Galectin-9 Activates and Expands Human T-Helper 1 Cells
et al. (2013) Galectin-9 Activates and Expands Human T-Helper 1 Cells. PLoS
ONE 8(5): e65616. doi:10.1371/journal.pone.0065616
Galectin-9 Activates and Expands Human T-Helper 1 Cells
Marloes J. M. Gooden 0
Valerie R. Wiersma 0
Douwe F. Samplonius 0
Jurjen Gerssen 0
Robert J. van Ginkel 0
Hans W. Nijman 0
Mitsuomi Hirashima 0
Toshiro Niki 0
Paul Eggleton 0
Wijnand Helfrich 0
Edwin Bremer 0
Kjetil Tasken, University of Oslo, Norway
0 1 Department of Surgery, Translational Surgical Oncology, University Medical Center Groningen (UMCG), University of Groningen , Groningen , The Netherlands , 2 Department of Obstetrics and Gynecology, University Medical Center Groningen (UMCG), University of Groningen , Groningen , The Netherlands, 3 GalPharma Co. , Ltd., Kagawa, Japan, 4 Department of Immunology and Immunopathology, Kagawa University Faculty of Medicine, Kagawa, Japan, 5 University of Exeter Medical School , Exeter , United Kingdom
Galectin-9 (Gal-9) is known for induction of apoptosis in IFN-c and IL-17 producing T-cells and amelioration of autoimmunity in murine models. On the other hand, Gal-9 induced IFN-c positive T-cells in a sarcoma mouse model and in food allergy, suggesting that Gal-9 can have diametric effects on T-cell immunity. Here, we aimed to delineate the immunomodulatory effect of Gal-9 on human resting and ex vivo activated peripheral blood lymphocytes. Treatment of resting lymphocytes with low concentrations of Gal-9 (5-30 nM) induced apoptosis in ,60% of T-cells after 1 day, but activated the surviving Tcells. These viable T-cells started to expand after 4 days with up to 6 cell divisions by day 7 and an associated shift from nave towards central memory and IFN-c producing phenotype. In the presence of T-cell activation signals (anti-CD3/IL-2) Gal-9 did not induce T-cell expansion, but shifted the CD4/CD8 balance towards a CD4-dominated T-cell response. Thus, Gal-9 activates resting T-cells in the absence of typical T-cell activating signals and promotes their transition to a TH1/C1 phenotype. In the presence of T-cell activating signals T-cell immunity is directed towards a CD4-driven response by Gal-9. Thus, Gal-9 may specifically enhance reactive immunological memory.
Funding: This work was supported by Dutch Cancer Society grants RUG 2009-4355 (E.B.), RUG2009-4542/RUG2011-5206 (E.B/W.H.) and RUG2007-3784 (W.H.), the
Netherlands Organization for Scientific Research (E.B.), the Melanoma Research Alliance (E.B.), the Alexander von Humboldt Foundation (E.B.) and the European
Communitys Seventh Framework Programme (FP7/20072013) under grant agreement (grant number 215009) (P.E.). The funders had no role in study design,
data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have the following interests: Drs. Niki and Hirashima are board members of GalPharma Co., Ltd. The authors have a product
in development: stable-form galectin-9. Name of the patent: NOVEL MODIFIED GALECTIN 9 PROTEINS AND USE THEREOF. Patent numbers: EP1736541,
JP4792390, US8268324. There are no further patents, products in development or marketed products to declare. This does not alter the authors adherence to all
the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.
. These authors contributed equally to this work.
" These authors also contributed equally to this work.
