Investigating iRHOM2-Associated Transcriptional Changes in Tylosis With Esophageal Cancer.

Background and Aims: Survival rates for esophageal squamous cell carcinoma (ESCC) are extremely low due to the late diagnosis of most cases. An understanding of the early molecular processes that lead to ESCC may facilitate opportunities for early diagnosis; however, these remain poorly defined. Tylosis with esophageal cancer (TOC) is a rare syndrome associated with a high lifetime risk of ESCC and germline mutations in RHBDF2, encoding iRhom2. Using TOC as a model of ESCC predisposition, this study aimed to identify early-stage transcriptional changes in ESCC development. Methods: Esophageal biopsies were obtained from control and TOC individuals, the latter undergoing surveillance endoscopy, and adjacent diagnostic biopsies were graded as having no dysplasia or malignancy. Bulk RNA-Seq was performed, and findings were compared with sporadic ESCC vs normal RNA-Seq datasets. Results: Multiple transcriptional changes were identified in TOC samples, relative to controls, and many were detected in ESCC. Accordingly, pathway analyses predicted an enrichment of cancer-associated processes linked to cellular proliferation and metastasis, and several transcription factors were predicted to be associated with TOC and ESCC, including negative enrichment of GRHL2. Subsequently, a filtering strategy revealed 22 genes that were significantly dysregulated in both TOC and ESCC. Moreover, Keratin 17, which was upregulated in TOC and ESCC, was also found to be overexpressed at the protein level in 'normal' TOC esophagus tissue. Conclusion: Transcriptional changes occur in TOC esophagus prior to the onset of dysplasia, many of which are associated with ESCC. These findings support the utility of TOC to help reveal the early molecular processes that lead to sporadic ESCC.

Perinatal thymic-derived CD8αß-expressing γδ T cells are innate IFN-γ producers that expand in IL-7R-STAT5B-driven neoplasms.

The contribution of γδ T cells to immune responses is associated with rapid secretion of interferon-γ (IFN-γ). Here, we show a perinatal thymic wave of innate IFN-γ-producing γδ T cells that express CD8αß heterodimers and expand in preclinical models of infection and cancer. Optimal CD8αß+ γδ T cell development is directed by low T cell receptor signaling and through provision of interleukin (IL)-4 and IL-7. This population is pathologically relevant as overactive, or constitutive, IL-7R-STAT5B signaling promotes a supraphysiological accumulation of CD8αß+ γδ T cells in the thymus and peripheral lymphoid organs in two mouse models of T cell neoplasia. Likewise, CD8αß+ γδ T cells define a distinct subset of human T cell acute lymphoblastic leukemia pediatric patients. This work characterizes the normal and malignant development of CD8αß+ γδ T cells that are enriched in early life and contribute to innate IFN-γ responses to infection and cancer.

IL-27 maintains cytotoxic Ly6C+ γδ T cells that arise from immature precursors.

In mice, γδ-T lymphocytes that express the co-stimulatory molecule, CD27, are committed to the IFNγ-producing lineage during thymic development. In the periphery, these cells play a critical role in host defense and anti-tumor immunity. Unlike αß-T cells that rely on MHC-presented peptides to drive their terminal differentiation, it is unclear whether MHC-unrestricted γδ-T cells undergo further functional maturation after exiting the thymus. Here, we provide evidence of phenotypic and functional diversity within peripheral IFNγ-producing γδ T cells. We found that CD27+ Ly6C- cells convert into CD27+Ly6C+ cells, and these CD27+Ly6C+ cells control cancer progression in mice, while the CD27+Ly6C- cells cannot. The gene signatures of these two subsets were highly analogous to human immature and mature γδ-T cells, indicative of conservation across species. We show that IL-27 supports the cytotoxic phenotype and function of mouse CD27+Ly6C+ cells and human Vδ2+ cells, while IL-27 is dispensable for mouse CD27+Ly6C- cell and human Vδ1+ cell functions. These data reveal increased complexity within IFNγ-producing γδ-T cells, comprising immature and terminally differentiated subsets, that offer new insights into unconventional T-cell biology.

