RESUMO
BACKGROUND: Anti-CD19 chimeric antigen receptor (CAR) T-cell therapy has shown remarkable clinical efficacy in B-cell cancers. However, CAR T cells can induce substantial toxic effects, and the manufacture of the cells is complex. Natural killer (NK) cells that have been modified to express an anti-CD19 CAR have the potential to overcome these limitations. METHODS: In this phase 1 and 2 trial, we administered HLA-mismatched anti-CD19 CAR-NK cells derived from cord blood to 11 patients with relapsed or refractory CD19-positive cancers (non-Hodgkin's lymphoma or chronic lymphocytic leukemia [CLL]). NK cells were transduced with a retroviral vector expressing genes that encode anti-CD19 CAR, interleukin-15, and inducible caspase 9 as a safety switch. The cells were expanded ex vivo and administered in a single infusion at one of three doses (1×105, 1×106, or 1×107 CAR-NK cells per kilogram of body weight) after lymphodepleting chemotherapy. RESULTS: The administration of CAR-NK cells was not associated with the development of cytokine release syndrome, neurotoxicity, or graft-versus-host disease, and there was no increase in the levels of inflammatory cytokines, including interleukin-6, over baseline. The maximum tolerated dose was not reached. Of the 11 patients who were treated, 8 (73%) had a response; of these patients, 7 (4 with lymphoma and 3 with CLL) had a complete remission, and 1 had remission of the Richter's transformation component but had persistent CLL. Responses were rapid and seen within 30 days after infusion at all dose levels. The infused CAR-NK cells expanded and persisted at low levels for at least 12 months. CONCLUSIONS: Among 11 patients with relapsed or refractory CD19-positive cancers, a majority had a response to treatment with CAR-NK cells without the development of major toxic effects. (Funded by the M.D. Anderson Cancer Center CLL and Lymphoma Moonshot and the National Institutes of Health; ClinicalTrials.gov number, NCT03056339.).
Assuntos
Antígenos CD19 , Células Matadoras Naturais/transplante , Leucemia Linfocítica Crônica de Células B/terapia , Linfoma não Hodgkin/terapia , Receptores de Antígenos Quiméricos/antagonistas & inibidores , Idoso , Aloenxertos , Terapia Baseada em Transplante de Células e Tecidos , Feminino , Sangue Fetal , Vetores Genéticos , Humanos , Células Matadoras Naturais/imunologia , Leucemia Linfocítica Crônica de Células B/imunologia , Linfoma não Hodgkin/imunologia , Masculino , Pessoa de Meia-Idade , Indução de Remissão/métodos , Retroviridae/genética , Condicionamento Pré-TransplanteRESUMO
Immune checkpoint therapy has resulted in remarkable improvements in the outcome for certain cancers. To broaden the clinical impact of checkpoint targeting, we devised a strategy that couples targeting of the cytokine-inducible Src homology 2-containing (CIS) protein, a key negative regulator of interleukin 15 (IL-15) signaling, with fourth-generation "armored" chimeric antigen receptor (CAR) engineering of cord blood-derived natural killer (NK) cells. This combined strategy boosted NK cell effector function through enhancing the Akt/mTORC1 axis and c-MYC signaling, resulting in increased aerobic glycolysis. When tested in a lymphoma mouse model, this combined approach improved NK cell antitumor activity more than either alteration alone, eradicating lymphoma xenografts without signs of any measurable toxicity. We conclude that targeting a cytokine checkpoint further enhances the antitumor activity of IL-15-secreting armored CAR-NK cells by promoting their metabolic fitness and antitumor activity. This combined approach represents a promising milestone in the development of the next generation of NK cells for cancer immunotherapy.
Assuntos
Sangue Fetal/citologia , Imunoterapia Adotiva , Interleucina-15/genética , Células Matadoras Naturais/efeitos dos fármacos , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas Supressoras da Sinalização de Citocina/antagonistas & inibidores , Aerobiose , Animais , Antígenos CD19/imunologia , Linfoma de Burkitt/patologia , Linfoma de Burkitt/terapia , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Técnicas de Inativação de Genes , Glicólise , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Interleucina-15/metabolismo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/transplante , Alvo Mecanístico do Complexo 1 de Rapamicina/fisiologia , Camundongos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Receptores de Antígenos Quiméricos , Transdução de Sinais/fisiologia , Proteínas Supressoras da Sinalização de Citocina/genética , Proteínas Supressoras da Sinalização de Citocina/fisiologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Farrerol, a dihydroflavone isolated from Rhododendron dauricum L., can inhibit vascular smooth muscle cell (VSMC) proliferation and exert a protective effect on H2O2-induced vascular endothelial cells injury. In this study, we investigated the effects of farrerol on VSMC phenotypic modulation and balloon injury-induced vascular neointimal formation and explored the underlying mechanisms. Serum-starved rat thoracic aorta SMCs (RASMCs) were first pretreated with farrerol (3, 10, and 30 µM, respectively), U0126 (a MEK kinase inhibitor), and SB203580 (a p38 kinase inhibitor), and followed by treatment with serum (10% FBS). The expression of several VSMC-specific markers, including α-SMA, SM22α, and OPN, were analyzed by western blot. Phosphorylation of extracellular signal-regulated protein kinase 1/2 (ERK 1/2) and p38 mitogen-activated protein kinase (MAPK) was also investigated. Farrerol inhibited the serum-induced transition of RASMCs from the contractile to the synthetic phenotype, and this was associated with a decrease in α-SMA and SM22α expression, and an increase in OPN expression. Farrerol also inhibited serum-induced phosphorylation of ERK1/2 and p38MAPK in RASMCs. Moreover, U0126 and SB203580 both inhibited the serum-induced phenotypic transition of RASMCs. These findings indicate that farrerol can maintain the contractile phenotype of VSMCs partly via inactivating the ERK1/2 and p38 MAPK signaling pathways. Using a rat model of carotid artery balloon injury, inhibition of VSMC phenotypic transition and suppression of neointimal formation were confirmed in vivo following the perivascular application of farrerol. Our results suggested that farrerol could be a promising lead compound for the treatment of vascular proliferative diseases.
