Inhibition of activation induced CD154 on CD4+ CD25- cells: a valid surrogate for human Treg suppressor function

Natural Regulatory T cells (Tregs) are defined by stable expression of the cell surface proteins CD4 and CD25, low surface expression of CD127 and expression of the transcription factor FOXP3. The contribution of Treg to the prevention of autoimmunity and the maintenance of immune homoestasis is the subject of ongoing interest, as alterations in Treg numbers and function are implicated in a wide range of diseases. The in vitro benchmark for determining Treg function is suppression of proliferation of unmatched effector T cells in a mixed lymphocyte reaction (MLR) over a 3–6-day time period. As an alternative to this assay, we show that a 7-h CD154 expression assay is rapid, simple and provides a reliable readout of suppressor function. Using multiple Treg-like cell types including natural (n)Treg, inducible (i)Treg and Treg cell lines, we show that suppression of CD154 expression is a surrogate for suppression of proliferation. We propose this as a suitable alternative to the MLR assay, as it is rapid and may be more amenable to high-throughput screening, analysing large cohorts of clinical samples or assaying transiently suppressive populations. Keywords: regulatory T cells; functional assays; iTreg; nTregDanika Hill, Nicola Eastaff-Leung, Suzanne Bresatz-Atkins, Noel Warner, Joyce Ruitenberg, Dorren Krumbiegel, Steve Pederson, Natasha McInnes, Cheryl Y. Brown, Timothy Sadlon and Simon C. Barr

PI16 is expressed by a subset of human memory Treg with enhanced migration to CCL17 and CCL20

The peptidase inhibitor PI16 was shown previously by microarray analysis to be over-expressed by CD4-positive/CD25-positive Treg compared with CD4-positive/CD25-negative Th cells. Using a monoclonal antibody to the human PI16 protein, we found that PI16-positive Treg have a memory (CD45RO-positive) phenotype and express higher levels of FOXP3 than PI16-negative Treg. PI16-positive Treg are functional in suppressor assays in vitro with potency similar to PI16-negative Treg. Further phenotyping of the PI16-positive Treg revealed that the chemokine receptors CCR4 and CCR6 are expressed by more of the PI16-positive/CD45RO-positive Treg compared with PI16-negative/CD45RO-positive Treg or Th cells. PI16-positive Treg showed enhanced in vitro migration towards the inflammatory chemokines CCL17 and CCL20, suggesting they can migrate to sites of inflammation. We conclude that PI16 identifies a novel distinct subset of functional memory Treg which can migrate to sites of inflammation and regulate the pro-inflammatory response at those sites.Ian C. Nicholson, Christos Mavrangelos, Daniel R.G. Bird, Suzanne Bresatz-Atkins, Nicola G. Eastaff-Leung, Randall H. Grose, Batjargal Gundsambuu, Danika Hill, Debbrah J. Millard, Timothy J. Sadlon, Sarah To, Heddy Zola, Simon C. Barry, Doreen Krumbiege