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1.
Nat Immunol ; 25(5): 764-777, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38609546

RESUMEN

The linear ubiquitin assembly complex (LUBAC) consists of HOIP, HOIL-1 and SHARPIN and is essential for proper immune responses. Individuals with HOIP and HOIL-1 deficiencies present with severe immunodeficiency, autoinflammation and glycogen storage disease. In mice, the loss of Sharpin leads to severe dermatitis due to excessive keratinocyte cell death. Here, we report two individuals with SHARPIN deficiency who manifest autoinflammatory symptoms but unexpectedly no dermatological problems. Fibroblasts and B cells from these individuals showed attenuated canonical NF-κB responses and a propensity for cell death mediated by TNF superfamily members. Both SHARPIN-deficient and HOIP-deficient individuals showed a substantial reduction of secondary lymphoid germinal center B cell development. Treatment of one SHARPIN-deficient individual with anti-TNF therapies led to complete clinical and transcriptomic resolution of autoinflammation. These findings underscore the critical function of the LUBAC as a gatekeeper for cell death-mediated immune dysregulation in humans.


Asunto(s)
Síndromes de Inmunodeficiencia , Proteínas del Tejido Nervioso , Ubiquitinas , Humanos , Síndromes de Inmunodeficiencia/genética , Síndromes de Inmunodeficiencia/inmunología , Femenino , Masculino , FN-kappa B/metabolismo , Ubiquitina-Proteína Ligasas/genética , Inflamación/inmunología , Inflamación/genética , Linfocitos B/inmunología , Mutación con Pérdida de Función , Fibroblastos/metabolismo , Fibroblastos/inmunología , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Animales , Ratones , Alelos
2.
Immunity ; 56(10): 2373-2387.e8, 2023 10 10.
Artículo en Inglés | MEDLINE | ID: mdl-37714151

RESUMEN

Immunoglobulin A (IgA) maintains commensal communities in the intestine while preventing dysbiosis. IgA generated against intestinal microbes assures the simultaneous binding to multiple, diverse commensal-derived antigens. However, the exact mechanisms by which B cells mount broadly reactive IgA to the gut microbiome remains elusive. Here, we have shown that IgA B cell receptor (BCR) is required for B cell fitness during the germinal center (GC) reaction in Peyer's patches (PPs) and for generation of gut-homing plasma cells (PCs). We demonstrate that IgA BCR drove heightened intracellular signaling in mouse and human B cells, and as a consequence, IgA+ B cells received stronger positive selection cues. Mechanistically, IgA BCR signaling offset Fas-mediated death, possibly rescuing low-affinity B cells to promote a broad humoral response to commensals. Our findings reveal an additional mechanism linking BCR signaling, B cell fate, and antibody production location, which have implications for how intestinal antigen recognition shapes humoral immunity.


Asunto(s)
Linfocitos B , Ganglios Linfáticos Agregados , Ratones , Humanos , Animales , Antígenos/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Inmunoglobulina A , Mucosa Intestinal
3.
Immunity ; 45(3): 483-496, 2016 09 20.
Artículo en Inglés | MEDLINE | ID: mdl-27617678

RESUMEN

Broadly neutralizing antibodies (bnAbs) against the N332 supersite of the HIV envelope (Env) trimer are the most common bnAbs induced during infection, making them promising leads for vaccine design. Wild-type Env glycoproteins lack detectable affinity for supersite-bnAb germline precursors and are therefore unsuitable immunogens to prime supersite-bnAb responses. We employed mammalian cell surface display to design stabilized Env trimers with affinity for germline-reverted precursors of PGT121-class supersite bnAbs. The trimers maintained native-like antigenicity and structure, activated PGT121 inferred-germline B cells ex vivo when multimerized on liposomes, and primed PGT121-like responses in PGT121 inferred-germline knockin mice. Design intermediates have levels of epitope modification between wild-type and germline-targeting trimers; their mutation gradient suggests sequential immunization to induce bnAbs, in which the germline-targeting prime is followed by progressively less-mutated design intermediates and, lastly, with native trimers. The vaccine design strategies described could be utilized to target other epitopes on HIV or other pathogens.


