IMMUNOTHERAPY FOR CANCER

Abstract

Disclosed is a composition comprising an immunogenic composition for use in treatment of squamous cell carcinoma in combination with myeloid-derived suppressor cell-inhibiting agents as well as a corresponding method of treatment.

Claims

1. A method of treating or delaying progression of squamous cell carcinoma (SCC) of the lungs in an individual, comprising: administering a first composition comprising a dendritic cell vaccine (DC vaccine) in combination with a second composition comprising a myeloid-derived suppressor cell (MDSC)-inhibiting agent or an inhibitor of MDSC effector functions to an individual in need thereof.

2. The method of claim 1, wherein the individual is further characterized in having: (a) a fraction of living cells in peripheral blood mononuclear cells which are monocytic MDSCs (M-MDSCs) of at least 0.08%, (b) a fraction of living cells in tumor tissue which are M-MDSCs of at least 0.005%, (c) a level of LOX-1 in serum or plasma above 75 pg/ml, (d) a percentage of LOX-1-expressing polymorphonuclear MDSCs (PMN-MDSC) among all PMN-MDSC of at least 5%, (e) a fraction of living cells in tumor tissue which are PMN-MDSCs of at least 2%, (f) a fraction of living Treg cells in peripheral blood mononuclear cells of at most 1.8%, (g) a fraction of living Treg cells in tumor tissue of at most 10% of CD4+ T cells, (h) a fraction of CD3ζ among CD4+ T cells in peripheral blood mononuclear cells is reduced compared to control CD4+ T cells of healthy individuals by a factor of at least 0.9, (i) a fraction of CD3ζ among CD8+ T cells in peripheral blood mononuclear cells is reduced compared to control CD8+ T cells of healthy individuals by a factor of at least 0.75, and/or (j) a relative expression of Arginase 1 in peripheral blood mononuclear cells of patients compared to control peripheral blood mononuclear cells of healthy individuals is increased at least by a factor of 2.5.

3. The method of claim 2, wherein the fraction of living cells in peripheral blood mononuclear cells which are monocytic MDSCs (M-MDSCs) is at least 0.1%, the fraction of living cells in tumor tissue which are M-MDSCs at least 0.008%, the level of LOX-1 in serum or plasma is above 100 pg/ml, the percentage of LOX-1-expressing polymorphonuclear MDSCs (PMN-MDSC) among all PMN-MDSC is at least 10%, the fraction of living Treg cells in peripheral blood mononuclear cells is at most 1.5%, and the fraction of living Treg cells in tumor tissue is at most 9% of CD4+ T cells.

4. The method of claim 1, wherein the M-MDSCs have a CD14+CD15-CD33hiHLA-DR-/lo phenotype.

5. The method of claim 1, wherein the PMN-MDSCs have a suppressive CD14-CD15+CD11b+ phenotype.

6. The method of claim 5, wherein the PMN-MDSCs express LOX-1.

7. The method of claim 1, wherein the DC vaccines have been prepared with an antigen source selected from tumor associated peptide(s), whole antigens from DNA or RNA, whole antigen-protein, idiotype protein, tumor lysate, whole tumor cells or viral vector-delivered whole antigen.

8. The method of claim 7, wherein the antigen source is whole tumor cells that have been prepared by high hydrostatic pressure.

9. The method of claim 1, wherein the second composition comprises carboplatin plus paclitaxel, pemetrexed plus carboplatin, gemcitabine plus cisplatin, pemetrexed plus cisplatin or vinorelbine plus carboplatin.

10. The method of claim 1, wherein the MDSC-inhibiting agent blocks or inhibits differentiation and/or maturation of MDSCs, blocks or inhibits migration of MDSCs, induces depletion of MDSCs and/or apoptosis of MDSCs, inhibits expansion of MDSCs, or inhibits MDSC effector functions.

