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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 αβ-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., (© 2024. The Author(s).)

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 CD8αβ heterodimers and expand in preclinical models of infection and cancer. Optimal CD8αβ + 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 CD8αβ + γδ T cells in the thymus and peripheral lymphoid organs in two mouse models of T cell neoplasia. Likewise, CD8αβ + 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 CD8αβ + γδ T cells that are enriched in early life and contribute to innate IFN-γ responses to infection and cancer., (© 2024. The Author(s).)

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., (© 2024 The Authors; Published by the American Association for Cancer Research.)

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., (© 2024 The Authors.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Sanz, Mann, Ryan, Bosque, Pennington, Hackstein and Soriano-Sarabia.)

The assessment of the cellular heterogeneity and abundance in bulk tissue samples is essential for characterising cellular and organismal states. Computational approaches to estimate cellular abundance from bulk RNA-Seq datasets have variable performances, often requiring benchmarking matrices to select the best performing methods for individual studies. However, such benchmarking investigations are difficult to perform and assess in typical applications because of the absence of gold standard/ground-truth cellular measurements. Here we describe Decosus, an R package that integrates seven methods and signatures for deconvoluting cell types from gene expression profiles (GEP). Benchmark analysis on a range of datasets with ground-truth measurements revealed that our integrated estimates consistently exhibited stable performances across datasets than individual methods and signatures. We further applied Decosus to characterise the immune compartment of skin samples in different settings, confirming the well-established Th1 and Th2 polarisation in psoriasis and atopic dermatitis, respectively. Secondly, we revealed immune system-related UV-induced changes in sun-exposed skin. Furthermore, a significant motivation in the design of Decosus is flexibility and the ability for the user to include new gene signatures, algorithms, and integration methods at run time., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Anene, Taggart, Harwood, Pennington and Wang.)

The study of a desmoglein 2 murine model of arrhythmogenic cardiomyopathy revealed cardiac inflammation as a key early event leading to fibrosis. Arrhythmogenic cardiomyopathy (AC) is an inherited heart muscle disorder leading to ventricular arrhythmias and heart failure due to abnormalities in the cardiac desmosome. We examined how loss of desmoglein 2 (Dsg2) in the young murine heart leads to development of AC. Apoptosis was an early cellular phenotype, and RNA sequencing analysis revealed early activation of inflammatory-associated pathways in Dsg2-null (Dsg2 -/- ) hearts at postnatal day 14 (2 weeks) that were absent in the fibrotic heart of adult mice (10 weeks). This included upregulation of iRhom2/ADAM17 and its associated pro-inflammatory cytokines and receptors such as TNFα, IL6R and IL-6. Furthermore, genes linked to specific macrophage populations were also upregulated. This suggests cardiomyocyte stress triggers an early immune response to clear apoptotic cells allowing tissue remodelling later on in the fibrotic heart. Our analysis at the early disease stage suggests cardiac inflammation is an important response and may be one of the mechanisms responsible for AC disease progression., (© 2021. Crown.)

Murine γδ 17 cells, which are T cells that bear the γδ T cell receptor (TCRγδ) and secrete interleukin-17A (IL-17A), are generated in the thymus and are critical for various immune responses. Although strong TCRγδ signals are required for the development of interferon-γ (IFN-γ)-secreting γδ cells (γδ IFN cells), the generation of γδ 17 cells requires weaker TCRγδ signaling. Here, we demonstrated that constrained activation of the kinase Syk downstream of TCRγδ was required for the thymic development of γδ 17 cells. Increasing or decreasing Syk activity by stimulating TCRγδ or inhibiting Syk, respectively, substantially reduced γδ 17 cell numbers. This delimited Syk activity optimally engaged the phosphoinositide 3-kinase (PI3K)-Akt signaling pathway, which maintained the expression of master regulators of the IL-17 program, RORγt and c-Maf. Inhibition of PI3K not only abrogated γδ 17 cell development but also augmented the development of a distinct, previously undescribed subset of γδ T cells. These CD8 + Ly6a + γδ T cells had a type-I IFN gene expression signature and expanded in response to stimulation with IFN-β. Collectively, these studies elucidate how weaker TCRγδ signaling engages distinct signaling pathways to specify the γδ 17 cell fate and identifies a role for type-I IFNs in γδ T cell development., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

