Flow cytometric analysis of innate lymphoid cells: challenges and solutions

IntroductionThe three groups of helper innate lymphoid cells (ILCs), namely ILC1, ILC2 and ILC3, have been identified by flow cytometry by combinations of cell surface markers. Here, we review various ways ILCs are currently identified, focusing on potential problems and their solutions. The first step to identify all ILCs is to exclude other lymphocytes and myeloid cells by their lineage-specific markers (Lin). However, the Lin cocktail varies in various studies, and the definition of Lin- population containing ILCs is often ambiguous, resulting in contamination of Lin+ cells, particularly T cells.MethodWe have designed combinations of cell surface markers to identify ILC populations in various tissues of B6 mice by flow cytometry. To minimize T cell contamination, TCR/CD3ϵ antibodies were used separately from the Lin cocktail. ILCs identified by surface markers are confirmed by the expression of the transcription factors GATA3, RORγt, T-bet and Eomes.ResultILC1s in the B6 mouse liver are identified by Lin-NKp46+NK1.1+TCR/CD3ϵ−CD49a+CD49b−. However, defining ILC1s in other tissues remains a challenge. ILC2s in the lung are identified by Lin−TCR/CD3ϵ− Thy1+CD127+ST2+ whereas ILC2s in the small intestine and liver are identified by Lin−TCR/CD3ϵ−Thy1+GATA3+RORγt−. ILC3s in B6 mouse spleen, liver, lung and small intestine are identified by Lin−TCR/CD3ϵ− Thy1+CD127+RORγt+.DiscussionThe ILC population is heterogeneous and the strategies to identify ILCs have to be designed for each ILC population and tissue. Excluding T cells in all cases is crucial, and a combination of transcription factors GATA3, RORγt, T-bet, and Eomes should be used to identify ILCs. Using CD3ϵ/TCRs in a different fluorochrome not in Lin cocktail minimizes contamination of T cells specifically identify individual ILC populations in various tissues

A role for DNA hypomethylation and histone acetylation in maintaining allele-specific expression of mouse NKG2A in developing and mature NK cells.

The repertoire of receptors that is expressed by NK cells is critical for their ability to kill virally infected or transformed cells. However, the molecular mechanisms that determine whether and when NK receptor genes are transcribed during hemopoiesis remain unclear. In this study, we show that hypomethylation of a CpG-rich region in the mouse NKG2A gene is associated with transcription of NKG2A in ex vivo NK cells and NK cell lines. This observation was extended to various developmental stages of NK cells sorted from bone marrow, in which we demonstrate that the CpGs are methylated in the NKG2A-negative stages (hemopoietic stem cells, NK progenitors, and NKG2A-negative NK cells), and hypomethylated specifically in the NKG2A-positive NK cells. Furthermore, we provide evidence that DNA methylation is important in maintaining the allele-specific expression of NKG2A. Finally, we show that acetylated histones are associated with the CpG-rich region in NKG2A positive, but not negative, cell lines, and that treatment with the histone deacetylase inhibitor trichostatin A alone is sufficient to induce NKG2A expression. Treatment with the methyltransferase inhibitor 5-azacytidine only is insufficient to induce transcription, but cotreatment with both drugs resulted in a significantly greater induction, suggesting a cooperative role for DNA methylation and histone acetylation status in regulating gene expression. These results enhance our understanding of the formation and maintenance of NK receptor repertoires in developing and mature NK cells

Nucleophilic substitution reactions of 1-methoxy-6-nitroindole-3-carbaldehyde 1

金沢大学大学院自然科学研究科生理活性物質科学金沢大学薬学部1-Methoxy-6-nitroindole-3-carbaldehyde is proved to be a versatile substrate for the nucleophilic substitution reactions providing 2,3,6-trisubstituted indole derivatives. Preparation of a novel pyrimido[1,2-a]indole derivative is also reported

Nucleophilic Substitution Reaction in Indole Chemistry: 1-Methoxy-6-nitroindole-3-carbaldehyde as a Versatile Building Block for 2,3,6-Trisubstituted Indoles

金沢大学医薬保健研究域薬学系金沢大学名誉教

Nucleophilic substitution reaction in indole chemistry: 1-methoxy-6-nitroindole-3-carbaldehyde as a versatile building block for 2,3,6-trisubstituted indoles

1-Methoxy-6-nitroindole-3-carbaldehyde is proved to be a versatile electrophile and reacts regioselectively at the 2-position with various types of nucleophiles providing 2,3,6-trisubstituted indole derivatives. The reaction is applicable for the preparation of a novel pyrimido[1,2-a]indole derivative. © 2009 The Japan Institute of Heterocyclic Chemistry All rights reserved

