RESUMEN
The envelope glycoprotein trimer mediates HIV-1 entry into cells. The trimer is flexible, fluctuating between closed and more open conformations and sometimes sampling the fully open, CD4-bound form. We hypothesized that conformational flexibility and transient exposure of non-neutralizing, immunodominant epitopes could hinder the induction of broadly neutralizing antibodies (bNAbs). We therefore modified soluble Env trimers to stabilize their closed, ground states. The trimer variants were indeed stabilized in the closed conformation, with a reduced ability to undergo receptor-induced conformational changes and a decreased exposure of non-neutralizing V3-directed antibody epitopes. In rabbits, the stabilized trimers induced similar autologous Tier-1B or Tier-2 NAb titers to those elicited by the corresponding wild-type trimers but lower levels of V3-directed Tier-1A NAbs. Stabilized, closed trimers might therefore be useful components of vaccines aimed at inducing bNAbs.
Asunto(s)
Vacunas contra el SIDA/química , Vacunas contra el SIDA/inmunología , Animales , Anticuerpos Neutralizantes , Epítopos/química , Proteína gp41 de Envoltorio del VIH/química , Proteína gp41 de Envoltorio del VIH/genética , VIH-1 , Interacciones Hidrofóbicas e Hidrofílicas , Inmunoglobulina G/química , Modelos Moleculares , Mutagénesis , Conformación Proteica , Conejos , Productos del Gen env del Virus de la Inmunodeficiencia Humana/químicaRESUMEN
Signaling via the inducible costimulator ICOS fuels the stepwise development of follicular helper T cells (TFH cells). However, a signaling pathway unique to ICOS has not been identified. We found here that the kinase TBK1 associated with ICOS via a conserved motif, IProx, that shares homology with the tumor-necrosis-factor receptor (TNFR)-associated factors TRAF2 and TRAF3. Disruption of this motif abolished the association of TBK1 with ICOS, TRAF2 and TRAF3, which identified a TBK1-binding consensus. Alteration of this motif in ICOS or depletion of TBK1 in T cells severely impaired the differentiation of germinal center (GC) TFH cells and the development of GCs, interfered with B cell differentiation and disrupted the development of antibody responses, but the IProx motif and TBK1 were dispensable for the early differentiation of TFH cells. These results reveal a previously unknown ICOS-TBK1 signaling pathway that specifies the commitment of GC TFH cells.
Asunto(s)
Linfocitos B/fisiología , Linfocitos T CD4-Positivos/fisiología , Centro Germinal/inmunología , Proteína Coestimuladora de Linfocitos T Inducibles/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Formación de Anticuerpos/genética , Diferenciación Celular/genética , Células Cultivadas , Proteína Coestimuladora de Linfocitos T Inducibles/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal , Factor 2 Asociado a Receptor de TNF/genética , Factor 3 Asociado a Receptor de TNF/genéticaRESUMEN
Immunization results in the differentiation of CD8+ T cells, such that they acquire effector abilities and convert into a memory pool. Priming of T cells takes place via an immunological synapse formed with an antigen-presenting cell (APC). By disrupting synaptic stability at different times, we found that the differentiation of CD8+ T cells required cell interactions beyond those made with APCs. We identified a critical differentiation period that required interactions between primed T cells. We found that T cell-T cell synapses had a major role in the generation of protective CD8+ T cell memory. T cell-T cell synapses allowed T cells to polarize critical secretion of interferon-γ (IFN-γ) toward each other. Collective activation and homotypic clustering drove cytokine sharing and acted as regulatory stimuli for T cell differentiation.
