Protocol for Flow Cytometric Detection of Immune Cell Infiltration in the Epidermis and Dermis of a Psoriasis Mouse Model.

Psoriasis is an incurable chronic inflammatory skin disorder. The imiquimod (IMQ)-induced mouse model of psoriasis is the most widely used model for drug discovery and pre-clinical studies of psoriasis. The inflamed and thickened skin frequently compromises the quality of single-cell suspensions generated from IMQ-induced skin lesions, which has an impact on subsequent analyses by flow cytometry. This protocol details the complete procedure for the establishment of a mouse model of psoriasis and flow cytometric detection of immune cells in the inflamed epidermis and dermis. For complete details on the use and execution of this protocol, please refer to Lou et al. (2020).

T-Cell Lymphoblastic Lymphoma/Leukemia Presenting as a Diffuse Viral Exanthem-like Reaction: A Clinical and Histopathological Challenge.

Cutaneous involvement by leukemia, or leukemia cutis, is a rare manifestation of leukemic disorders, most frequently occurring in children. The skin findings, which usually include multiple violaceous or erythematous nodules on the face, most often follow the classic presenting signs and symptoms of leukemia and occur in patients with an established primary diagnosis. Patients with T-cell acute lymphoblastic leukemia and associated leukemia cutis typically present with a solitary firm red to bluish nodule, often with an accompanying mediastinal mass, that can produce respiratory symptoms. In this article, we report a case of a patient with primary T-cell acute lymphoblastic leukemia/lymphoma presenting with a diffuse exanthem mimicking a viral illness with an associated SET-NUP214 translocation.

Blocking LFA-1 Aggravates Cardiac Inflammation in Experimental Autoimmune Myocarditis.

The lymphocyte function-associated antigen 1 (LFA-1) is a member of the beta2-integrin family and plays a pivotal role for T cell activation and leukocyte trafficking under inflammatory conditions. Blocking LFA-1 has reduced or aggravated inflammation depending on the inflammation model. To investigate the effect of LFA-1 in myocarditis, mice with experimental autoimmune myocarditis (EAM) were treated with a function blocking anti-LFA-1 antibody from day 1 of disease until day 21, the peak of inflammation. Cardiac inflammation was evaluated by measuring infiltration of leukocytes into the inflamed cardiac tissue using histology and flow cytometry and was assessed by analysis of the heart weight/body weight ratio. LFA-1 antibody treatment severely enhanced leukocyte infiltration, in particular infiltration of CD11b+ monocytes, F4/80+ macrophages, CD4+ T cells, Ly6G+ neutrophils, and CD133+ progenitor cells at peak of inflammation which was accompanied by an increased heart weight/body weight ratio. Thus, blocking LFA-1 starting at the time of immunization severely aggravated acute cardiac inflammation in the EAM model.

The distribution of T-cell subsets and the expression of immune checkpoint receptors and ligands in patients with newly diagnosed and relapsed acute myeloid leukemia.

BACKGROUND: Phenotypic characterization of immune cells in the bone marrow (BM) of patients with acute myeloid leukemia (AML) is lacking. METHODS: T-cell infiltration was quantified on BM biopsies from 13 patients with AML, and flow cytometry was performed on BM aspirates (BMAs) from 107 patients with AML who received treatment at The University of Texas MD Anderson Cancer Center. The authors evaluated the expression of inhibitory receptors (programmed cell death protein 1 [PD1], cytotoxic T-lymphocyte antigen 4 [CTLA4], lymphocyte-activation gene 3 [LAG3], T-cell immunoglobulin and mucin-domain containing-3 [TIM3]) and stimulatory receptors (glucocorticoid-induced tumor necrosis factor receptor-related protein [GITR], OX40, 41BB [a type 2 transmembrane glycoprotein receptor], inducible T-cell costimulatory [ICOS]) on T-cell subsets and the expression of their ligands (41BBL, B7-1, B7-2, ICOSL, PD-L1, PD-L2, and OX40L) on AML blasts. Expression of these markers was correlated with patient age, karyotype, baseline next-generation sequencing for 28 myeloid-associated genes (including P53), and DNA methylation proteins (DNA methyltransferase 3α, isocitrate dehydrogenase 1[IDH1], IDH2, Tet methylcytosine dioxygenase 2 [TET2], and Fms-related tyrosine kinase 3 [FLT3]). RESULTS: On histochemistry evaluation, the T-cell population in BM appeared to be preserved in patients who had AML compared with healthy donors. The proportion of T-regulatory cells (Tregs) in BMAs was higher in patients with AML than in healthy donors. PD1-positive/OX40-positive T cells were more frequent in AML BMAs, and a higher frequency of PD1-positive/cluster of differentiation 8 (CD8)-positive T cells coexpressed TIM3 or LAG3. PD1-positive/CD8-positive T cells were more frequent in BMAs from patients who had multiply relapsed AML than in BMAs from those who had first relapsed or newly diagnosed AML. Blasts in BMAs from patients who had TP53-mutated AML were more frequently positive for PD-L1. CONCLUSIONS: The preserved T-cell population, the increased frequency of regulatory T cells, and the expression of targetable immune receptors in AML BMAs suggest a role for T-cell-harnessing therapies in AML.

