RESUMO
T cells play a central role in adaptive immunity by recognizing peptide Ags presented by MHC molecules (pMHC) via their clonotypic TCRs. αßTCRs are heterodimers, consisting of TCRα and TCRß subunits that are composed of variable (Vα, Vß) and constant (Cα, Cß) domains. Whereas the Vα, Vß, and Cß domains adopt typical Ig folds in the extracellular space, the Cα domain lacks a top ß sheet and instead has two loosely associated top strands (C- and F-strands) on its surface. Previous results suggest that this unique Ig-like fold mediates homotypic TCR interactions and influences signaling in vitro. To better understand why evolution has selected this unique structure, we asked, what is the fitness cost for development and function of mouse CD4+ T cells bearing a mutation in the Cα C-strand? In both TCR retrogenic and transgenic mice we observed increased single-positive thymocytes bearing mutant TCRs compared with those expressing wild-type TCRs. Furthermore, our analysis of mutant TCR transgenic mice revealed an increase in naive CD4+ T cells experiencing strong tonic TCR signals, increased homeostatic survival, and increased recruitment of responders to cognate pMHC class II upon immunization compared with the wild-type. The mutation did not, however, overtly impact CD4+ T cell proliferation or differentiation after immunization. We interpret these data as evidence that the unique Cα domain has evolved to fine-tune TCR signaling, particularly in response to weak interactions with self-pMHC class II.
Assuntos
Reparo do DNA , Receptores de Antígenos de Linfócitos T , Animais , Camundongos , Membrana Celular , Timócitos , Camundongos TransgênicosRESUMO
Bacterial infections are the primary cause of pathogenic sepsis. An uropathogenic E. coli (UPEC) model of monomicrobial sepsis represents a useful tool for interrogating the host immune response to this pathogen. Here, we present a protocol for inducing monomicrobial sepsis in mice using UPEC. We describe steps for preparing the bacteria, delivering UPEC into mice, and monitoring the mice post-infection. We then detail procedures for measuring cytokine response and detecting immune cell subsets using flow cytometry. For complete details on the use and execution of this protocol, please refer to Martin et al.1.
RESUMO
CD4+ T cell activation is driven by five-module receptor complexes. The T cell receptor (TCR) is the receptor module that binds composite surfaces of peptide antigens embedded within MHCII molecules (pMHCII). It associates with three signaling modules (CD3γε, CD3δε, and CD3ζζ) to form TCR-CD3 complexes. CD4 is the coreceptor module. It reciprocally associates with TCR-CD3-pMHCII assemblies on the outside of a CD4+ T cells and with the Src kinase, LCK, on the inside. Previously, we reported that the CD4 transmembrane GGXXG and cytoplasmic juxtamembrane (C/F)CV+C motifs found in eutherian (placental mammal) CD4 have constituent residues that evolved under purifying selection (Lee et al., 2022). Expressing mutants of these motifs together in T cell hybridomas increased CD4-LCK association but reduced CD3ζ, ZAP70, and PLCγ1 phosphorylation levels, as well as IL-2 production, in response to agonist pMHCII. Because these mutants preferentially localized CD4-LCK pairs to non-raft membrane fractions, one explanation for our results was that they impaired proximal signaling by sequestering LCK away from TCR-CD3. An alternative hypothesis is that the mutations directly impacted signaling because the motifs normally play an LCK-independent role in signaling. The goal of this study was to discriminate between these possibilities. Using T cell hybridomas, our results indicate that: intracellular CD4-LCK interactions are not necessary for pMHCII-specific signal initiation; the GGXXG and (C/F)CV+C motifs are key determinants of CD4-mediated pMHCII-specific signal amplification; the GGXXG and (C/F)CV+C motifs exert their functions independently of direct CD4-LCK association. These data provide a mechanistic explanation for why residues within these motifs are under purifying selection in jawed vertebrates. The results are also important to consider for biomimetic engineering of synthetic receptors.
