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Title: The Th9 axis reduces the oxidative stress and promotes the survival of malignant T-cells in
cutaneous T-cell lymphoma patients
Author: Sushant Kumar1, Bhavuk Dhamija
1, Soumitra Marathe
1, Sarbari Ghosh
1, Alka Dwivedi
1,
Atharva Karulkar1, Neha Sharma
2, Manju Sengar
2, Epari Sridhar
3, Avinash Bonda
2, Jayashree
Thorat2, Prashant Tembhare
3, Tanuja Shet
3, Sumeet Gujral
3, Bhausaheb Bagal
2, Siddhartha
Laskar4, Hasmukh Jain
2, Rahul Purwar*
1
Affiliations:
1Department of Biosciences & Bioengineering, Indian Institute of Technology Bombay, Mumbai,
Maharashtra, India, 400076
2Medical oncology, Tata Memorial Hospital, Mumbai, Maharashtra, India, 400012
3Pathology, Tata Memorial Hospital, Mumbai, Maharashtra, India, 400012
4Radiation oncology, Tata Memorial Hospital, Mumbai, Maharashtra, India, 400012
Running Title: Th9 axis promotes T-cell survival in CTCL
Keywords: Interleukin-9, T helper 9 cells, Cutaneous Lymphocyte Antigen, Reactive oxygen
species, Cell survival, Apoptosis
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Corresponding Author: Rahul Purwar, Biosciences and Bioengineering Department, IIT
Bombay, Powai, Mumbai, Maharashtra, India, 400076; e-mail: [email protected]; Phone:
+(91-22)25767737.
Conflict of interests: Authors declare no potential conflicts of interest.
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Abstract
Immune dysfunction is critical in pathogenesis of cutaneous T-cell lymphoma (CTCL). Few
studies have reported abnormal cytokine profile and dysregulated T-cell functions during the onset
and progression of certain types of lymphoma. However, the presence of IL-9 producing Th9 cells
and their role in tumor cell metabolism and survival remain unexplored. With this clinical study,
we performed multidimensional blood endotyping of CTCL patients before and after standard
photo/chemo-therapy and revealed distinct immune hallmarks of the disease. Importantly, there
was a higher frequency of “skin homing” Th9 cells in CTCL patients with early (T1, T2) and
advanced-stage disease (T3, T4). However, advanced-stage CTCL patients had severely impaired
frequency of skin-homing Th1 and Th17 cells, indicating attenuated immunity. Treatment of
CTCL patients with standard photo/chemotherapy decreased the skin homing Th9 cells and
increased the Th1 and Th17 cells. Interestingly, T-cells of CTCL patients express IL-9 receptor
(IL-9R), and there was negligible IL-9R expression on T-cells of healthy donors. Mechanistically,
IL-9/IL-9R interaction on CD3+
T-cells of CTCL patients and Jurkat cells reduced oxidative stress,
lactic acidosis, and apoptosis and ultimately increased their survival. In conclusion, co-expression
of IL-9 and IL-9R on T-cells in CTCL patients indicates the autocrine positive feedback loop of
Th9 axis in promoting the survival of malignant T-cells by reducing the oxidative stress.
Implications: The critical role of Th9 axis in CTCL pathogenesis indicates that strategies targeting
Th9 cells might harbor significant potential in developing robust CTCL therapy.
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Introduction
Cutaneous T-cell lymphoma (CTCL) represents a diverse group of non-Hodgkin lymphomas
derived from mature malignant T-cells that traffic to the human skin (1,2). Based on Surveillance,
Epidemiology, and End Results (SEER) registry data, the incidence of CTCL is 6.4 per million
individual in USA and the highest incidence has been reported among African- Americans (3).
According to the World Health Organization/European Organization for Research and Treatment
of Cancer (WHO-EORTC) classification, primary CTCL include multiple variants with distinct
clinical manifestations (4). The pathogenesis of CTCL is not clear, however, the immune
dysregulation is believed to have a vital role in the pathogenesis of lymphoma (5-7). Few studies
reported the abnormal production of various cytokines during the onset of certain lymphomas (8).
Interleukin 9 (IL-9) is a pleiotropic cytokine and IL-9/ IL-9 receptor (IL-9R) interaction promotes
T-cell growth (9) and is responsible for diverse functions in inflammatory and immune responses
(10,11). Multiple cell types including Th2, Th17, Treg, mast cells, dendritic cells and natural killer
(NK)/T-cell have been reported to secrete IL-9 (12-16). Later, IL-9 producing Th9 cells, a distinct
T helper (Th) subset was described. The Th9 cells are reported to be “skin tropic” as a majority of
Th9 cells express cutaneous lymphocyte antigen positive (CLA+) (17) and their presence was
demonstrated in the various skin diseases including psoriasis and atopic dermatitis patients
(17,18). Increased levels of IL-9 were demonstrated in biopsies or sera in multiple types of
peripheral T-cell lymphomas (PTCL) including adult T-cell leukemia (ATLL), anaplastic large
cell lymphoma (ALCL), Hodgkin lymphoma (HL) and nasal NK/T-cell lymphoma patients (19-
24). A recent study reported the presence of CD3+ IL-9 producing T-cells in the dermis and
dermo-epidermal junction in mycosis fungoides (MF) skin lesions (19). However, the presence of
Th9 cells as well as IL-9 production by malignant T-cells in an early and advanced-stage of CTCL
patients have not been reported so far.
