RESUMEN
Suboptimal functional persistence limits the efficacy of adoptive T-cell therapies. CD28-based chimeric antigen receptors (CAR) impart potent effector function to T cells but with a limited lifespan. We show here that the genetic disruption of SUV39H1, which encodes a histone-3, lysine-9 methyl-transferase, enhances the early expansion, long-term persistence, and overall antitumor efficacy of human CAR T cells in leukemia and prostate cancer models. Persisting SUV39H1-edited CAR T cells demonstrate improved expansion and tumor rejection upon multiple rechallenges. Transcriptional and genome accessibility profiling of repeatedly challenged CAR T cells shows improved expression and accessibility of memory transcription factors in SUV39H1-edited CAR T cells. SUV39H1 editing also reduces expression of inhibitory receptors and limits exhaustion in CAR T cells that have undergone multiple rechallenges. Our findings thus demonstrate the potential of epigenetic programming of CAR T cells to balance their function and persistence for improved adoptive cell therapies. SIGNIFICANCE: T cells engineered with CD28-based CARs possess robust effector function and antigen sensitivity but are hampered by limited persistence, which may result in tumor relapse. We report an epigenetic strategy involving disruption of the SUV39H1-mediated histone-silencing program that promotes the functional persistence of CD28-based CAR T cells. See related article by López-Cobo et al., p. 120. This article is featured in Selected Articles from This Issue, p. 5.
Asunto(s)
Leucemia , Receptores Quiméricos de Antígenos , Masculino , Humanos , Linfocitos T , Receptores de Antígenos de Linfocitos T , Histonas/metabolismo , Antígenos CD28/genética , Antígenos CD28/metabolismo , Inmunoterapia Adoptiva , Leucemia/metabolismo , Metilación , Ensayos Antitumor por Modelo de Xenoinjerto , Metiltransferasas/genética , Metiltransferasas/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismoRESUMEN
Further advances in cell engineering are needed to increase the efficacy of chimeric antigen receptor (CAR) and other T cell-based therapies1-5. As T cell differentiation and functional states are associated with distinct epigenetic profiles6,7, we hypothesized that epigenetic programming may provide a means to improve CAR T cell performance. Targeting the gene that encodes the epigenetic regulator ten-eleven translocation 2 (TET2)8 presents an interesting opportunity as its loss may enhance T cell memory9,10, albeit not cause malignancy9,11,12. Here we show that disruption of TET2 enhances T cell-mediated tumour rejection in leukaemia and prostate cancer models. However, loss of TET2 also enables antigen-independent CAR T cell clonal expansions that may eventually result in prominent systemic tissue infiltration. These clonal proliferations require biallelic TET2 disruption and sustained expression of the AP-1 factor BATF3 to drive a MYC-dependent proliferative program. This proliferative state is associated with reduced effector function that differs from both canonical T cell memory13,14 and exhaustion15,16 states, and is prone to the acquisition of secondary somatic mutations, establishing TET2 as a guardian against BATF3-induced CAR T cell proliferation and ensuing genomic instability. Our findings illustrate the potential of epigenetic programming to enhance T cell immunity but highlight the risk of unleashing unchecked proliferative responses.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Proliferación Celular , Proteínas de Unión al ADN , Dioxigenasas , Inmunoterapia Adoptiva , Activación de Linfocitos , Receptores Quiméricos de Antígenos , Linfocitos T , Humanos , Masculino , Diferenciación Celular/genética , Dioxigenasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Inmunoterapia Adoptiva/métodos , Inmunoterapia Adoptiva/normas , Receptores Quiméricos de Antígenos/inmunología , Receptores Quiméricos de Antígenos/metabolismo , Leucemia/inmunología , Neoplasias de la Próstata/inmunología , Linfocitos T/citología , Linfocitos T/inmunología , Linfocitos T/patología , Epigénesis Genética , Memoria Inmunológica , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismoRESUMEN
Chimeric antigen receptors (CARs) are receptors for antigen that direct potent immune responses. Tumor escape associated with low target antigen expression is emerging as one potential limitation of their efficacy. Here we edit the TRAC locus in human peripheral blood T cells to engage cell-surface targets through their T cell receptor-CD3 complex reconfigured to utilize the same immunoglobulin heavy and light chains as a matched CAR. We demonstrate that these HLA-independent T cell receptors (HIT receptors) consistently afford high antigen sensitivity and mediate tumor recognition beyond what CD28-based CARs, the most sensitive design to date, can provide. We demonstrate that the functional persistence of HIT T cells can be augmented by constitutive coexpression of CD80 and 4-1BBL. Finally, we validate the increased antigen sensitivity afforded by HIT receptors in xenograft mouse models of B cell leukemia and acute myeloid leukemia, targeting CD19 and CD70, respectively. Overall, HIT receptors are well suited for targeting cell surface antigens of low abundance.
