RESUMEN
BACKGROUND: Understanding the host genetic architecture and viral immunity contributes to the development of effective vaccines and therapeutics for controlling the COVID-19 pandemic. Alterations of immune responses in peripheral blood mononuclear cells play a crucial role in the detrimental progression of COVID-19. However, the effects of host genetic factors on immune responses for severe COVID-19 remain largely unknown. METHODS: We constructed a computational framework to characterize the host genetics that influence immune cell subpopulations for severe COVID-19 by integrating GWAS summary statistics (N = 969,689 samples) with four independent scRNA-seq datasets containing healthy controls and patients with mild, moderate, and severe symptom (N = 606,534 cells). We collected 10 predefined gene sets including inflammatory and cytokine genes to calculate cell state score for evaluating the immunological features of individual immune cells. RESULTS: We found that 34 risk genes were significantly associated with severe COVID-19, and the number of highly expressed genes increased with the severity of COVID-19. Three cell subtypes that are CD16+monocytes, megakaryocytes, and memory CD8+T cells were significantly enriched by COVID-19-related genetic association signals. Notably, three causal risk genes of CCR1, CXCR6, and ABO were highly expressed in these three cell types, respectively. CCR1+CD16+monocytes and ABO+ megakaryocytes with significantly up-regulated genes, including S100A12, S100A8, S100A9, and IFITM1, confer higher risk to the dysregulated immune response among severe patients. CXCR6+ memory CD8+ T cells exhibit a notable polyfunctionality including elevation of proliferation, migration, and chemotaxis. Moreover, we observed an increase in cell-cell interactions of both CCR1+ CD16+monocytes and CXCR6+ memory CD8+T cells in severe patients compared to normal controls among both PBMCs and lung tissues. The enhanced interactions of CXCR6+ memory CD8+T cells with epithelial cells facilitate the recruitment of this specific population of T cells to airways, promoting CD8+T cell-mediated immunity against COVID-19 infection. CONCLUSIONS: We uncover a major genetics-modulated immunological shift between mild and severe infection, including an elevated expression of genetics-risk genes, increase in inflammatory cytokines, and of functional immune cell subsets aggravating disease severity, which provides novel insights into parsing the host genetic determinants that influence peripheral immune cells in severe COVID-19.
Asunto(s)
Linfocitos T CD8-positivos/virología , COVID-19/genética , COVID-19/patología , Monocitos/virología , Análisis de la Célula Individual/métodos , COVID-19/inmunología , Biología Computacional/métodos , Proteínas Ligadas a GPI/metabolismo , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Humanos , Células Progenitoras de Megacariocitos/inmunología , Células Progenitoras de Megacariocitos/virología , Monocitos/metabolismo , Sitios de Carácter Cuantitativo , Receptores CCR1/inmunología , Receptores CCR1/metabolismo , Receptores CXCR6/inmunología , Receptores CXCR6/metabolismo , Receptores de IgG/metabolismo , Análisis de Secuencia de ARN , Índice de Severidad de la EnfermedadRESUMEN
Radical surgery still represents the treatment choice for several malignancies. However, local and distant tumor relapses remain the major causes of treatment failure, indicating that a postsurgery consolidation treatment is necessary. Immunotherapy with checkpoint inhibitors has elicited impressive clinical responses in several types of human malignancies and may represent the ideal consolidation treatment after surgery. Here, we genetically engineered platelets from megakaryocyte (MK) progenitor cells to express the programmed cell death protein 1 (PD-1). The PD-1 platelet and its derived microparticle could accumulate within the tumor surgical wound and revert exhausted CD8+ T cells, leading to the eradication of residual tumor cells. Furthermore, when a low dose of cyclophosphamide (CP) was loaded into PD-1-expressing platelets to deplete regulatory T cells (Tregs), an increased frequency of reinvigorated CD8+ lymphocyte cells was observed within the postsurgery tumor microenvironment, directly preventing tumor relapse.