Versican Associates with Tumor Immune Phenotype and Limits T-cell Trafficking via Chondroitin Sulfate.

Immunotherapies for cancers of epithelial origin have limited efficacy, and a growing body of evidence links the composition of extracellular matrix (ECM) with the likelihood of a favorable response to treatment. The ECM may be considered an immunologic barrier, restricting the localization of cytotoxic immune cells to stromal areas and inhibiting their contact with tumor cells. Identifying ECM components of this immunologic barrier could provide targets that whether degraded in situ may support antitumor immunity and improve immunotherapy response. Using a library of primary triple-negative breast cancer tissues, we correlated CD8+ T-cell tumor contact with ECM composition and identified a proteoglycan, versican (VCAN), as a putative member of the immunologic barrier. Our analysis reveals that CD8+ T-cell contact with tumor associates with the location of VCAN expression, the specific glycovariant of VCAN [defined through the pattern of posttranslational attachments of glycosaminoglycans (GAG)], and the cell types that produce the variant. In functional studies, the isomers of chondroitin sulfate presented on VCAN have opposing roles being either supportive or inhibiting of T-cell trafficking, and removal of the GAGs ameliorates these effects on T-cell trafficking. Overall, we conclude that VCAN can either support or inhibit T-cell trafficking within the tumor microenvironment depending on the pattern of GAGs present, and that VCAN is a major component of the ECM immunologic barrier that defines the type of response to immunotherapy. SIGNIFICANCE: The response to immunotherapy has been poor toward solid tumors despite immune cells infiltrating into the tumor. The ECM has been associated with impacting T-cell infiltration toward the tumor and in this article we have identified VCAN and its structural modification, chondroitin sulfate as having a key role in T-cell invasion.

Deep characterization of human γδ T cell subsets defines shared and lineage-specific traits.

Under non-pathological conditions, human γδ T cells represent a small fraction of CD3+ T cells in peripheral blood (1-10%). They constitute a unique subset of T lymphocytes that recognize stress ligands or non-peptide antigens through MHC-independent presentation. Major human γδ T cell subsets, Vδ1 and Vδ2, expand in response to microbial infection or malignancy, but possess distinct tissue localization, antigen recognition, and effector responses. We hypothesized that differences at the gene, phenotypic, and functional level would provide evidence that γδ T cell subpopulations belong to distinct lineages. Comparisons between each subset and the identification of the molecular determinants that underpin their differences has been hampered by experimental challenges in obtaining sufficient numbers of purified cells. By utilizing a stringent FACS-based isolation method, we compared highly purified human Vδ1 and Vδ2 cells in terms of phenotype, gene expression profile, and functional responses. We found distinct genetic and phenotypic signatures that define functional differences in γδ T cell populations. Differences in TCR components, repertoire, and responses to calcium-dependent pathways suggest that Vδ1 and Vδ2 T cells are different lineages. These findings will facilitate further investigation into the ligand specificity and unique role of Vδ1 and Vδ2 cells in early immune responses.

Aging-induced isoDGR-modified fibronectin activates monocytic and endothelial cells to promote atherosclerosis.

