The R2 Genomics Analysis and Visualization Platform (http://r2.amc.nl) was used for the Kaplan-Meier overall survival curve for patients with high and low IL-25 expression. The ‘Mixed Lung Adenocarcinoma TCGA 2022 v32’ dataset was used for visualization with the Kaplan Scan tool of the R2 Genomics Analysis and Visualization Platform, determining the optimal survival cut-off based on statistical tests within the web application.

Peripheral blood, tumor sample, along with an adjacent non-invaded tissue sample and mediastinal lymph node sample were collected from 30 patients with NSCLC undergoing surgical resection at Cerrahpasa Hospital and Medipol Mega Hospital in Istanbul. Prior to sampling, none of the patients had received chemotherapy, radiation therapy or immunotherapy. All subjects gave written informed consent to participate in this study. The study protocol was approved by Istanbul Medipol University Ethics Committee. The study was conducted in accordance with the ethical guidelines of the Declaration of Helsinki. Details of the clinicopathological characteristics of these patients are summarized in Supplementary Table S1 . As a control, peripheral blood samples were taken from 14 sex- and age-matched healthy donors (HD).

Peripheral blood mononuclear cells (PBMCs) were obtained from blood samples by the Lymphocytes Separation Media (cat. no. LSM-A; Capricorn, Germany) density gradient centrifugation method. Briefly, whole blood was diluted by the addition of an equal volume of PBS, blotted slowly onto separation medium, and centrifuged at 2000 rpm for 20 minutes at room temperature without stopping or acceleration. Cells were harvested from the interface, washed twice in phosphate buffered saline (PBS), then counted and prepared for staining. Tumor tissues and adjacent noncancerous tissues were dissociated into single cell suspensions using the human tumor dissociation kit (cat. no. 130-095-929; Miltenyi, Germany) according to the manufacturer’s instructions. Mediastinal lymph node tissues were mechanically dissociated into single cell suspensions. Red blood cells were removed from single cell suspensions using an ammonium chloride solution prepared in-house. The cells were filtered using a 70 µm cell strainer and then washed with PBS+ 2% fetal bovine serum (FBS) for subsequent flow cytometric assays.

Wild-type (WT) C57BL/6J mice were provided by Medipol University Medical Research Center (Istanbul, Turkey). B6PL-Thy1.1 mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Six- to eight-week-old C57BL/6J mice of both sexes were used as recipients of LLc1 tumor cells (tumor-bearing mice). B6PL-Thy1.1 mice of both sexes, six to eight weeks old, were used as donor mice for ILC2 isolation. For animal experiments, mice of the same age and sex were randomly assigned to experimental groups. Mice were housed under controlled conditions of temperatures of ~18-23°C with 40-60% humidity and a 12/12 h reverse light/dark cycle. Mouse care and experimental procedures were performed according to federal guidelines and protocols approved by Istanbul Medipol University Animal Experiments Local Ethics Committee (IMU-HADYEK) with protocol number 43. Tumor-bearing mice were monitored three times a week and at shorter intervals depending on the condition of the mice. Mice showing signs of stress, discomfort, pain, lethargy, inability to properly groom themselves, or inability to obtain food and/or water were immediately sacrificed.

The murine Lewis lung carcinoma (LLc1) cell line was kindly provided by Prof. Dr. Güneş Esendağlı (Hacettepe University-Ankara/Turkey) at passage number 6 from the original stock obtained from ATCC (CRL-1642). The cells were tested negative for mycoplasma with the EZ-PCR Mycoplasma Detection kit (Biological Industries, cat. 20-700-20). Upon receipt, the cells were expanded, and aliquots were frozen to allow use of low passage cells throughout the study. Thawed cells were maintained in RPMI 1640 medium (Sigma-Aldrich) supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 10 mM HEPES, non-essential amino acids (Sigma-Aldrich), 10% FBS and 1% penicillin–streptomycin (Gibco) (complete RPMI medium) at 37°C in a humidified atmosphere with 5% CO₂. Before injection, LLc1 cells (70–80% confluency) were harvested. Briefly, the cells were washed with PBS and detached using 0.25% trypsin-EDTA. Trypsin was neutralized with medium containing 10% FBS. After centrifugation, the cells were resuspended in PBS.

