Signalling mechanisms driving homeostatic and inflammatory effects of interleukin-15 on tissue lymphocytes.

There is an intriguing dichotomy in the function of cytokine interleukin-15-at low levels, it is required for the homeostasis of the immune system, yet when it is upregulated in response to pathogenic infections or in autoimmunity, IL-15 drives inflammation. IL-15 associates with the IL-15Rα within both myeloid and non-haematopoietic cells, where IL-15Rα trans-presents IL-15 in a membrane-bound form to neighboring cells. Alongside homeostatic maintenance of select lymphocyte populations such as NK cells and tissue-resident T cells, when upregulated, IL-15 also promotes inflammatory outcomes by driving effector function and cytotoxicity in NK cells and T cells. As chronic over-expression of IL-15 can lead to autoimmunity, IL-15 expression is tightly regulated. Thus, blocking dysregulated IL-15 and its downstream signalling pathways are avenues for immunotherapy. In this review we discuss the molecular pathways involved in IL-15 signalling and how these pathways contribute to both homeostatic and inflammatory functions in IL-15-dependent mature lymphoid populations, focusing on innate, and innate-like lymphocytes in tissues.

Breast Cancer Stem Cell-Derived Tumors Escape from γδ T-cell Immunosurveillance In Vivo by Modulating γδ T-cell Ligands.

There are no targeted therapies for patients with triple-negative breast cancer (TNBC). TNBC is enriched in breast cancer stem cells (BCSC), which play a key role in metastasis, chemoresistance, relapse, and mortality. γδ T cells hold great potential in immunotherapy against cancer and might provide an approach to therapeutically target TNBC. γδ T cells are commonly observed to infiltrate solid tumors and have an extensive repertoire of tumor-sensing mechanisms, recognizing stress-induced molecules and phosphoantigens (pAgs) on transformed cells. Herein, we show that patient-derived triple-negative BCSCs are efficiently recognized and killed by ex vivo expanded γδ T cells from healthy donors. Orthotopically xenografted BCSCs, however, were refractory to γδ T-cell immunotherapy. We unraveled concerted differentiation and immune escape mechanisms: xenografted BCSCs lost stemness, expression of γδ T-cell ligands, adhesion molecules, and pAgs, thereby evading immune recognition by γδ T cells. Indeed, neither promigratory engineered γδ T cells, nor anti-PD-1 checkpoint blockade, significantly prolonged overall survival of tumor-bearing mice. BCSC immune escape was independent of the immune pressure exerted by the γδ T cells and could be pharmacologically reverted by zoledronate or IFNα treatment. These results pave the way for novel combinatorial immunotherapies for TNBC.

Tissue environment, not ontogeny, defines murine intestinal intraepithelial T lymphocytes.

Tissue-resident intestinal intraepithelial T lymphocytes (T-IEL) patrol the gut and have important roles in regulating intestinal homeostasis. T-IEL include both induced T-IEL, derived from systemic antigen-experienced lymphocytes, and natural T-IEL, which are developmentally targeted to the intestine. While the processes driving T-IEL development have been elucidated, the precise roles of the different subsets and the processes driving activation and regulation of these cells remain unclear. To gain functional insights into these enigmatic cells, we used high-resolution, quantitative mass spectrometry to compare the proteomes of induced T-IEL and natural T-IEL subsets, with naive CD8+ T cells from lymph nodes. This data exposes the dominant effect of the gut environment over ontogeny on T-IEL phenotypes. Analyses of protein copy numbers of >7000 proteins in T-IEL reveal skewing of the cell surface repertoire towards epithelial interactions and checkpoint receptors; strong suppression of the metabolic machinery indicating a high energy barrier to functional activation; upregulated cholesterol and lipid metabolic pathways, leading to high cholesterol levels in T-IEL; suppression of T cell antigen receptor signalling and expression of the transcription factor TOX, reminiscent of chronically activated T cells. These novel findings illustrate how T-IEL integrate multiple tissue-specific signals to maintain their homeostasis and potentially function.

