Dendritic cells (DC) are mediators between innate and adaptive immune responses to pathogens and tumors. DC development is determined by signaling through the receptor tyrosine kinase Fms-like ...tyrosine kinase 3 (FLT3) in bone marrow myeloid progenitors. Recently the naming conventions for DC phenotypes have been updated to distinguish between "Conventional" DCs (cDCs) and plasmacytoid DCs (pDCs). Activating mutations of FLT3, including Internal Tandem Duplication (FLT3-ITD), are associated with poor prognosis for acute myeloid leukemia (AML) patients. Having a shared myeloid lineage it can be difficult to distinguish
DCs from AML tumor cells. To date, there is little information on the effects of FLT3-ITD in DC biology. To further elucidate this relationship we utilized CITE-seq technology in combination with flow cytometry and multiplex immunoassays to measure changes to DCs in human and mouse tissues. We examined the cDC phenotype and frequency in bone marrow aspirates from patients with AML to understand the changes to cDCs associated with FLT3-ITD. When compared to healthy donor (HD) we found that a subset of FLT3-ITD+ AML patient samples have overrepresented populations of cDCs and disrupted phenotypes. Using a mouse model of FLT3-ITD+ AML, we found that cDCs were increased in percentage and number compared to control wild-type (WT) mice. Single cell RNA-seq identified FLT3-ITD+ cDCs as skewed towards a cDC2 T-bet
phenotype, previously shown to promote Th17 T cells. We assessed the phenotypes of CD4
T cells in the AML mice and found significant enrichment of both Treg and Th17 CD4
T cells in the bone marrow and spleen compartments.
stimulation of CD4
T cells also showed increased Th17 phenotype in AML mice. Moreover, co-culture of AML mouse-derived DCs and naïve OT-II cells preferentially skewed T cells into a Th17 phenotype. Together, our data suggests that FLT3-ITD+ leukemia-associated cDCs polarize CD4
T cells into Th17 subsets, a population that has been shown to be negatively associated with survival in solid tumor contexts. This illustrates the complex tumor microenvironment of AML and highlights the need for further investigation into the effects of FLT3-ITD mutations on DC phenotypes and their downstream effects on Th polarization.
In conventional αβ T cells, the Tec family tyrosine kinase Itk is required for signaling downstream of the T cell receptor (TCR). Itk also regulates αβ T cell development, lineage commitment, and ...effector function. A well established feature of Itk⁻/⁻ mice is their inability to generate T helper type 2 (Th2) responses that produce IL-4, IL-5, and IL-13; yet these mice have spontaneously elevated levels of serum IgE and increased numbers of germinal center B cells. Here we show that the source of this phenotype is γδ T cells, as normal IgE levels are observed in Itk⁻/⁻Tcrd⁻/⁻ mice. When stimulated through the γδ TCR, Itk⁻/⁻ γδ T cells produce high levels of Th2 cytokines, but diminished IFNγ. In addition, activated Itk⁻/⁻ γδ T cells up-regulate costimulatory molecules important for B cell help, suggesting that they may directly promote B cell activation and Ig class switching. Furthermore, we find that γδ T cells numbers are increased in Itk⁻/⁻ mice, most notably the Vγ1.1⁺Vδ6.3⁺ subset that represents the dominant population of γδ NKT cells. Itk⁻/⁻ γδ NKT cells also have increased expression of PLZF, a transcription factor required for αβ NKT cells, indicating a common molecular program between αβ and γδ NKT cell lineages. Together, these data indicate that Itk signaling regulates γδ T cell lineage development and effector function and is required to control IgE production in vivo.