Asunto(s)
Leucemia Mieloide Aguda , Receptores Quiméricos de Antígenos , Animales , Antígenos CD19 , Antígenos de Histocompatibilidad , Humanos , Inmunoterapia Adoptiva , Ratones , Receptores de Antígenos de Linfocitos T , Receptores Quiméricos de Antígenos/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Adoptive T cell therapy (ACT) is a promising strategy for treating cancer, but it often fails because of cell intrinsic regulatory programs that limit the degree or duration of T cell function. In this study, we found that ectopic expression of microRNA-200c (miR-200c) markedly enhanced the antitumor activity of CD8+ cytotoxic T lymphocytes (CTLs) during ACT in multiple mouse models. CTLs transduced with miR-200c exhibited reduced apoptosis during engraftment and enhanced in vivo persistence, accompanied by up-regulation of the transcriptional regulator T cell factor 1 (TCF1) and the inflammatory cytokine tumor necrosis factor (TNF). miR-200c elicited these changes by suppressing the transcription factor Zeb1 and thereby inducing genes characteristic of epithelial cells. Overexpression of one of these genes, Epcam, was sufficient to augment therapeutic T cell responses against both solid and liquid tumors. These results identify the miR-200cEpCAM axis as an avenue for improving ACT and demonstrate that select genetic perturbations can produce phenotypically distinct T cells with advantageous therapeutic properties.
Asunto(s)
Molécula de Adhesión Celular Epitelial , Inmunoterapia Adoptiva , MicroARNs , Neoplasias Experimentales/inmunología , Animales , Línea Celular Tumoral , Tratamiento Basado en Trasplante de Células y Tejidos , Molécula de Adhesión Celular Epitelial/genética , Regulación Neoplásica de la Expresión Génica , Ratones Endogámicos C57BL , Ratones Transgénicos , MicroARNs/genética , Linfocitos TRESUMEN
Chimeric antigen receptors (CARs) are synthetic antigen receptors that reprogram T cell specificity, function and persistence1. Patient-derived CAR T cells have demonstrated remarkable efficacy against a range of B-cell malignancies1-3, and the results of early clinical trials suggest activity in multiple myeloma4. Despite high complete response rates, relapses occur in a large fraction of patients; some of these are antigen-negative and others are antigen-low1,2,4-9. Unlike the mechanisms that result in complete and permanent antigen loss6,8,9, those that lead to escape of antigen-low tumours remain unclear. Here, using mouse models of leukaemia, we show that CARs provoke reversible antigen loss through trogocytosis, an active process in which the target antigen is transferred to T cells, thereby decreasing target density on tumour cells and abating T cell activity by promoting fratricide T cell killing and T cell exhaustion. These mechanisms affect both CD28- and 4-1BB-based CARs, albeit differentially, depending on antigen density. These dynamic features can be offset by cooperative killing and combinatorial targeting to augment tumour responses to immunotherapy.
