Full-Length Immune Repertoire Reconstruction and Profiling at the Transcriptome Level Using Long-Read Sequencing.

BACKGROUND: Due to the diversity of the immune repertoire (IR), reconstructing full-length IR using traditional short-read sequencing has proven challenging. METHODS: A full-length IR sequencing (FLIRseq) work flow was developed with linear rolling circle amplification and nanopore sequencing. Its accuracy and quantification ability were verified by plasmid mixtures and commercial B-cell receptor/T-cell receptor sequencing (BCR/TCR-seq) based on short reads. IRs in tissues and the peripheral blood from 8 patients with acute lymphoblastic leukemia, 3 patients with allergic diseases, 4 patients with psoriasis, and 5 patients with prostate cancer were analyzed using FLIRseq. RESULTS: FLIRseq reads had lower mismatch rates and gap rates, and higher identify rates than nanopore reads (all P < 2.2 × -16). The relative quantification of components by FLIRseq was consistent with the actual quantification (P > 0.05). FLIRseq had superiority over BCR/TCR-seq, providing the long complementarity-determining region 3, B-cell isotype, and the rarely used V gene sequence. FLIRseq observed an increase in clonotype diversity (P < 0.05) and a decrease in the percentage of abnormal BCRs/TCRs in patients with leukemia in remission. For patients with allergic diseases or psoriasis, FLIRseq provided direct insights into V(D)J recombination and specific immunoglobulin classes. Compared with that in prostate cancer tissues, the full-length V segment of the biased T-cell receptor ß chain from lymphocytes in psoriatic tissues showed a more consistent AlphaFold2-predicted protein structure (P < 0.05). CONCLUSIONS: FLIRseq enables unbiased and comprehensive analyses of direct V(D)J recombination and immunoglobulin classes, thereby contributing to characterizing pathogenic mechanisms, monitoring minimal residual disease, and customizing adoptive cell therapy.

Enhanced quality of hESC-derived melanocytes through modified concentration of endothelin-1.

The study investigated the effectiveness of EDN1 and EDN3 cytokines in the differentiation of melanocytes from hESCs. The findings showed that 100 nM EDN1 was more effective in promoting hESC to CD117+/TYR+ melanoblasts compared to 100 nM EDN3. Additionally, maintaining melanoblasts is beneficial for preserving the ability to proliferate. The study found that 10 nM EDN1 helped maintain the proliferation of melanoblasts without over maturing them into melanocytes in the late stage of differentiation. Thus, using 100 nM EDN1 in the initial stage and 10 nM EDN1 in the late stage proved to be an efficient and cost-effective method for obtaining hESC-derived melanocytes. The preliminary results suggest that EDN1 promotes melanoblast formation during the initial differentiation stage through its binding to both the EDNRB receptor and EDNRA receptor. This study provides a valuable tool for studying the development of human melanocytes and modelling the biology of disease.

Human embryonic stem cell-derived melanocytes exhibit limited immunogenicity.

Although surgical interventions have become optional for refractory vitiligo, grafting related injuries is inevitable. Embryonic stem cell (ESC) derivatives can be used in transplantation to address this issue, but the immune rejection due to allogeneic transplantation is of great concern. To investigate the immunogenicity of ESC derived melanocytes (ES-MC), we established a co-culture system of ES-MC and allogeneic PBMC. The results showed that ES-MC were similar to human primary melanocytes, with low expression of immune related molecules, and limited capability of stimulating allogeneic lymphocytes in vitro. Taken together, our findings confirm that ES-MC are of limited immunogenicity, providing new insights into the application of ES-MC in the regenerative medicine such as treating vitiligo.

Role of hippo pathway and cuproptosis-related genes in immune infiltration and prognosis of skin cutaneous melanoma.

Melanoma is the most lethal type of skin cancer with an increasing incidence. Cuproptosis is the most recently identified copper-dependent form of cell death that relies on mitochondrial respiration. The hippocampal (Hippo) pathway functions as a tumor suppressor by regulating Yes-associated protein/transcriptional coactivator with PDZ-binding motif (YAP/TAZ) activity. However, its role in cuproptosis remains unknown. In addition, the correlation of cuproptosis-related genes and Hippo pathway-related genes with tumor prognosis warrants further investigation. In the present study, we explored the correlation of cuproptosis-related genes and Hippo pathway-related genes with the prognosis of melanoma through analysis of data from a public database and experimental verification. We found eight Hippo pathway-related genes that were downregulated in melanoma and exhibited predictive value for prognosis. There was a significant positive correlation between cuproptosis-related genes and Hippo pathway-related genes in skin cutaneous melanoma. YAP1 expression was positively correlated with ferredoxin 1 (FDX1) expression in the GSE68599 dataset and A2058 cells. Moreover, YAP1 was positively and negatively correlated with M2 macrophages and regulatory T cell infiltration, respectively. In conclusion, the present study demonstrated the prognostic value of Hippo pathway-related genes (particularly YAP1) in melanoma, revealing the correlation between the expression of Hippo pathway-related genes and immune infiltration. Thus, the present findings may provide new clues on the prognostic assessment of patients with melanoma and a new target for the immunotherapy of this disease.

