A novel function for FOXP3 in humans: intrinsic regulation ... · 4 degradation of FOXP3, promotion of Th17 cell development and blockade of Treg differentiation.14 Beyond this “tug-of-war”
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A novel function for FOXP3 in humans: intrinsic regulation of conventional T cells
Alicia N. McMurchy,1 Jana Gillies,1 Maria Concetta Gizzi2,3, Michela Riba3, Jose Manuel
Garcia-Manteiga3, Davide Cittaro3, Dejan Lazarevic3, Sara Di Nunzio2 , Ignazio S. Piras4,
Alessandro Bulfone4, Maria Grazia Roncarolo2, 5, Elia Stupka3, Rosa Bacchetta,2* and Megan K.
Levings1*
1Department of Surgery, University of British Columbia & Child and Family Research Institute, Vancouver, Canada; 2San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Division of Regenerative Medicine, Stem Cells, and Gene Therapy, San Raffaele Scientific Institute, Milan, Italy; 3Center for Translational Genomics and Bioinformatics, San Raffaele Scientific Institute, Milan, Italy; 4 CRS4 - Biomedicine Sector Center, Technology Park Polaris, Pula (CA), Italy; 5 San Raffaele Vita-Salute University, Milan, Italy.
* co-senior authors
Correspondence: Megan Levings, Department of Surgery, University of British Columbia, Room A4-186, 950 West 28th Ave. Vancouver, B.C. V5Z 4H4; tel: 604-875-2000 ext 4686; fax: 604-875-2373; email: [email protected] Short title: The role of FOXP3 in conventional T cells Scientific Section Designation: IMMUNOBIOLOGY
Blood First Edition Paper, prepublished online November 20, 2012; DOI 10.1182/blood-2012-05-431023
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Figure Legends
Figure 1: FOXP3 null CD4+CD25- Tconv cell clones proliferate to a greater extent and
produce more IFN-γ and IL-2 than wild type Tconv cell clones. Tconv cell clones from an
individual who is hemizygous for a null mutation in FOXP3 were labeled with CFSE and
stimulated with different ratios of anti-CD3/anti-CD28-coated beads. After 4 days, Tconv clones
were stained with anti-CD4 and anti-FOXP3 antibodies (236A/E7) and read on a BD
FACSCanto. (A) Activation-induced FOXP3 expression in WT but not FOXP3 null Tconv cell
clones. Two representative WT and two FOXP3 null Tconv clones are shown for clones that
were stimulated with 1 anti-CD3/anti-CD28 coated bead per 32 cells for 4 days. (B) Shows one
representative experiment; numbers in each plot represent division index. (C) The average
division index of multiple WT (n = 7) and FOXP3 null (n = 11) Tconv clones. (D) The division
indices (DI) of the FOXP3+ and FOXP3- populations are given within each plot of two
representative WT Tconv clones stimulated with 1 anti-CD3/anti-CD28 coated bead per 32 cells
for 4 days. (E) Average IFN-γ and IL-2 production by WT (n = 7) and FOXP3 null (n = 11)
Tconv clones (5 x 105 cells/mL). Supernatants from cultures were collected 20 hours after T cell
activation and analyzed by ELISA. (F) Percent suppression of IFN-γ production in Tconv clone
co-cultures. FOXP3 null clones were labeled with CFSE and co-cultured at the indicated ratios
with WT (black) or FOXP3 null (grey) Tconv clones labeled with CPD. Co-cultures were
activated with anti-CD3/anti-CD28-coated beads (1 bead : 32 cells). After 4 days, co-cultures
were re-stimulated with PMA and ionomycin and stained for IFN-γ. Shown is the average
percent suppression against 3 different FOXP3 null responders by each WT or null clone. The
average FOXP3 expression on day 4 for the WT clones is also shown (n =3).
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Figure 2. Gene expression analysis of FOXP3 WT and null Tconv clones. Total RNA from T
cell clones was hybridized to a GeneChip Human Gene 1.0 ST array. (A) Hierarchical clustering
revealed differential expression of 274 RefSeq annotated genes with a p.value <0.05 (Benjamini
correction). (B) Differentially expressed genes were subject to Gene Set Enrichment Analysis
after cross-validation using randomization from background genes. (C) Differential expression of
6 genes was confirmed by quantitative RT-PCR. Mean values ± SEM determined in triplicate are
shown.
Figure 3: CD4+CD25- T cells transduced with siRNA against FOXP3 proliferate to a
greater extent than CD4+CD25- T cells transduced with control siRNA against luciferase.
CD4+CD25- T cells transduced with siFOXP3 or siLuc were purified as ΔLNGFR+ cells, then
labeled with CFSE and stimulated with different ratios of anti-CD3/anti-CD28-coated beads.
Transduced T cells were stained with anti-CD4, anti-ΔLNGFR, and anti-FOXP3 antibodies. (A)
Representative FACS plots of FOXP3 expression in purified siFOXP3 and siLuc-transduced T
cells 3 days after activation at a 1:32 bead:cell ratio. (B) FOXP3 expression in ΔLNGFR+
siFOXP3-transduced T cells and control siLuc-transduced T cells following anti-CD3/anti-CD28
bead stimulation (n =4). (C) Average division index of siFOXP3-transduced T cells and control
siLuc-transduced T cells (n = 4). (D) CD4+CD25- T cells transduced with siFOXP3 or siLuc
were stimulated with different ratios of anti-CD3/anti-CD28-coated beads at 5 x 105 cells/mL.