IL-15 and PIM kinases direct the metabolic programming of intestinal intraepithelial lymphocytes.

Intestinal intraepithelial lymphocytes (IEL) are an abundant population of tissue-resident T cells that protect and maintain the intestinal barrier. IEL respond to epithelial cell-derived IL-15, which is complexed to the IL-15 receptor α chain (IL-15/Rα). IL-15 is essential both for maintaining IEL homeostasis and inducing IEL responses to epithelial stress, which has been associated with Coeliac disease. Here, we apply quantitative mass spectrometry to IL-15/Rα-stimulated IEL to investigate how IL-15 directly regulates inflammatory functions of IEL. IL-15/Rα drives IEL activation through cell cycle regulation, upregulation of metabolic machinery and expression of a select repertoire of cell surface receptors. IL-15/Rα selectively upregulates the Ser/Thr kinases PIM1 and PIM2, which are essential for IEL to proliferate, grow and upregulate granzyme B in response to inflammatory IL-15. Notably, IEL from patients with Coeliac disease have high PIM expression. Together, these data indicate PIM kinases as important effectors of IEL responses to inflammatory IL-15.

Mechanisms of activation of innate-like intraepithelial T lymphocytes.

Intraepithelial T lymphocytes (T-IEL) contain subsets of innate-like T cells that evoke innate and adaptive immune responses to provide rapid protection at epithelial barrier sites. In the intestine, T-IEL express variable T cell antigen receptors (TCR), with unknown antigen specificities. Intriguingly, they also express multiple inhibitory receptors, many of which are normally found on exhausted or antigen-experienced T cells. This pattern suggests that T-IEL are antigen-experienced, yet it is not clear where, and in what context, T-IEL encounter TCR ligands. We review recent evidence indicating TCR antigens for intestinal innate-like T-IEL are found on thymic or intestinal epithelium, driving agonist selection of T-IEL. We explore the contributions of the TCR and various co-stimulatory and co-inhibitory receptors in activating T-IEL effector functions. The balance between inhibitory and activating signals may be key to keeping these highly cytotoxic, rapidly activated cells in check, and key to harnessing their immune surveillance potential.

Isolation, Characterization, and Culture of Intestinal Intraepithelial Lymphocytes.

Intestinal intraepithelial lymphocytes (IEL) comprise distinct groups of innate-like and memory T cells that collectively form one of the largest T cell compartments in the body. IEL are located within the intestinal epithelium and are the first immune cells in the gut to interact with the food, microbiota, and pathogens that the gut is continually exposed to. IEL can respond rapidly to external insults to protect the small intestinal epithelium but are also considered regulatory cells that are important to maintain the homeostasis of the gut. However, the mechanisms of IEL activation and their interactions within the epithelium remain largely elusive. Indeed, IEL are not commonly evaluated even in studies of gut immunology, potentially because they are perceived as being difficult to isolate and study. In this protocol, we present a simplified method to isolate IEL from the murine small intestine and provide representative data for flow cytometric analyses of the different IEL subsets. We also outline two procedures for culturing IEL, which can permit functional studies and coculture with epithelial cells. These strategies should make studies of this large but enigmatic T cell compartment more accessible and open up understanding of homeostatic mechanisms in the intestine, and tissue-associated immunity.

Loss of adenomatous polyposis coli function renders intestinal epithelial cells resistant to the cytokine IL-22.

