The impact of TCR affinity on resident memory T cell formation during influenza virus infection
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ABSTRACT: Resident memory T (TRM) cells in the lung are vital for heterologous protection against influenza A virus (IAV). Environmental factors are necessary to establish lung TRM, however the role of T cell intrinsic factors like T cell receptor (TCR) signal strength have not been elucidated. Here we investigated the impact of TCR signal strength on the generation and maintenance of lung TRM cells after IAV infection. We inserted high and low affinity OT-I epitopes into IAV and infected mice after transfer of OT-I T cells. We uncovered a bias in TRM formation in the lung elicited by lower affinity TCR stimulation. TCR affinity did not impact the overall phenotype or long-term maintenance of lung TRM cells. Overall, these findings demonstrate that TRM formation is negatively correlated with increased TCR signal strength. Lower affinity cells may have an advantage in forming TRM to ensure diversity in the antigen-specific repertoire in tissues.
Project description:IL-2 inducible tyrosine kinase (Itk) is a Tec family non-receptor tyrosine kinase that is known to regulate T cell receptor signal strength (TcR) and T cell development and differentiation. TcR signal strength and antigen affinity are parameters known to regulate the development of CD8+ T cell memory. However, the intersection between TcR signal strength and antigen affinity on CD8+ memory T cell development remains unclear. Therefore, we compared the transcriptomes of WT and Itk-/- OT-1/Rag-/- CD8+ T cells at day 0 and day 7 following infection with Listeria monocytogenes expressing the SIINFEKL (N4) or SIITFEKL (T4) OVA peptide variant (different affinities for TcR) by RNA sequencing.
Project description:Lung tissue-resident memory CD8+ T-cells (Trm) are critical for heterosubtypic immunity against influenza virus-(IAV)-reinfection. How these cells surveil the lung, respond to infection and interact with other cells remains unresolved. Here, we used mouse models to identify and enrich lung Trm in combination with intravital and static imaging to assess spatiotemporal dynamics of IAV-induced lung TRM before and after recall-infection. CD69+CD103+ Trm localize to sites of prior influenza infection where they exhibit substantially slower movement properties than non-Trm that also surveil the lung. After rechallenge, lung Trm form tight clusters in an antigen-dependent manner. IAV-specific Trm express several factors that regulate myeloid cell biology and their protective immune responses are accompanied by activation and migration of dendritic cells to draining lymph nodes and recruitment of inflammatory monocytes. Overall, these data reveal the dynamic landscapes of lung Trm cells associated with early protective immunity against IAV infection.
Project description:Lung resident memory (Trm) CD8 T cells induced by influenza A virus (IAV), are pivotal for providing heterosubtypic immunity, but are not maintained long term, causing gradual loss of protection. This contrasts sharply with long-term maintenance of Trm induced by localized infections of the skin and other tissues. Here we show that the decline in lung Trm is determined by an imbalance between apoptosis and lung recruitment/conversion to Trm of circulating memory cells. At the cellular level, circulating effector memory (Tem) rather than central memory (Tcm) cells are the precursors for conversion to lung Trm. Time-dependent changes in expression of genes critical for Trm differentiation together with enrichment of Tcm diminish the capacity of circulating memory CD8 T cells to form Trm, explaining why IAV-induced Trm are not stably maintained over time. Importantly, systemic booster immunization, through increasing the number of circulating Tem cells, induces an increase in lung Trm pool, providing a new rational for future IAV vaccines.
Project description:Here we provide a framework to qualtitatively evaluate the tyrosine phosphorylation-mediated signaling pathways triggered by antigen-independent antibody and antigen-specific OVA peptide-MHC using Jurkat T cells expressing OT-1 T cell receptors. Our data suggest that pTyr-mediated regulatory axis triggered by OVA antigen-specific activation of TCR closely resembled that of antigen-independent stimulation using anti-TCR antibody, albeit OVA could likely induce a relatively stronger signaling effect. While data from this study do not invalidate previous studies of T cell signaling using antibody-based stimulation, our data revealed potential advantages of using peptide-MHC tetramers in studying T cell signaling. Antigen-specific activation of the OT-1 TCR using a panel of peptide-MHC tetramers (OVA, T4 and G4) generated data that correlates well with the corresponding binding affinity of the peptide-MHC, consistent with predicted signaling strength. Importantly, the apparent correlation between signaling strength and peptide-MHC affinity enables us to fine-tune the signaling strength of T cell stimulation in future studies, allowing a more precise control of the experimental parameter instead of a more binary antibody-based activation. Overall, we demonstrate the utility of BOOST to reveal new biological insights in the immune system.
Project description:Affinity and dose of T cell receptor (TCR) interaction with antigens govern the magnitude of CD4+ T cell responses, but questions remain regarding the quantitative translation of TCR engagement into downstream signals. We find that while the response of CD4+ T cells to antigenic stimulation is bimodal, activated cells exhibit analog responses proportional to signal strength. Gene expression output reflects TCR signal strength, providing a signature of T cell activation. Expression changes rely on a pre-established enhancer landscape and quantitative acetylation at AP-1 binding sites. Finally, we show that graded expression of activation genes depends on ERK pathway activation, suggesting that an ERK-AP-1 axis translates TCR signal strength into proportional activation of enhancers and genes essential for T cell function.