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 β1 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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

Deep sequencing of B cell receptor (BCR) heavy chains from a cohort of 31 COVID-19 patients from the UK reveals a stereotypical naive immune response to SARS-CoV-2 which is consistent across patients. Clonal expansion of the B cell population is also observed and may be the result of memory bystander effects. There was a strong convergent sequence signature across patients, and we identified 1,254 clonotypes convergent between at least four of the COVID-19 patients, but not present in healthy controls or individuals following seasonal influenza vaccination. A subset of the convergent clonotypes were homologous to known SARS and SARS-CoV-2 spike protein neutralizing antibodies. Convergence was also demonstrated across wide geographies by comparison of data sets between patients from UK, USA, and China, further validating the disease association and consistency of the stereotypical immune response even at the sequence level. These convergent clonotypes provide a resource to identify potential therapeutic and prophylactic antibodies and demonstrate the potential of BCR profiling as a tool to help understand patient responses., Competing Interests: JO, AL, OC, SS, JG, JD, RM, and DF are employees of Alchemab Therapeutics Limited. RB-R is a founder of and consultant to Alchemab Therapeutics Limited. GK is a consultant to Alchemab Therapeutics Limited. CC is a member of the AstraZeneca External Science Panel and has research grants from Roche, Genentech, AstraZeneca, and Servier that are administered by the University of Cambridge. JB was employed by Illumina, Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Galson, Schaetzle, Bashford-Rogers, Raybould, Kovaltsuk, Kilpatrick, Minter, Finch, Dias, James, Thomas, Lee, Betley, Cavlan, Leech, Deane, Seoane, Caldas, Pennington, Pfeffer and Osbourn.)

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.

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., (© 2018 John Wiley & Sons Ltd.)

[This corrects the article DOI: 10.1371/journal.pmed.1002352.].

CD4 (+) CD8 (+) "double positive" (DP) thymocytes differentiate into diverse αβ T cell sub-types using mechanistically distinct programs. For example, conventional αβ T cells develop from DP cells after partial-agonist T cell receptor (TCR) interactions with self-peptide/MHC, whereas unconventional αβ T cells, such as TCRαβ (+) CD8αα (+) intraepithelial lymphocytes (IELs), require full-agonist TCR interactions. Despite this, DP cells appear homogeneous, and it remains unclear how distinct TCR signalling instructs distinct developmental outcomes. Moreover, whether TCR signals at earlier stages of development, for example in CD4 (-) CD8 (-) double negative (DN) cells, impact on later fate decisions is presently unknown. Here, we assess four strains of mice that display altered TCR signal strength in DN cells, which correlates with altered generation of unconventional TCRαβ (+) CD8αα (+) IELs. FVB/n mice (compared to C57BL/6 animals) and mice with altered preTCRα (pTα) expression, both displayed weaker TCR signalling in DN cells, an inefficient DN-to-DP transition, and reduced contribution of TCRαβ (+) CD8αα (+) IELs to gut epithelium. Conversely, TCRαβ (+) CD8αα (+) IEL development was favoured in mice with increased TCR signal strength in DN cells. Collectively, these data suggest TCR signal strength in DN cells directly impacts on subsequent DP cell differentiation, fundamentally altering the potential of thymocyte progenitors to adopt conventional versus unconventional T cell fates.

Despite a growing appreciation of γδ T cell contributions to numerous immune responses, the mechanisms that underpin their thymic development remain poorly understood. Here, using precursor/product relationships, we identify thymic stages in two distinct developmental pathways that generate γδ T cells pre-committed to subsequent secretion of either IL-17A or IFNγ. Importantly, this framework for tracking γδ T cell development has permitted definitive assessment of TCRγδ signal strength in commitment to γδ T cell effector fate; increased TCRγδ signal strength profoundly prohibited the development of all IL-17A-secreting γδ T cells, regardless of Vγ usage, but promoted the development of γδ progenitors along the IFNγ pathway. This clarifies the recently debated role of TCRγδ signal strength in commitment to distinct γδ T cell effector fates and proposes an alternate methodology for the study of γδ T cell development., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

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 αβ 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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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., Competing Interests: The authors have declared that no competing interests exist.