Interleukin-22 (IL-22) is a critical immune defence cytokine that maintains intestinal homeostasis and promotes wound healing and tissue regeneration, which can support the growth of colorectal tumours. Mutations in the adenomatous polyposis coli gene (Apc) are a major driver of familial colorectal cancers (CRCs). How IL-22 contributes to APC-mediated tumorigenesis is poorly understood. To investigate IL-22 signalling in wild-type (WT) and APC-mutant cells, we performed RNA sequencing (RNAseq) of IL-22-treated murine small intestinal epithelial organoids. In WT epithelia, antimicrobial defence and cellular stress response pathways were most strongly induced by IL-22. Surprisingly, although IL-22 activates signal transducer and activator of transcription 3 (STAT3) in APC-mutant cells, STAT3 target genes were not induced. Our analyses revealed that ApcMin/Min cells are resistant to IL-22 due to reduced expression of the IL-22 receptor, and increased expression of inhibitors of STAT3, particularly histone deacetylases (HDACs). We further show that IL-22 increases DNA damage and genomic instability, which can accelerate cellular transition from heterozygosity (ApcMin/+) to homozygosity (ApcMin/Min) to drive tumour formation. Our data reveal an unexpected role for IL-22 in promoting early tumorigenesis while excluding a function for IL-22 in transformed epithelial cells.

A Cholesterol-Based Allostery Model of T Cell Receptor Phosphorylation.

Signaling through the T cell receptor (TCR) controls adaptive immune responses. Antigen binding to TCRαß transmits signals through the plasma membrane to induce phosphorylation of the CD3 cytoplasmic tails by incompletely understood mechanisms. Here we show that cholesterol bound to the TCRß transmembrane region keeps the TCR in a resting, inactive conformation that cannot be phosphorylated by active kinases. Only TCRs that spontaneously detached from cholesterol could switch to the active conformation (termed primed TCRs) and then be phosphorylated. Indeed, by modulating cholesterol binding genetically or enzymatically, we could switch the TCR between the resting and primed states. The active conformation was stabilized by binding to peptide-MHC, which thus controlled TCR signaling. These data are explained by a model of reciprocal allosteric regulation of TCR phosphorylation by cholesterol and ligand binding. Our results provide both a molecular mechanism and a conceptual framework for how lipid-receptor interactions regulate signal transduction.

Glucose and glutamine fuel protein O-GlcNAcylation to control T cell self-renewal and malignancy.

Sustained glucose and glutamine transport are essential for activated T lymphocytes to support ATP and macromolecule biosynthesis. We found that glutamine and glucose also fuel an indispensable dynamic regulation of intracellular protein O-GlcNAcylation at key stages of T cell development, transformation and differentiation. Glucose and glutamine are precursors of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), a substrate for cellular glycosyltransferases. Immune-activated T cells contained higher concentrations of UDP-GlcNAc and increased intracellular protein O-GlcNAcylation controlled by the enzyme O-linked-ß-N-acetylglucosamine (O-GlcNAc) glycosyltransferase as compared with naive cells. We identified Notch, the T cell antigen receptor and c-Myc as key controllers of T cell protein O-GlcNAcylation via regulation of glucose and glutamine transport. Loss of O-GlcNAc transferase blocked T cell progenitor renewal, malignant transformation and peripheral T cell clonal expansion. Nutrient-dependent signaling pathways regulated by O-GlcNAc glycosyltransferase are thus fundamental for T cell biology.

Intestinal intraepithelial lymphocyte activation promotes innate antiviral resistance.

Unrelenting environmental challenges to the gut epithelium place particular demands on the local immune system. In this context, intestinal intraepithelial lymphocytes (IEL) compose a large, highly conserved T cell compartment, hypothesized to provide a first line of defence via cytolysis of dysregulated intestinal epithelial cells (IEC) and cytokine-mediated re-growth of healthy IEC. Here we show that one of the most conspicuous impacts of activated IEL on IEC is the functional upregulation of antiviral interferon (IFN)-responsive genes, mediated by the collective actions of IFNs with other cytokines. Indeed, IEL activation in vivo rapidly provoked type I/III IFN receptor-dependent upregulation of IFN-responsive genes in the villus epithelium. Consistent with this, activated IEL mediators protected cells against virus infection in vitro, and pre-activation of IEL in vivo profoundly limited norovirus infection. Hence, intraepithelial T cell activation offers an overt means to promote the innate antiviral potential of the intestinal epithelium.

Cholesterol and sphingomyelin drive ligand-independent T-cell antigen receptor nanoclustering.

