RESUMEN
There is pressing urgency to understand the pathogenesis of the severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2), which causes the disease COVID-19. SARS-CoV-2 spike (S) protein binds angiotensin-converting enzyme 2 (ACE2), and in concert with host proteases, principally transmembrane serine protease 2 (TMPRSS2), promotes cellular entry. The cell subsets targeted by SARS-CoV-2 in host tissues and the factors that regulate ACE2 expression remain unknown. Here, we leverage human, non-human primate, and mouse single-cell RNA-sequencing (scRNA-seq) datasets across health and disease to uncover putative targets of SARS-CoV-2 among tissue-resident cell subsets. We identify ACE2 and TMPRSS2 co-expressing cells within lung type II pneumocytes, ileal absorptive enterocytes, and nasal goblet secretory cells. Strikingly, we discovered that ACE2 is a human interferon-stimulated gene (ISG) in vitro using airway epithelial cells and extend our findings to in vivo viral infections. Our data suggest that SARS-CoV-2 could exploit species-specific interferon-driven upregulation of ACE2, a tissue-protective mediator during lung injury, to enhance infection.
Asunto(s)
Células Epiteliales Alveolares/metabolismo , Enterocitos/metabolismo , Células Caliciformes/metabolismo , Interferón Tipo I/metabolismo , Mucosa Nasal/citología , Peptidil-Dipeptidasa A/genética , Adolescente , Células Epiteliales Alveolares/inmunología , Enzima Convertidora de Angiotensina 2 , Animales , Betacoronavirus/fisiología , COVID-19 , Línea Celular , Células Cultivadas , Niño , Infecciones por Coronavirus/virología , Enterocitos/inmunología , Células Caliciformes/inmunología , Infecciones por VIH/inmunología , Humanos , Gripe Humana/inmunología , Interferón Tipo I/inmunología , Pulmón/citología , Pulmón/patología , Macaca mulatta , Ratones , Mycobacterium tuberculosis , Mucosa Nasal/inmunología , Pandemias , Peptidil-Dipeptidasa A/metabolismo , Neumonía Viral/virología , Receptores Virales/genética , SARS-CoV-2 , Serina Endopeptidasas/metabolismo , Análisis de la Célula Individual , Tuberculosis/inmunología , Regulación hacia ArribaRESUMEN
Chimeric antigen receptor (CAR) T cells targeting CD19+ hematologic malignancies have rapidly emerged as a promising, novel therapy. In contrast, results from the few CAR T-cell studies for infectious diseases such as HIV-1 have been less convincing. These challenges are likely due to the low level of antigen present in antiretroviral therapy (ART)-suppressed patients in contrast to those with hematologic malignancies. Using our well-established nonhuman primate model of ART-suppressed HIV-1 infection, we tested strategies to overcome these limitations and challenges. We first optimized CAR T-cell production to maintain central memory subsets, consistent with current clinical paradigms. We hypothesized that additional exogenous antigen might be required in an ART-suppressed setting to aid expansion and persistence of CAR T cells. Thus, we studied 4 simian/HIV-infected, ART-suppressed rhesus macaques infused with virus-specific CD4CAR T cells, followed by supplemental infusion of cell-associated HIV-1 envelope (Env). Env boosting led to significant and unprecedented expansion of virus-specific CAR+ T cells in vivo; after ART treatment interruption, viral rebound was significantly delayed compared with controls (P = .014). In 2 animals with declining CAR T cells, rhesusized anti-programmed cell death protein 1 (PD-1) antibody was administered to reverse PD-1-dependent immune exhaustion. Immune checkpoint blockade triggered expansion of exhausted CAR T cells and concordantly lowered viral loads to undetectable levels. These results show that supplemental cell-associated antigen enables robust expansion of CAR T cells in an antigen-sparse environment. To our knowledge, this is the first study to show expansion of virus-specific CAR T cells in infected, suppressed hosts, and delay/control of viral recrudescence.