Assuntos
Proteína Tirosina Quinase p56(lck) Linfócito-Específica , Placenta , Gravidez , Animais , Feminino , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Placenta/metabolismo , Transdução de Sinais/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Complexo Receptor-CD3 de Antígeno de Linfócitos T/metabolismo , Fosforilação , Antígenos CD4 , Mamíferos/metabolismoRESUMO
Human sepsis is a complex disease that manifests with a diverse range of phenotypes and inherent variability among individuals, making it hard to develop a comprehensive animal model. Despite this difficulty, numerous models have been developed that capture many key aspects of human sepsis. The robustness of these models is vital for conducting pre-clinical studies to test and develop potential therapeutics. In this article, we describe four different models of murine sepsis that can be used to address different scientific questions relevant to the pathology and immune response during and after a septic event. Basic Protocol 1 details a non-synchronous cecal ligation and puncture (CLP) model of sepsis, where mice are subjected to polymicrobial exposure through surgery at different time points within 2 weeks. This variation in sepsis onset establishes each mouse at a different state of inflammation and cytokine levels that mimics the variability observed in humans when they present in the clinic. This model is ideal for studying the long-term impact of sepsis on the host. Basic Protocol 2 is also a type of polymicrobial sepsis, where injection of a specific amount of cecal slurry from a donor mouse into the peritoneum of recipient mice establishes immediate inflammation and sepsis without any need for surgery. Basic Protocol 3 describes infecting mice with a defined gram-positive or -negative bacterial strain to model a subset of sepsis observed in humans infected with a single pathogen. Basic Protocol 4 describes administering LPS to induce sterile endotoxemia. This form of sepsis is observed in humans exposed to bacterial toxins from the environment. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Non-synchronous cecal ligation and puncture Basic Protocol 2: Cecal slurry model of murine sepsis Basic Protocol 3: Monomicrobial model of murine sepsis Basic Protocol 4: LPS model of murine sepsis.
Assuntos
Lipopolissacarídeos , Sepse , Humanos , Animais , Camundongos , Lipopolissacarídeos/toxicidade , Modelos Animais de Doenças , Instituições de Assistência Ambulatorial , InflamaçãoRESUMO
BACKGROUNDSepsis remains a major clinical challenge for which successful treatment requires greater precision in identifying patients at increased risk of adverse outcomes requiring different therapeutic approaches. Predicting clinical outcomes and immunological endotyping of septic patients generally relies on using blood protein or mRNA biomarkers, or static cell phenotyping. Here, we sought to determine whether functional immune responsiveness would yield improved precision.METHODSAn ex vivo whole-blood enzyme-linked immunosorbent spot (ELISpot) assay for cellular production of interferon γ (IFN-γ) was evaluated in 107 septic and 68 nonseptic patients from 5 academic health centers using blood samples collected on days 1, 4, and 7 following ICU admission.RESULTSCompared with 46 healthy participants, unstimulated and stimulated whole-blood IFN-γ expression was either increased or unchanged, respectively, in septic and nonseptic ICU patients. However, in septic patients who did not survive 180 days, stimulated whole-blood IFN-γ expression was significantly reduced on ICU days 1, 4, and 7 (all P < 0.05), due to both significant reductions in total number of IFN-γ-producing cells and amount of IFN-γ produced per cell (all P < 0.05). Importantly, IFN-γ total expression on days 1 and 4 after admission could discriminate 180-day mortality better than absolute lymphocyte count (ALC), IL-6, and procalcitonin. Septic patients with low IFN-γ expression were older and had lower ALCs and higher soluble PD-L1 and IL-10 concentrations, consistent with an immunosuppressed endotype.CONCLUSIONSA whole-blood IFN-γ ELISpot assay can both identify septic patients at increased risk of late mortality and identify immunosuppressed septic patients.TRIAL REGISTRYN/A.FUNDINGThis prospective, observational, multicenter clinical study was directly supported by National Institute of General Medical Sciences grant R01 GM-139046, including a supplement (R01 GM-139046-03S1) from 2022 to 2024.
