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T cell-mediated immunity plays an important role in controlling SARS-CoV-2 infection, but the repertoire of naturally processed and presented viral epitopes on class I human leukocyte antigen (HLA-I) remains uncharacterized. Here, we report the first HLA-I immunopeptidome of SARS-CoV-2 in two cell lines at different times post infection using mass spectrometry. We found HLA-I peptides derived not only from canonical open reading frames (ORFs) but also from internal out-of-frame ORFs in spike and nucleocapsid not captured by current vaccines. Some peptides from out-of-frame ORFs elicited T cell responses in a humanized mouse model and individuals with COVID-19 that exceeded responses to canonical peptides, including some of the strongest epitopes reported to date. Whole-proteome analysis of infected cells revealed that early expressed viral proteins contribute more to HLA-I presentation and immunogenicity. These biological insights, as well as the discovery of out-of-frame ORF epitopes, will facilitate selection of peptides for immune monitoring and vaccine development.
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Epítopos de Linfocito T/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Sistemas de Lectura Abierta/genética , Péptidos/inmunología , Proteoma/inmunología , SARS-CoV-2/inmunología , Células A549 , Alelos , Secuencia de Aminoácidos , Animales , Presentación de Antígeno/inmunología , COVID-19/inmunología , COVID-19/virología , Femenino , Células HEK293 , Humanos , Cinética , Masculino , Ratones , Péptidos/química , Linfocitos T/inmunologíaRESUMEN
Understanding the hallmarks of the immune response to SARS-CoV-2 is critical for fighting the COVID-19 pandemic. We assessed antibody and T cell reactivity in convalescent COVID-19 patients and healthy donors sampled both prior to and during the pandemic. Healthy donors examined during the pandemic exhibited increased numbers of SARS-CoV-2-specific T cells, but no humoral response. Their probable exposure to the virus resulted in either asymptomatic infection without antibody secretion or activation of preexisting immunity. In convalescent patients, we observed a public and diverse T cell response to SARS-CoV-2 epitopes, revealing T cell receptor (TCR) motifs with germline-encoded features. Bulk CD4+ and CD8+ T cell responses to the spike protein were mediated by groups of homologous TCRs, some of them shared across multiple donors. Overall, our results demonstrate that the T cell response to SARS-CoV-2, including the identified set of TCRs, can serve as a useful biomarker for surveying antiviral immunity.
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Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , COVID-19/inmunología , Epítopos de Linfocito T/metabolismo , SARS-CoV-2/fisiología , Glicoproteína de la Espiga del Coronavirus/metabolismo , Adolescente , Adulto , Anticuerpos Antivirales/metabolismo , Infecciones Asintomáticas , Células Cultivadas , Convalecencia , Epítopos de Linfocito T/inmunología , Femenino , Humanos , Inmunidad , Memoria Inmunológica , Activación de Linfocitos , Masculino , Persona de Mediana Edad , Pandemias , Receptores de Antígenos de Linfocitos T/metabolismo , Glicoproteína de la Espiga del Coronavirus/inmunología , Adulto JovenRESUMEN
Appropriate immune responses require a fine balance between immune activation and attenuation. NLRC3, a non-inflammasome-forming member of the NLR innate immune receptor family, attenuates inflammation in myeloid cells and proliferation in epithelial cells. T lymphocytes express the highest amounts of Nlrc3 transcript where its physiologic relevance is unknown. We show that NLRC3 attenuated interferon-γ and TNF expression by CD4+ T cells and reduced T helper 1 (Th1) and Th17 cell proliferation. Nlrc3-/- mice exhibited increased and prolonged CD4+ T cell responses to lymphocytic choriomeningitis virus infection and worsened experimental autoimmune encephalomyelitis (EAE). These functions of NLRC3 were executed in a T-cell-intrinsic fashion: NLRC3 reduced K63-linked ubiquitination of TNF-receptor-associated factor 6 (TRAF6) to limit NF-κB activation, lowered phosphorylation of eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), and diminished glycolysis and oxidative phosphorylation. This study reveals an unappreciated role for NLRC3 in attenuating CD4+ T cell signaling and metabolism.