Asunto(s)
Vacunas contra el SIDA/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Anti-VIH/inmunología , Polisacáridos/inmunología , Secuencia de Aminoácidos , Animales , Linfocitos B/inmunología , Epítopos/inmunología , Infecciones por VIH/inmunología , VIH-1/inmunología , Inmunización/métodos , Ratones , Ratones Noqueados , Mutación/inmunología , Alineación de Secuencia , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología
4.
Eur J Immunol ; 53(11): e2249816, 2023 11.
Artículo en Inglés | MEDLINE | ID: mdl-36303448

RESUMEN

This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state-of-the-art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy, and functional characterization of mouse and human dendritic cells (DC) from lymphoid organs and various non-lymphoid tissues. This article provides protocols with top ticks and pitfalls for preparation and successful generation of mouse and human DC from different cellular sources, such as murine BM and HoxB8 cells, as well as human CD34+ cells from cord blood, BM, and peripheral blood or peripheral blood monocytes. We describe murine cDC1, cDC2, and pDC generation with Flt3L and the generation of BM-derived DC with GM-CSF. Protocols for human DC generation focus on CD34+ cell culture on OP9 cell layers for cDC1, cDC2, cDC3, and pDC subset generation and DC generation from peripheral blood monocytes (MoDC). Additional protocols include enrichment of murine DC subsets, CRISPR/Cas9 editing, and clinical grade human DC generation. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer-reviewed by leading experts and approved by all co-authors, making it an essential resource for basic and clinical DC immunologists.


Asunto(s)
Células Dendríticas , Monocitos , Animales , Ratones , Humanos , Antígenos CD34 , Fenotipo , Diferenciación Celular
5.
J Immunol ; 209(11): 2083-2092, 2022 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-36426970

RESUMEN

Costimulatory CD40 plays an essential role in autoimmune diseases, including experimental autoimmune encephalomyelitis (EAE), a murine model of human multiple sclerosis (MS). However, how CD40 drives autoimmune disease pathogenesis is not well defined. Here, we used a conditional knockout approach to determine how CD40 orchestrates a CNS autoimmune disease induced by recombinant human myelin oligodendrocyte glycoprotein (rhMOG). We found that deletion of CD40 in either dendritic cells (DCs) or B cells profoundly reduced EAE disease pathogenesis. Mechanistically, CD40 expression on DCs was required for priming pathogenic Th cells in peripheral draining lymph nodes and promoting their appearance in the CNS. By contrast, B cell CD40 was essential for class-switched MOG-specific Ab production, which played a crucial role in disease pathogenesis. In fact, passive transfer of MOG-immune serum or IgG into mice lacking CD40 on B cells but not DCs reconstituted autoimmune disease, which was associated with inundation of the spinal cord parenchyma by Ig and complement. These data demonstrate that CD40 supports distinct effector programs in B cells and DCs that converge to drive a CNS autoimmune disease and identify targets for intervention.


Asunto(s)
Enfermedades Autoinmunes del Sistema Nervioso , Enfermedades del Sistema Nervioso Central , Encefalomielitis Autoinmune Experimental , Humanos , Animales , Ratones , Antígenos CD40 , Recuento de Linfocitos , Células Dendríticas
6.
BMC Biol ; 21(1): 205, 2023 10 02.
Artículo en Inglés | MEDLINE | ID: mdl-37784185

RESUMEN

BACKGROUND: After the eradication of smallpox in China in 1979, vaccination with the vaccinia virus (VACV) Tiantan strain for the general population was stopped in 1980. As the monkeypox virus (MPXV) is rapidly spreading in the world, we would like to investigate whether the individuals with historic VACV Tiantan strain vaccination, even after more than 40 years, could still provide ELISA reactivity and neutralizing protection; and whether the unvaccinated individuals have no antibody reactivity against MPXV at all. RESULTS: We established serologic ELISA to measure the serum anti-MPXV titer by using immunodominant MPXV surface proteins, A35R, B6R, A29L, and M1R. A small proportion of individuals (born before 1980) with historic VACV Tiantan strain vaccination exhibited serum ELISA cross-reactivity against these MPXV surface proteins. Consistently, these donors also showed ELISA seropositivity and serum neutralization against VACV Tiantan strain. However, surprisingly, some unvaccinated young adults (born after 1980) also showed potent serum ELISA activity against MPXV proteins, possibly due to their past infection by some self-limiting Orthopoxvirus (OPXV). CONCLUSIONS: We report the serum ELISA cross-reactivity against MPXV surface protein in a small proportion of individuals both with and without VACV Tiantan strain vaccination history. Combined with our serum neutralization assay against VACV and the recent literature about mice vaccinated with VACV Tiantan strain, our study confirmed the anti-MPXV cross-reactivity and cross-neutralization of smallpox vaccine using VACV Tiantan strain. Therefore, it is necessary to restart the smallpox vaccination program in high risk populations.