The T-cell antigen receptor (TCR) exists in monomeric and nanoclustered forms independently of antigen binding. Although the clustering is involved in the regulation of T-cell sensitivity, it is unknown how the TCR nanoclusters form. We show that cholesterol is required for TCR nanoclustering in T cells and that this clustering enhances the avidity but not the affinity of the TCR-antigen interaction. Investigating the mechanism of the nanoclustering, we found that radioactive photocholesterol specifically binds to the TCRß chain in vivo. In order to reduce the complexity of cellular membranes, we used a synthetic biology approach and reconstituted the TCR in liposomes of defined lipid composition. Both cholesterol and sphingomyelin were required for the formation of TCR dimers in phosphatidylcholine-containing large unilamellar vesicles. Further, the TCR was localized in the liquid disordered phase in giant unilamellar vesicles. We propose a model in which cholesterol and sphingomyelin binding to the TCRß chain causes TCR dimerization. The lipid-induced TCR nanoclustering enhances the avidity to antigen and thus might be involved in enhanced sensitivity of memory compared with naive T cells. Our work contributes to the understanding of the function of specific nonannular lipid-membrane protein interactions.

Butyrophilins: an emerging family of immune regulators.

Butyrophilins (Btns) and butyrophilin-like (Btnl) molecules are emerging as novel regulators of immune responses in mice and humans. Several clues point to their probable importance: many of the genes are located within the MHC; they are structurally related to B7-co-stimulatory molecules; they are functionally implicated in T cell inhibition and in the modulation of epithelial cell-T cell interactions; and they are genetically associated with inflammatory diseases. Nonetheless, initial immersion into the current literature can uncover confusion over even basic information such as gene names and expression patterns, and seemingly conflicting data regarding the biological activities of different family members. This review addresses each of these issues, concluding with the attractive potential of Btn and Btnl molecules to act as specific attenuators of tissue-associated inflammatory responses.

Increased sensitivity of antigen-experienced T cells through the enrichment of oligomeric T cell receptor complexes.

Although memory T cells respond more vigorously to stimulation and they are more sensitive to low doses of antigen than naive T cells, the molecular basis of this increased sensitivity remains unclear. We have previously shown that the T cell receptor (TCR) exists as different-sized oligomers on the surface of resting T cells and that large oligomers are preferentially activated in response to low antigen doses. Through biochemistry and electron microscopy, we now showed that previously stimulated and memory T cells have more and larger TCR oligomers at the cell surface than their naive counterparts. Reconstitution of cells and mice with a point mutant of the CD3ζ subunit, which impairs TCR oligomer formation, demonstrated that the increased size of TCR oligomers was directly responsible for the increased sensitivity of antigen-experienced T cells. Thus, we propose that an "avidity maturation" mechanism underlies T cell antigenic memory.

Butyrophilin-like 1 encodes an enterocyte protein that selectively regulates functional interactions with T lymphocytes.

Although local regulation of T-cell responses by epithelial cells is increasingly viewed as important, few molecules mediating such regulation have been identified. Skint1, a recently identified member of the Ig-supergene family expressed by thymic epithelial cells and keratinocytes, specifies the murine epidermal intraepithelial lymphocyte (IEL) repertoire. Investigating whether Skint1-related molecules might regulate IEL in other compartments, this study focuses on buytrophilin-like 1 (Btnl1), which is conspicuously similar to Skint1 and primarily restricted to small intestinal epithelium. Btnl1 protein is mostly cytoplasmic, but surface expression can be induced, and in vivo Btnl1 can be detected adjacent to the IEL. In a newly developed culture system, enforced epithelial cell expression of Btnl1 attenuated the cells' response to activated IEL, as evidenced by suppression of IL-6 and other inflammatory mediators. These findings offer a unique perspective on emerging genetic data that Btnl genes may comprise novel and important local regulators of gut inflammation.

Structural characterization of the TCR complex by electron microscopy.