BackgroundIn Japan, the number of patients with aggressive hematological malignancies (PHMs) admitted at the palliative care unit (PCU) in their end-of-life (EOL) stage was fewer than that of ...patients with solid tumors due to several reasons. The assessment of patient characteristics and the methods of survival prediction among PHMs in the EOL stage are warranted. ObjectivesThis study aimed to identify the current medical status and the method of survival prediction among PHMs treated at the PCU. Setting/Subjects/MeasurementsWe retrospectively analyzed the clinical data of 25 PHMs treated at our PCU between January 2017 and December 2020. The association between survival time and the palliative prognostic score (PAP) and palliative prognostic index (PPI) was analyzed. ResultsThe average age of the PHMs was higher than that of patients with lung cancer as a control. The median survival time of the PHMs was shorter than the control group. Most PHMs could not receive standard chemotherapy, and the most common cause of death was disease-related organ failure. Significant associations were observed between the survival time and each PAP/PPI value in patients with malignant lymphoma, but not in those with leukemia. ConclusionThe PHMs in the PCU had a lower median survival time than the control group. These results were induced by the result of patient selection to avoid treatment-related severe toxicity. The survival prediction using the PAP and PPI was less accurate in patients with leukemia.
Novel therapies capable of targeting drug resistant clonogenic MM cells are required for more effective treatment of multiple myeloma. This study investigates the cytotoxicity of natural killer cell ...lines against bulk and clonogenic multiple myeloma and evaluates the tumor burden after NK cell therapy in a bioluminescent xenograft mouse model.
The cytotoxicity of natural killer cell lines was evaluated against bulk multiple myeloma cell lines using chromium release and flow cytometry cytotoxicity assays. Selected activating receptors on natural killer cells were blocked to determine their role in multiple myeloma recognition. Growth inhibition of clonogenic multiple myeloma cells was assessed in a methylcellulose clonogenic assay in combination with secondary replating to evaluate the self-renewal of residual progenitors after natural killer cell treatment. A bioluminescent mouse model was developed using the human U266 cell line transduced to express green fluorescent protein and luciferase (U266eGFPluc) to monitor disease progression in vivo and assess bone marrow engraftment after intravenous NK-92 cell therapy.
Three multiple myeloma cell lines were sensitive to NK-92 and KHYG-1 cytotoxicity mediated by NKp30, NKp46, NKG2D and DNAM-1 activating receptors. NK-92 and KHYG-1 demonstrated 2- to 3-fold greater inhibition of clonogenic multiple myeloma growth, compared with killing of the bulk tumor population. In addition, the residual colonies after treatment formed significantly fewer colonies compared to the control in a secondary replating for a cumulative clonogenic inhibition of 89-99% at the 20:1 effector to target ratio. Multiple myeloma tumor burden was reduced by NK-92 in a xenograft mouse model as measured by bioluminescence imaging and reduction in bone marrow engraftment of U266eGFPluc cells by flow cytometry.
This study demonstrates that NK-92 and KHYG-1 are capable of killing clonogenic and bulk multiple myeloma cells. In addition, multiple myeloma tumor burden in a xenograft mouse model was reduced by intravenous NK-92 cell therapy. Since multiple myeloma colony frequency correlates with survival, our observations have important clinical implications and suggest that clinical studies of NK cell lines to treat MM are warranted.
BackgroundAcute Myeloid Leukemia (AML) is the most common adult leukemia and has a very poor prognosis. With a 5-year survival of under 30% (seer.cancer.gov), most people diagnosed with AML will die ...from the disease. AML is caused by an uncontrolled proliferation of poorly differentiated myeloid precursor cells which results from a combination of three classes of mutations that affect proliferation, differentiation and epigenetic state. For this reason, drugs targeting epigenetic modifications are being actively studied in AML. AML has been shown to avoid immune recognition though inhibiting the function of multiple cell types, especially T cells1 2 and therefore immune checkpoint blockade presents a