The galectin family is a group of glycan-binding proteins
characterized by conserved carbohydrate recognition domains
(CRDs) that bind glycosylated proteins. Galectins are involved in
various processes including embryonic development, tumor
biology and regulation of the immune system . Within this
family, Galectin-9 (Gal-9) has gained attention as a multifaceted
player in adaptive and innate immunity, in particular in T-cell
development and homeostasis . The most prominent effects
reported for Gal-9 are the induction of apoptosis in subsets of
differentiated T-cells, particularly in CD4+ T-helper 1 (TH1) and
T-helper 17 (TH17) cells [3,4,5,6,7], and a stimulatory effect on
regulatory T-cell (Treg) activity [6,8]. In view of these
immunomodulatory effects, Gal-9 has been tested as a potential
therapeutic agent for various autoimmune diseases. Treatment
with Gal-9 ameliorated disease in mouse models of experimental
autoimmune encephalomyelitis , arthritis  and diabetes
[10,11], by reducing the number of autoreactive TH1 and TH17
cells and decreasing circulating IFN-c concentrations. In contrast,
treatment with Gal-9 stimulated anti-tumor T-cell immune
responses in a sarcoma bearing mouse model . Here,
recombinant Gal-9 induced cytotoxic T-cells (CTLs) and
increased IFN-c concentrations. In addition, in a recent study
focused on food-allergy treatment of ex vivo activated human
Tcells with Gal-9 promoted TH1 generation as well as IFN-c
production . These data imply that Gal-9 can have a
Januslike dual activity; inhibiting immunity in autoimmune disease on
the one side and stimulating immunity in cancer and allergy on the
The immunomodulatory effects of Gal-9 were initially
attributed to signaling via T-cell immunoglobulin and mucin domain-3
(TIM-3) , a prominent T-cell inhibitory receptor and a marker
for T-cell exhaustion that is currently being evaluated as a target
for antibody-based therapy in cancer . However, it has
become clear that, aside from TIM-3, Gal-9 can signal via other
receptors on T-cells , like protein disulfide isomerase [16,17],
CD40  and possibly other, yet unidentified receptors. Indeed,
the outcome of Gal-9 signaling on T-cells likely depends on the
specific receptor being activated by Gal-9 as well as the presence of
additional (T-cell) skewing stimuli. In this respect, most
experimental murine autoimmune models used to evaluate therapeutic
effects of Gal-9 rely on specific antibodies or disease inducing
peptides in combination with infection stimulating adjuvants and/
or bacteria [3,19,9]. In contrast, the CTL stimulatory effects via
dendritic cell (DC) activation and induction of IFN-c found in a
sarcoma did not require additional skewing stimuli . Together,
this suggests that the outcome of Gal-9 signaling varies greatly,
depending on experimental conditions and/or the balance of
immunity in specific disease settings.
Here, we aimed to establish the effect of Gal-9 treatment on
freshly isolated and non-skewed human peripheral blood immune
cells in the absence of other stimuli. In line with current thinking,
Gal-9 triggered cell death in .95% of T-cells at high
concentrations. However, at lower doses, Gal-9 activated and strongly
expanded surviving T-cells in a TIM-3-independent manner. In
addition, the T-cell expansion induced by Gal-9 was characterized
by a shift from a nave towards a central memory (Tcm) and IFN-c
producing TH1 phenotype. Further, a shift in monocytes towards a
DC-phenotype was detected. However, monocyte depletion did
not affect T-cell activation, indicating that Gal-9 had a direct effect
on T-cells. In the presence of anti-CD3/IL-2 T-cell activation
signals, Gal-9 did not trigger expansion of T-cells, but shifted the
normal CD8/CD4 balance towards a predominant CD4+
phenotype. Taken together, these data indicate that Gal-9 has
diverse immunomodulatory effects depending on concentration
and skewing signals available and is therefore likely to have a
disease-specific role that needs to be evaluated in depth for both
autoimmunity and cancer.
Materials and Methods
Antibodies & reagents
The following fluorophore-conjugated anti-human antibodies
from Immunotools (Friesoythe) were used in this study;
anti-CD3FITC (MEM-57), anti-CD3-Dy647 (MEM-57), anti-CD4-PE
(MEM-241), anti-CD4-PE/Dy647 (MEM-241), anti-CD8-Pe/
Dy47 (MEM-31), anti-CD14-Dy647 (MEM-15), anti-CD19-APC
(LT19), anti-CD25-PE (MEM-181), anti-CD62L-FITC
(LTTD180). The following anti-human antibodies were from
eBioscience; anti-CD8-PEcy7 (RPA-T8), anti-CD45RO-APC
(UCHL1), anti-CD56-APC (MEM188), anti-TIM-3-APC
(F382E2), anti-PD-1-PERCP (eBioI105), anti-CCR7-PerCP-Cy5.5
(3D12), anti-FoxP3-APC (236A/E7), anti-IL-2-PERCP-Cy5.5
(MQ1-17H12), anti-IL-4-PE-Cy7 (8D4-8),
anti-IFNy-PERCPCy5.5 (4S-B3), and anti-IL-17-PERCP-Cy5.5 (eBio64DEC17).
The anti-CD3-CyQ antibody was from IQ-products (IQP-519C).
