Systematic single amino acid affinity tuning of CD229 CAR T cells retains efficacy against multiple myeloma and eliminates on-target off-tumor toxicity.

T cells expressing chimeric antigen receptors (CARs) have shown remarkable therapeutic activity against different types of cancer. However, the wider use of CAR T cells has been hindered by the potential for life-threatening toxicities due to on-target off-tumor killing of cells expressing low amounts of the target antigen. CD229, a signaling lymphocyte-activation molecule (SLAM) family member, has previously been identified as a target for CAR T cell-mediated treatment of multiple myeloma (MM) due to its high expression on the surfaces of MM cells. CD229 CAR T cells have shown effective clearance of MM cells in vitro and in vivo. However, healthy lymphocytes also express CD229, albeit at lower amounts than MM cells, causing their unintended targeting by CD229 CAR T cells. To increase the selectivity of CD229 CAR T cells for MM cells, we used a single amino acid substitution approach of the CAR binding domain to reduce CAR affinity. To identify CARs with increased selectivity, we screened variant binding domains using solid-phase binding assays and biolayer interferometry and determined the cytotoxic activity of variant CAR T cells against MM cells and healthy lymphocytes. We identified a CD229 CAR binding domain with micromolar affinity that, when combined with overexpression of c-Jun, confers antitumor activity comparable to parental CD229 CAR T cells but lacks the parental cells' cytotoxic activity toward healthy lymphocytes in vitro and in vivo. The results represent a promising strategy to improve the efficacy and safety of CAR T cell therapy that requires clinical validation.

CD11c+ myeloid cell exosomes reduce intestinal inflammation during colitis.

Intercellular communication is critical for homeostasis in mammalian systems, including the gastrointestinal (GI) tract. Exosomes are nanoscale lipid extracellular vesicles that mediate communication between many cell types. Notably, the roles of immune cell exosomes in regulating GI homeostasis and inflammation are largely uncharacterized. By generating mouse strains deficient in cell-specific exosome production, we demonstrate deletion of the small GTPase Rab27A in CD11c+ cells exacerbated murine colitis, which was reversible through administration of DC-derived exosomes. Profiling RNAs within colon exosomes revealed a distinct subset of miRNAs carried by colon- and DC-derived exosomes. Among antiinflammatory exosomal miRNAs, miR-146a was transferred from gut immune cells to myeloid and T cells through a Rab27-dependent mechanism, targeting Traf6, IRAK-1, and NLRP3 in macrophages. Further, we have identified a potentially novel mode of exosome-mediated DC and macrophage crosstalk that is capable of skewing gut macrophages toward an antiinflammatory phenotype. Assessing clinical samples, RAB27A, select miRNAs, and RNA-binding proteins that load exosomal miRNAs were dysregulated in ulcerative colitis patient samples, consistent with our preclinical mouse model findings. Together, our work reveals an exosome-mediated regulatory mechanism underlying gut inflammation and paves the way for potential use of miRNA-containing exosomes as a novel therapeutic for inflammatory bowel disease.

High-throughput enrichment and isolation of megakaryocyte progenitor cells from the mouse bone marrow.

Megakaryocytes are a rare population of cells that develop in the bone marrow and function to produce platelets that circulate throughout the body and form clots to stop or prevent bleeding. A major challenge in studying megakaryocyte development, and the diseases that arise from their dysfunction, is the identification, classification, and enrichment of megakaryocyte progenitor cells that are produced during hematopoiesis. Here, we present a high throughput strategy for identifying and isolating megakaryocytes and their progenitor cells from a heterogeneous population of bone marrow samples. Specifically, we couple thrombopoietin (TPO) induction, image flow cytometry, and principal component analysis (PCA) to identify and enrich for megakaryocyte progenitor cells that are capable of self-renewal and directly differentiating into mature megakaryocytes. This enrichment strategy distinguishes megakaryocyte progenitors from other lineage-committed cells in a high throughput manner. Furthermore, by using image flow cytometry with PCA, we have identified a combination of markers and characteristics that can be used to isolate megakaryocyte progenitor cells using standard flow cytometry methods. Altogether, these techniques enable the high throughput enrichment and isolation of cells in the megakaryocyte lineage and have the potential to enable rapid disease identification and diagnoses ahead of severe disease progression.

