Tissue-specific shaping of the TCR repertoire and antigen specificity of iNKT cells

Tissue homeostasis is critically dependent on the function of tissue-resident lymphocytes, including lipid-reactive invariant natural killer T (iNKT) cells. Yet, if and how the tissue environment shapes the antigen specificity of iNKT cells remains unknown. By analysing iNKT cells from lymphoid tissues of mice and humans we demonstrate that their T cell receptor (TCR) repertoire is highly diverse and is distinct for cells from various tissues resulting in differential lipid-antigen recognition. Within peripheral tissues iNKT cell recent thymic emigrants exhibit a different TCR repertoire than mature cells, suggesting that the iNKT population is shaped after arrival to the periphery. Consistent with this, iNKT cells from different organs show distinct basal activation, proliferation and clonal expansion. Moreover, the iNKT cell TCR repertoire changes following immunisation and is shaped by age and environmental changes. Thus, post-thymic modification of the TCR-repertoire underpins the distinct antigen specificity for iNKT cells in peripheral tissues

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Recent advances in iNKT cell development

F1000Research ◽

10.12688/f1000research.21378.1 ◽

2020 ◽

Vol 9 ◽

pp. 127 ◽

Cited By ~ 5

Author(s):

Kristin Hogquist ◽

Hristo Georgiev

Keyword(s):

T Cell Receptor ◽

Signal Strength ◽

Cell Receptor ◽

Cell Development ◽

Inkt Cell ◽

Inkt Cells ◽

Effector Cells ◽

Receptor Signal ◽

Invariant Natural Killer ◽

Division Model

Recent studies suggest that murine invariant natural killer T (iNKT) cell development culminates in three terminally differentiated iNKT cell subsets denoted as NKT1, 2, and 17 cells. Although these studies corroborate the significance of the subset division model, less is known about the factors driving subset commitment in iNKT cell progenitors. In this review, we discuss the latest findings in iNKT cell development, focusing in particular on how T-cell receptor signal strength steers iNKT cell progenitors toward specific subsets and how early progenitor cells can be identified. In addition, we will discuss the essential factors for their sustenance and functionality. A picture is emerging wherein the majority of thymic iNKT cells are mature effector cells retained in the organ rather than developing precursors.

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Probing T-cell response by sequence-based probabilistic modeling

10.1101/2020.12.17.423283 ◽

2020 ◽

Author(s):

Barbara Bravi ◽

Vinod P. Balachandran ◽

Benjamin D. Greenbaum ◽

Aleksandra M. Walczak ◽

Thierry Mora ◽

...

Keyword(s):

T Cell ◽

Probabilistic Models ◽

Cell Receptor ◽

T Cell Response ◽

Specific Response ◽

Sequence Motifs ◽

Antigen Specificity ◽

Human T Cell ◽

Machine Learning Approach ◽

Tcr Repertoire

AbstractWith the increasing ability to use high-throughput next-generation sequencing to quantify the diversity of the human T cell receptor (TCR) repertoire, the ability to use TCR sequences to infer antigen-specificity could greatly aid potential diagnostics and therapeutics. Here, we use a machine-learning approach known as Restricted Boltzmann Machine to develop a sequence-based inference approach to identify antigen-specific TCRs. Our approach combines probabilistic models of TCR sequences with clone abundance information to extract TCR sequence motifs central to an antigen-specific response. We use this model to identify patient personalized TCR motifs that respond to individual tumor and infectious disease antigens, and to accurately discriminate specific from non-specific responses. Furthermore, the hidden structure of the model results in an interpretable representation space where TCRs responding to the same antigen cluster, correctly discriminating the response of TCR to different viral epitopes. The model can therefore be used to identify both experimental and condition specific responding TCRs, of great importance to advancing personalized immunotherapies.

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A Framework to Identify Antigen-Expanded T Cell Receptor Clusters Within Complex Repertoires

Frontiers in Immunology ◽

10.3389/fimmu.2021.735584 ◽

2021 ◽

Vol 12 ◽

Author(s):

Valentina Ceglia ◽

Erin J. Kelley ◽

Annalee S. Boyle ◽

Sandra Zurawski ◽

Heather L. Mead ◽

...