Streptavidin-Alexa488 was from Invitrogen. Recombinant Gal-9
(truncated form Gal-9(0)) and the physiologically occurring short
isoform of Gal-9 (Gal-9(S)) were produced as described before
. Gal-1, Gal-2, Gal-3, Gal-8 were purchased commercially
Isolation of peripheral blood mononuclear cells (PBMCs)
and activation of T-cells
Peripheral blood mononuclear cells (PBMCs) were obtained
from venous blood from healthy volunteers using standard density
gradient centrifugation (Lymphoprep). The ethics review board of
the Multi- Regional Ethics Committee approved the study
(MREC 06/Q2102/56), and all blood samples were obtained
with written consent from the healthy subjects. Activated T-cells
were generated by culturing PBMCs with anti-CD3 mAb (0.5 mg/
mL; 72 h, UCHT1, Immunotools) followed by 96 h IL-2 (100 ng/
mL, Immunotools). Monocyte depletion prior to treatment was
performed by magnetic-activated cell sorting (MACS) using
antiCD14-beads (Miltenyi Biotec), resulting in .99% depletion of
monocytes as verified by flow cytometry (data not shown). Cells
were cultured at 37uC exposed to 21% O2/5% CO2 in X-VIVO
medium (Lonza), a chemically defined and serum-free
hematopoietic cell medium, or RPMI-1640 (Lonza) supplemented with
10% v/v FBS (Thermo Scientific). Cell numbers were quantified
using a cell counter (Sysmex).
All flow cytometric analyses were performed on a BD Accuri C6
flow cytometer (BD Biosciences) and accessory CFlow Plus analysis
software. Positively and negatively stained populations were
calculated by quadrant dot plot analysis (for representative
dotplots of stainings see Figures S1S3). For cell surface marker
analysis with antibodies, viable cells were gated based on forward
and sideward scatter plot (for example see Figure S1B).
Assessment of apoptosis and Gal-9 binding
PBMCs were treated for 17 days with medium with or without
the indicated concentration of Gal-9 and analyzed for apoptosis
using flow cytometric staining for phosphatidylserine exposure
using an Annexin-V-FITC staining kit according to the
manufacturers protocol (Immunotools). In brief, cells were washed once
with calcium binding buffer, resuspended in calcium buffer with
Annexin-V, incubated for 10 min at 4 C, and analyzed by flow
cytometry. To determine if Gal-9 bound to PBMCs in a
CRDdependent manner, competitive binding experiments were
performed in which cells were incubated with biotinylated Gal-9 for 1
h, in the presence and absence of 40 mM a-lactose, followed by 3
washes to remove excess and non-bound Gal-9. Cell surface
binding was detected using streptavidin-Alexa488.
Phenotype assessment was determined by immunofluorescent
flow cytometric staining essentially as described before .
Briefly, distribution of cell populations was analyzed gating on
peripheral blood lymphocytes from forward scatter/sideward
scatter, detecting CD3+ and CD19+ populations. In addition,
CD32/CD192 cells were gated and analyzed for CD56-PE
binding. The balance of CD4 and CD8 was analyzed within the
viable cell population gated from forward scatter/sideward scatter
using anti-CD4-PE and anti-CD8-PEcy7. TEM staining was
performed on 0.56106 cells per condition by incubation of
PBMCs with APC-conjugated anti-CD45RO,
PERCP-Cy5-conjugated anti-CCR7, and FITC-conjugated anti-CD3 for 30 min at
room temperature in the dark. Alternatively, cells were stained
with FITC-conjugated anti-CD62L, CyQ-conjugated anti-CD3
and APC-conjugated CD45RO. Isotype-matched non-specific
antibodies were used as negative controls. Staining was analyzed
on viable cells on forward scatter/sideward scatter plot selected
within the CD3+ cell population.
Carboxyfluorescein succinimidyl ester (CFSE) analysis
To determine the number of cell divisions after Gal-9 treatment,
a CFSE cell proliferation kit was used according to manufacturers
protocol (CellTraceTM CFSE Cell Proliferation Kit, Invitrogen).
In brief, harvested PBMCs were stained with 2.5 mM CFSE in
0.1% w/v BSA/PBS for 10 min at 37uC. Subsequently, PBMCs
Figure 1. Gal-9 triggers TIM-3-independent cell death and PBMC expansion. A. Resting PBMCs were incubated with anti-TIM3-APC or
isotype control antibody or B. with streptavidin-Alexa488, biotinylated Gal-9+streptavidin-Alexa488 in the presence or absence of a-lactose. Cell
surface staining was evaluated by flow cytometry C. Resting PBMCs (n = 3) were treated with medium or 15 nM of recombinant Gal-9 for up to 7
days. Cell death was determined by Annexin-V staining. D. PBMCs (n = 8) treated as in (C) were analyzed for cell number. E. Resting PBMCs (n = 8)
were treated as in (C) in the presence of a-lactose or sucrose, and analyzed for cell number. F. Resting PBMCs (n = 3) were treated with medium or
15 nM of recombinant Gal-9, Gal-1, Gal-2, Gal-3, or Gal-8 for up to 7 days, after which cell number was determined. All graphs represent mean +/2
were incubated in fresh medium for 10 min on ice and excess dye
was removed by 3 washes. CFSE-stained cells were then used to
establish in vitro cell cultures. During a time course of 7 days, cells
were harvested and an additional staining was performed,
incubating CFSE-labeled PBMCs with anti-CD3-Dye647,
antiCD4-PE/Dye647, anti-CD8-PE/Dye647, anti-CD19-APC or
CD56-APC for 30 min at 4uC. Staining was analyzed gating on
the cell populations positive for the different phenotypic cellular
markers, in which their CFSE-staining pattern was detected.
