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LYMPHOID NEOPLASIA
Brief report
Galectin-1 inhibits the viability, proliferation, and Th1 cytokine production of
nonmalignant T cells in patients with leukemic cutaneous T-cell lymphoma
Filiberto Cedeno-Laurent,1,2 Rei Watanabe,1,2 Jessica E. Teague,1 Thomas S. Kupper,1,2 Rachael A. Clark,1,2 and
Charles J. Dimitroff1,2
1Department
of Dermatology, Brigham and Women’s Hospital, Boston, MA; and 2Harvard Medical School, Boston, MA
Tumor-derived galectin-1 (Gal-1), a
␤-galactoside–binding S-type lectin, has
been shown to encourage T-cell death
and promote T cell–mediated tumor immune escape. In this report, we show that
patients with leukemic cutaneous T-cell
lymphomas, known to have limited complexity of their T-cell repertoires, have a
predominant T helper type-2 (Th2) cytokine profile and significantly elevated
plasma levels of Gal-1 compared with
healthy controls. Circulating clonal malignant T cells were a major source of Gal-1.
The conditioned supernatant of cultured
malignant T cells induced a ␤-galactoside–
dependent inhibition of normal T-cell prolif-
eration and a Th2 skewing of cytokine production. These data implicate Gal-1 in
development of the Th2 phenotype in patients with advanced-stage cutaneous T-cell
lymphoma and highlight the Gal-1–Gal-1
ligand axis as a potential therapeutic target
for enhancing antitumor immune responses.
(Blood. 2012;119(15):3534-3538)
Introduction
Contraction of the T-cell repertoire and Th2 cytokine skewing are
2 immunosuppressive features linked to high morbidity and
mortality of patients with leukemic cutaneous T-cell lymphoma
(L-CTCL).1-5 Certain soluble factor(s) expressed by clonal malignant cells or dermal fibroblasts (eg, IL-18, eotaxins) could be
responsible for this immunologic signature; nevertheless, mechanistic evidence on how these cells elicit their immunoregulatory
function is still incomplete.6,7 Recently, the ␤-galactoside binding
protein, galectin-1 (Gal-1), has been implicated in the induction of
a Th2 signature and suppression of antitumor immune responses in
patients with Hodgkin lymphoma.8,9 Gal-1 functions as a homodimer with affinity for N-acetyllactosamine–bearing glycoproteins on effector T cells that, on engagement, induces proapoptotic
and/or immunosuppressive activities.10,11 Gal-1–binding interactions induce deletion of pro-inflammatory T helper (Th)–1 and
Th17 cells but spare naive, regulatory, and Th2 cells, leading to a
tolerogenic environment that favors tumor immune escape.12,13
Indeed, in patients with Hodgkin disease, increased Gal-1 expression is correlated with poor prognoses and implicated as a
biomarker of disease progression.14
Although a prior study shows that Gal-1 expression is elevated
in epidermotropic malignant T cells in patients with mycosis
fungoides,15 its presence and role in more advanced leukemic
forms of CTCL have not been addressed. Here we show that clonal
malignant T cells in patients with stage 3 or 4 CTCL exhibited a
Th2 cytokine signature and high intracellular Gal-1 expression.
Moreover, L-CTCL patients exhibited higher plasma Gal-1 levels
than healthy controls, and conditioned supernatants from primary
L-CTCL cell cultures significantly attenuated normal T-cell proliferation and diminished Th1 responses in a ␤-galactoside–
dependent manner. Collectively, these data suggest that elevated
levels of Gal-1 in L-CTCL patients may inhibit the proliferation of
nonmalignant T cells and favor Th2 skewing, leading to impaired
antitumor responses and increased susceptibility to infection.
These studies suggest that neutralization of Gal-1–Gal-1 ligand
interactions may be an effective strategy for enhancing antitumor
immune responses in L-CTCL patients.
Methods
PBMCs and plasma samples
All studies were performed in accordance with the Declaration of Helsinki and
approved by the review board of the Partners Human Research Committee.
