Epithelial damage and tissue γδ T cells promote a unique tumor-protective IgE response.

IgE is an ancient and conserved immunoglobulin isotype with potent immunological function. Nevertheless, the regulation of IgE responses remains an enigma, and evidence of a role for IgE in host defense is limited. Here we report that topical exposure to a common environmental DNA-damaging xenobiotic initiated stress surveillance by γδTCR+ intraepithelial lymphocytes that resulted in class switching to IgE in B cells and the accumulation of autoreactive IgE. High-throughput antibody sequencing revealed that γδ T cells shaped the IgE repertoire by supporting specific variable-diversity-joining (VDJ) rearrangements with unique characteristics of the complementarity-determining region CDRH3. This endogenous IgE response, via the IgE receptor FcεRI, provided protection against epithelial carcinogenesis, and expression of the gene encoding FcεRI in human squamous-cell carcinoma correlated with good disease prognosis. These data indicate a joint role for immunosurveillance by T cells and by B cells in epithelial tissues and suggest that IgE is part of the host defense against epithelial damage and tumor development.

Antibodies to Costimulatory Receptor 4-1BB Enhance Anti-tumor Immunity via T Regulatory Cell Depletion and Promotion of CD8 T Cell Effector Function.

The costimulatory receptor 4-1BB is expressed on activated immune cells, including activated T cells. Antibodies targeting 4-1BB enhance the proliferation and survival of antigen-stimulated T cells in vitro and promote CD8 T cell-dependent anti-tumor immunity in pre-clinical cancer models. We found that T regulatory (Treg) cells infiltrating human or murine tumors expressed high amounts of 4-1BB. Intra-tumoral Treg cells were preferentially depleted by anti-4-1BB mAbs in vivo. Anti-4-1BB mAbs also promoted effector T cell agonism to promote tumor rejection. These distinct mechanisms were competitive and dependent on antibody isotype and FcγR availability. Administration of anti-4-1BB IgG2a, which preferentially depletes Treg cells, followed by either agonistic anti-4-1BB IgG1 or anti-PD-1 mAb augmented anti-tumor responses in multiple solid tumor models. An antibody engineered to optimize both FcγR-dependent Treg cell depleting capacity and FcγR-independent agonism delivered enhanced anti-tumor therapy. These insights into the effector mechanisms of anti-4-1BB mAbs lay the groundwork for translation into the clinic.

Programmed cell death-1 receptor-mediated regulation of Tbet+NK1.1- innate lymphoid cells within the tumor microenvironment.

Innate lymphoid cells (ILCs) play a key role in tissue-mediated immunity and can be controlled by coreceptor signaling. Here, we define a subset of ILCs that are Tbet+NK1.1- and are present within the tumor microenvironment (TME). We show programmed death-1 receptor (PD-1) expression on ILCs within TME is found in Tbet+NK1.1- ILCs. PD-1 significantly controlled the proliferation and function of Tbet+NK1.1- ILCs in multiple murine and human tumors. We found tumor-derived lactate enhanced PD-1 expression on Tbet+NK1.1- ILCs within the TME, which resulted in dampened the mammalian target of rapamycin (mTOR) signaling along with increased fatty acid uptake. In line with these metabolic changes, PD-1-deficient Tbet+NK1.1- ILCs expressed significantly increased IFNγ and granzyme B and K. Furthermore, PD-1-deficient Tbet+NK1.1- ILCs contributed toward diminished tumor growth in an experimental murine model of melanoma. These data demonstrate that PD-1 can regulate antitumor responses of Tbet+NK1.1- ILCs within the TME.

Transcriptomic analysis of cutaneous squamous cell carcinoma reveals a multigene prognostic signature associated with metastasis.