Keyword(s):

T Cell ◽

T Cell Receptor ◽

T Cell Responses ◽

Cell Receptor ◽

Antigen Specificity ◽

Protein Antigens ◽

Cell Responses ◽

Tcr Repertoire ◽

Receptor Clusters ◽

Theoretical Sensitivity

Common approaches for monitoring T cell responses are limited in their multiplexity and sensitivity. In contrast, deep sequencing of the T Cell Receptor (TCR) repertoire provides a global view that is limited only in terms of theoretical sensitivity due to the depth of available sampling; however, the assignment of antigen specificities within TCR repertoires has become a bottleneck. This study combines antigen-driven expansion, deep TCR sequencing, and a novel analysis framework to show that homologous ‘Clusters of Expanded TCRs (CETs)’ can be confidently identified without cell isolation, and assigned to antigen against a background of non-specific clones. We show that clonotypes within each CET respond to the same epitope, and that protein antigens stimulate multiple CETs reactive to constituent peptides. Finally, we demonstrate the personalized assignment of antigen-specificity to rare clones within fully-diverse uncultured repertoires. The method presented here may be used to monitor T cell responses to vaccination and immunotherapy with high fidelity.

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Exogenous Activation of Invariant Natural Killer T Cells by α-Galactosylceramide Reduces Pneumococcal Outgrowth and Dissemination Postinfluenza

mBio ◽

10.1128/mbio.01440-16 ◽

2016 ◽

Vol 7 (6) ◽

Cited By ~ 11

Author(s):

Adeline Barthelemy ◽

Stoyan Ivanov ◽

Maya Hassane ◽

Josette Fontaine ◽

Béatrice Heurtault ◽

...

Keyword(s):

Mouse Model ◽

Influenza A Virus ◽

Bacterial Pneumonia ◽

Influenza A ◽

Bacterial Infections ◽

Pneumococcal Infection ◽

Clinical Development ◽

Inkt Cell ◽

Inkt Cells ◽

Invariant Natural Killer

ABSTRACT Influenza A virus infection can predispose to potentially devastating secondary bacterial infections. Invariant natural killer T (iNKT) cells are unconventional, lipid-reactive T lymphocytes that exert potent immunostimulatory functions. Using a mouse model of postinfluenza invasive secondary pneumococcal infection, we sought to establish whether α-galactosylceramide (α-GalCer [a potent iNKT cell agonist that is currently in clinical development]) could limit bacterial superinfection. Our results highlighted the presence of a critical time window during which α-GalCer treatment can trigger iNKT cell activation and influence resistance to postinfluenza secondary pneumococcal infection. Intranasal treatment with α-GalCer during the acute phase (on day 7) of influenza virus H3N2 and H1N1 infection failed to activate (gamma interferon [IFN-γ] and interleukin-17A [IL-17A]) iNKT cells; this effect was associated with a strongly reduced number of conventional CD103 + dendritic cells in the respiratory tract. In contrast, α-GalCer treatment during the early phase (on day 4) or during the resolution phase (day 14) of influenza was associated with lower pneumococcal outgrowth and dissemination. Less intense viral-bacterial pneumonia and a lower morbidity rate were observed in superinfected mice treated with both α-GalCer (day 14) and the corticosteroid dexamethasone. Our results open the way to alternative (nonantiviral/nonantibiotic) iNKT-cell-based approaches for limiting postinfluenza secondary bacterial infections. IMPORTANCE Despite the application of vaccination programs and antiviral drugs, influenza A virus (IAV) infection is responsible for widespread morbidity and mortality (500,000 deaths/year). Influenza infections can also result in sporadic pandemics that can be devastating: the 1918 pandemic led to the death of 50 million people. Severe bacterial infections are commonly associated with influenza and are significant contributors to the excess morbidity and mortality of influenza. Today’s treatments of secondary bacterial (pneumococcal) infections are still not effective enough, and antibiotic resistance is a major issue. Hence, there is an urgent need for novel therapies. In the present study, we set out to evaluate the efficacy of α-galactosylceramide (α-GalCer)—a potent agonist of invariant NKT cells that is currently in clinical development—in a mouse model of postinfluenza, highly invasive pneumococcal pneumonia. Our data indicate that treatment with α-GalCer reduces susceptibility to superinfections and, when combined with the corticosteroid dexamethasone, reduces viral-bacterial pneumonia.

