PI16 is expressed by a subset of human memory Treg with enhanced migration to CCL17 and CCL20
Introduction
Regulatory T cells (Treg) are critical for maintaining tolerance and immune homeostasis [1], and the manipulation of Treg numbers or function is a potential strategy for the treatment of autoimmune diseases and graft-versus-host disease [2], [3]. Expression of FOXP3 is a defining characteristic of Treg [4], [5], but viable Treg cannot be isolated based on their expression of this transcription factor [6], [7].
The expression of high levels of the interleukin 2 receptor alpha chain (CD25) by CD4-positive lymphocytes is widely used to identify Treg [8], [9], but CD25 expression is also upregulated during T cell activation [10], [11]. The expression of high levels of CD25 and low levels of the interleukin 7 receptor alpha chain (CD127) has also been used to isolate functional Treg [12], [13]. Again, this approach may be limited, as CD127 expression is reduced following T cell activation [14]. Neither of these combinations defines a population of CD4-positive lymphocytes in which all the cells express FOXP3. As FOXP3 is expressed by several functionally distinct subsets of CD4-positive lymphocytes [15], [16], additional cell surface markers are necessary to identify, isolate and characterize pure sub-populations of viable Treg.
In the course of genome wide expression profiling to identify genes regulated by FOXP3 in human Treg, and to discover novel candidate surface proteins specific to human Treg, we identified the peptidase inhibitor PI16 as over-expressed by in vitro expanded Treg derived from cord blood, compared with T helper (Th) cells from the same source [17].
PI16 (also called CRISP-9 or PSPBP) is a member of the cysteine-rich secretory protein (CRISP) family. The mature PI16 protein is a 436 amino acid polypeptide with three N-linked glycosylation motifs, which is predicted to localize to the plasma membrane through a GPI anchor [18]. PI16 has been identified as a binding protein for “prostate secretory protein of 94 amino acids” (PSP94), and serum levels of PSP94 and PI16 have been described as biomarkers of recurrence risk in prostate cancer [19], but the expression of PI16 by immune cells has not been reported previously.
The differential expression of PI16 by peripheral blood Treg compared with Th cells was confirmed by qRT-PCR, and cell surface expression was demonstrated by flow cytometry using polyclonal antibodies to human PI16 [17]. To evaluate the potential of PI16 as a marker for circulating Treg, we generated a mouse monoclonal antibody to the human PI16 protein. Here we describe the phenotype and function of PI16-expressing Treg in adult peripheral blood.
Section snippets
PBMC isolation
Peripheral blood was collected using lithium-heparin anticoagulant from healthy adult volunteers who gave informed consent, under ethics clearance from the Research Ethics Committee of the Children’s, Youth and Women’s Health Service, South Australia (REC 2007/11/2013). Mononuclear cells (PBMC) were isolated by density centrifugation over Lymphoprep (Axis-Shield, Oslo, Norway) using standard protocols.
Antibodies and chemokines
A mouse polyclonal antibody raised to full-length recombinant PI16 protein was purchased from
Distribution of PI16 expression by mononuclear cells
The monoclonal antibody CRCBT-02-001 was confirmed to react with PI16-transfected NIH3T3 mouse fibroblasts, but not untransfected NIH3T3 cells (Supplementary Fig. S1). Staining of PBMC with the CRCBT-002-01 monoclonal antibody and antibodies to CD4, CD8 and CD19 (Supplementary Fig. S2) indicated that the majority of PI16-expressing cells were CD4-positive lymphocytes (typically 20% of CD4-positive subset), with the remaining cells being CD8-positive T cells (approximately 10% of the CD8-bright
Discussion
Imbalances in the frequency and/or function of Treg have been reported in many diseases [23], [24], [25], [26]. The difficulty of using FOXP3 to isolate viable Treg for use in functional assays has lead to many candidate cell surface molecules being evaluated as surrogates for FOXP3 expression, but with limited success. As part of a gene discovery program, we identified PI16 as being over-expressed by cord-blood-derived Treg compared with Th cells [17]. We generated a monoclonal antibody to
Acknowledgment
This work was funded by the Co-operative Research Centre for Biomarker Translation.
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