PBMCs and plasma were obtained using Ficoll separation from venous blood
samples of patients with stage 3 or 4 CTCL (WHO/TNM classification) or
healthy controls seen at the Dana-Farber/Brigham and Women’s Cancer Center
(supplemental Table 1A-B, available on the Blood Web site; see the Supplemental Materials link at the top of the online article).
ELISA
The 96-well plates were coated with goat anti–human Gal-1 (R&D
Systems), and undiluted plasma samples and standard control, recombinant
human Gal-1 (PeproTech), were incubated for 1 hour at room temperature.
Bound Gal-1 was detected using rabbit anti–hGal-1-biotin/streptavidinHRP (LS Bio), followed by spectrophotometry analysis. ELISA reagents
were purchased from BD Biosciences.
Flow cytometry
PBMCs were stained with anti-CD3, CD4, CD7, CD8, CD45RO mAbs
(BD Biosciences), or anti-specific T-cell receptor (TCR)–v␤ clones
(Beckman Coulter). Intracellular Gal-1 detection was assessed with a
rabbit anti–hGal-1 Ab (LS Bio)/anti–rabbit IgG-FITC (BioLegend).
Gal-1 ligand detection was performed using Gal-1hFc as previously
reported.10,16,17
Submitted December 2, 2011; accepted February 23, 2012. Prepublished
online as Blood First Edition paper, March 1, 2012; DOI 10.1182/blood-201112-396457.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
The online version of this article contains a data supplement.
© 2012 by The American Society of Hematology
3534
BLOOD, 12 APRIL 2012 䡠 VOLUME 119, NUMBER 15
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BLOOD, 12 APRIL 2012 䡠 VOLUME 119, NUMBER 15
L-CTCL-DERIVED Gal-1 IS AN IMMUNOSUPPRESSANT
3535
Figure 1. Patients with L-CTCL exhibit a contracted T-cell repertoire and Th2 cytokine profile. (A) PBMCs from patients with stage 4 CTCL or healthy controls
(n ⫽ 3/group) were analyzed by flow cytometry. Representative plots show cells analyzed for CD3 and CD4 expression. Arrow indicates contraction of the CD3⫹/CD4⫺
population. (B) Th cells from patients with stage 4 CTCL show increased expression of a single malignant TCR-V␤ clone. Representative plot is shown. (C) PBMCs from
healthy controls or L-CTCL patients (n ⫽ 3/group) were stimulated ex vivo with PMA/ionomycin/brefeldin A, and intracellular levels of Th2 cytokines were analyzed by flow
cytometry. Representative plot of L-CTCL patients is shown. (D) Graphical representation of data from all L-CTCL and health control donors depicting mean percentage of
CD4⫹ cells expressing IL-4, IL-13, and IL-10. Statistically significant difference compared with healthy controls using Student paired t test: *P ⱕ .05, **P ⱕ .001. (E) PBMCs
from healthy controls or L-CTCL patients were stimulated ex vivo with PMA/ionomycin/brefeldin A, and intracellular levels of IL-10 were analyzed in CD8⫹ T cells by flow
cytometry. A representative plot is shown. (F) Graphical representation of undiluted plasma samples from patients with advanced-stage CTCL (triangles, n ⫽ 7) and healthy
controls (squares, n ⫽ 5) were analyzed for Gal-1 expression by ELISA. Experiments were performed in triplicate. (G) PBMCs from L-CTCL patients or healthy controls were
gated on CD3⫹/CD4⫹ and analyzed for specific TCR-V␤ expression and intracellular Gal-1. A representative plot is shown.
Cell death, cytokine expression, and proliferation assays
L-CTCL cells (⬎ 90% purity) from advanced-stage CTCL patients were
cultured for 24 hours in X-VIVO15 medium (Lonza Walkersville) at a
density of 8 ⫻ 106 cells/mL. T cells from healthy donor PBMCs were
activated for 48 hours with anti–human CD3 (OKT3, BioLegend) and
further incubated with L-CTCL–conditioned medium or with 0.25␮M
Gal-1hFc (with or without 50mM lactose) for an additional 24 hours. T cells
were stained with allophycocyanin–annexin V and propidium iodide or
alternatively were stimulated for additional 5 hours with phorbol myristate
acetate (PMA)/ionomycin/brefeldin A and analyzed for intracellular cytokines by FACS. Experiments included supernatants from 3 different
L-CTCL donors (supplemental Table 1B) and were performed in triplicate.
