LRIG1 regulates cadherin‐dependent contact inhibition directing epithelial homeostasis and pre‐invasive squamous cell carcinoma development

Epidermal growth factor receptor (EGFR) pathway activation is a frequent event in human carcinomas. Mutations in EGFR itself are, however, rare, and the mechanisms regulating EGFR activation remain elusive. Leucine‐rich immunoglobulin repeats‐1 (LRIG1), an inhibitor of EGFR activity, is one of four genes identified that predict patient survival across solid tumour types including breast, lung, melanoma, glioma, and bladder. We show that deletion of Lrig1 is sufficient to promote murine airway hyperplasia through loss of contact inhibition and that re‐expression of LRIG1 in human lung cancer cells inhibits tumourigenesis. LRIG1 regulation of contact inhibition occurs via ternary complex formation with EGFR and E‐cadherin with downstream modulation of EGFR activity. We find that LRIG1 LOH is frequent across cancers and its loss is an early event in the development of human squamous carcinomas. Our findings imply that the early stages of squamous carcinoma development are driven by a change in amplitude of EGFR signalling governed by the loss of contact inhibition. © 2012 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Deciphering new mechanisms on T‐cell costimulation by human mast cells

It is well established that full activation of T cells to recognize a specific antigen requires additional signals. These secondary signals are generated by the interaction of costimulatory molecules expressed on APCs. Classical APCs include DCs, macrophages, Langerhans cells, and B cells. However, in recent years, several haematopoietic and nonhaematopoietic cells have been described to express MHC class II antigens and, in appropriate conditions, costimulatory molecules. In this issue, Suurmond et al. [Eur. J. Immunol. 2016. 46: 1132–1141] show, for the first time, that human mast cells not only express costimulatory molecules of the TNF‐receptor and CD28 families, but can also costimulate T cells through a yet‐to‐be‐defined CD28‐independent interaction.     

Expression of non‐mast cell histidine decarboxylase in tumor‐associated microvessels in human esophageal squamous cell carcinomas

Histamine is produced by mast cells and many other types of cells. The role of histamine released from mast cells in promoting tumor angiogenesis has been intensively studied; however, the role of non‐mast cell histamine in regulating tumor angiogenesis has been largely ignored. In this study, tissue specimen sections from 43 patients with esophageal squamous cell carcinoma (ESCC) and normal esophageal biopsies from 17 heath individuals obtained from a high incidence area of north China were used to assess changes in microvessel density (MVD) and non‐mast cell L‐histidine decarboxylase (HDC) (the only rate‐limiting enzyme that catalyzes the formation of histamine from L‐histidine) expression in the tumor microenvironment by immunohistochemistry (IHC). In addition, the cellular characterization of non‐mast cell HDC‐positive cells in microvessels was examined by double IHC combined with HDC/CD34 and HDC/PCNA antibodies. These IHC analyses revealed a significantly increased HDC‐positive MVD in ESCC as compared with normal controls, which accounted for ～61% of CD34‐labeled general MVD in ESCC. Furthermore, IHC in serial sections and double IHC showed that most of these HDC‐positive cells were CD34‐positive endothelial cells in microvessels with an increased proliferative capacity. Thus, our results suggest that non‐mast cell histamine expressed in endothelial cells of microvessels could be an additional cellular source and might play a role in regulating angiogenesis in ESCC.     

Comparison of integrin,cadherin, and catenin expression in squamous cell carcinomas of the oral cavity

In addition to their role in maintenance of tissue integrity, cell adhesion molecules regulate the growth and differentiation of stratified squamous epithelia. Reduced expression of E-cadherin and the α₂β₁, α₃β₁ and α₆β₄ integrins is already reported to correlate with poor histological differentiation in oral squamous cell carcinomas. However, it is not clear how closely cadherin and integrin loss are related in any given tumour, nor whether cadherin loss is correlated with changes in expression of the cytoplasmic regulatory proteins known as catenins. Double-label immunofluorescence has been used to stain a panel of 22 oral squamous cell carcinomas with antibodies to ten proteins, including E- and P-cadherin, the major keratinocyte integrin subunits, and α-, β- and γ-catenin. Overall, E-cadherin expression and integrin expression correlated well with tumour grade, while P-cadherin staining was more variable. All tumours, regardless of differentiation status, showed reduced staining for at least two of the catenins, implying that the adhesive function of E- and P-cadherin could be impaired even when cadherin expression is normal. It is concluded that in all squamous cell carcinomas, regardless of degree of histological differentiation, there is some perturbed expression of cell adhesion molecules and that integrin and E-cadherin loss are closely related. © 1998 John Wiley & Sons, Ltd.     