Evolution of Metastases in Space and Time under Immune Selection.

We examined how the immune microenvironment molds tumor evolution at different metastatic organs in a longitudinal dataset of colorectal cancer. Through multiplexed analyses, we showed that clonal evolution patterns during metastatic progression depend on the immune contexture at the metastatic site. Genetic evidence of neoantigen depletion was observed in the sites with high Immunoscore and spatial proximity between Ki67+ tumor cells and CD3+ cells. The immunoedited tumor clones were eliminated and did not recur, while progressing clones were immune privileged, despite the presence of tumor-infiltrating lymphocytes. Characterization of immune-privileged metastases revealed tumor-intrinsic and tumor-extrinsic mechanisms of escape. The lowest recurrence risk was associated with high Immunoscore, occurrence of immunoediting, and low tumor burden. We propose a parallel selection model of metastatic progression, where branched evolution could be traced back to immune-escaping clones. The findings could inform the understanding of cancer dissemination and the development of immunotherapeutics.

T-Lymphoblastic Leukemia/Lymphoma With Annular Skin Rash and Epidermotropism.

Leukemia cutis is uncommon in patients with acute lymphoblastic leukemia. It typically presents with dermal papules or subcutaneous nodules, with no epidermal or upper papillary dermal involvement on histopathology. We present an unusual clinical presentation of leukemia cutis, with annular plaques and epidermotropism.

Progressive hypoventilation due to mixed CD8+ and CD4+ lymphocytic polymyositis following tremelimumab - durvalumab treatment.

BACKGROUND: The combination of CTLA-4 and PD-L1 inhibitors has a manageable adverse effect profile, although rare immune-related adverse events (irAE) can occur. CASE PRESENTATION: We describe an autoimmune polymyositis following a partial response to combination tremelimumab and durvalumab for the treatment of recurrent lung adenocarcinoma. Radiography revealed significant reduction in all metastases; however, the patient developed progressive neuromuscular hypoventilation due to lymphocytic destruction of the diaphragmatic musculature. Serologic testing revealed a low level of de novo circulating antibodies against striated muscle fiber. Immunohistochemistry revealed type II muscle fiber atrophy with a mixed CD8+ and CD4+ lymphocyte infiltrate, indicative of inflammatory myopathy. CONCLUSIONS: This case supports the hypothesis that muscle tissue is a target for lymphocytic infiltration in immune checkpoint inhibitor-associated polymyositis. Further insights into the autoimmune mechanism of PM will hopefully contribute to the prevention and treatment of this phenomenon.

Dietary Apigenin Exerts Immune-Regulatory Activity in Vivo by Reducing NF-κB Activity, Halting Leukocyte Infiltration and Restoring Normal Metabolic Function.

The increasing prevalence of inflammatory diseases and the adverse effects associated with the long-term use of current anti-inflammatory therapies prompt the identification of alternative approaches to reestablish immune balance. Apigenin, an abundant dietary flavonoid, is emerging as a potential regulator of inflammation. Here, we show that apigenin has immune-regulatory activity in vivo. Apigenin conferred survival to mice treated with a lethal dose of Lipopolysaccharide (LPS) restoring normal cardiac function and heart mitochondrial Complex I activity. Despite the adverse effects associated with high levels of splenocyte apoptosis in septic models, apigenin had no effect on reducing cell death. However, we found that apigenin decreased LPS-induced apoptosis in lungs, infiltration of inflammatory cells and chemotactic factors' accumulation, re-establishing normal lung architecture. Using NF-κB luciferase transgenic mice, we found that apigenin effectively modulated NF-κB activity in the lungs, suggesting the ability of dietary compounds to exert immune-regulatory activity in an organ-specific manner. Collectively, these findings provide novel insights into the underlying immune-regulatory mechanisms of dietary nutraceuticals in vivo.

Regulatory T-cell infiltration in tongue squamous cell carcinoma.