Assuntos
Cromonas/farmacologia , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Músculo Liso Vascular/efeitos dos fármacos , Lesões do Sistema Vascular/tratamento farmacológico , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Animais , Aorta/efeitos dos fármacos , Aorta/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Masculino , Contração Muscular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Neointima/metabolismo , Fosforilação , Compostos Fitoquímicos/farmacologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Lesões do Sistema Vascular/metabolismo , Lesões do Sistema Vascular/patologiaRESUMO
Inflammation and reactive oxygen species (ROS) are important factors in the pathogenesis of atherosclerosis (AS). 5,2'-dibromo-2,4',5'-trihydroxydiphenylmethanone (TDD), possess anti-atherogenic properties; however, its underlying mechanism of action remains unclear. Therefore, we sought to understand the therapeutic molecular mechanism of TDD in inflammatory response and oxidative stress in EA.hy926 cells. Microarray analysis revealed that the expression of homeobox containing 1 (HMBOX1) was dramatically upregulated in TDD-treated EA.hy926 cells. According to the gene ontology (GO) analysis of microarray data, TDD significantly influenced the response to lipopolysaccharide (LPS); it suppressed the LPS-induced adhesion of monocytes to EA.hy926 cells. Simultaneously, TDD dose-dependently inhibited the production or expression of IL-6, IL-1ß, MCP-1, TNF-α, VCAM-1, ICAM-1 and E-selectin as well as ROS in LPS-stimulated EA.hy926 cells. HMBOX1 knockdown using RNA interference attenuated the anti-inflammatory and anti-oxidative effects of TDD. Furthermore, TDD inhibited LPS-induced NF-κB and MAPK activation in EA.hy926 cells, but this effect was abolished by HMBOX1 knockdown. Overall, these results demonstrate that TDD activates HMBOX1, which is an inducible protective mechanism that inhibits LPS-induced inflammation and ROS production in EA.hy926 cells by the subsequent inhibition of redox-sensitive NF-κB and MAPK activation. Our study suggested that TDD may be a potential novel agent for treating endothelial cells dysfunction in AS.
Assuntos
Anti-Inflamatórios/farmacologia , Proteínas de Homeodomínio/metabolismo , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Lipopolissacarídeos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Aterosclerose/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Humanos , Inflamação/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , NF-kappa B/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
The establishment of long-lived pathogen-specific T cells is a fundamental property of the adaptive immune response. However, the mechanisms underlying long-term persistence of antigen-specific CD4+ T cells are not well-defined. Here we identify a subset of memory CD4+ T cells capable of effluxing cellular toxins, including rhodamine (Rho), through the multidrug efflux protein MDR1 (also known as P-glycoprotein and ABCB1). Drug-effluxing CD4+ T cells were characterized as CD161+CD95+CD45RA-CD127hiCD28+CD25int cells with a distinct chemokine profile and a Th1-polarized pro-inflammatory phenotype. CD4+CD161+Rho-effluxing T cells proliferated vigorously in response to stimulation with anti-CD3/CD28 beads and gave rise to CD161- progeny in vitro. These cells were also capable of self-renewal and maintained their phenotypic and functional characteristics when cultured with homeostatic cytokines. Multidrug-effluxing CD4+CD161+ T cells were enriched within the viral-specific Th1 repertoire of healthy donors and patients with acute myeloid leukemia (AML) and survived exposure to daunorubicin chemotherapy in vitro. Multidrug-effluxing CD4+CD161+ T cells also resisted chemotherapy-induced cytotoxicity in vivo and underwent significant expansion in AML patients rendered lymphopenic after chemotherapy, contributing to the repopulation of anti-CMV immunity. Finally, after influenza vaccination, the proportion of influenza-specific CD4+ T cells coexpressing CD161 was significantly higher after 2 years compared with 4 weeks after immunization, suggesting CD161 is a marker for long-lived antigen-specific memory T cells. These findings suggest that CD4+CD161+ T cells with rapid efflux capacity contribute to the maintenance of viral-specific memory T cells. These data provide novel insights into mechanisms that preserve antiviral immunity in patients undergoing chemotherapy and have implications for the development of novel immunotherapeutic approaches.