RESUMEN
Anti-programmed death-1 (anti-PD-1) immunotherapy reinvigorates CD8 T cell responses in patients with cancer but PD-1 is also expressed by other immune cells, including follicular helper CD4 T cells (Tfh) which are involved in germinal centre responses. Little is known, however, about the effects of anti-PD-1 immunotherapy on noncancer immune responses in humans. To investigate this question, we examined the impact of anti-PD-1 immunotherapy on the Tfh-B cell axis responding to unrelated viral antigens. Following influenza vaccination, a subset of adults receiving anti-PD-1 had more robust circulating Tfh responses than adults not receiving immunotherapy. PD-1 pathway blockade resulted in transcriptional signatures of increased cellular proliferation in circulating Tfh and responding B cells compared with controls. These latter observations suggest an underlying change in the Tfh-B cell and germinal centre axis in a subset of immunotherapy patients. Together, these results demonstrate dynamic effects of anti-PD-1 therapy on influenza vaccine responses and highlight analytical vaccination as an approach that may reveal underlying immune predisposition to adverse events.
Asunto(s)
Vacunas contra la Influenza , Adulto , Humanos , Inmunidad Humoral , Estaciones del Año , Linfocitos T Colaboradores-Inductores , VacunaciónRESUMEN
TCF-1 is a key transcription factor in progenitor exhausted CD8 T cells (Tex). Moreover, this Tex cell subset mediates responses to PD-1 checkpoint pathway blockade. However, the role of the transcription factor TCF-1 in early fate decisions and initial generation of Tex cells is unclear. Single-cell RNA sequencing (scRNA-seq) and lineage tracing identified a TCF-1+Ly108+PD-1+ CD8 T cell population that seeds development of mature Tex cells early during chronic infection. TCF-1 mediated the bifurcation between divergent fates, repressing development of terminal KLRG1Hi effectors while fostering KLRG1Lo Tex precursor cells, and PD-1 stabilized this TCF-1+ Tex precursor cell pool. TCF-1 mediated a T-bet-to-Eomes transcription factor transition in Tex precursors by promoting Eomes expression and drove c-Myb expression that controlled Bcl-2 and survival. These data define a role for TCF-1 in early-fate-bifurcation-driving Tex precursor cells and also identify PD-1 as a protector of this early TCF-1 subset.
Asunto(s)
Linfocitos T CD8-positivos/metabolismo , Redes Reguladoras de Genes , Factor 1 de Transcripción de Linfocitos T/metabolismo , Transcripción Genética , Animales , Linfocitos T CD8-positivos/inmunología , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Enfermedad Crónica , Perfilación de la Expresión Génica , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Ratones , Receptor de Muerte Celular Programada 1/metabolismo , Factor 1 de Transcripción de Linfocitos T/genética , Virosis/genética , Virosis/inmunología , Virosis/virologíaRESUMEN
Exhausted CD8+ T (Tex) cells in chronic infections and cancer have limited effector function, high co-expression of inhibitory receptors and extensive transcriptional changes compared with effector (Teff) or memory (Tmem) CD8+ T cells. Tex cells are important clinical targets of checkpoint blockade and other immunotherapies. Epigenetically, Tex cells are a distinct immune subset, with a unique chromatin landscape compared with Teff and Tmem cells. However, the mechanisms that govern the transcriptional and epigenetic development of Tex cells remain unknown. Here we identify the HMG-box transcription factor TOX as a central regulator of Tex cells in mice. TOX is largely dispensable for the formation of Teff and Tmem cells, but it is critical for exhaustion: in the absence of TOX, Tex cells do not form. TOX is induced by calcineurin and NFAT2, and operates in a feed-forward loop in which it becomes calcineurin-independent and sustained in Tex cells. Robust expression of TOX therefore results in commitment to Tex cells by translating persistent stimulation into a distinct Tex cell transcriptional and epigenetic developmental program.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Epistasis Genética , Proteínas de Homeodominio/metabolismo , Transcripción Genética , Animales , Calcineurina/metabolismo , Señalización del Calcio , Retroalimentación Fisiológica , Femenino , Regulación de la Expresión Génica/inmunología , Genotipo , Memoria Inmunológica , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Factores de Transcripción NFATC/metabolismo , Escape del TumorRESUMEN
Despite the success of monotherapies based on blockade of programmed cell death 1 (PD-1) in human melanoma, most patients do not experience durable clinical benefit. Pre-existing T-cell infiltration and/or the presence of PD-L1 in tumours may be used as indicators of clinical response; however, blood-based profiling to understand the mechanisms of PD-1 blockade has not been widely explored. Here we use immune profiling of peripheral blood from patients with stage IV melanoma before and after treatment with the PD-1-targeting antibody pembrolizumab and identify pharmacodynamic changes in circulating exhausted-phenotype CD8 T cells (Tex cells). Most of the patients demonstrated an immunological response to pembrolizumab. Clinical failure in many patients was not solely due to an inability to induce immune reinvigoration, but rather resulted from an imbalance between T-cell reinvigoration and tumour burden. The magnitude of reinvigoration of circulating Tex cells determined in relation to pretreatment tumour burden correlated with clinical response. By focused profiling of a mechanistically relevant circulating T-cell subpopulation calibrated to pretreatment disease burden, we identify a clinically accessible potential on-treatment predictor of response to PD-1 blockade.