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., Competing Interests: The authors declare no conflict of interest.

The mouse thymus produces discrete γδ T cell subsets that make either interferon-γ (IFN-γ) or interleukin 17 (IL-17), but the role of the T cell antigen receptor (TCR) in this developmental process remains controversial. Here we show that Cd3g(+/-) Cd3d(+/-) (CD3 double-haploinsufficient (CD3DH)) mice have reduced TCR expression and signaling strength on γδ T cells. CD3DH mice had normal numbers and phenotypes of αβ thymocyte subsets, but impaired differentiation of fetal Vγ6(+) (but not Vγ4(+)) IL-17-producing γδ T cells and a marked depletion of IFN-γ-producing CD122(+) NK1.1(+) γδ T cells throughout ontogeny. Adult CD3DH mice showed reduced peripheral IFN-γ(+) γδ T cells and were resistant to experimental cerebral malaria. Thus, TCR signal strength within specific thymic developmental windows is a major determinant of the generation of proinflammatory γδ T cell subsets and their impact on pathophysiology.

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., (©2015 American Association for Cancer Research.)

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.

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.

The thymus generates T cells that are generally functionally immature and thus require peripheral activation for differentiation into effector lymphocytes. Notable exceptions to this rule are murine γδ T cells, many of which have been shown to acquire their functional potential during thymic development from late embryonic stages. Here, we review the underlying ontogenic processes and molecular differentiation mechanisms of murine γδ T cells, focusing on the transcriptional control of IFN-γ and IL-17 expression. We propose that functional commitment of γδ T cells occurs in "developmental windows" defined by the molecular composition of the thymic microenvironment, such as T-cell receptor (TCR), TCR coreceptor ligands, and cytokines. We further discuss the similarities and particularities of functional development of γδ T cells in mice and humans, while highlighting some key unresolved issues for future investigation., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

IL-17-producing CD27(-) γδ cells (γδ(27-) cells) are widely viewed as innate immune cells that make critical contributions to host protection and autoimmunity. However, factors that promote them over IFN-γ-producing γδ(27+) cells are poorly elucidated. Moreover, although human IL-17-producing γδ cells are commonly implicated in inflammation, such cells themselves have proved difficult to isolate and characterize. Here, murine γδ(27-) T cells and thymocytes are shown to be rapidly and substantially expanded by IL-7 in vitro and in vivo. This selectivity owes in substantial part to the capacity of IL-7 to activate STAT3 in such cells. Additionally, IL-7 promotes strong responses of IL-17-producing γδ cells to TCR agonists, thus reemphasizing the cells' adaptive and innate potentials. Moreover, human IL-17-producing γδ cells are also substantially expanded by IL-7 plus TCR agonists. Hence, IL-7 has a conserved potential to preferentially regulate IL-17-producing γδ cells, with both biological and clinical implications.

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., (© 2012 The Authors. Immunology © 2012 Blackwell Publishing Ltd.)

