RESUMEN
As COVID-19 continues, an increasing number of patients develop long COVID symptoms varying in severity that last for weeks, months, or longer. Symptoms commonly include lingering loss of smell and taste, hearing loss, extreme fatigue, and "brain fog." Still, persistent cardiovascular and respiratory problems, muscle weakness, and neurologic issues have also been documented. A major problem is the lack of clear guidelines for diagnosing long COVID. Although some studies suggest that long COVID is due to prolonged inflammation after SARS-CoV-2 infection, the underlying mechanisms remain unclear. The broad range of COVID-19's bodily effects and responses after initial viral infection are also poorly understood. This workshop brought together multidisciplinary experts to showcase and discuss the latest research on long COVID and chronic inflammation that might be associated with the persistent sequelae following COVID-19 infection.
Asunto(s)
COVID-19 , Síndrome Post Agudo de COVID-19 , Humanos , SARS-CoV-2 , Inflamación , Progresión de la EnfermedadRESUMEN
Natural killer (NK) cells are a subset of innate lymphoid cells (ILC) capable of recognizing stressed and infected cells through multiple germ line-encoded receptor-ligand interactions. Missing-self recognition involves NK cell sensing of the loss of host-encoded inhibitory ligands on target cells, including MHC class I (MHC-I) molecules and other MHC-I-independent ligands. Mouse cytomegalovirus (MCMV) infection promotes a rapid host-mediated loss of the inhibitory NKR-P1B ligand Clr-b (encoded by Clec2d) on infected cells. Here we provide evidence that an MCMV m145 family member, m153, functions to stabilize cell surface Clr-b during MCMV infection. Ectopic expression of m153 in fibroblasts augments Clr-b cell surface levels. Moreover, infections using m153-deficient MCMV mutants (Δm144-m158 and Δm153) show an accelerated and exacerbated Clr-b downregulation. Importantly, enhanced loss of Clr-b during Δm153 mutant infection reverts to wild-type levels upon exogenous m153 complementation in fibroblasts. While the effects of m153 on Clr-b levels are independent of Clec2d transcription, imaging experiments revealed that the m153 and Clr-b proteins only minimally colocalize within the same subcellular compartments, and tagged versions of the proteins were refractory to coimmunoprecipitation under mild-detergent conditions. Surprisingly, the Δm153 mutant possesses enhanced virulence in vivo, independent of both Clr-b and NKR-P1B, suggesting that m153 potentially targets additional host factors. Nevertheless, the present data highlight a unique mechanism by which MCMV modulates NK ligand expression.IMPORTANCE Cytomegaloviruses are betaherpesviruses that in immunocompromised individuals can lead to severe pathologies. These viruses encode various gene products that serve to evade innate immune recognition. NK cells are among the first immune cells that respond to CMV infection and use germ line-encoded NK cell receptors (NKR) to distinguish healthy from virus-infected cells. One such axis that plays a critical role in NK recognition involves the inhibitory NKR-P1B receptor, which engages the host ligand Clr-b, a molecule commonly lost on stressed cells ("missing-self"). In this study, we discovered that mouse CMV utilizes the m153 glycoprotein to circumvent host-mediated Clr-b downregulation, in order to evade NK recognition. These results highlight a novel MCMV-mediated immune evasion strategy.
