Human antimicrobial cytotoxic T lymphocytes, defined by NK receptors and antimicrobial proteins, kill intracellular bacteria
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE115686 | GEO | 2018/08/31
REPOSITORIES: GEO
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Project description:Human CD8+ cytotoxic T lymphocytes (CTLs) contribute to antimicrobial defense against intracellular pathogens through secretion of cytotoxic granule proteins granzyme B, perforin, and granulysin. However, CTLs are heterogeneous in the expression of these proteins, and the subset(s) responsible for antimicrobial activity is unclear. Studying human leprosy, we found that the subset of CTLs coexpressing all three cytotoxic molecules is increased in the resistant form of the disease, can be expanded by interleukin-15 (IL-15), and is differentiated from naïve CD8+ T cells by Langerhans cells. RNA sequencing analysis identified that these CTLs express a gene signature that includes an array of surface receptors typically expressed by natural killer (NK) cells. We determined that CD8+ CTLs expressing granzyme B, perforin, and granulysin, as well as the activating NK receptor NKG2C, represent a population of "antimicrobial CTLs" (amCTLs) capable of T cell receptor (TCR)-dependent and TCR-independent release of cytotoxic granule proteins that mediate antimicrobial activity.
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Project description:Human antimicrobial cytotoxic T lymphocytes, defined by NK receptors and antimicrobial proteins, kill intracellular bacteria
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Project description:When killer lymphocytes recognize infected cells, perforin delivers cytotoxic proteases (granzymes) into the target cell to trigger apoptosis. What happens to intracellular bacteria during this process is unclear. Human, but not rodent, cytotoxic granules also contain granulysin, an antimicrobial peptide. Here, we show that granulysin delivers granzymes into bacteria to kill diverse bacterial strains. In Escherichia coli, granzymes cleave electron transport chain complex I and oxidative stress defense proteins, generating reactive oxygen species (ROS) that rapidly kill bacteria. ROS scavengers and bacterial antioxidant protein overexpression inhibit bacterial death. Bacteria overexpressing a GzmB-uncleavable mutant of the complex I subunit nuoF or strains that lack complex I still die, but more slowly, suggesting that granzymes disrupt multiple vital bacterial pathways. Mice expressing transgenic granulysin are better able to clear Listeria monocytogenes. Thus killer cells play an unexpected role in bacterial defense.
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Project description:The mechanistic target of rapamycin (mTOR) signaling, which is an essential metabolic pathway that influences T cell differentiation, can be limited under conditions of metabolic stress e.g. during the cell starvation. As we observed reduced CD8 expression upon cell starvation in vitro, we sorted human cytotoxic T lymphocytes derived from six donors into low CD8 expression and high CD8 expressing T lymphocytes using flow cytometry. Their transcriptome was analyzed in order to investigate the two CD8 subsets. Our data suggests a functional diversity of CD8low and CD8high T cells with regard to cytotoxic effector function that might be useful to study these cells also in tumor sections or under conditions when cytotoxic T cells need to adapt to a changing microenvironment.
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Project description:Protozoan infections are a serious global health problem. Natural killer (NK) cells and cytolytic T lymphocytes (CTLs) eliminate pathogen-infected cells by releasing cytolytic granule contents--granzyme (Gzm) proteases and the pore-forming perforin (PFN)--into the infected cell. However, these cytotoxic molecules do not kill intracellular parasites. CD8(+) CTLs protect against parasite infections in mice primarily by secreting interferon (IFN)-?. However, human, but not rodent, cytotoxic granules contain the antimicrobial peptide granulysin (GNLY), which selectively destroys cholesterol-poor microbial membranes, and GNLY, PFN and Gzms rapidly kill intracellular bacteria. Here we show that GNLY delivers Gzms into three protozoan parasites (Trypanosoma cruzi, Toxoplasma gondii and Leishmania major), in which the Gzms generate superoxide and inactivate oxidative defense enzymes to kill the parasite. PFN delivers GNLY and Gzms into infected cells, and GNLY then delivers Gzms to the intracellular parasites. Killer cell-mediated parasite death, which we term 'microbe-programmed cell death' or 'microptosis', is caspase independent but resembles mammalian apoptosis, causing mitochondrial swelling, transmembrane potential dissipation, membrane blebbing, phosphatidylserine exposure, DNA damage and chromatin condensation. GNLY-transgenic mice are protected against infection by T. cruzi and T. gondii, and survive infections that are lethal to wild-type mice. Thus, GNLY-, PFN- and Gzm-mediated elimination of intracellular protozoan parasites is an unappreciated immune defense mechanism.