TH1, TH2, TH17 and regulatory T-cell (Treg)
To determine the effect of Gal-9 treatment on TH1, TH2, TH17,
PBMCs were treated for 7 days with medium supplemented with
or without 15 nM Gal-9, then washed and subsequently
stimulated with phorbol 12-myristate 13-acetate (PMA) for 4 h.
Subsequently, cells were washed in wash buffer (PBS, 5% v/v
FBS, 0.1% w/v sodium azide) and stained with FITC-conjugated
anti-CD3 for 15 min at room temperature. Cells were fixed with
Reagent A (Caltag) for 10 min. After washing, cells were
resuspended in permeabilization Reagent B (Caltag) and labeled
with PERCP-Cy5-conjugated anti-IL-2, PERCP-Cy5-conjugated
anti-IL-4, PERCP-Cy5-conjugated anti-IFN-c, or
PERCP-Cy5conjugated anti-IL-17 for 20 min in the dark. Cells were analyzed
for T-cell phenotype after 2 subsequent washes with PBS.
Cytokine staining was performed on CD3+-gated cells. Treg
staining was performed by cell surface staining of PBMCs with
PE-conjugated anti-CD4, FITC-conjugated anti-CD3, fixation/
permeabilization as for cytokine staining above, and subsequent
intracellular staining for the transcription factor FoxP3 with
APCconjugated anti-FoxP3. Staining for FoxP3 was assessed within the
CD3 and CD4 double-positive population of cells.
Analysis of cytokine secretion
ELISAs were used to quantify the secretion of IFN-c and IL-17
from PBMCs treated with and without Gal-9. In brief, in vitro
experiments were set-up as described above and supernatant of
treated cells was collected at day 7. The IFN-c (Immunotools) and
IL-17 (Thermo Scientific) ELISAs were performed according to
Statistical analysis was performed by one-way ANOVA
followed by Tukey-Kramer post-test or, where appropriate, by
two-sided unpaired Students t-test using Prism software. p,0.05
was defined as a statistically significant difference. Where indicated
* = p,0.05; ** = p,0.01; *** = p,0.001.
Galectin-9 triggers cell death but also expands resting
peripheral blood cells
Initial evidence suggested that the predominant effect of
treatment of T-cells with Gal-9 is the induction of apoptotic cell
death in T-helper 1 (TH1) and T-helper 17 (TH17) cells via the
receptor TIM-3 . However, flow cytometric analysis on resting
PBMCs revealed that expression of TIM-3 was negligible, whereas
ex vivo activated T-cells did express TIM-3 (Figure 1A and Figure
S1A). Despite the lack of cell surface-expressed TIM-3 Gal-9
bound to resting PBMCs (Figure 1B). Binding of Gal-9 was
inhibited in a lectin-specific manner by co-incubation with 40 mM
a-lactose (Figure 1B), but not by an anti-TIM-3 blocking antibody
(data not shown). Thus, the binding of Gal-9 was carbohydrate
recognition domain (CRD)-dependent and TIM-3-independent.
Treatment of PBMCs with Gal-9 at a concentration of 150 nM,
falling within the previously published range, eliminated the vast
majority of cells within 1 day (Figure S1B). However, a 10-fold
lower concentration of Gal-9 (15 nM) only triggered cell death in
,half of the cells (Figure S1B). Quantification of cell death during
prolonged treatment of up to 7 days, revealed that the percentage
of apoptotic cells in Gal-9 treated conditions gradually declined
from ,60% at day 1 to that of medium control within 4 days
(Figure 1C). In line with this, treatment with 15 nM Gal-9
decreased cell counts compared to control at days 13 (Figure 1D).
This initial depletion was followed by strong PBMC expansion, as
evidenced by a 3-fold increase in cell count at day 7 from 16106
cells/ml to ,36106 cells/ml in Gal-9 treated conditions (Figure
1D). This PBMC expansion by Gal-9 was inhibited by
cotreatment with a-lactose, but not sucrose, and thus
CRDdependent (Figure 1E).