BACKGROUND AND AIMS: Aging is the primary risk factor for cardiovascular disease (CVD), but the mechanisms underlying age-linked atherosclerosis remain unclear. We previously observed that long-lived vascular matrix proteins can acquire 'gain-of-function' isoDGR motifs that might play a role in atherosclerotic pathology. METHODS: IsoDGR-specific mAb were generated and used for ELISA-based measurement of motif levels in plasma samples from patients with coronary artery diseases (CAD) and non-CAD controls. Functional consequences of isoDGR accumulation in age-damaged fibronectin were determined by bioassay for capacity to activate monocytes, macrophages, and endothelial cells (signalling activity, pro-inflammatory cytokine expression, and recruitment/adhesion potential). Mice deficient in the isoDGR repair enzyme PCMT1 were used to assess motif distribution and macrophage localisation in vivo. RESULTS: IsoDGR-modified fibronectin and fibrinogen levels in patient plasma were significantly enhanced in CAD and further associated with smoking status. Functional assays demonstrated that isoDGR-modified fibronectin activated both monocytes and macrophages via integrin receptor 'outside in' signalling, triggering an ERK:AP-1 cascade and expression of pro-inflammatory cytokines MCP-1 and TNFα to drive additional recruitment of circulating leukocytes. IsoDGR-modified fibronectin also induced endothelial cell expression of integrin ß1 to further enhance cellular adhesion and matrix deposition. Analysis of murine aortic tissues confirmed accumulation of isoDGR-modified proteins co-localised with CD68+ macrophages in vivo. CONCLUSIONS: Age-damaged fibronectin features isoDGR motifs that increase binding to integrins on the surface of monocytes, macrophages, and endothelial cells. Subsequent activation of 'outside-in' signalling elicits a range of potent cytokines and chemokines that drive additional leukocyte recruitment to the developing atherosclerotic matrix.

Distinct metabolic programs established in the thymus control effector functions of γδ T cell subsets in tumor microenvironments.

Metabolic programming controls immune cell lineages and functions, but little is known about γδ T cell metabolism. Here, we found that γδ T cell subsets making either interferon-γ (IFN-γ) or interleukin (IL)-17 have intrinsically distinct metabolic requirements. Whereas IFN-γ+ γδ T cells were almost exclusively dependent on glycolysis, IL-17+ γδ T cells strongly engaged oxidative metabolism, with increased mitochondrial mass and activity. These distinct metabolic signatures were surprisingly imprinted early during thymic development and were stably maintained in the periphery and within tumors. Moreover, pro-tumoral IL-17+ γδ T cells selectively showed high lipid uptake and intracellular lipid storage and were expanded in obesity and in tumors of obese mice. Conversely, glucose supplementation enhanced the antitumor functions of IFN-γ+ γδ T cells and reduced tumor growth upon adoptive transfer. These findings have important implications for the differentiation of effector γδ T cells and their manipulation in cancer immunotherapy.

iRHOM2: A Regulator of Palmoplantar Biology, Inflammation, and Viral Susceptibility.

The palmoplantar epidermis is a specialized area of the skin that undergoes high levels of mechanical stress. The palmoplantar keratinization and esophageal cancer syndrome, tylosis with esophageal cancer, is linked to mutations in RHBDF2 encoding the proteolytically inactive rhomboid protein, iRhom2. Subsequently, iRhom2 was found to affect palmoplantar thickening to modulate the stress keratin response and to mediate context-dependent stress pathways by p63. iRhom2 is also a direct regulator of the sheddase, ADAM17, and the antiviral adaptor protein, stimulator of IFN genes. In this perspective, the pleiotropic functions of iRhom2 are discussed with respect to the skin, inflammation, and the antiviral response.

Comparable Vδ2 Cell Functional Characteristics in Virally Suppressed People Living with HIV and Uninfected Individuals.

Crosstalk between innate and adaptive pathways is a critical component to developing an effective, lasting immune response. Among natural effector cells, innate-like γδ T cells promote immunity by facilitating communication between the two compartments and exerting cytotoxic effector functions. Dysregulation of γδ T cell populations is a byproduct of primary Humanimmunodeficiency virus (HIV) infection. This is most pronounced in the depletion and loss of function within cells expressing a Vγ9Vδ2 TCR (Vδ2 cells). Whether or not prolonged viral suppression mediated by antiretroviral therapy (ART) can reverse these effects has yet to be determined. In this study, we present evidence of similar Vδ2 cell functional responses within a cohort of people living with HIV (PLWH) that has been stably suppressed for >1 year and uninfected donors. Through the use of aminobisphosphonate drugs, we were able to generate a comprehensive comparison between ex vivo and expanded Vδ2 cells within each group. Both groups had largely similar compositions of memory and effector phenotypes, post-expansion TCR repertoire diversity, and cytotoxic capabilities. Our findings support the notion that ART promotes the recovery of Vδ2 polyfunctionality and provides insight for strategies aiming to reconstitute the full immune response after infection with HIV.