Asunto(s)
Interacciones Huésped-Patógeno/genética , Células Asesinas Naturales/virología , Lectinas Tipo C/genética , Muromegalovirus/genética , Subfamilia B de Receptores Similares a Lectina de Células NK/genética , Receptores Inmunológicos/genética , Proteínas de la Matriz Viral/genética , Animales , Regulación de la Expresión Génica/inmunología , Prueba de Complementación Genética , Infecciones por Herpesviridae , Interacciones Huésped-Patógeno/inmunología , Inmunidad Innata , Células Asesinas Naturales/inmunología , Lectinas Tipo C/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Muromegalovirus/inmunología , Muromegalovirus/patogenicidad , Células 3T3 NIH , Subfamilia B de Receptores Similares a Lectina de Células NK/inmunología , Receptores Inmunológicos/inmunología , Transducción de Señal , Carga Viral , Proteínas de la Matriz Viral/deficiencia , Proteínas de la Matriz Viral/inmunología , Replicación ViralRESUMEN
Adverse drug reactions (ADRs) are a major obstacle to drug development, and some of these, including hypersensitivity reactions to the HIV reverse transcriptase inhibitor abacavir (ABC), are associated with HLA alleles, particularly HLA-B*57:01. However, not all HLA-B*57:01+ patients develop ADRs, suggesting that in addition to the HLA genetic risk, other factors may influence the outcome of the response to the drug. To study HLA-linked ADRs in vivo, we generated HLA-B*57:01-Tg mice and show that, although ABC activated Tg mouse CD8+ T cells in vitro in a HLA-B*57:01-dependent manner, the drug was tolerated in vivo. In immunocompetent Tg animals, ABC induced CD8+ T cells with an anergy-like phenotype that did not lead to ADRs. In contrast, in vivo depletion of CD4+ T cells prior to ABC administration enhanced DC maturation to induce systemic ABC-reactive CD8+ T cells with an effector-like and skin-homing phenotype along with CD8+ infiltration and inflammation in drug-sensitized skin. B7 costimulatory molecule blockade prevented CD8+ T cell activation. These Tg mice provide a model for ABC tolerance and for the generation of HLA-B*57:01-restricted, ABC-reactive CD8+ T cells dependent on both HLA genetic risk and immunoregulatory host factors.
Asunto(s)
Didesoxinucleósidos/efectos adversos , Tolerancia a Medicamentos/genética , Tolerancia a Medicamentos/inmunología , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/genética , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/inmunología , Antígenos HLA-B/genética , Animales , Fármacos Anti-VIH/efectos adversos , Linfocitos T CD8-positivos/inmunología , Modelos Animales de Enfermedad , Hipersensibilidad a las Drogas/genética , Hipersensibilidad a las Drogas/inmunología , Femenino , Humanos , Técnicas In Vitro , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Inhibidores de la Transcriptasa Inversa/efectos adversosRESUMEN
The molecular mechanism through which the interaction of a clonotypic αß T-cell receptor (TCR) with a peptide-loaded major histocompatibility complex (p/MHC) leads to T-cell activation is not yet fully understood. Here we exploit a high-affinity TCR (B4.2.3) to examine the structural changes that accompany binding to its p/MHC ligand (P18-I10/H2-Dd). In addition to conformational changes in complementarity-determining regions (CDRs) of the TCR seen in comparison of unliganded and bound X-ray structures, NMR characterization of the TCR ß-chain dynamics reveals significant chemical shift effects in sites removed from the MHC-binding site. Remodelling of electrostatic interactions near the Cß H3 helix at the membrane-proximal face of the TCR, a region implicated in interactions with the CD3 co-receptor, suggests a possible role for an allosteric mechanism in TCR signalling. The contribution of these TCR residues to signal transduction is supported by mutagenesis and T-cell functional assays.