Briefly, for in vivo induction of ILC2s, donor mice (B6PL-Thy1.1) were hydrodynamically injected with 10 µg of a plasmid encoding murine IL25 (pCMV3-mIL25, Sino Biological) as previously described (16). Mice were sacrificed three days post-injection, spleens and mesenteric lymph nodes (MLN) were harvested and mechanically dissociated into single cell suspensions. Red blood cells from spleen single cell suspensions were lysed using an ammonium chloride solution prepared in-house. MLN and spleen single cell suspensions were filtered through a 70µm cell strainer and washed with PBS+2% FBS. Lineage-positive cells were depleted from the resulting cell suspensions using the mouse direct lineage cell depletion kit (cat. 130-110-470, Miltenyi Biotec) according to the manufacturer’s instructions. The flow through containing lineage negative (Lin-) cells was washed with PBS+2% FBS before antibody staining and fluorescence-activated cell sorting (FACS) for CD45+ Lin- ICOS+ KLRG1+ ILC2s. FACS purified ILC2s were washed in PBS before injecting intravenously (i.v.) via the tail vein into tumor-bearing C57BL/6J recipient mice. Each injection consisted of 500,000 ILC2s in PBS. Each mouse received 3 injections per week for 4 weeks, and tissues were collected 5 days after the final injection for assessment of immune cell composition, which included 5 and 6 mice per group. For the survival experiment (5 and 4 mice per group), mice continued to be monitored after the final ILC2 injection until natural death or euthanasia. Mice were euthanized when reaching humane endpoint (showing clinical signs of stress, discomfort, pain, lethargy, inability to properly groom themselves, inability to obtain food and/or water, hunching, weight loss above 20% of body weight, or a tumor volume above 1000mm³). For the tumor burden experiment, a total of 10 and 9 mice per group pooled from 3 independent experiments were assessed. For the experiment assessing the migration of ILC2s which included 3 mice per group, each mouse received a single injection of ILC2s, and tissues were collected 24h later. All ILC2s for adoptive transfers were prepared fresh using the above protocol.

Tumor-bearing WT C57BL/6J mice were partially lymphodepleted by injecting cyclophosphamide (CTX, Sigma-Aldrich) intraperitoneally (i.p.) as a single dose at 200 mg/kg.

6 - 8-week-old WT C57BL/6J mice were inoculated subcutaneously (s.c.) in the axilla area of the right forelimb with 1x10⁵ LLc1 cells in 100 µl sterile PBS per mouse. Tumor growth was assessed three times per week by measuring tumor dimensions with calipers. Tumor volume was calculated using the following formula: V = (W² × L)/2, W= Width; L= Length.

At the time of natural death or euthanasia as indicated in ‘adoptive transfer of ILC2 cells’, left and right lobes of the lungs were collected from mice (a total of 9 and 6 mice per group pooled from 2 independent experiments) and immediately placed separately in fixative solution (10% buffered formaldehyde). Fixed lung tissues were then embedded in paraffin. Paraffin blocks were then sliced into 4µm sections, which were stained with hematoxylin and eosin (H&E). Images from whole left and right lobe sections were taken using the Zeiss Axio Zoom V16 confocal microscope with Plan-NeoFluar Z objective set on brightfield mode. Tumor and non-tumor regions were manually defined on images taken from the left and right lobes using QuPath software (version 0.4.3). Percent lung metastatic area was calculated by dividing tumor lung area by total lung tissue area x 100 using QuPath software. Number of tumor nodules were manually counted.

