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  • br Conclusions When the authors

    2021-06-09


    Conclusions When the authors joined the field of oxysterol analysis around the turn of the century, oxysterols and cholestenoic acids were mostly considered as uninteresting intermediates of cholesterol metabolism. Some oxysterols had been shown to have biological activity, but the most studied oxysterol, 25-HC, was detected at low levels in biological samples, and was often suspected to be an artefact of sample handling [62]. Despite notable exceptions [3], [62], [76], [77], [78], [79] few researchers were interested in the biochemistry of oxysterols. However, in the last 5–10 years we have seen a paradigm shift, with a huge surge of interest in oxysterol biochemistry. There have been major developments in MS technology and methods for oxysterol analysis, but problems still are evident. Oxysterols can be formed from cholesterol in air, so there is always the danger of their artefactual formation through sample handling. There are a huge number of isomeric oxysterols. We now know that not only is 7α,26-diHC a biologically relevant molecule on the pathway to bile ribitol biosynthesis, but is its epimer, 7β,26-diHC, is also biologically relevant. Similarly, both 24S-HC and 24R-HC can be found in biological samples [80]. The analyst must be aware of the huge potential for isomeric structures or important molecules will be missed or misidentified and if chromatographic separation is inadequate quantitation will be inaccurate. Despite these reservations the future for cholesterolomics is bright.
    Acknowledgements This work was supported by the UK Biotechnology and Biological Sciences Research Council (BBSRC, grant numbers BB/I001735/1 and BB/N015932/1 to WJG, BB/L001942/1 to YW), the Welsh Government (Life Sciences Research Network Wales award to YW) and the European Regional Development Fund/Welsh Government-funded BEACON research program. JAK was supported by a PhD studentship from Imperial College Healthcare Charities. Members of the European Network for Oxysterol Research (ENOR, http://oxysterols.com/) are thanked for informative discussions.
    Introduction In response to T cell-dependent (TD) antigen stimulation, antigen-specific B cells migrate to the periphery of B cell follicles and interfollicular zones of secondary lymphoid organs, where they interact with cognate T helper cells within 1–3 days (Kerfoot et al., 2011, Okada et al., 2005, Qi et al., 2008). After then, B cells can follow one of three alternative fates by differentiating into extrafollicular plasma cells, follicular germinal center (GC) B cells, or recirculating early memory B cells (McHeyzer-Williams et al., 2012). GC B cells undergo massive clonal expansion, somatic hypermutation, and class switch recombination, after which selected clones undergo differentiation into memory B cells and long-lived plasma cells (Allen et al., 2007, Victora and Nussenzweig, 2012). T follicular helper (TFH) cells express the chemokine receptor CXCR5 and costimulatory molecule PD-1 at high levels and specialize in providing help to B cells during the humoral immune response (Crotty, 2011). Antigen-presenting dendritic cells mediate the initiation of TFH cell differentiation at early time points after immunization (Choi et al., 2011, Goenka et al., 2011). Interactions with cognate B cells, especially GC B cells within GCs, are critical for further polarization, maintenance, and function of TFH cells at later stages of the immune response (Choi et al., 2011, Kerfoot et al., 2011, Kitano et al., 2011). Thus, the reciprocal development of GC B cells and TFH cells is crucial for establishment of the GC reaction, including the formation of high-affinity antibody and the generation of long-lived plasma cells. The Bcl6 transcriptional repressor is a master regulator of the GC reaction required for development of both GC B cells and TFH cells, respectively (Dent et al., 1997, Fukuda et al., 1997, Johnston et al., 2009, Nurieva et al., 2009, Ye et al., 1997, Yu et al., 2009). In addition, Bcl6 plays a key role in suppressing inflammatory cytokine expression in macrophages (Toney et al., 2000). Bcl6-deficient (Bcl6−/−) mice in addition to failing to form GCs are sickly and die within weeks from a lethal inflammatory syndrome primarily driven by macrophages with secondary contributions from Th2 and Th17 cells (Mondal et al., 2010, Toney et al., 2000). However, from a mechanistic standpoint the function of Bcl6 is poorly understood outside of the context of already established GC B cells. Bcl6 enables proliferation and tolerance of DNA damage by silencing DNA damage sensing and cell cycle checkpoint genes, and it also delays plasma cell differentiation by repression of critical GC exit and plasma cell differentiation genes (Bunting and Melnick, 2013, Klein and Dalla-Favera, 2008). However, Bcl6−/− mice display a complete loss of GC formation with no evidence of the capacity to establish nascent GC clusters (Dent et al., 1997, Fukuda et al., 1997, Ye et al., 1997), suggesting that Bcl6 might have biological functions prior to GC formation. Indeed, Bcl6 protein is upregulated in early GC-committed B cells (i.e., “pre-GC B cells”) outside GCs 3–5 days after immunization (Kerfoot et al., 2011, Kitano et al., 2011) and plays an essential role in maintaining interactions with TFH cell as well as subsequent migration and clustering into GC structures, at least in part through repressing the expression of Gpr183, encoding the G protein-coupled receptor Ebi2 (Kitano et al., 2011, Shaffer et al., 2000).