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Generation of a Jurkat-based fluorescent reporter cell line to evaluate lipid antigen interaction with the human iNKT cell receptor

Scientific Reports ◽

10.1038/s41598-019-43529-4 ◽

2019 ◽

Vol 9 (1) ◽

Author(s):

Piotr Humeniuk ◽

Sabine Geiselhart ◽

Claire Battin ◽

Tonya Webb ◽

Peter Steinberger ◽

...

Keyword(s):

Cell Line ◽

Cell Receptor ◽

Inkt Cell ◽

Reporter Cell Line ◽

Reporter Cell ◽

Fluorescent Reporter ◽

Lipid Antigen ◽

Antigen Interaction

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Cancer Immunotherapeutic Potential of NKTT320, a Novel, Invariant, Natural Killer T Cell-Activating, Humanized Monoclonal Antibody

International Journal of Molecular Sciences ◽

10.3390/ijms21124317 ◽

2020 ◽

Vol 21 (12) ◽

pp. 4317 ◽

Cited By ~ 1

Author(s):

Nishant P. Patel ◽

Peng Guan ◽

Devika Bahal ◽

Tanwir Hashem ◽

Felix Scheuplein ◽

...

Keyword(s):

Monoclonal Antibody ◽

T Cell ◽

Natural Killer ◽

Cell Activation ◽

Cell Receptor ◽

Inkt Cell ◽

Humanized Monoclonal Antibody ◽

Cancer Immunotherapeutic ◽

Invariant Natural Killer ◽

Natural Killer T

Invariant natural killer T cells (iNKTs) directly kill tumor cells and trans-activate the anti-tumor functions of dendritic cells (DC), natural killer (NK) cells, and T and B cells. As such, iNKTs serve as a powerful tool for use in cell-based cancer immunotherapy. iNKT cell activation commonly requires engagement of the invariant T cell receptor (iTCR) by CD1d presenting glycolipid antigens. However, transformed cells often down-regulate CD1d expression, which results in a reduction of iNKT cell anti-tumor functions. One approach to circumvent this critical barrier to iNKT cell activation is to develop an agonistic antibody that binds directly to the iTCR without the requirement for CD1d-mediated antigen presentation. To this end, we have characterized the iNKT cell stimulatory properties of NKTT320, a novel, recombinant, humanized, monoclonal antibody that binds selectively and with high affinity to human iTCRs. Strikingly, immobilized NKTT320 mediated robust iNKT cell activation (upregulation of CD25 and CD69) and proliferation (carboxyfluorescein succinimidyl ester (CFSE) dilution), as well as Th1 and Th2 cytokine production. Additionally, iNKTs stimulated by plate-bound NKTT320 exhibited increased intracellular levels of granzyme B and degranulation (exposure of CD107 on the cell surface). Furthermore, both soluble and immobilized NKTT320 induced iNKT cell-mediated activation of bystander immune cells, suggesting that this novel anti-iTCR antibody facilitates both direct and indirect iNKT cell cytotoxicity. These studies are significant, as they provide a framework by which iNKT cell anti-cancer functions could be enhanced for therapeutic purposes.

