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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 bone fide 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. Ex vivo 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.
Assuntos
Leucemia Mieloide Aguda , Tirosina Quinase 3 Semelhante a fms , Animais , Humanos , Camundongos , Células Dendríticas/patologia , Tirosina Quinase 3 Semelhante a fms/genética , Homeostase , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Mutação , Microambiente Tumoral/genéticaRESUMO
Background: In 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. Objectives: This study aimed to identify the current medical status and the method of survival prediction among PHMs treated at the PCU. Setting/Subjects/Measurements: We 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. Results: The 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. Conclusion: The 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.
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Dendritic cells (DC) are mediators of 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 leukemia patients. To date, there is little information on the effects of FLT3-ITD in DC biology. We examined the cDC phenotype and frequency in bone marrow aspirates from patients with acute myeloid leukemia (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. Furthermore, 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.
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PURPOSE: Targeted therapeutics are a goal of medicine. Methods for targeting T-cell lymphoma lack specificity for the malignant cell, leading to elimination of healthy cells. The T-cell receptor (TCR) is designed for antigen recognition. T-cell malignancies expand from a single clone that expresses one of 48 TCR variable beta (Vß) genes, providing a distinct therapeutic target. We hypothesized that a mAb that is exclusive to a specific Vß would eliminate the malignant clone while having minimal effects on healthy T cells. EXPERIMENTAL DESIGN: We identified a patient with large granular T-cell leukemia and sequenced his circulating T-cell population, 95% of which expressed Vß13.3. We developed a panel of anti-Vß13.3 antibodies to test for binding and elimination of the malignant T-cell clone. RESULTS: Therapeutic antibody candidates bound the malignant clone with high affinity. Antibodies killed engineered cell lines expressing the patient TCR Vß13.3 by antibody-dependent cellular cytotoxicity and TCR-mediated activation-induced cell death, and exhibited specific killing of patient malignant T cells in combination with exogenous natural killer cells. EL4 cells expressing the patient's TCR Vß13.3 were also killed by antibody administration in an in vivo murine model. CONCLUSIONS: This approach serves as an outline for development of therapeutics that can treat clonal T-cell-based malignancies and potentially other T-cell-mediated diseases. See related commentary by Varma and Diefenbach, p. 4024.
Assuntos
Linfoma de Células T , Receptores de Antígenos de Linfócitos T , Humanos , Camundongos , Animais , Rituximab , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/imunologiaRESUMO
Many acute myeloid leukemia (AML) patients exhibit hallmarks of immune exhaustion, such as increased myeloid-derived suppressor cells, suppressive regulatory T cells and dysfunctional T cells. Similarly, we have identified the same immune-related features, including exhausted CD8+ T cells (TEx) in a mouse model of AML. Here we show that inhibitors that target bromodomain and extra-terminal domain (BET) proteins affect tumor-intrinsic factors but also rescue T cell exhaustion and ICB resistance. Ex vivo treatment of cells from AML mice and AML patients with BET inhibitors (BETi) reversed CD8+ T cell exhaustion by restoring proliferative capacity and expansion of the more functional precursor-exhausted T cells. This reversal was enhanced by combined BETi and anti-PD1 treatment. BETi synergized with anti-PD1 in vivo, resulting in the reduction of circulating leukemia cells, enrichment of CD8+ T cells in the bone marrow, and increase in expression of Tcf7, Slamf6, and Cxcr5 in CD8+ T cells. Finally, we profiled the epigenomes of in vivo JQ1-treated AML-derived CD8+ T cells by single-cell ATAC-seq and found that JQ1 increases Tcf7 accessibility specifically in Tex cells, suggesting that BETi likely acts mechanistically by relieving repression of progenitor programs in Tex CD8+ T cells and maintaining a pool of anti-PD1 responsive CD8+ T cells.
Assuntos
Linfócitos T CD8-Positivos , Leucemia Mieloide Aguda , Animais , Camundongos , Leucemia Mieloide Aguda/metabolismo , Linfócitos T ReguladoresRESUMO
Acute myeloid leukemia (AML) is a cancer of myeloid-lineage cells with limited therapeutic options. We previously combined ex vivo drug sensitivity with genomic, transcriptomic, and clinical annotations for a large cohort of AML patients, which facilitated discovery of functional genomic correlates. Here, we present a dataset that has been harmonized with our initial report to yield a cumulative cohort of 805 patients (942 specimens). We show strong cross-cohort concordance and identify features of drug response. Further, deconvoluting transcriptomic data shows that drug sensitivity is governed broadly by AML cell differentiation state, sometimes conditionally affecting other correlates of response. Finally, modeling of clinical outcome reveals a single gene, PEAR1, to be among the strongest predictors of patient survival, especially for young patients. Collectively, this report expands a large functional genomic resource, offers avenues for mechanistic exploration and drug development, and reveals tools for predicting outcome in AML.