Asunto(s)
Reacciones Cruzadas , Monkeypox virus , Vacuna contra Viruela , Vacunación , Animales , Humanos , Ratones , Adulto Joven , Formación de Anticuerpos , Pueblos del Este de Asia , Proteínas de la Membrana , Viruela/prevención & control , Virus Vaccinia , Vacuna contra Viruela/inmunología , Vacuna contra Viruela/uso terapéutico , China
7.
Nat Immunol ; 12(9): 898-907, 2011 Aug 14.
Artículo en Inglés | MEDLINE | ID: mdl-21841785

RESUMEN

Regulatory T cells (T(reg) cells) are essential for self-tolerance and immune homeostasis. Lack of effector T cell (T(eff) cell) function and gain of suppressive activity by T(reg) cells are dependent on the transcriptional program induced by Foxp3. Here we report that repression of SATB1, a genome organizer that regulates chromatin structure and gene expression, was crucial for the phenotype and function of T(reg) cells. Foxp3, acting as a transcriptional repressor, directly suppressed the SATB1 locus and indirectly suppressed it through the induction of microRNAs that bound the SATB1 3' untranslated region. Release of SATB1 from the control of Foxp3 in T(reg) cells caused loss of suppressive function, establishment of transcriptional T(eff) cell programs and induction of T(eff) cell cytokines. Our data support the proposal that inhibition of SATB1-mediated modulation of global chromatin remodeling is pivotal for maintaining T(reg) cell functionality.


Asunto(s)
Ensamble y Desensamble de Cromatina/inmunología , Factores de Transcripción Forkhead/inmunología , Regulación de la Expresión Génica , Proteínas de Unión a la Región de Fijación a la Matriz/inmunología , Autotolerancia , Linfocitos T Reguladores/inmunología , Regiones no Traducidas 3'/genética , Regiones no Traducidas 3'/inmunología , Animales , Diferenciación Celular/efectos de los fármacos , Ensamble y Desensamble de Cromatina/efectos de los fármacos , Citometría de Flujo , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Perfilación de la Expresión Génica , Genoma Humano , Estudio de Asociación del Genoma Completo , Humanos , Lentivirus , Activación de Linfocitos/efectos de los fármacos , Proteínas de Unión a la Región de Fijación a la Matriz/genética , Proteínas de Unión a la Región de Fijación a la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , MicroARNs/inmunología , MicroARNs/metabolismo , MicroARNs/farmacología , Interferencia de ARN , ARN Interferente Pequeño/inmunología , ARN Interferente Pequeño/metabolismo , ARN Interferente Pequeño/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Autotolerancia/efectos de los fármacos , Autotolerancia/genética , Autotolerancia/inmunología , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/metabolismo , Transducción Genética
8.
J Med Virol ; 95(2): e28440, 2023 02.
Artículo en Inglés | MEDLINE | ID: mdl-36573441

RESUMEN

Emergence of various circulating SARS-CoV-2 variants of concern (VOCs) promotes the identification of pan-sarbecovirus vaccines and broadly neutralizing antibodies (bNAbs). Here, to characterize monoclonal antibodies cross-reactive against both SARS-CoV-1 and SARS-CoV-2 and to search the criterion for bNAbs against all emerging SARS-CoV-2, we isolated several SARS-CoV-1-cross-reactive monoclonal antibodies (mAbs) from a wildtype SARS-CoV-2 convalescent donor. These antibodies showed broad binding capacity and cross-neutralizing potency against various SARS-CoV-2 VOCs, including B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), and B.1.617.2 (Delta), but failed to efficiently neutralize Omicron variant and its sublineages. Structural analysis revealed how Omicron sublineages, but not other VOCs, efficiently evade an antibody family cross-reactive against SARS-CoV-1 through their escape mutations. Further evaluation of a series of SARS-CoV-1/2-cross-reactive bNAbs showed a negative correlation between the neutralizing activities against SARS-CoV-1 and SARS-CoV-2 Omicron variant. Together, these results suggest the necessity of using cross-neutralization against SARS-CoV-1 and SARS-CoV-2 Omicron as criteria for rational design and development of potent pan-sarbecovirus vaccines and bNAbs.