BACKGROUND: Metastasis of cutaneous squamous cell carcinoma (cSCC) is uncommon. Current staging methods are reported to have sub-optimal performances in metastasis prediction. Accurate identification of patients with tumors at high risk of metastasis would have a significant impact on management. OBJECTIVE: To develop a robust and validated gene expression profile signature for predicting primary cSCC metastatic risk using an unbiased whole transcriptome discovery-driven approach. METHODS: Archival formalin-fixed paraffin-embedded primary cSCC with perilesional normal tissue from 237 immunocompetent patients (151 nonmetastasizing and 86 metastasizing) were collected retrospectively from four centers. TempO-seq was used to probe the whole transcriptome and machine learning algorithms were applied to derive predictive signatures, with a 3:1 split for training and testing datasets. RESULTS: A 20-gene prognostic model was developed and validated, with an accuracy of 86.0%, sensitivity of 85.7%, specificity of 86.1%, and positive predictive value of 78.3% in the testing set, providing more stable, accurate prediction than pathological staging systems. A linear predictor was also developed, significantly correlating with metastatic risk. LIMITATIONS: This was a retrospective 4-center study and larger prospective multicenter studies are now required. CONCLUSION: The 20-gene signature prediction is accurate, with the potential to be incorporated into clinical workflows for cSCC.

Coexistence of psoriasis and cutaneous T-cell lymphoma.

It has been reported that individuals with psoriasis are at an increased risk of developing cutaneous T-cell lymphoma (CTCL). However, the increased risk of lymphoma in these patients has been questioned because CTCL in its early stages may be incorrectly labelled as psoriasis, thus introducing potential for misclassification bias. We retrospectively reviewed patients with a confirmed diagnosis of CTCL seen in a tertiary cutaneous lymphoma clinic (n = 115) over a 5-year period and found that 6 (5.2%) patients had clinical evidence of coexisting psoriasis. This demonstrates that there is a small cohort of individuals who develop both psoriasis and CTCL.

Proteomic Profiling of Archived Tissue of Primary Melanoma Identifies Proteins Associated with Metastasis.

Formalin-fixed paraffin embedded (FFPE) clinical tissues represent an abundant and unique resource for translational proteomic studies. In the US, melanoma is the 5th and 6th most common cancer in men and women, respectively, affecting over 230,000 people annually and metastasising in 5-15% of cases. Median survival time for distant metastatic melanoma is 6-9 months with a 5-year-survival of < 15%. In this study, 24 primary FFPE tumours which have metastasised (P-M) and 24 primary FFPE tumours which did not metastasise (P-NM) were subjected to proteomic profiling. In total, 2750 proteins were identified, of which 16 were significantly differentially expressed. Analysis of TCGA data demonstrated that expression of the genes encoding for 6 of these 16 proteins had a significant effect on survival in cutaneous melanoma. Pathway analysis of the proteomics data revealed mechanisms likely involved in the process of melanoma metastasis, including cytoskeleton rearrangement, extracellular changes and immune system alterations. A machine learning prediction model scoring an AUC of 0.922, based on these 16 differentially expressed proteins was able to accurately classify samples into P-M and P-NM. This study has identified potential biomarkers and key processes relating to melanoma metastasis using archived clinical samples, providing a basis for future studies in larger cohorts.

Characteristics of immunosuppressive regulatory T cells in cutaneous squamous cell carcinomas and role in metastasis.