For proliferation assays, CFSE-labeled T cells were cultured in L-CTCL–
conditioned medium plus IL-2 (25 U/mL, PeproTech; with or without
50mM lactose) or with 0.25␮M Gal-1hFc for an additional 72 hours
followed by FACS analysis.
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3536
CEDENO-LAURENT et al
BLOOD, 12 APRIL 2012 䡠 VOLUME 119, NUMBER 15
Figure 2. Gal-1 secreted from L-CTCL cells dampens T-cell proliferation and promotes Th2 cytokine skewing. (A) Intracellular Gal-1 expression in Th cells from patients with
L-CTCL or healthy controls (n ⫽ 3/group) was analyzed by flow cytometry and presented as a contour plot. Arrows indicate Gal-1 high, intermediate, and low (“lo”) populations.
(B) Graphical representation of intracellular Gal-1 levels quantified by mean fluorescence intensity (MFI). Statistically significant difference compared with healthy controls using Student
paired t test. (C) Graphical representation of Gal-1hi Th cells in stage 4 CTCL patients or healthy controls. Statistically significant difference compared with healthy controls using Student
paired t test. (D) PBMCs from healthy donors were gated on CD8⫹, and expression of Gal-1 ligands (Gal-1hFc binding) on CD45RO⫹ memory cells was determined by flow cytometry.
Coexpression of CD7 in CD45RO⫹/⫺ CD8⫹ T cells was also analyzed. Representative plots are shown. (E) Activated T cells from healthy donors (n ⫽ 3) were incubated with conditioned
medium from primary L-CTCL cultures (with or without lactose) for 24 hours and then analyzed for annexin V binding. Statistically significant difference compared with lactose-treated
control using Student paired t test: **P ⱕ .001. (F) Alternatively, activated T cells were labeled with CFSE, incubated with L-CTCL–conditioned medium (with or without lactose) or with
0.25␮M Gal-1hFc (with or without lactose) for 3 days, and CFSE dilution was analyzed by flow cytometry. Activated T cells from healthy donors (n ⫽ 3) were incubated with conditioned
medium from primary L-CTCL cell cultures (with or without lactose; G) or with 0.25␮M Gal-1hFc (with or without lactose; H) for 24 hours and then stained and analyzed for CD3 and
intracellular IFN-␥, IL-4, IL-10, and IL-13. All experiments were performed with L-CTCL–conditioned medium from 3 different donors and performed in triplicate. Statistically significant
difference compared with lactose-treated control using Student paired t test: *P ⱕ .05, **P ⱕ .001.
Results and discussion
Prior reports show that L-CTCL patients have contracted T-cell repertoires, a marked Th1/Th2 cytokine imbalance, and are highly suscep-
tible to opportunistic infections.3 Recently, these findings have been
associated with increased Gal-1 levels in patients with Hodgkin
lymphoma.8 To study Gal-1’s role in the pathogenesis of these immunosuppressive features, we studied circulating T cells in L-CTCL patients
with elevated CD4/CD8 ratios and clonal malignant T cells that could be
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BLOOD, 12 APRIL 2012 䡠 VOLUME 119, NUMBER 15
L-CTCL-DERIVED Gal-1 IS AN IMMUNOSUPPRESSANT
identified by staining with anti–TCR-V␤ mAbs (Figure 1A-B). Clonal
T cells in L-CTCL patients expressed a distinctive Th2 signature
characterized by elevated expression of IL-4 and IL-13 (Figure 1C-D;
supplemental Figure 1A). Notably, we also found increased levels of
IL-10 in clonal malignant CD4⫹ cells and in CD8⫹ T cells of L-CTCL
patients compared with healthy controls (Figure 1C-E).