promising therapy for any immune-targeted strategy; however, clinical trials to date have shown very modest efficacy.3–5 T cell exhaustion in cancer has been shown to be a regulated process involving transcriptional and epigenetic changes.6–9 BRD4 has been shown to be important for maintaining this exhaustion state.10 11 It stands to reason that drugs designed to target epigenetic pathways in tumors will have effects on T cell populations present in the tumor microenvironment. In these studies, we investigated the effects of the BET inhibitor (BETi) JQ1 on T cell exhaustion and checkpoint responsiveness in a murine model of AML.MethodsThe AML mouse model bears FLT3-ITD and deletion of TET2 restricted to the myeloid lineage. For in vitro studies, splenocytes were stimulated with anti-CD3 and either JQ1, anti-PD1 or both and proliferation and differentiation status were assessed by flow cytometry. For in vivo studies, treatment consisted of 2 weeks with JQ1, anti-PD1 or both.ResultsThis mouse model of AML exhibits an expansion of terminally exhausted T cells and impaired proliferative capacity after stimulation through the TCR (figure 1). Ex vivo treatment with BETi and anti-PD1 reverses CD8+ T cell exhaustion via rescue of proliferative dysfunction and expansion of more functional precursor exhausted T cells (TPEx-CD8, PD1+, TCF1+, TIM3-) (figure 2). Finally, we show that BETi synergizes with anti-PD1 in vivo leading to a reduction of tumor cells in multiple organ sites, and enrichment of CD8+ T cells in the bone marrow (figure 3).ConclusionsUsing an AML mouse model that exhibits leukemia-induced immune exhaustion, we demonstrate the pre-clinical efficacy of combining BETi and anti-PD1 therapy in the treatment of AML.ReferencesLamble AJ, Lind EF. Targeting the immune microenvironment in acute myeloid leukemia: a focus on T Cell immunity. Front Oncol 2018;8:213.Lamble AJ, Kosaka Y, Laderas T, Maffit A, Kaempf A, Brady LK, et al. Reversible suppression of T cell function in the bone marrow microenvironment of acute myeloid leukemia. Proc Natl Acad Sci U S A. 2020;117(25):14331–41.Boddu P, Kantarjian H, Garcia-Manero G, Allison J, Sharma P, Daver N. The emerging role of immune checkpoint based approaches in AML and MDS. Leuk Lymphoma 2018;59(4):790–802.Bewersdorf JP, Shallis RM, Zeidan AM. Immune checkpoint inhibition in myeloid malignancies: moving beyond the PD-1/PD-L1 and CTLA-4 pathways. Blood Rev 2020:100709.Daver N, Garcia-Manero G, Basu S, Boddu PC, Alfayez M, Cortes JE, et al. Efficacy, safety, and biomarkers of response to azacitidine and nivolumab in relapsed/Refractory acute myeloid leukemia: a nonrandomized, Open-Label, Phase II Study. Cancer Discov 2019;9(3):370–83.Beltra JC, Manne S, Abdel-Hakeem MS, Kurachi M, Giles JR, Chen Z, et al. Developmental Relationships of Four Exhausted CD8(+) T Cell subsets reveals underlying transcriptional and epigenetic landscape control mechanisms. Immunity 2020;52(5):825–41 e8.Khan O, Giles JR, McDonald S, Manne S, Ngiow SF, Patel KP, et al. TOX transcriptionally and epigenetically programs CD8(+) T cell exhaustion. Nature 2019;571(7764):211–8.Pauken KE, Sammons MA, Odorizzi PM, Manne S, Godec J, Khan O, et al. Epigenetic stability of exhausted T cells limits durability of reinvigoration by PD-1 blockade. Science 2016;354(6316):1160–5.Abdel-Hakeem MS, Manne S, Beltra JC, Stelekati E, Chen Z, Nzingha K, et al. Epigenetic scarring of exhausted T cells hinders memory differentiation upon eliminating chronic antigenic stimulation. Nat Immunol 2021;22(8):1008–19.Milner JJ, Toma C, Quon S, Omilusik K, Scharping NE, Dey A, et al. Bromodomain protein BRD4 directs and sustains CD8 T cell differentiation during infection. J Exp Med 2021;218(8).Kagoya Y, Nakatsugawa M, Yamashita Y, Ochi T, Guo T, Anczurowski M, et al. BET bromodomain inhibition enhances T cell persistence and function in adoptive immunotherapy models. J Clin Invest 2016;126(9):3479–94.Ethics ApprovalThis study has been approved by the OHSU IACUC committee protocol IP00000907 “Immune-based therapeutic approaches for acute myeloid leukemia” Evan Lind PI.Abstract 734 Figure 1T cell exhaustion in the AML mouse model. (A) Cytotoxic T cells show an exhausted phenotype in mice with AML. Spleen cultures from mice with AML or WT controls were stained with antibodies to CD3, CD8, TIM3, PD1, and TCF1. Left shows percent of TPEX CD8 T cells. Right panel shows TEX CD8 T cells. N = 12 animals per group. (B) Proliferative defect in T cells in mice