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Autoinmunidad/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Inmunidad Innata/inmunología , Péptidos y Proteínas de Señalización Intercelular/inmunología , Coriomeningitis Linfocítica/inmunología , Virus de la Coriomeningitis Linfocítica/inmunología , Proteínas Adaptadoras Transductoras de Señales , Animales , Autoinmunidad/genética , Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular , Encefalomielitis Autoinmune Experimental/genética , Factores Eucarióticos de Iniciación , Humanos , Inmunidad Innata/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Coriomeningitis Linfocítica/genética , Coriomeningitis Linfocítica/microbiología , Virus de la Coriomeningitis Linfocítica/fisiología , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/inmunología , FN-kappa B/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/inmunología , Fosfoproteínas/metabolismo , Factor 6 Asociado a Receptor de TNF/genética , Factor 6 Asociado a Receptor de TNF/inmunología , Factor 6 Asociado a Receptor de TNF/metabolismo , Células TH1/inmunología , Células TH1/metabolismo , Células Th17/inmunología , Células Th17/metabolismoRESUMEN
COVID-19 is a systemic inflammatory disease initiated by SARS-CoV-2 virus infection. Multiple vaccines against the Wuhan variant of SARS-CoV-2 have been developed including a whole virion beta-propiolactone-inactivated vaccine based on the B.1.1 strain (CoviVac). Since most of the population has been vaccinated by targeting the original or early variants of SARS-CoV-2, the emergence of novel mutant variants raises concern over possible evasion of vaccine-induced immune responses. Here, we report on the mechanism of protection by CoviVac, a whole virion-based vaccine, against the Omicron variant. CoviVac-immunized K18-hACE2 Tg mice were protected against both prototype B.1.1 and BA.1-like (Omicron) variants. Subsequently, vaccinated K18-hACE2 Tg mice rapidly cleared the infection via cross-reactive T-cell responses and cross-reactive, non-neutralizing antibodies recognizing the Omicron variant Spike protein. Thus, our data indicate that efficient protection from SARS-CoV-2 variants can be achieved by the orchestrated action of cross-reactive T cells and non-neutralizing antibodies.
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COVID-19 , Melfalán , SARS-CoV-2 , gammaglobulinas , Animales , Humanos , Ratones , Vacunas de Productos Inactivados , Formación de Anticuerpos , COVID-19/prevención & control , Linfocitos T , Virión , Anticuerpos ampliamente neutralizantes , Anticuerpos Neutralizantes , Anticuerpos AntiviralesRESUMEN
Vaccine-induced mucosal immunity and broad protective capacity against various severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants remain inadequate. Formyl peptide receptor-like 1 inhibitory protein (FLIPr), produced by Staphylococcus aureus, can bind to various Fcγ receptor subclasses. Recombinant lipidated FLIPr (rLF) was previously found to be an effective adjuvant. In this study, we developed a vaccine candidate, the recombinant Delta SARS-CoV-2 spike (rDS)-FLIPr fusion protein (rDS-F), which employs the property of FLIPr binding to various Fcγ receptors. Our study shows that rDS-F plus rLF promotes rDS capture by dendritic cells. Intranasal vaccination of mice with rDS-F plus rLF increases persistent systemic and mucosal antibody responses and CD4/CD8 T-cell responses. Importantly, antibodies induced by rDS-F plus rLF vaccination neutralize Delta, Wuhan, Alpha, Beta, and Omicron strains. Additionally, rDS-F plus rLF provides protective effects against various SARS-CoV-2 variants in hamsters by reducing inflammation and viral loads in the lung. Therefore, rDS-F plus rLF is a potential vaccine candidate to induce broad protective responses against various SARS-CoV-2 variants.IMPORTANCEMucosal immunity is vital for combating pathogens, especially in the context of respiratory diseases like COVID-19. Despite this, most approved vaccines are administered via injection, providing systemic but limited mucosal protection. Developing vaccines that stimulate both mucosal and systemic immunity to address future coronavirus mutations is a growing trend. However, eliciting strong mucosal immune responses without adjuvants remains a challenge. In our study, we have demonstrated that using a recombinant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike-formyl peptide receptor-like 1 inhibitory protein (FLIPr) fusion protein as an antigen, in combination with recombinant lipidated FLIPr as an effective adjuvant, induced simultaneous systemic and mucosal immune responses through intranasal immunization in mice and hamster models. This approach offered protection against various SARS-CoV-2 strains, making it a promising vaccine candidate for broad protection. This finding is pivotal for future broad-spectrum vaccine development.