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Regulação Leucêmica da Expressão Gênica , Memória Imunológica , Influenza Humana/prevenção & controle , Leucemia Mieloide Aguda/imunologia , Subfamília B de Receptores Semelhantes a Lectina de Células NK/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/imunologia , Antibióticos Antineoplásicos/farmacologia , Anticorpos/farmacologia , Transporte Biológico , Antígenos CD4/genética , Antígenos CD4/imunologia , Linfócitos T CD4-Positivos/classificação , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD4-Positivos/virologia , Citomegalovirus/efeitos dos fármacos , Citomegalovirus/crescimento & desenvolvimento , Citomegalovirus/imunologia , Daunorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Imunofenotipagem , Vacinas contra Influenza/administração & dosagem , Influenza Humana/imunologia , Influenza Humana/virologia , Leucemia Mieloide Aguda/patologia , Leucemia Mieloide Aguda/virologia , Subfamília B de Receptores Semelhantes a Lectina de Células NK/imunologia , Orthomyxoviridae/efeitos dos fármacos , Orthomyxoviridae/crescimento & desenvolvimento , Orthomyxoviridae/imunologia , Rodaminas/metabolismo , Rodaminas/farmacologia , Transdução de Sinais , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Células Th1/patologiaRESUMO
Cord blood (CB) offers a number of advantages over other sources of hematopoietic stem cells, including a lower rate of chronic graft-versus-host disease (cGVHD) in the presence of increased HLA disparity. Recent research in experimental models of autoimmunity and in patients with autoimmune or alloimmune disorders has identified a functional group of interleukin-10 (IL-10)-producing regulatory B cells (Bregs) that negatively regulate T-cell immune responses. At present, however, there is no consensus on the phenotypic signature of Bregs, and their prevalence and functional characteristics in CB remain unclear. Here, we demonstrate that CB contains an abundance of B cells with immunoregulatory function. Bregs were identified in both the naive and transitional B-cell compartments and suppressed T-cell proliferation and effector function through IL-10 production as well as cell-to-cell contact involving CTLA-4. We further show that the suppressive capacity of CB-derived Bregs can be potentiated through CD40L signaling, suggesting that inflammatory environments may induce their function. Finally, there was robust recovery of IL-10-producing Bregs in patients after CB transplantation, to higher frequencies and absolute numbers than seen in the peripheral blood of healthy donors or in patients before transplant. The reconstituting Bregs showed strong in vitro suppressive activity against allogeneic CD4(+) T cells, but were deficient in patients with cGVHD. Together, these findings identify a rich source of Bregs and suggest a protective role for CB-derived Bregs against cGVHD development in CB recipients. This advance could propel the development of Breg-based strategies to prevent or ameliorate this posttransplant complication.
Assuntos
Linfócitos B Reguladores/imunologia , Transplante de Células-Tronco de Sangue do Cordão Umbilical/efeitos adversos , Sangue Fetal/imunologia , Doença Enxerto-Hospedeiro/prevenção & controle , Neoplasias Hematológicas/terapia , Interleucina-10/metabolismo , Ativação Linfocitária/imunologia , Adulto , Idoso , Linfócitos T CD4-Positivos/imunologia , Feminino , Doença Enxerto-Hospedeiro/etiologia , Neoplasias Hematológicas/complicações , Humanos , Masculino , Pessoa de Meia-Idade , Transdução de Sinais , Adulto JovemRESUMO
The ability of cord blood transplantation (CBT) to prevent relapse depends partly on donor natural killer (NK) cell alloreactivity. NK effector function depends on specific killer-cell immunoglobulin-like receptors (KIR) and HLA interactions. Thus, it is important to identify optimal combinations of KIR-HLA genotypes in donors and recipients that could improve CBT outcome. We studied clinical data, KIR and HLA genotypes, and NK-cell reconstitution in CBT patients (n = 110). Results were validated in an independent cohort (n = 94). HLA-KIR genotyping of recipient germline and transplanted cord blood (CB) grafts predicted for large differences in outcome. Patients homozygous for HLA-C2 group alleles had higher 1-year relapse rate and worse survival after CBT than did HLA-C1/C1 or HLA-C1/C2 (HLA-C1/x) patients: 67.8% vs 26.0% and 15.0% vs 52.9%, respectively. This inferior outcome was associated with delayed posttransplant recovery of NK cells expressing the HLA-C2-specific KIR2DL1/S1 receptors. HLA-C1/x patients receiving a CB graft with the combined HLA-C1-KIR2DL2/L3/S2 genotype had lower 1-year relapse rate (6.7% vs 40.1%) and superior survival (74.2% vs 41.3%) compared with recipients of grafts lacking KIR2DS2 or HLA-C1 HLA-C2/C2 patients had lower relapse rate (44.7% vs 93.4%) and better survival (30.1% vs 0%) if they received a graft with the combined HLA-C2-KIR2DL1/S1 genotype. Relapsed/refractory disease at CBT, recipient HLA-C2/C2 genotype, and donor HLA-KIR genotype were independent predictors of outcome. Thus, we propose the inclusion of KIR genotyping in graft selection criteria for CBT. HLA-C1/x patients should receive an HLA-C1-KIR2DL2/L3/S2 CB graft, while HLA-C2/C2 patients may benefit from an HLA-C2-KIR2DL1/S1 graft.
