RESUMEN
Overcoming immune-mediated resistance to PD-1 blockade remains a major clinical challenge. Enhanced efficacy has been demonstrated in melanoma patients with combined nivolumab (anti-PD-1) and relatlimab (anti-LAG-3) treatment, the first in its class to be FDA approved. However, how these two inhibitory receptors synergize to hinder anti-tumor immunity remains unknown. Here, we show that CD8+ T cells deficient in both PD-1 and LAG-3, in contrast to CD8+ T cells lacking either receptor, mediate enhanced tumor clearance and long-term survival in mouse models of melanoma. PD-1- and LAG-3-deficient CD8+ T cells were transcriptionally distinct, with broad TCR clonality and enrichment of effector-like and interferon-responsive genes, resulting in enhanced IFN-γ release indicative of functionality. LAG-3 and PD-1 combined to drive T cell exhaustion, playing a dominant role in modulating TOX expression. Mechanistically, autocrine, cell-intrinsic IFN-γ signaling was required for PD-1- and LAG-3-deficient CD8+ T cells to enhance anti-tumor immunity, providing insight into how combinatorial targeting of LAG-3 and PD-1 enhances efficacy.
Asunto(s)
Antígenos CD , Linfocitos T CD8-positivos , Interferón gamma , Proteína del Gen 3 de Activación de Linfocitos , Ratones Endogámicos C57BL , Receptor de Muerte Celular Programada 1 , Receptor de Muerte Celular Programada 1/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Animales , Interferón gamma/metabolismo , Ratones , Antígenos CD/metabolismo , Comunicación Autocrina , Humanos , Melanoma/inmunología , Melanoma/tratamiento farmacológico , Femenino , Línea Celular Tumoral , Melanoma Experimental/inmunología , Agotamiento de Células TRESUMEN
Exhausted CD8 T (Tex) cells in chronic viral infection and cancer have sustained co-expression of inhibitory receptors (IRs). Tex cells can be reinvigorated by blocking IRs, such as PD-1, but synergistic reinvigoration and enhanced disease control can be achieved by co-targeting multiple IRs including PD-1 and LAG-3. To dissect the molecular changes intrinsic when these IR pathways are disrupted, we investigated the impact of loss of PD-1 and/or LAG-3 on Tex cells during chronic infection. These analyses revealed distinct roles of PD-1 and LAG-3 in regulating Tex cell proliferation and effector functions, respectively. Moreover, these studies identified an essential role for LAG-3 in sustaining TOX and Tex cell durability as well as a LAG-3-dependent circuit that generated a CD94/NKG2+ subset of Tex cells with enhanced cytotoxicity mediated by recognition of the stress ligand Qa-1b, with similar observations in humans. These analyses disentangle the non-redundant mechanisms of PD-1 and LAG-3 and their synergy in regulating Tex cells.
Asunto(s)
Antígenos CD , Linfocitos T CD8-positivos , Antígenos de Histocompatibilidad Clase I , Proteína del Gen 3 de Activación de Linfocitos , Subfamília D de Receptores Similares a Lectina de las Células NK , Receptor de Muerte Celular Programada 1 , Animales , Antígenos CD/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Ratones , Receptor de Muerte Celular Programada 1/metabolismo , Subfamília D de Receptores Similares a Lectina de las Células NK/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Subfamília C de Receptores Similares a Lectina de Células NK/metabolismo , Ratones Endogámicos C57BL , Proteínas del Grupo de Alta Movilidad/metabolismo , Proteínas del Grupo de Alta Movilidad/genética , Citotoxicidad Inmunológica , Proliferación Celular , Células Asesinas Naturales/metabolismo , Células Asesinas Naturales/inmunologíaRESUMEN
Naïve CD8+ T cells can differentiate into effector (Teff), memory (Tmem) or exhausted (Tex) T cells. These developmental pathways are associated with distinct transcriptional and epigenetic changes that endow cells with different functional capacities and therefore therapeutic potential. The molecular circuitry underlying these developmental trajectories and the extent of heterogeneity within Teff, Tmem and Tex populations remain poorly understood. Here, we used the lymphocytic choriomeningitis virus model of acute-resolving and chronic infection to address these gaps by applying longitudinal single-cell RNA-sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) analyses. These analyses uncovered new subsets, including a subpopulation of Tex cells expressing natural killer cell-associated genes that is dependent on the transcription factor Zeb2, as well as multiple distinct TCF-1+ stem/progenitor-like subsets in acute and chronic infection. These data also revealed insights into the reshaping of Tex subsets following programmed death 1 (PD-1) pathway blockade and identified a key role for the cell stress regulator, Btg1, in establishing the Tex population. Finally, these results highlighted how the same biological circuits such as cytotoxicity or stem/progenitor pathways can be used by CD8+ T cell subsets with highly divergent underlying chromatin landscapes generated during different infections.