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The role of IL-9 in tumor development depends on multiple factors including the nature of tumor
and tumor microenvironment. We and others have reported the anti-tumor role of Th9 cells in a
murine model of melanoma, which were mediated by promoting CD8+ cytotoxic T-cells and mast
cell functions (25,26). Unlike melanoma, the pro-tumor activity of IL-9 has been reported in
hematological cancers using murine models or cell lines. For example, transgenic mice over-
expressing IL-9 have been shown to develop thymic lymphoma at the age of 3-9 months (27).
Moreover, IL-9 stimulation of mouse thymic lymphoma cells induced by N-methyl-N-nitrosourea
or by X-ray irradiation showed increased proliferative responses (28). IL-9 promoted the
proliferation of human HL cell lines in dose-dependent manner, and ablation of IL-9 by IL-9
specific antisense oligomer inhibited the proliferation of HL cells (29). In primary T-cell
lymphoma mouse models, IL-9/IL-9R interaction activated STAT proteins and contributed to in
vivo growth of tumor (30-32). However, the roles of IL-9 in malignant T-cells survival in CTCL
patients have not been probed.
We performed blood endotyping of early and advanced-stage CTCL patients (Mycosis Fungoides
(MF), CD30+ lymphoproliferative disease (CD30
+ LPD), subcutaneous panniculitis-like T-cell
lymphoma (SPTL) and CTCL, not otherwise specified (NOS)) focusing on three major
components of “skin-homing” and “systemic” T-cell mediated immune responses: 1) T-cell
cytokine profile, 2) T-cell activation and 3) T-cell subsets. We demonstrated the increased
frequency of “skin-homing” Th9 cells in early as well as advanced-stage CTCL. Interestingly, we
observed the co-expression of IL-9 and IL-9R on malignant T-cells of CTCL patients.
Functionally, IL-9/IL-9R interaction reduced the toxic reactive oxygen species (ROS) production,
lactic acidosis and apoptosis, and ultimately increased the survival of malignant T-cells. In
conclusion, Th9 axis promotes malignant T-cell survival by reducing the oxidative stress of T-cells
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in CTCL patients.
Materials and Methods
Study protocol and blood samples
The protocol of this study was approved by the Institute Ethics Committee (IEC) of Tata Memorial
Hospital and IIT Bombay and is in compliance with the Declaration of Helsinki. The clinical trial
(CTRI/2017/04/008356) is registered in the Clinical Trial Registry of India (CTRI). In this study
we recruited primary CTCL patients, mainly MF, CD30+ LPD, SPTL and CTCL-NOS. Table 1
describes the patient’s details, risk status, symptoms, clinical examination, staging and treatment
history of CTCL patients at the baseline. In brief, de novo and previously treated adult patients (≥
18 years) suffering from CTCL (n=17) were recruited, provided they were not on any active
immunosuppression at least 4 weeks prior to the sampling. Among these, six patients of CTCL
were enrolled as a follow-up study after standard photo/chemotherapy. The treatment details and
patient information of follow-up patients are described in Supplementary Table S1. CTCL
diagnoses were established in accordance with the WHO-EORTC classification. Good clinical
practice guidelines were followed and written consent was obtained from all patients for blood
sample collection. Whole blood was collected from 17 CTCL patients (10 early stage and 7
advanced-stage patients) and 19 healthy donors in BD Vacutainer® EDTA tubes.
Surface marker and intracellular cytokine analysis by flow cytometry
The PBMCs from peripheral blood were isolated by ficoll-histopaque (Sigma-Aldrich, Missouri,
USA; 10771) density gradient centrifugation method. Cells (1x106 cells/tube) were washed in
FACS staining buffer (2% FBS in PBS) and resuspended in 50 µl staining buffer. The
fluorochrome-conjugated surface antibodies (CD3, CD4, CD8, CLA, CCR7, L-selectin, CD45RA,
CD45RO, CD25, and CD69) were added in different combinations in tubes and mixed gently by
tapping. Similarly, fluorochrome-conjugated IL-9R antibody was added to Jurkat cells (Source:
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National centre for cell science, Pune, India) and sorted T-cells. All tubes were incubated for 40
minutes in dark on ice and then washed twice with staining buffer and then resuspended in 250 µl
staining buffer for acquisition by BD FACSVerse or BD FACS Aria Fusion III (BD Biosciences;
CA, USA) and analyses were done using BD FACSuite software.
For intracellular staining, the PBMCs were cultured in T-cell media (Iscove's Modified Dulbecco's
Medium (IMDM) with 10% heat inactivated fetal bovine serum (FBS), 1% penicillin/streptomycin
and 1% L-glutamine). PBMCs were stimulated in 1 ml of T-cell media in 24-well plate with PMA
(Merck, Darmstadt, Germany; P8139, 10 ng/ml) and Ionomycin (Invitrogen, CA, USA; I24222,
500 ng/ml) in the presence of Brefeldin-A (Golgi Plug- BD Biosciences, CA, USA; 555028, 0.75
µl/ml) for 5 h at 37 °C. The PBMCs were first stained with fluorochrome-conjugated surface
antibodies (CD4 and CLA) and then surface stained cells were washed twice with staining buffer
and cells were resuspended in 250 µl 1X Cytofix/Cytoperm buffer (BD Biosciences, CA, USA;
555028), mixed gently and incubated at room temperature for 20 minutes. After incubation, cells
were washed twice with 500 µl perm/wash buffer. Fluorochrome-conjugated antibodies for
intracellular marker (IL-9, IL4, IL17, and IFNγ) were added in different combinations in the
respective tubes and mixed gently by tapping in perm/wash buffer (total volume not exceeding 100
µl). All tubes were incubated for 30 minutes in dark at room temperature and then washed twice
with perm/wash buffer and re-suspended in 250 µl staining buffer for acquisition by BD
FACSVerse or BD FACS Aria Fusion III (BD Biosciences) and the analyses were done using BD
FACSuite software.
