Immune cell multiomics analysis reveals contribution of oxidative phosphorylation to B-cell functions and organ damage of lupus.

OBJECTIVE: Systemic lupus erythematosus (SLE) is the prototypical systemic autoimmune disease. While the long-term prognosis has greatly improved, better long-term survival is still necessary. The type I interferon (IFN) signature, a prominent feature of SLE, is not an ideal therapeutic target or outcome predictor. To explore immunological pathways in SLE more precisely, we performed transcriptomic, epigenomic and genomic analyses using 19 immune cell subsets from peripheral blood. METHODS: We sorted 19 immune cell subsets and identified the mRNA expression profiles and genetic polymorphisms in 107 patients with SLE and 92 healthy controls. Combined differentially expressed genes and expression quantitative trait loci analysis was conducted to find key driver genes in SLE pathogenesis. RESULTS: We found transcriptomic, epigenetic and genetic importance of oxidative phosphorylation (OXPHOS)/mitochondrial dysfunction in SLE memory B cells. Particularly, we identified an OXPHOS-regulating gene, PRDX6 (peroxiredoxin 6), as a key driver in SLE B cells. Prdx6-deficient B cells showed upregulated mitochondrial respiration as well as antibody production. We revealed OXPHOS signature was associated with type I IFN signalling-related genes (ISRGs) signature in SLE memory B cells. Furthermore, the gene sets related to innate immune signalling among ISRGs presented correlation with OXPHOS and these two signatures showed associations with SLE organ damage as well as specific clinical phenotypes. CONCLUSION: This work elucidated the potential prognostic marker for SLE. Since OXPHOS consists of the electron transport chain, a functional unit in mitochondria, these findings suggest the importance of mitochondrial dysfunction as a key immunological pathway involved in SLE.

Priming treatment with T-cell redirecting bispecific antibody ERY974 reduced cytokine induction without losing cytotoxic activity in vitro by changing the chromatin state in T cells.

CD3 bispecific constructs are anticipated to become an important form of cancer immunotherapy, but they frequently cause cytokine release syndrome (CRS) that is difficult to manage in clinical contexts. A combination of intra-patient dose escalation and immunosuppressive treatment is widely used to mitigate CRS. Studies suggest that CRS after subsequent doses of CD3 bispecific constructs is less severe than after the priming dose, and that step-up dosing reduces cytokine levels in animals and humans. However, the mechanism underlying the reduced cytokine induction after priming treatment with CD3 bispecific constructs is unclear. To understand human T-cell activation and chromatin states after priming treatment with CD3 bispecific construct targeting CD3ɛ and glypican 3 (ERY974), we examined cytokine levels, cytokine mRNA expression, CD3ɛ expression, CD3-mediated signal transduction, T cell activation markers, cytotoxicity against target cells, and chromatin states in T cells after ERY974 priming treatment or negative control. The second ERY974 treatment decreased cytokines on Day 8, and ERY974 priming treatment changed the chromatin state in T cells. CD3ɛ expression, CD3-mediated signal transduction, T cell activation markers, and cytotoxicity were similar between the priming treatment with ERY974 and negative control. The present study suggests that chromatin state changes in T cells after the priming treatment was a pivotal factor in the mitigation of cytokine release after the second ERY974 treatment.

Integrative Network Analysis Combined with Quantitative Phosphoproteomics Reveals Transforming Growth Factor-beta Receptor type-2 (TGFBR2) as a Novel Regulator of Glioblastoma Stem Cell Properties.