Asunto(s)
Antígenos Virales/inmunología , Infecciones por VIH/inmunología , VIH-1/inmunología , Huésped Inmunocomprometido , Receptores de Antígenos de Linfocitos T/inmunología , Receptores Quiméricos de Antígenos/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Animales , Terapia Antirretroviral Altamente Activa/efectos adversos , Terapia Antirretroviral Altamente Activa/métodos , Modelos Animales de Enfermedad , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , Inhibidores de Puntos de Control Inmunológico/farmacología , Proteínas de Punto de Control Inmunitario/genética , Proteínas de Punto de Control Inmunitario/metabolismo , Macaca mulatta , Virus de la Inmunodeficiencia de los Simios/inmunología , Linfocitos T/efectos de los fármacosRESUMEN
Lupus nephritis (LN) is a major contributor to morbidity and mortality in lupus patients, but the mechanisms of kidney damage remain unclear. In this study, we introduce, to our knowledge, novel models of LN designed to resemble the polygenic nature of human lupus by embodying three key genetic alterations: the Sle1 interval leading to anti-chromatin autoantibodies; Mfge8-/- , leading to defective clearance of apoptotic cells; and either C1q-/- or C3-/- , leading to low complement levels. We report that proliferative glomerulonephritis arose only in the presence of all three abnormalities (i.e., in Sle1.Mfge8 -/- C1q -/- and Sle1.Mfge8 -/- C3 -/- triple-mutant [TM] strains [C1q -/-TM and C3-/- TM, respectively]), with structural kidney changes resembling those in LN patients. Unexpectedly, both TM strains had significant increases in autoantibody titers, Ag spread, and IgG deposition in the kidneys. Despite the early complement component deficiencies, we observed assembly of the pathogenic terminal complement membrane attack complex in both TM strains. In C1q-/- TM mice, colocalization of MASP-2 and C3 in both the glomeruli and tubules indicated that the lectin pathway likely contributed to complement activation and tissue injury in this strain. Interestingly, enhanced thrombin activation in C3-/- TM mice and reduction of kidney injury following attenuation of thrombin generation by argatroban in a serum-transfer nephrotoxic model identified thrombin as a surrogate pathway for complement activation in C3-deficient mice. These novel mouse models of human lupus inform the requirements for nephritis and provide targets for intervention.
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Enfermedades por Deficiencia de Complemento Hereditario/genética , Riñón/patología , Nefritis Lúpica/inmunología , Animales , Anticuerpos Antinucleares/sangre , Antígenos de Superficie/genética , Activación de Complemento , Complemento C1q/genética , Complemento C3/genética , Modelos Animales de Enfermedad , Glomerulonefritis , Enfermedades por Deficiencia de Complemento Hereditario/inmunología , Humanos , Nefritis Lúpica/genética , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de la Leche/genética , Herencia MultifactorialRESUMEN
Adoptive transfer of freshly isolated natural occurring CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg) prevents graft-versus-host disease (GVHD) in several animal models and following hematopoietic cell transplantation (HCT) in clinical trials. Donor-derived Treg have been mainly used, as they share the same MHC with CD4(+) and CD8(+) conventional T cells (Tcon) that are primarily responsible for GVHD. Third party-derived Treg are a promising alternative for cellular therapy, as they can be prepared in advance, screened for pathogens and activity, and banked. We explored MHC disparities between Treg and Tcon in HCT to evaluate the impact of different Treg populations in GVHD prevention and survival. Third-party Treg and donor Treg are equally suppressive in ex vivo assays, whereas both donor and third-party but not host Treg protect from GVHD in allogeneic HCT, with donor Treg being the most effective. In an MHC minor mismatched transplantation model (C57BL/6 â BALB/b), donor and third-party Treg were equally effective in controlling GVHD. Furthermore, using an in vivo Treg depletion mouse model, we found that Treg exert their main suppressive activity in the first 2 d after transplantation. Third-party Treg survive for a shorter period of time after adoptive transfer, but despite the shorter survival, they control Tcon proliferation in the early phases of HCT. These studies provide relevant insights on the mechanisms of Treg-mediated protection from GVHD and support for the use of third-party Treg in clinical trials.