Assuntos
Leucemia Mieloide Aguda , Diferenciação Celular , Estudos de Coortes , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Receptores de Superfície Celular/genética , TranscriptomaRESUMO
Blinatumomab is currently approved for use as a single agent in relapsed and refractory acute lymphoblastic leukemia (ALL). Cytotoxicity is mediated via signaling through the T-cell receptor (TCR). There is now much interest in combining blinatumomab with targeted therapies, particularly in Philadelphia chromosome-positive ALL (Ph+ ALL). However, some second- and third-generation ABL inhibitors also potently inhibit Src family kinases that are important in TCR signaling. We combined ABL inhibitors and dual Src/ABL inhibitors with blinatumomab in vitro from both healthy donor samples and primary samples from patients with Ph+ ALL. Blinatumomab alone led to both T-cell proliferation and elimination of target CD19+ cells and enhanced production of interferon-γ (IFN-γ). The addition of the ABL inhibitors imatinib or nilotinib to blinatumomab did not inhibit T-cell proliferation or IFN-γ production. However, the addition of dasatinib or ponatinib inhibited T-cell proliferation and IFN-γ production. Importantly, there was no loss of CD19+ cells treated with blinatumomab plus dasatinib or ponatinib in healthy samples or samples with a resistant ABL T315I mutation by dasatinib in combination with blinatumomab. These in vitro findings bring pause to the excitement of combination therapies, highlighting the importance of maintaining T-cell function with targeted therapies.
Assuntos
Anticorpos Biespecíficos/farmacologia , Antineoplásicos/farmacologia , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Ativação Linfocitária/efeitos dos fármacos , Proteínas de Neoplasias/antagonistas & inibidores , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Linfócitos T/imunologia , Quinases da Família src/antagonistas & inibidores , Linfócitos B , Dasatinibe/farmacologia , Humanos , Mesilato de Imatinib/farmacologia , Imidazóis/farmacologia , Testes de Liberação de Interferon-gama , Células Jurkat , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Mutação de Sentido Incorreto , Proteínas de Neoplasias/fisiologia , Fosforilação/efeitos dos fármacos , Mutação Puntual , Leucemia-Linfoma Linfoblástico de Células Precursoras/enzimologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-abl/genética , Piridazinas/farmacologia , Pirimidinas/farmacologia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/enzimologia , Linfócitos T/metabolismo , Células Tumorais Cultivadas , Quinases da Família src/fisiologiaRESUMO
A 77-year-old Japanese woman with a 21-year history of seropositive, erosive rheumatoid arthritis (RA) and a 10-year history of methotrexate (MTX) therapy was admitted with malaise and mild consciousness disturbance. Laboratory data showed hypercalcemia, acute kidney injury, normocytic anaemia, and thrombocytopenia. As we first assumed drug-induced toxicity by MTX and eldecalcitol, both were discontinued and leucovorin rescue therapy and calcitonin were administered. However, her condition continued to worsen. Serum protein electrophoresis showed only a small M-peak, immunoelectrophoresis of both the serum and urine demonstrated Bence-Jones kappa (κ) type monoclonal protein without immunoglobulin heavy chain, and bone marrow examination revealed proliferation of plasma cells. We diagnosed her with Bence-Jones κ type multiple myeloma (MM) and transferred her to the department of haematology of a higher order medical institution. Conclusively, the diagnosis of immunoglobulin (Ig) D-κ type MM, a rare variant of this disorder, was determined in accordance with serum immunofixation. Several previous studies have suggested that pre-existing RA is a risk factor for MM. Although IgD MM is characterised by its clinical severity and poor prognosis compared to other subtypes, it is often misdiagnosed or mistaken as light chain type MM, as in the present case, because of the low level of IgD M-protein, resulting in delayed diagnosis. Physicians must take MM into consideration as a differential diagnosis when inactive RA patients present with inexplicable elevated calcium, renal failure, anaemia, and bone lesion symptoms and should be aware of IgD MM to establish the correct diagnosis promptly.