Asunto(s)
Antígenos de Neoplasias/inmunología , Antígenos de Neoplasias/metabolismo , Leucemia/inmunología , Receptores Quiméricos de Antígenos/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Escape del Tumor/inmunología , Ligando 4-1BB/inmunología , Animales , Antígenos CD28/inmunología , Citotoxicidad Inmunológica , Femenino , Inmunoterapia Adoptiva , Leucemia/patología , Masculino , Ratones , Ratones Endogámicos NOD , Recurrencia Local de Neoplasia/inmunología , Linfocitos T/citologíaRESUMEN
In the version of this article originally published, there was an error in the legend for Extended Data Fig. 7. The legend for panel f was originally: "f, FACS analysis of IL7R-, CD62L- and CD45RA- expression on TRAC-1928ζ and TRAC-1XX CAR T cells at day 63 post CAR infusion (representative for at least n = 3 mice per group in one independent experiment)." The legend should have been: "f, FACS analysis of IL7R+, CD62L+ and CD45RA+ expression on TRAC-1928ζ and TRAC-1XX CAR T cells at day 63 post CAR infusion (representative for at least n = 3 mice per group in one independent experiment)." The error has been corrected in the HTML and PDF versions of this article.
RESUMEN
Chimeric antigen receptors (CARs) are synthetic receptors that target and reprogram T cells to acquire augmented antitumor properties1. CD19-specific CARs that comprise CD28 and CD3ζ signaling motifs2 have induced remarkable responses in patients with refractory leukemia3-5 and lymphoma6 and were recently approved by the US Food and Drug Administration7. These CARs program highly performing effector functions that mediate potent tumor elimination4,8 despite the limited persistence they confer on T cells3-6,8. Extending their functional persistence without compromising their potency should improve current CAR therapies. Strong T cell activation drives exhaustion9,10, which may be accentuated by the redundancy of CD28 and CD3ζ signaling11,12 as well as the spatiotemporal constraints imparted by the structure of second-generation CARs2. Thus, we hypothesized that calibrating the activation potential of CD28-based CARs would differentially reprogram T cell function and differentiation. Here, we show that CARs encoding a single immunoreceptor tyrosine-based activation motif direct T cells to different fates by balancing effector and memory programs, thereby yielding CAR designs with enhanced therapeutic profiles.
Asunto(s)
Linaje de la Célula , Inmunoterapia , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/inmunología , Animales , Calibración , Línea Celular , Masculino , Ratones , Dominios Proteicos , Receptores de Antígenos de Linfocitos T/químicaRESUMEN
The effector potential of NK cells is counterbalanced by their sensitivity to inhibition by "self" MHC class I molecules in a process called "education." In humans, interactions between inhibitory killer immunoglobulin-like receptors (KIR) and human MHC (HLA) mediate NK cell education. In HLA-B(∗)27:05(+) transgenic mice and in patients undergoing HLA-mismatched hematopoietic cell transplantation (HCT), NK cells derived from human CD34(+) stem cells were educated by HLA from both donor hematopoietic cells and host stromal cells. Furthermore, mature human KIR3DL1(+) NK cells gained reactivity after adoptive transfer to HLA-B(∗)27:05(+) mice or bone marrow chimeric mice where HLA-B(∗)27:05 was restricted to either the hematopoietic or stromal compartment. Silencing of HLA in primary NK cells diminished NK cell reactivity, while acquisition of HLA from neighboring cells increased NK cell reactivity. Altogether, these findings reveal roles for cell-extrinsic HLA in driving NK cell reactivity upward, and cell-intrinsic HLA in maintaining NK cell education.