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Linfocitos T CD8-positivos/inmunología , Melanoma/tratamiento farmacológico , Melanoma/inmunología , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/inmunología , Carga Tumoral/inmunología , Anticuerpos Monoclonales Humanizados/administración & dosificación , Anticuerpos Monoclonales Humanizados/inmunología , Anticuerpos Monoclonales Humanizados/farmacocinética , Anticuerpos Monoclonales Humanizados/uso terapéutico , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/metabolismo , Femenino , Humanos , Antígeno Ki-67/inmunología , Antígeno Ki-67/metabolismo , Masculino , Melanoma/irrigación sanguínea , Melanoma/patología , Estadificación de Neoplasias , Fenotipo , Resultado del TratamientoRESUMEN
Antibodies capable of neutralizing HIV-1 often target variable regions 1 and 2 (V1V2) of the HIV-1 envelope, but the mechanism of their elicitation has been unclear. Here we define the developmental pathway by which such antibodies are generated and acquire the requisite molecular characteristics for neutralization. Twelve somatically related neutralizing antibodies (CAP256-VRC26.01-12) were isolated from donor CAP256 (from the Centre for the AIDS Programme of Research in South Africa (CAPRISA)); each antibody contained the protruding tyrosine-sulphated, anionic antigen-binding loop (complementarity-determining region (CDR) H3) characteristic of this category of antibodies. Their unmutated ancestor emerged between weeks 30-38 post-infection with a 35-residue CDR H3, and neutralized the virus that superinfected this individual 15 weeks after initial infection. Improved neutralization breadth and potency occurred by week 59 with modest affinity maturation, and was preceded by extensive diversification of the virus population. HIV-1 V1V2-directed neutralizing antibodies can thus develop relatively rapidly through initial selection of B cells with a long CDR H3, and limited subsequent somatic hypermutation. These data provide important insights relevant to HIV-1 vaccine development.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Anti-VIH/inmunología , Proteínas gp160 de Envoltorio del VIH/química , Proteínas gp160 de Envoltorio del VIH/inmunología , Vacunas contra el SIDA/química , Vacunas contra el SIDA/inmunología , Secuencia de Aminoácidos , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/genética , Anticuerpos Neutralizantes/aislamiento & purificación , Afinidad de Anticuerpos/genética , Afinidad de Anticuerpos/inmunología , Linfocitos B/citología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Sitios de Unión/inmunología , Antígenos CD4/inmunología , Antígenos CD4/metabolismo , Linaje de la Célula , Regiones Determinantes de Complementariedad/química , Regiones Determinantes de Complementariedad/genética , Regiones Determinantes de Complementariedad/inmunología , Mapeo Epitopo , Epítopos de Linfocito B/química , Epítopos de Linfocito B/inmunología , Evolución Molecular , Anticuerpos Anti-VIH/química , Anticuerpos Anti-VIH/genética , Anticuerpos Anti-VIH/aislamiento & purificación , Infecciones por VIH/inmunología , VIH-1/química , VIH-1/inmunología , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Pruebas de Neutralización , Estructura Terciaria de Proteína , Hipermutación Somática de Inmunoglobulina/genéticaRESUMEN
The role of Ab and B cells in preventing infection is established. In contrast, the role of B cell responses in containing chronic infections remains poorly understood. IgG2a (IgG1 in humans) can prevent acute infections, and T-bet promotes IgG2a isotype switching. However, whether IgG2a and B cell-expressed T-bet influence the host-pathogen balance during persisting infections is unclear. We demonstrate that B cell-specific loss of T-bet prevents control of persisting viral infection. T-bet in B cells controlled IgG2a production, as well as mucosal localization, proliferation, glycosylation, and a broad transcriptional program. T-bet controlled a broad antiviral program in addition to IgG2a because T-bet in B cells was important, even in the presence of virus-specific IgG2a. Our data support a model in which T-bet is a universal controller of antiviral immunity across multiple immune lineages.