SARS-CoV-2 innate effector associations and viral load in early nasopharyngeal infection.

COVID-19 causes severe disease with poor outcomes. We tested the hypothesis that early SARS-CoV-2 viral infection disrupts innate immune responses. These changes may be important for understanding subsequent clinical outcomes. We obtained residual nasopharyngeal swab samples from individuals who requested COVID-19 testing for symptoms at drive-through COVID-19 clinical testing sites operated by the University of Utah. We applied multiplex immunoassays, real-time polymerase chain reaction assays and quantitative proteomics to 20 virus-positive and 20 virus-negative samples. ACE-2 transcripts increased with infection (OR =17.4, 95% CI [CI] =4.78-63.8) and increasing viral N1 protein transcript load (OR =1.16, CI =1.10-1.23). Transcripts for two interferons (IFN) were elevated, IFN-λ1 (OR =71, CI =7.07-713) and IFN-λ2 (OR =40.2, CI =3.86-419), and closely associated with viral N1 transcripts (OR =1.35, CI =1.23-1.49 and OR =1.33 CI =1.20-1.47, respectively). Only transcripts for IP-10 were increased among systemic inflammatory cytokines that we examined (OR =131, CI =1.01-2620). We found widespread discrepancies between transcription and translation. IFN proteins were unchanged or decreased in infected samples (IFN-γ OR =0.90 CI =0.33-0.79, IFN-λ2,3 OR =0.60 CI =0.48-0.74) suggesting viral-induced shut-off of host antiviral protein responses. However, proteins for IP-10 (OR =3.74 CI =2.07-6.77) and several interferon-stimulated genes (ISG) increased with viral load (BST-1 OR =25.1, CI =3.33-188; IFIT1 OR =19.5, CI =4.25-89.2; IFIT3 OR =245, CI =15-4020; MX-1 OR =3.33, CI =1.44-7.70). Older age was associated with substantial modifications of some effects. Ambulatory symptomatic patients had an innate immune response with SARS-CoV-2 infection characterized by elevated IFN, proinflammatory cytokine and ISG transcripts, but there is evidence of a viral-induced host shut-off of antiviral responses. Our findings may characterize the disrupted immune landscape common in patients with early disease.

SARS-CoV-2 Innate Effector Associations and Viral Load in Early Nasopharyngeal Infection.

To examine innate immune responses in early SARS-CoV-2 infection that may change clinical outcomes, we compared nasopharyngeal swab data from 20 virus-positive and 20 virus-negative individuals. Multiple innate immune-related and ACE-2 transcripts increased with infection and were strongly associated with increasing viral load. We found widespread discrepancies between transcription and translation. Interferon proteins were unchanged or decreased in infected samples suggesting virally-induced shut-off of host anti-viral protein responses. However, IP-10 and several interferon-stimulated gene proteins increased with viral load. Older age was associated with modifications of some effects. Our findings may characterize the disrupted immune landscape of early disease.

Limited efficacy of BMS-911543 in a murine model of Janus kinase 2 V617F myeloproliferative neoplasm.

