High-Throughput Screening of Functional Neo-Antigens and Their Specific T-Cell Receptors via the Jurkat Reporter System Combined with Droplet Microfluidics.

T-cell receptor (TCR)-engineered T cells can precisely recognize a broad repertoire of targets derived from both intracellular and surface proteins of tumor cells. TCR-T adoptive cell therapy has shown safety and promising efficacy in solid tumor immunotherapy. However, antigen-specific functional TCR screening is time-consuming and expensive, which limits its application clinically. Here, we developed a novel integrated antigen-TCR screening platform based on droplet microfluidic technology, enabling high-throughput peptide-major histocompatibility complex (pMHC)-to-TCR paired screening with a high sensitivity and low background signal. We introduced DNA barcoding technology to label peptide antigen candidate-loaded antigen-presenting cells and Jurkat reporter cells to check the specificity of pMHC-TCR candidates. Coupled with the next-generation sequencing pipeline, interpretation of the DNA barcodes and the gene expression level of the Jurkat T-cell activation pathway provided a clear peptide-MHC-TCR recognition relationship. Our proof-of-principle study demonstrates that the platform could achieve pMHC-TCR paired high-throughput screening, which is expected to be used in the cross-reactivity and off-target high-throughput paired testing of candidate pMHC-TCRs in clinical applications.

A Novel Proteogenomic Integration Strategy Expands the Breadth of Neo-Epitope Sources.

Tumor-specific antigens can activate T cell-based antitumor immune responses and are ideal targets for cancer immunotherapy. However, their identification is still challenging. Although mass spectrometry can directly identify human leukocyte antigen (HLA) binding peptides in tumor cells, it focuses on tumor-specific antigens derived from annotated protein-coding regions constituting only 1.5% of the genome. We developed a novel proteogenomic integration strategy to expand the breadth of tumor-specific epitopes derived from all genomic regions. Using the colorectal cancer cell line HCT116 as a model, we accurately identified 10,737 HLA-presented peptides, 1293 of which were non-canonical peptides that traditional database searches could not identify. Moreover, we found eight tumor neo-epitopes derived from somatic mutations, four of which were not previously reported. Our findings suggest that this new proteogenomic approach holds great promise for increasing the number of tumor-specific antigen candidates, potentially enlarging the tumor target pool and improving cancer immunotherapy.

Prpf31 is essential for the survival and differentiation of retinal progenitor cells by modulating alternative splicing.

Dysfunction of splicing factors often result in abnormal cell differentiation and apoptosis, especially in neural tissues. Mutations in pre-mRNAs processing factor 31 (PRPF31) cause autosomal dominant retinitis pigmentosa, a progressive retinal degeneration disease. The transcriptome-wide splicing events specifically regulated by PRPF31 and their biological roles in the development and maintenance of retina are still unclear. Here, we showed that the differentiation and viability of retinal progenitor cells (RPCs) are severely perturbed in prpf31 knockout zebrafish when compared with other tissues at an early embryonic stage. At the cellular level, significant mitotic arrest and DNA damage were observed. These defects could be rescued by the wild-type human PRPF31 rather than the disease-associated mutants. Further bioinformatic analysis and experimental verification uncovered that Prpf31 deletion predominantly causes the skipping of exons with a weak 5' splicing site. Moreover, genes necessary for DNA repair and mitotic progression are most enriched among the differentially spliced events, which may explain the cellular and tissular defects in prpf31 mutant retinas. This is the first time that Prpf31 is demonstrated to be essential for the survival and differentiation of RPCs during retinal neurogenesis by specifically modulating the alternative splicing of genes involved in DNA repair and mitosis.

BCAS2 is essential for hematopoietic stem and progenitor cell maintenance during zebrafish embryogenesis.

Hematopoietic stem and progenitor cells (HSPCs) originate from the hemogenic endothelium via the endothelial-to-hematopoietic transition, are self-renewing, and replenish all lineages of blood cells throughout life. BCAS2 (breast carcinoma amplified sequence 2) is a component of the spliceosome and is involved in multiple biological processes. However, its role in hematopoiesis remains unknown. We established a bcas2 knockout zebrafish model by using transcription activator-like effector nucleases. The bcas2 -/- zebrafish showed severe impairment of HSPCs and their derivatives during definitive hematopoiesis. We also observed significant signs of HSPC apoptosis in the caudal hematopoietic tissue of bcas2 -/- zebrafish, which may be rescued by suppression of p53. Furthermore, we show that the bcas2 deletion induces an abnormal alternative splicing of Mdm4 that predisposes cells to undergo p53-mediated apoptosis, which provides a mechanistic explanation of the deficiency observed in HSPCs. Our findings revealed a novel and vital role for BCAS2 during HSPC maintenance in zebrafish.

Identification and structural characterization of two peroxisome proliferator activated receptors and their transcriptional changes at different developmental stages and after feeding with different fatty acids.

Peroxisome proliferator activated receptors beta1 (PPARß1) and beta2 (PPARß2) were investigated in loach (Misgurnus anguillicaudatus). The PPARß1 and PPARß2 were widely distributed in loach tissues. Multiple alignments of deduced amino acid sequences revealed homologous characteristics of the two subtypes of PPARß with 88% identity. PPARß1 was markedly expressed in the liver, about 100-fold higher than liver PPARß2. The two subtypes in unfertilized ovum (UO) showed the highest transcriptions in early life stages, and there were great divergences in expression between unfertilized and fertilized stages. The regulation of PPARß1 and PPARß2 in response to dietary fatty acids was studied in liver of loach fed with diets containing fish oil (FO, rich in n-3 highly unsaturated fatty acid) or soybean oil (SO, rich in 18:2n-6) for 75days. Results showed that hepatic transcription of PPARß1 in the SO group was higher than in the FO group. However, PPARß2 expression was similar. The differences of molecular characterization, tissue expressions in early life stages, and transcriptional regulation by lipid resources indicated that PPARß1 and PPARß2 were functionally different. This is the first report of differential expression of PPARß1 and PPARß2 in various tissues and early life stages of loach are regulated by lipid resources. These results will stimulate further studies to better understand the functional characterization of PPARß1 and PPARß2.