To determine whether Gal-1 could be contributing to the
immunologic abnormalities observed in patients with L-CTCL, we
quantified plasma Gal-1 levels in L-CTCL patients and compared
them with healthy controls. As reported for other solid and
hematologic malignancies,18,19 Gal-1 plasma levels were significantly elevated in patients with active L-CTCL and diminished
during remission (Figure 1F; supplemental Table 1A). Moreover,
analyses of intracellular Gal-1 and Gal-1 immunoblots of CTCL
HUT78 cell lysates (supplemental Figure 1B) indicated that the
malignant T clones were the main Gal-1 source in the PBMC
compartment of L-CTCL patients (Figure 1G). In addition, we
found that Gal-1 was expressed in circulating T cells at 3 distinct
levels (low, intermediate, and high); and although approximately
50% of T cells in healthy volunteers express Gal-1, their expression
was in the low to intermediate range, compared with clonal
malignant cells, which expressed intermediate to predominantly
high Gal-1 levels (Figure 2A-C; supplemental Figure 1C). Even in
L-CTCL patient 10, who possessed the lowest Gal-1 expression,
virtually no T cells expressed Gal-1 in the low range (supplemental
Figure 1C).
To determine whether Gal-1 overexpression by lymphoma cells
induces Th2 cytokine skewing in L-CTCL patients, we studied the
ability of ␤-galactoside-binding lectins secreted from L-CTCL
cells to alter the viability and cytokine production of nonmalignant
T cells. Using a Gal-1-human immunoglobulin fusion protein
(Gal-1hFc) that functionally mimics native Gal-1,10,17,20 we first
validated Gal-1’s affinity for effector T cells and found that Gal-1
predominantly binds to memory CD45RO⫹ T cells coexpressing
CD7 in a ␤-galactoside–dependent manner (Figure 2D). This
selective binding activity to the effector/memory T-cell compartment strengthened our hypothesis that Gal-1 secreted from clonal
malignant T cells might promote deletion/immunoregulation of
CD8⫹ T cells, promoting abnormally high CD4/CD8 ratios found
in patients with advanced-stages of CTCL. Notably, L-CTCL cells
have been reported to be insensitive to Gal-1–mediated apoptosis,
as they lack CD7, the most remarkable Gal-1 ligand linked to cell
death induction.21,22 To demonstrate that clonal malignant T cells
induce apoptosis and/or regulate the proliferation of nonmalignant
T cells through ␤-galactoside–mediated interactions, we cultured
activated T cells from healthy volunteers with conditioned supernatants of clonal malignant T cells (⬎ 90% purity) or with Gal-1hFc
(with or without competitive inhibitor, lactose) for 24 hours. Our
results show that, although only a minority of T cells cultured with
L-CTCL–conditioned medium underwent apoptosis (annexin V⫹/
propodium iodide⫹), the percentage of T cells positive by annexin
V staining was significantly decreased on addition of ␤-galactoside–
binding lectin inhibitor, lactose (P ⱕ .01, Figure 2E; supplemental
Figure 1D). Moreover, the addition of lactose restored the proliferative capacity of CFSE-labeled T cells incubated with L-CTCL–
conditioned medium or with Gal-1hFc (Figure 2F), suggesting that
3537
a ␤-galactoside–binding lectin secreted by clonal malignant T cells
yielded antiproliferative activity. L-CTCL cells lack CD7, a bona
fide Gal-1 proapoptotic ligand (supplemental Figure 2A), allowing
them to escape Gal-1–mediated cell death, and favoring their
uncontrolled expansion.21-23 Gal-1–mediated effects are not limited
to apoptosis. Prior studies have shown that Gal-1 induces the
production of Th2 and tolerogenic cytokines.10 To determine
whether Gal-1 in L-CTCL–conditioned medium could promote
Th2 skewing of nonmalignant T cells, we incubated T cells from
healthy donors with L-CTCL–conditioned medium or with Gal1hFc and assessed cytokine expression after 24 hours. Compared
with lactose controls, we found that CTCL-conditioned medium
significantly suppressed the production of IFN-␥⫹ T cells (P ⱕ .05)
while markedly increasing the level of IL-4⫹ (P ⱕ .01), IL-10⫹
(P ⱕ .01), and IL-13⫹ T cells (P ⱕ .05). Of note, control Gal-1
incubations elicited a similar induction of Th2 cytokines in T cells
(Figure 2G; supplemental Figure 2B).