All antibodies were procured from BD Biosciences (San Jose, CA, USA), BioLegend (San Diego,
CA, USA) and Thermo Fisher Scientific (Waltham, MA, USA).
Cytokine analysis by ELISA
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In TCR stimulation (recall assay), the PBMCs were cultured in T-cell media and were stimulated
using anti-CD3/CD28 Dynabeads Human T-cell activator (Bead: cell ratio= 1:1, Gibco, MA, USA;
11131D) and incubated for 48 h. Post 48 h, IL-9 production was quantified in the 100 µl of cell
free supernatant using the human IL-9 ELISA kit (Invitrogen, CA, USA; 88-7958-88) as per
manufacturer’s instructions.
T-cell sorting
The PBMCs (1x106 cells/tube) were washed using FACS staining buffer (2% FBS in PBS) and
resuspended in 50 µl staining buffer. The Fluorochrome-conjugated CD3 antibodies were added in
tubes and mixed gently by tapping. The tubes were incubated for 40 minutes in dark on ice and
then washed twice with staining buffer and then re-suspended in 250 µl staining buffer for sorting
by BD FACS Aria Fusion III (BD Biosciences; San Jose, CA, USA) and purity analyses were done
using BD FACSDiva software.
Cell survival analysis
Jurkat cells were procured from National centre for cell science, Pune, India and mycoplasma
testing was performed using PCR mycoplasma detection kit (abm Inc., Canada; G238) as per
manufacturer’s instruction. No additional cell line authentication was performed. Jurkat cells (0.1
million/ml) were suspended in RPMI media with 10% FBS and 1% penicillin/streptomycin and
seeded in a 24 well plate. Similarly, sorted T-cells of CTCL patient and healthy donors (0.1
million/ml) were seeded in T-cell media in a 24 well plate. Cells were cultured for 72 h in the
presence and absence of cytokines (IL-9: 20 ng/ml and IFNγ: 2 ng/ml). Post incubation, cells were
harvested and stained for trypan blue and live cells were counted.
Flow cytometric apoptosis assay
0.1 million/ml Jurkat cells were seeded in 12 well plates and cultured in 5 % CO2 incubator at
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37 °C for 72 h in the presence and absence of cytokines (IL-9: 20 ng/ml and IFNγ: 2 ng/ml) At the
end of the incubation period, cells were harvested and washed with Annexin V binding buffer
(1X). The apoptotic cells were stained using the FITC Annexin V Apoptosis Detection Kit (BD
Pharmingen, San Diego, CA, USA; 556547) as per manufacturer’s instructions and acquisition
was performed by BD FACSVerse (BD Biosciences). The analyses of FACS data were done using
BD FACSuite software.
Measurement of oxidative stress
Measurement of intracellular ROS was carried out using 2, 7-dichlorodihydrofluorescein diacetate
(H2DCF-DA), a dye that after hydrolysis by intracellular esterases reacts with superoxide,
hydroxyl or oxygen radicals and forms fluorescent green product dichlorofluorescein (DCF). After
24 h treatment of cytokines (IL-9, 20 ng/ml and IFNγ, 2 ng/ml), cells were collected, washed with
PBS and stained with 1µM H2DCFDA for 30 minutes at 37 °C and ROS levels are measured by
BD FACSVerse or BD FACS Aria Fusion III (BD Biosciences).
Lactate measurement in supernatant
Jurkat cells and sorted T-cells from patients and healthy individuals were cultured in the presence
and absence of cytokines (IL-9: 20 ng/ml and IFNγ: 2 ng/ml). Post 24 h, the supernatant was
collected and lactate was measured through the spectro-fluorimetric method. Lactate
dehydrogenase (1 U/ml) was used to convert lactate to pyruvate, further generating NADH which
was measured at 340 nm using ELISA plate reader. Incubation was carried out in 96-well ELISA
plates for 1 h at 37°C. The pyruvate generated was trapped using hydrazine present in glycine-
hydrazine buffer (pH -9.0), to prevent reverse production of lactate.
Statistical analysis of individual immune features
For studies described in Figures 1-6, comparative data were analyzed by using Student’s t-test.
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The specific statistical test chosen for each experiment is mentioned in the figure legend. Prism
7.02 software was used to perform the statistical analysis. In the figures, * stands for a p-value
<0.05, ** for p<0.01, *** for p<0.001 and ns for non-significant.
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Results
Increased skin-homing Th9 cells in blood of CTCL patients as compared to healthy controls.