Assuntos
Transplante de Células-Tronco de Sangue do Cordão Umbilical , Genótipo , Antígenos HLA/genética , Neoplasias Hematológicas , Receptores KIR/genética , Doadores não Relacionados , Adulto , Idoso , Aloenxertos , Intervalo Livre de Doença , Feminino , Técnicas de Genotipagem , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/mortalidade , Neoplasias Hematológicas/terapia , Humanos , Masculino , Pessoa de Meia-Idade , Taxa de SobrevidaRESUMO
The past several years have seen tremendous advances in the engineering of immune effector cells as therapy for cancer. While chimeric antigen receptors (CARs) have been used extensively to redirect the specificity of autologous T cells against hematological malignancies with striking clinical results, studies of CAR-modified natural killer (NK) cells have been largely preclinical. In this review, we focus on recent advances in NK cell engineering, particularly on preclinical evidence suggesting that NK cells may be as effective as T cells in recognizing and killing targets after genetic modification. We will discuss strategies to introduce CARs into both primary NK cells and NK cell lines in an effort to provide antigen specificity, the challenges of manufacturing engineered NK cells, and evidence supporting the effectiveness of this approach from preclinical and early-phase clinical studies using CAR-engineered NK cells. CAR-NK cells hold great promise as a novel cellular immunotherapy against refractory malignancies. Notably, NK cells can provide an "off-the-shelf" product, eliminating the need for a personalized and patient-specific product that plagues current CAR-T cell therapies. The ability to more potently direct NK cell-mediated cytotoxicity against refractory tumors through the expression of CAR is likely to contribute to the recent paradigm shift in cancer treatment.
Assuntos
Engenharia Genética , Imunoterapia Adotiva , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Neoplasias/imunologia , Neoplasias/terapia , Animais , Biomarcadores , Terapia Baseada em Transplante de Células e Tecidos/métodos , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Engenharia Genética/métodos , Humanos , Imunoterapia Adotiva/efeitos adversos , Imunoterapia Adotiva/métodos , Células Matadoras Naturais/citologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Linfócitos T/imunologia , Linfócitos T/metabolismo , Pesquisa Translacional BiomédicaRESUMO
Multiple myeloma (MM) is a disease with known immune dysregulation. Natural killer (NK) cells have shown preclinical activity in MM. We conducted a first-in-human study of umbilical cord blood-derived (CB) NK cells for MM patients undergoing high dose chemotherapy and autologous haematopoietic stem cell transplantation (auto-HCT). Patients received lenalidomide (10 mg) on days -8 to -2, melphalan 200 mg/m2 on day -7, CB-NK cells on day -5 and auto-HCT on day 0. Twelve patients were enrolled, three on each of four CB-NK cell dose levels: 5 × 106 , 1 × 107 , 5 × 107 and 1 × 108 CB-NK cells/kg. Ten patients had either high-risk chromosomal changes or a history of relapsed/progressed disease. There were no infusional toxicities and no graft-versus-host disease. One patient failed to engraft due to poor autologous graft quality and was rescued with a back-up autologous graft. Overall, 10 patients achieved at least a very good partial response as their best response, including eight with near complete response or better. With a median follow-up of 21 months, four patients have progressed or relapsed, two of whom have died. CB-NK cells were detected in vivo in six patients, with an activated phenotype (NKG2D+ /NKp30+ ). These data warrant further development of this novel cellular therapy.
Assuntos
Sangue Fetal/imunologia , Transplante de Células-Tronco Hematopoéticas/métodos , Células Matadoras Naturais/transplante , Mieloma Múltiplo/terapia , Transferência Adotiva/métodos , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Separação Celular/métodos , Terapia Combinada , Feminino , Citometria de Fluxo/métodos , Sobrevivência de Enxerto , Humanos , Lenalidomida , Masculino , Melfalan/administração & dosagem , Pessoa de Meia-Idade , Talidomida/administração & dosagem , Talidomida/análogos & derivados , Resultado do TratamentoRESUMO
Adoptive transfer of T lymphocytes expressing a CD19-specific chimeric antigen receptor (CAR.CD19) induces complete tumor regression in patients with lymphoid malignancies. Although in vivo persistence of CAR-T cells correlates with clinical responses, it remains unknown whether specific cell subsets within the CAR-T-cell product correlate with their subsequent in vivo expansion and persistence. We analyzed 14 patients with B-cell malignancies infused with autologous CAR.CD19-redirected T cells expanded ex vivo using IL-2, and found that their in vivo expansion only correlated with the frequency within the infused product of a CD8(+)CD45RA(+)CCR7(+) subset, whose phenotype is closest to "T-memory stem cells." Preclinical models showed that increasing the frequency of CD8(+)CD45RA(+)CCR7(+) CAR-T cells in the infused line by culturing the cells with IL-7 and IL-15 produced greater antitumor activity of CAR-T cells mediated by increased resistance to cell death, following repetitive encounters with the antigen, while preserving their migration to secondary lymphoid organs. This trial was registered at www.clinicaltrials.gov as #NCT00586391 and #NCT00709033.