SPMLD: A skin pathological image dataset for non-melanoma with detailed lesion area annotation.

Skin tumors are the most common tumors in humans and the clinical characteristics of three common non-melanoma tumors (IDN, SK, BCC) are similar, resulting in a high misdiagnosis rate. The accurate differential diagnosis of these tumors needs to be judged based on pathological images. However, a shortage of experienced dermatological pathologists leads to bias in the diagnostic accuracy of these skin tumors in China. In this paper, we establish a skin pathological image dataset, SPMLD, for three non-melanoma to achieve automatic and accurate intelligent identification for them. Meanwhile, we propose a lesion-area-based enhanced classification network with the KLS module and an attention module. Specifically, we first collect thousands of H&E-stained tissue sections from patients with clinically and pathologically confirmed IDN, SK, and BCC from a single-center hospital. Then, we scan them to construct a pathological image dataset of these three skin tumors. Furthermore, we mark the complete lesion area of the entire pathology image to better learn the pathologist's diagnosis process. In addition, we applied the proposed network for lesion classification prediction on the SPMLD dataset. Finally, we conduct a series of experiments to demonstrate that this annotation and our network can effectively improve the classification results of various networks. The source dataset and code are available at https://github.com/efss24/SPMLD.git.

Low Immunogenicity of Keratinocytes Derived from Human Embryonic Stem Cells.

Epidermal transplantation is a common and widely used surgical technique in clinical medicine. Derivatives of embryonic stem cells have the potential to serve as a source of transplantable cells. However, allograft rejection is one of the main challenges. To investigate the immunogenicity of keratinocytes derived from human embryonic stem cells (ESKCs), we conducted a series of in vivo and in vitro experiments. The results showed that ESKCs have low HLA molecule expression, limited antigen presentation capabilities, and a weak ability to stimulate the proliferation and secretion of inflammatory factors in allogeneic PBMCs in vitro. In humanized immune mouse models, ESKCs elicited weak transplant rejection responses in the host. Overall, we found that ESKCs have low immunogenicity and may have potential applications in the field of regenerative medicine.

The Treatment of Refractory Vitiligo With Autologous Cultured Epithelium Grafting: A Real-World Retrospective Cohort Study.

BACKGROUND: Surgical intervention is the main therapy for refractory vitiligo. We developed a modified autologous cultured epithelial grafting (ACEG) technique for vitiligo treatment. Between January 2015 and June 2019, a total of 726 patients with vitiligo underwent ACEG in China, with patient characteristics and clinical factors being meticulously documented. Using a generalized linear mixed model, we were able to assess the association between these characteristics and the repigmentation rate. RESULTS: ACEG demonstrated a total efficacy rate of 82.81% (1754/2118) in treating 726 patients, with a higher repigmentation rate of 64.87% compared to conventional surgery at 52.69%. Notably, ACEG showed a better response in treating segmental vitiligo, lesions on lower limbs, age ≤ 18, and stable period > 3 years. A keratinocyte:melanocyte ratio below 25 was found to be advantageous too. Single-cell RNA sequencing analysis revealed an increase in melanocyte count and 2 subclusters of keratinocytes after ACEG, which remained higher in repigmented sites even after 1 year. CONCLUSIONS: ACEG is a promising therapy for refractory vitiligo. Patient age, clinical type, lesion site, and stability before surgery influence repigmentation in ACEG. The mechanism of repigmentation after ACEG treatment is likely not confined to the restoration of melanocyte populations. It may also involve an increase in the number of keratinocytes that support melanocyte function within the affected area. These keratinocytes may aid the post-transplant survival and function of melanocytes by secreting cytokines and extracellular matrix components. TRIAL REGISTRATION: registered with Chictr.org.cn (ChiCTR2100051405).

Single-cell sequencing reveals increased LAMB3-positive basal keratinocytes and ZNF90-positive fibroblasts in autologous cultured epithelium.

