the th9 axis reduces oxidative stress and promotes the survival … · 2020. 1. 29. · t-cell...

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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

on July 18, 2021. © 2020 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on January 29, 2020; DOI: 10.1158/1541-7786.MCR-19-0894

http://mcr.aacrjournals.org/

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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.



mailto:[email protected]


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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




17

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-




19

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.




24

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)




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,




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.




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


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.


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


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


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




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


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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