Various other immunomodulatory members of the galectin
family, i.e. Galectin-8, which is a tandem-repeat Galectin like
Gal9, as well as Galectin-1, Galectin-2 (prototypic Galectins) and
Galectin-3 (chimeric Galectin) did not trigger significant increase
of PBMC counts at these concentrations (Figure 1F), nor
significant induction of apoptosis (data not shown). Thus, in
addition to initial induction of apoptosis, Gal-9 triggers expansion
of peripheral blood cells.
Galectin-9 and the short isoform of Gal-9 (Gal-9(S))
dosedependently activate T-cells
To specify the stimulatory effect of Gal-9 on the PBMC
fraction, the relative proportion of T-cells, B-cells and NK-cells
was analyzed in medium and Gal-9 treated PBMCs. In medium
control, the percentages of all lymphocyte populations remained
stable from day 07 (Figure 2A). In contrast, treatment with Gal-9
reduced the percentage of CD3+ positive T-cells during the first 3
days, followed by a concomitant increase in T-cells and decrease in
B-cells and NK-cells from day 4 onwards (Figure 2B). Further, in
the viable population of CD3+ T-cells, treatment with 15 nM
Gal9 also triggered T-cell activation, as evidenced by the upregulation
of surface CD25-expression from ,50% at day 1 to a maximum of
,80% after 57 days (Figure 2C, representative dot-plots in
Figure S1C). Activation of T-cells by Gal-9 was inhibited by
cotreatment with a-lactose and thus CRD-dependent (Figure 2D).
Other galectin family members did not trigger T-cell activation
(Figure S1D). Interestingly, during this time-course, the expression
of TIM-3 was induced in only 2030% of T-cells after 47 days of
treatment with Gal-9 (Figure 2E). These T-cells did not become
double-positive for TIM-3 and PD-1 (Figure S2E), a marker
profile associated with T-cell exhaustion . Thus, Gal-9
interacted with T-cells via an as yet unidentified receptor, resulting
in apoptosis of part of the T-cells. However, the remaining
population of viable T-cells underwent activation as well as strong
In addition to lymphocytes, the isolated PBMC fraction
contains monocytes. These monocytes responded to Gal-9
treatment with a marked stretching and loss of CD14 expression,
indicative of a shift towards a DC-phenotype (Figure S2A).
Importantly, depletion of monocytes from the PBMC population
prior to Gal-9 treatment did not affect Gal-9-induced activation of
T-cells, as determined by CD3/CD25 double staining (Figure 2F).
Thus, although Gal-9 activated monocytes, this was not required
for activation and expansion of T-cells.
In addition to recombinant Gal-9, the short isoform of Gal-9
(Gal-9(S)) was included to evaluate whether naturally occurring
isoforms of Gal-9 could have similar immunomodulatory effects.
Both Gal-9 and Gal-9(S) dose-dependently triggered activation
and expansion of T-cells, with maximal effects of recombinant
Gal-9 at 15 nM (0.5 mg/ml) and Gal-9(S) at 30 nM (1 mg/ml)
(Figure 2G and 2H). Thus, both recombinant Gal-9 and Gal-9(S)
triggered dose-dependent activation of T-cells at low
Galectin-9 treatment results in expansion of CD4+ T-cells
The effect of treatment of T-cells with Gal-9 on cell division was
evaluated by determining T-cell proliferation by CFSE staining. In
control conditions, the CD3+ T-cells did not divide during the 7
day time-course, as evidenced by a single CFSE fluorescence peak
(Figure 3A). In contrast, treatment with Gal-9 triggered up to 6 cell
divisions within the CD3+ T-cell population (Figure 3B), with
,7% of T-cells having divided 6 times at day 7 (Figure 3C). In line
with the data on apoptosis and cell counts, T-cell division was first
detected after 3 days of Gal-9 treatment (Figure 3D).
Further analysis within the Gal-9 treated populations identified
that most dividing T-cells were CD4+ (,76% of total PBMCs at
day 7, Figure 3E), although the remaining CD8+ T-cells (,20%)
also divided in the final days (Figure 3F). In line with this, Gal-9
treatment induced a steady increase in the percentage of CD4+
Tcells during the 7 day time-course, whereas the percentage of
CD8+ T-cells initially decreased, but returned to base levels at day
7 (Figure 3G; representative dot-plot in Figure S2B). Again, Gal-9
induced effects were CRD-specific as a-lactose completely
inhibited cell division in both CD4+ and CD8+ T-cells (Figure
3E and 3F).