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Enfermedad Injerto contra Huésped/prevención & control , Trasplante de Células Madre Hematopoyéticas , Linfocitos T Reguladores/inmunología , Traslado Adoptivo , Animales , Biomarcadores/sangre , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Proliferación Celular , Factores de Transcripción Forkhead/sangre , Expresión Génica , Enfermedad Injerto contra Huésped/inmunología , Enfermedad Injerto contra Huésped/patología , Prueba de Histocompatibilidad , Subunidad alfa del Receptor de Interleucina-2/sangre , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/trasplante , Donantes de Tejidos , Trasplante Homólogo , Irradiación Corporal TotalRESUMEN
Microglia play an important role in receptor-mediated phagocytosis in the CNS. In brain abscess and other CNS infections, invading bacteria undergo opsonization with Igs or complement. Microglia recognize these opsonized pathogens by Fc or complement receptors triggering phagocytosis. In this study, we investigated the role of Fcα/µR, the less-studied receptor for IgM and IgA, in microglial phagocytosis. We showed that primary microglia, as well as N9 microglial cells, express Fcα/µR. We also showed that anti-Staphylococcus aureus IgM markedly increased the rate of microglial S. aureus phagocytosis. To unequivocally test the role of Fcα/µR in IgM-mediated phagocytosis, we performed experiments in microglia from Fcα/µR(-/-) mice. Surprisingly, we found that IgM-dependent phagocytosis of S. aureus was similar in microglia derived from wild-type or Fcα/µR(-/-) mice. We hypothesized that IgM-dependent activation of complement receptors might contribute to the IgM-mediated increase in phagocytosis. To test this, we used immunologic and genetic inactivation of complement receptor 3 components (CD11b and CD18) as well as C3. IgM-, but not IgG-mediated phagocytosis of S. aureus was reduced in wild-type microglia and macrophages following preincubation with an anti-CD11b blocking Ab. IgM-dependent phagocytosis of S. aureus was also reduced in microglia derived from CD18(-/-) and C3(-/-) mice. Taken together, our findings implicate complement receptor 3 and C3, but not Fcα/µR, in IgM-mediated phagocytosis of S. aureus by microglia.
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Complemento C3/inmunología , Inmunoglobulina M/inmunología , Antígeno de Macrófago-1/inmunología , Microglía/inmunología , Fagocitosis/inmunología , Animales , Anticuerpos Bloqueadores/farmacología , Antígeno CD11b/inmunología , Antígenos CD18/genética , Antígenos CD18/inmunología , Línea Celular , Complemento C3/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores Fc/biosíntesis , Receptores Fc/genética , Receptores Fc/inmunología , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/inmunologíaRESUMEN
Complement activation contributes to inflammation in many diseases, yet it also supports physiologic apoptotic cells (AC) clearance and its downstream immunosuppressive effects. The roles of individual complement components in AC phagocytosis have been difficult to dissect with artificially depleted sera. Using human in vitro systems and the novel antibody complement C1s inhibitor TNT003, we uncoupled the role of the enzymatic activation of the classical pathway from the opsonizing role of C1q in mediating a) the phagocytosis of early and late AC, and b) the immunosuppressive capacity of early AC. We found that C1s inhibition had a small impact on the physiologic clearance of early AC, leaving their immunosuppressive properties entirely unaffected, while mainly inhibiting the phagocytosis of late apoptotic/secondary necrotic cells. Our data suggest that C1s inhibition may represent a valuable therapeutic strategy to control classical pathway activation without causing significant AC accumulation in diseases without defects in AC phagocytosis.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Apoptosis/efectos de los fármacos , Activación de Complemento/efectos de los fármacos , Complemento C1q/efectos de los fármacos , Complemento C1s/antagonistas & inhibidores , Citocinas/efectos de los fármacos , Citofagocitosis/efectos de los fármacos , Tolerancia Inmunológica/efectos de los fármacos , Macrófagos/efectos de los fármacos , Apoptosis/inmunología , Activación de Complemento/inmunología , Complemento C1q/inmunología , Complemento C1q/metabolismo , Complemento C1s/metabolismo , Complemento C3b/efectos de los fármacos , Complemento C3b/inmunología , Complemento C3b/metabolismo , Citocinas/inmunología , Citofagocitosis/inmunología , Humanos , Técnicas In Vitro , Interleucina-1beta/efectos de los fármacos , Interleucina-1beta/inmunología , Interleucina-6/inmunología , Células Jurkat , Macrófagos/inmunología , Fagocitosis/efectos de los fármacos , Fagocitosis/inmunología , Factor de Necrosis Tumoral alfa/efectos de los fármacos , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
PURPOSE OF REVIEW: Systemic lupus erythematosus (SLE) is characterized by autoantibodies directed against nuclear autoantigens normally concealed from immune recognition in healthy individuals. Here, we summarize recently identified mechanisms of abnormal cell death leading to exposure and aberrant processing of nucleoprotein self antigens, and discuss their role in the SLE pathogenesis. RECENT FINDINGS: During the past few years, the unveiling of several new forms of cell death has expanded our understanding beyond the simple view of 'apoptotic' versus 'necrotic' cell death. SLE patients show abnormalities in cell death at several levels, including increased rates of apoptosis, necrosis, and autophagy, as well as reduced clearance of dying cells. These abnormalities lead to an increased autoantigen burden and antigen modifications, such as nucleic acid oxidation that increases the inflammatory properties of self antigens. Recent investigations have highlighted the role of opsonins in determining the immunogenic versus tolerogenic characteristics of self antigens. SUMMARY: Dysregulation of different forms of programmed cell death contributes to increased exposure, availability, and immunogenic characteristics of intracellular self antigens, which all participate in development of lupus autoimmunity. As our understanding of abnormalities of cell death in SLE advances, potential therapeutic opportunities await human implementation.
