RESUMO
Posttransplantation cyclophosphamide (PTCy), given on days +3 and +4, reduces graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (HCT), but its immunologic underpinnings are not fully understood. In a T-cell-replete, major histocompatibility complex-haploidentical murine HCT model (B6C3F1âB6D2F1), we previously showed that PTCy rapidly induces suppressive mechanisms sufficient to prevent GVHD induction by non-PTCy-exposed donor splenocytes infused on day +5. Here, in PTCy-treated mice, we found that depleting Foxp3+ regulatory T cells (Tregs) in the initial graft but not the day +5 splenocytes did not worsen GVHD, yet depleting Tregs in both cellular compartments led to fatal GVHD induced by the day +5 splenocytes. Hence, Tregs were necessary to control GVHD induced by new donor cells, but PTCy's impact on Tregs appeared to be indirect. Therefore, we hypothesized that myeloid-derived suppressor cells (MDSCs) play a complementary role. Functionally suppressive granulocytic and monocytic MDSCs were increased in percentages in PTCy-treated mice, and MDSC percentages were increased after administering PTCy to patients undergoing HLA-haploidentical HCT. PTCy increased colony-stimulating factors critical for MDSC development and rapidly promoted the generation of MDSCs from bone marrow precursors. MDSC reduction via anti-Gr1 treatment in murine HCT did not worsen histopathologic GVHD but resulted in decreased Tregs and inferior survival. The clinical implications of these findings, including the potential impact of expanded MDSCs after PTCy on engraftment and cytokine release syndrome, remain to be elucidated. Moreover, the indirect effect that PTCy has on Tregs, which in turn play a necessary role in GVHD prevention by initially transplanted or subsequently infused T cells, requires further investigation.
Assuntos
Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Células Supressoras Mieloides , Camundongos , Animais , Células Supressoras Mieloides/patologia , Ciclofosfamida/uso terapêutico , Transplante de Células-Tronco Hematopoéticas/métodos , Doença Enxerto-Hospedeiro/patologia , Linfócitos T ReguladoresRESUMO
Relapse limits the therapeutic efficacy both of chimeric antigen receptor (CAR) T cells and allogeneic hematopoietic cell transplantation (allo-HCT). Patients may undergo these therapies sequentially to prevent or treat relapsed malignancy. However, direct integration of the 2 therapies has been avoided over concerns for potential induction of graft-versus-host disease (GVHD) by allogeneic CAR T cells. We have shown in murine T-cell-replete MHC-haploidentical allo-HCT that suppressive mechanisms induced immediately after posttransplant cyclophosphamide (PTCy), given on days +3/+4, prevent GVHD induction by alloreactive T cells infused as early as day +5. Therefore, we hypothesized that allogeneic CAR T cells given in a similarly integrated manner in our murine MHC-haploidentical allo-HCT model may safely exert antitumor effects. Indeed, allogeneic anti-CD19 CAR T cells given early after (day +5) PTCy or even prior to (day 0) PTCy cleared leukemia without exacerbating the cytokine release syndrome occurring from the MHC-haploidentical allo-HCT or interfering with PTCy-mediated GVHD prevention. Meanwhile, CAR T-cell treatment on day +9 or day +14 was safe but less effective, suggesting a limited therapeutic window. CAR T cells infused before PTCy were not eliminated, but surviving CAR T cells continued to proliferate highly and expand despite PTCy. In comparison with infusion on day +5, CAR T-cell infusion on day 0 demonstrated superior clinical efficacy associated with earlier CAR T-cell expansion, higher phenotypic CAR T-cell activation, less CD4+CD25+Foxp3+ CAR T-cell recovery, and transcriptional changes suggesting increased activation of CD4+ CAR T cells and more cytotoxic CD8+ CAR T cells. This study provides mechanistic insight into PTCy's impact on graft-versus-tumor immunity and describes novel approaches to integrate CAR T cells and allo-HCT that may compensate for deficiencies of each individual approach.
Assuntos
Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Leucemia , Humanos , Camundongos , Animais , Ciclofosfamida/farmacologia , Ciclofosfamida/uso terapêutico , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Linfócitos T CD4-Positivos/patologia , Leucemia/tratamento farmacológicoRESUMO
Efficient clearance of apoptotic cells by phagocytosis, also known as efferocytosis, is fundamental to developmental biology, organ physiology, and immunology. Macrophages use multiple mechanisms to detect and engulf apoptotic cells, but the signaling pathways that regulate the digestion of the apoptotic cell cargo, such as the dynamic Ca2+ signals, are poorly understood. Using an siRNA screen, we identify TRPM7 as a Ca2+-conducting ion channel essential for phagosome maturation during efferocytosis. Trpm7-targeted macrophages fail to fully acidify or digest their phagosomal cargo in the absence of TRPM7. Through perforated patch electrophysiology, we demonstrate that TRPM7 mediates a pH-activated cationic current necessary to sustain phagosomal acidification. Using mice expressing a genetically-encoded Ca2+ sensor, we observe that phagosome maturation requires peri-phagosomal Ca2+-signals dependent on TRPM7. Overall, we reveal TRPM7 as a central regulator of phagosome maturation during macrophage efferocytosis.
