Poster Presentation Australasian Cytometry Society 43rd Annual Conference and Workshop

Parallel analysis of multiple human memory CD4+ T cell subsets within antigen-specific responses using cell proliferation dyes (#104)

Laura Cook 1 2 3 , John Zaunders 2 3 , Nabila Seddiki 3 4 , David van Bockel 2 3 , Anthony D Kelleher 2 3 , Cynthia Mee Ling Munier 2 3
  1. Department of Microbiology and Immunology, University of Melbourne, at the Peter Doherty Institute for Infection and Immunity, Parkville, VIC, Australia
  2. Immunovirology and Pathogenesis Program, The Kirby Institute, Sydney, NSW, Australia
  3. St Vincent's Centre for Applied Medical Research, St Vincent's Hospital, Sydney, NSW, Australia
  4. IDMIT Department/IBFJ, Immunology of Viral Infections and Autoimmune Diseases (IMVA), INSERM U1184, CEA, Université Paris Sud, Paris, France

Background. Assays to study antigen-specific CD4+ T-cell populations are in high demand, particularly those that do not require a priori knowledge of immunogenic epitopes or their HLA restriction elements. In this regard, a broad group of assays, termed activation induced marker (AIM) assays, have proven to be extremely useful and have been widely used to analyse T-cell responses to various disease antigens. One such assay that was developed in our lab, the OX40 assay, uses activation induced co-expression of CD25 (IL-2Rα) and OX40 (CD134) after 44-48h incubation with antigen to define antigen-specific CD4+ T-cells. We aimed to combine the OX40 assay with cell sorting and labelling with cell proliferation dyes to enable precise tracking of human CD4+ memory T-cell subsets within in vitro recall responses.

Methods. We utilised CFSE-like fluorescent dyes to label memory cell populations immediately after sorting from unstimulated PBMCs and analysed their contribution to antigen-driven early activation and subsequent cell proliferation by flow cytometry.

Results. We isolated and analysed memory non-Tregs, CD39+ Tregs and CD39neg Tregs; although any three memory CD4+ T-cell populations able to be isolated by cell surface markers could potentially be tracked. Labelled cell populations could be detected after PBMC reconstitution at ex vivo proportions. In concordance with our previous publication (1), after 44h activation the antigen-specific CD25+OX40+ T-cells were primarily comprised of CD39+ Tregs (average = 45%, non-Tregs = 30%, CD39neg Tregs = 4%), but after 5 days the proliferative response was almost entirely comprised of memory non-Tregs, with negligible cell proliferation occurring within memory Treg populations.

Conclusion. This adaption of the OX40 assay enables parallel analysis of the relative contribution of several CD4+ memory T-cell subsets to recall responses detected by activation marker expression and cell proliferation. This will enable more precise studies of immune memory generated following natural infection or vaccination.

  1. Seddiki, N.*, Cook, L.*, Hsu, D. C., Phetsouphanh, C., Brown, K., Xu, Y., Kerr, S. J., Cooper, D. A., Munier, C. M., Pett, S., Ananworanich, J., Zaunders, J. and Kelleher, A. D., Human antigen-specific CD4+ CD25+ CD134+ CD39+ T cells are enriched for regulatory T cells and comprise a substantial proportion of recall responses. Eur J Immunol 2014. 44: 1644-1661