Project description:Affinity and dose of T cell receptor (TCR) interaction with antigens govern the magnitude of CD4+ T cell responses, but questions remain regarding the quantitative translation of TCR engagement into downstream signals. We find that while the response of CD4+ T cells to antigenic stimulation is bimodal, activated cells exhibit analog responses proportional to signal strength. Gene expression output reflects TCR signal strength, providing a signature of T cell activation. Expression changes rely on a pre-established enhancer landscape and quantitative acetylation at AP-1 binding sites. Finally, we show that graded expression of activation genes depends on ERK pathway activation, suggesting that an ERK-AP-1 axis translates TCR signal strength into proportional activation of enhancers and genes essential for T cell function. CD4+ T cells from transgenic AND mice were sequenced under the conditions indicated. Replicates are included for each type of data (RNA-Seq, ChIP-Seq), and are numbered accordingly. The No Peptide condition serves as the untreated control for the peptide-treated samples, and inputs are provided for ChIP-Sequencing samples.
Project description:Variable strengths of T cell receptor (TCR) signaling can produce divergent outcomes for T cell development and function. The mechanisms leading to different outcomes are incompletely understood, but may include distinct activation thresholds for different transcription factors as well as distinct sensitivities among target genes to transcription factors. IRF4 is one transcription factor implicated in responses to variable TCR signal strength. IRF4 expression increases uniformly with increasing TCR signal strength (i.e., analog), but it is unclear how IRF4 induced distinct genes at different levels, rather than different amounts of the same genes. Here, we analyzed global gene expression in TH2 cells and used ChIP-seq to define the relationship between TCR signal strength, enhancer occupancy and transcriptional activity for BATF/IRF4-dependent genes. We show that enhancers exhibit a spectrum of affinity for the BATF/IRF4 ternary complex mediate graded responsiveness of individual genes to increasing TCR signal strength. Differential gene induction by BATF and IRF4 occurs through interaction with enhancer elements of different affinity for BATF/IRF4 complexes. The increased resolution of factor binding site identified using ChIP-exo allowed the identification of a novel AICE2 motif binding BATF/IRF4 with higher affinity and that this may explain the protective role of a single nucleotide polymorphism in the CTLA-4 locus known to decrease the incidence of autoimmune diseases.
Project description:Variable strengths of signaling via the T cell antigen receptor (TCR) can produce divergent outcomes, but the mechanism of this remains obscure. The abundance of the transcription factor IRF4 increases with TCR signal strength, but how this would induce distinct types of responses is unclear. We compared the expression of genes in the TH2 subset of helper T cells to enhancer occupancy by the BATF–IRF4 transcription factor complex at varying strengths of TCR stimulation. Genes dependent on BATF–IRF4 clustered into groups with distinct TCR sensitivities. Enhancers exhibited a spectrum of occupancy by the BATF–IRF4 ternary complex that correlated with the sensitivity of gene expression to TCR signal strength. DNA sequences immediately flanking the previously defined AICE motif controlled the affinity of BATF–IRF4 for direct binding to DNA. Analysis by the chromatin immunoprecipitation–exonuclease (ChIP-exo) method allowed the identification of a previously unknown high-affinity AICE2 motif at a human single-nucleotide polymorphism (SNP) of the gene encoding the immunomodulatory receptor CTLA-4 that was associated with resistance to autoimmunity. Thus, the affinity of different enhancers for the BATF–IRF4 complex might underlie divergent signaling outcomes in response to various strengths of TCR signaling
Project description:Tissue resident memory (Trm) represent a newly described memory T cell population. We have previously characterized a population of Trm that persists within the brain following acute virus infection. Although capable of providing marked protection against a subsequent local challenge, brain Trm do not undergo recall expansion following dissociation from the tissue. Furthermore, these Trm do not depend on the same survival factors as the circulating memory T cell pool as assessed either in vivo or in vitro. To gain greater insight into this population of cells we compared the gene-expression profiles of Trm isolated from the brain to circulating memory T cells isolated from the spleen following an acute virus infection. Trm displayed altered expression of genes involved in chemotaxis, expressed a distinct set of transcription factors and overexpressed several inhibitory receptors. Cumulatively, these data indicates that Trm are a distinct memory T cell population disconnected from the circulating memory T cell pool and displaying a unique molecular signature which likely results in optimal survival and function within their local environment. 13 samples were analyzed: 5 replicates of memory OT-I CD8+.CD103- T cells isolated from the spleen of mice on day 20 p.i. with VSV-OVA. 5 replicates of memory OT-I CD8+CD103+ T cells isolated from the brain of mice on day 20 p.i. with VSV-OVA; and 3 replicates of memory OT-I.CD8+ CD103- T cells isolated from the brain of mice on day 20 p.i. with VSV-OVA
Project description:To understand the impact of TCR affinity during priming on memory formation, we performed transcriptional profiling of OT-1 cells at different time points after stimulation with high- or low-affinity ligands. Our findings indicate that low-affinity stimulaation is associated with earlier memory formation.