11. The method of claim 10, wherein the inhibitor of differentiation or maturation of MDSCs is selected from all-trans-retinoic acid, Curcumin derivatives; wherein the inhibitor of migration of MDSCs is selected from Zolendronic acid, anti-glycan antibodies and CSF-1R inhibitors; wherein the inducer of depletion or apoptosis of MDSCs is selected from tyrosine kinase inhibitors; wherein the inhibitor of expansion of MDSCs is selected from Bevacizumab, Celecoxib and Pimozide; and wherein the inhibitor of MDSC effector functions is an inducer of oxidative stress.

12. The method according to claim 11, wherein the tyrosine kinase inhibitor is selected from Sunitinib, anti-Gr1 antibodies, IL4Rα aptamer, Gemcitabine, Cisplatin, Paclitaxel, 17-Dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) or 5-fluorouracil (5-FU); and wherein the inducer of oxidative stress is selected from N-hydroxyl-L-Arginine (NOHA), Nitroaspirin, N-acetyl cysteine (NAC), CpG oligodeoxy-nucleotides, Bardoxolone methyl (CDDO-Me), Withaferin A or Stattic.

13. A method of treating or delaying progression of non-small cell lung cancer (NSCLC) in an individual, comprising: administering a first composition comprising a dendritic cell vaccine (DC vaccine) in combination with a second composition comprising a myeloid-derived suppressor cell (MDSC)-inhibiting agent or an inhibitor of MDSC effector functions to an individual in need thereof, wherein the individual is further characterized in having an immunosuppressive tumor microenvironment caused by the presence of MDSCs.

14. The method of claim 13, wherein the individual is further characterized in having: (a) a fraction of living cells in peripheral blood mononuclear cells which are monocytic MDSCs (M-MDSCs) of at least 0.08%, (b) a fraction of living cells in tumor tissue which are M-MDSCs, of at least 0.005%, (c) a level of LOX-1 in serum or plasma above 75 pg/ml, (d) a percentage of LOX-1-expressing PMN-MDSC among all PMN-MDSC of at least 5%, (e) a fraction of living cells in tumor tissue which are PMN-MDSCs of at least 2%, (f) a fraction of living Treg cells in peripheral blood mononuclear cells of at most 1.8%, (g) a fraction of living Treg cells in tumor tissue of at most 10% of CD4+ cells, (h) a fraction of CD3ζ among CD4+ T cells in peripheral blood mononuclear cells is reduced compared to control cells of healthy individuals by a factor of at least 0.9, (i) a fraction of CD3ζ among CD8+ T cells in peripheral blood mononuclear cells is reduced compared to control cells of healthy individuals by a factor of at least 0.75, and/or (j) a relative expression of Arginase 1 in peripheral blood mononuclear cells of patients compared to control peripheral blood mononuclear cells of healthy individuals is increased at least by a factor of 2.5.

15. The method of claim 14, wherein the fraction of living cells in peripheral blood mononuclear cells which are monocytic MDSCs (M-MDSCs) is at least 0.1%, the fraction of living cells in tumor tissue which are M-MDSCs, is at least 0.008%, the level of LOX-1 in serum or plasma is above 100 pg/ml, the percentage of LOX-1-expressing PMN_MDSC is at least 10%, the fraction of living cells in tumor tissue which are PMN-MDSCs is at least 2.5%, the fraction of living Treg cells in peripheral blood mononuclear cells is at most 1.5%, the fraction of living Treg cells in tumor tissue is at most 9% of CD4+ cells, the fraction of CD3ζ among CD4+ T cells in peripheral blood mononuclear cells is reduced compared to control cells of healthy individuals by a factor of at least 0.8, and the fraction of CD3ζ among CD8+ T cells in peripheral blood mononuclear cells is reduced compared to control cells of healthy individuals by a factor of at least by a factor of 0.65.

Description

DESCRIPTION OF THE FIGURES

[0064] FIGS. 1A-C: Viability and origin of cells present in the tumors and healthy tissue.

