Targeting human plasma cells using small molecule regulated BCMA CAR T cells eliminates circulating antibodies in humanized mice.

Pathogenic long-lived plasma cells (LLPCs) secrete autoreactive antibodies, exacerbating autoimmune diseases and complicating solid organ transplantation. Targeted elimination of the autoreactive B-cell pool represents a promising therapeutic strategy, yet current treatment modalities fall short in depleting mature plasma cells. Here, we demonstrate that chimeric antigen receptor (CAR) T cells, targeting BCMA utilizing a split-receptor design, offer a controlled and effective therapeutic strategy against LLPCs.

Generation, expansion, gene delivery, and single-cell profiling in rhesus macaque plasma B cells.

A key step in developing engineered B cells for therapeutic purposes is evaluation in immunocompetent, large-animal models. Therefore, we developed methods to purify, expand, and differentiate non-human primate (NHP; rhesus macaque) B cells. After 7 days in culture, B cells expanded 10-fold, differentiated into a plasma cell phenotype (CD38, CD138), and secreted immunoglobulin G.

Blunting specific T-dependent antibody responses with engineered "decoy" B cells.

Antibody inhibitors pose an ongoing challenge to the treatment of subjects with inherited protein deficiency disorders, limiting the efficacy of both protein replacement therapy and corrective gene therapy. Beyond their central role as producers of serum antibody, B cells also exhibit many unique properties that could be exploited in cell therapy applications, notably including antigen-specific recognition and the linked capacity for antigen presentation. Here we employed CRISPR-Cas9 to demonstrate that ex vivo antigen-primed Blimp1-knockout "decoy" B cells, incapable of differentiation into plasma cells, participated in and downregulated host antigen-specific humoral responses after adoptive transfer.

Human plasma cells engineered to secrete bispecifics drive effective in vivo leukemia killing.

Bispecific antibodies are an important tool for the management and treatment of acute leukemias. As a next step toward clinical translation of engineered plasma cells, we describe approaches for secretion of bispecific antibodies by human plasma cells. We show that human plasma cells expressing either fragment crystallizable domain-deficient anti-CD19 × anti-CD3 (blinatumomab) or anti-CD33 × anti-CD3 bispecific antibodies mediate T cell activation and direct T cell killing of B acute lymphoblastic leukemia or acute myeloid leukemia cell lines in vitro.

BCR signaling is required for posttransplant lymphoproliferative disease in immunodeficient mice receiving human B cells.

Posttransplant lymphoproliferative disease (PTLD) is a major therapeutic challenge that has been difficult to study using human cells because of a lack of suitable models for mechanistic characterization. Here, we show that ex vivo-differentiated B cells isolated from a subset of healthy donors can elicit pathologies similar to PTLD when transferred into immunodeficient mice. The primary driver of PTLD-like pathologies were IgM-producing plasmablasts with Epstein-Barr virus (EBV) genomes that expressed genes commonly associated with EBV latency.

Human plasma cells engineered to secrete bispecifics drive effective leukemia killing.

Bispecific antibodies are an important tool for the management and treatment of acute leukemias. Advances in genome-engineering have enabled the generation of human plasma cells that secrete therapeutic proteins and are capable of long-term engraftment in humanized mouse models. As a next step towards clinical translation of engineered plasma cells (ePCs) towards cancer therapy, here we describe approaches for the expression and secretion of bispecific antibodies by human plasma cells.

SEC-seq: association of molecular signatures with antibody secretion in thousands of single human plasma cells.

The secreted products of cells drive many functions in vivo; however, methods to link this functional information to surface markers and transcriptomes have been lacking. By accumulating secretions close to secreting cells held within cavity-containing hydrogel nanovials, we demonstrate workflows to analyze the amount of IgG secreted from single human B cells and link this information to surface markers and transcriptomes from the same cells. Measurements using flow cytometry and imaging flow cytometry corroborate the association between IgG secretion and CD38/CD138.

A lentiviral vector B cell gene therapy platform for the delivery of the anti-HIV-1 eCD4-Ig-knob-in-hole-reversed immunoadhesin.

Barriers to effective gene therapy for many diseases include the number of modified target cells required to achieve therapeutic outcomes and host immune responses to expressed therapeutic proteins. As long-lived cells specialized for protein secretion, antibody-secreting B cells are an attractive target for foreign protein expression in blood and tissue. To neutralize HIV-1, we developed a lentiviral vector (LV) gene therapy platform for delivery of the anti-HIV-1 immunoadhesin, eCD4-Ig, to B cells.