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Subgrupos de Linfocitos B/inmunología , Linfocitos B/inmunología , Coriomeningitis Linfocítica/inmunología , Proteínas de Dominio T Box/inmunología , Animales , Separación Celular , Enfermedad Crónica , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Inmunoglobulina G/inmunología , Virus de la Coriomeningitis Linfocítica , Ratones , Ratones Transgénicos , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
UNLABELLED: The epitopes defined by HIV-1 broadly neutralizing antibodies (bNAbs) are valuable templates for vaccine design, and studies of the immunological development of these antibodies are providing insights for vaccination strategies. In addition, the most potent and broadly reactive of these bNAbs have potential for clinical use. We previously described a family of 12 V1V2-directed neutralizing antibodies, CAP256-VRC26, isolated from an HIV-1 clade C-infected donor at years 1, 2, and 4 of infection (N. A. Doria-Rose et al., Nature 509:55-62, 2014, http://dx.doi.org/10.1038/nature13036). Here, we report on the isolation and characterization of new members of the family mostly obtained at time points of peak serum neutralization breadth and potency. Thirteen antibodies were isolated from B cell culture, and eight were isolated using trimeric envelope probes for differential single B cell sorting. One of the new antibodies displayed a 10-fold greater neutralization potency than previously published lineage members. This antibody, CAP256-VRC26.25, neutralized 57% of diverse clade viral isolates and 70% of clade C isolates with remarkable potency. Among the viruses neutralized, the median 50% inhibitory concentration was 0.001 µg/ml. All 33 lineage members targeted a quaternary epitope focused on V2. While all known bNAbs targeting the V1V2 region interact with the N160 glycan, the CAP256-VRC26 antibodies showed an inverse correlation of neutralization potency with dependence on this glycan. Overall, our results highlight the ongoing evolution within a single antibody lineage and describe more potent and broadly neutralizing members with potential clinical utility, particularly in areas where clade C is prevalent. IMPORTANCE: Studies of HIV-1 broadly neutralizing antibodies (bNAbs) provide valuable information for vaccine design, and the most potent and broadly reactive of these bNAbs have potential for clinical use. We previously described a family of V1V2-directed neutralizing antibodies from an HIV-1 clade C-infected donor. Here, we report on the isolation and characterization of new members of the family mostly obtained at time points of peak serum neutralization breadth and potency. One of the new antibodies, CAP256-VRC26.25, displayed a 10-fold greater neutralization potency than previously described lineage members. It neutralized 57% of diverse clade viral isolates and 70% of clade C isolates with remarkable potency: the median 50% inhibitory concentration was 0.001 µg/ml. Our results highlight the ongoing evolution within a single antibody lineage and describe more potent and broadly neutralizing members with potential clinical utility, particularly in areas where clade C is prevalent.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Reacciones Cruzadas , Anticuerpos Anti-VIH/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/inmunología , Anticuerpos Neutralizantes/genética , Anticuerpos Neutralizantes/aislamiento & purificación , Mapeo Epitopo , Femenino , Anticuerpos Anti-VIH/genética , Anticuerpos Anti-VIH/aislamiento & purificación , Proteína gp120 de Envoltorio del VIH/inmunología , Humanos , Concentración 50 Inhibidora , Datos de Secuencia Molecular , Análisis de Secuencia de ADNRESUMEN
Antibody PG9 is a prototypical member of a class of V1/V2-directed antibodies that effectively neutralizes diverse strains of HIV-1. We analyzed strain-specific resistance to PG9 using sequence and structural information. For multiply resistant strains, mutations in a short segment of V1/V2 resulted in gain of sensitivity to PG9 and related V1/V2 neutralizing antibodies, suggesting both a common mechanism of HIV-1 resistance to and a common mode of recognition by this class of antibodies.