A large proportion of CLA+ skin resident effector T-cells are known to secrete IL-9 with distinct
Th9 phenotype in healthy and inflamed skin (17). In this study, we quantified skin-homing (CLA+)
and systemic (CLA-) Th1 cells (CD4
+ IFNγ
+ IL-9
- IL4
- IL17
-), Th2 cells (CD4
+ IL4
+ IFNγ
- IL-9
-
IL17-), Th9 cells (CD4
+ IL-9
+ IFNγ
- IL4
- IL17
-) and Th17 cells (CD4
+ IL17
+ IL4
- IFNγ
- IL-9
-) in
primary CTCL (early and advanced) patients. Figure 1A represents the gating strategy to quantify
the skin-homing (CLA+) and systemic (CLA
-) Th9 cells and Th1 cells. Similar strategies were
used for gating Th2 and Th17 cells. In early as well as advanced-stage CTCL patients, there was
an increased frequency of skin-homing (CLA+) Th9 cells as compared to healthy donors (Figure
1D). However, higher numbers of systemic (CLA-) Th9 cells were found in advanced-stage CTCL
patients as compared to healthy donors (Figure 1H).
There was a significantly lower frequency of skin-homing Th1 cells (Figure 1B) and skin-homing
Th17 cells (Figure 1E) in advanced-stage CTCL patients as compared to healthy donors. There
was no difference in the frequency of systemic Th1 cells (Figure 1F), skin-homing and systemic
Th2 cells (Figure 1C and 1G) and systemic Th17 cells (Figure 1I) among healthy donors and
CTCL patients. Collectively, this data suggest that advanced CTCL patients have attenuated skin
immunity (Th1 and Th17 responses), and Th9 cells are increased in early and advanced-stage
CTCL patients.
CTCL patient derived T-cells secrete increased levels of IL-9 and express high levels of IL-
9R on their surface
Th9 cells are the major source of IL-9, which signals through a γC family receptor (IL-9R) on
target cells. We examined if patient derived T-cells secrete increased levels of IL-9 upon T-cell
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activation. The IL-9 production was quantified by ELISA in the cell-free culture supernatant of
PBMCs isolated from CTCL patients and healthy subjects upon TCR stimulation using anti-
CD3/CD28 coated dynabeads. Similar to flow cytometry data, IL-9 production was higher in
CTCL patients as compared to healthy donors (Figure 2A).
Next, we examined the surface expression of IL-9R on malignant T-cells. CD3+T-cells were
sorted from CTCL patients as well as healthy individuals. We observed a higher expression of IL-
9R on CTCL patient T-cells and negligible expression of IL-9R on healthy T-cells (Figure 2B).
Similarly, there was increased IL-9R expression on Jurkat cells (a T-cell lymphoma cell line). This
data collectively suggest the co-expression of IL-9 and IL-9R expression on malignant T-cells.
IL-9 promotes the survival of T-cells of CTCL patients and Jurkat cells
We further examined the functional relevance of the co-expression of IL-9 and IL-9R on
malignant T-cells. IL-9 promoted T-cell survival of CTCL patients, Jurkat cells and had no impact
on healthy T-cells (Figure 3A). Since IFNγ is known to be pro-apoptotic and anti-proliferative
(34) and has the ability to induce oxidative stress (35) in various cancer cells, we also examined
the role of IL-9 in IFNγ milieu to further strengthen our observation. IFNγ inhibited the cell
survival and IL-9 reversed the IFNγ mediated inhibition of cell survival in CTCL patients and
Jurkat cells (Figure 3A). Next, we examined the mechanism of IL-9 mediated increase in tumor
cell survival. IL-9 reduced the Jurkat cell apoptosis as quantified by Annexin-V/7-AAD staining
through flow cytometry (Figure 3B).
IL-9 reduces oxidative stress and lactic acidosis of T-cells of CTCL patients and Jurkat cells
Since reduced toxic ROS levels are described to promote healthy T-cell survival (33), we
examined if IL-9/IL-9R interaction impacts metabolic alterations, which would be beneficial for
the survival of malignant T-cells. Sorted CD3+ T-cells from CTCL patients and healthy donors
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were cultured in the presence and absence of IL-9 and/or IFN and ROS levels were quantified by
DCFDA staining using flow cytometry. Interestingly, IL-9 alone and/or in presence of IFN
significantly reduced the ROS levels inside the T-cells of CTCL patients and Jurkat cells, however
there was no significant reduction in ROS levels in healthy T-cells (Figure 4A).
Since lactic acidosis promotes ROS release in cancer cells and increases oxidative stress inside the
cells (36), we quantified extracellular lactate in cell-free supernatant of CTCL patients, Jurkat cells
and healthy donors. IL-9 decreased the extracellular lactate concentration in T-cells of CTCL
patients and Jurkat cells, however, had no impact on lactate production in healthy donors (Figure
4B). Collectively this data suggest that IL-9 promotes cell survival by reducing the intracellular
toxic ROS levels, lactic acidosis and apoptosis.
T-cell subsets and immune activation profile of CTCL patients and healthy donors
An earlier report shows increased TMM in few CTCL patients and depletion of TMM cells in both
the circulation and the skin of CTCL patients treated with alemtuzumab (37). Very recently,
migratory memory T-cells (TMM) were demonstrated to be the connecting link between skin and
lymph nodes and critical to the pathogenesis of L-CTCL, a malignancy of central memory T-cells
(TCM) (38). Here, we quantified the relative percentage of TMM and TCM in CTCL patients as
compared to healthy donors. There was an increase in percentage of skin-homing (CLA+) as well
as systemic (CLA-) TMM (CCR7
+ L-Selectin
-) in CTCL patients as compared to healthy donors.
However, we observed increased frequency of skin-homing TCM (CCR7+L- Selectin
+) in
advanced-stage CTCL patients as compared to healthy donors (Figure 5A-D). In addition,
decreased numbers of CD4+ naïve T-cells (CD4
+ Tnaïve) and increased CD4
+ effector T-cells (CD4
+
Teff) were found in CTCL patients as compared to the healthy donors (Figure 5E and 5F).