Asunto(s)
Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Comunicación Celular/inmunología , Diferenciación Celular/inmunología , Sinapsis Inmunológicas , Subgrupos de Linfocitos T/inmunología , Animales , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Citocinas/inmunología , Citocinas/metabolismo , Memoria Inmunológica , Ratones , Ratones Noqueados , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/metabolismoRESUMEN
Apoptosis, inflammation, and wound healing are critical pathophysiological events associated with various liver diseases. Currently, there is a lack of in vivo approaches to study hepatocyte apoptosis-induced liver injury and repair. To address this critical knowledge gap, we developed a unique genetically modified mouse model, namely, 3xTg-iHAP (3-Transgene with inducible Hepatocyte Apoptosis Phenotype) in this study. The 3xTg-iHAP mice possess three transgenes including Alb-Cre, Rosa26-rtTA, and tetO-Fasl on a B6 background. These mice are phenotypically normal, viable, and fertile. After subcutaneous administration of a single dose of doxycycline (5 mg/kg, Dox) to 3xTg-iHAP mice, we observed a complete histological spectrum of sterile liver wound-healing responses: asymptomatic hepatocyte apoptosis at 8 h, necrotic liver injury and sterile inflammation at 48 h, followed by hepatocyte mitosis and regeneration within 7 days. During the injury phase, the mice exhibited an increase in biomarkers of ALT, CXCL1, and IL-6 in peripheral blood and α-SMA protein in liver tissues. Conversely, the mice displayed a decrease in these markers in the recovery phase. Remarkably, this model shows that the sterile liver injury following elevated hepatocyte apoptosis is associated with an increase in myeloid cells in the liver. Within 7 days post-Dox administration, the liver of Dox-treated 3xTg-iHAP mice displays a normal histological structure, indicating completion of wound-healing. Together, we established a novel mouse model of injury and regeneration induced by hepatocyte apoptosis. This tool provides a robust in vivo platform for studying the pathophysiology of sterile liver inflammation, regeneration, and new therapeutic interventions for liver diseases.
RESUMEN
Most anti-cancer modalities are designed to directly kill cancer cells deploying mechanisms of action (MOAs) centered on the presence of a precise target on cancer cells. The efficacy of these approaches is limited because the rapidly evolving genetics of neoplasia swiftly circumvents the MOA generating therapy-resistant cancer cell clones. Other modalities engage endogenous anti-cancer mechanisms by activating the multi-cellular network (MCN) surrounding neoplastic cells in the tumor microenvironment (TME). These modalities hold a better chance of success because they activate numerous types of immune effector cells that deploy distinct cytotoxic MOAs. This in turn decreases the chance of developing treatment-resistance. Engagement of the MCN can be attained through activation of immune effector cells that in turn kill cancer cells or when direct cancer killing is complemented by the production of proinflammatory factors that secondarily recruit and activate immune effector cells. For instance, adoptive cell therapy (ACT) supplements cancer cell killing with the release of homeostatic and pro-inflammatory cytokines by the immune cells and damage associated molecular patterns (DAMPs) by dying cancer cells. The latter phenomenon, referred to as immunogenic cell death (ICD), results in an exponential escalation of anti-cancer MOAs at the tumor site. Other approaches can also induce exponential cancer killing by engaging the MCN of the TME through the release of DAMPs and additional pro-inflammatory factors by dying cancer cells. In this commentary, we will review the basic principles that support emerging paradigms likely to significantly improve the efficacy of anti-cancer therapy.
Asunto(s)
Antineoplásicos , Neoplasias , Humanos , Neoplasias/terapia , Antineoplásicos/uso terapéutico , Citocinas/metabolismo , Microambiente TumoralRESUMEN
Advancing personalized medicine in brain cancer relies on innovative strategies, with mRNA vaccines emerging as a promising avenue. While the initial use of mRNA vaccines was in oncology, their stunning success in COVID-19 resulted in widespread attention, both positive and negative. Regardless of politically biased opinions, which relate more to the antigenic source than form of delivery, we feel it is important to objectively review this modality as relates to brain cancer. This class of vaccines trigger robust immune responses through MHC-I and MHC-II pathways, in both prophylactic and therapeutic settings. The mRNA platform offers advantages of rapid development, high potency, cost-effectiveness, and safety. This review provides an overview of mRNA vaccine delivery technologies, tumor antigen identification, combination therapies, and recent therapeutic outcomes, with a particular focus on brain cancer. Combinatorial approaches are vital to maximizing mRNA cancer vaccine efficacy, with ongoing clinical trials exploring combinations with adjuvants and checkpoint inhibitors and even adoptive cell therapy. Efficient delivery, neoantigen identification, preclinical studies, and clinical trial results are highlighted, underscoring mRNA vaccines' potential in advancing personalized medicine for brain cancer. Synergistic combinatorial therapies play a crucial role, emphasizing the need for continued research and collaboration in this area.