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Anticuerpos Neutralizantes/química , Anticuerpos Anti-VIH/química , Proteína gp120 de Envoltorio del VIH/química , VIH-1/química , Secuencia de Aminoácidos , Anticuerpos Neutralizantes/inmunología , Anticuerpos Anti-VIH/inmunología , Proteína gp120 de Envoltorio del VIH/inmunología , VIH-1/inmunología , Humanos , Relación Estructura-ActividadRESUMEN
Identifying novel molecular mechanisms of exhausted CD8 T cells (T ex ) is a key goal of improving immunotherapy of cancer and other diseases. However, high-throughput interrogation of in vivo T ex can be costly and inefficient. In vitro models of T ex are easily customizable and quickly generate high cellular yield, offering an opportunity to perform CRISPR screening and other high-throughput assays. We established an in vitro model of chronic stimulation and benchmarked key phenotypic, functional, transcriptional, and epigenetic features against bona fide in vivo T ex . We leveraged this model of in vitro chronic stimulation in combination with pooled CRISPR screening to uncover transcriptional regulators of T cell exhaustion. This approach identified several transcription factors, including BHLHE40. In vitro and in vivo validation defined a role for BHLHE40 in regulating a key differentiation checkpoint between progenitor and intermediate subsets of T ex . By developing and benchmarking an in vitro model of T ex , we demonstrate the utility of mechanistically annotated in vitro models of T ex , in combination with high-throughput approaches, as a discovery pipeline to uncover novel T ex biology.
RESUMEN
Identifying molecular mechanisms of exhausted CD8 T cells (Tex) is a key goal of improving immunotherapy of cancer and other diseases. However, high-throughput interrogation of in vivo Tex can be costly and inefficient. In vitro models of Tex are easily customizable and quickly generate high cellular yield, enabling CRISPR screening and other high-throughput assays. We established an in vitro model of chronic stimulation and benchmarked key phenotypic, functional, transcriptional, and epigenetic features against bona fide in vivo Tex. We leveraged this model of in vitro chronic stimulation in combination with CRISPR screening to identify transcriptional regulators of T cell exhaustion. This approach identified several transcription factors, including BHLHE40. In vitro and in vivo validation defined a role for BHLHE40 in regulating a key differentiation checkpoint between progenitor and intermediate Tex subsets. By developing and benchmarking an in vitro model of Tex, then applying high-throughput CRISPR screening, we demonstrate the utility of mechanistically annotated in vitro models of Tex.
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Agotamiento de Células T , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Linfocitos T CD8-positivos , Diferenciación Celular , EpigenómicaRESUMEN
In response to infection or immunization, antibodies are produced that provide protection against re-exposure with the same pathogen. These antibodies can persist at high titers for decades and are maintained by bone marrow-resident long-lived plasma cells (LLPC). However, the durability of antibody responses to immunization varies amongst vaccines. It is unknown what factors contribute to the differential longevity of serum antibody responses and whether heterogeneity in LLPC contributes to this phenomenon. While LLPC differentiation has been studied extensively in mice, little is known about this population in humans or non-human primates (NHP). Here, we use multi-omic single-cell profiling to identify and characterize the LLPC compartment in NHP. We identify LLPC biomarkers including the marker CD102 and show that CD102 in combination with CD31 identifies LLPC in NHP bone marrow. Additionally, we find that CD102 is expressed by LLPC in mouse and humans. These results further our understanding of the LLPC compartment in NHP, identify biomarkers of LLPC, and provide tissue-specific single cell references for future studies.