Assuntos
Células-Tronco Adultas/fisiologia , Antígenos CD19/genética , Memória Imunológica , Interleucina-15/farmacologia , Interleucina-7/farmacologia , Linfoma/terapia , Linfócitos T/fisiologia , Transferência Adotiva/métodos , Células-Tronco Adultas/efeitos dos fármacos , Células-Tronco Adultas/metabolismo , Células-Tronco Adultas/transplante , Animais , Antígenos CD19/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Terapia Genética/métodos , Humanos , Ativação Linfocitária/efeitos dos fármacos , Linfoma/genética , Linfoma/imunologia , Camundongos , Camundongos SCID , Camundongos Transgênicos , Receptores de Antígenos/genética , Receptores de Antígenos/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Linfócitos T/transplanteRESUMO
A subset of regulatory B cells (Bregs) in mice negatively regulate T-cell immune responses through the secretion of regulatory cytokines such as IL-10 and direct cell-cell contact and have been linked to experimental models of autoimmunity, inflammation, and cancer. However, the regulatory function of Bregs in human disease is much less clear. Here we demonstrate that B cells with immunoregulatory properties are enriched within both the CD19(+)IgM(+)CD27(+) memory and CD19(+)CD24(hi)CD38(hi) transitional B-cell subsets in healthy human donors. Both subsets suppressed the proliferation and interferon-γ production of CD3/CD28-stimulated autologous CD4(+) T cells in a dose-dependent manner, and both relied on IL-10 secretion as well as cell-cell contact, likely mediated through CD80 and CD86, to support their full suppressive function. Moreover, after allogeneic stem cell transplantation, Bregs from patients with chronic graft-versus-host disease (cGVHD) were less frequent and less likely to produce IL-10 than were Bregs from healthy donors and patients without cGVHD. These findings suggest that Bregs may be involved in the pathogenesis of cGVHD and support future investigation of regulatory B cell-based therapy in the treatment of this disease.
Assuntos
Subpopulações de Linfócitos B/imunologia , Linfócitos B Reguladores/imunologia , Doença Enxerto-Hospedeiro/imunologia , Imunoglobulina M/imunologia , Memória Imunológica , ADP-Ribosil Ciclase 1/metabolismo , Antígenos CD19/metabolismo , Subpopulações de Linfócitos B/metabolismo , Linfócitos B Reguladores/metabolismo , Antígeno B7-1/metabolismo , Antígeno B7-2/metabolismo , Antígeno CD24/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Estudos de Casos e Controles , Comunicação Celular , Células Cultivadas , Doença Crônica , Citocinas/biossíntese , Doença Enxerto-Hospedeiro/metabolismo , Humanos , Imunomodulação , Imunofenotipagem , Mediadores da Inflamação/metabolismo , Interleucina-10/biossíntese , Ativação Linfocitária/imunologia , Contagem de Linfócitos , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Membro 7 da Superfamília de Receptores de Fatores de Necrose TumoralRESUMO
Regulatory T cells (Tregs) play a fundamental role in the maintenance of self-tolerance and immune homeostasis. Defects in Treg function and/or frequencies have been reported in multiple disease models. Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder affecting upper and lower motor neurons. Compelling evidence supports a neuroprotective role for Tregs in this disease. Indeed, rapid progression in ALS patients is associated with decreased FoxP3 expression and Treg frequencies. Thus, we propose that strategies to restore Treg number and function may slow disease progression in ALS. In this study, we developed a robust, Good Manufacturing Practice (GMP)-compliant procedure to enrich and expand Tregs from ALS patients. Tregs isolated from these patients were phenotypically similar to those from healthy individuals but were impaired in their ability to suppress T-cell effector function. In vitro expansion of Tregs for 4 weeks in the presence of GMP-grade anti-CD3/CD28 beads, interleukin (IL)-2 and rapamcyin resulted in a 25- to 200-fold increase in their number and restored their immunoregulatory activity. Collectively, our data facilitate and support the implementation of clinical trials of adoptive therapy with ex vivo expanded and highly suppressive Tregs in patients with ALS.