Developmental origins of murine γδ T-cell subsets.

Murine γδ T cells display diverse responses to pathogens and tumours through early provision of pro-inflammatory cytokines such as interleukin-17A (IL-17) and interferon-γ (IFN-γ). Although it is now clear that acquisition of these cytokine-secreting effector fates is to a great extent developmentally pre-programmed in the thymus, the stages through which γδ progenitor cells transition, and the underlying mechanistic processes that govern these commitment events, are still largely unclear. Here, we review recent progress in the field, with particular consideration of how TCR-γδ signalling impacts on developmental programmes initiated before TCR-γδ expression.

Thymic Determinants of γδ T Cell Differentiation.

γd T cells have emerged as major sources of the proinflammatory cytokines interleukin-17 (IL-17) and interferon-γ (IFNγ) in multiple models of infection, cancer and autoimmune disease. However, unlike their αß T cell counterparts that require peripheral activation for effector cell differentiation, γδ T cells instead can be 'developmentally programmed' in the thymus to generate discrete γδ T cell effector subsets with distinctive molecular signatures. Nonetheless, recent studies have presented conflicting viewpoints on the signals involved in thymic γδ T cell development and differentiation, namely on the role of both T cell receptor (TCR)-dependent and TCR-independent factors. Here we review the current data and the ongoing controversies.

IFNγ Regulates Activated Vδ2+ T Cells through a Feedback Mechanism Mediated by Mesenchymal Stem Cells.

γδ T cells play a role in a wide range of diseases such as autoimmunity and cancer. The majority of circulating human γδ T lymphocytes express a Vγ9Vδ2+ (Vδ2+) T cell receptor (TCR) and following activation release pro-inflammatory cytokines. In this study, we show that IFNγ, produced by Vδ2+ cells, activates mesenchymal stem cell (MSC)-mediated immunosupression, which in turn exerts a negative feedback mechanism on γδ T cell function ranging from cytokine production to proliferation. Importantly, this modulatory effect is limited to a short period of time (<24 hours) post-T cell activation, after which MSCs can no longer exert their immunoregulatory capacity. Using genetically modified MSCs with the IFNγ receptor 1 constitutively silenced, we demonstrate that IFNγ is essential to this process. Activated γδ T cells induce expression of several factors by MSCs that participate in the depletion of amino acids. In particular, we show that indolamine 2,3-dioxygenase (IDO), an enzyme involved in L-tryptophan degradation, is responsible for MSC-mediated immunosuppression of Vδ2+ T cells. Thus, our data demonstrate that γδ T cell responses can be immuno-modulated by different signals derived from MSC.

Heterogeneous yet stable Vδ2(+) T-cell profiles define distinct cytotoxic effector potentials in healthy human individuals.