However, there was comparable frequency of CD8+ naïve T-cells (CD8
+ Tnaïve) and CD8
+ effector
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T-cells (CD4+
Teff) in CTCL patients and healthy donors (Figure 5G and 5H).
To understand the activation state of T-cells (CD4+/CD8
+) in CTCL patients as compared to
healthy donors, the expression and frequency of CD69+ and CD25+ T-cells were quantified. CD69
is an activation marker which appears very early while CD25 appears a bit late on the surface of
lymphocyte’s plasma membrane. Interestingly, the frequency of CD8+CD69
+CD25
- T-cells was
higher in CTCL patients as compared to healthy donors (Figure 5K) and no difference was
observed in the frequency of CD4+CD69
+CD25
- T-cells CTCL patients as compared to healthy
donors (Figure 5I). Further, CD69- CD25
+ T-cells (CD4
+ & CD8
+) frequency was higher in CTCL
patients as compared to healthy donors (Figure 5J and 5L). This data indicate that CTCL showed
increased early activation in CD8+T-cells.
Patients under standard photo/chemotherapy exhibit attenuated skin-homing Th9 cells and
re-established Th1 and Th17 immunity
Finally, we examined the cytokine profile in six follow-up CTCL patients (MF early-stage (n=2),
CD30+ LPD advanced-stage (n=1) and SPTL advanced-stage (n=3)) who were under standard
photo/chemotherapy. The treatment and patients’ clinical details are provided in Supplementary
Table S1. Interestingly, in these patients there was a significant reduction in the frequency of skin-
homing Th9 cells. However, the frequency of Th1 and Th17 cells significantly increased upon
treatment in follow-up patients (Figure 6A).
Discussion
With this study, we have observed a distinct cytokine profile in early and advanced-stage CTCL
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patients. Early-stage CTCL patients had an increased frequency of skin-homing Th9 cells and
comparable numbers of other effector Th cells as compared to the healthy individuals. In contrast,
patients with advanced disease had increased frequency of Th9 cells (skin-homing and systemic)
and impaired frequency of skin-homing Th1 and Th17 cells. This indicates that skin-homing Th9
cells may play a role in initiation as well as in maintenance of the disease in CTCL. The disease
progression (advanced-stage CTCL: T3, T4) led to an impaired anti-tumor immunity, which might
have contributed to the infection and persistence of lymphoma in these patients. In addition, there
was elevated IL-9 production in CTCL patients as compared to healthy donors. Co-elevation of
Th9 frequency and IL-9 production indicate that Th9 cells are the major source of IL-9 in CTCL
patients. A recent study has reported the presence of CD3+ IL-9 producing T-cells in skin biopsy
of MF lesion, one of the major subtypes of CTCL (19). In this study, we have demonstrated the
presence of skin-homing Th9 and elevated IL-9 production across different types of CTCL
including MF, CD30+ LPD, SPTL and CTCL-NOS. Importantly, we observed attenuated Th1
immunity in a large cohort of patients with advanced disease. Further, there was an increase in
Th1 cell frequency upon photo/chemotherapy treatment. A recent study has reported that increase
in Th1 response reduces the malignant T-cell burden (39). Our observation of impaired Th17 in
advanced CTCL directly correlates with a previous study where deficiency of RORγ, a
transcription factor required for differentiation of IL-17, was shown to promote rapid development
of T-cell lymphoma (40). Finally, we observed the reversal of cytokine profile, increase in
frequency of skin-homing Th1 and Th17 cells as well as a significant decrease in Th9 cells upon
standard photo/chemotherapy in a subset of follow-up CTCL patients. Collectively, this reveals
the interplay of cytokines in pathogenesis of CTCL and posits that strategies promoting the Th1
and Th17 responses and inhibiting Th9 cells might be beneficial and effective in treating CTCL.
Next, we demonstrated the role of IL-9 in tumorigenesis and delineated the underlying
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mechanisms. Our study revealed the co-expression of IL-9 and IL-9R on patient derived T-cells,
indicating the presence of autocrine role of IL-9 on tumor cells. Various studies, using cell lines
and murine models, have reported the roles of IL-9 in the pathogenesis of different cancers
including lung cancer, breast cancer, thyroid cancer and leukemia (41-44).
We observed that IL-9 significantly decreased the rate of apoptosis and ROS levels (oxidative
stress) inside the T-cells of CTCL patients, thus indicating the role of IL-9 in maintaining a
steady-state ROS. Concurrently, IL-9 increased the malignant T-cell survival, which ultimately
resulted in high cell numbers. The state of redox balance in CTCL and the effect of IL-9 in
regulating ROS were not known previously. The ROS are chemically reactive oxygen-containing
species that are generated directly or indirectly from free oxygen. In cancer cells, ROS is relatively
higher as compared to the normal cells, which help them in inducing tumorigenesis (45).