Assuntos
Transferência Adotiva/normas , Esclerose Lateral Amiotrófica/patologia , Separação Celular , Terapia Baseada em Transplante de Células e Tecidos/normas , Cultura Primária de Células , Linfócitos T Reguladores/patologia , Transferência Adotiva/métodos , Esclerose Lateral Amiotrófica/imunologia , Estudos de Casos e Controles , Separação Celular/métodos , Separação Celular/normas , Terapia Baseada em Transplante de Células e Tecidos/métodos , Células Cultivadas , Fidelidade a Diretrizes/normas , Humanos , Tolerância Imunológica , Interleucina-2/metabolismo , Cultura Primária de Células/métodos , Cultura Primária de Células/normas , Linfócitos T Reguladores/imunologiaRESUMO
Activation of the inward rectifier potassium current (IK1) channel has been reported to be associated with suppression of ventricular arrhythmias. In this study, we tested the hypothesis that opening of the IK1 channel with zacopride (ZAC) was involved in the modulation of tissue repair after myocardial infarction. Sprague-Dawley rats were subject to coronary artery ligation and ZAC was administered intraperitoneally (15 µg/kg/day) for 28 days. Compared with the ischemia group, treatment with ZAC significantly reduced the ratio of heart/body weight and the cross-sectional area of cardiomyocytes, suggesting less cardiac hypertrophy. ZAC reduced the accumulation of collagen types I and III, accompanied with decrease of collagen area, which were associated with a reduction of collagen deposition in the fibrotic myocardium. Echocardiography showed improved cardiac function, evidenced by the reduced left ventricular end-diastolic dimension and left ventricular end-systolic dimension, and the increased ejection fraction and fractional shortening in ZAC-treated animals (all P < 0.05 vs. ischemia group). In coincidence with these changes, ZAC up-regulated the protein level of the IK1 channel and down-regulated the phosphorylation of mammalian target of rapamycin (mTOR) and 70-kDa ribosomal protein S6 (p70S6) kinase. Administration of chloroquine alone, an IK1 channel antagonist, had no effect on all the parameters measured, but significantly blocked the beneficial effects of ZAC on cardiac repair. In conclusion, opening of the IK1 channel with ZAC inhibits maladaptive tissue repair and improves cardiac function, potentially mediated by the inhibition of ischemia-activated mTOR-p70S6 signaling pathway via the IK1 channel. So the development of pharmacological agents specifically targeting the activation of the IK1 channel may protect the heart against myocardial ischemia-induced cardiac dysfunction.
Assuntos
Infarto do Miocárdio/fisiopatologia , Canais de Potássio Corretores do Fluxo de Internalização/fisiologia , Cicatrização , Animais , Benzamidas/farmacologia , Peso Corporal , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Colágeno/metabolismo , Hemodinâmica , Masculino , Infarto do Miocárdio/metabolismo , Tamanho do Órgão , Fosforilação , Canais de Potássio Corretores do Fluxo de Internalização/efeitos dos fármacos , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismoRESUMO
Autologous T cells expressing a CD19-specific chimeric antigen receptor (CD19.CAR) are active against B-cell malignancies, but it is unknown whether allogeneic CD19.CAR T cells are safe or effective. After allogeneic hematopoietic stem cell transplantation (HSCT), infused donor-derived virus-specific T cells (VSTs) expand in vivo, persist long term, and display antiviral activity without inducing graft-vs-host disease; therefore, we determined whether donor VSTs, engineered to express CD19.CAR, retained the characteristics of nonmanipulated allogeneic VSTs while gaining antitumor activity. We treated 8 patients with allogeneic (donor-derived) CD19.CAR-VSTs 3 months to 13 years after HSCT. There were no infusion-related toxicities. VSTs persisted for a median of 8 weeks in blood and up to 9 weeks at disease sites. Objective antitumor activity was evident in 2 of 6 patients with relapsed disease during the period of CD19.CAR-VST persistence, whereas 2 patients who received cells while in remission remain disease free. In 2 of 3 patients with viral reactivation, donor CD19.CAR-VSTs expanded concomitantly with VSTs. Hence CD19.CAR-VSTs display antitumor activity and, because their number may be increased in the presence of viral stimuli, earlier treatment post-HSCT (when lymphodepletion is greater and the incidence of viral infection is higher) or planned vaccination with viral antigens may enhance disease control.
