Neoantigen-targeted therapies have typically been based upon personalized neoantigen-specific vaccines; however, in this issue of JCI, van der Lee et al. describe the development of a potential ...cellular immunotherapy targeting a "public" neoantigen derived from nucleophosmin 1 (NPM1), which is mutated in approximately 30% of acute myeloid leukemias (AMLs). The authors use reverse immunology to predict, and biochemically confirm, NPM1-derived neoepitopes (ΔNPM1) and then generate high-avidity T cell clones and retrovirally transduced T cell populations that kill NPM1-mutated AML. This study provides a general approach to adoptive cellular therapy that can be applied to targeting other tumors with public neoantigens.
The study of tumour-specific antigens (TSAs) as targets for antitumour therapies has accelerated within the past decade. The most commonly studied class of TSAs are those derived from non-synonymous ...single-nucleotide variants (SNVs), or SNV neoantigens. However, to increase the repertoire of available therapeutic TSA targets, 'alternative TSAs', defined here as high-specificity tumour antigens arising from non-SNV genomic sources, have recently been evaluated. Among these alternative TSAs are antigens derived from mutational frameshifts, splice variants, gene fusions, endogenous retroelements and other processes. Unlike the patient-specific nature of SNV neoantigens, some alternative TSAs may have the advantage of being widely shared by multiple tumours, allowing for universal, off-the-shelf therapies. In this Opinion article, we will outline the biology, available computational tools, preclinical and/or clinical studies and relevant cancers for each alternative TSA class, as well as discuss both current challenges preventing the therapeutic application of alternative TSAs and potential solutions to aid in their clinical translation.
Regulatory B cells control inflammation and autoimmunity in mice, including the recently identified IL-10–competent B10 cell subset that represents 1% to 3% of spleen B cells. In this study, a ...comparable IL-10–competent B10 cell subset was characterized in human blood. B10 cells were functionally identified by their ability to express cytoplasmic IL-10 after 5 hours of ex vivo stimulation, whereas progenitor B10 (B10pro) cells required 48 hours of in vitro stimulation before they acquired the ability to express IL-10. B10 and B10pro cells represented 0.6% and approximately 5% of blood B cells, respectively. Ex vivo B10 and B10pro cells were predominantly found within the CD24hiCD27+ B-cell subpopulation that was able to negatively regulate monocyte cytokine production through IL-10–dependent pathways during in vitro functional assays. Blood B10 cells were present in 91 patients with rheumatoid arthritis, systemic lupus erythematosus, primary Sjögren syndrome, autoimmune vesiculobullous skin disease, or multiple sclerosis, and were expanded in some cases as occurs in mice with autoimmune disease. Mean B10 + B10pro-cell frequencies were also significantly higher in patients with autoimmune disease compared with healthy controls. The characterization of human B10 cells will facilitate their identification and the study of their regulatory activities during human disease.
Acute graft-versus-host disease (aGVHD) is the most common complication for patients undergoing allogeneic stem cell transplantation. Despite extremely aggressive therapy targeting donor T cells, ...patients with grade III or greater aGVHD of the lower GI tract, who do not respond to therapy with corticosteroids, have a dismal prognosis. Thus, efforts to improve understanding of the function of local immune and non-immune cells in regulating the inflammatory process in the GI tract during aGVHD are needed. Here, we demonstrate, using murine models of allogeneic BMT, that type 2 innate lymphoid cells (ILC2s) in the lower GI tract are sensitive to conditioning therapy and show very limited ability to repopulate from donor bone marrow. Infusion of donor ILC2s was effective in reducing the lethality of aGVHD and in treating lower GI tract disease. ILC2 infusion was associated with reduced donor proinflammatory Th1 and Th17 cells, accumulation of donor myeloid-derived suppressor cells (MDSCs) mediated by ILC2 production of IL-13, improved GI tract barrier function, and a preserved graft-versus-leukemia (GVL) response. Collectively, these findings suggest that infusion of donor ILC2s to restore gastrointestinal tract homeostasis may improve treatment of severe lower GI tract aGVHD.