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Artrite Reumatoide/complicações , Medula Óssea/patologia , Mieloma Múltiplo/diagnóstico , Idoso , Artrite Reumatoide/imunologia , Proteína de Bence Jones/urina , Feminino , Humanos , Imunoglobulina D/sangue , Imunoglobulina D/urina , Cadeias kappa de Imunoglobulina/sangue , Cadeias kappa de Imunoglobulina/urina , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/patologia , Proteínas do Mieloma/análiseRESUMO
Acute myeloid leukemia (AML) is the most common acute leukemia in adults, with approximately four new cases per 100,000 persons per year. Standard treatment for AML consists of induction chemotherapy with remission achieved in 50 to 75% of cases. Unfortunately, most patients will relapse and die from their disease, as 5-y survival is roughly 29%. Therefore, other treatment options are urgently needed. In recent years, immune-based therapies have led to unprecedented rates of survival among patients with some advanced cancers. Suppression of T cell function in the tumor microenvironment is commonly observed and may play a role in AML. We found that there is a significant association between T cell infiltration in the bone marrow microenvironment of newly diagnosed patients with AML and increased overall survival. Functional studies aimed at establishing the degree of T cell suppression in patients with AML revealed impaired T cell function in many patients. In most cases, T cell proliferation could be restored by blocking the immune checkpoint molecules PD-1, CTLA-4, or TIM3. Our data demonstrate that AML establishes an immune suppressive environment in the bone marrow, in part through T cell checkpoint function.
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Medula Óssea/metabolismo , Leucemia Mieloide Aguda/metabolismo , Linfócitos T/metabolismo , Microambiente Tumoral/fisiologia , Medula Óssea/imunologia , Antígeno CTLA-4/metabolismo , Proliferação de Células , Citocinas/metabolismo , Receptor Celular 2 do Vírus da Hepatite A/metabolismo , Humanos , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/terapia , Receptor de Morte Celular Programada 1/metabolismo , Linfócitos T/imunologiaRESUMO
Acute myeloid leukemia (AML) remains difficult to treat due to mutational heterogeneity and the development of resistance to therapy. Targeted agents, such as MEK inhibitors, may be incorporated into treatment; however, the impact of MEK inhibitors on the immune microenvironment in AML is not well understood. A greater understanding of the implications of MEK inhibition on immune responses may lead to a greater understanding of immune evasion and more rational combinations with immunotherapies. This study describes the impact of trametinib on both T cells and AML blast cells by using an immunosuppressive mouse model of AML and primary patient samples. We also used a large AML database of functional drug screens to understand characteristics of trametinib-sensitive samples. In the mouse model, trametinib increased T-cell viability and restored T-cell proliferation. Importantly, we report greater proliferation in the CD8+CD44+ effector subpopulation and impaired activation of CD8+CD62L+ naive cells. Transcriptome analysis revealed that trametinib-sensitive samples have an inflammatory gene expression profile, and we also observed increased programmed cell death ligand 1 (PD-L1) expression on trametinib-sensitive samples. Finally, we found that trametinib consistently reduced PD-L1 and PD-L2 expression in a dose-dependent manner on the myeloid population. Altogether, our data present greater insight into the impact of trametinib on the immune microenvironment and characteristics of trametinib-sensitive patient samples.
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Imunomodulação , Leucemia Mieloide Aguda/etiologia , Leucemia Mieloide Aguda/metabolismo , Sistema de Sinalização das MAP Quinases , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Antineoplásicos/farmacologia , Biomarcadores , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Perfilação da Expressão Gênica/métodos , Humanos , Imunomodulação/efeitos dos fármacos , Imunofenotipagem , Leucemia Mieloide Aguda/patologia , Ativação Linfocitária/imunologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
Mesenchymal stromal cells (MSCs) modulate immune responses through cell contact-dependent or paracrine mechanisms and are themselves known to have low immunogenicity. Given the increasing use of both natural killer (NK) cells and MSCs in cell-based therapies, we investigated the interaction between the two cell types using the NK cell lines, KHYG-1 and NK-92, and human bone marrow-derived MSCs. NK lines were cocultured with MSCs, either directly or in a transwell system, and the effects on proliferation, interferon-gamma (IFN-γ) production, and cytolytic activity of NK cells were analyzed. Cytotoxicity was measured in a 4 h chromium release assay. MSCs did not affect the proliferation of NK cell lines but reduced IFN-γ production by KHYG-1, but not NK-92, when cocultured directly at 10:1 NK:MSC ratio. MSCs suppressed K562 lysis by both KHYG-1 and NK-92 cells in contact-free transwell cocultures but only reduced cytotoxicity of KHYG-1 and not NK-92 cells when cells were in direct contact in coculture. Immunosuppressive effects of MSCs were mediated by indoleamine-2,3-dioxygenase (IDO) and prostaglandin E2 (PGE2) secreted by MSCs and were abrogated in the presence of IDO and PGE2 inhibitors. In the presence of MSCs, granule polarization was suppressed and induced respectively, in KHYG-1 and NK-92. Consistent with this, MSCs were susceptible to lysis by NK-92 but not KHYG-1. These studies indicate the differential crosstalk between MSCs and two highly cytotoxic NK lines and may be important when designing future cell therapy protocols with these two cell types.