Asunto(s)
Autoantígenos/metabolismo , Trasplante de Células Madre de Sangre del Cordón Umbilical , Antígeno HLA-B27/metabolismo , Neoplasias Hematológicas/terapia , Células Asesinas Naturales/inmunología , Receptores KIR3DL1/metabolismo , Células del Estroma/inmunología , Animales , Antígenos CD34/metabolismo , Diferenciación Celular , Células Cultivadas , Quimerismo , Espacio Extracelular/metabolismo , Antígeno HLA-B27/genética , Neoplasias Hematológicas/inmunología , Humanos , Isoantígenos/inmunología , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , ARN Interferente Pequeño/genéticaRESUMEN
T cell engineering is a powerful means to rapidly generate anti-tumor T cells. The costimulatory properties of second-generation chimeric antigen receptors (CARs) determine the overall potency of adoptively transferred T cells. Using an in vivo "stress test" to challenge CD19-targeted T cells, we studied the functionality and persistence imparted by seven different CAR structures providing CD28 and/or 4-1BB costimulation. One configuration, which uses two signaling domains (CD28 and CD3ζ) and the 4-1BB ligand, provided the highest therapeutic efficacy, showing balanced tumoricidal function and increased T cell persistence accompanied by an elevated CD8/CD4 ratio and decreased exhaustion. Remarkably, induction of the IRF7/IFNß pathway was required for optimal anti-tumor activity. Thus, 1928z-41BBL T cells possess strikingly potent intrinsic and immunomodulatory qualities.
Asunto(s)
Antígenos CD28/inmunología , Neoplasias Hematológicas/inmunología , Receptores de Antígenos/inmunología , Linfocitos T/inmunología , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunología , Antígenos CD19/inmunología , Antígenos CD19/metabolismo , Antígenos CD28/biosíntesis , Antígenos CD28/genética , Neoplasias Hematológicas/patología , Neoplasias Hematológicas/terapia , Humanos , Inmunoterapia Adoptiva , Factor 7 Regulador del Interferón/metabolismo , Interferón gamma/metabolismo , Cinética , Activación de Linfocitos/inmunología , Receptores de Antígenos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Transducción de Señal , Linfocitos T/patología , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/biosíntesis , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/genéticaRESUMEN
NK cells are regulated by inhibiting and activating cell surface receptors. Most inhibitory receptors recognize MHC class I Ags and protect healthy cells from NK cell-mediated autoaggression. However, certain activating receptors, including the human activating killer cell Ig-like receptor (KIR) 2DS1, also recognize MHC class I. This fact raises the question of how NK cells expressing such activating receptors are tolerized to host tissues. We investigated whether the presence of HLA-C2, the cognate ligand for 2DS1, induces tolerance in 2DS1-expressing NK cells. Anti-HLA-C2 activity could be detected in vitro in some 2DS1 positive NK clones irrespective of the presence or absence of HLA-C2 ligand in the donor. The frequency of anti-HLA-C2 reactivity was high in donors homozygous for HLA-C1. Surprisingly, no significant difference was seen in the frequency of anti-HLA-C2 cytotoxicity in donors heterozygous for HLA-C2 and donors without HLA-C2 ligand. However, donors homozygous for HLA-C2, compared with all other donors, had significantly reduced frequency of anti-HLA-C2 reactive clones. The 2DS1 positive clones that express inhibitory KIR for self-HLA class I were commonly noncytotoxic, and anti-HLA-C2 cytotoxicity was nearly exclusively restricted to 2DS1 single positive clones lacking inhibitory KIR. 2DS1 single positive NK clones with anti-HLA-C2 reactivity were also present posttransplantation in HLA-C2 positive recipients of hematopoietic stem cell transplants from 2DS1 positive donors. These results demonstrate that many NK cells with anti-HLA-C2 reactivity are present in HLA-C1 homozygous and heterozygous donors with 2DS1. In contrast, 2DS1 positive clones from HLA-C2 homozygous donors are frequently tolerant to HLA-C2.