For surface staining, both human and murine single cell suspensions were first stained with a viability marker (Fixable Viability Kit, Biolegend) according to manufacturer’s instructions to exclude dead cells, then Fc gamma receptors were blocked for 15 minutes with heat-inactivated human serum prepared in-house or anti-mouse CD16/32 (TruStain FcX, Biolegend) for human samples and mouse samples respectively, in PBS+2% FBS, and then incubated with the specific fluorochrome-labeled antibodies (all purchased from Biolegend unless otherwise noted) for 20 minutes at 4°C in the dark in PBS+2% FBS. For intracellular transcription factor staining, cells were fixed and permeabilized using a True-Nuclear Transcription Factor buffer kit (Biolegend) for 30 minutes at 4°C in the dark, washed twice with Perm buffer, and then stained for 30 minutes in Perm buffer. For intracellular cytokine staining on murine in vivo-induced ILC2 cells, freshly sorted cells were stained ex vivo or after stimulation with carrier-free recombinant mouse IL25 (Biolegend) at a concentration of 100 ng/ml in complete RPMI medium for 36 h. Both ex vivo and IL25-stimulated cells were cultured in complete RPMI medium in the presence of phorbol-12-myristate-13-acetate (500 ng/ml) (Sigma-Aldrich) and ionomycin (250 ng/ml) (Sigma-Aldrich) for 4 h at 37°C immediately prior to staining. Brefeldin A (Biolegend) was added in the last 3 h of the culture. Cells were then surface stained as indicated above. Cells were then fixed, permeabilized and stained using True-Nuclear Transcription Factor buffer kit (Biolegend) according to manufacturer’s instructions. After staining, cells were washed with PBS+2% FBS, then resuspended in PBS+2% FBS for acquisition on a flow cytometer. Acquisition of human samples was performed on BD Influx. Acquisition of murine samples was performed on BD Symphony A1. Analyses of acquired data were performed using Flow Jo v10.9. Cell sorting was performed using BD Influx with >98% purity.

For human samples, single-cell suspensions were stained with Zombie UV fixable viability kit (Biolegend), FITC-conjugated Hematopoietic Lineage cocktail prepared in-house (CD2 (TS1/8), CD3 (UCHT1), CD11b (ICRF44), CD14 (HCD14), CD15(HI98), CD16 (3G8), CD19 (HIB19), CD34 (561), CD56 (HCD56), CD123(6H6), CD20 (2H7), FcϵRIα AER-37 (CRA-1)), PE-conjugated anti-CD127 (A019D5), APC/Cy7–conjugated anti-CD45 (2D1)), APC-conjugated anti-CD294 (CRTH2) (BM16), PE/Cy7-conjugated anti-HLA-DR (L243), BV421-conjugated anti-CD86 (BU63), BV510-conjugated anti-PD-L1 (29E.2A3), PE/Dazzle™ 594-conjugated anti-CD25 (BC96) and BUV661-conjugated anti-CD4 (SK3) or appropriate isotype controls.

For mouse experiments, the following antibodies were used for surface staining of ILC2 cells: FITC-conjugated Lineage cocktail prepared in-house (anti-CD3 (17A2), anti-CD5 (53-7.3), anti-B220 (RA3-6B2), anti-CD11b (M1/70), anti-CD11c (N418), anti-NK1.1 (PK136), anti-TER-119 (TER-119), anti-Gr1 (RB6-8C5), anti-CD170 (S17007L), anti-FcϵRIα (Mar.01), anti-CD19 (6D5), anti-TCRβ (H57-597), anti-TCRγ/δ (UC7-13D5), PE-conjugated anti-CD45 (30.F11), APC-conjugated anti-KLRG (2F1/KLRG1), PE/Cy7 conjugated anti-CD278 (ICOS) (C398.4A), PerCP/Cyanine5.5-conjugated anti-CD90.1 (Thy1.1) (OX-7), Zombie UV fixable viability dye. For intracellular staining of ILC2 cells, the following antibodies were used: PE-conjugated anti-IL-5 (TRFK5), APC/Cy7-conjugated anti-IL-13 (eBioscience, clone eBio13A), PE/Dazzle 594-conjugated anti-IL-4 (11B11), and PE/Cy7-conjugated anti-GATA3 (eBioscience, clone TWAJ) or appropriate isotype controls. For immune cell composition of tissues obtained from tumor-bearing mice, the following antibodies were used for surface staining: PE/Cy7-conjugated anti-CD3 (eBioscience, clone 145-2C11), BV785-conjugated anti-CD4 (GK1.5), BV510-conjugated anti-CD8 (GK1.5), AF594-conjugated anti-CD11b (M1/70), FITC-conjugated anti-CD11c (N418), PE-conjugated anti-Ly6C (HK1.4), APC-conjugated anti-Ly6G (1A8), PE/Cy5-conjugated anti-MHCII (M5/114.15.2), Zombie UV fixable viability dye. For intracellular staining, AF700-conjugated anti-FOXP3 (MF-14) was used.