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License to Kill: When iNKT Cells Are Granted the Use of Lethal Cytotoxicity

International Journal of Molecular Sciences ◽

10.3390/ijms21113909 ◽

2020 ◽

Vol 21 (11) ◽

pp. 3909

Author(s):

Angélica Díaz-Basabe ◽

Francesco Strati ◽

Federica Facciotti

Keyword(s):

Immune Responses ◽

Inkt Cell ◽

Cell Cytotoxicity ◽

Future Perspectives ◽

Inkt Cells ◽

Cytotoxic Potential ◽

Lipid Antigens ◽

Cancer Immunotherapies ◽

Cluster Of Differentiation ◽

Invariant Natural Killer

Invariant Natural Killer T (iNKT) cells are a non-conventional, innate-like, T cell population that recognize lipid antigens presented by the cluster of differentiation (CD)1d molecule. Although iNKT cells are mostly known for mediating several immune responses due to their massive and diverse cytokine release, these cells also work as effectors in various contexts thanks to their cytotoxic potential. In this Review, we focused on iNKT cell cytotoxicity; we provide an overview of iNKT cell subsets, their activation cues, the mechanisms of iNKT cell cytotoxicity, the specific roles and outcomes of this activity in various contexts, and how iNKT killing functions are currently activated in cancer immunotherapies. Finally, we discuss the future perspectives for the better understanding and potential uses of iNKT cell killing functions in tumor immunosurveillance.

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Distinct bioenergetic features of human invariant natural killer T (iNKT) cells enable retained functions in nutrient-deprived states

10.1101/2021.04.29.442021 ◽

2021 ◽

Author(s):

Priya Khurana ◽

Chakkapong Burudpakdee ◽

Stephan A. Grupp ◽

Ulf H. Beier ◽

David M. Barrett ◽

...

Keyword(s):

Natural Killer ◽

Metabolic Flux ◽

Cytokine Production ◽

Inkt Cell ◽

Flux Analysis ◽

Inkt Cells ◽

Effector Functions ◽

Functional Features ◽

Invariant Natural Killer ◽

Natural Killer T

ABSTRACTInvariant natural killer T (iNKT) cells comprise a unique subset of lymphocytes that are primed for activation and possess innate NK-like functional features. Currently, iNKT cell-based immunotherapies remain in early clinical stages, and little is known about the ability of these cells to survive and retain effector functions within the solid tumor microenvironment (TME) long-term. In conventional T cells (TCONV), cellular metabolism is linked to effector functions and their ability to adapt to the nutrient-poor TME. In contrast, the bioenergetic requirements of iNKT cells – particularly those of human iNKT cells – at baseline and upon stimulation are not well understood; neither is how these requirements affect cytokine production or anti-tumor effector functions. We find that unlike TCONV, human iNKT cells are not dependent upon glucose or glutamine for cytokine production and cytotoxicity upon stimulation with anti-CD3 and anti-CD28. Additionally, transcriptional profiling revealed that stimulated human iNKT cells are less glycolytic than TCONV and display higher expression of fatty acid oxidation (FAO) and adenosine monophosphate-activated protein kinase (AMPK) pathway genes. Furthermore, stimulated iNKT cells displayed higher mitochondrial mass and membrane potential relative to TCONV. Real-time Seahorse metabolic flux analysis revealed that stimulated human iNKT cells utilize fatty acids as substrates for oxidation more than stimulated TCONV. Together, our data suggest that human iNKT cells possess different bioenergetic requirements from TCONV and display a more memory-like metabolic program relative to effector TCONV. Importantly, iNKT cell-based immunotherapeutic strategies could co-opt such unique features of iNKT cells to improve their efficacy and longevity of anti-tumor responses.

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Enhancing the antitumor functions of invariant natural killer T cells using a soluble CD1d-CD19 fusion protein

Blood Advances ◽

10.1182/bloodadvances.2018028886 ◽

2019 ◽

Vol 3 (5) ◽

pp. 813-824 ◽

Cited By ~ 1

Author(s):

Rupali Das ◽

Peng Guan ◽

Susan J. Wiener ◽

Nishant P. Patel ◽

Trevor G. Gohl ◽

...

