STAT3/5-Dependent IL9 Overexpression Contributes to Neoplastic Cell Survival in Mycosis Fungoides

Abstract

Purpose: Sustained inflammation is a key feature of mycosis fungoides (MF), the most common form of cutaneous T-cell lymphoma (CTCL). Resident IL9-producing T cells have been found in skin infections and certain inflammatory skin diseases, but their role in MF is currently unknown.

Experimental design: We analyzed lesional skin from patients with MF for the expression of IL9 and its regulators. To determine which cells were producing IL9, high-throughput sequencing was used to identify malignant clones and Vb-specific antibodies were employed to visualize malignant cells in histologic preparations. To explore the mechanism of IL9 secretion, we knocked down STAT3/5 and IRF4 by siRNA transfection in CTCL cell lines receiving psoralen+UVA (PUVA) ± anti-IL9 antibody. To further examine the role of IL9 in tumor development, the EL-4 T-cell lymphoma model was used in C57BL/6 mice.

Results: Malignant and reactive T cells produce IL9 in lesional skin. Expression of the Th9 transcription factor IRF4 in malignant cells was heterogeneous, whereas reactive T cells expressed it uniformly. PUVA or UVB phototherapy diminished the frequencies of IL9- and IL9r-positive cells, as well as STAT3/5a and IRF4 expression in lesional skin. IL9 production was regulated by STAT3/5 and silencing of STAT5 or blockade of IL9 with neutralizing antibodies potentiated cell death after PUVA treatment in vitro IL9-depleted mice exhibited a reduction of tumor growth, higher frequencies of regulatory T cells, and activated CD4 and CD8 T lymphocytes.

Conclusions: Our results suggest that IL9 and its regulators are promising new targets for therapy development in mycosis fungoides. Clin Cancer Res; 22(13); 3328-39. ©2016 AACR.

Figure 1

Expression of IL9, IL9r, and STAT3 in patients with MF. Paraffin-embedded sections were stained for IL9, IL9r, or STAT3. Representative micrographs are shown; magnified areas are indicated. A, normal skin samples from control individuals. B, lesional skin from a patient with MF (archived materials ST2, patient A2), view of 20× (scale bars as indicated). Pautrier microabscess is shown in the magnified area of the IL9 stain.

Figure 2

Malignant and reactive T cells produce IL9. Representative micrographs of two-color IHC (blue/red) in lesional skin of MF patient (ST2, Patient A4) CD3/IL9 (A) and STAT3/IL9 (B); magnified areas are indicated (detailed scoring of double stains are shown in ST2). C, average copy number by V-J pairing of TCRβ sequences by high-throughput sequencing of the CDR3 region (outstanding clone is indicated with red arrow). Immunohistochemical stain of the malignant clone based on sequencing results. The Vb22 antibody was used to identify the TCRβV02-01 gene product (D).

Figure 3

STAT3/STAT5 pathway regulates IL9 production in MyLa2000 cells. A, MyLa2000 cells were treated with 180nmol/L of ruxolitinib and cultured for up to 48 hours. IL9 expression was quantified by qPCR at indicated time points; not treated (NT) cells were used as controls. MyLa2000 cells were transfected with siRNA targeting STAT3 (B) and STAT5 (C). IL9 expression was quantified by qPCR at indicated time points; fold change is depicted with unspecific siRNA-treated cells (siCTRL) as reference control. D, simultaneous STAT3 and STAT5 silencing. IL9 expression was quantified by ELISA at 48 and 72 hours; siCTRL cells were used as reference control. E, IL32 expression was quantified by qPCR as control to examine the specificity of the silencing effect on IL9 expression. Experiments were done in triplicates: statistical significance was assessed by Dunnet post test; P values are shown.

Figure 4

IRF4 and PU.1 expression in patients with MF. Representative micrographs of two-color IHC are shown. The transcription factors IRF4 (A) or PU.1 (B) were stained together with CD3; magnified areas are indicated (detailed scoring of double stains are shown in ST2). C, malignant cells stained with the Vb22 antibody together with IRF4 (Patient A4 is shown in A–C.). Expression of IRF4 in MyLa2000 and Hut78 cells quantified by qPCR (D) and siRNA was used to silence IRF4. IL9 expression was determined after 24 and for up to 96 hours of transfection (E). Experiments were done in triplicates; statistical significance was assessed by Student unpaired t test; P values are shown.

Figure 5

Patients under photo(chemo)therapy treatment exhibit reduced IL9 production. Samples from the archived materials from patients with MF (ST2) were analyzed for IL9 (A) and IL9r (B) expression at baseline and after 12 weeks at clinical remission (micrographs from patient A1 are shown). The average of positive cells in three 40× fields per sample is presented: adjusted to the unit of area (mm²). Biopsies from the PUVA trial (ST3) were analyzed by qPCR for expression of IL9 (C) and STAT3 (D) at baseline and 6 weeks after start of treatment. E, MyLa2000 cells were transfected with siSTAT5 or siCTRL 24 hours before treated with PUVA in vitro (1 µmol/L 8-MOP, 0.4 J/cm²). F, MyLa2000 cells were given PUVA followed by incubation in media supplemented with IFNα2b (1,500 U/mL), goat polyclonal anti-human IL9 antibody (20 µg/mL) or the two combined. E and F, viability was assessed by Annexin V/PI incorporation after 48 hours of culture. Experiments were done in triplicates; statistical significance was assessed by Student paired t test (A–D), or Dunnet post test (E and F); P values are shown.

Figure 6

Depletion of IL9 improves antitumor immune response. Mouse T-cell lymphoma cell line EL4 (10⁴ cells) were injected intradermally into the backs of C57BL/6 mice. Polyclonal goat anti-IL9 antibodies (20 µg) or isotype control was given together with the cells and repeatedly every 48 hours (n = 10 mice per experimental group; A). Tumor growth was assessed daily for 14 days using a Mitutoyo Mini-caliper and tumor diameter mean is depicted (B). Mice were sacrificed when tumor size reached 1 cm of diameter; survival is shown (C). Mice were sacrificed 14 days after cell injections and inguinal lymph nodes were taken to analyze Treg cells by flow cytometry (D) and activated CD4 and CD8 T cells (E; n = 6 mice per experimental group). The percentage of positive cells is presented. Figures shown are representative of two independent experiments. Statistical significance was assessed by Student t test; P values are shown.