A maximum of 1g of tumor and lung tissue was rinsed with PBS, cut into small pieces using scissors and placed in a gentleMACS C tube (Miltenyi Biotec) in 5 ml RPMI1640 containing 100 µl 10X collagenase/hyaluronidase enzyme mixture (cat. 07912, Stemcell). The C tubes were then placed on a gentleMACS dissociator (Miltenyi Biotec) and the appropriate programs were run according to the mouse tumor dissociation kit protocol and mouse lung dissociation kit protocol (both from Miltenyi Biotec) for tumors and lungs respectively. The samples were then incubated for 45 min at 37°C while rotating. The digested fragments were manually dissociated further by mashing them with a syringe plunger onto a 70 µm cell strainer and the cells were washed in PBS+ 2% FBS. Erythrocytes were removed with an ammonium chloride solution prepared in-house. Mononuclear leukocytes from tumors were isolated by performing a Histopaque 1083 (Sigma-Aldrich) density gradient centrifugation according to the manufacturer’s instructions. Obtained single cell suspensions were then washed in cold PBS+ 2% FBS buffer before staining. Liver tissue was dissociated by following the ‘preparation of single cell suspensions from mouse liver’ protocol from Miltenyi Biotec. Cells were then washed in cold PBS+ 2% FBS buffer before staining. Spleen and lymph nodes were mechanically dissociated then filtered through a 70-µm cell strainer. Red blood cells from the spleen were removed using ammonium chloride solution prepared in-house. Samples were washed in cold PBS+ 2% FBS buffer prior to staining.

Statistical analyses and representations were performed using Prism (GraphPad Version 9.0) and are detailed in the figure legends.

We examined the survival probability of NSCLC patients with high and low IL25 gene expression by applying the R2 Genomics Analysis and Visualization Platform to the large publicly available TCGA dataset. Patients with high IL25 expression as determined by the Kaplan Scan tool were found to have significantly reduced overall survival (p-value 0.03) as compared to IL25-low patients ( Figure 1A ). IL-25 being a known activator of ILC2 cells, we sought to determine the frequencies of ILC2 cells in NSCLC patients. We obtained fresh surgically resected human primary NSCLC tumors, as well as adjacent normal lung tissue, tumor-draining mediastinal lymph nodes (LNs) and peripheral blood (PB) from the same patients. As a control, we also obtained PB from HDs. All samples from NSCLC patients and HDs were analyzed for their relative abundance in total ILC helpers as well as ILC2 cells by flow cytometry. Total helper ILCs were identified as live CD45⁺ Lin^- CD127⁺ cells, among which ILC2 cells were identified as CRTH2⁺ cells, in line with the literature and previous studies (1, 8, 9). Representative gating strategies for total ILC helpers and ILC2 cells are shown in Figure 1B .

As shown in Figure 1C , there was no significant difference between the frequencies of circulating total ILC helpers in NSCLC patients and HDs. No substantial difference in the frequency of total ILC helpers was observed between tumors and their normal counterparts ( Figure 1C ). Of note, ILC helpers were found to be more highly represented in patients’ lungs than in their LNs or PB ( Figure 1C ), consistent with the known tissue distribution of ILCs (1, 17). Next, we compared the relative abundance of ILC2 cells within total ILC helpers ( Figure 1D ). NSCLC patients had significantly higher frequencies of circulating ILC2 cells as compared to HDs ( Figure 1D ). There was no significant difference between the frequencies of ILC2 cells in tumors and their normal counterparts or patient PB ( Figure 1D ). Of note, ILC2 cells were less represented in mediastinal LNs compared to other tissues ( Figure 1D ), consistent with the known tissue distribution of ILC2s (1, 17). Calculation of ILC2 absolute numbers per mg of tissue revealed higher numbers of ILC2 cells in tumors than their normal counterparts ( Figure 1E ).