Personalized medicine holds tremendous promise for improving safety and efficacy of drug therapies by optimizing treatment regimens. Rapidly developed patient-derived xenografts (pdx) could be a ...helpful tool for analyzing the effect of drugs against an individual's tumor by growing the tumor in an immunodeficient animal. Severe combined immunodeficiency (SCID) mice enable efficient in vivo expansion of vital tumor cells and generation of personalized xenografts. However, they are not amenable to large-scale rapid screening, which is critical in identifying new compounds from large compound libraries. The development of a zebrafish model suitable for pdx could facilitate large-scale screening of drugs targeted against specific malignancies. Here, we describe a novel strategy for establishing a zebrafish model for drug testing in leukemia xenografts. We used chronic myelogenous leukemia and acute myeloid leukemia for xenotransplantation into SCID zebrafish to evaluate drug screening protocols. We showed the in vivo efficacy of the ABL inhibitor imatinib, MEK inhibitor U0126, cytarabine, azacitidine and arsenic trioxide. We performed corresponding in vitro studies, demonstrating that combination of MEK- and FLT3-inhibitors exhibit an enhanced effect in vitro. We further evaluated the feasibility of zebrafish for transplantation of primary human hematopoietic cells that can survive at 15 day-post-fertilization. Our results provide critical insights to guide development of high-throughput platforms for evaluating leukemia.
Capillary electrophoresis (CE) is a promising technique for single-cell analysis, but its use in biological studies has been limited by low throughput. This paper presents an automated platform ...employing microfabricated cell traps and a three-channel system for rapid buffer exchange for fast single-cell CE. Cells loaded with fluorescein and Oregon green were analyzed at a throughput of 3.5 cells/min with a resolution of 2.3 ± 0.6 for the fluorescein and Oregon green. Cellular protein kinase B (PKB) activity, as measured by immunofluorescence staining of phospho-PKB, was not altered, suggesting that this stress-activated kinase was not upregulated during the CE experiments and that basal cell physiology was not perturbed prior to cell lysis. The activity of sphingosine kinase (SK), which is often upregulated in cancer, was measured in leukemic cells by loading a sphingosine-fluorescein substrate into cells. Sphingosine fluorescein (SF), sphingosine-1-phosphate fluorescein (S1PF), and a third fluorescent species were identified in single cells. A single-cell throughput of 2.1 cells/min was achieved for 219 total cells. Eighty-eight percent of cells possessed upregulated SK activity, although subpopulations of cells with markedly different SK activity relative to that of the population average were readily identified. This system was capable of stable and reproducible separations of biological compounds in hundreds of adherent and nonadherent cells, enabling measurements of previously uncharacterized biological phenomena.
Objective: While T lymphocytes have been employed as a cancer immunotherapy, the development of effective and specific T-cell-based therapeutics remains challenging. A key obstacle is the difficulty ...in identifying T cells reactive to cancer-associated antigens. The objective of this research was to develop a versatile platform for single cell analysis and isolation that can be applied in immunology research and clinical therapy development. Methods: An automated microscopy and cell sorting system was developed to track the proliferative behavior of single-cell human primary CD4+ lymphocytes in response to stimulation using allogeneic lymphoblastoid feeder cells. Results: The system identified single human T lymphocytes with a sensitivity of 98% and specificity of 99% and possessed a cell collection efficiency of 86%. Time-lapse imaging simultaneously tracked 4,534 alloreactive T cells on a single array; 19% of the arrayed cells formed colonies of ≥2 cells. From the array, 130 clonal colonies were isolated and 7 grew to colony sizes of >10,000 cells, consistent with the known proliferative capacity of T cells in vitro and their tendency to become exhausted with prolonged stimulation. The isolated colonies underwent ELISA assay to detect interferon-γ secretion and Sanger sequencing to determine T cell receptor β sequences with a 100% success rate. Conclusion: The platform is capable of both identification and isolation of proliferative T cells in an automated manner. Significance: This novel technology enables the identification of TCR sequences based on T cell proliferation which is expected to speed the development of future cancer immunotherapies.