Asunto(s)
Antígenos HLA-C/inmunología , Tolerancia Inmunológica/inmunología , Células Asesinas Naturales/inmunología , Receptores KIR/inmunología , Receptores KIR/metabolismo , Antígenos CD/inmunología , Antígenos CD/metabolismo , Regulación hacia Abajo/inmunología , Antígenos HLA-C/metabolismo , Trasplante de Células Madre Hematopoyéticas , Humanos , Interleucina-15/inmunología , Interleucina-15/metabolismo , Células Asesinas Naturales/metabolismo , Receptor Leucocitario Tipo Inmunoglobulina B1 , Ligandos , Receptores Inmunológicos/inmunología , Receptores Inmunológicos/metabolismo , Donantes de TejidosRESUMEN
The human liver is enriched in NK cells which are potent effectors of the innate immune system. We have determined that liver NK cells freshly isolated from surgical specimens from patients with hepatic malignancy have less cytolytic activity than autologous blood NK cells. This difference was due to a higher proportion of CD16(-) NK cells in the liver and reduced cytotoxicity by CD16(+) liver NK cells compared with their blood counterparts. CD16(+) liver NK cells had similar expression of activating NK receptors and had similar intracellular granzyme B and perforin content compared with CD16(+) blood NK cells. CD16(+) liver NK cells contained a reduced fraction of cells with inhibitory killer Ig-like receptors specific for self-MHC class I (self-killer Ig-related receptor (KIR)) and an increased fraction of self-KIR(neg)NKG2A(pos) and self-KIR(neg)NKG2A(neg) cells. Using single-cell analysis of intracellular IFN-gamma production and cytotoxicity assays, we determined that CD16(+) liver NK cells expressing self-KIR were more responsive to target cells than those cells that did not express self-KIR molecules. CD16(+) liver NK cells gained cytolytic function when stimulated with IL-2 or cultured with LPS or poly(I:C)-activated autologous liver Kupffer cells. Thus, the human liver contains NK cell subsets which have reduced effector function, but under appropriate inflammatory conditions become potent killers.
Asunto(s)
Citotoxicidad Inmunológica , Células Asesinas Naturales/inmunología , Hígado/inmunología , Receptores KIR/análisis , Autoinmunidad , Células Sanguíneas/inmunología , Humanos , Inmunidad Innata , Receptores de IgGRESUMEN
TNF-like weak inducer of apoptosis (TWEAK), a member of the TNF superfamily, is a prominent inducer of proinflammatory cytokines in vitro and in vivo. We previously found that kidney cells display the TWEAK receptor Fn14, and that TWEAK stimulation of mesangial cells and podocytes induces a potent proinflammatory response. Several of the cytokines up-regulated in the kidney in response to TWEAK are instrumental in Lupus nephritis; we therefore hypothesized that TWEAK/Fn14 interactions may be important in the cascade(s) leading to renal damage in systemic Lupus erythematosus. In this study, we analyzed the effects of Fn14 deficiency in the chronic graft-vs-host model of SLE, and the benefits of treatment with an anti-TWEAK mAb in this mouse model. We found that anti-nuclear Ab titers were no different between C57BL/6 Fn14 wild-type and deficient mice injected with alloreactive bm12 splenocytes. However, kidney disease was significantly less severe in Fn14 knockout mice. Furthermore, kidney IgG deposition, IL-6, MCP-1, RANTES, and IP-10, as well as macrophage infiltration, were significantly decreased in Fn14-deficient mice with induced lupus. Similarly, mice with induced Lupus treated with an anti-TWEAK neutralizing mAb had significantly diminished kidney expression of IL-6, MCP-1, IL-10, as well as proteinuria, but similar autoantibody titers, as compared with control-treated mice. We conclude that TWEAK is an important mediator of kidney damage that acts by promoting local inflammatory events, but without impacting adaptive immunity in this experimental LN model. Thus, TWEAK blockade may be a novel therapeutic approach to reduce renal damage in SLE.