Human γδ T cells display potent responses to pathogens and malignancies. Of particular interest are those expressing a γδ T-cell receptor (TCR) incorporating TCRδ-chain variable-region-2 [Vδ2(+)], which are activated by pathogen-derived phosphoantigens (pAgs), or host-derived pAgs that accumulate in transformed cells or in cells exposed to aminobisphosphonates. Once activated, Vδ2(+) T cells exhibit multiple effector functions that have made them attractive candidates for immunotherapy. Despite this, clinical trials have reported mixed patient responses, highlighting a need for better understanding of Vδ2(+) T-cell biology. Here, we reveal previously unappreciated functional heterogeneity between the Vδ2(+) T-cell compartments of 63 healthy individuals. In this cohort, we identify distinct "Vδ2 profiles" that are stable over time; that do not correlate with age, gender, or history of phosphoantigen activation; and that develop after leaving the thymus. Multiple analyses suggest these Vδ2 profiles consist of variable proportions of two dominant but contrasting Vδ2(+) T-cell subsets that have divergent transcriptional programs and that display mechanistically distinct cytotoxic potentials. Importantly, an individual's Vδ2 profile predicts defined effector capacities, demonstrated by contrasting mechanisms and efficiencies of killing of a range of tumor cell lines. In short, these data support patient stratification to identify individuals with Vδ2 profiles that have effector mechanisms compatible with tumor killing and suggest that tailored Vδ2-profile-specific activation protocols may maximize the chances of future treatment success.

The emerging Protumor role of γδ T lymphocytes: implications for cancer immunotherapy.

Tumor-infiltrating lymphocytes are key mediators of tumor immune surveillance and are important prognostic indicators in cancer progression. Among the various lymphocyte subsets implicated in protection against cancer are γδ T lymphocytes, which can kill tumor cells and secrete potent antitumor cytokines. By contrast, recent reports have revealed an unexpected series of protumor functions of γδ T cells in mouse models and human patients. In particular, specific γδ T-cell subsets are capable of recruiting immunosuppressive myeloid populations, inhibiting antitumor responses, and enhancing angiogenesis, thus promoting cancer progression. A common mediator of such functions appears to be the cytokine IL17, whose pathogenic effects can override the antitumor immune response orchestrated by IFNγ. Here, we review these studies and discuss their implications for the manipulation of γδ T cells in cancer immunotherapy.

Murine CD27(-) Vγ6(+) γδ T cells producing IL-17A promote ovarian cancer growth via mobilization of protumor small peritoneal macrophages.

Cancer-associated inflammation mobilizes a variety of leukocyte populations that can inhibit or enhance tumor cell growth in situ. These subsets include γδ T cells, which can infiltrate tumors and typically provide large amounts of antitumor cytokines, such as IFN-γ. By contrast, we report here that in a well-established transplantable (ID8 cell line) model of peritoneal/ovarian cancer, γδ T cells promote tumor cell growth. γδ T cells accumulated in the peritoneal cavity in response to tumor challenge and could be visualized within solid tumor foci. Functional characterization of tumor-associated γδ T cells revealed preferential production of interleukin-17A (IL-17), rather than IFN-γ. Consistent with this finding, both T cell receptor (TCR)δ-deficient and IL-17-deficient mice displayed reduced ID8 tumor growth compared with wild-type animals. IL-17 production by γδ T cells in the tumor environment was essentially restricted to a highly proliferative CD27((-)) subset that expressed Vγ6 instead of the more common Vγ1 and Vγ4 TCR chains. The preferential expansion of IL-17-secreting CD27((-)) Vγ6((+)) γδ T cells associated with the selective mobilization of unconventional small peritoneal macrophages (SPMs) that, in comparison with large peritoneal macrophages, were enriched for IL-17 receptor A, and for protumor and proangiogenic molecular mediators, which were up-regulated by IL-17. Importantly, SPMs were uniquely and directly capable of promoting ovarian cancer cell proliferation. Collectively, this work identifies an IL-17-dependent lymphoid/myeloid cross-talk involving γδ T cells and SPMs that promotes tumor cell growth and thus counteracts cancer immunosurveillance.

Cytokines and chemokines: At the crossroads of cell signalling and inflammatory disease.