Chimeric antigen receptor T (CAR‐T) cell immunotherapies have seen success in treating hematological malignancies in recent years; however, the results can be highly variable. Single cell ...heterogeneity plays a key role in the variable efficacy of CAR‐T cell treatments yet is largely unexplored. A major challenge is to understand the killing behavior and phenotype of individual CAR‐T cells, which are able to serially kill targets. Thus, a platform capable of measuring time‐dependent CAR‐T cell mediated killing and then isolating single cells for downstream assays would be invaluable in characterizing CAR‐T cells. An automated microraft array platform was designed to track CD19 CAR‐T cell killing of CD19+ target cells and CAR‐T cell motility over time followed by CAR‐T cell collection based on killing behavior. The platform demonstrated automated CAR‐T cell counting with up to 98% specificity and 96% sensitivity, and single cells were isolated with 89% efficiency. On average, 2.3% of single CAR‐T cells were shown to participate in serial‐killing of target cells, killing a maximum of three target cells in a 6 h period. The cytotoxicity and motility of >7000 individual CAR‐T cells was tracked across four microraft arrays. The automated microraft array platform measured temporal cell‐mediated cytotoxicity, CAR‐T cell motility, CAR‐T cell death, and CAR‐T cell to target cell distances, followed by the capability to sort any desired CAR‐T cell. The pipeline has the potential to further our understanding of T cell‐based cancer immunotherapies and improve cell‐therapy products for better patient outcomes.
Older adults suffer a disproportionate burden of influenza-related morbidity and mortality typically attributed to defects in the aging immune system collectively known as immunosenescence. While the ...age-related decline in the adaptive immune system has been well characterized, little is known about how aging affects the principal site of influenza infection-the nasal epithelium. In human nasal epithelial cell cultures (hNECs) from older adults, we found similar or increased levels of cytokines during influenza infection compared with hNECs from younger individuals. However, hNECs from older individuals demonstrated decreased mRNA expression for several key proteins that affect clearance of infected cells, including MHC-I and transporter associated with antigen presentation (TAP). These findings were confirmed at the level of protein expression. In vivo studies corroborated the in vitro differences in MHC-I and TAP gene expression and also revealed important decreases in the expression of key influenza-specific antiviral mediators MX1 and IFITM1. Furthermore, epithelial cell-cytotoxic T lymphocyte co-cultures demonstrate that CTL cytotoxic activity is dose-dependent on MHC-I antigen presentation. Taken together, these results indicate that aging is associated with important changes in the nasal epithelium, including antigen presentation and antiviral pathways, which may contribute to increased severity of disease in older adults through impaired clearance of infected cells.
CD8 T cells recognize infected and cancerous cells via their T-cell receptor (TCR), which binds peptide-MHC complexes on the target cell. The affinity of the interaction between the TCR and ...peptide-MHC contributes to the antigen sensitivity, or functional avidity, of the CD8 T cell. In response to peptide-MHC stimulation, the TCR-CD3 complex and CD8 co-receptor are downmodulated. We quantified CD3 and CD8 downmodulation following stimulation of human CD8 T cells with CMV, EBV, and HIV peptides spanning eight MHC restrictions, observing a strong correlation between the levels of CD3 and CD8 downmodulation and functional avidity, regardless of peptide viral origin. In TCR-transduced T cells targeting a tumor-associated antigen, changes in TCR-peptide affinity were sufficient to modify CD3 and CD8 downmodulation. Correlation analysis and generalized linear modeling indicated that CD3 downmodulation was the stronger correlate of avidity. CD3 downmodulation, simply measured using flow cytometry, can be used to identify high-avidity CD8 T cells in a clinical context.
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