BACKGROUND: Non-melanoma skin cancer is the most common cancer worldwide, and cutaneous squamous cell carcinomas (SCCs) account for substantial morbidity and mortality because of their potential for metastasis. SCCs are surrounded by an immune cell infiltrate containing regulatory T cells (Tregs). The aim of this study was to characterise Tregs in SCCs and investigate whether increased Treg numbers in primary skin SCCs are associated with subsequent metastasis. METHODS: Lymphocytes were extracted from freshly excised skin SCC tumours and corresponding peripheral blood and normal skin. Flow cytometry was used for T-cell analysis and cell sorting. Tritiated thymidine based lymphocyte proliferation assays and interferon γ (IFNγ) ELISPOT assays were used to assess peritumoral lymphocyte function in vitro. Immunohistochemistry was performed on primary cutaneous SCC sections from tumours that subsequently metastasised and from those that did not after 5-year follow-up. FINDINGS: Increased frequencies of CD3+CD4+CD25hiCD127loFOXP3+ Tregs were found in SCCs (21·5% of CD4+ immune infiltrate, n=60 tumours) compared with corresponding peripheral blood (5·4%) and normal skin (7·6%). SCC Tregs expressed significantly higher levels of the co-stimulatory molecules OX40 (37·2% of FOXP3+ cell population, n=10 tumours) and 4-1BB (12·6%, n=9) than peritumoral non-regulatory T cells and Tregs from peripheral blood and normal skin (p=0·0005). The inhibitory receptor CTLA4 and the transcription factor Helios were expressed at high levels in peritumoral Tregs. SCC Tregs significantly suppressed phytohaemagglutinin-stimulated peritumoral CD4+ T-cell proliferation (p=0·005, n=10), peritumoral CD8+ T-cell proliferation (p=0·015, n=9), and IFNγ secretion by CD4+ effector T cells (p=0·026, n=10). Increased in-vitro proliferation of phytohaemagglutinin-stimulated peritumoral CD4+ T cells was shown after the addition of anti-OX40 antibodies (p=0·0078, n=9 tumours) and anti-4-1BB antibodies (p=0·0039, n=9). Immunohistochemistry showed fewer CD8+ T cells in SCCs that metastasised (n=29) than in non-metastatic SCCs (n=26) (28·5% of immune infiltrate vs 44·6%%, p<0·0001) and more FOX3+ Tregs (28·5% of immune infiltrate vs 49·3%, p<0·0001). INTERPRETATION: Our study shows that immunosuppressive Tregs are present in the immune infiltrate of cutaneous SCCs, and contribute to ineffective anti-tumour immune responses, thereby permitting SCC development and promoting metastasis. FUNDING: Wellcome Trust, National Institute for Health Research.

Variants of the melanocortin-1 receptor: do they matter clinically?

The melanocortin 1 receptor (MC1R) gene encodes for a seven-pass transmembrane receptor primarily expressed on melanocytes and melanoma cells. Single nucleotide polymorphisms (SNPs, also termed variants) in MC1R frequently cause red hair, fair skin and are associated with melanoma and keratinocyte-derived skin cancer development. Activation of wild-type (WT) MC1R in skin assists cutaneous photoprotection whereas reduced MC1R signalling, seen with MC1R variants, impairs ultraviolet radiation (UVR)-protective responses. As ancestral humans migrated out of Africa, the evolutionary advantage of MC1R variants may have related to improved cutaneous vitamin D synthesis and higher birthweight reported with certain MC1R variants. Reduced photoprotection secondary to MC1R dysfunction involves pigmentary and non-pigmentary mechanisms (reduced DNA repair, effects on cell proliferation and possibly immunological parameters), leading to clonal expansion of mutated cells within skin and subsequent carcinogenesis. Recent investigations suggest an association between MC1R genotype and vitiligo, with preliminary evidence that a MC1R agonist, [Nle4-D-Phe7]-alpha-MSH, in combination with UVB, assists repigmentation. Future development of compounds to correct defective MC1R responses secondary to MC1R variants could result in photoprotective benefits for fair-skinned individuals and reduce their skin cancer risk.

Impaired expression of metallothioneins contributes to allergen-induced inflammation in patients with atopic dermatitis.

Regulation of cutaneous immunity is severely compromised in inflammatory skin disease. To investigate the molecular crosstalk underpinning tolerance versus inflammation in atopic dermatitis, we utilise a human in vivo allergen challenge study, exposing atopic dermatitis patients to house dust mite. Here we analyse transcriptional programmes at the population and single cell levels in parallel with immunophenotyping of cutaneous immunocytes revealed a distinct dichotomy in atopic dermatitis patient responsiveness to house dust mite challenge. Our study shows that reactivity to house dust mite was associated with high basal levels of TNF-expressing cutaneous Th17 T cells, and documents the presence of hub structures where Langerhans cells and T cells co-localised. Mechanistically, we identify expression of metallothioneins and transcriptional programmes encoding antioxidant defences across all skin cell types, that appear to protect against allergen-induced inflammation. Furthermore, single nucleotide polymorphisms in the MTIX gene are associated with patients who did not react to house dust mite, opening up possibilities for therapeutic interventions modulating metallothionein expression in atopic dermatitis.

CD8+CD103+ tissue-resident memory T cells convey reduced protective immunity in cutaneous squamous cell carcinoma.