Assuntos
Antígenos CD19/imunologia , Linfócitos B/imunologia , Transplante de Células-Tronco Hematopoéticas , Leucemia Linfocítica Crônica de Células B/terapia , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Linfócitos T/transplante , Adenoviridae/imunologia , Adulto , Antígenos CD19/genética , Antineoplásicos/uso terapêutico , Linfócitos B/patologia , Criança , Citomegalovirus/imunologia , Feminino , Expressão Gênica , Herpesvirus Humano 4/imunologia , Humanos , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/patologia , Leucemia Linfocítica Crônica de Células B/virologia , Masculino , Pessoa de Meia-Idade , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/virologia , Engenharia de Proteínas , Recidiva , Linfócitos T/imunologia , Linfócitos T/virologia , Transplante HomólogoRESUMO
BACKGROUND: Cellular therapies could play a role in cancer treatment and regenerative medicine if it were possible to quickly eliminate the infused cells in case of adverse events. We devised an inducible T-cell safety switch that is based on the fusion of human caspase 9 to a modified human FK-binding protein, allowing conditional dimerization. When exposed to a synthetic dimerizing drug, the inducible caspase 9 (iCasp9) becomes activated and leads to the rapid death of cells expressing this construct. METHODS: We tested the activity of our safety switch by introducing the gene into donor T cells given to enhance immune reconstitution in recipients of haploidentical stem-cell transplants. Patients received AP1903, an otherwise bioinert small-molecule dimerizing drug, if graft-versus-host disease (GVHD) developed. We measured the effects of AP1903 on GVHD and on the function and persistence of the cells containing the iCasp9 safety switch. RESULTS: Five patients between the ages of 3 and 17 years who had undergone stem-cell transplantation for relapsed acute leukemia were treated with the genetically modified T cells. The cells were detected in peripheral blood from all five patients and increased in number over time, despite their constitutive transgene expression. A single dose of dimerizing drug, given to four patients in whom GVHD developed, eliminated more than 90% of the modified T cells within 30 minutes after administration and ended the GVHD without recurrence. CONCLUSIONS: The iCasp9 cell-suicide system may increase the safety of cellular therapies and expand their clinical applications. (Funded by the National Heart, Lung, and Blood Institute and the National Cancer Institute; ClinicalTrials.gov number, NCT00710892.).
Assuntos
Caspase 9/genética , Genes Transgênicos Suicidas , Doença Enxerto-Hospedeiro/terapia , Imunoterapia Adotiva , Linfócitos T/transplante , Proteínas de Ligação a Tacrolimo/genética , Adolescente , Apoptose , Caspase 9/metabolismo , Criança , Pré-Escolar , Feminino , Técnicas de Transferência de Genes , Humanos , Leucemia/terapia , Masculino , Compostos Orgânicos/uso terapêutico , Recidiva , Transplante de Células-Tronco , Linfócitos T/imunologiaRESUMO
Activating IK1 channels is considered to be a promising antiarrhythmic strategy. Zacopride has been identified as a selective IK1 channel agonist and can suppress triggered arrhythmias. Whether this drug also exerts a beneficial effect on cardiac remodeling is unknown, and the present study sought to address this question. Cardiac remodeling was induced through coronary ligation-induced myocardial infarction (MI) in male Sprague-Dawley rats. Zacopride (15 µg/kg) was administered (intraperitoneally) daily for 28 days after MI to determine whether it could attenuate MI-induced cardiac remodeling. A 4-week treatment with zacopride attenuated post-MI cardiac remodeling, as shown by the reduced left ventricular end-diastolic dimension and left ventricular end-systolic dimension and the increased ejection fraction and fractional shortening in zacopride-treated animals compared with animals treated with vehicle (all P < 0.05). Furthermore, zacopride significantly decreased myocardial collagen deposition, cardiomyocyte hypertrophy, the plasma level of brain natriuretic peptide, and cardiomyocyte ultrastructural injury. Zacopride also upregulated the expression of the IK1 channel protein and downregulated the expression of phosphorylated p70S6 kinase (p-p70S6K) and mTOR. These beneficial effects of zacopride were partially abolished by the IK1 channel blocker chloroquine. We conclude that the activation of IK1 channel by zacopride attenuates post-MI cardiac remodeling by suppressing mTOR-p70S6 kinase signaling.
Assuntos
Antiarrítmicos/uso terapêutico , Benzamidas/uso terapêutico , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Infarto do Miocárdio/tratamento farmacológico , Canais de Potássio Corretores do Fluxo de Internalização/agonistas , Remodelação Ventricular/efeitos dos fármacos , Animais , Antiarrítmicos/administração & dosagem , Benzamidas/administração & dosagem , Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Cloroquina/sangue , Cloroquina/farmacologia , Ecocardiografia , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/metabolismo , Ventrículos do Coração/ultraestrutura , Masculino , Microscopia Eletrônica de Transmissão , Infarto do Miocárdio/diagnóstico por imagem , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/ultraestrutura , Canais de Potássio Corretores do Fluxo de Internalização/antagonistas & inibidores , Ratos Sprague-DawleyRESUMO
Osteoarthritis (OA) is the most prevalent degenerative musculoskeletal disease, severely impacting the function of patients and potentially leading to disability, especially among the elderly population. Natural products (NPs), obtained from components or metabolites of plants, animals, microorganisms etc., have gained significant attention as important conservative treatments for various diseases. Recently, NPs have been well studied in preclinical and clinical researches, showing promising potential in the treatment of OA. In this review, we summed up the main signaling pathways affected by NPs in OA treatment, including NF-κB, MAPKs, PI3K/AKT, SIRT1, and other pathways, which are related to inflammation, anabolism and catabolism, and cell death. In addition, we described the therapeutic effects of NPs in different OA animal models and the current clinical studies in OA patients. At last, we discussed the potential research directions including in-depth analysis of the mechanisms and new application strategies of NPs for the OA treatment, so as to promote the basic research and clinical transformation in the future. We hope that this review may allow us to get a better understanding about the potential bioeffects and mechanisms of NPs in OA therapy, and ultimately improve the effectiveness of NPs-based clinical conservative treatment for OA patients.