Supernatants were collected 20 hours later and analyzed for IFN-γ and IL-2 by ELISA (n = 4).
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Figure 4: Enriched Th17 cells express high levels of FOXP3 upon activation. (A) Sorting
procedure for human Th1 (CD4+CD25-CXCR3+CCR4-CCR6-) and Th17 (CD4+CD25-CXCR3-
CCR4+CCR6+) cells. CD4+ T cells were isolated from human PBMCs and depleted of CD25+
cells prior to sorting. (B) Phenotype of Th1-enriched and Th17-enriched cells after two weeks
expansion with APC and anti-CD3 antibodies. T cells were re-stimulated and IL-17A and IFN-γ
production were determined by intracellular cytokine staining. The cytokine profiles from one
representative donor of 6 are shown. (C) Representative plots of FOXP3 expression in Th1-
enriched and Th17-enriched cells from one donor of 4 on days 1, 3, and 12 post re-activation
with anti-CD3/anti-CD28-coated beads (1 bead : 32 cells) are shown. (D) Average FOXP3
expression in Th1-enriched and Th17-enriched cells after re-activation with anti-CD3/anti-
CD28-coated beads (1 bead : 32 cells) over 14 days. Each time point represents the average
FOXP3 expression from 2-5 different donors. ** indicates significant difference in FOPX3
expression between Th1-enriched and Th17-enriched cells 14 days after re-activation (p =
0.0028). There is no difference at day 0 (p = 0.1080). † indicates a significant difference in
FOXP3 expression between day 0 and 14 in Th17-enriched cells (p = 0.0126). (E)
Representative (left panel) and average (right panel; n = 3 for IL-17, n = 2 for IFN-γ) FOXP3
expression in IL-17A+, IL-17A-, IFN-γ+, and IFN-γ- subsets within Th17-enriched cultures 12
days after re-stimulation with anti-CD3/anti-CD28 coated beads (1 bead : 32 cells).
Figure 5: FOXP3-deficient human Th17 cells have a greater expansion potential than
control Th17 cells. Th1 and Th17 cells transduced with siFOXP3 or control siLuc were
stimulated with anti-CD3/anti-CD28-coated beads (1 bead : 32 cells) in the presence of IL-2. (A)
FOXP3 expression in siFOXP3 and control siLuc-transduced Th1 and Th17 cells over two
28
weeks (n = 4). (B&C) At the indicated time points, cells were collected and stained with
viability dye and anti-ΔLNGFR. Live, ΔLNGFR+ cells were counted by flow cytometry with
counting beads. Fold expansion was determined by dividing the number of cells at each time
point by the number of live, unstimulated cells counted by the same method on day 1. One
representative experiment of 4 is shown in (B) and the average fold expansion of siFOXP3-
transduced T cells over control siLuc-transduced T cells on day 8 after activation is shown in
(C).
Figure 6: Cytokine production by FOXP3-deficient Th17 and Th1 cell lines. Th1 and Th17
cells transduced with siFOXP3 or control siLuc and purified based on ΔLNGFR expression were
re-stimulated with anti-CD3/anti-CD28-coated beads (1 bead : 32 cells) in the presence of IL-2.
(A) On day 8 after stimulation, cells were washed and re-plated at 1 x 106 cells/mL, and
supernatants were collected 48 hours later for analysis by cytometric bead array. Each dot
represents the fold change in cytokine production by siFOXP3-transduced T cells relative to
control siLuc-transduced T cells for one donor. (B&C) On days 0, 8, and 11 of the expansion,
Th1 and Th17 cells were re-stimulated with PMA and ionomycin and stained intracellularly for
IFN-γ and IL-17A. Analysis was conducted on ΔLNGFR+ cells. (B) Representative plots of IL-
17A and IFN-γ expression 11 days after activation. (C) The average fold changes in the percent
of IFN-γ+IL-17- Th17 cells (n = 4), IFN-γ+ of IL-17+ Th17 cells (n = 4), and IFN-γ+ Th1 cells (n
= 3) in siFOXP3 relative to control siLuc over the course of the experiment. At day 8 after
activation, the fold change in the percent of IFN-γ+IL-17- Th17 cells is significant (p = 0.0436)
and at day 11 after activation, the fold change in the percent of IFN-γ+ of IL-17+ is significant (p
= 0.0033).
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Figure 7: Cell surface marker expression in FOXP3-deficient Th1 and Th17 cells. Th1 and
Th17 cells transduced with siFOXP3 or control siLuc and purified based on ΔLNGFR expression
were re-stimulated with anti-CD3/anti-CD28-coated beads (1 bead : 32 cells) in the presence of
IL-2. At the indicated days after activation, cell surface marker expression was determined by
flow cytometry. Analysis was conducted on ΔLNGFR+ cells. The top panels of A-D show mean
fluorescence intensities (MFIs) of one representative experiment. The bottom panels of A-D
show the average fold change in MFI of siFOXP3 relative to siLuc (MFI siFOXP3/ MFI siLuc).