Asunto(s)
COVID-19 , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo , Vacunas , Humanos , SARS-CoV-2 , Anticuerpos Neutralizantes , Anticuerpos Monoclonales , Anticuerpos ampliamente neutralizantes , Anticuerpos Antivirales , Glicoproteína de la Espiga del Coronavirus
9.
Proc Natl Acad Sci U S A ; 117(40): 24957-24963, 2020 10 06.
Artículo en Inglés | MEDLINE | ID: mdl-32963096

RESUMEN

B lymphocytes acquire self-reactivity as an unavoidable byproduct of antibody gene diversification in the bone marrow and in germinal centers (GCs). Autoreactive B cells emerging from the bone marrow are silenced in a series of well-defined checkpoints, but less is known about how self-reactivity that develops by somatic mutation in GCs is controlled. Here, we report the existence of an apoptosis-dependent tolerance checkpoint in post-GC B cells. Whereas defective GC B cell apoptosis has no measurable effect on autoantibody development, disruption of post-GC apoptosis results in accumulation of autoreactive memory B cells and plasma cells, antinuclear antibody production, and autoimmunity. The data presented shed light on mechanisms that regulate immune tolerance and the development of autoantibodies.


Asunto(s)
Apoptosis/genética , Autoinmunidad/genética , Genes de Inmunoglobulinas/genética , Tolerancia Inmunológica/genética , Animales , Anticuerpos Antinucleares/inmunología , Apoptosis/inmunología , Autoanticuerpos/inmunología , Autoinmunidad/inmunología , Linfocitos B/inmunología , Genes de Inmunoglobulinas/inmunología , Centro Germinal/inmunología , Humanos , Memoria Inmunológica/genética , Memoria Inmunológica/inmunología , Ratones , Células Plasmáticas/inmunología
10.
Proc Natl Acad Sci U S A ; 111(52): 18667-72, 2014 Dec 30.
Artículo en Inglés | MEDLINE | ID: mdl-25512519

RESUMEN

Activation-induced cytidine deaminase (AID) initiates class switch recombination (CSR) and somatic hypermutation (SHM) by deaminating cytosine residues in immunoglobulin genes (Igh, Igκ, and Igλ). At a lower frequency, AID also causes collateral DNA damage at non-Ig loci, including genes that are rearranged or mutated in B-cell lymphoma. Precisely how AID is recruited to these off-target sites is not entirely understood. To gain further insight into how AID selects its targets, we compared AID-mediated translocations in two different cell types, B cells and mouse embryonic fibroblasts (MEFs). AID targets a distinct set of hotspots in the two cell types. In both cases, hotspots are concentrated in highly transcribed but stalled genes. However, transcription alone is insufficient to recruit AID activity. Comparison of genes similarly transcribed in B cells and MEFs but targeted in only one of the two cell types reveals a common set of epigenetic features associated with AID recruitment in both cells. AID target genes are enriched in chromatin modifications associated with active enhancers (such as H3K27Ac) and marks of active transcription (such as H3K36me3) in both fibroblasts and B cells, indicating that these features are universal mediators of AID recruitment.


Asunto(s)
Linfocitos B/enzimología , Citidina Desaminasa , Embrión de Mamíferos/enzimología , Epigénesis Genética , Marcación de Gen , Transcripción Genética/fisiología , Animales , Linfocitos B/citología , Línea Celular , Citidina Desaminasa/genética , Citidina Desaminasa/metabolismo , Embrión de Mamíferos/citología , Fibroblastos/citología , Fibroblastos/enzimología , Histonas/genética , Histonas/metabolismo , Inmunoglobulinas/biosíntesis , Inmunoglobulinas/genética , Ratones , Ratones Noqueados
11.
Eur J Immunol ; 44(10): 2990-3002, 2014 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-25042334

RESUMEN

Foxp3 specifies the Treg cell lineage and is indispensable for immune tolerance. Accordingly, rare Foxp3 mutations cause lethal autoimmunity. The mechanisms precipitating more prevalent human autoimmune diseases are poorly understood, but involve a combination of genetic and environmental factors. Many autoimmune diseases associate with a partial Treg-cell dysfunction, yet mouse models reflecting such complex pathophysiological processes are rare. Around 95% of Foxp3(+) Treg cells can be specifically depleted in bacterial artifical chromosome (BAC)-transgenic Depletion of REGulatory T cells (DEREG) mice through diphtheria toxin (DT) treatment. However, Treg-cell depletion fails to cause autoimmunity in adult DEREG mice for unclear reasons. By crossing Foxp3(GFP) knock-in mice to DEREG mice, we introduced additional genetic susceptibility that does not affect untreated mice. Strikingly, DT treatment of DEREG × Foxp3(GFP) mice rapidly causes autoimmunity characterized by blepharitis, tissue damage, and autoantibody production. This inflammatory disease is associated with augmented T-cell activation, increased Th2 cytokine production and myeloproliferation, and is caused by defective Treg-cell homeostasis, preventing few DT-insensitive Treg cells from repopulating the niche after Treg-cell depletion. Our study provides important insights into self-tolerance. We further highlight DEREG × Foxp3(GFP) mice as a model to investigate the role of environmental factors in precipitating autoimmunity. This may help to better understand and treat human autoimmunity.


