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An optimized method to differentiate mouse follicular helper T cells in vitro

Cellular & Molecular Immunology volume 17, pages 779–781 (2020)Cite this article

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Upon priming, naive CD4+ helper T (Th) cells differentiate into distinct subsets with specialized functions. The differentiation of Th subsets is driven not only by signals from the T cell receptor (TCR) and costimulatory receptors but is also critically dependent on the specific cytokine milieu. By mimicking such conditions, robust methods have been developed for the in vitro differentiation of type 1 and type 2 Th (Th1 and Th2) cells, and more recently, IL-17-producing Th (Th17) cells and regulatory T (Treg) cells,1 which greatly support the research and applications of these Th subsets. Follicular helper T (Tfh) cells represent another Th subset that specializes in supporting the germinal center (GC) response and regulating the generation of memory B cells and long-lived plasma cells.2 However, current methods for in vitro Tfh differentiation are not optimal. Even in the best practice, only 20% of polarized cells showed the expression of CXCR5, the key Tfh functional marker.3 Here, we report an optimized in vitro differentiation method that generates 50–75% CXCR5+ cells with enhanced B cell helper function. We demonstrate that the priming of antigen-presenting cells (APCs) by lipopolysaccharide (LPS) and the increase of the APC:T cell ratio were key to efficiently generating Tfh cells in vitro.

Our second attempt was to increase the APC:T cell ratio since a high dose of antigens has been shown to promote Tfh differentiation.6 By maintaining a consistent number of splenocytes and gradually reducing the number of OT-II cells, APC:T cell ratios were increased from 5:1 (the conventional method) to 50:1 and 500:1. Correspondingly, we observed a more than two-fold increase in CXCR5 expression, which showed a positive correlation with the APC:T cell ratio. When LPS-treated splenocytes and at an APC:T cell ratio of 500:1 were used, over 70% of OT-II cells displayed the CXCR5+ PD-1+ phenotype (Fig. 1a, b). By testing the OVA peptide concentration ranging from 0.1 to 1 µg/mL (high APC:T cell ratio at 500:1) or from 1 to 10 µg/mL (low APC:T cell ratio at 5:1), we found that peptide concentrations had a negligible impact on Tfh cell differentiation (Fig. 1c), suggesting that signaling other than TCR, presumably costimulatory signaling, is the limiting factor for Tfh differentiation in the APC:T cell coculture system.

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Acknowledgements

The study is supported by the National Key Research and Development Program of China (2017YFC0909003), the Australian National Health and Medical Research Council (GNT1147769) and the Bellberry-Viertel Senior Medical Research Fellowship to D.Y.

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Authors and Affiliations

  1. Department of Immunology and Infectious Disease, John Curtin School of Medical Research, The Australian National University, Canberra, ACT, Australia

    Xin Gao, Hao Wang, Zhian Chen, Pengcheng Zhou & Di Yu

  2. China-Australia Centre for Personalized Immunology, Shanghai Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China

    Di Yu

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  1. Xin Gao
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  2. Hao Wang
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Contributions

D.Y. and X.G. conceived and designed the study. X.G. performed the experiments and analyzed the data with the help of W.H., Z.C. and P.Z. X.G. and D.Y. wrote the paper. All authors approved the final paper.

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Correspondence to Di Yu.

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Gao, X., Wang, H., Chen, Z. et al. An optimized method to differentiate mouse follicular helper T cells in vitro. Cell Mol Immunol 17, 779–781 (2020). https://doi.org/10.1038/s41423-019-0329-7

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