Deep immunophenotyping at the single-cell level identifies a combination of anti-IL-17 and checkpoint blockade as an effective treatment in a preclinical model of data-guided personalized immunotherapy.

Background: Although immune checkpoint blockade is effective for several malignancies, a substantial number of patients remain refractory to treatment. The future of immunotherapy will be a personalized approach adapted to each patient's cancer-immune interactions in the tumor microenvironment (TME) to prevent suppression of antitumor immune responses. To demonstrate the feasibility of this kind of approach, we developed combination therapy for a preclinical model guided by deep immunophenotyping of the TME.

Vertical flow array chips reliably identify cell types from single-cell mRNA sequencing experiments.

Single-cell mRNA sequencing offers an unbiased approach to dissecting cell types as functional units in multicellular tissues. However, highly reliable cell typing based on single-cell gene expression analysis remains challenging because of the lack of methods for efficient sample preparation for high-throughput sequencing and evaluating the statistical reliability of the acquired cell types. Here, we present a highly efficient nucleic reaction chip (a vertical flow array chip (VFAC)) that uses porous materials to reduce measurement noise and improve throughput without a substantial increase in reagent.

Non-biased and efficient global amplification of a single-cell cDNA library.

Analysis of single-cell gene expression promises a more precise understanding of molecular mechanisms of a living system. Most techniques only allow studies of the expressions for limited numbers of gene species. When amplification of cDNA was carried out for analysing more genes, amplification biases were frequently reported.

Quantitative analysis of gene expression in a single cell by qPCR.

We developed a quantitative PCR method featuring a reusable single-cell cDNA library immobilized on beads for measuring the expression of multiple genes in a single cell. We used this method to analyze multiple cDNA targets (from several copies to several hundred thousand copies) with an experimental error of 15.9% or less.

Fukuyama-type congenital muscular dystrophy (FCMD) and alpha-dystroglycanopathy.

Fukuyama-type congenital muscular dystrophy (FCMD), Walker-Warburg syndrome (WWS), and muscle-eye-brain (MEB) disease are clinically similar autosomal recessive disorders characterized by congenital muscular dystrophy, lissencephaly, and eye anomalies. Through positional cloning, we identified the gene for FCMD and MEB, which encodes the fukutin protein and the protein O-linked mannose beta1, 2-N-acetylglucosaminy ltransferase (POMGnT1), respectively. Recent studies have revealed that posttranslational modification of alpha-dystroglycan is associated with these congenital muscular dystrophies with brain malformations.

Loss-of-function of an N-acetylglucosaminyltransferase, POMGnT1, in muscle-eye-brain disease.

Muscle-eye-brain disease (MEB), an autosomal recessive disorder, is characterized by congenital muscular dystrophy, brain malformation, and ocular abnormalities. Previously, we found that MEB is caused by mutations in the gene encoding the protein O-linked mannose beta1,2-N-acetylglucosaminyltransferase 1 (POMGnT1), which is responsible for the formation of the GlcNAcbeta1-2Man linkage of O-mannosyl glycan. Although 13 mutations have been identified in patients with MEB, only the protein with the most frequently observed splicing site mutation has been studied.

Worldwide distribution and broader clinical spectrum of muscle-eye-brain disease.

Muscle-eye-brain disease (MEB), an autosomal recessive disorder prevalent in Finland, is characterized by congenital muscular dystrophy, brain malformation and ocular abnormalities. Since the MEB phenotype overlaps substantially with those of Fukuyama-type congenital muscular dystrophy (FCMD) and Walker-Warburg syndrome (WWS), these three diseases are thought to result from a similar pathomechanism. Recently, we showed that MEB is caused by mutations in the protein O-linked mannose beta1,2-N-acetylglucosaminyltransferase 1 (POMGnT1) gene.