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Proteínas Bacterianas , Vacunas contra la COVID-19 , COVID-19 , Inmunidad Mucosa , Lípidos , Proteínas Recombinantes de Fusión , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Animales , Cricetinae , Ratones , Adyuvantes Inmunológicos , Anticuerpos Antivirales/inmunología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , COVID-19/inmunología , COVID-19/prevención & control , COVID-19/virología , Vacunas contra la COVID-19/administración & dosificación , Vacunas contra la COVID-19/química , Vacunas contra la COVID-19/genética , Vacunas contra la COVID-19/inmunología , Células Dendríticas/inmunología , Modelos Animales de Enfermedad , Receptores de IgG/clasificación , Receptores de IgG/inmunología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , SARS-CoV-2/clasificación , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/metabolismo , Staphylococcus aureus , Desarrollo de Vacunas , Carga ViralRESUMEN
Cytomegalovirus (CMV)-seropositive adults have large T cell responses to a wide range of CMV proteins; these responses have been associated with chronic inflammation and frailty in people with or without HIV infection. We analyzed the relationships between chronic HIV infection, frailty, and the breadth and polyfunctionality of CD4 and CD8 T cell responses to CMV. Peripheral blood mononuclear cells from 42 men (20 without HIV and 22 with virologically suppressed HIV) in the Multicenter AIDS Cohort Study (MACS) were stimulated with peptide pools spanning 19 CMV open reading frames (ORFs). As measured by flow cytometry and intracellular cytokine staining for IFN-γ, TNF-α, and IL-2, CD8 T cells from men with HIV responded to significantly more CMV ORFs than those from men without HIV. This was primarily due to a broader response to ORFs that are expressed during the late phase of CMV replication. The number of ORFs to which a participant's T cells responded was positively correlated with the sum of all that individual's T cell responses; these correlations were weaker in men with than without HIV. Polyfunctional CMV-specific CD4 responses (production of more than one cytokine) were significantly lower in men with than without HIV. Frailty status did not substantially affect the breadth or magnitude of the CMV-specific T cell responses. These results suggest that immune control of CMV infection is affected more by chronic HIV infection than by frailty. The differences between men with and without HIV were similar to those reported between young and older adults without HIV. IMPORTANCE: T cell responses to chronic cytomegalovirus (CMV) infection have significant biological and clinical implications in HIV infection and aging. Here, we systematically analyzed the breadth, magnitude, and polyfunctionality of T cell responses to multiple CMV antigens in men with and without HIV in the Multicenter AIDS Cohort Study (MACS), a longstanding study of the natural and treated history of HIV-1 infection in men who have sex with men. We found that the breadth and polyfunctionality of T cell responses to CMV were different between men with chronic, treated HIV and those without HIV. The reason for these differences is unknown, but these findings suggest that people with treated HIV may have more frequent CMV reactivation than people without HIV. Differences between people with and without HIV also resembled differences reported between young and older adults without HIV, supporting a role for the immune responses to CMV in the aging process.
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Rapid clonal expansion of antigen-specific T cells is a fundamental feature of adaptive immune responses. It enables the outgrowth of an individual T cell into thousands of clonal descendants that diversify into short-lived effectors and long-lived memory cells. Clonal expansion is thought to be programmed upon priming of a single naive T cell and then executed by homogenously fast divisions of all of its descendants. However, the actual speed of cell divisions in such an emerging "T cell family" has never been measured with single-cell resolution. Here, we utilize continuous live-cell imaging in vitro to track the division speed and genealogical connections of all descendants derived from a single naive CD8+ T cell throughout up to ten divisions of activation-induced proliferation. This comprehensive mapping of T cell family trees identifies a short burst phase, in which division speed is homogenously fast and maintained independent of external cytokine availability or continued T cell receptor stimulation. Thereafter, however, division speed diversifies, and model-based computational analysis using a Bayesian inference framework for tree-structured data reveals a segregation into heritably fast- and slow-dividing branches. This diversification of division speed is preceded already during the burst phase by variable expression of the interleukin-2 receptor alpha chain. Later it is accompanied by selective expression of memory marker CD62L in slower dividing branches. Taken together, these data demonstrate that T cell clonal expansion is structured into subsequent burst and diversification phases, the latter of which coincides with specification of memory versus effector fate.