BACKGROUND: Tumor infiltrating lymphocytes play a key role in antitumor responses; however, while several memory T-cell subtypes have been reported in inflammatory and neoplastic conditions, the proportional representation of the different subsets of memory T cells and their functional significance in cancer is unclear. Keratinocyte skin cancer is one of the most common cancers globally, with cutaneous squamous cell cancer (cSCC) among the most frequent malignancies capable of metastasis. METHODS: Memory T-cell subsets were delineated in human cSCCs and, for comparison, in non-lesional skin and blood using flow cytometry. Immunohistochemistry was conducted to quantify CD103+ cells in primary human cSCCs which had metastasized (P-M) and primary cSCCs which had not metastasized (P-NM). TIMER2.0 (timer.cistrome.org) was used to analyze TCGA cancer survival data based on ITGAE expression. Immunofluorescence microscopy was performed to determine frequencies of CD8+CD103+ cells in P-M and P-NM cSCCs. RESULTS: Despite intertumoral heterogeneity, most cSCC T cells were CCR7-/CD45RA- effector/resident memory (TRM) lymphocytes, with naive, CD45RA+/CCR7- effector memory re-expressing CD45RA, CCR7+/L-selectin+ central memory and CCR7+/L-selectin- migratory memory lymphocytes accounting for smaller T-cell subsets. The cSCC CD8+ T-cell population contained a higher proportion of CD69+/CD103+ TRMs than that in non-lesional skin and blood. These cSCC CD69+/CD103+ TRMs exhibited increased IL-10 production, and higher CD39, CTLA-4 and PD-1 expression compared with CD103- TRMs in the tumor. CD103+ cells were more frequent in P-M than P-NM cSCCs. Analysis of TCGA data demonstrated that high expression of ITGAE (encoding CD103) was associated with reduced survival in primary cutaneous melanoma, breast carcinoma, renal cell carcinoma, kidney chromophobe cancer, adrenocortical carcinoma and lower grade glioma. Immunofluorescence microscopy showed that the majority of CD103 was present on CD8+ T cells and that CD8+CD103+ cells were significantly more frequent in P-M than P-NM cSCCs. CONCLUSION: These results highlight CD8+CD103+ TRMs as an important functional T-cell subset associated with poorer clinical outcome in this cancer.

ENTPD1 (CD39) Expression Inhibits UVR-Induced DNA Damage Repair through Purinergic Signaling and Is Associated with Metastasis in Human Cutaneous Squamous Cell Carcinoma.

UVR and immunosuppression are major risk factors for cutaneous squamous cell carcinoma (cSCC). Regulatory T cells promote cSCC carcinogenesis, and in other solid tumors, infiltrating regulatory T cells and CD8+ T cells express ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1) (also known as CD39), an ectoenzyme that catalyzes the rate-limiting step in converting extracellular adenosine triphosphate (ATP) to extracellular adenosine (ADO). We previously showed that extracellular purine nucleotides influence DNA damage repair. In this study, we investigate whether DNA damage repair is modulated through purinergic signaling in cSCC. We found increased ENTPD1 expression on T cells within cSCCs when compared with the expression on T cells from blood or nonlesional skin, and accordingly, concentrations of derivative extracellular adenosine diphosphate (ADP), adenosine monophosphate (AMP), and ADO are increased in tumors compared with those in normal skin. Importantly, ENTPD1 expression is significantly higher in human cSCCs that metastasize than in those that are nonmetastatic. We also identify in a mouse model that ENTPD1 expression is induced by UVR in an IL-27-dependent manner. Finally, increased extracellular ADO is shown to downregulate the expression of NAP1L2, a nucleosome assembly protein we show to be important for DNA damage repair secondary to UVR. Together, these data suggest a role for ENTPD1 expression on skin-resident T cells to regulate DNA damage repair through purinergic signaling to promote skin carcinogenesis and metastasis.

Subclonal Evolution of Cancer-Related Gene Mutations in p53 Immunopositive Patches in Human Skin.

