Epigenetic modulators of B cell fate identified through coupled phenotype-transcriptome analysis

Abstract

High-throughput methodologies are the cornerstone of screening approaches to identify novel compounds that regulate immune cell function. To identify novel targeted therapeutics to treat immune disorders and haematological malignancies, there is a need to integrate functional cellular information with the molecular mechanisms that regulate changes in immune cell phenotype. We facilitate this goal by combining quantitative methods for dissecting complex simultaneous cell phenotypic effects with genomic analysis. This combination strategy we term Multiplexed Analysis of Cells sequencing (MAC-seq), a modified version of Digital RNA with perturbation of Genes (DRUGseq). We applied MAC-seq to screen compounds that target the epigenetic machinery of B cells and assess altered humoral immunity by measuring changes in proliferation, survival, differentiation and transcription. This approach revealed that polycomb repressive complex 2 (PRC2) inhibitors promote antibody secreting cell (ASC) differentiation in both murine and human B cells in vitro. This is further validated using T cell-dependent immunization in mice. Functional dissection of downstream effectors of PRC2 using arrayed CRISPR screening uncovered novel regulators of B cell differentiation, including Mybl1, Myof, Gas7 and Atoh8. Together, our findings demonstrate that integrated phenotype-transcriptome analyses can be effectively combined with drug screening approaches to uncover the molecular circuitry that drives lymphocyte fate decisions.

An immunohistochemical atlas of necroptotic pathway expression

“…Immunisation was performed as previously described (Kong et al , 2022). 8–10-week-old Ripk3 +/+ or Ripk3 -/- littermate mice received a single intraperitoneal injection of 100Lµg 4-hydroxy-3-nitrophenylacetyl hapten coupled to keyhole limpet hemocyanin (NP-KLH; produced in-house) at a ratio of 21:1 with alum.…”

“…Immunisation was performed as previously described (116). 8-10-week-old female C57BL/6J wild-type mice received a single intraperitoneal injection of 100 µg 4-hydroxy-principles expressed in the Helsinki Declaration.…”

High-throughput CRISPR technology: a novel horizon for solid organ transplantation

“…Plasma cells are the primary source of antibodies, and the mechanism underlying their divergence from B cells has been thoroughly investigated ( 191 – 193 ). Disruptions in the typical antibody-secreting cell (ASC) formation pathway can cause immune-related diseases ( 132 ). The MAC-seq, which combines genetic analysis with quantitative methodologies, has been used to screen for drugs impacting the epigenetic machinery of B cells while assessing alterations in humoral immunity ( 132 ).…”

Histone demethylases in the regulation of immunity and inflammation

“…The role of KDMs in the development and function of B lymphocytes B lymphocytes were essential effectors in humoral immunity, and epigenetic regulation is required for the proliferation and differentiation of B cells [73][74][75][76]. Upon activation by thymusdependent antigen, naïve B cells (nB) transform into plasmablasts (PB) to form germinal centers (GC), from which antibody-secreting plasma cells and memory B cells were developed.…”