Assuntos
Interferon gama , Sepse , Humanos , Interferon gama/metabolismo , Imunoadsorventes/uso terapêutico , Estudos Prospectivos , BiomarcadoresRESUMO
CD4+ T cell activation is driven by 5-module receptor complexes. The T cell receptor (TCR) is the receptor module that binds composite surfaces of peptide antigens embedded within MHCII molecules (pMHCII). It associates with three signaling modules (CD3γε, CD3δε, and CD3ζζ) to form TCR-CD3 complexes. CD4 is the coreceptor module. It reciprocally associates with TCR-CD3-pMHCII assemblies on the outside of a CD4+ T cells and with the Src kinase, LCK, on the inside. Previously, we reported that the CD4 transmembrane GGXXG and cytoplasmic juxtamembrane (C/F)CV+C motifs found in eutherian (placental mammal) CD4 have constituent residues that evolved under purifying selection (Lee, et al., 2022). Expressing mutants of these motifs together in T cell hybridomas increased CD4-LCK association but reduced CD3ζ, ZAP70, and PLCγ1 phosphorylation levels, as well as IL-2 production, in response to agonist pMHCII. Because these mutants preferentially localized CD4-LCK pairs to non-raft membrane fractions, one explanation for our results was that they impaired proximal signaling by sequestering LCK away from TCR-CD3. An alternative hypothesis is that the mutations directly impacted signaling because the motifs normally play an LCK-independent role in signaling. The goal of this study was to discriminate between these possibilities. Using T cell hybridomas, our results indicate that: intracellular CD4-LCK interactions are not necessary for pMHCII-specific signal initiation; the GGXXG and (C/F)CV+C motifs are key determinants of CD4-mediated pMHCII-specific signal amplification; the GGXXG and (C/F)CV+C motifs exert their functions independently of direct CD4-LCK association. These data provide a mechanistic explanation for why residues within these motifs are under purifying selection in jawed vertebrates. The results are also important to consider for biomimetic engineering of synthetic receptors.
RESUMO
Uropathogenic E. coli (UPEC) is a primary organism responsible for urinary tract infections and a common cause of sepsis. Microbially experienced laboratory mice, generated by cohousing with pet store mice, exhibit increased morbidity and mortality to polymicrobial sepsis or lipopolysaccharide challenge. By contrast, cohoused mice display significant resistance, compared with specific pathogen-free mice, to a monomicrobial sepsis model using UPEC. CD115+ monocytes mediate protection in the cohoused mice, as depletion of these cells leads to increased mortality and UPEC pathogen burden. Further study of the cohoused mice reveals increased TNF-α production by monocytes, a skewing toward Ly6ChiCD115+ "classical" monocytes, and enhanced egress of Ly6ChiCD115+ monocytes from the bone marrow. Analysis of cohoused bone marrow also finds increased frequency and number of myeloid multipotent progenitor cells. These results show that a history of microbial exposure impacts innate immunity in mice, which can have important implications for the preclinical study of sepsis.
Assuntos
Infecções por Escherichia coli , Sepse , Infecções Urinárias , Escherichia coli Uropatogênica , Camundongos , Animais , Monócitos , Escherichia coli , Imunidade Inata , Receptores Proteína Tirosina QuinasesRESUMO
CD4+ T cells use T cell receptor (TCR)-CD3 complexes, and CD4, to respond to peptide antigens within MHCII molecules (pMHCII). We report here that, through ~435 million years of evolution in jawed vertebrates, purifying selection has shaped motifs in the extracellular, transmembrane, and intracellular domains of eutherian CD4 that enhance pMHCII responses, and covary with residues in an intracellular motif that inhibits responses. Importantly, while CD4 interactions with the Src kinase, Lck, are viewed as key to pMHCII responses, our data indicate that CD4-Lck interactions derive their importance from the counterbalancing activity of the inhibitory motif, as well as motifs that direct CD4-Lck pairs to specific membrane compartments. These results have implications for the evolution and function of complex transmembrane receptors and for biomimetic engineering.