Asunto(s)
Enfermedades Autoinmunes/inmunología , Autoinmunidad/inmunología , Activación de Linfocitos/inmunología , Linfocitos T Reguladores/inmunología , Animales , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Factores de Transcripción Forkhead/inmunología , Técnicas de Sustitución del Gen , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos
12.
Blood ; 121(4): 679-91, 2013 Jan 24.
Artículo en Inglés | MEDLINE | ID: mdl-23160472

RESUMEN

We have recently identified T cells as important mediators of ischemic brain damage, but the contribution of the different T-cell subsets is unclear. Forkhead box P3 (FoxP3)-positive regulatory T cells (Tregs) are generally regarded as prototypic anti-inflammatory cells that maintain immune tolerance and counteract tissue damage in a variety of immune-mediated disorders. In the present study, we examined the role of Tregs after experimental brain ischemia/reperfusion injury. Selective depletion of Tregs in the DEREG mouse model dramatically reduced infarct size and improved neurologic function 24 hours after stroke and this protective effect was preserved at later stages of infarct development. The specificity of this detrimental Treg effect was confirmed by adoptive transfer experiments in wild-type mice and in Rag1(-/-) mice lacking lymphocytes. Mechanistically, Tregs induced microvascular dysfunction in vivo by increased interaction with the ischemic brain endothelium via the LFA-1/ICAM-1 pathway and platelets and these findings were confirmed in vitro. Ablation of Tregs reduced microvascular thrombus formation and improved cerebral reperfusion on stroke, as revealed by ultra-high-field magnetic resonance imaging at 17.6 Tesla. In contrast, established immunoregulatory characteristics of Tregs had no functional relevance. We define herein a novel and unexpected role of Tregs in a primary nonimmunologic disease state.


Asunto(s)
Isquemia Encefálica/inmunología , Microvasos/fisiopatología , Accidente Cerebrovascular/metabolismo , Linfocitos T Reguladores/metabolismo , Traslado Adoptivo , Animales , Plaquetas/inmunología , Plaquetas/metabolismo , Encéfalo/inmunología , Encéfalo/metabolismo , Encéfalo/patología , Isquemia Encefálica/genética , Isquemia Encefálica/terapia , Comunicación Celular , Modelos Animales de Enfermedad , Células Endoteliales/inmunología , Células Endoteliales/metabolismo , Depleción Linfocítica , Masculino , Ratones , Ratones Noqueados , Microvasos/patología , Accidente Cerebrovascular/inmunología , Accidente Cerebrovascular/terapia , Linfocitos T Reguladores/inmunología
13.
Eur J Immunol ; 43(10): 2543-53, 2013 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-23784881

RESUMEN

Vaccination is one of the oldest yet still most effective methods to prevent infectious diseases. However, eradication of intracellular pathogens and treatment of certain diseases like cancer requiring efficient cytotoxic immune responses remain a medical challenge. In mice, a successful approach to induce strong cytotoxic CD8⁺ T-cell (CTL) reactions is to target antigens to DCs using specific antibodies against surface receptors in combination with adjuvants. A major drawback for translating this strategy into one for the clinic is the lack of analogous targets in human DCs. DC-SIGN (DC-specific-ICAM3-grabbing-nonintegrin/CD209) is a C-type lectin receptor with potent endocytic capacity and a highly restricted expression on human immature DCs. Therefore, DC-SIGN represents an ideal candidate for DC targeting. Using transgenic mice that express human DC-SIGN under the control of the murine CD11c promoter (hSIGN mice), we explored the efficacy of anti-DC-SIGN antibodies to target antigens to DCs and induce protective immune responses in vivo. We show that anti-DC-SIGN antibodies conjugated to OVA induced strong and persistent antigen-specific CD4⁺ and CD8⁺ T-cell responses, which efficiently protected from infection with OVA-expressing Listeria monocytogenes. Thus, we propose DC targeting via DC-SIGN as a promising strategy for novel vaccination protocols against intracellular pathogens.


Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Moléculas de Adhesión Celular/metabolismo , Células Dendríticas/inmunología , Lectinas Tipo C/metabolismo , Listeria monocytogenes/inmunología , Receptores de Superficie Celular/metabolismo , Adyuvantes Inmunológicos/genética , Adyuvantes Inmunológicos/metabolismo , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/metabolismo , Antígeno CD11c/genética , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/inmunología , Humanos , Inmunidad Activa , Inmunidad Celular , Inmunomodulación , Lectinas Tipo C/genética , Lectinas Tipo C/inmunología , Listeria monocytogenes/genética , Ratones , Ratones Transgénicos , Ovalbúmina/genética , Ovalbúmina/metabolismo , Regiones Promotoras Genéticas/genética , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/inmunología , Transgenes/genética , Vacunación
14.
J Immunol ; 187(4): 1684-91, 2011 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-21746966

RESUMEN

CD4(+)CD25(++)Foxp3(+) regulatory T cells (Tregs) control self-reactive cells to maintain peripheral tolerance. Treg homeostasis has to be controlled tightly to ensure balanced Treg-mediated suppression. One mechanism that regulates the CD4(+) T cell pool is activation-induced cell death (AICD). This is mimicked in vitro by TCR restimulation-induced expression of the death ligand CD95L (FasL/APO-1L/CD178) in expanded T cells. These cells express the death receptor CD95 (Fas/APO-1), and binding of CD95L to CD95 results in AICD. In contrast, Tregs do not undergo AICD upon TCR (re)stimulation in vitro despite a functional CD95 cell death pathway. In this study, we show that human and murine Tregs express low levels of CD95L upon stimulation. Knockdown of the transcriptional repressor Foxp3 partially rescues CD95L expression and AICD in human Tregs. Moreover, upon stimulation Foxp3-mutant Tregs from Scurfy mice express CD95L similar to conventional T cells. We further addressed whether exogenous CD95 stimulation provides a mechanism of Treg homeostatic control in vivo in mice. Triggering of CD95 reduced Treg numbers systemically as reflected by in vivo imaging and decreased GFP(+) Treg numbers ex vivo. Our study reveals that Foxp3 negatively regulates CD95L expression in Tregs and demonstrates that Tregs are susceptible to homeostatic control by CD95 stimulation.


Asunto(s)
Proteína Ligando Fas/inmunología , Factores de Transcripción Forkhead/inmunología , Regulación de la Expresión Génica/inmunología , Linfocitos T Reguladores/inmunología , Animales , Muerte Celular/genética , Muerte Celular/inmunología , Proteína Ligando Fas/genética , Proteína Ligando Fas/metabolismo , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Regulación de la Expresión Génica/genética , Ratones , Ratones Noqueados , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T Reguladores/metabolismo
15.
Cell Rep ; 42(5): 112503, 2023 05 30.
Artículo en Inglés | MEDLINE | ID: mdl-37178120

RESUMEN

Striking antibody evasion by emerging circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants drives the identification of broadly neutralizing antibodies (bNAbs). However, how a bNAb acquires increased neutralization breadth during antibody evolution is still elusive. Here, we identify a clonally related antibody family from a convalescent individual. One of the members, XG005, exhibits potent and broad neutralizing activities against SARS-CoV-2 variants, while the other members show significant reductions in neutralization breadth and potency, especially against the Omicron sublineages. Structural analysis visualizing the XG005-Omicron spike binding interface reveals how crucial somatic mutations endow XG005 with greater neutralization potency and breadth. A single administration of XG005 with extended half-life, reduced antibody-dependent enhancement (ADE) effect, and increased antibody product quality exhibits a high therapeutic efficacy in BA.2- and BA.5-challenged mice. Our results provide a natural example to show the importance of somatic hypermutation during antibody evolution for SARS-CoV-2 neutralization breadth and potency.