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Células da Medula Óssea/imunologia , Imunomodulação , Células Matadoras Naturais/imunologia , Células-Tronco Mesenquimais/imunologia , Células da Medula Óssea/citologia , Humanos , Células K562 , Células Matadoras Naturais/citologia , Células-Tronco Mesenquimais/citologiaRESUMO
BACKGROUND: 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. DESIGN AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.
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Citotoxicidade Imunológica , Imunoterapia Adotiva , Células Matadoras Naturais/imunologia , Mieloma Múltiplo/terapia , Animais , Linhagem Celular Tumoral , Células Clonais , Citometria de Fluxo , Genes Reporter , Humanos , Imunoensaio , Fatores Imunológicos/administração & dosagem , Injeções Intravenosas , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/transplante , Luciferases/genética , Medições Luminescentes , Camundongos , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/patologia , Receptores de Células Matadoras Naturais/antagonistas & inibidores , Receptores de Células Matadoras Naturais/imunologia , Transplante Heterólogo , Ensaio Tumoral de Célula-TroncoRESUMO
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.
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Imunoterapia Adotiva/métodos , Leucemia/terapia , Cromossomo Filadélfia , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Linfócitos T Citotóxicos/metabolismo , Linfócitos T Citotóxicos/transplante , Animais , Proliferação de Células , Células Cultivadas , Citotoxicidade Imunológica/fisiologia , Modelos Animais de Doenças , Humanos , Células K562 , Leucemia/genética , Leucemia/imunologia , Leucemia/patologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/fisiologia , Transplante HeterólogoRESUMO
BACKGROUND AIMS: There is increasing interest in using γδ T cells (GDTC) for cancer immunotherapy. Most studies have been concerned with the Vδ2 subset in blood, for which several expansion protocols exist. We have developed a protocol to expand Vδ1 and Vδ2 preferentially from human blood. We have characterized these subsets and their specificities for leukemic targets. METHODS: GDTC were isolated from the peripheral blood mononuclear cells (PBMC) of healthy donors via positive magnetic cell sorting; their proliferation in vitro was induced by exposure to the mitogen concanavalin A (Con A). CD107 and cytotoxicity (Cr(51)-release and flow cytometric) assays were performed. GDTC clones and target cells were immunophenotyped via flow cytometry. RESULTS: Longer initial exposure to Con A typically resulted in higher Vδ1 prevalence. Vδ1 were activated by and cytotoxic to B-cell chronic lymphocytic leukemia (B-CLL)-derived MEC1 cells, whereas Vδ2 also responded to MEC1 but more so to the Philadelphia chromosome-positive [Ph+] leukemia cell line EM-enhanced green fluorescent protein (2eGFPluc). Vδ2 clone cytotoxicity against EM-2eGFPluc correlated with Vδ2 T-cell antigen receptor (TCR) and receptor found on Natural Killer cells and many T-cells (NKG2D), whereas Vδ1 clone cytotoxicity versus MEC1 correlated with Vδ1 TCR, CD56 and CD95 expression. Vδ1 also killed Epstein-Barr Virus (EBV)-negative B-CLL-derived TMD2 cells. Immunophenotyping revealed reduced HLA-ABC expression on EM-2eGFPluc, whereas MEC1 and TMD2 exhibited higher Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAILR1). CONCLUSIONS: Our ability to expand peripheral Vδ1 cells and show their cytotoxicity to B-CLL-derived cell lines suggests that this novel approach to the cellular treatment of B-CLL may be feasible.
Assuntos
Leucemia Linfocítica Crônica de Células B/terapia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Subpopulações de Linfócitos T/imunologia , Anexina A5 , Apoptose , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Concanavalina A/farmacologia , Citometria de Fluxo , Humanos , Imunoterapia , Leucemia Linfocítica Crônica de Células B/imunologia , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/metabolismoRESUMO
In conventional alphabeta T cells, the Tec family tyrosine kinase Itk is required for signaling downstream of the T cell receptor (TCR). Itk also regulates alphabeta 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 gammadelta T cells, as normal IgE levels are observed in Itk(-/-)Tcrd(-/-) mice. When stimulated through the gammadelta TCR, Itk(-/-) gammadelta T cells produce high levels of Th2 cytokines, but diminished IFNgamma. In addition, activated Itk(-/-) gammadelta 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 gammadelta T cells numbers are increased in Itk(-/-) mice, most notably the Vgamma1.1(+)Vdelta6.3(+) subset that represents the dominant population of gammadelta NKT cells. Itk(-/-) gammadelta NKT cells also have increased expression of PLZF, a transcription factor required for alphabeta NKT cells, indicating a common molecular program between alphabeta and gammadelta NKT cell lineages. Together, these data indicate that Itk signaling regulates gammadelta T cell lineage development and effector function and is required to control IgE production in vivo.