Murine ILC2 cells have been reported to express MHC Class II and to be able to function as antigen-presenting cells (APCs) (18–21). In humans, only one study reported MHC Class II and CD80 expression on ILC2 cells as being elevated in patients with acute exacerbation of chronic pulmonary obstructive pulmonary disease (AECOPD) (22). Moreover, ILC2 cells stimulated by IL-33 were found to upregulate PD-L1 (23). PD-L1 is a ligand for PD-1, which is a known checkpoint inhibitor of CD4⁺ T cells that plays important roles in diminishing anti-tumor immunity. We therefore wanted to assess whether ILC2 cells in NSCLC patients may have immunosuppressive capacities by interacting directly with CD4⁺ T cells via MHC II, CD86 and PD-L1. To this end, we assessed the expression of HLA-DR, CD86 and PD-L1 on ILC2 cells in the various tissues obtained from NSCLC patients as well as in HDs ( Supplementary Figure 1 ). ILC2 cells in PB of HDs and tumors of NSCLC patients did not express HLA-DR, CD86 nor PD-L1 ( Supplementary Figure 1 ). In NSCLC patients, the lack of expression of HLA-DR, CD86 and PD-L1 by ILC2 cells was irrespective of the tissue of origin (data not shown). ILC2 cells from NSCLC and HD samples expressed CD25 ( Supplementary Figure 1 ), consistent with the literature (24). CD25 expression by ILC2 cells in samples obtained from NSCLC patients was irrespective of the tissue of origin (data not shown).

Next, we assessed the frequencies of total CD4⁺ T lymphocytes and CD4⁺ CD25^hi Tregs by flow cytometry ( Figure 2 ). Circulating CD4⁺ T cells were present in significantly lower amount in NSCLC patients compared to HDs ( Figure 2A ). The frequency of CD4⁺ T cells was significantly higher in tumors than in matched normal lung tissues or patient PB. There was no significant difference between frequencies of CD4⁺ T cells in tumors and LNs. Circulating Treg frequencies were found to be significantly elevated in NSCLC patients compared to HDs ( Figure 2B ). Tumor-infiltrating Tregs were present in significantly higher amount if compared with normal tissues. Tregs were represented the highest in LNs.

In summary, we report that high IL25 expression in NSCLC patients is associated with reduced survival. NSCLC patients bear a systemic increase of ILC2 cells, resulting in high numbers of ILC2 cells in tumors. Frequencies of Tregs are also increased in patients compared to HDs, with a preferential accumulation in tumors and tumor-draining LNs. Taken together, these findings suggest that the IL25/ILC2 axis and Tregs may concomitantly contribute to a tumor-promoting environment in human NSCLC.

In order to test our hypothesis that the IL25/ILC2 axis bears tumor-promoting properties, we decided to examine the effect of adoptive transfer of IL25-activated ILC2 cells in lung tumor-bearing mice using the well-established Lewis lung carcinoma (LLc1) heterotopic model of lung cancer. The LLc1 tumor cell line is known to be highly tumorigenic but weakly metastatic in mice.

The obtention of IL25-activated ILC2 cells for adoptive transfer purposes was achieved by endogenous ILC2 cell expansion in vivo by hydrodynamic gene delivery (hgd) of an IL25-encoding plasmid (pCMV-IL25) as previously described (16). Such ILC2 cells were identified as CD45⁺ Lin^- KLRG1⁺ ICOS⁺ cells and were recovered from mesenteric LNs (MLNs) and spleen as previously described (16). A representative example of the in vivo expansion and phenotypic profile of such ILC2 cells following hgd is shown in Figure 3A . These cells were high producers of IL-4 and IL-13, but not IL-5 ( Figures 3B, C ). Their cytokine production profile remained the same when cells were restimulated with murine IL-25 in vitro ( Figures 3B, C ). Moreover, these cells expressed the master transcription factor GATA3 ( Figure 3D ). Altogether, these results show that CD45⁺ Lin^- KLRG1⁺ ICOS⁺ cells obtained after hgd with IL-25-encoding plasmid were true ILC2 cells that expressed type 2 cytokines and GATA3. Moreover, their expansion by IL-25 and high KLRG1 expression reminisce of the inflammatory subset of ILC2 cells (iILC2) previously described (5).

In order to temporarily free up niches for adoptively transferred cells, we decided to partially lymphodeplete recipient mice by administering cyclophosphamide (CTX) i.p. one day prior to adoptive transfer. First, we wanted to assess the migration potential of IL25-activated ILC2 cells adoptively transferred i.v. into tumor-bearing mice. To this end, we transferred Thy1.1⁺ ILC2 cells into tumor-bearing Thy1.2⁺ recipient mice two days after CTX or PBS vehicle injection ( Figure 4A ). The recipient mice were sacrificed 24h after ILC2 transfer and various organs were collected for staining with anti-Thy1.1 antibody ( Figure 4A ). As shown in Figure 4B , transferred IL-25-activated ILC2 cells migrated to all tissues of recipient mice, including tumors. In mice that were not lymphodepleted prior to ILC2 adoptive transfer, the percentage of transferred cells was very low in all tissues, and almost undetectable in PB and secondary lymphoid organs ( Figure 4B , lower panels). Lymphodepletion with CTX prior to adoptive transfer enabled a 3-fold increase in the frequency of transferred cells that could be recovered in tumors ( Figure 4B , 0.083% in vehicle-treated mice vs. 0.28% in CTX-treated mice).