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Apoptosis/inmunología , Muerte Celular/inmunología , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/patología , Apoptosis/genética , Autofagia/genética , Autofagia/inmunología , Muerte Celular/genética , Humanos , Lupus Eritematoso Sistémico/genética , MicroARNs/genética , Modelos Biológicos , Necrosis , Proteínas Opsoninas/sangre , Receptores de Superficie Celular/inmunologíaRESUMEN
Regulatory T cell (Treg) immunotherapy is a promising strategy for the treatment of graft rejection responses and autoimmune disorders. Our and other laboratories have shown that the transfer of highly purified CD4(+)CD25(+)Foxp3(+) natural Treg can prevent lethal graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation across both major and minor histocompatibility barriers. However, recent evidence suggests that the Treg suppressive phenotype can become unstable, a phenomenon that can culminate in Treg conversion into IL-17-producing cells. We hypothesized that the intense proinflammatory signals released during an ongoing alloreaction might redirect a fraction of the transferred Treg to the Th17 cell fate, thereby losing immunosuppressive potential. We therefore sought to evaluate the impact of Il17 gene ablation on Treg stability and immunosuppressive capacity in a major MHC mismatch model. We show that although Il17 gene ablation results in a mildly enhanced Treg immunosuppressive ability in vitro, such improvement is not observed when IL-17-deficient Treg are used for GVHD suppression in vivo. Similarly, when we selectively blocked IL-1 signaling in Treg, that was shown to be necessary for Th17 conversion, we did not detect any improvement on Treg-mediated GVHD suppressive ability in vivo. Furthermore, upon ex vivo reisolation of transferred wild-type Treg, we detected little or no Treg-mediated IL-17 production upon GVHD induction. Our results indicate that blocking Th17 conversion does not affect the GVHD suppressive ability of highly purified natural Treg in vivo, suggesting that IL-17 targeting is not a valuable strategy to improve Treg immunotherapy after hematopoietic cell transplantation.
Asunto(s)
Trasplante de Médula Ósea/métodos , Enfermedad Injerto contra Huésped/inmunología , Inmunoterapia Adoptiva/métodos , Interleucina-17/genética , Linfocitos T Reguladores/trasplante , Animales , Enfermedad Injerto contra Huésped/genética , Humanos , Interleucina-17/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Linfocitos T Reguladores/inmunología , Células Th17/inmunologíaRESUMEN
Notch signaling promotes T cell pathogenicity and graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (allo-HCT) in mice, with a dominant role for the Delta-like Notch ligand DLL4. To assess whether Notch's effects are evolutionarily conserved and to identify the mechanisms of Notch signaling inhibition, we studied antibody-mediated DLL4 blockade in a nonhuman primate (NHP) model similar to human allo-HCT. Short-term DLL4 blockade improved posttransplant survival with durable protection from gastrointestinal GVHD in particular. Unlike prior immunosuppressive strategies tested in the NHP GVHD model, anti-DLL4 interfered with a T cell transcriptional program associated with intestinal infiltration. In cross-species investigations, Notch inhibition decreased surface abundance of the gut-homing integrin α4ß7 in conventional T cells while preserving α4ß7 in regulatory T cells, with findings suggesting increased ß1 competition for α4 binding in conventional T cells. Secondary lymphoid organ fibroblastic reticular cells emerged as the critical cellular source of Delta-like Notch ligands for Notch-mediated up-regulation of α4ß7 integrin in T cells after allo-HCT. Together, DLL4-Notch blockade decreased effector T cell infiltration into the gut, with increased regulatory to conventional T cell ratios early after allo-HCT. Our results identify a conserved, biologically unique, and targetable role of DLL4-Notch signaling in intestinal GVHD.