Ex vivo engineered human plasma cells exhibit robust protein secretion and long-term engraftment in vivo.

Due to their unique longevity and capacity to secrete high levels of protein, plasma B cells have the potential to be used as a cell therapy for protein replacement. Here, we show that ex vivo engineered human plasma cells exhibit single-cell RNA profiles, scanning electron micrograph ultrastructural features, and in vivo homing capacity of long-lived plasma cells. After transferring human plasma cells to immunodeficient mice in the presence of the human cytokines BAFF and IL-6, we observe increases in retention of plasma cells in the bone marrow, with engraftment exceeding a year.

Transcriptomic analysis of pathways associated with ITGAV/alpha(v) integrin-dependent autophagy in human B cells.

Macroautophagy/autophagy proteins have been linked with the development of immune-mediated diseases including lupus, but the mechanisms for this are unclear due to the complex roles of these proteins in multiple immune cell types. We have previously shown that a form of noncanonical autophagy induced by ITGAV/alpha(v) integrins regulates B cell activation by viral and self-antigens, in mice. Here, we investigate the involvement of this pathway in B cells from human tissues.

An optimized measles virus glycoprotein-pseudotyped lentiviral vector production system to promote efficient transduction of human primary B cells.

Measles virus envelope pseudotyped LV (MV-LV) can achieve high B cell transduction rates (up to 50%), but suffers from low titers. To overcome current limitations, we developed an optimized MV-LV production protocol that achieved consistent B cell transduction efficiency up to 75%. We detail this protocol along with analytical assays to assess the results of MV-LV mediated B cell transduction, including flow cytometry for B cell phenotypic characterization and measurement of transduction efficiency, and ddPCR for VCN analysis.

Mast cell surfaceome characterization reveals CD98 heavy chain is critical for optimal cell function.

Background: Mast cells are involved in many distinct pathologic conditions, suggesting that they recognize and respond to various stimuli and thus require a rich repertoire of cell surface proteins. However, mast cell surface proteomes have not been comprehensively characterized.

Objective: We aimed to further characterize the mast cell surface proteome to obtain a better understanding of how mast cells function in health and disease.

Activated interleukin-7 receptor signaling drives B-cell acute lymphoblastic leukemia in mice.

Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is a high-risk subtype of B-ALL often associated with genetic variants that alter cytokine receptor signaling, including mutations in the interleukin-7 receptor (IL7R). To investigate whether IL7R variants are leukemia-initiating, we built mouse models expressing activated Il7r (aIL7R). B-cell intrinsic aIL7R mice developed spontaneous B-ALL, demonstrating sufficiency of Il7r activating mutations in leukemogenesis.

Multiplexed Functional Assessment of Genetic Variants in CARD11.

Genetic testing has increased the number of variants identified in disease genes, but the diagnostic utility is limited by lack of understanding variant function. CARD11 encodes an adaptor protein that expresses dominant-negative and gain-of-function variants associated with distinct immunodeficiencies. Here, we used a "cloning-free" saturation genome editing approach in a diploid cell line to simultaneously score 2,542 variants for decreased or increased function in the region of CARD11 associated with immunodeficiency.

Recommendations for the collection and use of multiplexed functional data for clinical variant interpretation.

Variants of uncertain significance represent a massive challenge to medical genetics. Multiplexed functional assays, in which the functional effects of thousands of genomic variants are assessed simultaneously, are increasingly generating data that can be used as additional evidence for or against variant pathogenicity. Such assays have the potential to resolve variants of uncertain significance, thereby increasing the clinical utility of genomic testing.

Folliculin Interacting Protein 1 Maintains Metabolic Homeostasis during B Cell Development by Modulating AMPK, mTORC1, and TFE3.

Folliculin interacting protein 1 (Fnip1) is a cytoplasmic protein originally discovered through its interaction with the master metabolic sensor 5' AMP-activated protein kinase (AMPK) and Folliculin, a protein mutated in individuals with Birt-Hogg-Dubé Syndrome. In response to low energy, AMPK stimulates catabolic pathways such as autophagy to enhance energy production while inhibiting anabolic pathways regulated by the mechanistic target of rapamycin complex 1 (mTORC1). We previously found that constitutive disruption of in mice resulted in a lack of peripheral B cells because of a block in B cell development at the pre-B cell stage.