Evidence for high bi-allelic expression of activating Ly49 receptors

Stochastic expression is a hallmark of the Ly49 family that encode the main MHC class-I-recognizing receptors of mouse natural killer (NK) cells. This highly polygenic and polymorphic family includes both activating and inhibitory receptor genes and is one of genome's fastest evolving loci. The inhibitory Ly49 genes are expressed in a stochastic mono-allelic manner, possibly under the control of an upstream bi-directional early promoter and show mono-allelic DNA methylation patterns. To date, no studies have directly addressed the transcriptional regulation of the activating Ly49 receptors. Our study shows differences in DNA methylation pattern between activating and inhibitory genes in C57BL/6 and F1 hybrid mouse strains. We also show a bias towards bi-allelic expression of the activating receptors based on allele-specific single-cell RT–PCR in F1 hybrid NK cells for Ly49d and Ly49H expression in Ly49h+/− mice. Furthermore, we have identified a region of high sequence identity with possible transcriptional regulatory capacity for the activating Ly49 genes. Our results also point to a likely difference between NK and T-cells in their ability to transcribe the activating Ly49 genes. These studies highlight the complex regulation of this rapidly evolving gene family of central importance in mouse NK cell function

Leucine-rich alpha-2 glycoprotein as a marker of mucosal healing in inflammatory bowel disease

Leucine-rich alpha-2 glycoprotein (LRG) may be a novel serum biomarker for patients with inflammatory bowel disease. The association of LRG with the endoscopic activity and predictability of mucosal healing (MH) was determined and compared with those of C-reactive protein (CRP) and fecal markers (fecal immunochemical test [FIT] and fecal calprotectin [Fcal]) in 166 ulcerative colitis (UC) and 56 Crohn's disease (CD) patients. In UC, LRG was correlated with the endoscopic activity and could predict MH, but the performance was not superior to that of fecal markers (areas under the curve [AUCs] for predicting MH: LRG: 0.61, CRP: 0.59, FIT: 0.75, and Fcal: 0.72). In CD, the performance of LRG was equivalent to that of CRP and Fcal (AUCs for predicting MH: LRG: 0.82, CRP: 0.82, FIT: 0.70, and Fcal: 0.88). LRG was able to discriminate patients with MH from those with endoscopic activity among UC and CD patients with normal CRP levels. LRG was associated with endoscopic activity and could predict MH in both UC and CD patients. It may be particularly useful in CD

Collapsin response mediator protein 1 mediates Reelin signaling in cortical neuronal migration

Collapsin response mediator protein 1 ( CRMP1) is one of the CRMP family members that mediates signal transduction of axon guidance molecules. Here, we show evidence that CRMP1 is involved in Reelin ( Reln) signaling to regulate neuronal migration in the cerebral cortex. In crmp1(-/-) mice, radial migration of cortical neurons was retarded. This phenotype was not observed in the sema3A(-/-) and crmp1(+/+); sema3A(+/+) cortices. However, CRMP1 was colocalized with disabled- 1 ( Dab1), an adaptor protein in Reln signaling. In the Reln(rl/rl) cortex, CRMP1 and Dab1 were expressed at a higher level, yet tyrosine phosphorylated at a lower level. Loss of crmp1 in a dab1 heterozygous background led to the disruption of hippocampal lamination, a Reeler- like phenotype. In addition to axon guidance, CRMP1 regulates neuronal migration by mediating Reln signaling

Alterations of the genes involved in the PI3K and estrogen-receptor pathways influence outcome in human epidermal growth factor receptor 2-positive and hormone receptor-positive breast cancer patients treated with trastuzumab-containing neoadjuvant chemotherapy

Background Chemotherapy with trastuzumab is widely used for patients with human epidermal growth factor receptor 2-positive (HER2+) breast cancer, but a significant number of patients with the tumor fail to respond, or relapse. The mechanisms of recurrence and biomarkers that indicate the response to the chemotherapy and outcome are not fully investigated. Methods Genomic alterations were analyzed using single-nucleotide polymorphism arrays in 46 HER2 immunohistochemistry (IHC) 3+ or 2+/fluorescent in situ hybridization (FISH)+ breast cancers that were treated with neoadjuvant chemotherapy with paclitaxel, cyclophosphamid, epirubicin, fluorouracil, and trastuzumab. Patients were classified into two groups based on presence or absence of alterations of 65 cancer-associated genes, and the two groups were further classified into four groups based on genomic HER2 copy numbers or hormone receptor status (HR+/−). Pathological complete response (pCR) and relapse-free survival (RFS) rates were compared between any two of the groups. Results and discussion The pCR rate was 54% in 37 patients, and the RFS rate at 3 years was 72% (95% CI, 0.55-0.89) in 42 patients. The analysis disclosed 8 tumors with nonamplified HER2 and 38 tumors with HER2 amplification, indicating the presence of discordance in tumors diagnosed using current HER2 testing. The 8 patients showed more difficulty in achieving pCR (P=0.019), more frequent relapse (P=0.018), and more frequent alterations of genes in the PI3K pathway (P=0.009) than the patients with HER2 amplification. The alterations of the PI3K and estrogen receptor (ER) pathway genes generally indicated worse RFS rates. The prognostic significance of the alterations was shown in patients with a HR+ tumor, but not in patients with a HR- tumor when divided. Alterations of the PI3K and ER pathway genes found in patients with a HR+ tumor with poor outcome suggested that crosstalk between the two pathways may be involved in resistance to the current chemotherapy with trastuzumab. Conclusions We recommend FISH analysis as a primary HER2 testing because patients with IHC 2+/3+ and nonamplified HER2 had poor outcome. We also support concurrent use of trastuzumab, lapatinib, and cytotoxic and anti-hormonal agents for patients having HR+ tumors with alterations of the PI3K and ER pathway genes