Glioblastoma is one of the most malignant brain tumors with poor prognosis and their development and progression are known to be driven by glioblastoma stem cells. Although glioblastoma stem cells lose their cancer stem cell properties during cultivation in serum-containing medium, little is known about the molecular mechanisms regulating signaling alteration in relation to reduction of stem cell-like characteristics. To elucidate the global phosphorylation-related signaling events, we performed a SILAC-based quantitative phosphoproteome analysis of serum-induced dynamics in glioblastoma stem cells established from the tumor tissues of the patient. Among a total of 2876 phosphorylation sites on 1584 proteins identified in our analysis, 732 phosphorylation sites on 419 proteins were regulated through the alteration of stem cell-like characteristics. The integrative computational analyses based on the quantified phosphoproteome data revealed the relevant changes of phosphorylation levels regarding the proteins associated with cytoskeleton reorganization such as Rho family GTPase and Intermediate filament signaling, in addition to transforming growth factor-ß receptor type-2 (TGFBR2) as a prominent upstream regulator involved in the serum-induced phosphoproteome regulation. The functional association of transforming growth factor-ß receptor type-2 with stem cell-like properties was experimentally validated through signaling perturbation using the corresponding inhibitors, which indicated that transforming growth factor-ß receptor type-2 could play an important role as a novel cell fate determinant in glioblastoma stem cell regulation.

Quantitative phosphoproteomics-based molecular network description for high-resolution kinase-substrate interactome analysis.

MOTIVATION: Phosphorylation-dependent cellular signaling is known to play a diverse role in regulating multiple cellular processes such as proliferation, differentiation and apoptosis. Recent technological advances in mass spectrometry-based phosphoproteomics have enabled us to measure network-wide signaling dynamics in a comprehensive and quantitative manner. As conventional protein-protein interaction (PPI) information-based network analysis is insufficient to systematically analyze phosphorylation site-dependent complex interaction dynamics, here we develop and evaluate a platform to provide a high-resolution molecular network description for kinase-substrate interactome analysis. RESULTS: In this study, we developed a Cytoscape-based bioinformatical platform named 'Post Translational Modification mapper (PTMapper)' to integrate PPI data with publicly available kinase-substrate relations at the resolution of phosphorylated amino acid residues. The previous phosphoproteome data on EGF-induced cellular signaling in glioblastoma stem cells was applied to evaluate our platform, leading to discovery of phosphorylation-dependent crucial signaling modulation in the p70S6K1-related pathway. Our study revealed that high-resolution cellular network description of phosphorylation-site dependent kinase-substrate signaling regulation should accelerate phosphoproteomics-based exploration of novel drug targets in the context of each disease-related signaling. AVAILABILITY AND IMPLEMENTATION: PTMapper and the example data for construction of phosphorylation site-oriented networks are available at https://github.com/y-narushima/PTMapper CONTACT: moyama@ims.u-tokyo.ac.jp SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Human recombinant Fab fragment from combinatorial libraries of a B-cell lymphoma patient recognizes core protein of chondroitin sulphate proteoglycan 4.

CD antigens are well known as therapeutic targets of B-cell lymphoma. To isolate therapeutic antibodies that recognize novel targets other than CD antigens, we constructed a phage display combinatorial antibody Fab library from bone marrow lymphocytes of B-cell lymphoma patient. To eliminate antibodies reactive with known B-cell lymphoma antigen, non-hematopoietic and patient's sera reactive HeLaS3 cells was selected as a target of whole cell panning. Five rounds of panning against live HeLaS3 cells retrieved single Fab clone, termed AHSA (Antibody to HeLa Surface Antigen). Using phage display random peptide library, LSYLEP was identified as an epitope sequence of AHSA. LC-MS/MS analysis of AHSA-precipitated HeLaS3 cell lysates detected several fragments corresponding to the sequence of chondroitin sulphate proteoglycan 4 (CSPG4) core protein. Since LSYLEP sequence was at the position of 313-318 of CSPG4, we considered that CSPG4 was AHSA-associated antigen. Double staining of CSPG4-postive MDA-MB-435S cells with AHSA and anti-CSPG4 rabbit antibody showed identical staining position, and reduced AHSA reactivity was observed in CSPG4-siRNA treated MDA-MB-435S cells. In conclusion, we retrieved a human Fab from antibody library of B-cell lymphoma patient, and identified CSPG4 as a recognizing antigen. AHSA may have potential benefits for development of CSPG4-targeting theranostics for B-cell lymphoma.