The who's who of T‐cell differentiation: Human memory T‐cell subsets

Following antigen encounter and subsequent resolution of the immune response, a single naïve T cell is able to generate multiple subsets of memory T cells with different phenotypic and functional properties and gene expression profiles. Single‐cell technologies, first and foremost flow cytometry, have revealed the complex heterogeneity of the memory T‐cell compartment and its organization into subsets. However, a consensus has still to be reached, both at the semantic (nomenclature) and phenotypic level, regarding the identification of these subsets. Here, we review recent developments in the characterization of the heterogeneity of the memory T‐cell compartment, and propose a unified classification of both human and nonhuman primate T cells on the basis of phenotypic traits and in vivo properties. Given that vaccine studies and adoptive cell transfer immunotherapy protocols are influenced by these recent findings, it is important to use uniform methods for identifying and discussing functionally distinct subsets of T cells.     

miR-204-5p inhibits cell proliferation and induces cell apoptosis in esophageal squamous cell carcinoma by regulating Nestin

Esophageal cancer (EC) is a highly malignant gastrointestinal tumor, and esophageal squamous cell carcinoma (ESCC) is one of the most common histological types of EC. MicroRNAs (miRNAs) are small noncoding RNAs closely related to tumorigenesis and tumor progression. In addition, Nestin is an intermediate filament protein (class VI) and contributes to the progression of numerous tumors. However, the correlation between miRNAs and Nestin in ESCC remains unclear. This study aimed to investigate the relationship between miR-204-5p and Nestin in ESCC. First, Nestin-related miRNAs in ESCC were explored using RNA sequencing. In ESCC tissues and cell lines, the expression of miR-204-5p was decreased detected by quantitative real-time polymerase chain reaction (qPCR), whereas Nestin protein level was upregulated identified by Western blotting (WB). Besides, Nestin was the direct target of miR-204-5p in ESCC determined via the luciferase reported assay. Moreover, miR-204-5p regulated Nestin to inhibit ESCC cell proliferation detected by the colony formation assay and promote ESCC cell apoptosis identified using the flow cytometry and TUNEL assay. Furthermore, miR-204-5p suppressed tumorigenesis in vivo evaluated by the murine xenograft tumor model. In conclusion, these results indicated that miR-204-5p inhibited cell proliferation and induced cell apoptosis in ESCC through targeting Nestin, which might provide novel therapeutic targets for ESCC therapy.     

Differentiating keratoacanthoma from squamous cell carcinoma by the use of apoptotic and cell adhesion markers

AIMS: Keratoacanthomas (KA) are well-differentiated squamoproliferative skin lesions that grow rapidly and regress spontaneously. In contrast, squamous cell carcinomas (SCC) can have variable differentiation, inexorably progress and on occasion metastasize. Distinguishing between KA and SCC using haematoxylin and eosin-stained sections from an initial biopsy can often be difficult. There is also some debate as to whether KA is simply a variety of well-differentiated SCC or a distinct entity. METHODS AND RESULTS: Initial biopsy sections from 25 cases of SCC and 20 of KA were labelled with markers for both the initiation (the cytolytic receptor P2X7) and end-stage (terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling) of apoptosis, telomerase-associated protein (TP1) and the cell adhesion protein E-cadherin. As this was a retrospective study, the clinical outcome of each case was known. This resulted in a unique labelling pattern of each marker for SCC and KA, allowing a differential diagnosis between the two conditions. The simplest marker to use for this purpose was anti-P2X7. Sections from five cases that were initially very difficult to diagnose were correctly identified as SCC using this method. CONCLUSIONS: These results support the view that KA has a different pathogenesis and biochemistry from that of SCC, and is a distinct entity. Anti-P2X7 labelling, using routine immunohistochemical techniques, provides a method for differentially diagnosing these conditions.     