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Plaquetas/inmunología , Ingeniería Genética/métodos , Inmunoterapia/métodos , Melanoma/terapia , Receptor de Muerte Celular Programada 1/inmunología , Animales , Plaquetas/metabolismo , Linfocitos T CD8-positivos/inmunología , Línea Celular , Ciclofosfamida/administración & dosificación , Ciclofosfamida/uso terapéutico , Expresión Génica , Células HEK293 , Humanos , Inmunosupresores/administración & dosificación , Inmunosupresores/uso terapéutico , Células Progenitoras de Megacariocitos/inmunología , Células Progenitoras de Megacariocitos/metabolismo , Melanoma/inmunología , Ratones Endogámicos C57BL , Receptor de Muerte Celular Programada 1/genéticaRESUMEN
Stem cell-derived platelets have the potential to replace donor platelets for transfusion. Defining the platelet-producing megakaryocytes (MKs) within the heterogeneous MK culture may help to optimize the in vitro generation of platelets. Using 2 human stem cell models of megakaryopoiesis, we identified novel MK populations corresponding to distinct maturation stages. An immature, low granular (LG) MK pool (defined by side scatter on flow cytometry) gives rise to a mature high granular (HG) pool, which then becomes damaged by apoptosis and glycoprotein Ib α chain (CD42b) shedding. We define an undamaged HG/CD42b+ MK subpopulation, which endocytoses fluorescently labeled coagulation factor V (FV) from the media into α-granules and releases functional FV+CD42b+ human platelet-like particles in vitro and when infused into immunodeficient mice. Importantly, these FV+ particles have the same size distribution as infused human donor platelets and are preferentially incorporated into clots after laser injury. Using drugs to protect HG MKs from apoptosis and CD42b shedding, we also demonstrate that apoptosis precedes CD42b shedding and that apoptosis inhibition enriches the FV+ HG/CD42b+ MKs, leading to increased platelet yield in vivo, but not in vitro. These studies identify a transition between distinct MK populations in vitro, including one that is primed for platelet release. Technologies to optimize and select these platelet-ready MKs may be important to efficiently generate functional platelets from in vitro-grown MKs.
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Plaquetas/citología , Células de la Médula Ósea/inmunología , Factor V/genética , Células Progenitoras de Megacariocitos/citología , Megacariocitos/citología , Animales , Apoptosis/efectos de los fármacos , Arteriolas/efectos de los fármacos , Arteriolas/inmunología , Arteriolas/lesiones , Biomarcadores/sangre , Plaquetas/inmunología , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Diferenciación Celular , Linaje de la Célula/inmunología , Endocitosis , Factor V/inmunología , Factor V/farmacología , Citometría de Flujo , Expresión Génica , Humanos , Inmunofenotipificación , Rayos Láser , Células Progenitoras de Megacariocitos/inmunología , Megacariocitos/inmunología , Ratones , Ratones SCID , Complejo GPIb-IX de Glicoproteína Plaquetaria/genética , Complejo GPIb-IX de Glicoproteína Plaquetaria/inmunologíaRESUMEN
Platelets, like stromal cells, present antigen only via MHC class I, but the immune potential of their progenitors has not been explored in humans. We derived CD34(+)CD117(+)CD41(+)CD151(+) megakaryocyte progenitors (MKp) in vitro from mobilized peripheral blood hematopoietic stem and progenitor cells (HSPC) of normal subjects using culture conditions akin to bone marrow niche, or organs that support extramedullary hematopoiesis. The MKp expressed MHC Class II in contrast to platelets and functioned as professional APC before they matured further. Moreover, MKp constitutively expressed mRNA encoding mediators for human Th17 expansion, including IL-1, IL-18, IL-6, TGFß, IL-23, BAFF, and COX2. MKp also expressed high levels of type I interferon and IRF5 mRNA. In contrast to platelets, MKp augmented the expansion of Th17, Th1, and potent Th17/Th1 double-positive cells in normal PBMC and CD4 line T cells from normal subjects or lupus patients. The Th cell augmentation involved pre-committed memory cells, and was significant although modest, because only non-cognate MKp-T cell interactions could be studied, under non-polarizing conditions. Importantly, the MKp-mediated expansion was observed in the presence or absence of direct MKp-T cell contact. Furthermore, MKp augmented Th17 responses against Candida albicans, a serious opportunistic pathogen. These results indicate an immunologic role of MKp in situations associated with extramedullary hematopoiesis and mobilization of HSPC.