Assuntos
Sinalização do Cálcio , Fagocitose , Canais de Cátion TRPM , Animais , Macrófagos/metabolismo , Camundongos , Fagocitose/fisiologia , Fagossomos/metabolismo , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismoRESUMO
OBJECTIVE: Brown adipose tissue (BAT) is specialized in thermogenesis. The conversion of energy into heat in brown adipocytes proceeds via stimulation of ß-adrenergic receptor (ßAR)-dependent signaling and activation of mitochondrial uncoupling protein 1 (UCP1). We have previously demonstrated a functional role for pannexin-1 (Panx1) channels in white adipose tissue; however, it is not known whether Panx1 channels play a role in the regulation of brown adipocyte function. Here, we tested the hypothesis that Panx1 channels are involved in brown adipocyte activation and thermogenesis. METHODS: In an immortalized brown pre-adipocytes cell line, Panx1 currents were measured using patch-clamp electrophysiology. Flow cytometry was used for assessment of dye uptake and luminescence assays for adenosine triphosphate (ATP) release, and cellular temperature measurement was performed using a ratiometric fluorescence thermometer. We used RNA interference and expression plasmids to manipulate expression of wild-type and mutant Panx1. We used previously described adipocyte-specific Panx1 knockout mice (Panx1Adip-/-) and generated brown adipocyte-specific Panx1 knockout mice (Panx1BAT-/-) to study pharmacological or cold-induced thermogenesis. Glucose uptake into brown adipose tissue was quantified by positron emission tomography (PET) analysis of 18F-fluorodeoxyglucose (18F-FDG) content. BAT temperature was measured using an implantable telemetric temperature probe. RESULTS: In brown adipocytes, Panx1 channel activity was induced either by apoptosis-dependent caspase activation or by ß3AR stimulation via a novel mechanism that involves Gßγ subunit binding to Panx1. Inactivation of Panx1 channels in cultured brown adipocytes resulted in inhibition of ß3AR-induced lipolysis, UCP-1 expression, and cellular thermogenesis. In mice, adiponectin-Cre-dependent genetic deletion of Panx1 in all adipose tissue depots resulted in defective ß3AR agonist- or cold-induced thermogenesis in BAT and suppressed beigeing of white adipose tissue. UCP1-Cre-dependent Panx1 deletion specifically in brown adipocytes reduced the capacity for adaptive thermogenesis without affecting beigeing of white adipose tissue and aggravated diet-induced obesity and insulin resistance. CONCLUSIONS: These data demonstrate that Gßγ-dependent Panx1 channel activation is involved in ß3AR-induced thermogenic regulation in brown adipocytes. Identification of Panx1 channels in BAT as novel thermo-regulatory elements downstream of ß3AR activation may have therapeutic implications.
Assuntos
Tecido Adiposo Marrom/metabolismo , Conexinas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Termogênese/fisiologia , Adipócitos Marrons/metabolismo , Adiponectina/metabolismo , Tecido Adiposo Marrom/patologia , Tecido Adiposo Branco/metabolismo , Animais , Temperatura Baixa , Conexinas/genética , Fluordesoxiglucose F18 , Resistência à Insulina , Lipólise , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Obesidade/metabolismo , Transdução de Sinais , Termogênese/genética , TranscriptomaRESUMO
Toll-like receptors (TLRs) sense pathogen-associated molecular patterns to activate the production of inflammatory mediators. TLR4 recognizes lipopolysaccharide (LPS) and drives the secretion of inflammatory cytokines, often contributing to sepsis. We report that transient receptor potential melastatin-like 7 (TRPM7), a non-selective but Ca2+-conducting ion channel, mediates the cytosolic Ca2+ elevations essential for LPS-induced macrophage activation. LPS triggered TRPM7-dependent Ca2+ elevations essential for TLR4 endocytosis and the subsequent activation of the transcription factor IRF3. In a parallel pathway, the Ca2+ signaling initiated by TRPM7 was also essential for the nuclear translocation of NFκB. Consequently, TRPM7-deficient macrophages exhibited major deficits in the LPS-induced transcriptional programs in that they failed to produce IL-1ß and other key pro-inflammatory cytokines. In accord with these defects, mice with myeloid-specific deletion of Trpm7 are protected from LPS-induced peritonitis. Our study highlights the importance of Ca2+ signaling in macrophage activation and identifies the ion channel TRPM7 as a central component of TLR4 signaling.