CONCLUSION: In tongue squamous cell carcinoma (SCC), high levels of regulatory T-cell (Treg) infiltration in tumor nests are observed in the cases with poor prognosis. OBJECTIVES: The role of Tregs in head and neck cancers remains unclear. The aim of this study was to observe the distribution of Tregs in different stages of tongue SCC and estimate the effects on prognosis. METHODS: Thirty-four cases with tongue SCC were examined immunohistochemically for CD4, CD8, and Forkhead box P3 (Foxp3). Immunoreactive cells were counted in cancer stroma and nest regions, and relationships between cell numbers and disease-free survival rates were analyzed. RESULTS: In the 34 cases, univariate analysis for disease-free survival indicated high-level infiltration of Tregs (CD4(+)Foxp3+) into both cancer nests and stroma and presence of helper T (CD4(+)Foxp3-) cells in cancer stroma as potential predictors of significantly worse prognosis. In early-stage cases (stage I/II), high-level infiltration of Tregs in cancer nests correlated significantly with poor disease-free survival rate. Multivariate analysis for disease-free survival found no independent variables.

Development and function of human innate immune cells in a humanized mouse model.

Mice repopulated with human hematopoietic cells are a powerful tool for the study of human hematopoiesis and immune function in vivo. However, existing humanized mouse models cannot support development of human innate immune cells, including myeloid cells and natural killer (NK) cells. Here we describe two mouse strains called MITRG and MISTRG, in which human versions of four genes encoding cytokines important for innate immune cell development are knocked into their respective mouse loci. The human cytokines support the development and function of monocytes, macrophages and NK cells derived from human fetal liver or adult CD34(+) progenitor cells injected into the mice. Human macrophages infiltrated a human tumor xenograft in MITRG and MISTRG mice in a manner resembling that observed in tumors obtained from human patients. This humanized mouse model may be used to model the human immune system in scenarios of health and pathology, and may enable evaluation of therapeutic candidates in an in vivo setting relevant to human physiology.

Effects of ICOSLG expressed in mouse hematological neoplasm cell lines in the GVL reaction.

As a member of the B7 family, inducible co-stimulator ligand (ICOSLG) expressed on tumor cell has been reported to have an important role in tumor immunity. In this study, we sought to determine whether the expression of ICOSLG in mouse hematological malignancy cells influences GVL reaction after mouse allogeneic BMT. In our study, we analyzed the expression of ICOSLG in six mice hematological malignancy cell lines for the first time, and found that FBL3, A20 and P388 cells expressed high levels of ICOSLG. Then, we chose A20 cells as targets to construct a GVL model and study the effects on the GVL reaction by silencing the ICOSLG gene. The survival was analyzed. We found that in GVL model, mortality of interference groups was significantly delayed compared with the control group (P=0.0005). Our results indicate that knockdown of ICOSLG of mouse leukemic cells may significantly enhance GVL effect after allogeneic BMT.

Bcl-2 inhibits the innate immune response during early pathogenesis of murine congenital muscular dystrophy.

Laminin α2 (LAMA2)-deficient congenital muscular dystrophy is a severe, early-onset disease caused by abnormal levels of laminin 211 in the basal lamina leading to muscle weakness, transient inflammation, muscle degeneration and impaired mobility. In a Lama2-deficient mouse model for this disease, animal survival is improved by muscle-specific expression of the apoptosis inhibitor Bcl-2, conferred by a MyoD-hBcl-2 transgene. Here we investigated early disease stages in this model to determine initial pathological events and effects of Bcl-2 on their progression. Using quantitative immunohistological and mRNA analyses we show that inflammation occurs very early in Lama2-deficient muscle, some aspects of which are reduced or delayed by the MyoD-hBcl-2 transgene. mRNAs for innate immune response regulators, including multiple Toll-like receptors (TLRs) and the inflammasome component NLRP3, are elevated in diseased muscle compared with age-matched controls expressing Lama2. MyoD-hBcl-2 inhibits induction of TLR4, TLR6, TLR7, TLR8 and TLR9 in Lama2-deficient muscle compared with non-transgenic controls, and leads to reduced infiltration of eosinophils, which are key death effector cells. This congenital disease model provides a new paradigm for investigating cell death mechanisms during early stages of pathogenesis, demonstrating that interactions exist between Bcl-2, a multifunctional regulator of cell survival, and the innate immune response.

Prognostic and predictive impact of intra- and peritumoral immune infiltrates.