Asunto(s)
Enfermedad Injerto contra Huésped , Trasplante de Células Madre Hematopoyéticas , Ratones , Humanos , Animales , Trasplante Homólogo , Receptores Notch/metabolismo , Transducción de Señal , Enfermedad Injerto contra Huésped/metabolismo , PrimatesRESUMEN
In APCs, the protein tyrosine kinase Syk is required for signaling of several immunoreceptors, including the BCR and FcR. We show that conditional ablation of the syk gene in dendritic cells (DCs) abrogates FcgammaR-mediated cross priming of diabetogenic T cells in RIP-mOVA mice, a situation phenocopied in wild-type RIP-mOVA mice treated with the selective Syk inhibitor R788. In addition to blocking FcgammaR-mediated events, R788 also blocked BCR-mediated Ag presentation, thus broadly interrupting the humoral contributions to T cell-driven autoimmunity. Indeed, oral administration of R788 significantly delayed spontaneous diabetes onset in NOD mice and successfully delayed progression of early-established diabetes even when treatment was initiated after the development of glucose intolerance. At the DC level, R788 treatment was associated with reduced insulin-specific CD8 priming and decreased DC numbers. At the B cell level, R788 reduced total B cell numbers and total Ig concentrations. Interestingly, R788 increased the number of IL-10-producing B cells, thus inducing a tolerogenic B cell population with immunomodulatory activity. Taken together, we show by genetic and pharmacologic approaches that Syk in APCs is an attractive target in T cell-mediated autoimmune diseases such as type 1 diabetes.
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Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/terapia , Marcación de Gen/métodos , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Tirosina Quinasas/genética , Aminopiridinas , Animales , Reactividad Cruzada/genética , Reactividad Cruzada/inmunología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Células Dendríticas/patología , Diabetes Mellitus Tipo 1/enzimología , Femenino , Inmunoglobulina G/fisiología , Inmunoglobulina M/fisiología , Insulina/inmunología , Insulina/metabolismo , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Péptidos y Proteínas de Señalización Intracelular/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Ratones Transgénicos , Morfolinas , Ovalbúmina/genética , Ovalbúmina/inmunología , Oxazinas/uso terapéutico , Proteínas Tirosina Quinasas/deficiencia , Proteínas Tirosina Quinasas/fisiología , Piridinas/uso terapéutico , Pirimidinas , Receptores de Antígenos de Linfocitos B/antagonistas & inhibidores , Receptores de Antígenos de Linfocitos B/fisiología , Receptores de IgG/antagonistas & inhibidores , Receptores de IgG/fisiología , Quinasa SykRESUMEN
Organ infiltration by donor T cells is critical to the development of acute graft-versus-host disease (aGVHD) in recipients after allogeneic hematopoietic stem cell transplant (allo-HCT). However, deconvoluting the transcriptional programs of newly recruited donor T cells from those of tissue-resident T cells in aGVHD target organs remains a challenge. Here, we combined the serial intravascular staining technique with single-cell RNA sequencing to dissect the tightly connected processes by which donor T cells initially infiltrate tissues and then establish a pathogenic tissue residency program in a rhesus macaque allo-HCT model that develops aGVHD. Our results enabled creation of a spatiotemporal map of the transcriptional programs controlling donor CD8+ T cell infiltration into the primary aGVHD target organ, the gastrointestinal (GI) tract. We identified the large and small intestines as the only two sites demonstrating allo-specific, rather than lymphodepletion-driven, T cell infiltration. GI-infiltrating donor CD8+ T cells demonstrated a highly activated, cytotoxic phenotype while simultaneously developing a canonical tissue-resident memory T cell (TRM) transcriptional signature driven by interleukin-15 (IL-15)/IL-21 signaling. We found expression of a cluster of genes directly associated with tissue invasiveness, including those encoding adhesion molecules (ITGB2), specific chemokines (CCL3 and CCL4L1) and chemokine receptors (CD74), as well as multiple cytoskeletal proteins. This tissue invasion transcriptional signature was validated by its ability to discriminate the CD8+ T cell transcriptome of patients with GI aGVHD from those of GVHD-free patients. These results provide insights into the mechanisms controlling tissue occupancy of target organs by pathogenic donor CD8+ TRM cells during aGVHD in primate transplant recipients.