Asunto(s)
Neoplasias Encefálicas , Vacunas contra el Cáncer , Neoplasias , Humanos , Medicina de Precisión/métodos , Inmunoterapia/métodos , Neoplasias Encefálicas/terapia , Neoplasias Encefálicas/tratamiento farmacológico , ARN Mensajero/genética , Neoplasias/terapiaRESUMEN
MicroRNA (miRNA)-deficient helper T cells exhibit abnormal IFN-γ production and decreased proliferation. However, the contributions of individual miRNAs to this phenotype remain poorly understood. We conducted a screen for miRNA function in primary T cells and identified individual miRNAs that rescue the defects associated with miRNA deficiency. Multiple members of the miR-17 and miR-92 families enhanced miRNA-deficient T cell proliferation whereas miR-29 largely corrected their aberrant interferon-γ (IFN-γ) expression. Repression of IFN-γ production by miR-29 involved direct targeting of both T-bet and Eomes, two transcription factors known to induce IFN-γ production. Although not usually expressed at functionally relevant amounts in helper T cells, Eomes was abundant in miRNA-deficient cells and was upregulated after miR-29 inhibition in wild-type cells. These results demonstrate that miR-29 regulates helper T cell differentiation by repressing multiple target genes, including at least two that are independently capable of inducing the T helper 1 (Th1) cell gene expression program.
Asunto(s)
Interferón gamma/metabolismo , MicroARNs/metabolismo , Proteínas de Dominio T Box/metabolismo , Linfocitos T Colaboradores-Inductores/metabolismo , Animales , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Citocinas/genética , Citocinas/inmunología , Citocinas/metabolismo , Regulación de la Expresión Génica/inmunología , Interferón gamma/genética , Interferón gamma/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , MicroARNs/genética , MicroARNs/inmunología , Proteínas/genética , Proteínas de Unión al ARN , Proteínas de Dominio T Box/inmunología , Linfocitos T Colaboradores-Inductores/citología , Linfocitos T Colaboradores-Inductores/inmunologíaRESUMEN
Natural infections expose the immune system to escalating antigen and inflammation over days to weeks, whereas nonlive vaccines are single bolus events. We explored whether the immune system responds optimally to antigen kinetics most similar to replicating infections, rather than a bolus dose. Using HIV antigens, we found that administering a given total dose of antigen and adjuvant over 1-2 wk through repeated injections or osmotic pumps enhanced humoral responses, with exponentially increasing (exp-inc) dosing profiles eliciting >10-fold increases in antibody production relative to bolus vaccination post prime. Computational modeling of the germinal center response suggested that antigen availability as higher-affinity antibodies evolve enhances antigen capture in lymph nodes. Consistent with these predictions, we found that exp-inc dosing led to prolonged antigen retention in lymph nodes and increased Tfh cell and germinal center B-cell numbers. Thus, regulating the antigen and adjuvant kinetics may enable increased vaccine potency.