Autologous cultured epithelium grafting (ACEG) presents a promising treatment for refractory vitiligo, yet concerns regarding infections and immunological reactions hinder its surgical use due to serum and feeder dependencies. Addressing this, we culture autologous epithelium under serum- and feeder-free (SFF) conditions, comparing its safety and efficacy with serum- and feeder-dependent (SFD) conditions in stable vitiligo patients, and we discover no significant differences in repigmentation between the SFF and SFD grafts. Single-cell RNA transcriptomics on SFF- and SFD-cultured epithelium alongside healthy skin reveal increased populations of LAMB3+ basal keratinocytes and ZNF90+ fibroblasts in the SFF sheets. Functional analyses showcase active cellular metabolism in LAMB3+ basal keratinocytes, vital in extracellular matrix homeostasis, while ZNF90+ fibroblasts demonstrate increased differentiation, essential in collagen formation for cell adhesion. Importantly, these cell populations in SFF sheets exhibit enhanced interactions with melanocytes compared to SFD sheets. Further, knockdown experiments of LAMB3 in keratinocytes and ZNF90 in fibroblasts lead to a downregulation in melanocyte ligand-receptor-related genes. Overall, SFF sheets demonstrate comparable efficacy to SFD sheets, offering superior safety. LAMB3+ basal keratinocytes and ZNF90+ fibroblasts act as potential drivers behind repigmentation in ACEG under SFF conditions. This study provides translational insights into ACEG repigmentation and potential therapeutic targets for vitiligo.

Icaritin inhibits PLK1 to activate DNA damage response in NK/T cell lymphoma and increases sensitivity to GELOX regime.

Natural killer/T cell lymphoma (NKTCL) is a highly aggressive subtype of non-Hodgkin lymphoma. Gemcitabine, oxaliplatin, and L-asparaginase (GELOX) is one of the first-line chemotherapy regimens of NKTCL. Yet, the prognosis of NKTCL is poor. Icaritin is an herb-derived monomer from icariin with antitumor effects. We found that icaritin induced proliferation inhibition and apoptosis of NKTCL both in vitro and in vivo. Moreover, icaritin inhibited the dissemination of NKTCL in vivo. RNA sequencing revealed the Polo-like kinase 1 (PLK1) gene and DNA damage response (DDR) as the targets of icaritin. Mechanistically, icaritin inhibited PLK1 to promote checkpoint kinase 2 (Chk2) homodimerization and its T387 phosphorylation, which further activated p53, leading to the activation of the DDR pathway. Moreover, inhibiting PLK1 increased Forkhead box O3a nuclear localization, the latter of which activated ataxia telangiectasia mutated (ATM), an early sensor of DNA damage. Then ATM phosphorylated Chk2 T68 and initiated Chk2 activation. Remarkably, the combined treatment of icaritin and GELOX achieved better antitumor efficacy than single treatment in vivo. In summary, our results proved the efficacy of icaritin treating NKTCL, provided insights into its antitumor molecular mechanism, and revealed the application value of icaritin in facilitating clinical NKTCL treatment.

Comprehensive Analysis of Cuproptosis-Related Genes in Immune Infiltration and Prognosis in Melanoma.

Skin cutaneous melanoma (SKCM, hereafter referred to as melanoma) is the most lethal skin cancer with increasing incidence. Regulated cell death plays an important role in tumorigenesis and serves as an important target for almost all treatment strategies. Cuproptosis is the most recently identified copper-dependent regulated cell death form that relies on mitochondria respiration. However, its role in tumorigenesis remains unknown. The correlation of cuproptosis-related genes with tumor prognosis is far to be understood, either. In the present study, we explored the correlation between cuproptosis-related genes with the prognosis of melanoma by accessing and analyzing a public database and found 11 out 12 genes were upregulated in melanoma tissues and three genes (LIPT1, PDHA1, and SLC31A1) have predictive value for the prognosis. The subgroup of melanoma patients with higher cuproptosis-related gene expression showed longer overall survival than those with lower gene expression. We chose LIPT1 for further exploration. LIPT1 expression was increased in melanoma biopsies and was an independent favorable prognostic indicator for melanoma patients. Moreover, LIPT1 expression was positively correlated with PD-L1 expression and negatively associated with Treg cell infiltration. The melanoma patients with higher LIPT1 expression showed longer overall survival than those with lower LIPT1 expression after receiving immunotherapy, indicating the prognostic predictive value of LIPT1. Finally, a pan-cancer analysis indicated that LIPT1 was differentially expressed in diverse cancers as compared to normal tissues and correlated with the expression of multiple immune checkpoints, especially PD-L1. It could serve as a favorable prognosis indicator in some cancer types. In conclusion, our study demonstrated the prognostic value of cuproptosis-related genes, especially LIPT1, in melanoma, and revealed the correlation between LIPT1 expression and immune infiltration in melanoma, thus providing new clues on the prognostic assessment of melanoma patients and providing a new target for the immunotherapy of melanoma.