are exploring utility of including additional co-stimulatory molecules such as ICOS, OX40 and CD27 or various combinations of multiple co-stimulatory domains. Clinical and preclinical evidence implicates the co-stimulatory signal in several aspects of CAR-T cell therapy including response kinetics, persistence and durability, and toxicity profiles each of which impact the safety and anti-tumor efficacy of this immunotherapy. Herein we provide an overview of CAR-T cell co-stimulation by the prototypical receptors and discuss current and emerging strategies to modulate co-stimulatory signals to enhance CAR-T cell function.

The use of flow cytometry to diagnose hematological malignancies has become routine due to its ability to often differentiate between morphologically similar diseases based on antigens expressed on the surface of malignant cells. In an attempt to expand on the utility of flow cytometry in the study of B-cell malignancies we have used the most reliable quantitative methodology, QIFI (quantitative indirect immunofluorescence assay), to study the expression of CD5, CD10, CD11c, CD19, CD20, CD22, CD23, and CD79b in 384 cases of several common B-lineage malignancies, including: B-ALL, CLL, SLL, hairy cell leukemia, diffuse large B-cell lymphoma, and follicular lymphoma. The impetus behind this extensive, single institution study of surface antigens was two-fold: evaluating similarities and differences of antigen expression between B-cell neoplasms and finding additional clinical utility for the quantitative flow cytometric data generated. Our results show that each distinct malignant histology has its own quantitative pattern of surface antigen expression. In most cases, these quantitative patterns do not increase the ability of flow cytometry to distinguish between them. However, a high expression of specific antigens on a given B-cell malignancy may potentially identify optimal therapeutic targets for current and/or future monoclonal antibody-based therapies.

Significance: MicroRNAs limit gene expression by recruiting a large protein complex known as the RNA-induced silencing complex (RISC) to target mRNAs. While attempting to understand physiological regulation of RISC assembly, we found that most healthy adult tissues retain a reserve of microRNAs not stably associated with target mRNA. Recruitment of microRNAs to large mRNA-containing complexes was accompanied by an increase in their ability to repress targets and was regulated in part by phosphoinositide-3 kinase–RAC-alpha serine/threonine-protein kinase–mechanistic target of rapamycin pathway-dependent enhancement of the glycine-tryptophan protein of 182 kDa protein expression. Data presented here suggest that in vivo, many expressed microRNAs exist in an inactive reserve, allowing resting cells to use microRNAs to dynamically regulate the translation of target mRNAs in their environment.

Purpose: Targeting malignant B cells using rituximab (anti-CD20) has improved the efficacy of chemotherapy regimens used to treat patients with non-Hodgkin's lymphoma. Despite the promising clinical results obtained using rituximab, many patients relapse with therapy-resistant disease following rituximab-based treatments. We have created a cell line model of rituximab resistance using three B-cell non-Hodgkin's lymphoma–derived cell lines (Raji, RL, and SUDHL-4). In an attempt to define strategies to overcome rituximab resistance, we sought to determine the chemotherapy sensitivity of our rituximab-resistant cell lines (RRCL).