Asunto(s)
Vacunas contra el SIDA/administración & dosificación , Anticuerpos Antivirales/biosíntesis , Linfocitos B/efectos de los fármacos , Centro Germinal/efectos de los fármacos , Proteína gp120 de Envoltorio del VIH/administración & dosificación , Vacunación/métodos , Adyuvantes Inmunológicos/administración & dosificación , Animales , Afinidad de Anticuerpos , Linfocitos B/citología , Linfocitos B/inmunología , Células CHO , Cricetulus , Esquema de Medicación , Femenino , Centro Germinal/citología , Centro Germinal/inmunología , Células HEK293 , Proteína gp120 de Envoltorio del VIH/biosíntesis , Humanos , Inmunogenicidad Vacunal , Bombas de Infusión Implantables , Lípido A/administración & dosificación , Lípido A/análogos & derivados , Ratones , Ratones Endogámicos C57BL , Presión Osmótica , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/biosíntesis , Vacunación/instrumentaciónRESUMEN
Follicular CD4(+) Th (Tfh) cells provide B cell help in germinal center reactions that support class switching, somatic hypermutation, and the generation of high-affinity Abs. In this article, we show that deficiency in NFAT1 and NFAT2 in CD4(+) T cells leads to impaired germinal center reactions upon viral infection because of reduced Tfh cell differentiation and defective expression of proteins involved in T/B interactions and B cell help, including ICOS, PD-1, and SLAM family receptors. Genome-wide chromatin immunoprecipitation data suggest that NFAT proteins likely directly participate in regulation of genes important for Tfh cell differentiation and function. NFAT proteins are important TCR and Ca(2+)-dependent regulators of T cell biology, and in this article we demonstrate a major positive role of NFAT family members in Tfh differentiation.
Asunto(s)
Diferenciación Celular/inmunología , Regulación de la Expresión Génica/inmunología , Factores de Transcripción NFATC/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Virosis/inmunología , Animales , Inmunoprecipitación de Cromatina , Centro Germinal/inmunología , Activación de Linfocitos/inmunología , Ratones , Ratones Transgénicos , Linfocitos T Colaboradores-Inductores/citologíaRESUMEN
T follicular helper (Tfh) cells are essential providers of help to B cells. The transcription factor B-cell CLL/lymphoma 6 (Bcl6) is a lineage-defining regulator of Tfh cells and germinal center B cells. In B cells, Bcl6 has the potential to recruit distinct transcriptional corepressors through its BTB domain or its poorly characterized middle domain (also known as RDII), but in Tfh cells the roles of the Bcl6 middle domain have yet to be clarified. Mimicked acetylation of the Bcl6 middle domain (K379Q) in CD4 T cells results in significant reductions in Tfh differentiation in vivo. Blimp1 (Prdm1) is a potent inhibitor of Tfh cell differentiation. Although Bcl6 K379Q still bound to the Prdm1 cis-regulatory elements in Tfh cells, Prdm1 expression was derepressed. This was a result of the failure of Bcl6 K379Q to recruit metastasis-associated protein 3 (MTA3). The loss of Bcl6 function in Bcl6 K379Q-expressing CD4 T cells could be partially rescued by abrogating Prdm1 expression. In addition to Prdm1, we found that Bcl6 recruits MTA3 to multiple genes involved in Tfh cell biology, including genes important for cell migration, cell survival, and alternative differentiation pathways. Thus, Bcl6 middle domain mediated repression is a major mechanism of action by which Bcl6 controls CD4 T-cell fate and function.