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Linfocitos T CD8-positivos/citología , Linaje de la Célula , Animales , Antígenos CD/inmunología , Biomarcadores , Linfocitos T CD8-positivos/inmunología , Diferenciación Celular/inmunología , División Celular , Ratones , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: Cockroach allergy contributes to morbidity among urban children with asthma. Few trials address the effect of subcutaneous immunotherapy (SCIT) with cockroach allergen among these at-risk children. OBJECTIVES: We sought to determine whether nasal allergen challenge (NAC) responses to cockroach allergen would improve following 1 year of SCIT. METHODS: Urban children with asthma, who were cockroach-sensitized and reactive on NAC, participated in a year-long randomized double-blind placebo-controlled SCIT trial using German cockroach extract. The primary endpoint was the change in mean Total Nasal Symptom Score (TNSS) during NAC after 12 months of SCIT. Changes in nasal transcriptomic responses during NAC, skin prick test wheal size, serum allergen-specific antibody production, and T-cell responses to cockroach allergen were assessed. RESULTS: Changes in mean NAC TNSS did not differ between SCIT-assigned (n = 28) versus placebo-assigned (n = 29) participants (P = .63). Nasal transcriptomic responses correlated with TNSS, but a treatment effect was not observed. Cockroach serum-specific IgE decreased to a similar extent in both groups, while decreased cockroach skin prick test wheal size was greater among SCIT participants (P = .04). A 200-fold increase in cockroach serum-specific IgG4 was observed among subjects receiving SCIT (P < .001) but was unchanged in the placebo group. T-cell IL-4 responses following cockroach allergen stimulation decreased to a greater extent among SCIT versus placebo (P = .002), while no effect was observed for IL-10 or IFN-γ. CONCLUSIONS: A year of SCIT failed to alter NAC TNSS and nasal transcriptome responses to cockroach allergen challenge despite systemic effects on allergen-specific skin tests, induction of serum-specific IgG4 serum production and down-modulation of allergen-stimulated T-cell responses.
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In the recent mpox outbreak, people living with HIV (PLWH) were at high risk both for contracting infection and for suffering a more severe disease course. We studied cellular and humoral immune responses elicited by mpox infection (n = 5; n = 3 PLWH) or smallpox vaccination (n = 17; all PLWH) in a cohort of men who have sex with men. All PLWH were successfully treated, with stable CD4 counts and undetectable HIV viral loads. 11/17 vaccinated individuals had received childhood smallpox vaccination. In this group of individuals, both two-dose MVA-vaccination and natural infection evoked mpox-specific immune responses mediated by B cells as well as CD4 and CD8 T cells. This study improves our understanding of smallpox vaccination mediated cross-reactivity to other orthopox viruses, and the long-lasting durability of childhood smallpox vaccination mediated immune responses including in PLWH.
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This study longitudinally evaluated the immune response in individuals over a year after receiving three doses of an inactivated SARS-CoV-2 vaccine, focusing on reactions to Omicron breakthrough infections. From 63 blood samples of 37 subjects, results showed that the third booster enhanced the antibody response against Alpha, Beta, and Delta VOCs but was less effective against Omicron. Although antibody titres decreased post-vaccination, SARS-CoV-2-specific T-cell responses, both CD4+ and CD8+, remained stable. Omicron breakthrough infections significantly improved neutralization against various VOCs, including Omicron. However, the boost in antibodies against WT, Alpha, Beta, and Delta variants was more pronounced. Regarding T cells, breakthrough infection predominantly boosted the CD8+ T-cell response, and the intensity of the spike protein-specific T-cell response was roughly comparable between WT and Omicron BA.5.
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Anticuerpos Antivirales , Infección Irruptiva , Vacunas contra la COVID-19 , COVID-19 , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Infección Irruptiva/epidemiología , Infección Irruptiva/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , COVID-19/prevención & control , COVID-19/inmunología , Vacunas contra la COVID-19/inmunología , Vacunas contra la COVID-19/administración & dosificación , Inmunización Secundaria , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/inmunología , Linfocitos T/inmunología , Vacunas de Productos Inactivados/inmunología , Vacunas de Productos Inactivados/administración & dosificaciónRESUMEN
Little is known about the efficacy of COVID-19 vaccines during acute lymphoblastic leukemia therapy (ALL); data for COVID-19 vaccine immune responses in pediatric leukemia remain sparse. We conducted a single center study of patients aged 5-25 years undergoing ALL chemotherapy who received COVID-19 vaccination. Twenty-one patients were enrolled; efficacy was evaluable in 20. Twenty were vaccinated while receiving chemotherapy. Twenty received the BNT162b2 mRNA vaccine. Spike reactive antibodies (S-IgG) and/or T-cells (SRT) were detected in 16 of 20 (80%) vaccinated patients; 13 (65%) and 9 (45%) were positive for S-IgG and SRT, respectively. Six (30%) showed both spike reactive B and T-cell responses. Eleven of the 13 with S-IgG positivity were negative for anti-Nucleocapsid IgG, an antibody profile consistent with a vaccine induced immune response. All 13S-IgG+ patients showed neutralizing antibodies. SRT included CD4+ (7) and CD8+ (6) T-cells; both CD4+ and CD8+ SRT were seen in 4. SRT were multifunctional (producing multiple cytokines) in most patients (8 of 9); 4 showed SRT with triple cytokine and B-cell co-stimulatory responses, indicating a multimodal adaptive immune response. Immune responses were seen among patients vaccinated in the settings of lymphopenia (6 of 12) intensive chemotherapy (3 of 4), and Peg allergy (6 of 8). Sequencing revealed public CD4+ and CD8+ TCR sequences reactive to epitopes across the spike protein. In conclusion, COVID-19 vaccination induced B and/or T-cell responses in a majority of children and young adults undergoing ALL chemotherapy.