γδ cells have been conserved across ∼450 million years of evolution, from which they share the distinction, alongside αβ T cells and B cells, of forming antigen receptors by somatic gene recombination. However, much about these cells remains unclear. Indeed, although γδ cells display 'innate-like' characteristics exemplified by rapid tissue-localised responses to stress-associated stimuli, their huge capacity for T cell receptor (TCR)γδ diversity also suggests 'adaptive-like' potential. Clarity requires a better understanding of TCRγδ itself, not only through identification of TCR ligands, but also by correlating thymic TCRγδ signalling with commitment to γδ effector fates. Here, we propose that thymic TCRγδ-ligand engagement versus ligand-independent signalling differentially imprints innate-like versus adaptive-like characteristics on developing γδ cells, which fundamentally dictate their peripheral effector properties., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Whether thymocytes adopt an αβ or a γδ T cell fate in the thymus is determined at the β selection checkpoint by the relatively weak or strong signals that are delivered by either the pre-T cell receptor (preTCR) or the γδ TCR, respectively. Signal initiation at the β selection checkpoint is thought to be independent of ligand engagement of these receptors. Some reports have suggested that receptor oligomerization, which is thought to be mediated by either the immunoglobulin (Ig)-like domain of the preTCRα (pTα) chain or the variable domain of TCRδ, is a unifying mechanism that initiates signaling in early CD4(-)CD8(-) double-negative (DN) thymocyte progenitors. Here, we demonstrate that the extracellular regions of pTα and TCRδ that are implicated in mediating receptor oligomerization were not required for signal initiation from the preTCR or TCRγδ. Indeed, a truncated TCRγδ that lacked all of its extracellular Ig-like domains still formed a signaling-competent TCR that drove cells through the β selection checkpoint. These observations suggest that signal initiation in DN thymocytes is simply a consequence of the surface-pairing of TCR chains, with signal strength being a function of the abundances of surface TCRs. Thus, processes that regulate the surface abundances of TCR complexes in DN cells, such as oligomerization-induced endocytosis, would be predicted to have a major influence in determining whether cells adopt an αβ versus γδ T cell fate.

A best evidence topic in congenital cardiac surgery was written according to a structured protocol. The question addressed was whether neonatal thymectomy in patients undergoing cardiac surgery has any adverse immunological consequences. Altogether 164 papers were found using the reported search, of which nine papers represented the best evidence to answer the clinical question. The author, journal, date and country of publication, patient group studied, study type, relevant outcomes, results and study weaknesses were tabulated. The thymus is the major production site of T cells, whose stocks are built-up during foetal and early postnatal life. However, its function diminishes after the first years of life, and although thymic output is maintained into adulthood, the thymus mostly degenerates into fatty tissue in elderly adults. To date, there has been no general consensus with regard to the importance of this organ during childhood and adulthood. As a consequence, during cardiac surgery in neonates, partial or total thymectomy is routinely performed to enable better access to the heart and great vessels to correct congenital heart defects, suggesting that it may be dispensable during childhood and adulthood. Interestingly, current best available evidence from nine case-control studies suggests that neonatal thymectomy affects peripheral T-cell populations both in the short- as well as long-term and results in premature immunosenescence. However, the impact of these changes on the risk of infectious diseases or malignancy has not been thoroughly evaluated by any of these studies. Maintenance of a registry of patients undergoing neonatal thymectomy and further studies to assess the functional or clinical consequences of this practice would be valuable.

The production of cytokines such as interferon-gamma and interleukin 17 by alphabeta and gammadelta T cells influences the outcome of immune responses. Here we show that most gammadelta T lymphocytes expressed the tumor necrosis factor receptor family member CD27 and secreted interferon-gamma, whereas interleukin 17 production was restricted to CD27(-) gammadelta T cells. In contrast to the apparent plasticity of alphabeta T cells, the cytokine profiles of these distinct gammadelta T cell subsets were essentially stable, even during infection. These phenotypes were established during thymic development, when CD27 functions as a regulator of the differentiation of gammadelta T cells at least in part by inducing expression of the lymphotoxin-beta receptor and genes associated with trans-conditioning and interferon-gamma production. Thus, the cytokine profiles of peripheral gammadelta T cells are predetermined mainly by a mechanism involving CD27.

The mouse CD8alpha+ DC subset excels at cross-presentation of antigen, which can elicit robust CTL responses. A receptor allowing specific antigen targeting to this subset and its equivalent in humans would therefore be useful for the induction of antitumor CTLs. Here, we have characterized a C-type lectin of the NK cell receptor group that we named DC, NK lectin group receptor-1 (DNGR-1). DNGR-1 was found to be expressed in mice at high levels by CD8+ DCs and at low levels by plasmacytoid DCs but not by other hematopoietic cells. Human DNGR-1 was also restricted in expression to a small subset of blood DCs that bear similarities to mouse CD8alpha+ DCs. The selective expression pattern and observed endocytic activity of DNGR-1 suggested that it could be used for antigen targeting to DCs. Consistent with this notion, antigen epitopes covalently coupled to an antibody specific for mouse DNGR-1 were selectively cross-presented by CD8alpha+ DCs in vivo and, when given with adjuvants, induced potent CTL responses. When the antigens corresponded to tumor-expressed peptides, treatment with the antibody conjugate and adjuvant could prevent development or mediate eradication of B16 melanoma lung pseudometastases. We conclude that DNGR-1 is a novel, highly specific marker of mouse and human DC subsets that can be exploited for CTL cross-priming and tumor therapy.