[0065] (FIG. 1A) Viability of all isolated cells isolated from tumor (Tu) and non-tumor healthy lung tissue (NTu) was measured by DAPI staining followed by flow cytometry top panel). The percentage of epithelial cells in AC or SCC tumor (Tu) and non-tumor healthy lung tissue (NTu) was measured by flow cytometry after immune-staining for cytokeratin bottom panel).

[0066] (FIG. 1B) The percentage of lymphocytes in tumor (Tu) and non-tumor healthy lung tissue (NTu) was measured by flow cytometry after immune-staining for CD45.

[0067] (FIG. 1C) Gating strategy for flow cytometry.

[0068] FIGS. 2A-C: The immune cell infiltration in NSCLC tumors and non-tumoral tissue in adenocarcinoma (AC) and squamous cell carcinoma (SCC).

[0069] (FIG. 2A) The percentage of cDC (CD11c+ HLA-DR+, top left panel), monocytes (CD14+ HLA-DR+, top right panel), mast cells (CD117+, bottom left panel) and B cells (CD19/20+, bottom right panel) from live cells in AC or SCC tumor (Tu) and non-tumor healthy lung tissue (NTu) was estimated by flow cytometry.

[0070] (FIG. 2B) The percentage of NK cells (CD3− CD56+, top left panel), T regs (CD4+ CD25+FoxP3+, top right panel), CD4+ T cells (CD4+, bottom left panel) and CD8+ T cells (CD8+, bottom right panel) from live cells in AC or SCC tumor (Tu) and non-tumor healthy lung tissue (NTu) was estimated by flow cytometry.

[0071] (FIG. 2C) The percentage of CD4+ T cells from CD3+ cells (CD4+, top left panel), of CD4+ T cells from CD3+ cells (CD8+, top right panel), memory CD4+ T cells (CD45RO.sup.+ CD4+, bottom left panel) and memory CD8+ T cells (CD45RO+CD8+, bottom right panel) from live cells in AC or SCC tumor (Tu) and non-tumor healthy lung tissue (NTu) was estimated by flow cytometry.

[0072] FIG. 3: Intratumoral INF-γ producing CD4.sup.+ and CD8.sup.+ T cells were significantly inhibited in SSC tumors compared to non-tumoral tissue and tumors in AC patients.

[0073] The percentage of IFN-γ.sup.+ cells from CD4.sup.+ T cells (top panel) and IFN-γ.sup.+ cells from CD8.sup.+ T cells from live cells in AC or SCC tumor (Tu) and non-tumor healthy lung tissue (NTu) was measured by flow cytometry.

[0074] FIGS. 4A-B: Cytokine production in tumor and non-tumoral tissue of NSCLC patients.

[0075] FIG. 4A: After cell stimulation with PMA+ ionomycin for 24 h, the production of GM-CSF, IL-1β, IL-2 in AC or SCC tumor (Tu) and non-tumor healthy lung tissue (NTu) were determined by Luminex

[0076] FIG. 4B: After cell stimulation with PMA+ ionomycin for 24 h, the production of TNF-α, IL-23, IL-10 in AC or SCC tumor (Tu) and non-tumor healthy lung tissue (NTu) were determined by Luminex.

[0077] FIGS. 5A-C: Presence of M-MDSC and Tregs in tumors, and their influence on the tumor microenvironment. Inhibition of CD3ζ expression in T cells and mRNA for ARG1 in PBMC.

[0078] (FIG. 5A) The percentage of M-MDSC (CD15.sup.− CD14.sup.+ CD33.sup.hi HLA-DR.sup.−/lo, left panel) and Tregs (right panel) among live PBMC from healthy aged-matched controls patients, AC patients and SCC patients was measured by flow cytometry.

[0079] (FIG. 5B) The amount of CD3ζ.sup.+ CD4.sup.+ T cells (left panel) and CD3ζ.sup.+ CD8.sup.+ T cells (right panel) among live PBMC from healthy aged-matched controls patients, AC patients and SCC patients was measured by flow cytometry.