However, ROS generation higher than the steady-state level can be toxic even for the cancer cells
(46). Once the balance is disturbed, it can lead to oxidative stress inside the cells. Our data
indicates the role of IL-9 in maintaining ROS levels in CTCL to promote the malignant T-cell
survival. Rapid increases in intracellular ROS may lead to cellular transformation, DNA damage
and activation of p53, which can ultimately lead to apoptosis (46). ROS levels have been shown to
be elevated in various cell types when the extracellular environment is rich in lactate (36). The
decrease in ROS thus could be due to the less acidic environment (reduced lactate levels)
generated by IL-9. We also observed that IL-9 stimulation reduced the extracellular lactate
levels in the T-cell culture supernatant of CTCL patients, indicating less acidosis. This could be
due to the fact that Warburg effect is energetically inefficient as compared to oxidative
phosphorylation, and could limit the tumor growth in glucose depleted environment, where the
cells would then start utilizing lactate as an alternative nutrient source (47,48). Thus, the alteration
in redox balance caused by elevated IL-9 helps in the overall survival and maintenance of
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malignant T-cells in CTCL, generating a protumor response. These findings suggest a possible
role of IL-9 in reducing the oxidative stress, which otherwise could be detrimental for the cancer
cells and can lead to apoptosis instead of helping in tumorigenesis. Another interesting observation
was identifying the unique immune hallmarks of CTCL (summarized in Figure 6B). In addition to
cytokine profile, we examined the skin-homing and systemic immune-phenotyping focusing on T-
cell subsets (Tnaive, Teff, TMM and TCM) and T-cell (CD4/CD8) activation profile (CD69/CD25).
Surprisingly, in our study, we have found an increase in skin-homing as well as systemic TMM
cells in CTCL as compared to healthy donors. In addition, there was decreased frequency of CD4+
Tnaïve and increased frequency of CD4+Teff in CTCL patients as compared to healthy donors.
Interestingly, the frequency of CD8+CD69
+CD25
- T-cells was higher in CTCL patients and
percentage of CD69- CD25
+ T-cells (CD4
+ & CD8
+) was higher in CTCL patients as compared to
healthy donors.
In conclusion, this study provides first evidence of the presence of increased skin-homing Th9
cells and elevated IL-9 in CTCL. Moreover, it demonstrates the autocrine positive feedback loop
of Th9 axis in CTCL and delineates the role of IL-9 in reducing the oxidative stress and rate of
apoptosis to promote the survival of malignant T-cells (graphical abstract: Figure 6C). Strategies
targeting Th9 cells and inhibition of IL-9 may harbor significant potential in the development of
novel effective therapeutics for these difficult-to-treat malignancies.
Acknowledgements
This work was supported by Department of Science and Technology (DST) grant (RD/0119-
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DST0000-10), Indian Council of medical research (ICMR) grant (RD/0119-ICMR000-001), Tata
trust fund (RD/0117TATAE00-001), Bristol-Myers Squibb (15BMS001) and intramural fund of
IIT Bombay to RP. This work was partially supported by a grant from Department of
Biotechnology (DBT), Government of India awarded to Wadhwani Research Centre for
Bioengineering (WRCB), IIT Bombay (BT/INF/22/SP23026/2017). We would like to thank the
individuals (patients and healthy volunteers) who donated the blood for this study. We also thank
family members of the patients, the medical staff of Tata Memorial Hospital and Central FACS
facility of BSBE, IIT Bombay for their support.
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Authors’ Contributions
R.P. and H.J. conceptualized and supervised the project. R.P. and S.K. designed the protocol and
experiments. H.J., N.S., M.S., E.S., A.B., J.T., P.T., T.S., S.G., B.B. and S.L. recruited patients
and provided the blood samples. S.K., S.M., and A.K. collected the sample from TMH. S.K.
performed PBMCs isolation, Surface and intracellular staining, Sample acquisition, T-cell sorting,
Gating and ELISA. S.K. and B.D. performed lactate assays, measurement of ROS and cell
viability analysis. B.D. performed flow cytometric apoptosis assay. S.M., S.B., A.K. and A.D.
helped in performing experiments. S.K. performed data analysis. S.K. prepared the figures. S.K.,
B.D. and R.P. interpreted the results and wrote the manuscript.
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Figure 1: Increased skin homing Th9 cells in CTCL patients with early and advanced stage
disease: Human peripheral blood mononuclear cells (PBMCs) of CTCL patients (early (T1, T2):
n=10, advanced disease (T3, T4): n=7) and healthy individuals (n=19) were stimulated with
PMA/ionomycin in presence of brefeldin-A and stained as described in materials and methods. (A)
Representative dot plots and gating strategy for identification of Th9 and Th1 cells are depicted.
(B-I) Cumulative data of skin homing (CLA+: B-E) and systemic (CLA
-: F-I) Th9 cells, Th1 cells,
Th2 cells and Th17 cells are shown. Bar plots represent means ± standard error of mean. Statistical
significance was determined as compared to healthy donors by Student unpaired t test; P-values are
designated as ***<0.001, **<0.01, *<0.05.
Figure 2: T-cells of CTCL patients produce increased levels of IL-9 and express high levels of
IL-9R: (A) Isolated PBMCs from healthy donors (n=23) and CTCL patients (n=11; 6 early and 5
advanced disease) were stimulated using anti-CD3/CD28 coated Dynabeads (Bead to cell ratio;
1:1). Post 48 h incubation, IL-9 production was quantified by ELISA in the cell free supernatant as
per manufacturer instruction. Bar plots represent means ± standard error of mean. Statistical
significance was determined by comparing with healthy using Student unpaired t test. (B) Sorted
CD3+
T-cells from CTCL patients (n=3) and healthy donors (n=3) and Jurkat cells (n=3) were
stained for IL-9R and analyzed by flow cytometry. Representative histogram (upper panel) for
CTCL, healthy and Jurkat cells are shown and cumulative data is depicted in the lower panel.