Our genome-wide association study of celiac disease previously identified risk variants in the IL2-IL21 region. To identify additional risk variants, we genotyped 1,020 of the most strongly associated non-HLA markers in an additional 1,643 cases and 3,406 controls. Through joint analysis including the genome-wide association study data (767 cases, 1,422 controls), we identified seven previously unknown risk regions (P < 5 x 10(-7)). Six regions harbor genes controlling immune responses, including CCR3, IL12A, IL18RAP, RGS1, SH2B3 (nsSNP rs3184504) and TAGAP. Whole-blood IL18RAP mRNA expression correlated with IL18RAP genotype. Type 1 diabetes and celiac disease share HLA-DQ, IL2-IL21, CCR3 and SH2B3 risk regions. Thus, this extensive genome-wide association follow-up study has identified additional celiac disease risk variants in relevant biological pathways.

A fundamental issue in T cell development is what controls whether a thymocyte differentiates into a gammadelta T cell or an alphabeta T cell, each defined by their distinct T cell receptor. Most likely, lessons learned in studying that issue will also provide insight into how the thymus produces T cell subsets with distinct functional and regulatory potentials. Here we review recent experiments, focusing on three factors that regulate thymocyte differentiation up to and including the expression of the first products of antigen receptor gene rearrangements. Those factors are the archetypal developmental regulator Notch, intrinsic signals emanating from antigen-receptor complexes, and trans conditioning, which reflects communication between different subsets of thymocytes. We also review new findings on the positive selection of gammadelta T cells and on extrathymic T cell development.

In cellular immunology the critical balance between effector and regulatory mechanisms is highlighted by serious immunopathologies attributable to mutations in Foxp3, a transcription factor required for a major subset of regulatory T (Tr) cells. Thus, many studies have focused on the developmental origin of Tr cells, with the prevailing view that they emerge in the thymus from late-stage T-cell progenitors whose T-cell receptors (TCRs) engage high affinity (agonist) ligands. This study questions the completeness of that interpretation. Here we show that without any obvious effect on TCR-mediated selection, the normal differentiation of mouse gammabeta T cells into potent cytolytic and interferon-gamma-secreting effector cells is switched towards an aggregate regulatory phenotype by limiting the capacity of CD4+CD8+ T-cell progenitors to influence in trans early gammabeta cell progenitors. Unexpectedly, we found that the propensity of early TCR-alphabeta+ progenitors to differentiate into Foxp3+ Tr cells is also regulated in trans by CD4+CD8+ T-cell progenitor cells, before agonist selection.

Gammadelta T cells play critical roles in immune regulation, tumour surveillance and specific primary immune responses. Mature gammadelta cells derive from thymic precursors that also generate alphabeta T cells. Recent reports have highlighted the impact of the strength of signal received via the T cell receptor on T cell lineage commitment, and the importance of cross-talk between committed alphabeta thymocytes and bipotential progenitors for normal gammadelta T cell differentiation. Studies on T cell receptor-mediated selection of gammadelta cells have supported the view that these unconventional T cells are positively rather than negatively selected on cognate self antigen.

The thymus gives rise to two T cell lineages, alphabeta and gammadelta, that are thought to develop independently of one another. Hence, double positive (DP) thymocytes expressing CD4 and CD8 coreceptors are usually viewed simply as progenitors of CD4+ and CD8+ alphabeta T cells. Instead we report that DP cells regulate the differentiation of early thymocyte progenitors and gammadelta cells, by a mechanism dependent on the transcription factor RORgt, and the lymphotoxin (LT) beta receptor (LTbetaR). This finding provokes a revised view of the thymus, in which lymphoid tissue induction-type processes coordinate the developmental and functional integration of the two T cell lineages.