T cell receptor β‐chain repertoire analysis reveals intratumour heterogeneity of tumour‐infiltrating lymphocytes in oesophageal squamous cell carcinoma

Oesophageal squamous cell carcinoma (ESCC) has a generally poor prognosis, due to the lack of effective treatment methods. Immunotherapeutic approaches based on tumour‐infiltrating lymphocytes (TILs) have demonstrated that durable responses are produced in some patients with solid tumours, which suggests the potential feasibility of clinical application of immunotherapy for ESCC. However, many of the basic characteristics of TILs in ESCC are poorly understood, including clonality, specificity and spatial heterogeneity of the response of TILs, which depends on the interaction between antigens and T cell receptors (TCRs). We used ultra‐deep sequencing of rearranged genes in TCR β‐chain (TCRβ) to profile the basic characteristics of T cells in tumour tissues (four to six regions from each tumour) as well as matched adjacent normal tissue and peripheral blood from seven patients diagnosed with primary ESCC. We found that T cell clones within ESCCs were quite different from those of the peripheral blood and even the adjacent normal tissues in general. Although there was a relatively higher degree of overlap of intratumoural TCRβ repertoires than those between the tumour and other tissues, intratumoural TCRβ repertoires were spatially heterogeneous. Due to the restricted sampling, high‐throughput TCRβ sequencing could characterize the diversity and composition of a limited (compartment‐dependent) fraction of the respective T cell clones in any individual ESCC, expanding our understanding of immune behaviour and immune response and shedding more light on ESCC immunotherapy. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Therapeutic protein deimmunization by T‐cell epitope removal: antigen‐specific immune responses in vitro and in vivo

Hirudin III is an effective anti‐coagulant; however, in 40% of treated patients, a high‐titer of anti‐Hirudin III IgG antibodies is observed. Development of antibody responses requires the activation of helper T lymphocyte (HTL), which is dependent on peptide epitopes binding to HLA class II molecules. Based on computational prediction softwares, four new mutants of Hirudin III, T4K, S9G, V21G, and V21K, had been designed with the aim of reducing the binding affinity of these HTL epitopes. The constructed mutants have been purified and assayed for bioactivity. Finally in vitro and in vivo cell‐mediated responses were assessed and humoral immune assays were performed. All modified forms of Hirudin III were active, and showed significantly reduced human T‐cell responses. All mutants indicated lower human IFN‐γ level compared to native Hirudin, and V21K indicated lowest IFN‐γ level. Mice immunized with T4K and V21K showed a significant reduction in total antibody responses and mouse IFN‐γ levels. Mice immunized with V21K after 3rd immunization had lower T‐cell proliferation compared to native Hirudin and other mutants. Based on these results, V21K is proposed as the best alternate Hirudin III candidate with lowest antigenicity. These findings validate our rational design strategy aimed at providing new active analogs of therapeutic proteins with reduced immunogenicity.     

Interaction of KLRG1 with E‐cadherin: New functional and structural insights

The killer cell lectin‐like receptor G1 (KLRG1) is an inhibitory receptor expressed by memory T cells and NK cells in man and mice. It is frequently used as a cell differentiation marker and members of the cadherin family are ligands for KLRG1. The present study provides new insights into the interaction of mouse KLRG1 with E‐cadherin. Firstly, we demonstrate that co‐engagement of KLRG1 and CD3/TCR in a spatially linked manner was required for inhibition arguing against the notion that KLRG1‐ligation per se transmits inhibitory signals. Secondly, experiments with T cells carrying Y₇F‐mutant KLRG1 molecules with a replacement of the tyrosine residue to phenylalanine in the single ITIM indicated that the inhibitory activity of KLRG1 is counteracted to some degree by increased interaction of KLRG1⁺ T cells with E‐cadherin expressing target cells. Thirdly, we demonstrate that deletion of the first or the second external domain of E‐cadherin abolished reactivity in KLRG1‐reporter cell assays. Finally, we made the intriguing observation that KLRG1 formed multimeric protein complexes in T cells in addition to the previously described mono‐ and dimeric molecules.     

KiSS‐1 expression in oral squamous cell carcinoma and its prognostic significance

Downregulated expression of KiSS‐1 has been correlated with tumor progression, metastasis, and patient prognosis in various human malignancies. However, there is no information regarding the expression of KiSS‐1 in oral squamous cell carcinoma (OSCC). Our aims were to examine KiSS‐1 expression in OSCC tissue samples and cell lines and to determine its prognostic significance. KiSS‐1 expression was significantly lower in lymph node (LN) metastases than in primary tumor tissues. Five of six OSCC cell lines showed absence or relatively low expression of KiSS‐1. Correlations between KiSS‐1 expression and clinicopathological parameters were statistically assessed. There were significant correlations between KiSS‐1 expression and LN metastasis (p = 0.007), TNM stage (p = 0.024), and local recurrence (p = 0.012). In the Kaplan–Meier survival analysis, negative KiSS‐1 expression significantly correlated with poorer overall survival (OS) and disease‐free survival (DFS) (p = 0.000 and 0.000, respectively). Multivariate analysis using Cox regression modeling revealed that KiSS‐1 expression was an independent prognostic factor for both OS and DFS (p = 0.001 and 0.000, respectively). Our findings suggested that KiSS‐1 downregulation may play a role in tumor progression and metastasis of OSCC and may be a reliable biomarker for predicting clinical outcome in OSCC.     