Asunto(s)
Enfermedad Injerto contra Huésped/inmunología , Lupus Eritematoso Sistémico/inmunología , Nefritis Lúpica/inmunología , Receptores del Factor de Necrosis Tumoral/metabolismo , Factores de Necrosis Tumoral/metabolismo , Animales , Anticuerpos/administración & dosificación , Anticuerpos/farmacología , Anticuerpos/uso terapéutico , Autoanticuerpos/inmunología , Quimiocinas/efectos de los fármacos , Enfermedad Crónica , Citocina TWEAK , Citocinas/efectos de los fármacos , Citocinas/genética , Modelos Animales de Enfermedad , Femenino , Enfermedad Injerto contra Huésped/patología , Inmunoglobulina G/inmunología , Lupus Eritematoso Sistémico/complicaciones , Lupus Eritematoso Sistémico/patología , Nefritis Lúpica/tratamiento farmacológico , Nefritis Lúpica/patología , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteinuria/tratamiento farmacológico , Receptores del Factor de Necrosis Tumoral/deficiencia , Sensibilidad y Especificidad , Receptor de TWEAK , Factores de Necrosis Tumoral/deficienciaRESUMEN
Target Ag display is a necessary requirement for the expression of certain immune-mediated kidney diseases. We previously had shown that anti-DNA Abs that cross-react with alpha-actinin may be important in the pathogenesis of murine and human lupus nephritis; in murine models, we had found that a significant proportion of pathogenic serum and kidney-deposited Igs are alpha-actinin reactive. Furthermore, a pathogenic anti-DNA/alpha-actinin Ab showed enhanced binding to immortalized mesangial cells (MCs) derived from a lupus prone MRL-lpr/lpr mouse as compared with MCs from BALB/c mice which are not susceptible to spontaneous lupus, suggesting that kidney alpha-actinin expression may be contributing to nephritis. In the current study, we established that two isoforms of alpha-actinin that are present in the kidney, alpha-actinin 1 and alpha-actinin 4, can both be targeted by anti-alpha-actinin Abs. We found novel sequence polymorphisms between MRL-lpr/lpr and BALB/c in the gene for alpha-actinin 4. Moreover, alpha-actinin 4 and a splice variant of alpha-actinin 1 were both expressed at significantly higher levels (mRNA and protein) in MCs from the lupus prone MRL-lpr/lpr strain. Significantly, we were able to confirm these differences in intact kidney by examining glomerular Ig deposition of anti-alpha-actinin Abs. We conclude that enhanced alpha-actinin expression may determine the extent of Ig deposition in the Ab-mediated kidney disease in lupus. Modulation of Ag expression may be a promising approach to down-regulate immune complex formation in the target organ in individuals with circulating pathogenic Abs.
Asunto(s)
Actinina/fisiología , Anticuerpos Antinucleares/metabolismo , Sitios de Unión de Anticuerpos , Mesangio Glomerular/inmunología , Mesangio Glomerular/metabolismo , Actinina/biosíntesis , Actinina/genética , Actinina/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antinucleares/biosíntesis , Anticuerpos Antinucleares/sangre , Anticuerpos Monoclonales/metabolismo , Secuencia de Bases , Línea Celular , Reacciones Cruzadas , Mesangio Glomerular/citología , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos MRL lpr , Proteínas de Microfilamentos/biosíntesis , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/inmunología , Datos de Secuencia Molecular , Mutación Puntual , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Isoformas de Proteínas/fisiología , TransfecciónRESUMEN