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CD8+ T cells recognizing their cognate antigen are typically recruited as a polyclonal population consisting of multiple clonotypes with varying T-cell receptor (TCR) affinity to the target peptide-major histocompatibility complex (pMHC) complex. Advances in single-cell sequencing have increased accessibility toward identifying TCRs with matched antigens. Here we present the discovery of a monoclonal CD8+ T-cell population with specificity for a hepatitis C virus (HCV)-derived human leukocyte antigen (HLA) class I epitope (HLA-B*07:02 GPRLGVRAT) which was isolated directly ex vivo from an individual with an episode of acutely resolved HCV infection. This population was absent before infection and underwent expansion and stable maintenance for at least 2 years after infection as measured by HLA-multimer staining. Furthermore, the monoclonal clonotype was characterized by an unusually long dissociation time (half-life = 794 s and koff = 5.73 × 10-4) for its target antigen when compared with previously published results. A comparison with related populations of HCV-specific populations derived from the same individual and a second individual suggested that high-affinity TCR-pMHC interactions may be inherent to epitope identity and shape the phenotype of responses which has implications for rational TCR selection and design in the age of personalized immunotherapies.
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Linfocitos T CD8-positivos , Células Clonales , Hepacivirus , Hepatitis C , Receptores de Antígenos de Linfocitos T , Humanos , Linfocitos T CD8-positivos/inmunología , Hepatitis C/inmunología , Hepatitis C/virología , Hepacivirus/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Epítopos de Linfocito T/inmunologíaRESUMEN
Memory T cells are generated from naïve precursors undergoing proliferation during the initial immune response. Both naïve and memory T cells are maintained in a resting, quiescent state and respond to activation with a controlled proliferative burst and differentiation into effector cells. This similarity in the maintenance and response dynamics points to the preservation of key cellular fate programs; however, whether memory T cells have acquired intrinsic changes in these programs that may contribute to the enhanced immune protection in a recall response is not fully understood. Here we used a quantitative model-based analysis of proliferation and survival kinetics of in vitro-stimulated murine naïve and memory CD8+ T cells in response to homeostatic and activating signals to establish intrinsic similarities or differences within these cell types. We show that resting memory T cells display heightened sensitivity to homeostatic cytokines, responding to interleukin (IL)-2 in addition to IL-7 and IL-15. The proliferative response to αCD3 was equal in size and kinetics, demonstrating that memory T cells undergo the same controlled division burst and automated return to quiescence as naïve T cells. However, perhaps surprisingly, we observed reduced expansion of αCD3-stimulated memory T cells in response to activating signals αCD28 and IL-2 compared with naïve T cells. Overall, we demonstrate that although sensitivities to cytokine and costimulatory signals have shifted, fate programs regulating the scale of the division burst are conserved in memory T cells.