Keyword(s):

Fusion Protein ◽

B Cell ◽

Natural Killer ◽

Tumor Cells ◽

Inkt Cell ◽

Inkt Cells ◽

Cell Responses ◽

Invariant Natural Killer

Abstract Invariant natural killer T (iNKT) cells comprise a unique lineage of CD1d-restricted lipid-reactive T lymphocytes that potently kill tumor cells and exhibit robust immunostimulatory functions. Optimal tumor-directed iNKT cell responses often require expression of the antigen-presenting molecule CD1d on tumors; however, many tumor cells downregulate CD1d and thus evade iNKT cell recognition. We generated a soluble bispecific fusion protein designed to direct iNKT cells to the site of B-cell cancers in a tumor antigen-specific but CD1d-independent manner. This fusion protein is composed of a human CD1d molecule joined to a single chain antibody FV fragment specific for CD19, an antigen widely expressed on B-cell cancers. The CD1d-CD19 fusion protein binds specifically to CD19-expressing, but not CD19-negative cells. Once loaded with the iNKT cell lipid agonist α-galactosyl ceramide (αGC), the CD1d-CD19 fusion induces robust in vitro activation of and cytokine production by human iNKT cells. iNKT cells stimulated by the αGC-loaded CD1d-CD19 fusion also strongly transactivate T-, B-, and NK-cell responses and promote dendritic cell maturation. Importantly, the αGC-loaded fusion induces robust lysis of CD19+CD1d− Epstein-Barr virus immortalized human B-lymphoblastoid cell lines that are otherwise resistant to iNKT cell killing. Consistent with these findings; administration of the αGC-loaded fusion protein controlled the growth of CD19+CD1d− tumors in vivo, suggesting that it can “link” iNKT cells and CD19+CD1d− targets in a therapeutically beneficial manner. Taken together, these preclinical studies demonstrate that this B cell–directed fusion protein can be used to effectively induce iNKT cell antitumor responses in vitro and in vivo.

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Abstract 541: Specific Deletion of LDL Receptor--Related Protein on Macrophages Skews Cytokine Production by Invariant Natural Killer T Cells

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.34.suppl_1.541 ◽

2014 ◽

Vol 34 (suppl_1) ◽

Author(s):

Roman Covarrubias ◽

Amy S Major

Keyword(s):

Natural Killer ◽

Cell Activation ◽

Ldl Receptor ◽

Inkt Cell ◽

Related Protein ◽

Inkt Cells ◽

Glycolipid Antigen ◽

Invariant Natural Killer ◽

Natural Killer T

Invariant Natural Killer T (iNKT) cells are specialized lymphocytes that when activated can regulate chronic inflammatory conditions and atherosclerotic processes. The activation of iNKT cells occurs when glycolipid antigens bind the MHC class-I like molecule CD1d present on antigen presenting cells (APCs). The pathways by which glycolipid antigens target CD1d for presentation and activation of iNKT cells remain unclear, yet the expression of surface receptors associated with lipid homeostasis, such as the LDL receptor (LDLr), have been implicated in the modulation of iNKT cell activation. The LDLr has been shown to modulate this process by binding apoE-containing lipoproteins, which can carry antigenic glycolipids for iNKT cell activation. The LDL receptor-related protein (LRP), a transmembrane receptor from the LDL receptor family of proteins, shares structural homology with LDLr and can bind a number of ligands including apoE-containing lipoproteins. We hypothesized that LRP can play an active role in glycolipid antigen presentation and subsequent activation of iNKT cells. Here, we demonstrate that LRP is preferentially expressed at high levels on F4/80 + macrophages, when compared to other APCs. We also show that a specialized subset of macrophages expressing CD169, known for their ability to present glycolipid antigen to iNKT cells, have increased levels of LRP when compared to CD169 - macrophages. Using mice with a targeted deletion of LRP in macrophages, we observed decreased activation of iNKT cells in vitro (24, 48 hours) and normal IFN-gamma but blunted IL-4 response in vivo. Further flow cytometric analysis showed normal surface expression of CD1d in LRP-cKO macrophages as well as normal uptake of fluorescently labeled glycolipid in vitro . Additionally, analysis of the iNKT cell compartment in LRP-cKO mice revealed intact numbers and percentages of iNKT cells and no homeostatic disruption as evidenced by absence of programmed death-1 and LY-49. Collectively, these data suggest that macrophage LRP contributes to early iNKT cell activation by enhancing early IL-4 responses.

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