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Multiómica , Células Plasmáticas , Ratones , Animales , Formación de Anticuerpos , Anticuerpos , Primates , BiomarcadoresRESUMEN
The transcription factors T-bet and Eomesodermin (Eomes) regulate CD8 T cell exhaustion through undefined mechanisms. Here, we show that the subcellular localization of T-bet and Eomes dictate their regulatory activity in exhausted T cells (TEXs). TEXs had a higher ratio of nuclear Eomes:T-bet than memory T cells (TMEMs) during chronic lymphocytic choriomeningitis virus (LCMV) infection in preclinical cancer models and in human tumors. Biochemically, T-bet and Eomes compete for the same DNA sequences, including the Pdcd1 T-box. High nuclear T-bet strongly represses Pdcd1 transcription in TMEM, whereas low nuclear T-bet in TEX leads to a dominant effect of Eomes that acts as a weaker repressor of Pdcd1. Blocking PD-1 signaling in TEXs increases nuclear T-bet, restoring stronger repression of Pdcd1, and driving T-bet-associated gene expression programs of chemotaxis, homing, and activation. These data identify a mechanism whereby the T-bet-Eomes axis regulates exhaustion through their nuclear localization, providing insights into how these transcription factors regulate TEX biology.
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Linfocitos T CD8-positivos/metabolismo , Proteínas de Dominio T Box/metabolismo , Animales , Diferenciación Celular , Humanos , Ratones , Transducción de SeñalRESUMEN
Ebola virus disease in humans is highly lethal, with case fatality rates ranging from 25 to 90%. There is no licensed treatment or vaccine against the virus, underscoring the need for efficacious countermeasures. We ascertained that a human survivor of the 1995 Kikwit Ebola virus disease outbreak maintained circulating antibodies against the Ebola virus surface glycoprotein for more than a decade after infection. From this survivor we isolated monoclonal antibodies (mAbs) that neutralize recent and previous outbreak variants of Ebola virus and mediate antibody-dependent cell-mediated cytotoxicity in vitro. Strikingly, monotherapy with mAb114 protected macaques when given as late as 5 days after challenge. Treatment with a single human mAb suggests that a simplified therapeutic strategy for human Ebola infection may be possible.
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Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Neutralizantes/administración & dosificación , Anticuerpos Antivirales/administración & dosificación , Ebolavirus/inmunología , Fiebre Hemorrágica Ebola/prevención & control , Adulto , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/aislamiento & purificación , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/aislamiento & purificación , Ensayos Clínicos como Asunto , Brotes de Enfermedades , Femenino , Fiebre Hemorrágica Ebola/epidemiología , Humanos , Macaca , Masculino , Datos de Secuencia Molecular , SobrevivientesRESUMEN
Serum characterization and antibody isolation are transforming our understanding of the humoral immune response to viral infection. Here, we show that epitope specificities of HIV-1-neutralizing antibodies in serum can be elucidated from the serum pattern of neutralization against a diverse panel of HIV-1 isolates. We determined "neutralization fingerprints" for 30 neutralizing antibodies on a panel of 34 diverse HIV-1 strains and showed that similarity in neutralization fingerprint correlated with similarity in epitope. We used these fingerprints to delineate specificities of polyclonal sera from 24 HIV-1-infected donors and a chimeric siman-human immunodeficiency virus-infected macaque. Delineated specificities matched published specificities and were further confirmed by antibody isolation for two sera. Patterns of virus-isolate neutralization can thus afford a detailed epitope-specific understanding of neutralizing-antibody responses to viral infection.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Anti-VIH/inmunología , Infecciones por VIH/inmunología , VIH-1/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Mapeo Epitopo , Anticuerpos Anti-VIH/sangre , Infecciones por VIH/sangre , VIH-1/aislamiento & purificación , Humanos , Epítopos Inmunodominantes/química , Epítopos Inmunodominantes/inmunología , Macaca , Pruebas de Neutralización , Conformación Proteica , Suero/inmunologíaRESUMEN
HIV-1 uses a diverse N-linked-glycan shield to evade recognition by antibody. Select human antibodies, such as the clonally related PG9 and PG16, recognize glycopeptide epitopes in the HIV-1 V1-V2 region and penetrate this shield, but their ability to accommodate diverse glycans is unclear. Here we report the structure of antibody PG16 bound to a scaffolded V1-V2, showing an epitope comprising both high mannose-type and complex-type N-linked glycans. We combined structure, NMR and mutagenesis analyses to characterize glycan recognition by PG9 and PG16. Three PG16-specific residues, arginine, serine and histidine (RSH), were critical for binding sialic acid on complex-type glycans, and introduction of these residues into PG9 produced a chimeric antibody with enhanced HIV-1 neutralization. Although HIV-1-glycan diversity facilitates evasion, antibody somatic diversity can overcome this and can provide clues to guide the design of modified antibodies with enhanced neutralization.