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Linfocitos T CD8-positivos , Células T de Memoria , Animales , Ratones , Citocinas/metabolismo , División Celular , Diferenciación Celular , Memoria Inmunológica , Activación de LinfocitosRESUMEN
T cell recognition of a cognate peptide-major histocompatibility complex (pMHC) presented on the surface of infected or malignant cells is of the utmost importance for mediating robust and long-term immune responses. Accurate predictions of cognate pMHC targets for T cell receptors would greatly facilitate identification of vaccine targets for both pathogenic diseases and personalized cancer immunotherapies. Predicting immunogenic peptides therefore has been at the center of intensive research for the past decades but has proven challenging. Although numerous models have been proposed, performance of these models has not been systematically evaluated and their success rate in predicting epitopes in the context of human pathology has not been measured and compared. In this study, we evaluated the performance of several publicly available models, in identifying immunogenic CD8+ T cell targets in the context of pathogens and cancers. We found that for predicting immunogenic peptides from an emerging virus such as severe acute respiratory syndrome coronavirus 2, none of the models perform substantially better than random or offer considerable improvement beyond HLA ligand prediction. We also observed suboptimal performance for predicting cancer neoantigens. Through investigation of potential factors associated with ill performance of models, we highlight several data- and model-associated issues. In particular, we observed that cross-HLA variation in the distribution of immunogenic and non-immunogenic peptides in the training data of the models seems to substantially confound the predictions. We additionally compared key parameters associated with immunogenicity between pathogenic peptides and cancer neoantigens and observed evidence for differences in the thresholds of binding affinity and stability, which suggested the need to modulate different features in identifying immunogenic pathogen versus cancer peptides. Overall, we demonstrate that accurate and reliable predictions of immunogenic CD8+ T cell targets remain unsolved; thus, we hope our work will guide users and model developers regarding potential pitfalls and unsettled questions in existing immunogenicity predictors.
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COVID-19 , Neoplasias , Linfocitos T CD8-positivos/metabolismo , Simulación por Computador , Epítopos de Linfocito T , Humanos , PéptidosRESUMEN
BACKGROUND: Neoantigens are patient- and tumor-specific peptides that arise from somatic mutations. They stand as promising targets for personalized therapeutic cancer vaccines. The identification process for neoantigens has evolved with the use of next-generation sequencing technologies and bioinformatic tools in tumor genomics. However, in-silico strategies for selecting immunogenic neoantigens still have very low accuracy rates, since they mainly focus on predicting peptide binding to Major Histocompatibility Complex (MHC) molecules, which is key but not the sole determinant for immunogenicity. Moreover, the therapeutic potential of neoantigen-based vaccines may be enhanced using an optimal delivery platform that elicits robust de novo immune responses. METHODS: We developed a novel neoantigen selection pipeline based on existing software combined with a novel prediction method, the Neoantigen Optimization Algorithm (NOAH), which takes into account structural features of the peptide/MHC-I interaction, as well as the interaction between the peptide/MHC-I complex and the TCR, in its prediction strategy. Moreover, to maximize neoantigens' therapeutic potential, neoantigen-based vaccines should be manufactured in an optimal delivery platform that elicits robust de novo immune responses and bypasses central and peripheral tolerance. RESULTS: We generated a highly immunogenic vaccine platform based on engineered HIV-1 Gag-based Virus-Like Particles (VLPs) expressing a high copy number of each in silico selected neoantigen. We tested different neoantigen-loaded VLPs (neoVLPs) in a B16-F10 melanoma mouse model to evaluate their capability to generate new immunogenic specificities. NeoVLPs were used in in vivo immunogenicity and tumor challenge experiments. CONCLUSIONS: Our results indicate the relevance of incorporating other immunogenic determinants beyond the binding of neoantigens to MHC-I. Thus, neoVLPs loaded with neoantigens enhancing the interaction with the TCR can promote the generation of de novo antitumor-specific immune responses, resulting in a delay in tumor growth. Vaccination with the neoVLP platform is a robust alternative to current therapeutic vaccine approaches and a promising candidate for future personalized immunotherapy.
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Vacunas contra el Cáncer , Neoplasias , Vacunas , Humanos , Animales , Ratones , Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Péptidos , Receptores de Antígenos de Linfocitos T/metabolismo , Inmunoterapia/métodosRESUMEN
Co-existing chronic hepatitis B virus (CHB) infection and metabolic dysfunction associated steatotic liver disease (MASLD) can exert complex effects on hepatic metabolism, requiring mechanistic study. CHB participants were assessed for MASLD and the impact of hepatic steatosis/metabolic syndrome (MetS) on novel viral and immunological markers. In this prospective, cohort study, untreated CHB subjects were assessed for liver disease by non-invasive tests (i.e. FibroScan, controlled attenuation parameter, CAP). Subjects were tested for cytokines and IFN-γ ELISPOT assay to HBV Surface (S) and Core (C) proteins. Standard HBV serological, exploratory biomarkers and deep sequencing of HBV S and C genes were performed. In 53 subjects (median age 45 years [SD = 10.6], 35% F, 56% Asian, 20% Black, 3% White), 94% (50) HBeAg negative, 63% genotype B/C, mean HBV DNA 3.2 log10 IU/mL (SD = 1.8), quantitative HBsAg 2.9 log10 IU/mL (SD = 1.2) and HBV pgRNA 2.1 log10 copies/mL (SD = 1.3). In enrolled subjects, the mean ALT was 41.9 U/L (SD = 24.0), FibroScan was 5.7 kPa (SD = 1.9) and CAP was 306.4 dB/m (SD = 49.0). The mean BMI was 28.2 kg/m2 (SD = 4.2), 20% (11/53) had diabetes, 35% (19/53) dyslipidaemia and 24% (13/53) hypertension. Subjects with MetS and steatosis showed lower HBV markers (p < .01), higher HBV S diversity (p = .02) and greater frequency of HBV variants associated with host-anti-viral immune escape. Pro-inflammatory cytokine levels and HBV-specific cellular responses were higher in participants with hepatic steatosis. In CHB, MASLD/hepatic steatosis was associated with HBV variants and systemic immune responses potentially impacting liver disease progression despite low-level viraemia.
