Effector/memory CD4 T cells making either Th1 or Th2 cytokines commonly co-express T-bet and GATA-3

RESEARCH ARTICLE Effector/memory CD4 T cells making either Th1 or Th2 cytokines commonly co-express T-bet and GATA-3 Arundhoti Das☯, Vidya Ranganathan☯†, Danish Umar, Shipra Thukral¤, Anna George‡, Satyajit Rath‡, Vineeta Bal‡* National Institute of Immunology, New Delhi, India a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 ☯ These authors contributed equally to this work. † Deceased. ¤ Current address: BD Diagnostics, BD, India. ‡ These authors also contributed equally as senior authors on this work. * Abstract OPEN ACCESS Citation: Das A, Ranganathan V, Umar D, Thukral S, George A, Rath S, et al. (2017) Effector/memory CD4 T cells making either Th1 or Th2 cytokines commonly co-express T-bet and GATA-3. PLoS ONE 12(10): e0185932. https://doi.org/10.1371/ journal.pone.0185932 Editor: Hiroshi Shiku, Mie University Graduate School of Medicine, JAPAN Received: February 20, 2017 Accepted: September 21, 2017 Published: October 31, 2017 Copyright: © 2017 Das et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported in part by grants from the Department of Biotechnology, Government of India (to A.G. # BT/PR5138/BRB/ 10/1064/2012, to S.R. # BT/PR14592/BRB/10/858/ 2010, to V.B. # BT/PR7012/BRB/1159/2012); the Science and Engineering Research Board, Department of Science and Technology, Government of India (to A.G. # SR/SO/BB-0035/ Naïve CD4 T (NCD4T) cells post-activation undergo programming for inducible production of cytokines leading to generation of memory cells with various functions. Based on cytokine based polarization of NCD4T cells in vitro, programming for either ‘Th1’ (interferon-gamma [IFNg]) or ‘Th2’ (interleukin [IL]-4/5/13) cytokines is thought to occur via mutually exclusive expression and functioning of T-bet or GATA-3 transcription factors (TFs). However, we show that a high proportion of mouse and human memory-phenotype CD4 T (MCD4T) cells generated in vivo which expressed either Th1 or Th2 cytokines commonly co-expressed Tbet and GATA-3. While T-bet levels did not differ between IFNg-expressing and IL-4/5/13expressing MCD4T cells, GATA-3 levels were higher in the latter. These observations were also confirmed in MCD4T cells from FVB/NJ or aged C57BL/6 or IFNg-deficient mice. While MCD4T cells from these strains showed greater Th2 commitment than those from young C57BL/6 mice, pattern of co-expression of TF was similar. Effector T cells generated in vivo following immunization also showed TF co-expression in Th1 or Th2 cytokine producing cells. We speculated that the difference in TF expression pattern of MCD4T cells generated in vivo and those generated in cytokine polarized cultures in vitro could be due to relative absence of polarizing conditions during activation in vivo. We tested this by NCD4T cell activation in non-polarizing conditions in vitro. Anti-CD3 and anti-CD28-mediated priming of polyclonal NCD4T cells in vitro without polarizing milieu generated cells that expressed either IFNg or IL-4/5/13 but not both, yet both IFNg- and IL-4/5/13-expressing cells showed upregulation of both TFs. We also tested monoclonal T cell populations activated in nonpolarizing conditions. TCR-transgenic NCD4T cells primed in vitro by cognate peptide in non-polarizing conditions which expressed either IFNg or IL-4/5/13 also showed a high proportion of cells co-expressing TFs, and their cytokine commitment varied depending on genetic background or priming conditions, without altering pattern of TF co-expression. Thus, the model of mutually antagonistic differentiation programs driven by mutually PLOS ONE | https://doi.org/10.1371/journal.pone.0185932 October 31, 2017 1 / 25 In vivo generated memory CD4 cells are not polarized 2013, S.R. # SB/SO/HS/210/2013, to V.B. #SR/SO/ HS-0005/2011, EMR/2015/001074); and a fellowship from the Council for Scientific and Industrial Research, Government of India to A.D. and D.U. The National Institute of Immunology is supported by the Department of Biotechnology, Government of India. There were no sponsors for this work. Funding agencies had no role in either conceptualization or implementation of the work. exclusively expressed T-bet or GATA-3 does not completely explain natural CD4 T cell priming outcomes. Competing interests: The authors have declared that no competing interests exist. Peripheral naïve CD4 T cells are exposed over the animal’s lifetime to a variety of immunogens in a variety of micro-environmental contexts, leading to accumulation of a secondary/memory CD4 T cell pool diverse in both antigenic specificities and effector potential. During priming and differentiation, memory CD4 T cells are thought to commit to various alternative programs that enable them to make different effector cytokines upon restimulation. These include the so-called ‘Th1’ (IFNg), ‘Th2’ (IL-4-, IL-5, IL-13), ‘Th17’ (IL-17, IL-22), ‘Th9’ (IL-9) or the induced regulatory T (iTreg) programs. Cytokines [1–8], co-stimulatory and accessory molecules [9–11] as well as antigen-presenting cell (APC) types [12–14] make a significant contribution to the memory fate determination of NCD4T cells post-activation. In contrast, transcription factor (TF) Mina, a member of the jumonji C (JmjC) protein family described to confer Th2 bias [15], functions in T cell intrinsic fashion for cell fate determination postactivation. The mechanistic insights in the regulation and control of CD4 T cell memory fate determination programs [2,3,16] have commonly come from work in controlled conditions in vitro in which uniform microenvironments bring about relatively homogeneous differentiation and generation of memory cells [10–12]. This approach using polarizing conditions has shown that early production of IFNg or IL-4 during priming, either from APCs or from responding naive cells [17,18], leads to the induction of T-bet or GATA-3 respectively, and that these signaling pathways suppress each other, so that T-bet-expressing cells do not express GATA-3 and vice versa, leading to expression of either ‘Th1’ or ‘Th2’ cytokines from the primed cells [19–21]. Such polar-differentiated memory cells are resistant to de-differentiation and plasticity [19]. However, there are many examples of the existence of plasticity, particularly in memory cells differentiated in vivo [1,20–23]. Following infection or vaccination, individual memory cells appear to show binary choices of cytokine programs [24,25], in that they make either, say, ‘Th1’ cytokines or ‘Th2’ cytokines and only uncommonly both [23]. As memory cells accumulate in vivo as an outcome of extremely complex and heterogeneous priming conditions we were interested in asking the foll (...truncated)