This review builds on evidence that cell-mediated immune responses to bacteria, viruses, parasites, and tumors are an integration of conventional and unconventional T-cell activities. Whereas conventional T cells provide clonal antigen-specific responses, unconventional T cells profoundly regulate conventional T cells, often suppressing their activities such that immunopathology is limited. By extrapolation, immunopathologies and inflammatory diseases may reflect defects in regulation by unconventional T cells. To explore the function of unconventional T cells, several extensive gene expression analyses have been undertaken. These studies are reviewed in some detail, with emphasis on the mechanisms by which unconventional T cells may exert their regulatory functions. Highlighting the fundamental nature of T-cell integration, we also review emerging data that the development of conventional and unconventional T cells is also highly integrated.

Background: Pediatric radiologists serve an important role in the radiologic diagnosis, investigation, and in legal proceedings in cases of child abuse. The Society for Pediatric Radiology should evaluate and insure the adequacy of training of pediatric radiologists for this important role., Objective: The Society for Pediatric Radiology Committee on Child Abuse, 2002, conducted a 24-question survey to evaluate the scope and perceived adequacy of training received by pediatric radiology fellows regarding the radiologic diagnosis of child abuse and the associated legal process., Materials and Methods: Eighty-four surveys were mailed to radiologists who had completed a year in pediatric radiology fellowship training during the years 1999 and 2000. There were 33 surveys returned for an overall response of 39%., Results: Respondents' perception of adequacy of training was best for the radiologic diagnosis of child abuse. The majority perceived they were not well trained in the investigative and legal processes regarding child abuse. The majority would welcome standardized training., Conclusion: Current pediatric radiology training programs do not sufficiently prepare pediatric radiologists for their role in the legal system regarding child abuse. A standardized program to train pediatric radiologists about the imaging diagnosis of child abuse and their role in the legal system is recommended.

Lipopolysaccharide (LPS) is a powerful stimulator of macrophages and induces apoptosis in these cells. Using primary cultures of bone marrow-derived macrophages, we found that the autocrine production of tumor necrosis factor-alpha (TNF-alpha) has a major function in LPS-induced apoptosis. LPS activates PKC and regulates the different mitogen-activated protein kinases (MAPK). We aimed to determine its involvement either in the secretion of TNF-alpha or in the induction of apoptosis. Using specific inhibitors and mice with the gene for PKCepsilon disrupted, we found that LPS-induced TNF-alpha-dependent apoptosis is mostly mediated by PKCepsilon, which is not directly involved in the signaling mechanism of apoptosis but rather in the process of TNF-alpha secretion. In our cell model, all three MAPKs were involved in the regulation of TNF-alpha secretion, but at different levels. JNK mainly regulates TNF-alpha transcription and apoptosis, whereas ERK and p38 contribute to the regulation of TNF-alpha production, probably through posttranscriptional mechanisms. Only JNK activity is mediated by PKCepsilon in response to LPS and so plays a major role in TNF-alpha secretion and LPS-induced apoptosis. We demonstrated in macrophages that LPS involving PKCepsilon regulates JNK activity and produces TNF-alpha, which induces apoptosis.

Although T cell receptor (TCR)gammadelta+ and TCRalphabeta+ cells are commonly viewed as functionally independent, their relatedness and potential interdependence remain enigmatic. Here we have identified a gene profile that distinguishes mouse gammadelta cell populations from conventional alphabeta T cells. However, this profile was also expressed by sets of unconventional alphabeta T cells. Therefore, whereas TCR specificity determines the involvement of a T cell in an immune response, the cell's functional potential, as assessed by gene expression, does not segregate with the TCR. By monitoring the described gene profile, we show that gammadelta T cell development and function in TCRbeta-deficient mice was impaired because of the absence of alphabeta T cell progenitors. Thus, normal gammadelta cell development is dependent on the development of conventional alphabeta T cells.