OBJECTIVE: Cross-reactivity with kidney antigens is believed to be a critical determinant in the renal pathogenicity of anti-double-stranded DNA (anti-dsDNA) antibodies. Murine nephritogenic anti-dsDNA antibodies have been shown to cross-react with alpha-actinin, and anti-alpha-actinin antibodies have been found to be deposited in the kidneys of lupus mice with active nephritis. Furthermore, in humans with systemic lupus erythematosus (SLE), it has been found that a greater proportion of polyclonal IgG anti-dsDNA antibodies from patients with renal involvement bind to alpha-actinin than do those from patients without renal disease. We undertook this study to substantiate a direct link between cross-reactive anti-dsDNA/anti-alpha-actinin antibodies and the pathogenesis of lupus nephritis in humans. METHODS: A panel of 10 anti-dsDNA and/or anti-alpha-actinin antibodies was generated by Epstein-Barr virus transformation of lymphocytes from patients with SLE and was extensively characterized. Antibody binding was studied by enzyme-linked immunosorbent assay and Western blotting. Antibody potential for pathogenicity was assessed by measuring binding to isolated glomeruli and mesangial cells and by evaluation of histologic features of the kidney following injection in vivo. RESULTS: All anti-dsDNA antibodies isolated also bound alpha-actinin. Cross-reactive antibodies bound to mesangial cells and to isolated glomeruli ex vivo. Binding to glomeruli was not inhibited by DNase treatment, but could be abrogated by alpha-actinin. Furthermore, histopathologic abnormalities seen in mice injected intraperitoneally with a cross-reactive cell line included fusion of podocyte foot processes and subepithelial and subendothelial deposition. CONCLUSION: These studies provide strong support for the hypothesis that alpha-actinin is a major cross-reactive target for anti-dsDNA antibodies in SLE patients. Cross-reactive anti-dsDNA/anti-alpha-actinin antibodies from SLE patients are pathogenic and may contribute to the kidney lesions in lupus nephritis.
Asunto(s)
Actinina/inmunología , Anticuerpos Antinucleares/inmunología , ADN/inmunología , Lupus Eritematoso Sistémico/inmunología , Nefritis Lúpica/inmunología , Adulto , Animales , Autoanticuerpos/análisis , Western Blotting , Reacciones Cruzadas , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Mesangio Glomerular/inmunología , Humanos , Inmunoglobulina G/análisis , Inmunohistoquímica , Riñón/inmunología , Glomérulos Renales/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Persona de Mediana EdadRESUMEN
Previously, we showed that the BRCA1 protein interacts directly and functionally with estrogen receptor-alpha (ER-alpha), resulting in the inhibition of estradiol (E2)-stimulated ER-alpha transcriptional activity. The interaction sites were mapped to the N-terminus of BRCA1 (within amino acids (aa) 1-302) and the ligand-binding domain/activation function-2 (LBD/AF-2) region (within aa 282-420) of ER-alpha. In this study, we have further characterized the structure/function relationship for the BRCA1 : ER-alpha interaction. We found that the N-terminal RING domain (aa 20-64) is not required for the BRCA1 : ER-alpha interaction. We identified two separate contact points for ER-alpha, one within aa 1-100 and the other within aa 100-200 of BRCA1; and we showed that each of these BRCA1 peptides interacts with BRCA1 in vitro and in vivo. By using different fragments of the BRCA1 N-terminus, we found that aa 67-100 and 101-133 are required for the interaction with ER-alpha, but that aa 1-67 and 134-302 are dispensible. Previously, we showed that BRCA1 aa 1-302 does not inhibit E2-stimulated ER-alpha transcriptional activity but does bind to ER-alpha and acts as a dominant negative inhibitor of the full-length BRCA1 protein. Somewhat surprisingly, we found that BRCA1 aa 1-100 and BRCA1 aa 101-200 (but not aa 201-300) each inhibited ER-alpha activity, although not as efficiently as full-length BRCA1. Mutations within an HIV Rev-like nuclear export signal that resembles a nuclear receptor corepressor motif (aa 86-95) impaired the ability of both truncated (aa 1-100) and full-length (aa 1-1863) BRCA1 proteins to interact with and/or repress ER-alpha activity. Based on these findings, a partial BRCA1 : ER-alpha three-dimensional structure is proposed. The implications of these findings for understanding the BRCA1 : ER-alpha interaction are discussed.