Asunto(s)
COVID-19 , SARS-CoV-2 , Animales , Ratones , Anticuerpos , Anticuerpos ampliamente neutralizantes , Mutación/genética , Anticuerpos Antivirales , Anticuerpos Neutralizantes
16.
Eur J Immunol ; 41(3): 716-25, 2011 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-21312192

RESUMEN

"Suppressor T cells" were historically defined within the CD8(+) T-cell compartment and recent studies have highlighted several naturally occurring CD8(+) Foxp3(-) Treg populations. However, the relevance of CD8(+) Foxp3(+) T cells, which represent a minor population in both thymi and secondary lymphoid organs of nonmanipulated mice, remains unclear. We here demonstrate that de novo Foxp3 induction in peripheral CD8(+) Foxp3(-) T cells is counter-regulated by DC-mediated co-stimulation via CD80/CD86. CD8(+) Foxp3(+) T cells fail to develop in TCR-transgenic mice with Rag1(-/-) background, similar to classical CD4(+) Foxp3(+) Tregs. Notably, both naturally occurring and induced CD8(+) Foxp3(+) T cells express bona fide Treg markers including CD25, GITR, CTLA4 and CD103, and show defective IFN-γ production upon restimulation when compared with their CD8(+) Foxp3(-) counterparts. However, utilizing DEREG transgenic mice for the isolation of Foxp3(+) cells by eGFP reporter expression, we demonstrate that induced CD8(+) Foxp3(+) T cells similar to activated CD8(+) Foxp3(-) T cells only mildly suppress T-cell proliferation and IFN-γ production. We therefore categorize CD8(+) Foxp3(+) T cells as a tightly controlled population sharing certain developmental and phenotypic properties with classical CD4(+) Foxp3(+) Tregs, but lacking potent suppressive activity.


Asunto(s)
Linfocitos T CD8-positivos/inmunología , Linfocitos T Reguladores/inmunología , Animales , Antígeno B7-1/metabolismo , Antígeno B7-2/metabolismo , Antígenos CD28/metabolismo , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/metabolismo , Diferenciación Celular , Proliferación Celular , Células Dendríticas/inmunología , Factores de Transcripción Forkhead/metabolismo , Técnicas In Vitro , Interferón gamma/biosíntesis , Activación de Linfocitos , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Fenotipo , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/metabolismo , Factor de Crecimiento Transformador beta/metabolismo
17.
bioRxiv ; 2022 Dec 13.
Artículo en Inglés | MEDLINE | ID: mdl-36561175

RESUMEN

Striking antibody evasion by emerging circulating SARS-CoV-2 variants drives the identification of broadly neutralizing antibodies (bNAbs). However, how a bNAb acquires increased neutralization breadth during antibody evolution is still elusive. Here, we identified a clonally-related antibody family from a convalescent individual. One of the members, XG005, exhibited potent and broad neutralizing activities against SARS-CoV-2 variants, while the other members showed significant reductions in neutralization breadth and potency, especially against the Omicron sublineages. Structural analysis visualizing the XG005-Omicron spike binding interface revealed how crucial somatic mutations endowed XG005 with greater neutralization potency and breadth. A single administration of XG005 with extended half-life, reduced antibody-dependent enhancement (ADE) effect, and increased antibody product quality, exhibited a high therapeutic efficacy in BA.2- and BA.5-challenged mice. Our results provided a natural example to show the importance of somatic hypermutation during antibody evolution for SARS-CoV-2 neutralization breadth and potency.

18.
Protein Cell ; 13(9): 655-675, 2022 09.
Artículo en Inglés | MEDLINE | ID: mdl-34554412

RESUMEN

New threats posed by the emerging circulating variants of SARS-CoV-2 highlight the need to find conserved neutralizing epitopes for therapeutic antibodies and efficient vaccine design. Here, we identified a receptor-binding domain (RBD)-binding antibody, XG014, which potently neutralizes ß-coronavirus lineage B (ß-CoV-B), including SARS-CoV-2, its circulating variants, SARS-CoV and bat SARSr-CoV WIV1. Interestingly, antibody family members competing with XG014 binding show reduced levels of cross-reactivity and induce antibody-dependent SARS-CoV-2 spike (S) protein-mediated cell-cell fusion, suggesting a unique mode of recognition by XG014. Structural analyses reveal that XG014 recognizes a conserved epitope outside the ACE2 binding site and completely locks RBD in the non-functional "down" conformation, while its family member XG005 directly competes with ACE2 binding and position the RBD "up". Single administration of XG014 is effective in protection against and therapy of SARS-CoV-2 infection in vivo. Our findings suggest the potential to develop XG014 as pan-ß-CoV-B therapeutics and the importance of the XG014 conserved antigenic epitope for designing broadly protective vaccines against ß-CoV-B and newly emerging SARS-CoV-2 variants of concern.