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Enfermedad Injerto contra Huésped , Trasplante de Células Madre Hematopoyéticas , Enfermedad Aguda , Animales , Linfocitos T CD8-positivos , Humanos , Macaca mulatta , Donantes de TejidosRESUMEN
Allogeneic hematopoietic stem cell transplantation (allo-HCT) is a common treatment for patients suffering from different hematological disorders. Allo-HCT in combination with hematopoietic stem cell (HSC) gene therapy is considered a promising treatment option for millions of patients with HIV+ and acute myeloid leukemia. Most currently available HSC gene therapy approaches target CD34-enriched cell fractions, a heterogeneous mix of mostly progenitor cells and only very few HSCs with long-term multilineage engraftment potential. As a consequence, gene therapy approaches are currently limited in their HSC targeting efficiency, very expensive consuming huge quantities of modifying reagents, and can lead to unwanted side effects in nontarget cells. We have previously shown that purified CD34+CD90+CD45RA- cells are enriched for multipotent HSCs with long-term multilineage engraftment potential, which can reconstitute the entire hematopoietic system in an autologous nonhuman primate transplant model. Here, we tested the feasibility of transplantation with purified CD34+CD90+CD45RA- cells in the allogeneic setting in a nonhuman primate model. METHODS: To evaluate the feasibility of this approach, CD34+CD90+CD45RA- cells from 2 fully major histocompatibility complex-matched, full sibling rhesus macaques were sort-purified, quality controlled, and transplanted. Engraftment and donor chimerism were evaluated in the peripheral blood and bone marrow of both animals. RESULTS: Despite limited survival due to infectious complications, we show that the large-scale sort-purification and transplantation of CD34+CD90+CD45RA- cells is technically feasible and leads to rapid engraftment of cells in bone marrow in the allogeneic setting and absence of cotransferred T cells. CONCLUSIONS: We show that purification of an HSC-enriched CD34+ subset can serve as a potential stem cell source for allo-HCTs. Most importantly, the combination of allo-HCT and HSC gene therapy has the potential to treat a wide array of hematologic and nonhematologic disorders.
RESUMEN
Controlling graft-versus-host disease (GVHD) remains a major unmet need in stem cell transplantation, and new, targeted therapies are being actively developed. CD28-CD80/86 costimulation blockade represents a promising strategy, but targeting CD80/CD86 with CTLA4-Ig may be associated with undesired blockade of coinhibitory pathways. In contrast, targeted blockade of CD28 exclusively inhibits T cell costimulation and may more potently prevent GVHD. Here, we investigated FR104, an antagonistic CD28-specific pegylated-Fab', in the nonhuman primate (NHP) GVHD model and completed a multiparameter interrogation comparing it with CTLA4-Ig, with and without sirolimus, including clinical, histopathologic, flow cytometric, and transcriptomic analyses. We document that FR104 monoprophylaxis and combined prophylaxis with FR104/sirolimus led to enhanced control of effector T cell proliferation and activation compared with the use of CTLA4-Ig or CTLA4-Ig/sirolimus. Importantly, FR104/sirolimus did not lead to a beneficial impact on Treg reconstitution or homeostasis, consistent with control of conventional T cell activation and IL-2 production needed to support Tregs. While FR104/sirolimus had a salutary effect on GVHD-free survival, overall survival was not improved, due to death in the absence of GVHD in several FR104/sirolimus recipients in the setting of sepsis and a paralyzed INF-γ response. These results therefore suggest that effectively deploying CD28 in the clinic will require close scrutiny of both the benefits and risks of extensively abrogating conventional T cell activation after transplant.
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Antígenos CD28/antagonistas & inhibidores , Enfermedad Injerto contra Huésped/prevención & control , Linfocitos T/inmunología , Abatacept/administración & dosificación , Animales , Anticuerpos Monoclonales/administración & dosificación , Modelos Animales de Enfermedad , Enfermedad Injerto contra Huésped/inmunología , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Activación de Linfocitos , Macaca mulatta , Sirolimus/administración & dosificación , Biología de SistemasRESUMEN
Allogeneic transplantation (allo-HCT) has led to the cure of HIV in one individual, raising the question of whether transplantation can eradicate the HIV reservoir. To test this, we here present a model of allo-HCT in SHIV-infected, cART-suppressed nonhuman primates. We infect rhesus macaques with SHIV-1157ipd3N4, suppress them with cART, then transplant them using MHC-haploidentical allogeneic donors during continuous cART. Transplant results in ~100% myeloid donor chimerism, and up to 100% T-cell chimerism. Between 9 and 47 days post-transplant, terminal analysis shows that while cell-associated SHIV DNA levels are reduced in the blood and in lymphoid organs post-transplant, the SHIV reservoir persists in multiple organs, including the brain. Sorting of donor-vs.-recipient cells reveals that this reservoir resides in recipient cells. Moreover, tetramer analysis indicates a lack of virus-specific donor immunity post-transplant during continuous cART. These results suggest that early post-transplant, allo-HCT is insufficient for recipient reservoir eradication despite high-level donor chimerism and GVHD.