Asunto(s)
Proteína BRCA1/química , Proteína BRCA1/metabolismo , Receptor alfa de Estrógeno/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Neoplasias de la Mama , Línea Celular Tumoral , Clonación Molecular , Secuencia Conservada , Dimerización , Receptor alfa de Estrógeno/química , Femenino , Humanos , Masculino , Modelos Moleculares , Neoplasias de la Próstata , Unión Proteica , Conformación ProteicaRESUMEN
Estrogen receptor-alpha (ER-alpha) is a nuclear transcriptional factor that is part of the nuclear receptor superfamily. In this study, we isolated and identified a new LXXLL-containing protein that interacts with the ER-alpha via a yeast two-hybrid assay. We have termed this protein estrogen receptor repressor-10 (ERR-10). The ERR-10 cDNA is predicted to encode a polypeptide of 94 amino acids, with a molecular mass of about 10 kDa. Although the ERR-10 mRNA transcript is expressed in a wide range of normal human tissues, higher expression levels are found in endocrinal tissues relative to other tissues. We have demonstrated, through immunoprecipitation, Western blot and GST pull-down assays, that ERR-10 associates with ER-alpha. Moreover, ERR-10 decreased 17beta-estrodial-induced activation of ER-alpha transcriptional activity in transient transfection assays of mammalian cells. The ERR-10 N-terminus, which resembles two LXXLL motifs, is essential for ER-alpha binding and repression activity. Estrogen modulation of estrogen-responsive gene expression was markedly blocked by ERR-10. These results suggest that ERR-10 is a novel mediator in ER transcriptional activation.
Asunto(s)
Receptor alfa de Estrógeno/metabolismo , Receptores de Estrógenos/metabolismo , Proteínas Represoras/metabolismo , Activación Transcripcional , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Secuencia de Bases , Western Blotting , Línea Celular , Línea Celular Tumoral , Estradiol/farmacología , Receptor alfa de Estrógeno/química , Expresión Génica/efectos de los fármacos , Humanos , Datos de Secuencia Molecular , Peso Molecular , Pruebas de Precipitina , Estructura Terciaria de Proteína , ARN Largo no Codificante , Proteínas Represoras/química , Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Distribución Tisular , Transcripción Genética , Técnicas del Sistema de Dos HíbridosRESUMEN
BACKGROUND & OBJECTIVE: Carcinoembryonic antigen(CEA), is highly expressed in many kinds of carcinomas, especially in colon carcinoma. Anti-CEA antibodies have great application in diagnosis and therapy of the patients with colon carcinoma. This study was designed to investigate the biodistribution and radioimmunodetection in nude mice bearing human colon carcinoma using 188Re-labeled anti-CEA chimeric antibody. METHODS: CEA chimeric antibody and its parent McAb C50 were labeled with 188Re by a stannous chloride reduction method. The radiochemical purity of them were determined by ITLC. The biodistribution and whole body gamma scintigraphy imaging of 188Re-CEA chimeric antibody in nude mice bearing human colon carcinoma were fulfilled. The effect of radioimmunoimage between these two antibodies was compared. RESULTS: 188Re-CEA chimeric antibody showed high radiochemical purity of more than 95%. The specific reactivity of 188Re-CEA chimeric antibody was 356 MBq/mg. Immunoreactivity of 188Re-CEA chimeric antibody was 61% as determined by ELISA. Tumor/kidney ratio (0.75) and tumor/blood ratio(0.99) of 188Re-CEA chimeric antibody were observed in 24 h; compared to the orther organs, the ration is over 1.78. Tumor/kidney ratio (1.02) and tumor/blood ratio (1.12) of 188Re-CEA chimeric antibody were observed in 48 h; compared to the other organs more than 2.08. High uptake of 188Re-CEA chimeric antibody was observed in 20 hours after injection. The radioimmunodetection effect of these two antibodies was approximately same. CONCLUSIONS: 188Re-CEA chimeric antibody showed high specific tumor uptake and could fast display clear image in the nude mice bearing human colon carcinoma. Comparing with McAb C50, CEA chimeric antibody has good effect in clinic because of reducing of immunogenicity.