Asunto(s)
Diferenciación Celular/inmunología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/inmunología , Proteínas de Neoplasias/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Inmunoprecipitación de Cromatina , Clonación Molecular , Cartilla de ADN/genética , Citometría de Flujo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutagénesis Sitio-Dirigida , Proteínas de Neoplasias/genética , Factor 1 de Unión al Dominio 1 de Regulación Positiva , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-bcl-6 , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Represoras/inmunología , Factores de Transcripción/metabolismoRESUMEN
UNLABELLED: Generating neutralizing antibodies (nAbs) is a major goal of many current HIV-1 vaccine efforts. To be of practical value, these nAbs must be both potent and cross-reactive in order to be capable of preventing the transmission of the highly diverse and generally neutralization resistant (Tier-2) HIV-1 strains that are in circulation. The HIV-1 envelope glycoprotein (Env) spike is the only target for nAbs. To explore whether Tier-2 nAbs can be induced by Env proteins, we immunized conventional mice with soluble BG505 SOSIP.664 trimers that mimic the native Env spike. Here, we report that it is extremely difficult for murine B cells to recognize the Env epitopes necessary for inducing Tier-2 nAbs. Thus, while trimer-immunized mice raised Env-binding IgG Abs and had high-quality T follicular helper (Tfh) cell and germinal center (GC) responses, they did not make BG505.T332N nAbs. Epitope mapping studies showed that Ab responses in mice were specific to areas near the base of the soluble trimer. These areas are not well shielded by glycans and likely are occluded on virions, which is consistent with the lack of BG505.T332N nAbs. These data inform immunogen design and suggest that it is useful to obscure nonneutralizing epitopes presented on the base of soluble Env trimers and that the glycan shield of well-formed HIV Env trimers is virtually impenetrable for murine B cell receptors (BCRs). IMPORTANCE: Human HIV vaccine efficacy trials have not generated meaningful neutralizing antibodies to circulating HIV strains. One possible hindrance has been the lack of immunogens that properly mimic the native conformation of the HIV envelope trimer protein. Here, we tested the first generation of soluble, native-like envelope trimer immunogens in a conventional mouse model. We attempted to generate neutralizing antibodies to neutralization-resistant circulating HIV strains. Various vaccine strategies failed to induce neutralizing antibodies to a neutralization-resistant HIV strain. Further analysis revealed that mouse antibodies targeted areas near the bottom of the soluble envelope trimers. These areas are not easily accessible on the HIV virion due to occlusion by the viral membrane and may have resulted from an absence of glycan shielding. Our results suggest that obscuring the bottom of soluble envelope trimers is a useful strategy to reduce antibody responses to epitopes that are not useful for virus neutralization.
Asunto(s)
Anticuerpos Neutralizantes/biosíntesis , Antígenos Virales/química , Anticuerpos Anti-VIH/biosíntesis , Infecciones por VIH/prevención & control , Inmunoglobulina G/biosíntesis , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Animales , Anticuerpos Neutralizantes/química , Especificidad de Anticuerpos , Antígenos Virales/inmunología , Linfocitos B/inmunología , Mapeo Epitopo , Epítopos/química , Epítopos/inmunología , Expresión Génica , Células HEK293 , Anticuerpos Anti-VIH/química , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/química , VIH-1/genética , VIH-1/inmunología , Humanos , Inmunización , Inmunoglobulina G/química , Ratones , Modelos Moleculares , Unión Proteica , Ingeniería de Proteínas , Multimerización de Proteína , Receptores de Antígenos de Linfocitos B/química , Receptores de Antígenos de Linfocitos B/inmunología , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Solubilidad , Productos del Gen env del Virus de la Inmunodeficiencia Humana/administración & dosificación , Productos del Gen env del Virus de la Inmunodeficiencia Humana/química , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Generation of a robust immunological memory response is essential for protection on subsequent encounters with the same pathogen. The magnitude and quality of the memory CD8 T-cell population are shaped and influenced by the strength and duration of the initial antigenic stimulus as well as by inflammatory cytokines. Although chemokine receptors have been established to play a role in recruitment of effector CD8 T cells to sites of inflammation, their contribution to determination of T-cell fate and shaping of the long-lived memory T-cell population is not fully understood. Here, we investigated whether reduced access to antigen and inflammation through alterations in expression of inflammatory and homeostatic chemokine receptors has an impact on generation of effector and memory CD8 T cells. We found that in mice infected with lymphocytic choriomeningitis virus, colocalization of virus-specific CD8 T cells with antigen in spleen is dependent on expression of the inflammatory chemokine receptor, CXCR3. In addition, absence of CXCR3 expression on CD8 T cells leads to formation of fewer short-lived effector cells and more memory precursor cells. Furthermore, the memory CD8 T-cell population derived from CXCR3-deficient cells has fewer cells of the effector memory phenotype and exhibits a recall response of greater magnitude than that of WT cells. These data demonstrate that CD8 T-cell positioning relative to antigen and inflammatory cytokines in secondary lymphoid organs affects the balance of effector and memory T-cell formation and has both a quantitative and qualitative impact on the long-lived memory CD8 T-cell population.