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Reservorios de Enfermedades/virología , Trasplante de Células Madre Hematopoyéticas , Complejo Mayor de Histocompatibilidad , Virus de la Inmunodeficiencia de los Simios/fisiología , Trasplante Haploidéntico , Animales , Terapia Antirretroviral Altamente Activa , Linfocitos T CD8-positivos/inmunología , ADN Viral/metabolismo , Macaca mulatta , ARN Viral/metabolismo , Síndrome de Inmunodeficiencia Adquirida del Simio/tratamiento farmacológico , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Trasplante HomólogoAsunto(s)
Trasplante de Médula Ósea/métodos , Linfocitos T Reguladores/trasplante , Trasplante de Médula Ósea/efectos adversos , Técnicas de Cultivo de Célula , Enfermedad Injerto contra Huésped/inmunología , Enfermedad Injerto contra Huésped/prevención & control , Humanos , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/inmunologíaRESUMEN
Hematopoietic (Hem) and endothelial (End) lineages derive from a common progenitor cell, the hemangioblast: specifically, the human cord blood (CB) CD34+KDR+ cell fraction comprises primitive Hem and End cells, as well as hemangioblasts. In humans, the potential therapeutic role of Hem and End progenitors in ischemic heart disease is subject to intense investigation. Particularly, the contribution of these cells to angiogenesis and cardiomyogenesis in myocardial ischemia is not well established. In our studies, we induced myocardial infarct (MI) in the immunocompromised NOD-SCID mouse model, and monitored the effects of myocardial transplantation of human CB CD34+ cells on cardiac function. Specifically, we compared the therapeutic effect of unseparated CD34+ cells vs. PBS and mononuclear cells (MNCs); moreover, we compared the action of the CD34+KDR+ cell subfraction vs. the CD34+KDR- subset. CD34+ cells significantly improve cardiac function after MI, as compared with PBS/MNCs. Similar beneficial actions were obtained using a 2-log lower number of CD34+KDR+ cells, while the same number of CD34+KDR- cells did not have any effects. The beneficial effect of CD34+KDR+ cells may mostly be ascribed to their notable resistance to apoptosis and to their angiogenic action, since cardiomyogenesis was limited. Altogether, our results indicate that, within the CD34+ cell population, the CD34+KDR+ fraction is responsible for the improvement in cardiac hemodynamics and hence represents the candidate active CD34+ cell subset.
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Antígenos CD34/metabolismo , Trasplante de Células , Infarto del Miocardio/fisiopatología , Infarto del Miocardio/terapia , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Animales , Apoptosis , Comunicación Autocrina/efectos de los fármacos , Fusión Celular , Linaje de la Célula , Técnicas de Cocultivo , Medio de Cultivo Libre de Suero/farmacología , Sangre Fetal/citología , Sangre Fetal/metabolismo , Ventrículos Cardíacos/patología , Ventrículos Cardíacos/fisiopatología , Hemodinámica/fisiología , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Infarto del Miocardio/patología , Factores de Tiempo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Función VentricularRESUMEN
OBJECTIVE: To understand the roles of microRNAs (miRNAs) in proliferative lupus nephritis (LN). METHODS: A high-throughput analysis of the miRNA pattern of the kidneys of LN patients and controls was performed by molecular digital detection. Urinary miRNAs were measured by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Target gene expression in human mesangial cells was evaluated by arrays and qRT-PCR. Human epidermal growth factor receptor 2 (HER-2) was analyzed by immunohistochemistry in kidney samples from LN patients and in a murine model of lupus. Urinary levels of HER-2, monocyte chemotactic protein 1 (MCP-1), and vascular cell adhesion molecule 1 (VCAM-1) were measured by enzyme-linked immunosorbent assay. RESULTS: Levels of the miRNAs miR-26a and miR-30b were decreased in the kidneys and urine of LN patients. In vitro these miRNAs controlled mesangial cell proliferation, and their expression was regulated by HER-2. HER-2 was overexpressed in lupus-prone NZM2410 mice and in the kidneys of patients with LN, but not in other mesangioproliferative glomerulonephritides. HER-2 was found to be up-regulated by interferon-α and interferon regulatory factor 1. Urinary HER-2 was increased in LN and reflected disease activity, and its levels correlated with those of 2 other recognized LN biomarkers, MCP-1 and VCAM-1. CONCLUSION: The kidney miRNA pattern is broadly altered in LN, which contributes to uncontrolled cell proliferation. Levels of the miRNAs miR-26a and miR-30b are decreased in the kidneys and urine of LN patients, and they directly regulate the cell cycle in mesangial cells. The levels of these miRNAs are controlled by HER-2, which is overexpressed in NZM2410 mice and in the kidneys and urine of LN patients. HER-2, miR-26a, and miR-30b are thus potential LN biomarkers, and blocking HER-2 may be a promising new strategy to decrease cell proliferation and damage in this disease.