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Células Presentadoras de Antígenos/inmunología , Regulación de la Expresión Génica , Antígenos de Histocompatibilidad Clase II/inmunología , Células Progenitoras de Megacariocitos/inmunología , Células TH1/inmunología , Células Th17/inmunología , Células Presentadoras de Antígenos/citología , Antígenos CD/inmunología , Plaquetas/citología , Plaquetas/inmunología , Comunicación Celular/inmunología , Citocinas/inmunología , Humanos , Células Progenitoras de Megacariocitos/citología , Células TH1/citología , Células Th17/citologíaRESUMEN
In search of autoantigen-presenting cells that prime the pathogenic autoantibody-inducing Th cells of lupus, we found that CD41(+)CD151(+) cells among Lineage(-) (Lin(-)) CD117(+) (c-Kit(+)) CX3CR1(-) splenocytes depleted of known APCs were most proficient in presenting nuclear autoantigens from apoptotic cells to induce selectively an autoimmune Th17 response in different lupus-prone mouse strains. The new APCs have properties resembling megakaryocyte and/or bipotent megakaryocyte/erythroid progenitors of bone marrow, hence they are referred to as MM cells in this study. The MM cells produce requisite cytokines, but they require contact for optimal Th17 induction upon nucleosome feeding, and can induce Th17 only before undergoing differentiation to become c-Kit(-)CD41(+) cells. The MM cells expand up to 10-fold in peripheral blood of lupus patients and 49-fold in spleens of lupus mice preceding disease activity; they accelerate lupus in vivo and break tolerance in normal mice, inducing autoimmune Th17 cells. MM cells also cause Th17 skewing to foreign Ag in normal mice without Th17-polarizing culture conditions. Several molecules in MM cells are targets for blocking of autoimmunization. This study advances our understanding of lupus pathogenesis and Th17 differentiation biology by characterizing a novel category of APC.
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Células Presentadoras de Antígenos/inmunología , Lupus Eritematoso Sistémico/inmunología , Activación de Linfocitos/inmunología , Células Progenitoras de Megacariocitos/inmunología , Células Th17/inmunología , Adulto , Animales , Presentación de Antígeno/inmunología , Autoanticuerpos/inmunología , Autoantígenos/inmunología , Diferenciación Celular/inmunología , Separación Celular , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Perfilación de la Expresión Génica , Humanos , Ratones , Ratones Mutantes , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Th17/citologíaRESUMEN
BACKGROUND: The multifunctional protein semaphorin 7A (Sema7A) may have regulatory effects on blood cell differentiation via its receptors ß1-integrin and plexin C1. As thrombocytopenia can be treated with transfusion of ex vivo CD34(+) cell-derived megakaryocytes, we investigated the effect of Sema7A on differentiation of CD34(+) progenitor cells into megakaryocytes and platelets. METHODS: Megakaryocytes and platelets were differentiated with a specific cytokine cocktail (CC) from CD34(+) progenitor cells in the presence or absence of Sema7A. Expression of cell markers CD41, CD42a and CD61 or detection of the activation of the signal mediator focal adhesion kinase (FAK) was performed by flow cytometry, cytokine secretion by Luminex technology, and megakaryocyte cell density and morphology by microscopic studies. Sema7A levels in vivo were assessed by real-time PCR and ELISA in hematological patients undergoing chemotherapy. RESULTS: CD34(+) progenitor cells expressed the receptors for Sema7A. Expression of CD41, CD42a and CD61 was markedly reduced in the presence of Sema7A, after CC-dependent platelet production from CD34(+) progenitor cells. As revealed by microscopic analysis, megakaryocyte cell density was significantly lower in the presence of Sema7A as compared with controls. Blocking of CD29 abrogated the Sema7A-mediated inhibition. Sema7A activated FAK in CD34(+) progenitor cells and significantly increased secretion of the proinflammatory cytokines IL-6, IL-8 and GM-CSF. Finally, Sema7A levels were up-regulated in 50% of patients after chemotherapy. CONCLUSIONS: Sema7A markedly reduces the production rates of megakaryocytes and platelets from CD34(+) progenitor cells. Hence, up-regulation of Sema7A may be a major risk factor for a reduced platelet repopulation after hematopoietic stem cell transplantation.