Assuntos
Imunoglobulina E/biossíntese , Proteínas Tirosina Quinases/fisiologia , Receptores de Antígenos de Linfócitos T gama-delta , Linfócitos T/enzimologia , Animais , Linfócitos B , Linhagem da Célula , Citocinas/biossíntese , Switching de Imunoglobulina , Ativação Linfocitária , Camundongos , Camundongos Knockout , Células T Matadoras Naturais/citologia , Proteínas Tirosina Quinases/deficiência , Proteínas Tirosina Quinases/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta , Linfócitos T/citologia , Células Th2RESUMO
NF-kappaB-inducing kinase (NIK) is responsible for activation of the non-canonical p100 processing pathway of NF-kappaB activation. This kinase has been shown to be critical for activation of this pathway after signaling through several TNF family members including CD40. The functional importance of this pathway in CD40 and TLR-induced dendritic cell (DC) differentiation was studied in vivo in the alymphoplasia (Aly) mouse. The Aly mouse expresses a mutant NIK molecule that prohibits the induction of the non-canonical pathway. We show that while MHC class II presentation and in vivo migration of Aly DCs is intact, these cells are unable to cross-prime CD8+ T cells to exogenous Ag. Gene expression array analysis of DCs matured in vivo indicates multiple defects in Ag processing pathways after maturation and provide a global view of the genes that are regulated by the NF-kappaB2 pathway in DCs. These experiments indicate a possible role for NIK in mediating cross-priming of soluble Ag. In addition, our findings explain the profound immune unresponsiveness of the Aly mouse.
Assuntos
Antígenos/imunologia , Apresentação Cruzada/imunologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Subunidade p52 de NF-kappa B/metabolismo , Transdução de Sinais/imunologia , Transporte Ativo do Núcleo Celular , Animais , Linhagem Celular , Membrana Celular/imunologia , Membrana Celular/metabolismo , Movimento Celular , Células Dendríticas/citologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Doenças Linfáticas/imunologia , Doenças Linfáticas/metabolismo , Camundongos , Mutação/genética , Fenótipo , Solubilidade , Receptores Toll-Like/metabolismo , Regulação para CimaRESUMO
The Tec family of tyrosine kinases consists of five members (Itk, Rlk, Tec, Btk, and Bmx) that are expressed predominantly in hematopoietic cells. The exceptions, Tec and Bmx, are also found in endothelial cells. Tec kinases constitute the second largest family of cytoplasmic protein tyrosine kinases. While B cells express Btk and Tec, and T cells express Itk, Rlk, and Tec, all four of these kinases (Btk, Itk, Rlk, and Tec) can be detected in mast cells. This chapter will focus on the biochemical and cell biological data that have been accumulated regarding Itk, Rlk, Btk, and Tec. In particular, distinctions between the different Tec kinase family members will be highlighted, with a goal of providing insight into the unique functions of each kinase. The known functions of Tec kinases in T cell and mast cell signaling will then be described, with a particular focus on T cell receptor and mast cell Fc epsilon RI signaling pathways.
Assuntos
Mastócitos/enzimologia , Proteínas Tirosina Quinases/imunologia , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais/imunologia , Linfócitos T/enzimologia , Animais , Ativação Enzimática/imunologia , Humanos , Mastócitos/imunologia , Linfócitos T/imunologiaRESUMO
The Tec family tyrosine kinase, Itk, was initially characterized as a crucial component of T-cell receptor signaling pathways resulting in phospholipase C-gamma1 activation and actin polymerization. In 1999, a seminal report by Fowell, Locksley and colleagues demonstrated that, in CD4+ T cells, Itk-dependent signals are differentially required for T-helper (Th)2 versus Th1 differentiation and effector function. These findings launched a series of in vitro and in vivo studies addressing the molecular defects of Itk-/- CD4+ T cells, and the impaired immune responses of intact Itk-deficient mice. While demonstrating a bias against Th2 differentiation, overall these experiments have indicated that the most significant failing is an inability of Itk-/- CD4+ T cells to produce Th2 cytokines in a recall response, rather than an absolute defect in Th2 differentiation by T cells lacking Itk. In this review, we discuss the pathways by which Itk might impact the differentiation of Th cells.