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The continuous evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been accompanied by the emergence of viral mutations that pose a great challenge to existing vaccine strategies. It is not fully understood with regard to the role of mutations on the SARS-CoV-2 spike protein from emerging viral variants in T cell immunity. In the current study, recombinant eukaryotic plasmids were constructed as DNA vaccines to express the spike protein from multiple SARS-CoV-2 strains. These DNA vaccines were used to immunize BALB/c mice, and cross-T cell responses to the spike protein from these viral strains were quantitated using interferon-γ (IFN-γ) Elispot. Peptides covering the full-length spike protein from different viral strains were used to detect epitope-specific IFN-γ+ CD4+ and CD8+ T cell responses by fluorescence-activated cell sorting. SARS-CoV-2 Delta and Omicron BA.1 strains were found to have broad T cell cross-reactivity, followed by the Beta strain. The landscapes of T cell epitopes on the spike protein demonstrated that at least 30 mutations emerging from Alpha to Omicron BA.5 can mediate the escape of T cell immunity. Omicron and its sublineages have 19 out of these 30 mutations, most of which are new, and a few are inherited from ancient circulating variants of concerns. The cross-T cell immunity between SARS-CoV-2 prototype strain and Omicron strains can be attributed to the T cell epitopes located in the N-terminal domain (181-246 aa [amino acids], 271-318 aa) and C-terminal domain (1171-1273 aa) of the spike protein. These findings provide in vivo evidence for optimizing vaccine manufacturing and immunization strategies for current or future viral variants.
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COVID-19 , Vacunas de ADN , Animales , Ratones , Humanos , Epítopos de Linfocito T/genética , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/genética , Inmunidad Celular , Mutación , Interferón gamma , Anticuerpos Antivirales , Anticuerpos NeutralizantesRESUMEN
BACKGROUND: Infection with the Epstein-Barr virus (EBV) elicits a complex T-cell response against a broad range of viral proteins. Hence, identifying potential differences in the cellular immune response of patients with different EBV-associated diseases or different courses of the same disorder requires interrogation of a maximum number of EBV antigens. Here, we tested three novel EBV-derived antigen formulations for their ability to reactivate virus-specific T cells ex vivo in patients with EBV-associated infectious mononucleosis (IM). METHODS: We comparatively analyzed EBV-specific CD4+ and CD8+ T-cell responses to three EBV-derived antigen formulations in 20 pediatric patients during the early phase of IM: T-activated EBV proteins (BZLF1, EBNA3A) and EBV-like particles (EB-VLP), both able to induce CD4+ and CD8+ T-cell responses ex vivo, as well as an EBV-derived peptide pool (PP) covering 94 well-characterized CD8+ T-cell epitopes. We assessed the specificity, magnitude, kinetics, and functional characteristics of EBV-specific immune responses at two sequential time points (v1 and v2) within the first six weeks after IM symptom onset (Tonset). RESULTS: All three tested EBV-derived antigen formulations enabled the detection of EBV-reactive T cells during the early phase of IM without prior T-cell expansion in vitro. EBV-reactive CD4+ and CD8+ T cells were mainly mono-functional (CD4+: mean 64.92%, range 56.15-71.71%; CD8+: mean 58.55%, range 11.79-85.22%) within the first two weeks after symptom onset (v1) with IFN-γ and TNF-secreting cells representing the majority of mono-functional EBV-reactive T cells. By contrast, PP-reactive CD8+ T cells were primarily bi-functional (>60% at v1 and v2), produced IFN-γ and TNF and had more tri-functional than mono-functional components. We observed a moderate correlation between viral load and EBNA3A, EB-VLP, and PP-reactive CD8+ T cells (rs = 0.345, 0.418, and 0.356, respectively) within the first two weeks after Tonset, but no correlation with the number of detectable EBV-reactive CD4+ T cells. CONCLUSIONS: All three EBV-derived antigen formulations represent innovative and generic recall antigens suitable for monitoring EBV-specific T-cell responses ex vivo. Their combined use facilitates a thorough analysis of EBV-specific T-cell immunity and allows the identification of functional T-cell signatures linked to disease development and severity.