Statistical significance was determined by paired t test. P-values are designated as ***<0.001,
**<0.01, *<0.05.
Figure 3: IL-9 promotes survival of T-cells of CTCL patient and Jurkat cells and reduces the
apoptosis in Jurkat cells: CD3+ sorted T-cells from CTCL patients and healthy donors and Jurkat
cells were cultured in presence of cytokines (IL-9 and/or IFNγ) or left untreated (control). (A)
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25
Numbers of live cells were quantified to assess the cell survival (n=3). (B) Representative images
of Jurkat cells apoptosis after IFNγ and IL-9 treatment for 72h. Apoptotic cells were stained for
Annexin and 7-AAD and analyzed by flow cytometry. Live cells are shown in the lower left
quadrant (7-AAD-/Annexin
-); early apoptotic cells are shown in the lower right quadrant (7-AAD
-
/Annexin+); apoptotic cells are shown in the upper right quadrant (7-AAD
+/Annexin
+) and upper
left quadrant (7-AAD+/Annexin
-). Percentage of dead cells (7-AAD
+/Annexin
+ and 7-
AAD+/Annexin
-) were plotted and cumulative data of four individual experiments are depicted.
Statistical significance was determined by comparing with control using Student paired t test; P-
values are designated as ***<0.001, **<0.01, *<0.05.
Figure 4: IL-9 reduces toxic ROS levels and lactic acidosis of T-cells of CTCL patient and
Jurkat cells: CD3+ sorted T-cells from CTCL patients and healthy donors and Jurkat cells were
cultured in presence of cytokines (IL-9 and/or IFNγ) or left untreated (control). (A) ROS levels
were quantified by H2DCFDA staining using flow cytometry. Upper panel depicts a representative
histogram for each condition. Lower panel demonstrate the cumulative data of CTCL patients
(n=4), healthy donors (n=3) and Jurkat cells (n=8). (B) Lactate concentration was measured in cell-
free supernatant upon appropriate cytokine treatment as shown in figure in CTCL (n=4), healthy
donors (n=4) and Jurkat cells (n=5). Bar plots represent means ± standard error of mean. Statistical
significance was determined by comparing with control using Student paired t test; P-values are
designated as ***<0.001, **<0.01, *<0.05.
Figure 5: T-cell subsets and immune activation profile of CTCL patients and healthy donors:
Human PBMCs isolated from CTCL patients (early (T1, T2): n=9, advanced disease (T3, T4): n=6)
and healthy donors (n=19) were stained for surface markers (CD3, CD4, CD8, CD45RA,
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26
CD45RO, CLA, CCR7, L-Selectin, CD69 and CD25) as described in materials and methods. (A-L)
Cumulative data of skin homing TMM (CD4+ CLA
+ CCR7
+ L-Selectin
-) (A), systemic TMM (CD4
+
CLA- CCR7
+ L-Selectin
-) (B), skin homing TCM (CD4
+ CLA
+ CCR7
+ L-Selectin
+) (C), systemic
TCM (CD4+ CLA
- CCR7
+ L-Selectin
+) (D), CD4
+ Tnaive cells (CD4
+ CD45RA
+ CD45RO
-) (E),
CD4+ Teff cells (CD4
+ CD45RO
+ CD45RA
-) (F), CD8
+ Tnaïve cells (CD8
+ CD45RA
+ CD45RO
-)
(G), CD8+ Teff cells (CD8
+ CD45RO
+ CD45RA
-) (H), CD4
+ CD69
+ CD25
- T-cells (I), CD4
+CD69
-
CD25+
T-cells (J), CD8+ CD69
+ CD25
- T-cells (K) and CD4
+CD69
- CD25
+ T-cells (L) are shown
for healthy donors and CTCL patients. Bar plots represent means ± standard error of mean.
Statistical significance was determined by Student unpaired t test; P-values are designated as
***<0.001, **<0.01, *<0.05.
Figure 6: Cytokine profile after standard photo/chemotherapy, immune hallmarks of disease
and graphical abstract of the study. CTCL Patients were treated with standard
photo/chemotherapy as described in Supplementary table S1. (A) Skin homing (CLA+) and
systemic (CLA-) Th1, Th2, Th9 and Th17 cells were quantified. Cumulative data of 6 follow-up
patients are depicted. Statistical significance was determined by Student paired t test; P values are
shown. (B) Summary of blood endotyping represents hallmarks of immune features in CTCL. (C)
Graphical summary depict the role of Th9 cells in CTCL patient and healthy individual.
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Table 1: Patients information
S.
No Age Sex Diagnosis Stage Risk
Status Symptoms Clinical Examination Staging Treatment history
1 27 M MF
CT
CL
Ear
ly S
tag
e
T2A N0
M0,
Stage I B
Lymph Node swelling and
Rash
PS- 1, No Lymphadenopathy, No
organomegaly
PET CT scan - No nodal disease,
Bone Marrow- Not Done, CSF- Not
Done
PUVA
2 23 F MF T2 N0
M0,
Stage I B
Pruritus PS- 1, No Lymphadenopathy, No
organomegaly, Skin; Patches over trunk,
thighs, Upper Limbs
CT Scan- Multiple subcm
Retroperitoneal positive, Bone
Marrow - Not Done, CSF - Not Done
PUVA
3 50 M MF T2 N0
M0,
Stage I B
Excessive Dryness,
Occasional itching
PS- 1, No Lymphadenopathy, No
organomegaly.