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Quimiocina CCL2/orina , Riñón/metabolismo , Nefritis Lúpica/genética , Células Mesangiales/metabolismo , MicroARNs/metabolismo , Receptor ErbB-2/metabolismo , Molécula 1 de Adhesión Celular Vascular/orina , Adolescente , Animales , Niño , Modelos Animales de Enfermedad , Femenino , Glomerulonefritis Membranoproliferativa/genética , Glomerulonefritis Membranoproliferativa/metabolismo , Humanos , Masculino , Ratones , MicroARNs/orina , Receptor ErbB-2/orina , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Stem cells reside in specialized niches that regulate their self-renewal and differentiation. The vasculature is emerging as an important component of stem cell niches. Here, we show that the adult subventricular zone (SVZ) neural stem cell niche contains an extensive planar vascular plexus that has specialized properties. Dividing stem cells and their transit-amplifying progeny are tightly apposed to SVZ blood vessels both during homeostasis and regeneration. They frequently contact the vasculature at sites that lack astrocyte endfeet and pericyte coverage, a modification of the blood-brain barrier unique to the SVZ. Moreover, regeneration often occurs at these sites. Finally, we find that circulating small molecules in the blood enter the SVZ. Thus, the vasculature is a key component of the adult SVZ neural stem cell niche, with SVZ stem cells and transit-amplifying cells uniquely poised to receive spatial cues and regulatory signals from diverse elements of the vascular system.
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Células Madre Adultas/citología , Vasos Sanguíneos/citología , Encéfalo/citología , Ventrículos Laterales/citología , Células Madre Adultas/fisiología , Animales , Encéfalo/irrigación sanguínea , Encéfalo/fisiología , Diferenciación Celular , División Celular , Humanos , Ventrículos Laterales/irrigación sanguínea , Ventrículos Laterales/fisiología , Ratones , Ratones EndogámicosRESUMEN
Lymphocyte activation gene-3 (LAG-3) is a CD4-related transmembrane protein expressed by regulatory T cells that binds MHC II on APCs. It is shown in this study that during Treg:DC interactions, LAG-3 engagement with MHC class II inhibits DC activation. MHC II cross-linking by agonistic Abs induces an ITAM-mediated inhibitory signaling pathway, involving FcgammaRgamma and ERK-mediated recruitment of SHP-1 that suppresses dendritic cell maturation and immunostimulatory capacity. These data reveal a novel ITAM-mediated inhibitory signaling pathway in DCs triggered by MHC II engagement of LAG-3, providing a molecular mechanism in which regulatory T cells may suppress via modulating DC function.
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Antígenos CD/inmunología , Células Dendríticas/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Transducción de Señal/inmunología , Linfocitos T Reguladores/inmunología , Animales , Anticuerpos/farmacología , Antígenos CD/metabolismo , Antígenos CD4/inmunología , Antígenos CD4/metabolismo , Células Dendríticas/citología , Células Dendríticas/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Recubrimiento Inmunológico/efectos de los fármacos , Recubrimiento Inmunológico/inmunología , Ratones , Ratones Noqueados , Proteína Tirosina Fosfatasa no Receptora Tipo 6/inmunología , Proteína Tirosina Fosfatasa no Receptora Tipo 6/metabolismo , Receptores de IgG/inmunología , Receptores de IgG/metabolismo , Transducción de Señal/efectos de los fármacos , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/metabolismo , Proteína del Gen 3 de Activación de LinfocitosRESUMEN
The uptake of immune complexes by FcRs on APCs augments humoral and cellular responses to exogenous Ag. In this study, CD11c+ dendritic cells are shown to be responsible in vivo for immune complex-triggered priming of T cells. We examine the consequence of Ab-mediated uptake of self Ag by dendritic cells in the rat insulin promoter-membrane OVA model and identify a role for the inhibitory FcgammaRIIB in the maintenance of peripheral CD8 T cell tolerance. Effector differentiation of diabetogenic OT-I CD8+ T cells is enhanced in rat insulin promoter-membrane OVA mice lacking FcgammaRIIB, resulting in a high incidence of diabetes. FcgammaRIIB-mediated inhibition of CD8 T cell priming results from suppression of both DC activation and cross-presentation through activating FcgammaRs. Further FcgammaRIIB on DCs inhibited the induction of OVA-specific Th1 effectors, limiting Th1-type differentiation and memory T cell accumulation. In these MHC II-restricted responses, the presence of FcgammaRIIB only modestly affected initial CD4 T cell proliferative responses, suggesting that FcgammaRIIB limited effector cell differentiation primarily by inhibiting DC activation. Thus, FcgammaRIIB can contribute to peripheral tolerance maintenance by inhibiting DC activation alone or by also limiting processing of exogenously acquired Ag.