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Antígenos CD34/metabolismo , Antígenos CD/metabolismo , Plaquetas/metabolismo , Diferenciación Celular , Células Progenitoras de Megacariocitos/metabolismo , Megacariocitos/metabolismo , Semaforinas/metabolismo , Anticuerpos , Antígenos CD/genética , Antineoplásicos/farmacología , Biomarcadores/metabolismo , Plaquetas/efectos de los fármacos , Plaquetas/inmunología , Diferenciación Celular/efectos de los fármacos , Separación Celular/métodos , Células Cultivadas , Citocinas/metabolismo , Activación Enzimática , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Quinasa 1 de Adhesión Focal/metabolismo , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Regulación de la Expresión Génica , Humanos , Mediadores de Inflamación/metabolismo , Integrina beta1/inmunología , Integrina beta1/metabolismo , Integrina beta3/metabolismo , Células Progenitoras de Megacariocitos/efectos de los fármacos , Células Progenitoras de Megacariocitos/inmunología , Megacariocitos/efectos de los fármacos , Megacariocitos/inmunología , Fosforilación , Complejo GPIb-IX de Glicoproteína Plaquetaria/metabolismo , Glicoproteína IIb de Membrana Plaquetaria/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Semaforinas/genéticaRESUMEN
The purpose of this study was to explore the differences in megakaryocyte progenitor ex vivo expansion between CD34+ cells derived from human umbilical cord blood (CB) and bone marrow (BM). Mononuclear cells (MNCs) were obtained from CB or BM by Ficoll-Hypaque density gradient separation. CD34+ cells were purified by magnetic cell sorting (MACS). The selected CD34+ cells were seeded in serum-free conditions stimulated with thrombopoietin (TPO), TPO+interleukin 11 (IL-11), or TPO+IL11+heparin for 14 days. Amplification product (CD34+, CD41a+, and CD34+ CD41a+ cells) immunophenotypes, megakaryocyte apoptosis rates and the DNA content were measured by fluorescence-activated cell sorting (FACS). The colony-forming units of granulocytes and monocytes (CFU-GM), burst-forming units of erythrocytes (BFU-E), and colony-forming units of megakaryocytes (CFU-Mk) were also evaluated by the colony-forming units (CFU) assay. The results indicated that CD34+ cells derived from CB showed higher expansion ability of total cell counts, CD41a+ and CD34+ CD41a+ cells than those derived from BM for all days 14 of culture (p<0.05, respectively). There were no significant differences in CFU-GM, BFU-E, and total CFU-Mk counts between CB and BM-derived CD34+ cells on day 0 (p>0.05, respectively), but CB-derived CFU-Mk seemed mainly large colonies, and the number of large colonies was higher than that from BM (p<0.05) on day 0. There were no significant differences in expansion ability of CFU-GM between CB and BM-derived cells on days 7, 10, and 14 of culture (p > 0.05, respectively), but the expansion ability of BFU-E and CFU-Mk derived from CB cells was higher than that from BM (p<0.05, respectively). There were no significant differences in apoptosis rates of megakaryocyte from two source cells for days 14 of culture. Megakaryocytes derived from CB mostly showed the 2N DNA content (>90%) for days 14 of culture, while those cells derived from BM showed the increased DNA content, and 4N, 8N or more ploidy cells gradually increased with prolonging of culture time. It is concluded that CB-derived CD34+ cells have a greater proliferation potential than that derived from BM, which is therefore proven to be a better cell source for megakaryocyte progenitor expansion in vitro.