Leukocyte infiltrates into or around tumor cell nests are found in the context of protumorigenic inflammation and anticancer immunosurveillance. Hence, the detailed composition, density, architecture, and function of leukocyte infiltrates must be analyzed to understand their prognostic impact. The ectopic presence within tumors of high endothelial venule cells, which are normally characteristic for secondary lymphoid organs, correlates with a more pronounced infiltration by T lymphocytes and has a positive predictive impact on local advanced breast cancer treated with neoadjuvant chemotherapy. Recent progress in the field indicates that immune infiltrates of the primary tumors, as well as of metastases, are not only independent prognostic biomarkers but can also constitute predictive factors, suggesting that the pretherapeutic immune response can determine the efficacy of conventional chemotherapies. Moreover, accumulating evidence indicates that chemotherapy can stimulate anticancer immune responses coupled with an increased intratumoral lymphoid infiltration, which correlates with tumor mass reduction and patient survival. Improved methods for the automation of immunohistochemistry and digitalized image analyses will pave the way to an improved understanding of the complex interplay between cancer parenchyma, stroma, and immune effectors, as well as to the routine evaluation of immune-related parameters to the clinical management of cancer patients.

Human Th17 cells can be induced through head and neck cancer and have a functional impact on HNSCC development.

BACKGROUND: The T helper 17 (Th17) cells recently identified as distinct T helper cell lineage are characterised by their production of the proinflammatory cytokine interleukin 17. Although much effort has been made in understanding the function of Th17 cells in the pathogenesis of different diseases, their influence in carcinogenesis remain largely unknown. METHODS: We studied the prevalence and induction of Th17 cells in head and neck squamous cell carcinoma (HNSCC) patients by flow cytometry. To determine the migration mechanism of Th17 cells into primary tumours and metastasis of HNSCC, we performed chemotaxis assays. We analysed the proliferation and the angiogenesis-related proteins of HNSCCs in the presence of Th17 cells with MTT-based proliferation assay and an angiogenesis protein array. RESULTS: In this study, we showed that the prevalence of Th17 cells is elevated in peripheral blood of HNSCC patients. In addition, tumour tissue and tumour-draining lymph nodes are infiltrated by a huge number of Th17 cells representing an important fraction of the tumour-infiltrating lymphocytes (TILs). We further showed that Th17 cells can be induced and expanded in tumour microenvironment through cytokines produced by tumour cells and TILs, and in addition can be recruited to the tumour milieu through a CCR6/CCL20-dependent mechanism. Furthermore, we showed that the proliferation and angiogenesis of HNSCC are impaired in the presence of Th17 cells. CONCLUSION: We conclude that Th17 cells have a substantial impact on the carcinogenesis of HNSCCs and on their metastasis and could serve as a potential therapeutic target to modulate anti-tumour response in HNSCC.

Dense lymphocytic infiltrates associated with non-melanoma skin cancer in patients with chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a common hematologic malignancy associated with an increased risk of non-melanoma skin cancer. Basal cell carcinomas and squamous cell carcinomas in these patients may have an associated dense peritumoral leukemic infiltrate. This infiltrate can lead to the diagnosis of CLL and may also obscure tumor margins and pose a challenge in the assessment of perineural tumor spread. Immunohistochemical stains are useful in distinguishing leukemic B-cell infiltrates from tumor-reactive T-cell infiltrates. Leukemic cells of CLL are CD20+/CD23+/CD5+/CD43+/CD3-, whereas benign reactive infiltrates are composed of CD20-/CD23-/CD5+/CD43+/CD3+ T-cells. Given the paucity of symptoms in early stages of CLL, a dense lymphoid infiltrate surrounding a cutaneous neoplasm may serve as the first indication of CLL. We report a series of three cases of SCC with a coexisting infiltrate of CLL, including one with perineural involvement, one involving metastatic SCC, and one in which this histologic finding spurred the initial diagnosis of CLL.

Exacerbation of corneal scarring in HSV-1 gK-immunized mice correlates with elevation of CD8+CD25+ T cells in corneas of ocularly infected mice.

We have shown previously that exacerbation of corneal scarring (CS) in HSV-1 glycoprotein K (gK) immunized mice was associated with CD8+ T cells. In this study, we investigated the type and the nature of the immune responses that are involved in the exacerbation of CS in gK-immunized animals. BALB/c mice were vaccinated with baculovirus expressed gK, gD, or mock-immunized. Twenty-one days after the third immunization, mice were ocularly infected with 2 x 10(5) PFU/eye of virulent HSV-1 strain McKrae. Infiltration of the cornea by CD4+, CD8+, CD25+, CD4+CD25+, CD8+CD25+, CD19+, CD40+, CD40L+, CD62L+, CD95+, B7-1+, B7-2+, MHC-I+, and MHC-II+ cells was monitored by immunohistochemistry, qRT-PCR and FACS at various times post-infection (PI). This study demonstrated for the first time that the presence of CD8+CD25+ T cells in the cornea is correlated with exacerbation of CS in the gK-immunized group.