Next, in order to assess the effect of IL25-activated ILC2 cells on tumor growth, metastasis to the lung and overall survival, we transferred freshly sorted ILC2 cells or vehicle into lymphodepleted tumor-bearing mice twice per week for 4 weeks ( Figure 5A ). The purity of ILC2 cells after cell sorting was at or above 98% ( Supplementary Figure 2 ). Adoptive transfer of ILC2 cells had no effect on tumor growth in mice that did not receive CTX treatment ( Supplementary Figure 3 ). This is probably due to the fact that only very low numbers of ILC2 cells could reach the tumor in non-lymphodepleted tumor-bearing mice as stated above. Although initially the tumor volume was similar in mice that received IL25-activated ILC2 cells and controls, it started to become significantly higher in mice that received the cells as compared to those that received vehicle after the second week of treatment ( Figure 5B ). Overall, IL25-activated ILC2 adoptive transfer led to significant higher tumor burden ( Figure 5B ). Moreover, the survival of mice was significantly reduced when IL25-activated ILC2 cells were transferred ( Figure 5C ). Metastasis to the lung was assessed in CTX-treated mice that received IL25-activated ILC2 cells and those that received vehicle. Transfer of IL25-activated ILC2 cells led to a significantly higher metastasis burden both in overall percentage of tumor area and number of tumor nodules ( Figures 5D–F ).

Taken together, these results indicate that IL25-activated ILC2 cells promote tumor growth, increase the metastatic potential of tumor cells, and reduce the survival of tumor-bearing mice.

Next, we sought to determine the TME composition after IL25-activated ILC2 transfer in CTX-lymphodepleted tumor-bearing mice. To this end, we transferred freshly sorted ILC2 cells or vehicle into lymphodepleted tumor-bearing mice twice per week for 4 weeks. Recipient mice were sacrificed 5 days after the last ILC2 injection and tumors, spleens and lymph nodes (LNs) were collected for analysis ( Figure 6A ). As shown in Figure 6B , the percentages of CD4⁺, CD8⁺ and Foxp3⁺ Treg T lymphocyte subsets were similar in tumors of mice that received ILC2 cells or PBS. Figure 6C shows the percentages of myeloid cells in tumors. IL25-activated ILC2 cell transfer did not alter the frequencies of conventional dendritic cells (cDCs) nor granulocytic MDSCs (G-MDSCs), however there was a significant increase in the frequencies of monocytic MDSCs (M-MDSCs) in mice that received IL25-activated ILC2 cells as compared to controls ( Figure 6C ). This is in line with a previous report on IL-25-activated ILC2 cells promoting colorectal cancer by activating M-MDSCs (13). The accumulation of M-MDSCs in tumor-bearing mice that received IL25-activated ILC2 cells was not systemic but rather specific to tumors since there was no significant difference in their frequencies within spleens and LNs ( Supplementary Figure 4 and data not shown). This result is consistent with the higher migration potential of IL25-activated ILC2 cells into tumors as compared to other tissues ( Figure 4B ).

Overall, our results show that IL25-activated ILC2 cells preferentially migrate to tumors, and that they promote tumor growth and metastasis, as well as reduce survival of tumor-bearing mice. Moreover, we found that M-MDSCs accumulated into tumors following adoptive transfer with IL25-activated ILC2 cells, suggesting that these cells may actively recruit M-MDSCs into tumors. This may be due to the high production of IL-4 and IL-13 by IL25-activated ILC2 cells ( Figures 3B, C ) as it has previously been shown that ILC2s mediate the recruitment of MDSCs via IL-13 (25, 26) and that they induce the expansion and activation of M-MDSCs via the activation of STAT6 through the binding of the IL4Ralpha (13, 27).