In conclusion, our work suggests that L-CTCL–derived Gal-1
may impair the viability, proliferation, and Th1 responses of
nonmalignant T cells, leading to a systemic Th2 bias that favors
tumor survival and probably contributes to the observed susceptibility of these patients to infections. These studies highlight the
pivotal role of Gal-1 in modifying adaptive immune responses and
suggest that inhibition of Gal-1–Gal-1 ligand interactions may be
an effective strategy for enhancing both antitumor and antipathogen responses in patients with L-CTCL.
Acknowledgments
This work was supported by the National Institutes of Health/
National Cancer Institute (RO1 CA118124, C.J.D) and National
Institutes of Health/National Center for Complementary and Alternative Medicine (RO1 AT004268, C.J.D.), a Damon Runyon
Clinical Investigator Award (R.A.C.), National Institutes of Health/
National Institute of Arthritis, Musculoskeletal and Skin Diseases
(R01 AR056720; R.A.C.), National Institutes of Health/National
Cancer Institute (SPORE in Skin Cancer P50 CA9368305, T.S.K.),
and the National Institutes of Health/National Institute of Allergy
and Infectious Diseases (R01 A1025082, T.S.K.).
Authorship
Contribution: F.C.-L. designed the research, performed the experiments, analyzed the data, and wrote the paper; R.W. and J.E.T.
performed the experiments and analyzed the data; T.S.K. and
R.A.C. conceived the study, designed the research, and provided
the reagents; and C.J.D. conceived the study, designed the research,
provided the reagents, supervised all experimentation, analyzed the
data, and wrote the paper.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Charles J. Dimitroff, HIM, Rm 662, 77
Avenue Louis Pasteur, Boston, MA 02115; e-mail: cdimitroff@rics.
bwh.harvard.edu.
References
1. Yamanaka K, Fuhlbrigge RC, Mizutani H, et al. Restoration of peripheral blood T-cell repertoire complexity during remission in advanced cutaneous T cell
lymphoma. Arch Dermatol Res. 2010;302(6):453-459.
2. Yamanaka K, Yawalkar N, Jones DA, et al. Decreased T-cell receptor excision circles in cutaneous T-cell lymphoma. Clin Cancer Res. 2005;
11(16):5748-5755.
3. Yawalkar N, Ferenczi K, Jones DA, et al. Profound
loss of T-cell receptor repertoire complexity in cutaneous T-cell lymphoma. Blood. 2003;102(12):40594066.
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
3538
BLOOD, 12 APRIL 2012 䡠 VOLUME 119, NUMBER 15
CEDENO-LAURENT et al
4. Papadavid E, Economidou J, Psarra A, et al. The
relevance of peripheral blood T-helper 1 and 2 cytokine pattern in the evaluation of patients with mycosis
fungoides and Sezary syndrome. Br J Dermatol.
2003;148(4):709-718.
5. Vowels BR, Cassin M, Vonderheid EC, et al. Aberrant cytokine production by Sezary syndrome
patients: cytokine secretion pattern resembles
murine Th2 cells. J Invest Dermatol. 1992;99(1):
90-94.
6. Yamanaka K, Clark R, Dowgiert R, et al. Expression of interleukin-18 and caspase-1 in cutaneous
T-cell lymphoma. Clin Cancer Res. 2006;12(2):
376-382.
et al. Development of a nascent galectin-1 chimeric molecule for studying the role of leukocyte
galectin-1 ligands and immune disease modulation. J Immunol. 2010;185(8):4659-4672.
11. Cedeno-Laurent F, Opperman M, Barthel SR,
et al. Galectin-1 triggers an immunoregulatory
signature in Th cells functionally defined by IL-10
expression. J Immunol. 2012;188(7):3127-3137.