Galectin-9 expands central memory and T-helper 1 cells
As Gal-9 clearly activated and expanded T-cells, the relative
proportion of nave, central memory (TCM), and effector memory
(TEM) phenotypes in the PBMCs was evaluated (for representative
dot-plots of staining see Figure S2C). As expected, the majority of
T-cells in medium control were of a nave CCR7+/CD45RO2
phenotype (Figure 4A). However, upon treatment with Gal-9 or
Gal-9(S), the percentage of nave T-cells was strongly and
statistically significantly reduced (Figure 4A), with the majority
of T-cells acquiring a central CCR7+/CD45RO+ memory
phenotype (Figure 4B). This dramatic shift was confirmed by a
second staining for central memory using CD62L and CD45RO,
which revealed a similar increase in CD62L+/CD45RO+ TCM
(Figure S2D). In contrast, no significant changes were detected in
the CCR72/CD45RO+ TEM population (Figure 4C).
To characterize T-cells expanded by Gal-9 on a more
functional level, cytokine profiles were determined in medium
control and Gal-9 treated T-cells (see Figure S3 for representative
dot-plots). In line with the immunophenotyping data, there was a
marked and statistically significant increase in IL-2 producing
Tcells, indicative of TCM cells (Figure 4D). In addition, treatment
with Gal-9 resulted in a statistically significant increase in IFN-c
producing T-cells (Figure 4E), but not in IL-17 and IL-4
producing T-cells (Figure 4F and 4G, respectively). In line with
these findings, analysis of secreted cytokines within the supernatant
of Gal-9 treated cells revealed an ,100-fold increase in IFN-c
levels compared to medium control at day 7 (Figure 4H). Further,
a low but significant increase in IL-17 was also detected upon
treatment with Gal-9 (Figure 4H). In line with earlier findings,
treatment with 15 nM Gal-9(S) but not Gal-9 also triggered a
small but statistically significant increase in regulatory T-cells
(Tregs) after 7 days (Figure 4I). Thus, in resting PBMCs the
treatment with Gal-9 triggers a selective expansion of central
T-cell receptor-mediated activation in the presence of
Gal-9 shifts the balance towards CD4+ T-cells
The results above provide evidence that Gal-9 has a potent
immunomodulatory effect on resting T-cells. This TH1-stimulatory
effect is in line with a recent report in which CD3/CD28
costimulation of T-cells stimulated TH1 development . To
further characterize the modulatory effect of Gal-9 during T-cell
receptor (TCR)-stimulation, PBMCs were stimulated with
antiCD3 antibody for 3 days, followed by IL-2 stimulation for 4 days.
In the absence of Gal-9, this stimulation regime led to a slight
preferential induction of CD8+ T-cells compared to CD4+ T-cells
(Figure 5A and 5B; median CD4+ 42.7% vs. median CD8+ 47%).
However, treatment with Gal-9 or Gal-9(S) induced a dramatic
shift in percentages of CD8+ and CD4+ T-cells after 7 days of
activation (Figure 5A and 5B; median CD4+ 86.1% vs. median
CD8+ 7.2%). Of note, no significant increase in apoptotic cells or
cell counts was detected upon treatment with Gal-9 or Gal-9(S)
after 7 days of treatment (data not shown). TCR/IL-2 mediated
activation of T-cells in the presence of Gal-9 shifted T-cells toward
a central memory phenotype, as defined by immunophenotyping
for CD45RO/CCR7 or CD45RO/CD62L (Figure 5C), and
intracellular cytokine staining for IL-2 (Figure 5D). In contrast to
treatment of resting PBMCs, treatment of anti-CD3/IL-2
activated T-cells with Gal-9 did not trigger an increase in the
percentage of IFN-c producing cells (Figure 5E). Similarly, no
increase in IL-17 or IL-4 producing T-cells or regulatory T-cells
was observed (Figure 5F2H). Taken together, TCR-mediated
activation and IL-2 induced expansion of T-cells in the presence of
Gal-9 shifts the T-cell response towards a CD4+ helper response
that is further characterized by a central memory phenotype.