Metastatic metaplastic breast carcinoma mimicking pulmonary squamous cell carcinoma on fine‐needle aspiration

Metaplastic squamous cell carcinoma (SCC) of the breast is a rare type of breast cancer. Metastases to the lung, which can be a major site of second primary tumor development among breast cancer patients, are difficult to distinguish from primary SCC of the lung and present a unique challenge for pathologists. There are few available discriminating immunohistochemical markers as squamous differentiation typically leads to loss of expression of characteristic primary epithelial cell markers of both breast and lung origin. GATA protein binding 3 (GATA‐3) is a useful marker of breast origin in metastatic ductal and lobular carcinomas including poorly differentiated triple‐negative carcinomas and some metaplastic carcinomas. Here, we present a case of metastatic SCC presenting as a solitary lung mass with regional lymph node metastases and a single satellite lesion in a patient with a history of metaplastic SCC of the breast. In addition to the routine markers of squamous differentiation, the metastases were also positive for estrogen receptor (ER) and GATA‐3 on cytologic material obtained by transbronchial FNA. This suggests that immunoreactivity for ER and GATA‐3 may support a diagnosis of metastatic SCC in the context of a prior metaplastic SCC of the breast. Diagn. Cytopathol. 2015;43:844–849. © 2015 Wiley Periodicals, Inc.     

Fine‐needle aspiration cytology of basaloid squamous cell carcinoma and small cell carcinoma—A comparison study

The cytopathologic diagnosis of basaloid squamous cell carcinoma can be problematic as there are several components of the differential diagnosis that share common cytomorphologic features. In this study, we report the fine‐needle aspiration (FNA) findings of 16 basaloid squamous cell carcinoma cases and compare those cases to 16 cases of small cell carcinoma. To our knowledge, this is the largest series of basaloid squamous cell carcinoma FNA cases ever reported. The following cytomorphologic features were compared for both tumors: cohesive tissue fragments, single cells, adenoid cystic‐like features (cribriform pseudoglandular lumina with hyaline materials), necrosis, nuclear size, nuclear molding, nucleoli, cytoplasm, and the presence of single keratinized cells. Adenoid cystic‐like features and the presence of single keratinized cells were specific for basaloid squamous cell carcinoma (P < 0.05). Diagn. Cytopathol. 2013. © 2011 Wiley Periodicals, Inc.     

Tumor‐induced myeloid‐derived suppressor cell subsets exert either inhibitory or stimulatory effects on distinct CD8+ T‐cell activation events

Tumor growth coincides with an accumulation of myeloid‐derived suppressor cells (MDSCs), which exert immune suppression and which consist of two main subpopulations, known as monocytic (MO) CD11b⁺CD115⁺Ly6G^?Ly6C^high MDSCs and granulocytic CD11b⁺CD115^?Ly6G⁺Ly6C^int polymorphonuclear (PMN)‐MDSCs. However, whether these distinct MDSC subsets hamper all aspects of early CD8⁺ T‐cell activation — including cytokine production, surface marker expression, survival, and cytotoxicity — is currently unclear. Here, employing an in vitro coculture system, we demonstrate that splenic MDSC subsets suppress antigen‐driven CD8⁺ T‐cell proliferation, but differ in their dependency on IFN‐γ, STAT‐1, IRF‐1, and NO to do so. Moreover, MO‐MDSC and PMN‐MDSCs diminish IL‐2 levels, but only MO‐MDSCs affect IL‐2Rα (CD25) expression and STAT‐5 signaling. Unexpectedly, however, both MDSC populations stimulate IFN‐γ production by CD8⁺ T cells on a per cell basis, illustrating that some T‐cell activation characteristics are actually stimulated by MDSCs. Conversely, MO‐MDSCs counteract the activation‐induced change in CD44, CD62L, CD162, and granzyme B expression, while promoting CD69 and Fas upregulation. Together, these effects result in an altered CD8⁺ T‐cell adhesiveness to the extracellular matrix and selectins, sensitivity to FasL‐mediated apoptosis, and cytotoxicity. Hence, MDSCs intricately influence different CD8⁺ T‐cell activation events in vitro, whereby some parameters are suppressed while others are stimulated.