Inflammation occurs as a result of exposure of tissues and organs to harmful stimuli such as microbial pathogens, irritants, or toxic cellular components. The primary physical manifestations of inflammation are redness, swelling, heat, pain, and loss of function to the affected area. These processes involve the major cells of the immune system, including monocytes, macrophages, neutrophils, basophils, dendritic cells, mast cells, T-cells, and B-cells. However, examination of a range of inflammatory lesions demonstrates the presence of specific leukocytes in any given lesion. That is, the inflammatory process is regulated in such a way as to ensure that the appropriate leukocytes are recruited. These events are in turn controlled by a host of extracellular molecular regulators, including members of the cytokine and chemokine families that mediate both immune cell recruitment and complex intracellular signalling control mechanisms that characterise inflammation. This review will focus on the role of the main cytokines, chemokines, and their receptors in the pathophysiology of auto-inflammatory disorders, pro-inflammatory disorders, and neurological disorders involving inflammation.

Epigenetic and transcriptional signatures of stable versus plastic differentiation of proinflammatory γδ T cell subsets.

Two distinct subsets of γδ T cells that produce interleukin 17 (IL-17) (CD27(-) γδ T cells) or interferon-γ (IFN-γ) (CD27(+) γδ T cells) develop in the mouse thymus, but the molecular determinants of their functional potential in the periphery remain unknown. Here we conducted a genome-wide characterization of the methylation patterns of histone H3, along with analysis of mRNA encoding transcription factors, to identify the regulatory networks of peripheral IFN-γ-producing or IL-17-producing γδ T cell subsets in vivo. We found that CD27(+) γδ T cells were committed to the expression of Ifng but not Il17, whereas CD27(-) γδ T cells displayed permissive chromatin configurations at loci encoding both cytokines and their regulatory transcription factors and differentiated into cells that produced both IL-17 and IFN-γ in a tumor microenvironment.

Epithelial and dendritic cells in the thymic medulla promote CD4+Foxp3+ regulatory T cell development via the CD27-CD70 pathway.

CD4(+)Foxp3(+) regulatory T cells (Treg cells) are largely autoreactive yet escape clonal deletion in the thymus. We demonstrate here that CD27-CD70 co-stimulation in the thymus rescues developing Treg cells from apoptosis and thereby promotes Treg cell generation. Genetic ablation of CD27 or its ligand CD70 reduced Treg cell numbers in the thymus and peripheral lymphoid organs, whereas it did not alter conventional CD4(+)Foxp3(-) T cell numbers. The CD27-CD70 pathway was not required for pre-Treg cell generation, Foxp3 induction, or mature Treg cell function. Rather, CD27 signaling enhanced positive selection of Treg cells within the thymus in a cell-intrinsic manner. CD27 signals promoted the survival of thymic Treg cells by inhibiting the mitochondrial apoptosis pathway. CD70 was expressed on Aire(-) and Aire(+) medullary thymic epithelial cells (mTECs) and on dendritic cells (DCs) in the thymic medulla. CD70 on both mTECs and DCs contributed to Treg cell development as shown in BM chimera experiments with CD70-deficient mice. In vitro experiments indicated that CD70 on the CD8α(+) subset of thymic DCs promoted Treg cell development. Our data suggest that mTECs and DCs form dedicated niches in the thymic medulla, in which CD27-CD70 co-stimulation rescues developing Treg cells from apoptosis, subsequent to Foxp3 induction by TCR and CD28 signals.

Understanding the complexity of γδ T-cell subsets in mouse and human.

γδ T cells are increasingly recognized as having important functional roles in a range of disease scenarios such as infection, allergy, autoimmunity and cancer. With this has come realization that γδ cells are not a homogeneous population of cells with a single physiological role. Instead, ever increasing complexity in both phenotype and function is being ascribed to γδ cell subsets from various tissues and locations, and in both mouse and human. Here, we review this complexity by describing how diverse γδ cell subsets are generated in the murine thymus, and how these events relate to subsequent γδ subset function in the periphery. We then review the two major γδ cell populations in human, highlighting the several similarities of Vδ1(+) cells to certain murine γδ subsets, and describing the remarkable functional plasticity of human Vδ2(+) cells. A better understanding of this spectrum of γδ cell phenotypes should facilitate more targeted approaches to utilise their tremendous functional potential in the clinic.