Asunto(s)
Antígenos Virales , Linfocitos T CD4-Positivos , Linfocitos T CD8-positivos , Herpesvirus Humano 4 , Mononucleosis Infecciosa , Humanos , Mononucleosis Infecciosa/inmunología , Mononucleosis Infecciosa/virología , Antígenos Virales/inmunología , Herpesvirus Humano 4/inmunología , Niño , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD4-Positivos/inmunología , Femenino , Masculino , Adolescente , Preescolar , Epítopos de Linfocito T/inmunologíaRESUMEN
OBJECTIVE AND DESIGN: This observational study investigated the regulatory mechanism of Pim-1 in inflammatory signaling pathways. MATERIALS: THP-1, RAW 264.7, BV2, and Jurkat human T cell lines were used. TREATMENT: None. METHODS: Lipopolysaccharide (LPS) was used to induce inflammation, followed by PIM1 knockdown. Western blot, immunoprecipitation, immunofluorescence, and RT-PCR assays were used to assess the effect of PIM1 knockdown on LPS-induced inflammation. RESULTS: PIM1 knockdown in macrophage-like THP-1 cells suppressed LPS-induced upregulation of pro-inflammatory cytokines, inducible nitric oxide synthase, cyclooxygenase-2, phosphorylated Janus kinase, signal transducer and activator of transcription 3, extracellular signal-regulated kinase, c-Jun N-terminal kinase, p38, and nuclear factor kappa B p65 (NF-κB p65). It also suppressed upregulation of inhibitor of NF-κB kinase α/ß and enhanced the nuclear translocation of NF-κB p65. Moreover, it inhibited the upregulation of Nod-like receptor family pyrin domain-containing 3 (NLRP3) and cleavage of caspase-1 induced by co-treatment of LPS with adenosine triphosphate. Additionally, p-transforming growth factor-ß-activated kinase 1 (TAK1) interacted with Pim-1. All three members of Pim kinases (Pim-1, Pim-2, and Pim-3) were required for LPS-mediated inflammation in macrophages; however, unlike Pim-1 and Pim-3, Pim-2 functioned as a negative regulator of T cell activity. CONCLUSIONS: Pim-1 interacts with TAK1 in LPS-induced inflammatory responses and is involved in MAPK/NF-κB/NLRP3 signaling pathways. Additionally, considering the negative regulatory role of Pim-2 in T cells, further in-depth studies on their respective functions are needed.
RESUMEN
Regulatory T cells overwhelm conventional T cells in the tumor microenvironment (TME) thanks to a FOXP3-driven metabolic program that allows them to engage different metabolic pathways. Using a melanoma model of adoptive T cell therapy (ACT), we show that FOXP3 overexpression in mature CD8 T cells improved their antitumor efficacy, favoring their tumor recruitment, proliferation, and cytotoxicity. FOXP3-overexpressing (Foxp3UP) CD8 T cells exhibited features of tissue-resident memory-like and effector T cells, but not suppressor activity. Transcriptomic analysis of tumor-infiltrating Foxp3UP CD8 T cells showed positive enrichment in a wide variety of metabolic pathways, such as glycolysis, fatty acid (FA) metabolism, and oxidative phosphorylation (OXPHOS). Intratumoral Foxp3UP CD8 T cells exhibited an enhanced capacity for glucose and FA uptake as well as accumulation of intracellular lipids. Interestingly, Foxp3UP CD8 T cells compensated for the loss of mitochondrial respiration-driven ATP production by activating aerobic glycolysis. Moreover, in limiting nutrient conditions these cells engaged FA oxidation to drive OXPHOS for their energy demands. Importantly, their ability to couple glycolysis and OXPHOS allowed them to sustain proliferation under glucose restriction. Our findings demonstrate a hitherto unknown role for FOXP3 in the adaptation of CD8 T cells to TME that may enhance their efficacy in ACT.