Structural information on how the TCR transmits signals upon binding of its antigen peptide MHC molecule ligand is still lacking. The ectodomains of the TCRα/ß, CD3εγ and CD3εδ dimers, as well as the transmembrane domain of CD3ζ, have been characterized by X-ray crystallography and nuclear magnetic resonance (NMR). However, no structural data have been obtained for the entire TCR complex. In this study, we have purified the TCR from T cells under native conditions and used electron microscopy to derive a three-dimensional structure. The TCR complex appears as a pear-shaped structure of 180 × 120 × 65 . Furthermore, the use of mAbs has allowed to determine the orientation of the TCRα/ß and CD3 subunits and to suggest a model of interactions. Interestingly, the reconstructed TCR is larger than expected for a complex with a αßγεδεζζ stoichiometry. The accommodation of a second TCRαß to fill in the extra volume is discussed.

Epithelial decision makers: in search of the 'epimmunome'.

Frequent microbial and nonmicrobial challenges to epithelial cells trigger discrete pathways, promoting molecular changes such as the secretion of specific cytokines and chemokines and alterations to molecules displayed at the epithelial cell surface. In combination, these molecules impose key decisions on innate and adaptive immune cells. Depending on context, those decisions can be as diverse as those imposed by professional antigen-presenting cells, benefiting the host by balancing immune competence with the avoidance of immunopathology. Nonetheless, this potency of epithelial cells is also consistent with the causal contribution of epithelial dysregulation to myriad inflammatory diseases. This pathogenic axis provides an attractive target for tissue-specific clinical manipulation. In this context, a research goal should be to identify all molecules used by epithelial cells to instruct immune cells. We term this the 'epimmunome'.

Stoichiometry and intracellular fate of TRIM-containing TCR complexes.

BACKGROUND: Studying the stoichiometry and intracellular trafficking of the T cell antigen receptor (TCR) is pivotal in understanding its mechanisms of activation. The alphabetaTCR includes the antigen-binding TCRalphabeta heterodimer as well as the signal transducing CD3epsilongamma, CD3epsilondelta and zeta2 subunits. Although the TCR-interacting molecule (TRIM) is also part of the alphabetaTCR complex, it has not been included in most reports so far. RESULTS: We used the native antibody-based mobility shift (NAMOS) assay in a first dimension (1D) blue native (BN)-PAGE and a 2D BN-/BN-PAGE to demonstrate that the stoichiometry of the digitonin-solublized TRIM-containing alphabetaTCR is TCRalphabetaCD3epsilon2gammadeltazeta2TRIM2. Smaller alphabetaTCR complexes possess a TCRalphabeta CD3epsilon2gammadeltazeta2 stoichiometry. Complexes of these sizes were detected in T cell lines as well as in primary human and mouse T cells. Stimulating the alphabetaTCR with anti-CD3 antibodies, we demonstrate by confocal laser scanning microscopy that CD3epsilon colocalizes with zeta and both are degraded upon prolonged stimulation, possibly within the lysosomal compartment. In contrast, a substantial fraction of TRIM does not colocalize with zeta. Furthermore, TRIM neither moves to lysosomes nor is degraded. Immunoprecipitation studies and BN-PAGE indicate that TRIM also associates with the gammadeltaTCR. CONCLUSIONS: Small alphabetaTCR complexes have a TCRalphabeta CD3epsilon2gammadeltazeta2 stoichiometry; whereas those associated with one TRIM dimer are TCRalphabeta CD3epsilon2gammadeltazeta2TRIM2. TRIM is differentially processed compared to CD3 and zeta subunits after T cell activation and is not degraded. The gammadeltaTCR also associates with TRIM.

Detection of protein complex interactions via a Blue Native-PAGE retardation assay.

We describe the Blue Native (BN)-PAGE retardation assay for the detection of interactions of biomolecules with protein complexes. Potential interactors of proteins are included in the BN gel matrix, resulting in retardation of proteins that interact with the added molecule. After validation using the T-cell antigen receptor, we applied the assay for a general identification of dextran interactors in combination with mass spectroscopy. The proteomic screen revealed triosephosphate isomerase oligomer as a dextran-binding, high M(R) complex.