The signaling adaptor BCAP inhibits NLRP3 and NLRC4 inflammasome activation in macrophages through interactions with Flightless-1.

B cell adaptor for phosphoinositide 3-kinase (PI3K) (BCAP) is a signaling adaptor that activates the PI3K pathway downstream of B cell receptor signaling in B cells and Toll-like receptor (TLR) signaling in macrophages. BCAP binds to the regulatory p85 subunit of class I PI3K and is a large, multidomain protein. We used proteomic analysis to identify other BCAP-interacting proteins in macrophages and found that BCAP specifically associated with the caspase-1 pseudosubstrate inhibitor Flightless-1 and its binding partner leucine-rich repeat flightless-interacting protein 2.

Activated PIK3CD drives innate B cell expansion yet limits B cell-intrinsic immune responses.

Activated PI3K-delta syndrome (APDS) is an immunodeficiency caused by gain-of-function mutations in PIK3CD. This disease exhibits complex immune phenotypes including increased IgM, recurrent infection, and impaired vaccine responses. To better understand the impact of B cells in this disease, we generated an inducible model of the common APDS mutation (h-E1021K; referred to as aPIK3CD) and intercrossed these mice with B cell-specific Cre models.

Activated CARD11 accelerates germinal center kinetics, promoting mTORC1 and terminal differentiation.

Activating mutations in the adapter protein CARD11 associated with diffuse large B cell lymphomas (DLBCLs) are predicted to arise during germinal center (GC) responses, leading to inappropriate activation of NF-κB signaling. Here, we modeled the B cell-intrinsic impact of the L251P activating mutation in CARD11 (aCARD11) on the GC response. Global B cell aCARD11 expression led to a modest increase in splenic B cells and a severe reduction in B1 B cell numbers, respectively.

Deficient Follistatin-like 3 Secretion by Asthmatic Airway Epithelium Impairs Fibroblast Regulation and Fibroblast-to-Myofibroblast Transition.

Bronchial epithelial cells (BECs) from healthy children inhibit human lung fibroblast (HLF) expression of collagen and fibroblast-to-myofibroblast transition (FMT), whereas asthmatic BECs do so less effectively, suggesting that diminished epithelial-derived regulatory factors contribute to airway remodeling. Preliminary data demonstrated that secretion of the activin A inhibitor follistatin-like 3 (FSTL3) by healthy BECs was greater than that by asthmatic BECs. We sought to determine the relative secretion of FSTL3 and activin A by asthmatic and healthy BECs, and whether FSTL3 inhibits FMT.

Engineering Protein-Secreting Plasma Cells by Homology-Directed Repair in Primary Human B Cells.

The ability to engineer primary human B cells to differentiate into long-lived plasma cells and secrete a de novo protein may allow the creation of novel plasma cell therapies for protein deficiency diseases and other clinical applications. We initially developed methods for efficient genome editing of primary B cells isolated from peripheral blood. By delivering CRISPR/CRISPR-associated protein 9 (Cas9) ribonucleoprotein (RNP) complexes under conditions of rapid B cell expansion, we achieved site-specific gene disruption at multiple loci in primary human B cells (with editing rates of up to 94%).

Interferon response to respiratory syncytial virus by bronchial epithelium from children with asthma is inversely correlated with pulmonary function.

Background: Respiratory viral infection in early childhood, including that from respiratory syncytial virus (RSV), has been previously associated with the development of asthma.

Objective: We aimed to determine whether ex vivo RSV infection of bronchial epithelial cells (BECs) from children with asthma would induce specific gene expression patterns and whether such patterns were associated with lung function among BEC donors.

Methods: Primary BECs from carefully characterized children with asthma (n = 18) and matched healthy children without asthma (n = 8) were differentiated at an air-liquid interface for 21 days.

The A946T variant of the RNA sensor IFIH1 mediates an interferon program that limits viral infection but increases the risk for autoimmunity.

The single-nucleotide polymorphism rs1990760 in the gene encoding the cytosolic viral sensor IFIH1 results in an amino-acid change (A946T; IFIH1) that is associated with multiple autoimmune diseases. The effect of this polymorphism on both viral sensing and autoimmune pathogenesis remains poorly understood. Here we found that human peripheral blood mononuclear cells (PBMCs) and cell lines expressing the risk variant IFIH1 exhibited heightened basal and ligand-triggered production of type I interferons.