Asunto(s)
Sitio Alostérico/inmunología , Regiones Determinantes de Complementariedad/química , Receptores de Antígenos de Linfocitos T alfa-beta/química , Transducción de Señal/inmunología , Linfocitos T/inmunología , Animales , Regiones Determinantes de Complementariedad/metabolismo , Cristalografía por Rayos X , Complejo Mayor de Histocompatibilidad/inmunología , Ratones , Simulación de Dinámica Molecular , Mutagénesis , Péptidos/metabolismo , Unión Proteica/inmunología , Dominios Proteicos/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Linfocitos T/metabolismoRESUMEN
Drugs such as linezolid that inhibit bacterial protein synthesis may be beneficial in treating infections caused by toxigenic Staphylococcus aureus. As protein synthesis inhibitors have no effect on preformed toxins, neutralization of pathogenic exotoxins with anti-toxin antibodies may be beneficial in conjunction with antibacterial therapy. Herein, we evaluated the efficacy of human-mouse chimeric high-affinity neutralizing anti-staphylococcal enterotoxin B (SEB) antibodies in the treatment of experimental pneumonia caused by SEB-producing S. aureus. Since HLA class II transgenic mice mount a stronger systemic immune response following challenge with SEB and are more susceptible to SEB-induced lethal toxic shock than conventional mice strains, HLA-DR3 transgenic mice were used. Lethal pneumonia caused by SEB-producing S. aureus in HLA-DR3 transgenic mice was characterized by robust T cell activation and elevated systemic levels of several pro-inflammatory cytokines and chemokines. Prophylactic administration of a single dose of linezolid 30 min prior to the onset of infection attenuated the systemic inflammatory response and protected from mortality whereas linezolid administered 60 min after the onset of infection failed to confer significant protection. Human-mouse chimeric high-affinity neutralizing anti-SEB antibodies alone, but not polyclonal human IgG, mitigated this response and protected from death when administered immediately after initiation of infection. Further, anti-SEB antibodies as well as intact polyclonal human IgG, but not its Fab or Fc fragments, protected from lethal pneumonia when followed with linezolid therapy 60 min later. In conclusion, neutralization of superantigens with high-affinity antibodies may have beneficial effects in pneumonia.
Asunto(s)
Anticuerpos Antibacterianos/administración & dosificación , Anticuerpos Monoclonales Humanizados/administración & dosificación , Enterotoxinas/inmunología , Inmunización Pasiva , Neumonía/terapia , Infecciones Estafilocócicas/terapia , Staphylococcus aureus/efectos de los fármacos , Animales , Citocinas/genética , Citocinas/inmunología , Enterotoxinas/genética , Antígeno HLA-DR3/genética , Antígeno HLA-DR3/inmunología , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neumonía/genética , Neumonía/inmunología , Neumonía/microbiología , Infecciones Estafilocócicas/genética , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/genética , Staphylococcus aureus/inmunología , Linfocitos T/inmunologíaRESUMEN
T cell stimulation requires the input and integration of external signals. Signaling through the T cell receptor (TCR) is known to induce formation of the membrane-tethered CBM complex, comprising CARMA1, BCL10, and MALT1, which is required for TCR-mediated NF-κB activation. TCR signaling has been shown to activate NOTCH proteins, transmembrane receptors also implicated in NF-κB activation. However, the link between TCR-mediated NOTCH signaling and early events leading to induction of NF-κB activity remains unclear. In this report, we demonstrate a novel cytosolic function for NOTCH1 and show that it is essential to CBM complex formation. Using a model of skin allograft rejection, we show in vivo that NOTCH1 acts in the same functional pathway as PKCθ, a T cell-specific kinase important for CBM assembly and classical NF-κB activation. We further demonstrate in vitro NOTCH1 associates physically with PKCθ and CARMA1 in the cytosol. Unexpectedly, when NOTCH1 expression was abrogated using RNAi approaches, interactions between CARMA1, BCL10, and MALT1 were lost. This failure in CBM assembly reduced inhibitor of kappa B alpha phosphorylation and diminished NF-κB-DNA binding. Finally, using a luciferase gene reporter assay, we show the intracellular domain of NOTCH1 can initiate robust NF-κB activity in stimulated T cells, even when NOTCH1 is excluded from the nucleus through modifications that restrict it to the cytoplasm or hold it tethered to the membrane. Collectively, these observations provide evidence that NOTCH1 may facilitate early events during T cell activation by nucleating the CBM complex and initiating NF-κB signaling.