Normal sun-exposed skin contains numerous epidermal patches that stain positive for p53 protein (p53 immunopositive patches, PIPs), which are considered potential early precursors of skin cancer. Although the TP53 gene is mutated in many PIPs, it is unclear whether PIPs contain any other cancer-related mutations. Here we report that PIPs, predominantly <3,000 p53 immunopositive cells in size, within normal chronically exposed skin contain mutations in multiple genes that are mutated in cutaneous squamous cell cancers. These mutations in the PIPs were not detected within the non-PIP epidermis of corresponding normal chronically exposed skin. Although some of these genetic alterations are clonal in the PIPs, many of the mutations are subclonal within these lesions. Similar mutations are seen in later precancers (actinic keratoses and Bowen's disease). Our results demonstrate that PIPs in chronically exposed skin contain multiple mutations in cancer-related genes. In addition, the results indicate that the clonal evolution of mutations that are seen within later precancerous lesions and in established malignancy can also occur in PIPs within normal human skin.

Nanoscale dysregulation of collagen structure-function disrupts mechano-homeostasis and mediates pulmonary fibrosis.

Matrix stiffening with downstream activation of mechanosensitive pathways is strongly implicated in progressive fibrosis; however, pathologic changes in extracellular matrix (ECM) that initiate mechano-homeostasis dysregulation are not defined in human disease. By integrated multiscale biomechanical and biological analyses of idiopathic pulmonary fibrosis lung tissue, we identify that increased tissue stiffness is a function of dysregulated post-translational collagen cross-linking rather than any collagen concentration increase whilst at the nanometre-scale collagen fibrils are structurally and functionally abnormal with increased stiffness, reduced swelling ratio, and reduced diameter. In ex vivo and animal models of lung fibrosis, dual inhibition of lysyl oxidase-like (LOXL) 2 and LOXL3 was sufficient to normalise collagen fibrillogenesis, reduce tissue stiffness, and improve lung function in vivo. Thus, in human fibrosis, altered collagen architecture is a key determinant of abnormal ECM structure-function, and inhibition of pyridinoline cross-linking can maintain mechano-homeostasis to limit the self-sustaining effects of ECM on progressive fibrosis.

OX40+ Regulatory T Cells in Cutaneous Squamous Cell Carcinoma Suppress Effector T-Cell Responses and Associate with Metastatic Potential.

PURPOSE: Cutaneous squamous cell carcinoma (cSCC) is the most common human cancer with metastatic potential. Despite T cells accumulating around cSCCs, these tumors continue to grow and persist. To investigate reasons for failure of T cells to mount a protective response in cSCC, we focused on regulatory T cells (Tregs) as this suppressive population is well represented among the infiltrating lymphocytes. EXPERIMENTAL DESIGN: Flow cytometry was conducted on cSCC lymphocytes and in vitro functional assays were performed using sorted tumoral T cells. Lymphocyte subsets in primary cSCCs were quantified immunohistochemically. RESULTS: FOXP3(+) Tregs were more frequent in cSCCs than in peripheral blood (P < 0.0001, n = 86 tumors). Tumoral Tregs suppressed proliferation of tumoral effector CD4(+) (P = 0.005, n = 10 tumors) and CD8(+) T cells (P = 0.043, n = 9 tumors) and inhibited IFNγ secretion by tumoral effector T cells (P = 0.0186, n = 11 tumors). The costimulatory molecule OX40 was expressed predominantly on tumoral Tregs (P < 0.0001, n = 15 tumors) and triggering OX40 with an agonist anti-OX40 antibody overcame the suppression exerted by Tregs, leading to increased tumoral effector CD4(+) lymphocyte proliferation (P = 0.0098, n = 10 tumors). Tregs and OX40(+) lymphocytes were more abundant in primary cSCCs that metastasized than in primary cSCCs that had not metastasized (n = 48 and n = 49 tumors, respectively). CONCLUSIONS: Tregs in cSCCs suppress effector T-cell responses and are associated with subsequent metastasis, suggesting a key role for Tregs in cSCC development and progression. OX40 agonism reversed the suppressive effects of Tregs in vitro, suggesting that targeting OX40 could benefit the subset of cSCC patients at high risk of metastasis. Clin Cancer Res; 22(16); 4236-48. ©2016 AACR.