Asunto(s)
Linfocitos T CD8-positivos , Coriomeningitis Linfocítica , Humanos , Linfocitos T CD8-positivos/metabolismo , Transcriptoma , Virus de la Coriomeningitis Linfocítica , Epigénesis Genética , Cromatina/genética , Cromatina/metabolismo , Coriomeningitis Linfocítica/metabolismoRESUMEN
CD8+ T cell exhaustion is a major barrier to current anti-cancer immunotherapies. Despite this, the developmental biology of exhausted CD8+ T cells (Tex) remains poorly defined, restraining improvement of strategies aimed at "re-invigorating" Tex cells. Here, we defined a four-cell-stage developmental framework for Tex cells. Two TCF1+ progenitor subsets were identified, one tissue restricted and quiescent and one more blood accessible, that gradually lost TCF1 as it divided and converted to a third intermediate Tex subset. This intermediate subset re-engaged some effector biology and increased upon PD-L1 blockade but ultimately converted into a fourth, terminally exhausted subset. By using transcriptional and epigenetic analyses, we identified the control mechanisms underlying subset transitions and defined a key interplay between TCF1, T-bet, and Tox in the process. These data reveal a four-stage developmental hierarchy for Tex cells and define the molecular, transcriptional, and epigenetic mechanisms that could provide opportunities to improve cancer immunotherapy.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Epigénesis Genética/inmunología , Neoplasias/inmunología , Subgrupos de Linfocitos T/inmunología , Transcripción Genética/inmunología , Animales , Antígeno B7-H1/genética , Antígeno B7-H1/inmunología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/metabolismo , Células Cultivadas , Epigénesis Genética/genética , Factor Nuclear 1-alfa del Hepatocito/genética , Factor Nuclear 1-alfa del Hepatocito/inmunología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/inmunología , Humanos , Inmunoterapia/métodos , Ratones Endogámicos C57BL , Neoplasias/genética , Neoplasias/terapia , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/inmunología , Subgrupos de Linfocitos T/metabolismo , Transcripción Genética/genéticaRESUMEN
Patients with COVID-19 present with a wide variety of clinical manifestations. Thromboembolic events constitute a significant cause of morbidity and mortality in patients infected with SARS-CoV-2. Severe COVID-19 has been associated with hyperinflammation and pre-existing cardiovascular disease. Platelets are important mediators and sensors of inflammation and are directly affected by cardiovascular stressors. In this report, we found that platelets from severely ill, hospitalized COVID-19 patients exhibited higher basal levels of activation measured by P-selectin surface expression and had poor functional reserve upon in vitro stimulation. To investigate this question in more detail, we developed an assay to assess the capacity of plasma from COVID-19 patients to activate platelets from healthy donors. Platelet activation was a common feature of plasma from COVID-19 patients and correlated with key measures of clinical outcome including kidney and liver injury, and APACHEIII scores. Further, we identified ferritin as a pivotal clinical marker associated with platelet hyperactivation. The COVID-19 plasma-mediated effect on control platelets was highest for patients that subsequently developed inpatient thrombotic events. Proteomic analysis of plasma from COVID-19 patients identified key mediators of inflammation and cardiovascular disease that positively correlated with in vitro platelet activation. Mechanistically, blocking the signaling of the FcγRIIa-Syk and C5a-C5aR pathways on platelets, using antibody-mediated neutralization, IgG depletion or the Syk inhibitor fostamatinib, reversed this hyperactivity driven by COVID-19 plasma and prevented platelet aggregation in endothelial microfluidic chamber conditions. These data identified these potentially actionable pathways as central for platelet activation and/or vascular complications and clinical outcomes in COVID-19 patients. In conclusion, we reveal a key role of platelet-mediated immunothrombosis in COVID-19 and identify distinct, clinically relevant, targetable signaling pathways that mediate this effect.