RESUMEN
Staphylococcal enterotoxin B (SEB) is one of a family of toxins secreted by Staphylococcus aureus that act as superantigens, activating a large fraction of the T-cell population and inducing production of high levels of inflammatory cytokines that can cause toxic shock syndrome (TSS) and death. Extracellular engagement of the TCR of T-cells and class II MHC of antigen presenting cells by SEB triggers the activation of many intracellular signaling processes. We engineered chimeric antibodies to block the extracellular engagement of cellular receptors by SEB and used a statin to inhibit intracellular signaling. Chimeric human-mouse antibodies directed against different neutralizing epitopes of SEB synergistically inhibited its activation of human T-cells in vitro. In the in vivo model of lethal toxic shock syndrome (TSS) in HLA-DR3 transgenic mice, two of these antibodies conferred significant partial protection when administered individually, but offered complete protection in a synergistic manner when given together. Similarly, in vivo, lovastatin alone conferred only partial protection from TSS similar to single anti-SEB antibodies. However, used in combination with one chimeric neutralizing anti-SEB antibody, lovastatin provided complete protection against lethal TSS in HLA-DR3 transgenic mice. These experiments demonstrate that in vivo protection against lethal doses of SEB can be achieved by a statin of proven clinical safety and chimeric human-mouse antibodies, agents now widely used and known to be of low immunogenicity in human hosts.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Anticuerpos Neutralizantes/inmunología , Anticolesterolemiantes/uso terapéutico , Enterotoxinas/uso terapéutico , Lovastatina/uso terapéutico , Choque Séptico/prevención & control , Animales , Anticuerpos Antibacterianos/uso terapéutico , Anticuerpos Neutralizantes/uso terapéutico , Sinergismo Farmacológico , Enterotoxinas/genética , Enterotoxinas/inmunología , Antígeno HLA-DR3/genética , Antígeno HLA-DR3/inmunología , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Choque Séptico/inmunología , Choque Séptico/mortalidad , Superantígenos/inmunología , Tasa de SupervivenciaRESUMEN
Staphylococcal enterotoxin B (SEB), a shock-inducing exotoxin synthesized by Staphylococcus aureus, is an important cause of food poisoning and is a class B bioterrorism agent. SEB mediates antigen-independent activation of a major subset of the T-cell population by cross-linking T-cell receptors (TCRs) with class II major histocompatibility complex (MHC-II) molecules of antigen-presenting cells, resulting in the induction of antigen independent proliferation and cytokine secretion by a significant fraction of the T-cell population. Neutralizing antibodies inhibit SEB-mediated T-cell activation by blocking the toxin's interaction with the TCR or MHC-II and provide protection against the debilitating effects of this superantigen. We derived and searched a set of monoclonal mouse anti-SEB antibodies to identify neutralizing anti-SEB antibodies that bind to different sites on the toxin. A pair of non-cross-reactive, neutralizing anti-SEB monoclonal antibodies (MAbs) was found, and a combination of these antibodies inhibited SEB-induced T-cell proliferation in a synergistic rather than merely additive manner. In order to engineer antibodies more suitable than mouse MAbs for use in humans, the genes encoding the VL and VH gene segments of a synergistically acting pair of mouse MAbs were grafted, respectively, onto genes encoding the constant regions of human Igkappa and human IgG1, transfected into mammalian cells, and used to generate chimeric versions of these antibodies that had affinity and neutralization profiles essentially identical to their mouse counterparts. When tested in cultures of human peripheral blood mononuclear cells or splenocytes derived from HLA-DR3 transgenic mice, the chimeric human-mouse antibodies synergistically neutralized SEB-induced T-cell activation and cytokine production.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Enterotoxinas/antagonistas & inhibidores , Staphylococcus aureus/inmunología , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Antitoxinas/genética , Antitoxinas/inmunología , Sangre/inmunología , Células Cultivadas , Citocinas/metabolismo , Humanos , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Leucocitos Mononucleares/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Pruebas de Neutralización , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Bazo/inmunologíaRESUMEN