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Project description:Cytotoxic lymphocytes (CTL) have been reported to show a range of motility patterns from rapid long-range tracking to complete arrest, but how and whether these kinematics affect their ability to kill target cells is not known. Many in vitro killing assays utilize cell lines and tumour-derived cells as targets, which may be of limited relevance to the kinetics of CTL-mediated killing of somatic cells. Here, live-cell microscopy is used to examine the interactions of CTL and primary murine skin cells presenting antigens. We developed a qualitative and quantitative killing assay using extended-duration fluorescence time-lapse microscopy coupled with large-volume objective software-based data analysis to obtain population data of cell-to-cell interactions, motility and apoptosis. In vivo and ex vivo activated antigen-specific cytotoxic lymphocytes were added to primary keratinocyte targets in culture with fluorometric detection of caspase-3 activation in targets as an objective determinant of apoptosis. We found that activated CTL achieved contact-dependent apoptosis of non-tumour targets after a period of prolonged attachment - on average 21 hours - which was determined by target cell type, amount of antigen, and activation status of CTL. Activation of CTL even without engagement of the T cell receptor was sufficient to mobilise cells significantly above baseline, while the addition of cognate antigen further enhanced their motility. Highly activated CTL showed markedly increased vector displacement, and velocity, and lead to increased antigen-specific target cell death. These data show that the inherent kinematics of CTL correlate directly with their ability to kill non-tumour cells presenting cognate antigen.
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Project description:Ceragenins are a family of synthetic amphipathic molecules designed to mimic the properties of naturally occurring cationic antimicrobial peptides (CAMPs). Although ceragenins have potent antimicrobial activity, whether their mode of action is similar to that of CAMPs has remained elusive. Here, we reported the results of a comparative study of the bacterial responses to two well-studied CAMPs, LL37 and colistin, and two ceragenins with related structures, CSA13 and CSA131. Using transcriptomic and proteomic analyses, we found that Escherichia coli responded similarly to both CAMPs and ceragenins by inducing a Cpx envelope stress response. However, whereas E. coli exposed to CAMPs increased expression of genes involved in colanic acid biosynthesis, bacteria exposed to ceragenins specifically modulated functions related to phosphate transport, indicating distinct mechanisms of action between these two classes of molecules. Although traditional genetic approaches failed to identify genes that confer high-level resistance to ceragenins, using a Clustered Regularly Interspaced Short Palindromic Repeats interference (CRISPRi) approach we identified E. coli essential genes that when knocked down modify sensitivity to these molecules. Comparison of the essential gene-antibiotic interactions for each of the CAMPs and ceragenins identified both overlapping and distinct dependencies for their antimicrobial activities. Overall, this study indicated that, while some bacterial responses to ceragenins overlap those induced by naturally occurring CAMPs, these synthetic molecules target the bacterial envelope using a distinctive mode of action. IMPORTANCE The development of novel antibiotics is essential because the current arsenal of antimicrobials will soon be ineffective due to the widespread occurrence of antibiotic resistance. The development of naturally occurring cationic antimicrobial peptides (CAMPs) for therapeutics to combat antibiotic resistance has been hampered by high production costs and protease sensitivity, among other factors. The ceragenins are a family of synthetic CAMP mimics that kill a broad spectrum of bacterial species but are less expensive to produce, resistant to proteolytic degradation, and seemingly resistant to the development of high-level resistance. Determining how ceragenins function may identify new essential biological pathways of bacteria that are less prone to the development of resistance and will further our understanding of the design principles for maximizing the effects of synthetic CAMPs.