Activation of Janus kinase 2 (JAK2), frequently as a result of the JAK2(V617F) mutation, is a characteristic feature of the classical myeloproliferative neoplasms (MPNs) polycythemia vera, essential thrombocythemia, and myelofibrosis, and it is thought to be responsible for the constitutional symptoms associated with these diseases. BMS-911543 is a JAK2-selective inhibitor that induces apoptosis in JAK2-dependent cell lines and inhibits the growth of CD34(+) progenitor cells from patients with JAK2(V617F)-positive MPN. To explore the clinical potential of this inhibitor, we tested BMS-911543 in a murine retroviral transduction-transplantation model of JAK2(V617F) MPN. Treatment was initiated at two dose levels (3 mg/kg and 10 mg/kg) when the hematocrit exceeded 70%. Following the first week, white blood cell counts were reduced to normal in the high-dose group and were maintained well below the levels in vehicle-treated mice throughout the study. However, BMS-911543 had no effect on red blood cell parameters. After 42 days of treatment, the proportion of JAK2(V617F)-positive cells in hematopoietic tissues was identical or slightly increased compared with controls. Plasma concentrations of interleukin 6, interleukin 15, and tumor necrosis factor α were elevated in MPN mice and reduced in the high-dose treatment group, whereas other cytokines were unchanged. Inhibitor activity after dosing was confirmed in a cell culture assay using the plasma of dosed mice and phosphorylated signal transducer and activator of transcription 5 flow cytometry. Collectively, these results show that BMS-911543 has limited activity in this murine model of JAK2(V617F)-driven MPN and suggest that targeting JAK2 alone may be insufficient to achieve effective disease control.

Herbivorous rodents (Neotoma spp.) harbour abundant and active foregut microbiota.

Symbiotic gut microbes have facilitated the success of herbivorous mammals, which are generally grouped into foregut- and hindgut-fermenters. However, rodents are primarily herbivorous and exhibit a variety of gastrointestinal anatomies. Most rodents house microbes in hindgut chambers, such as the caecum and colon. Some rodents also exhibit stomach segmentation with a foregut chamber proximal to the stomach. For over a century, scientists have hypothesized that this foregut chamber houses a microbial community, yet this has never been explicitly examined. We investigated the capacity of each of the gut regions to house microbes by measuring size, pH, bacterial cell density, concentrations of microbial metabolites and digesta transit time in woodrats (Neotoma spp.). We also compared microbial communities across gut chambers, as well as faeces, using 16S rRNA sequencing. This allowed us to test the appropriateness of using faeces as a proxy for microbial communities of other gut chambers. We found that woodrats house foregut microbial communities with similar density and volatile fatty acid concentrations to rumen ecosystems. Resident microbial communities varied between gut chambers, and faecal bacterial communities were significantly different from caecal and colonic communities. The foregut microbiota may provide a number of physiological services to the host.

Methicillin-resistant Staphylococcus aureus-induced thrombo-inflammatory response is reduced with timely antibiotic administration.

Methicillin-resistant Staphylococcus aureus (MRSA) induces a pro-thrombotic and pro-inflammatory milieu. Although timely antibiotic administration in MRSAsepsis may improve outcomes by arresting bacterial growth, the effects of antibiotics on mitigating injurious thrombo-inflammatory cellular responses remains unexplored. Using a newly developed human whole blood model and an in vivo mouse model of MRSAinfection, we examined how antibiotics inhibit MRSAinduced thrombo-inflammatory pathways. Human whole blood was inoculated with MRSA. Thrombin generation and inflammatory cytokine synthesis was measured in the presence or absence of linezolid and vancomycin. C57BL/6 mice were injected with MRSA and the effect of vancomycin administration was examined. MRSAaccelerated thrombin generation in a time- and concentration-dependent manner andinduced the release of cytokines, including interleukin (IL)-6, IL-8, and monocyte chemotactic protein (MCP)-1. The increase in thrombin generation and inflammatory responses was mediated through the synthesis of tissue factor and cytokines, respectively, and the release of microparticles. The early administration of antibiotics restored normal thrombin generation patterns and significantly reduced the synthesis of cytokines. In contrast, when antibiotic administration was delayed, thrombin generation and cytokine synthesis were not significantly reduced. In mice infected with MRSA, early antibiotic administration reduced thrombin anti-thrombin complexes and cytokine synthesis, whereas delayed antibiotic administration did not. These data provide novel mechanistic evidence of the importance of prompt antibiotic administration in infectious syndromes.