WC1 is a transmembrane glycoprotein and member of the scavenger receptor cysteine rich (SRCR) family that is uniquely expressed on gammadelta T cells. The WC1 isoforms referred to as WC1.1, WC1.2, and WC1.3 are expressed on discrete subpopulations of gammadelta T cells with WC1.1 and WC1.2 expressed on mostly nonoverlapping gammadelta T cell populations. Studies have demonstrated a potential role for WC1 in modulating the response of gammadelta T cells but have not converged into a single accepted paradigm. Recent investigations that examined changing representation among mononuclear cells with age and patterns of proliferation and cytokine production by subsets bearing one or more of the previously identified variants of the WC1 molecule are summarized here. While the decrease in percentages within blood in the first year of life was found to be precipitous for WC1.1+ gammadelta T cells it was not for WC1.2+ cells. While both populations proliferated to mitogen stimulation there was a bias towards responses by WC1.2+ cells. In leptospira antigen-stimulated cultures and autologous mixed lymphocyte reaction (AMLR) cultures WC1.1+ cells proliferated and produced interferon-gamma (IFN-gamma) while WC1.2+ cells did to a much lower extent. This suggested functional differences related to the isoform of WC1 expressed. Under Th1-polarizing conditions, the WC1.1+ cells also made IFN-gamma whereas the vast majority of cells expressing WC1.2 did not. Despite the difference in IFN-gamma production, cells bearing either WC1 isoform had similar transcription levels of the high affinity IL-12 receptor subunit (IL-12Rbeta2) as well as of the transcription factors T-bet and GATA-3 when cultured with IL-12. Both populations transcribed low levels of IL-10 mRNA under Th1-polarizing conditions and TGF-beta transcripts were ubiquitously expressed by each of these cell types. Cloning and sequencing of the cytoplasmic tails of the WC1 isoforms revealed a consensus ITAM in all three isoforms but a DENY sequence adjacent to one of the SH-2 binding sites of WC1.1 only. The results suggest that WC1+ gammadelta T cells differentiated on the basis of WC1 isoform expression play distinct roles in immune responses that may be dictated by WC1 intracellular signaling.
Asunto(s)
Antígenos de Superficie/metabolismo , Glicoproteínas de Membrana/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Subgrupos de Linfocitos T/inmunología , Animales , Bovinos , Interferón gamma/biosíntesis , Isoformas de Proteínas/inmunologíaRESUMEN
WC1 molecules are transmembrane glycoproteins belonging to the scavenger receptor cysteine-rich family and uniquely expressed on gammadelta T cells. Although participation of WC1+ gammadelta T cells in immune responses is well established, very little is understood regarding the significance of expressing different forms of the WC1 molecule. Two forms previously identified by mAbs, i.e., WC1.1 and WC1.2, are expressed by largely nonoverlapping subpopulations of gammadelta T cells. In this study it was shown that expression of the WC1.1 coreceptor was the main indicator of proliferation and IFN-gamma production in response to autologous and bacterial Ags as well as for IFN-gamma production without proliferation in Th1-polarizing, IL-12-containing cultures. Nevertheless, after culture in either Th1-polarizing or neutral conditions, mRNA was present for both T-bet and GATA-3 as well as for IL-12Rbeta2 in WC1.1+ and WC1.2+ subpopulations, and neither produced IL-4 under any conditions. Although the steady decrease in the proportion of WC1.1+ cells, but not WC1.2+ cells, within PBMC with animal aging suggested that the two subpopulations may have different roles in immune regulation, cells bearing either WC1.1 or WC1.2 expressed mRNA for regulatory cytokines IL-10 and TGF-beta, with TGF-beta being constitutively expressed by ex vivo cells. Overall, the results demonstrate that the form of the WC1 coreceptor expressed on gammadelta T cells divides them into functional subsets according to IFN-gamma production and proliferative capacity to specific stimuli as well as with regard to representation within PBMC. Finally, evidence is provided for minor differences in the intracytoplasmic tail sequences of WC1.1 and WC1.2 that may affect signaling.