RESUMO
Glioblastoma (GBM) is an aggressive brain cancer with limited therapeutic options. Natural killer (NK) cells are innate immune cells with strong anti-tumor activity and may offer a promising treatment strategy for GBM. We compared the anti-GBM activity of NK cells engineered to express interleukin (IL)-15 or IL-21. Using multiple in vivo models, IL-21 NK cells were superior to IL-15 NK cells both in terms of safety and long-term anti-tumor activity, with locoregionally administered IL-15 NK cells proving toxic and ineffective at tumor control. IL-21 NK cells displayed a unique chromatin accessibility signature, with CCAAT/enhancer-binding proteins (C/EBP), especially CEBPD, serving as key transcription factors regulating their enhanced function. Deletion of CEBPD resulted in loss of IL-21 NK cell potency while its overexpression increased NK cell long-term cytotoxicity and metabolic fitness. These results suggest that IL-21, through C/EBP transcription factors, drives epigenetic reprogramming of NK cells, enhancing their anti-tumor efficacy against GBM.
Assuntos
Neoplasias Encefálicas , Proteína delta de Ligação ao Facilitador CCAAT , Glioblastoma , Interleucinas , Células Matadoras Naturais , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Glioblastoma/imunologia , Glioblastoma/genética , Glioblastoma/patologia , Glioblastoma/terapia , Interleucinas/genética , Interleucinas/metabolismo , Interleucinas/imunologia , Humanos , Animais , Camundongos , Proteína delta de Ligação ao Facilitador CCAAT/metabolismo , Proteína delta de Ligação ao Facilitador CCAAT/genética , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/terapia , Linhagem Celular Tumoral , Interleucina-15/genética , Interleucina-15/metabolismo , Interleucina-15/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
There is a pressing need for allogeneic chimeric antigen receptor (CAR)-immune cell therapies that are safe, effective and affordable. We conducted a phase 1/2 trial of cord blood-derived natural killer (NK) cells expressing anti-CD19 chimeric antigen receptor and interleukin-15 (CAR19/IL-15) in 37 patients with CD19+ B cell malignancies. The primary objectives were safety and efficacy, defined as day 30 overall response (OR). Secondary objectives included day 100 response, progression-free survival, overall survival and CAR19/IL-15 NK cell persistence. No notable toxicities such as cytokine release syndrome, neurotoxicity or graft-versus-host disease were observed. The day 30 and day 100 OR rates were 48.6% for both. The 1-year overall survival and progression-free survival were 68% and 32%, respectively. Patients who achieved OR had higher levels and longer persistence of CAR-NK cells. Receiving CAR-NK cells from a cord blood unit (CBU) with nucleated red blood cells ≤ 8 × 107 and a collection-to-cryopreservation time ≤ 24 h was the most significant predictor for superior outcome. NK cells from these optimal CBUs were highly functional and enriched in effector-related genes. In contrast, NK cells from suboptimal CBUs had upregulation of inflammation, hypoxia and cellular stress programs. Finally, using multiple mouse models, we confirmed the superior antitumor activity of CAR/IL-15 NK cells from optimal CBUs in vivo. These findings uncover new features of CAR-NK cell biology and underscore the importance of donor selection for allogeneic cell therapies. ClinicalTrials.gov identifier: NCT03056339 .
Assuntos
Transplante de Células-Tronco Hematopoéticas , Neoplasias , Receptores de Antígenos Quiméricos , Animais , Camundongos , Humanos , Receptores de Antígenos Quiméricos/genética , Interleucina-15 , Células Matadoras Naturais , Imunoterapia Adotiva/efeitos adversos , Antígenos CD19 , Proteínas Adaptadoras de Transdução de SinalRESUMO
Multiple factors in the design of a chimeric antigen receptor (CAR) influence CAR T-cell activity, with costimulatory signals being a key component. Yet, the impact of costimulatory domains on the downstream signaling and subsequent functionality of CAR-engineered natural killer (NK) cells remains largely unexplored. Here, we evaluated the impact of various costimulatory domains on CAR-NK cell activity, using a CD70-targeting CAR. We found that CD28, a costimulatory molecule not inherently present in mature NK cells, significantly enhanced the antitumor efficacy and long-term cytotoxicity of CAR-NK cells both in vitro and in multiple xenograft models of hematologic and solid tumors. Mechanistically, we showed that CD28 linked to CD3Z creates a platform that recruits critical kinases, such as LCK and ZAP70, initiating a signaling cascade that enhances CAR-NK cell function. Our study provides insights into how CD28 costimulation enhances CAR-NK cell function and supports its incorporation in NK-based CARs for cancer immunotherapy.