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Memoria Inmunológica , Receptores CXCR3/inmunología , Linfocitos T/inmunología , Animales , Linfocitos T CD8-positivos/virología , Citocinas/inmunología , Recuento de Linfocitos , Virus de la Coriomeningitis Linfocítica/inmunología , Ratones , Linfocitos T/citologíaRESUMEN
Immunodominance to nonneutralizing epitopes is a roadblock in designing vaccines against several diseases of high interest. One hypothetical possibility is that limited CD4 T cell help to B cells in a normal germinal center (GC) response results in selective recruitment of abundant, immunodominant B cells. This is a central issue in HIV envelope glycoprotein (Env) vaccine designs, because precursors to broadly neutralizing epitopes are rare. Here, we sought to elucidate whether modulating the quantity of T cell help can influence recruitment and competition of broadly neutralizing antibody precursor B cells at a physiological precursor frequency in response to Env trimer immunization. To do so, two new Env-specific CD4 transgenic (Tg) T cell receptor (TCR) mouse lines were generated, carrying TCR pairs derived from Env-protein immunization. Our results suggest that CD4 T cell help quantitatively regulates early recruitment of rare B cells to GCs.
Asunto(s)
Linfocitos B/inmunología , Linfocitos T CD4-Positivos/inmunología , Centro Germinal/inmunología , VIH-1/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Vacunas contra el SIDA/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Antígenos Virales/inmunología , Línea Celular , Epítopos/inmunología , Femenino , Células HEK293 , Anticuerpos Anti-VIH/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/virología , Humanos , Inmunización/métodos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Vacunación/métodosRESUMEN
DNA topoisomerase I is an essential nuclear enzyme involved in resolving the torsional stress associated with DNA replication, transcription, and chromatin condensation. Here we report the discovery of a seizure-suppressor mutant, top1(JS), which suppresses seizures in a Drosophila model of human epilepsy. A P-element mutagenesis screen using easily shocked seizure-sensitive mutant as a genetic background identified top1(JS), which plays a novel role in regulating nervous system excitability. Plasmid rescue, excision, complementation, and sequencing analyses verified that top1(JS) results from a P-element insertion in the 5' untranslated region. Quantitative reverse transcription analysis on wild-type and mutant fly heads showed that the top1(JS) mutation causes reduced transcription level in the CNS, suggesting a partial loss-of-function mutation. Electrophysiological experiments revealed normal seizure thresholds in top1(JS) mutants, which are different from other seizure suppressors identified previously, suggesting a novel mechanism underlying seizure suppression by top1(JS). The pharmacological camptothecin feeding experiment and cell death analysis suggested that the seizure suppression by top1(JS) may occur via increased neuronal apoptosis. Furthermore, overexpression of the DIAP1 (Drosophila inhibitor of apoptosis 1) gene rescues top1(JS) suppression, providing additional support for a neural apoptosis suppression mechanism. The top1(JS) mutation is the first viable partial loss-of-function mutation identified in higher eukaryotes, and the results presented here point to a novel function for topo I in construction and/or maintenance of circuits required for seizure propagation in vivo.
Asunto(s)
Alelos , ADN-Topoisomerasas de Tipo I/genética , Proteínas de Drosophila/genética , Mutación , Convulsiones/enzimología , Convulsiones/prevención & control , Animales , ADN-Topoisomerasas de Tipo I/fisiología , Drosophila , Proteínas de Drosophila/fisiología , Convulsiones/genéticaRESUMEN
An individual's T cell repertoire dynamically encodes their pathogen exposure history. To determine whether pathogen exposure signatures can be identified by documenting public T cell receptors (TCRs), we profiled the T cell repertoire of 666 subjects with known cytomegalovirus (CMV) serostatus by immunosequencing. We developed a statistical classification framework that could diagnose CMV status from the resulting catalog of TCRß sequences with high specificity and sensitivity in both the original cohort and a validation cohort of 120 different subjects. We also confirmed that three of the identified CMV-associated TCRß molecules bind CMV in vitro, and, moreover, we used this approach to accurately predict the HLA-A and HLA-B alleles of most subjects in the first cohort. As all memory T cell responses are encoded in the common format of somatic TCR recombination, our approach could potentially be generalized to a wide variety of disease states, as well as other immunological phenotypes, as a highly parallelizable diagnostic strategy.