12. Toscano MA, Bianco GA, Ilarregui JM, et al. Differential glycosylation of TH1, TH2 and TH-17
effector cells selectively regulates susceptibility to
cell death. Nat Immunol. 2007;8(8):825-834.
7. Miyagaki T, Sugaya M, Fujita H, et al. Eotaxins
and CCR3 interaction regulates the Th2 environment of cutaneous T-cell lymphoma. J Invest Dermatol. 2010;130(9):2304-2311.
13. Rubinstein N, Alvarez M, Zwirner NW, et al. Targeted inhibition of galectin-1 gene expression in
tumor cells results in heightened T cell-mediated
rejection: a potential mechanism of tumorimmune privilege. Cancer Cell. 2004;5(3):241251.
8. Juszczynski P, Ouyang J, Monti S, et al. The AP1dependent secretion of galectin-1 by Reed Sternberg cells fosters immune privilege in classical
Hodgkin lymphoma. Proc Natl Acad Sci U S A.
2007;104(32):13134-13139.
14. Kamper P, Ludvigsen M, Bendix K, et al. Proteomic analysis identifies galectin-1 as a predictive biomarker for relapsed/refractory disease in
classical Hodgkin lymphoma. Blood. 2011;
117(24):6638-6649.
9. Rodig SJ, Ouyang J, Juszczynski P, et al. AP1dependent galectin-1 expression delineates classical Hodgkin and anaplastic large cell lymphomas from other lymphoid malignancies with
shared molecular features. Clin Cancer Res.
2008;14(11):3338-3344.
15. Wollina U, Graefe T, Feldrappe S, et al. Galectin
fingerprinting by immuno- and lectin histochemistry in cutaneous lymphoma. J Cancer Res Clin
Oncol. 2002;128(2):103-110.
10. Cedeno-Laurent F, Barthel SR, Opperman MJ,
16. Cedeno-Laurent F, Dimitroff CJ. Galectin-1 research in T cell immunity: past, present and future. Clin Immunol. 2012;142(2):107-116.
17. Barthel SR, Antonopoulos A, Cedeno-Laurent F,
et al. Peracetylated 4-fluoro-glucosamine reduces the content and repertoire of N- and Oglycans without direct incorporation. J Biol Chem.
2011;286(24):21717-21731.
18. Gandhi MK, Moll G, Smith C, et al. Galectin-1 mediated suppression of Epstein-Barr virus specific
T-cell immunity in classic Hodgkin lymphoma.
Blood. 2007;110(4):1326-1329.
19. Le QT, Shi G, Cao H, et al. Galectin-1: a link between tumor hypoxia and tumor immune privilege. J Clin Oncol. 2005;23(35):8932-8941.
20. Cedeno-Laurent F, Opperman MJ, Barthel SR,
et al. Metabolic inhibition of galectin-1-binding
carbohydrates accentuates anti-tumor immunity.
J Invest Dermatol. 2012;132(2):410-420.
21. Rappl G, Abken H, Muche JM, et al. CD4⫹CD7leukemic T cells from patients with Sezary syndrome are protected from galectin-1-triggered
T cell death. Leukemia. 2002;16(5):840-845.
22. Pace KE, Hahn HP, Pang M, et al. CD7 delivers
a pro-apoptotic signal during galectin-1-induced T cell death. J Immunol. 2000;165(5):
2331-2334.
23. Roberts AA, Amano M, Felten C, et al. Galectin-1mediated apoptosis in mycosis fungoides: the
roles of CD7 and cell surface glycosylation. Mod
Pathol. 2003;16(6):543-551.
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2012 119: 3534-3538
doi:10.1182/blood-2011-12-396457 originally published
online March 1, 2012
Galectin-1 inhibits the viability, proliferation, and Th1 cytokine
production of nonmalignant T cells in patients with leukemic cutaneous
T-cell lymphoma
Filiberto Cedeno-Laurent, Rei Watanabe, Jessica E. Teague, Thomas S. Kupper, Rachael A. Clark
and Charles J. Dimitroff
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