No staging done. Biopsy showed no
evidence of Lymphoma
Off protocol. Referred to
dermatologist and cardiologist
opinion.
4 53 M MF T2 N0 M0
B0,
Stage I B
Erythematous Plaques on
chest abdomen and thighs
present.
PS-1, No Lymphadenopathy, No
organomegaly.
PET/CT - Not Done, Bone Marrow-
Uninvolved, CSF- Not Done
On observation
5 61 M MF T2 N0 M0
B0,
Stage I B
Itching, Macules in bilateral
hands, nape of neck, anterior
chest wall, forehead.
PS-1, No Lymphadenopathy, No
organomegaly.
USG abdomen-No disease, Bone
Marrow- Uninvolved, CSF-Not Done
TSET
6 31 F MF T1 N0
M0,
Stage I A
Hypo pigmented Patches on
trunk, limb and abdomen
PS-1, No Lymphadenopathy, No
organomegaly.
CT-Norma, BM- Not done, CSF-
Uninvolved
Referred to derma unit
7 55 M MF T1A N0
M0 Stage
I A
Swelling in right forearm PS-1, No Lymphadenopathy, No
organomegaly
CT- Right paratracheal and subcarinal
lymph nodes. BM- Likely to be
uninvolved. CSF- Not done
Observation
8 40 F MF T2A N0
M0
Stage I B
Hyper pigmented patch seen
in the left breast,
erythematous patch seen on
the left chest wall,
depigmented patch seen on
the lower abdominal wall,
legs (popliteal fossa)
PS1, No peripheral lymphadenopathy, No
hepatosplenomegaly chest clear. Macular
lesions, >10% BSA
PET/CT - Not Done, Bone Marrow-
Not Done, CSF- Not Done. USG
Abdomen- No nodes.
Patient is taking PUVA therapy
9 32 F MF HPR not
suggestive
of
Mycosis
Fungoides
White patch, non-itchy and
non-painful
Extensive macular, scaly lesion all over the
body involving chest, back, abdomen, B/L
upper limb and lower limb. No
lymphadenopathy. No organomegaly.
PECT/CT- Not done. USG Abdomen
Pelvis: No abnormality detected. Bone
marrow - Not done. HPR not
suggestive of Mycosis Fungoides.
Patient is under observation
10 55 F MF
CT
CL
A
dv
ance
d
Sta
ge
T2A N0
M0,
Stage I B
-- PS-1, No Lymphadenopathy, No
organomegaly
PET Scan- B/L Cervical Nodes, B/L
Axillary nodes, external iliac nodes,
B/L inguinal nodes
Chemotherapy- Gemcitabine.
Changed to CEOP after cycle of
Gemcitabine.
11 68 F MF T3 Nx M0
Stage II B
Red patches in B/L upper
limb, abdomen , chest, lower
limbs, nodular swelling in
lower abdomen and right
inguinal region
PS-3, No Lymphadenopathy, No
organomegaly
Not done TSET
12 55 M MF T3 N0 M0 Multiple maculopapular PS- 1, No Lymphadenopathy, No PET-Not available, Marrow- Gemcitabine Pall intent
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B0,
Stage II B
lesions, Lesion on left side of
the lip with desquamation
and serous discharge.
organomegaly uninvolved, CSF- Not done
13 57 F MF T4 Nx M0
B0,
Extensive
cutaneous
disease
Pruritus, Fissuring with
discharge all over the body,
associated with redness,
itching, flaking.
PS-2, Axillary Lymphadenopathy, Diffuse
erythroderma and scaling
PET- NA,BM-NA, CSF-NA INF plus MTX and RT opinion
on TSET
14 31 F SPTL T3B N0
M0
Fever, subcutaneous edema
over B/L Upper and lower
limbs, multiple nodules
palpable over left cervical,
B/L 15upper limbs, B/L
lower limb, back, anorexia
PS-2, No Lymphadenopathy,
Splenomegaly
PET- B/L axillary nodes,
B/L external iliac nodes, B/L inguinal
nodes, retroperitoneal nodes, BM-
Uninvolved , CSF- Not Done
6 cycles CHOP from 7/07/2018
15 63 M CTCL
:NOS
T3 N0 M0
B0,
Stage II B
Ulcerative lesion on right
axilla, reddish nodular lesion
over left lower limb
PS- 1, No Lymphadenopathy, No
organomegaly
CT- Right Axillary region, distal left
thigh, left inferior thyroid, mesenteric
nodes
Oral MTX
16 49 F CD30+
LPD
T4 N1
M0,
Stage III
A
Fever, weight Loss, Pruritus,
Lymph Node swelling
PS- 1, Lymphadenopathy- B/L axillary
nodes, No organomegaly, Skin; Diffuse
erythroderma, extensive scaling and
plaques
PET CT Scan- B/L axillary nodes :
2.4*1.4 cm, Inguinal node, Bone
Marrow - Not Done, CSF - Not Done
Chemotherapy-Methotrexate
17 45 M Cutaneous
ALCL
T3 N0
M0,
Stage II B
Skin Lesions on chest wall PS-0, No Lymphadenopathy, No
organomegaly.
CT/PET- Not Done, BM- Not Done,
CSF- Not Done
Observation
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Published OnlineFirst January 29, 2020.Mol Cancer Res Sushant Kumar, Bhavuk Dhamija, Soumitra Marathe, et al. patientssurvival of malignant T-cells in cutaneous T-cell lymphoma The Th9 axis reduces oxidative stress and promotes the
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