Asunto(s)
COVID-19 , SARS-CoV-2 , Enzima Convertidora de Angiotensina 2 , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Epítopos , Humanos , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/genética
19.
J Immunol ; 183(9): 5662-72, 2009 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-19812199

RESUMEN

Foxp3(+) regulatory T cells (Tregs) are crucial for preventing autoimmunity. We have demonstrated that depletion of Foxp3(+) Tregs results in the development of a scurfy-like disease, indicating that Foxp3(-) effector T cells are sufficient to induce autoimmunity. It has been postulated that nonfunctional Tregs carrying potentially self-reactive T cell receptors may contribute to scurfy (sf) pathogenesis due to enhanced recognition of self. Those cells, however, could not be identified in sf mutants due to the lack of Foxp3 protein expression. To address this issue, we crossed the natural sf mouse mutant with bacterial artificial chromosome transgenic DEREG (depletion of regulatory T cells) mice. Since DEREG mice express GFP under the control of an additional Foxp3 promoter, those crossings allowed proving the existence of "would-be" Tregs, which are characterized by GFP expression in the absence of functional Foxp3. Sf Tregs lost their in vitro suppressive capacity. This correlated with a substantial reduction of intracellular cAMP levels, whereas surface expression of Treg markers was unaffected. Both GFP(+) and GFP(-) sf cells produced high amounts of Th2-type cytokines, reflected also by enhanced Gata-3 expression, when tested in vitro. Nevertheless, sf Tregs could be induced in vitro, although with lower efficiency than DEREG Tregs. Transfer of GFP(+) sf Tregs, in contrast to GFP(-) sf T cells, into RAG1-deficient animals did not cause the sf phenotype. Taken together, natural and induced Tregs develop in the absence of Foxp3 in sf mice, which lack both suppressive activity and autoreactive potential, but rather display a Th2-biased phenotype.


Asunto(s)
Citocinas/biosíntesis , Factores de Transcripción Forkhead/deficiencia , Factores de Transcripción Forkhead/genética , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/patología , Células Th2/inmunología , Células Th2/patología , Animales , Diferenciación Celular/genética , Diferenciación Celular/inmunología , AMP Cíclico/deficiencia , AMP Cíclico/genética , Citocinas/deficiencia , Factores de Transcripción Forkhead/biosíntesis , Factor de Transcripción GATA3/biosíntesis , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/deficiencia , Proteínas Fluorescentes Verdes/genética , Linfopenia/genética , Linfopenia/inmunología , Linfopenia/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes , Ratones Transgénicos , Linfocitos T Reguladores/trasplante , Células Th2/metabolismo
20.
Cell Rep ; 34(5): 108699, 2021 02 02.
Artículo en Inglés | MEDLINE | ID: mdl-33485405

RESUMEN

Several potent neutralizing antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus have been identified. However, antibody-dependent enhancement (ADE) has not been comprehensively studied for SARS-CoV-2, and the relationship between enhancing versus neutralizing activities and antibody epitopes remains unknown. Here, we select a convalescent individual with potent IgG neutralizing activity and characterize his antibody response. Monoclonal antibodies isolated from memory B cells target four groups of five non-overlapping receptor-binding domain (RBD) epitopes. Antibodies to one group of these RBD epitopes mediate ADE of entry in Raji cells via an Fcγ receptor-dependent mechanism. In contrast, antibodies targeting two other distinct epitope groups neutralize SARS-CoV-2 without ADE, while antibodies against the fourth epitope group are poorly neutralizing. One antibody, XG014, potently cross-neutralizes SARS-CoV-2 variants, as well as SARS-CoV-1, with respective IC50 (50% inhibitory concentration) values as low as 5.1 and 23.7 ng/mL, while not exhibiting ADE. Therefore, neutralization and ADE of human SARS-CoV-2 antibodies correlate with non-overlapping RBD epitopes.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Acrecentamiento Dependiente de Anticuerpo , Epítopos/inmunología , Adolescente , Adulto , Anciano , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/uso terapéutico , Reacciones Antígeno-Anticuerpo , COVID-19/inmunología , COVID-19/virología , Línea Celular , Niño , Análisis por Conglomerados , Femenino , Humanos , Concentración 50 Inhibidora , Masculino , Persona de Mediana Edad , Dominios Proteicos/inmunología , SARS-CoV-2/aislamiento & purificación , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/inmunología , Adulto Joven , Tratamiento Farmacológico de COVID-19
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