with AML. Splenocytes were labeled with the proliferation dye CFSE. Whole spleen suspensions were stimulated with anti-CD3 or anti-CD3 and anti-CD28 for 3 days. FACs plots show proliferation of T cells in each conditionAbstract 734 Figure 2Treatment with JQ1 results in expansion of T cells with TPEX. (A) Example of proliferation (CFSE dilution) vs TCF-1 expression showing unstimulated, CD3 or CD3+JQ1 120 nM in in vitro 3-day culture. Results gated on CD8 T cells. (B) Summary of T cell proliferation from 4 independent experiments showing the percent proliferation of CD8 T cells with TPEX (PD1+ Tim-3- TCF-1+) (black line) or TEX (PD1+Tim-3+TCF1-) phenotype (red line). Statistics are unpaired T-Test for each treatment condition.Abstract 734 Figure 3In vivo treatment of FTL mice with the BETi JQ1. (A) Schematic overview of treatment protocol. (B) White blood cell counts at pre-treatment, 1 week and 2 weeks after JQ1, PD1 blockade or both. (C) Percent of CD8+ T cells of all CD3-gated T cells in the BM of treated animals. (D) A-C Percent of precursor-exhausted CD8+ T cells as a percent of all T cells in the spleen of treated animals. Results combined from 2 separate experiments n=7. D One experiment n=3.
Gamma delta T cells (GDTc) lyse a variety of hematological and solid tumour cells in vitro and in vivo, and are thus promising candidates for cellular immunotherapy. We have developed a protocol to ...expand human GDTc in vitro, yielding highly cytotoxic Vgamma9/Vdelta2 CD27/CD45RA double negative effector memory cells. These cells express CD16, CD45RO, CD56, CD95 and NKG2D. Flow cytometric, clonogenic, and chromium release assays confirmed their specific cytotoxicity against Ph(+) cell lines in vitro. We have generated a fluorescent and bioluminescent Ph(+) cell line, EM-2eGFPluc, and established a novel xenogeneic leukemia model. Intravenous injection of EM-2eGFPluc into NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice resulted in significant dose-dependent bone marrow engraftment; lower levels engrafted in blood, lung, liver and spleen. In vitro-expanded human GDTc injected intraperitoneally were found at higher levels in blood and organs compared to those injected intravenously; GDTc survived at least 33 days post-injection. In therapy experiments, we documented decreased bone marrow leukemia burden in mice treated with GDTc. Live GDTc were found in spleen and bone marrow at endpoint, suggesting the potential usefulness of this therapy.
SUMMARY
This paper proposes estimation methods for short circuit currents using phasor measurement unit (PMU) measurements (phasors). The methods follow the basic notion of representing the source ...side of a power system by an equivalent circuit with a voltage behind a back impedance, and employ a set of voltage and current phasors measured at substations during the normal variation of loads in their estimation. In order to improve the estimation accuracy of the proposed methods, the concept of using the changes between consecutive phasors is introduced. Furthermore, to make the methods applicable to a real‐world system, the concept of a reference phasor, used to remove the effects of system‐wide frequency variations and a filtering process to filter out the outlier phasors, is proposed and implemented. The validity and effectiveness of the proposed methods were checked and confirmed through experiments and field tests.
The characterization of cancer genomes has provided insight into somatically altered genes across tumors, transformed our understanding of cancer biology, and enabled tailoring of therapeutic ...strategies. However, the function of most cancer alleles remains mysterious, and many cancer features transcend their genomes. Consequently, tumor genomic characterization does not influence therapy for most patients. Approaches to understand the function and circuitry of cancer genes provide complementary approaches to elucidate both oncogene and non-oncogene dependencies. Emerging work indicates that the diversity of therapeutic targets engendered by non-oncogene dependencies is much larger than the list of recurrently mutated genes. Here we describe a framework for this expanded list of cancer targets, providing novel opportunities for clinical translation.
Members of the Cancer Target Discovery and Development Network synthesize recent insights into the different classes and characteristics of cancer therapeutic vulnerabilities.
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