Cutting Edge Cutting Edge
Cutting Edge: TGF-1 and IL-15 Induce FOXP3
Regulatory T Cells in the Presence of Antigen Stimulation
1
Rita Casetti,
2
* Chiara Agrati,* Marianne Wallace,
†
Alessandra Sacchi,* Federico Martini,*
Angelo Martino,* Alessandra Rinaldi,* and Miroslav Malkovsky
†
Several subsets of regulatory T cells (Tregs) have
been described and studied intensively, but the potential
regulatory role of T cells remains largely unclear.
Lymphocytes expressing TCR are involved in both
innate and adaptive immune responses, and their major
adult human peripheral blood subset (V9V2) dis-
plays a broad reactivity against microbial agents and
tumors. In this study we report that T lympho-
cytes with regulatory functions (V2 Tregs) are in-
duced in vitro in the presence of specific Ag stimula-
tion and cytokines (TGF-1 and IL-15). These cells
express FOXP3 and, similarly as Tregs, suppress
the proliferation of anti-CD3/anti-CD28 stimulated-
PBMC. Phenotypic and functional analyses of V2
Tregs will very likely improve our understanding about
the role of T cells in the pathogenesis of autoimmune,
infectious, and neoplastic diseases. The Journal of Immu-
nology, 2009, 183: 3574 –3577.
H
uman T cells are a lymphocyte population with
unusual tissue distribution and Ag recognition path-
ways. V9V2 T cells are a major subset of the circu-
lating T cell pool and are involved in immunity against
pathogens and malignancies (1–9). Unlike classical T cells,
V9V2 T cells recognize nonprocessed Ags such as pyro-
phosphomonoesters (10). This recognition is mediated by the
TCR and is not restricted by MHC molecules (11, 12). A reg-
ulatory role for T cells has already been shown in a number
of studies and is demonstrated by their influence on dendritic
cells (13), neutrophils (14), and B cells (15) in humans, and on
neutrophils (16) and macrophages (17, 18) in mice.
The immune system is responsible for protective response
against pathogens and tumors and maintains immune toler-
ance. Regulatory T cells (Tregs)
3
identified as Forkhead/winged
helix transcription factor box P3 (FOXP3)-expressing CD4
+
CD25
high
T cells represent a critical aspect in the balance of the
immune response, preventing the development of autoimmune
diseases (19) and preserving the efficiency of antitumor im-
mune responses (20). To date, FOXP3 is the master gene that
controls Treg development/function and is currently used as
the major Treg marker (21, 22). In addition, IL-10-producing
T regulatory type 1 (Tr1) cells and TGF--producing Th3 cells
induced from uncommitted peripheral CD4
+
T cells possess
suppressive functions similar to those of naturally occurring
Tregs (nTregs) (23, 24). The potential regulatory role of T
cells has been recently suggested but not yet clarified (17, 25,
26). In this study, we show for the first time that a subset of
FOXP3-expressing V2 T cells (V2 Tregs) is induced under
opportune Ag stimulation and cytokines.
Materials and Methods
Cell isolation and culture conditions
PBMC from healthy donors were isolated from fresh buffy coats by Lympholyte
(Cedarlane) density gradient centrifugation. Cells were cultured at a concen-
tration of 4 10
6
/ml in the presence of IL-2 (6.5 U/ml), IL-15 (10 ng/ml)
(both from Sigma-Aldrich), TGF-1 (1.7 ng/ml; Calbiochem) and isopentenyl
pyrophosphate (IPP; 20 g/ml; Sigma). On days 3, 6, and 9, half of the super-
natant volume was discarded and replaced with fresh medium containing cy-
tokines. Aliquots of PBMC and bead-sorted T cells (Miltenyi Biotec) were
used fresh or were frozen for use at later time points.
Flow cytometry
Cells were surface stained with anti-V2, anti-CD25, anti-CD69, anti-
CD45RA, anti-CD45RO, anti-CD27, and anti-CD127 (BD Biosciences),
fixed and permeabilized with saponin or Perm/Fix solution, and finally stained
intracellularly with anti-CTLA-4 (BD Biosciences) or FOXP3 (Biolegend)
according to the manufacturer’s instructions. The cells were analyzed using a
FACScalibur instrument with CellQuest software (BD Biosciences). Because
FOXP3 is expressed intracellularly, it is not possible to use it as a marker of the
Treg phenotype for purification by sorting V2
+
FOXP3
+
cells. Therefore,
V2
+
cells were purified from PBMC cultured to the point where the
V2
+
FOXP3
+
population represented 30% or more of the V2
+
cells (35.6
9.7; range, 30.1–50.4), using a FACSVantage-SE cell sorter (95% purity; BD
Biosciences).
*Laboratory of Cellular Immunology, National Institute for Infectious Diseases “Lazzaro
Spallanzani” Instituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy; and
†
De-
partment of Medical Microbiology and Immunology and University of Wisconsin Com-
prehensive Cancer Center, University of Wisconsin School of Medicine and Public Health,
Madison, WI 53792
Received for publication May 7, 2009. Accepted for publication July 16, 2009.
The costs of publication of this article were defrayed in part by the payment of page charges.
This article must therefore be hereby marked advertisement in accordance with 18 U.S.C.
Section 1734 solely to indicate this fact.
1
This study was supported by grants from the National Institutes of Health, U. S. De-
partment of Defense, and the Italian Ministry of Health.
2
Address correspondence and reprint requests to Dr. Rita Casetti, Laboratory of Cellular
Immunology, National Institute for Infectious Diseases “Lazzaro Spallanzani” Instituto di
Ricovero e Cura a Carattere Scientifico, Via Portuense 292, 00149 Rome, Italy. E-mail
address: casetti@inmi.it
3
Abbreviations used in this paper: Treg, regulatory T cell; CFDA-SE, carboxyfluorescein
diacetate succinimidyl ester; FOXP3, Forkhead box P3; IPP, isopentenyl pyrophosphate.
Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.0901334