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Project description:Ceragenins are a family of synthetic amphipathic molecules designed to mimic the properties of naturally-occurring cationic antimicrobial peptides (CAMPs). Although ceragenins have potent antimicrobial activity, whether their mode of action is similar to that of CAMPs has remained elusive. Here we report the results of a comparative study of the bacterial responses to two well-studied CAMPs, LL37 and colistin, and two ceragenins with related structures, CSA13 and CSA13.
2022-01-10 | PXD022149 | Pride
Project description:CD4+ cytotoxic T lymphocytes (CD4-CTLs) have been reported to play a protective role in several viral infections. However, little is known in humans about the biology of CD4-CTL generation, their functional properties, heterogeneity and clonal diversity, especially in relation to other well-described CD4+ memory T cell subsets. We performed single-cell RNA-seq in over 9000 cells to unravel CD4-CTL heterogeneity, transcriptional profile and clonality in humans. The single-cell differential gene expression analysis, revealed a spectrum of known transcripts, including several linked to cytotoxic and co-stimulatory function, and transcripts of unknown cytotoxicity-related function that are expressed at higher levels in the TEMRA subset, which is highly enriched for CD4-CTLs, compared to cells in the central and effector memory subsets (TCM, TEM). Simultaneous T cells antigen receptor (TCR) analysis in single-cells and bulk subsets revealed that CD4-TEMRA cells show marked clonal expansion compared to TCM and TEM cells and that the majority of CD4-TEMRA were dengue virus (DENV)-specific in subjects with previous DENV infection. The profile of CD4-TEMRA was highly heterogeneous across subjects, with four distinct clusters identified by the single-cell analysis. Most importantly, we identified distinct clusters of CD4-CTL effector and precursor cells in the TEMRA subset; the precursor cells shared TCR clonotypes with CD4-CTL effectors and were distinguished by high expression of the interleukin-7 receptor. Our identification of a CD4-CTL precursor population may allow further investigation of how CD4-CTLs arise in humans and thus could provide insights into the mechanisms that may be utilized to generate durable and effective CD4-CTL immunity.
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Project description:UnlabelledCD56+ natural killer (NK) and CD56+ T cells, from sputum or bronchoalveolar lavage of subjects with chronic obstructive pulmonary disease (COPD) are more cytotoxic to highly susceptible NK targets than those from control subjects. Whether the same is true in lung parenchyma, and if NK activity actually contributes to emphysema progression are unknown. To address these questions, we performed two types of experiments on lung tissue from clinically-indicated resections (n = 60). First, we used flow cytometry on fresh single-cell suspension to measure expression of cell-surface molecules (CD56, CD16, CD8, NKG2D and NKp44) on lung lymphocytes and of the 6D4 epitope common to MICA and MICB on lung epithelial (CD326+) cells. Second, we sequentially isolated CD56+, CD8+ and CD4+ lung lymphocytes, co-cultured each with autologous lung target cells, then determined apoptosis of individual target cells using Annexin-V and 7-AAD staining. Lung NK cells (CD56+ CD3-) and CD56+ T cells (CD56+ CD3+) were present in a range of frequencies that did not differ significantly between smokers without COPD and subjects with COPD. Lung NK cells had a predominantly "cytotoxic" CD56+ CD16+ phenotype; their co-expression of CD8 was common, but the percentage expressing CD8 fell as FEV1 % predicted decreased. Greater expression by autologous lung epithelial cells of the NKG2D ligands, MICA/MICB, but not expression by lung CD56+ cells of the activating receptor NKG2D, correlated inversely with FEV1 % predicted. Lung CD56+ lymphocytes, but not CD4+ or CD8+ conventional lung T cells, rapidly killed autologous lung cells without additional stimulation. Such natural cytotoxicity was increased in subjects with severe COPD and was unexplained in multiple regression analysis by age or cancer as indication for surgery. These data show that as spirometry worsens in COPD, CD56+ lung lymphocytes exhibit spontaneous cytotoxicity of autologous structural lung cells, supporting their potential role in emphysema progression.Trial registrationClinicalTrials.gov NCT00281229.
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