Asunto(s)
Citomegalovirus/inmunología , Antígenos HLA/inmunología , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Algoritmos , Estudios de Cohortes , Citomegalovirus/genética , Citomegalovirus/fisiología , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/inmunología , Antígenos HLA/genética , Antígenos HLA-A/genética , Antígenos HLA-A/inmunología , Antígenos HLA-B/genética , Antígenos HLA-B/inmunología , Prueba de Histocompatibilidad/métodos , Interacciones Huésped-Patógeno/inmunología , Humanos , Modelos Inmunológicos , Receptores de Antígenos de Linfocitos T/genética , Reproducibilidad de los Resultados , Linfocitos T/metabolismo , Linfocitos T/virologíaRESUMEN
SAP (SH2D1A) is required intrinsically in CD4 T cells to generate germinal center responses and long-term humoral immunity. SAP binds to SLAM family receptors, including SLAM, CD84, and Ly108 to enhance cytokine secretion and sustained T cell:B cell adhesion, which both improve T follicular helper (Tfh) cell aid to germinal center (GC) B cells. To understand the overlapping roles of multiple SLAM family receptors in germinal center responses, Slamf1Δ/Δ Slamf5Δ/Δ Slamf6Δ/Δ triple gene disruption (Slamf1,5,6Δ/Δ) mice were generated using CRISPR-Cas9 gene editing to eliminate expression of SLAM (CD150), CD84, and Ly108, respectively. Gene targeting was highly efficient, with 6 of 6 alleles disrupted in 14 of 23 pups and the majority of alleles disrupted in the remaining pups. NKT cell differentiation in Slamf1,5,6Δ/Δ mice was defective, but not completely absent. The remaining NKT cells exhibited substantially increased 2B4 (SLAMF4) expression. Surprisingly, there were no overt defects in germinal center responses to acute viral infections or protein immunizations in Slamf1,5,6Δ/Δ mice, unlike Sh2d1a-/- mice. Similarly, in the context of a competitive environment, SLAM family receptor expressing GC Tfh cell, GC B cell, and plasma cell responses exhibited no advantages over Slamf1,5,6Δ/Δ cells.
Asunto(s)
Técnicas de Silenciamiento del Gen , Células T Asesinas Naturales/inmunología , Células Plasmáticas/inmunología , Miembro 1 de la Familia de Moléculas Señalizadoras de la Activación Linfocitaria/inmunología , Familia de Moléculas Señalizadoras de la Activación Linfocitaria/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Edición Génica , Ratones , Familia de Moléculas Señalizadoras de la Activación Linfocitaria/genética , Miembro 1 de la Familia de Moléculas Señalizadoras de la Activación Linfocitaria/genéticaRESUMEN
Generating long-term humoral immunity is a crucial component of successful vaccines and requires interactions between T cells and B cells in germinal centers (GC). In GCs, a specialized subset of CD4+ helper T cells, called T follicular helper cells (Tfh), provide help to B cells; this help directs the magnitude and quality of the antibody response. Tfh cell help influences B cell survival, proliferation, somatic hypermutation, class switch recombination, and differentiation. Sustained contact between Tfh cells and B cells is necessary for the provision of help to B cells. SAP (Signaling lymphocytic activation molecule (SLAM)-associated protein, encoded by Sh2d1a) regulates the duration of T:B cell interactions and is required for long-term humoral immunity in animal models and in humans. SAP binds to SLAM family receptors and mediates signaling that affects cell adhesion, cytokine secretion, and TCR signaling strength. Therefore, the modulation of SAP and SLAM family receptor expression represents a major axis by which the quality and duration of an antibody response is controlled after vaccination.