Asunto(s)
Plaquetas/inmunología , COVID-19/inmunología , Complemento C5a/metabolismo , Receptor de Anafilatoxina C5a/metabolismo , Receptores de IgG/metabolismo , SARS-CoV-2/fisiología , Tromboembolia/inmunología , Adulto , Aminopiridinas/farmacología , Células Cultivadas , Femenino , Hospitalización , Humanos , Masculino , Morfolinas/farmacología , Activación Plaquetaria , Pirimidinas/farmacología , Índice de Severidad de la Enfermedad , Transducción de Señal , Quinasa Syk/antagonistas & inhibidoresRESUMEN
Patients with COVID-19 present with a wide variety of clinical manifestations. Thromboembolic events constitute a significant cause of morbidity and mortality in patients infected with SARS-CoV-2. Severe COVID-19 has been associated with hyperinflammation and pre-existing cardiovascular disease. Platelets are important mediators and sensors of inflammation and are directly affected by cardiovascular stressors. In this report, we found that platelets from severely ill, hospitalized COVID-19 patients exhibit higher basal levels of activation measured by P-selectin surface expression, and have a poor functional reserve upon in vitro stimulation. Correlating clinical features to the ability of plasma from COVID-19 patients to stimulate control platelets identified ferritin as a pivotal clinical marker associated with platelet hyperactivation. The COVID-19 plasma-mediated effect on control platelets was highest for patients that subsequently developed inpatient thrombotic events. Proteomic analysis of plasma from COVID-19 patients identified key mediators of inflammation and cardiovascular disease that positively correlated with in vitro platelet activation. Mechanistically, blocking the signaling of the FcγRIIa-Syk and C5a-C5aR pathways on platelets, using antibody-mediated neutralization, IgG depletion or the Syk inhibitor fostamatinib, reversed this hyperactivity driven by COVID-19 plasma and prevented platelet aggregation in endothelial microfluidic chamber conditions, thus identifying these potentially actionable pathways as central for platelet activation and/or vascular complications in COVID-19 patients. In conclusion, we reveal a key role of platelet-mediated immunothrombosis in COVID-19 and identify distinct, clinically relevant, targetable signaling pathways that mediate this effect. These studies have implications for the role of platelet hyperactivation in complications associated with SARS-CoV-2 infection. ONE-SENTENCE SUMMARY: The FcγRIIA and C5a-C5aR pathways mediate platelet hyperactivation in COVID-19.
RESUMEN
Pediatric COVID-19 following SARS-CoV-2 infection is associated with fewer hospitalizations and often milder disease than in adults. A subset of children, however, present with Multisystem Inflammatory Syndrome in Children (MIS-C) that can lead to vascular complications and shock, but rarely death. The immune features of MIS-C compared to pediatric COVID-19 or adult disease remain poorly understood. We analyzed peripheral blood immune responses in hospitalized SARS-CoV-2 infected pediatric patients (pediatric COVID-19) and patients with MIS-C. MIS-C patients had patterns of T cell-biased lymphopenia and T cell activation similar to severely ill adults, and all patients with MIS-C had SARS-CoV-2 spike-specific antibodies at admission. A distinct feature of MIS-C patients was robust activation of vascular patrolling CX3CR1+ CD8+ T cells that correlated with the use of vasoactive medication. Finally, whereas pediatric COVID-19 patients with acute respiratory distress syndrome (ARDS) had sustained immune activation, MIS-C patients displayed clinical improvement over time, concomitant with decreasing immune activation. Thus, non-MIS-C versus MIS-C SARS-CoV-2 associated illnesses are characterized by divergent immune signatures that are temporally distinct from one another and implicate CD8+ T cells in the clinical presentation and trajectory of MIS-C.