Gal-9 is a strong modulator of T-cell immunity known for its
apoptotic effects on TH1 and TH17 cells in autoimmunity, but also
for its stimulatory activity on CTLs and TH1 cells in cancer and
food allergy. These apparent diametrical outcomes of Gal-9
signaling on T-cell immunity suggests that the balance of
immunomodulatory signals is crucial for Gal-9 signaling. Data
presented in the current study indicate that single-agent treatment
of resting PBMCs with low Gal-9 doses is, after initial apoptotic
elimination, accompanied by activation and subsequent expansion
of CD4+ T-cells. Furthermore, Gal-9 treatment shifts T-cells from
a nave to a central memory phenotype and increases the
percentage of IFN-c producing T-cells. In activated (anti-CD3/
IL-2) T-cells, Gal-9 skews the CD4+/CD8+ balance towards a
As evident from the data, the reported differences in T-cell
responses upon Gal-9 treatment can be partly ascribed to the
amount of Gal-9 that is used. In particular, a concentration of
150 nM eliminated the vast majority of T-cells within one day,
whereas a dose-response analysis demonstrated that 1530 nM
Gal-9 is the optimal concentration for T-cell stimulatory effects. In
the presence of 15 nM Gal-9. C. Analysis of (A) and (B) showing percentage of CD3+ T-cells in the respective peak of all independent experiments
(mean +/2 SEM). D. Analysis of (B) showing the number of CFSE peaks of all independent experiments. E. Representative plots of 3 independent
experiments of resting PBMCs stained with CFSE and subsequently incubated in medium or 15 nM Gal-9 (+/2 lactose) for up to 7 days. At Day 7,
PBMCs were harvested, stained with the T-cell marker CD4, and CFSE peak pattern was analyzed within the CD-3+ cells by flow cytometry. F. As in (E)
but stained for the T-cell maker CD8. G. Resting PBMCs (n = 4) were treated for up to 7 days with medium or Gal-9 and analyzed for CD4 and CD8
distribution. All graphs represent mean +/2 SD unless stated otherwise.
previous reports, T-cell apoptosis was induced with relatively high
doses of up to 1000 nM Gal-9 [23,3]. Furthermore, a recent study
using murine T-cell clones also required high Gal-9 concentrations
to induce T-cell apoptosis (400 nM), whereas non-lethal doses had
T-cell stimulatory effects . In addition to concentration, most
studies were designed to evaluate Gal-9 activity at relatively short
incubation periods varying from 1h to 4 days [3,7,4,23,24]. In the
current study, Gal-9 effects were evaluated for up to 7 days.
Notably, significant T-cell expansion in the current study was only
seen after 4 days of treatment, whereas the induction of apoptosis
Figure 5. Gal-9 treatment of TCR-activated T-cells reverses CD8/CD4 distribution and shifts T-cells towards a central memory
phenotype. A2B. T-cells were activated by anti-CD3/IL-2, or additionally with 15 nM Gal-9 or Gal-9(S). After 7 days the percentage of CD4 (A) and
CD8 (B) were determined by flow cytometry. C. T-cells were activated as in (A) and percentage of T-cells with central memory phenotype was
determined. D2G. T-cells were activated as in A, after which T-cell cytokine production was analyzed by flow cytometry as described in M&M. The
percentage of IL-2 (n = 11) (D), IFN-c (E), IL-17 (F) and IL-4 (G) was determined. H. T-cells were activated as in (A), after which the percentage of
regulatory T-cells was determined. Unless indicated otherwise (n = 12).
occurred rapidly within 1 day. Of note, these effects were
independent of TIM-3, as freshly isolated T-cells lacked TIM-3
expression, and only ,35% of T-cells became TIM-3+ after 4-7
days of Gal-9 treatment. Such TIM-3 independent binding has
been described earlier [15,6,3,7], and several alternate binding
partners have been reported, e.g. CD40 , several adhesion
molecules [25,26], immunoglobulin E (IgE) , and protein
disulfide isomerase [16,17]. Whether the TIM-3 independent
binding observed in the current study can be attributed to binding
of Gal-9 to one of these alternate receptors, or an as yet
unidentified receptor, is subject of an ongoing study.
Binding of Gal-9 to resting blood lymphocytes activated T-cells
and shifted T-cell phenotype from nave toward IL-2 producing
central memory T-cells (CD62+/CCR7+/CD45R0+) and IFN-c
producing TH1 cells. This single-agent stimulatory effect of Gal-9
on resting T-cells has not been reported before and appears to
contrast with published studies that describe predominant
elimination of TH1 cells by Gal-9. However, our findings are in
line with several recent reports on the stimulatory effect of Gal-9
on activated TH1 cells [13,15], and the induction of central
memory cytokines by Gal-9 upon CD3/CD40 co-stimulation in
murine T-cells . The mechanism by which Gal-9 induces
Tcell activation and proliferation is currently unknown. However,
other lectins, such as concanavalin A, also potently stimulate T-cell
proliferation. Concanavalin A does so by directly interacting with
activating receptors, like CD3 . Within the Galectin family of
lectins, it has been reported that Galectin-1 can directly bind to
CD3 on T-cells . It was suggested that Galectin-1 mediated
ligation of the CD3-complex mimics antigen-induced TCR
signaling, which induces early events in T-cell activation
comparable to those elicited by agonistic anti-CD3 antibodies.