Asunto(s)
COVID-19/inmunología , Activación de Linfocitos , Síndrome de Respuesta Inflamatoria Sistémica/inmunología , Linfocitos T/inmunología , Adolescente , Adulto , Envejecimiento/inmunología , Niño , Preescolar , Femenino , Citometría de Flujo , Humanos , Leucopenia/inmunología , Masculino , Adulto JovenRESUMEN
Coronavirus disease 2019 (COVID-19) is currently a global pandemic, but human immune responses to the virus remain poorly understood. We used high-dimensional cytometry to analyze 125 COVID-19 patients and compare them with recovered and healthy individuals. Integrated analysis of ~200 immune and ~50 clinical features revealed activation of T cell and B cell subsets in a proportion of patients. A subgroup of patients had T cell activation characteristic of acute viral infection and plasmablast responses reaching >30% of circulating B cells. However, another subgroup had lymphocyte activation comparable with that in uninfected individuals. Stable versus dynamic immunological signatures were identified and linked to trajectories of disease severity change. Our analyses identified three immunotypes associated with poor clinical trajectories versus improving health. These immunotypes may have implications for the design of therapeutics and vaccines for COVID-19.
Asunto(s)
Linfocitos B/inmunología , Betacoronavirus/inmunología , Infecciones por Coronavirus/inmunología , Neumonía Viral/inmunología , Linfocitos T/inmunología , Inmunidad Adaptativa , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Antivirales/sangre , Subgrupos de Linfocitos B/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , COVID-19 , Citocinas/sangre , Femenino , Humanos , Memoria Inmunológica , Activación de Linfocitos , Masculino , Persona de Mediana Edad , Pandemias , Células Plasmáticas/inmunología , SARS-CoV-2 , Índice de Severidad de la Enfermedad , Subgrupos de Linfocitos T/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Factores de Tiempo , Adulto JovenRESUMEN
Pediatric COVID-19 following SARS-CoV-2 infection is associated with fewer hospitalizations and often milder disease than in adults. A subset of children, however, present with Multisystem Inflammatory Syndrome in Children (MIS-C) that can lead to vascular complications and shock, but rarely death. The immune features of MIS-C compared to pediatric COVID-19 or adult disease remain poorly understood. We analyzed peripheral blood immune responses in hospitalized SARS-CoV-2 infected pediatric patients (pediatric COVID-19) and patients with MIS-C. MIS-C patients had patterns of T cell-biased lymphopenia and T cell activation similar to severely ill adults, and all patients with MIS-C had SARS-CoV-2 spike-specific antibodies at admission. A distinct feature of MIS-C patients was robust activation of vascular patrolling CX3CR1+ CD8 T cells that correlated with use of vasoactive medication. Finally, whereas pediatric COVID-19 patients with acute respiratory distress syndrome (ARDS) had sustained immune activation, MIS-C patients displayed clinical improvement over time, concomitant with decreasing immune activation. Thus, non-MIS-C versus MIS-C SARS-CoV-2 associated illnesses are characterized by divergent immune signatures that are temporally distinct and implicate CD8 T cells in clinical presentation and trajectory of MIS-C.
RESUMEN
COVID-19 has become a global pandemic. Immune dysregulation has been implicated, but immune responses remain poorly understood. We analyzed 71 COVID-19 patients compared to recovered and healthy subjects using high dimensional cytometry. Integrated analysis of ~200 immune and >30 clinical features revealed activation of T cell and B cell subsets, but only in some patients. A subgroup of patients had T cell activation characteristic of acute viral infection and plasmablast responses could reach >30% of circulating B cells. However, another subgroup had lymphocyte activation comparable to uninfected subjects. Stable versus dynamic immunological signatures were identified and linked to trajectories of disease severity change. These analyses identified three "immunotypes" associated with poor clinical trajectories versus improving health. These immunotypes may have implications for therapeutics and vaccines.