Therefore, it is tentative to speculate that Gal-9 interacts with
activating receptors such as CD3 on the T-cell surface. This is
currently subject for further study in our laboratory.
Besides direct Gal-9 effects on T-cells, indirect T-cell
stimulation by Gal-9 via the activation of DCs and DC-like macrophages
was reported in sarcoma and melanoma bearing mouse models
[12,25]. Furthermore, Gal-9 induced the maturation of human
monocyte-derived dendritic cells, resulting in IL-12, IL-2 and
IFNc secretion . In line with these findings, monocytes did
undergo phenotypic changes towards DC-phenotype in the
current study. However, monocyte depletion did not affect the
activity of Gal-9 towards resting T-cells, which suggests that Gal-9
has a direct immunostimulatory effect on T-cells. Gal-9 was also
reported to enhance production of IFN-c by NK-cells , but
our analysis of lymphocyte distribution/activation in the current
study showed only an increase in T-cells and a decrease in
NKcells. Further, a clear increase in IFN-c producing T-cells was
detected. Thus, our data suggest that IFN-c is secreted by T-cells
and not NK-cells. In addition to TH1 cells, a statistically significant
increase in Treg cells by Gal-9(S) was detected, which is in line with
recent murine studies [32,33].
The here reported activating effect of low dose Gal-9 on resting
T-cells may be of relevance in certain human diseases. For
instance, elevated Gal-9 serum levels were detected in patients
with type 2 diabetes and chronic kidney disease . Here, serum
Gal-9 levels negatively correlated with renal function and
increased along with disease progression. Of note, aberrant
recruitment and activation of T-cells has been described in
diabetic nephropathy . Hence, Gal-9 may be involved in
Tcell activation, which contributes to kidney damage in diabetes
type 2. In addition, dietary supplementation of pre-biotic
oligosaccharides and Bifidobacterium breve reduced allergic symptoms
in a murine model of food allergy and in infants with atopic
dermatitis, as an effect of increased serum Gal-9 levels and
subsequent TH1 and Treg responses . Thus, depending on type
of disease and immunological environment, serum Gal-9 can have
both positive and negative effects on disease progression by the
activation of T-cells.
In conclusion, treatment of human resting blood T-cells with
Gal-9 induces apoptosis in a substantial proportion of the cells, but
also activates and expands IFN-c producing TH1 cells and central
memory T-cells in surviving population. In the presence of
activating signals (anti-CD3/IL2), the treatment with Gal-9 does
not expand T-cells, but skews the CD4+/CD8+ balance towards a
CD4+ phenotype. This study thus uncovers the stimulatory effect
of Gal-9 treatment on resting lymphocytes and highlights the
complexity of immunomodulatory signaling by Gal-9 on human
T-cells. Indeed, in various diseases settings the influence of Gal-9
on T-cell immunity will be determined by micro-environmental
concentrations of Gal-9 and other immune modulators as well as
the activation status of T-cells.
Figure S1 A. T-cells were activated with anti-CD3 (72h) and
IL2 (96h), after which cell surface expression of TIM-3 was analyzed
by flow cytometry. B. resting PBMCs were treated with medium,
15 nM or 150 nM of Gal-9 for 1 day. Representative fsc/ssc
dotplots of lymphocytes demonstrate that treatment with 150 nM of
Gal-9 shifts cells from a viable population (see medium; left panel)
to dead/fragmented distribution (150 nM Gal-9; right panel),
whereas at 15 nM ,50% of cells remain viable (15 nM Gal-9;
middle panel). C. representative flow cytometric dot-plots of
CD3/CD25 staining D. resting PBMCs were treated with
015 nM Gal-1, Gal-2, Gal-3, Gal-8 or Gal-9. E. representative flow
cytometric dot-plots of TIM-3/PD-1 staining in which cells were
pre-gated on presence of CD3.
Figure S2 A. resting PBMCs were treated with Gal-9 for 7 days
after which cell surface expression of CD14 in adhered monocytic
cells was analyzed by flow cytometry. B. representative flow
cytometric dot-plots of CD4/CD8 staining, C. CCR7/CD45RO
staining, D. CD62L/CD45RO staining. For B-D, cells were
pregated on presence of CD3.
Conceived and designed the experiments: DS PE EB WH. Performed the
experiments: VW MG DS JG. Analyzed the data: VW EB. Contributed
reagents/materials/analysis tools: MH TN PE RVG. Wrote the paper:
MG VW EB. Revised the paper: MG VW EB PE HN WH.
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