[0080] (FIG. 5C) The relative expression of ARG1 was measured by qPCR from mRNA extracted from PBMC from healthy control, AC patients and SCC patients

[0081] FIG. 6: Correlation analysis between the different types of immune suppressive cells in AC and SCC.

[0082] Correlation analysis between the number of M-MDSC (CD15.sup.− CD14.sup.+ CD33.sup.hi HLA-DR.sup.−/lo) and Tregs number in PBMC from AC patients (top left), SCC patients (top right) and healthy aged-matched controls patients (bottom left).

[0083] FIG. 7: Experimental protocol

[0084] FIGS. 8A-B: Intra-tumoral T cells and NK cells are functionally more suppressed in SCC than in AC NSCLC patients.

[0085] Tumor and non-tumoral cell suspensions were stimulated with PMA and ionomycin for 1 h before Brefelden was added for additional 3 h. Then cells were stained for intracellular IFN-γ and analyzed by flow cytometry;

[0086] (FIG. 8A-1) First aspect of gating strategy to detect IFN-γ-producing CD8.sup.+ and CD4.sup.+ T cells.

[0087] (FIG. 8A-2) Second aspect of gating strategy to detect IFN-γ-producing CD8.sup.+ and CD4.sup.+ T cells.

[0088] (FIG. 8B) Cytokine production determined after 24 h of stimulation with PMA+ionomycin by luminex. Graphs represent means of n=43 AC and n=39 SCC (* p<0.05; ** p<0.01, *** p<0.005 or **** p<0.005).

[0089] FIG. 9: Gating strategy to detect intro-tumoral Tregs, polymorphonuclear- MDSC (PNM-MDSC) (CD14.sup.−CD15.sup.+CD11b.sup.+) and monocytic-MDSC (M-MDSC) (CD15.sup.−CD14.sup.+CD33.sup.hi; HLA-DR.sup.−/lo).

[0090] FIGS. 10A-C: Tregs are more abundant in blood of AC patients whereas the number of MDSC is higher in blood of SCC patients

[0091] (FIG. 10A) Gating strategy to detect Tregs, PNM-MDSC (CD14.sup.−CD15.sup.+CD11b.sup.+) and M-MDSC (CD15.sup.−CD14.sup.+CD33.sup.hiHLLA-DR.sup.−/low) in the blood of NSCLC patients. Quantitative analysis of MDSC in PBMCs from cryopreserved PBMC

[0092] FIG. 10B) First aspect of correlation between the amount of Tregs and PMN-MDSC in PBMC of NSCLC patients from cryopreserved (B) and fresh (C) samples.

[0093] (FIG. 10C) Second aspect of correlation between the amount of Tregs and PMN-MDSC in PBMC of NSCLC patients from cryopreserved (B) and fresh (C) samples.

[0094] FIGS. 11A-F: Suppressive function of Tregs, but not MDSC is comparable in blood of AC and SCC NSCLC patients

[0095] (FIG. 11A) Treg suppression assay.

[0096] (FIG. 11B) M-MDSC suppression assay—magnetic beads isolation, or

[0097] (FIG. 11C) cell sorting, including IL-2 and IFN-γ secretion detected by ELISA. (FIG. 11D) Flow cytometric detection of CD3ζ expression in T cells from fresh samples of NSCLC patients.

[0098] (FIG. 11E) LOX-1 protein was detected in plasma samples by ELISA. Graphs represent means n=56 AC, n=52 SCC and n=41 control (* p<0.05; ** p<0.01,*** p<0.005 or **** p<0.005).

[0099] (FIG. 11F-1) A first aspect of quantifying LOX-1 positive PMN-MDSCs by flow cytometry. Graphs represent means n=3 AC, n=1 SCC and n=2 control.

[0100] (FIG. 11F-2) A second aspect of quantifying LOX-1 positive PMN-MDSCs by flow cytometry. Graphs represent means n=3 AC, n=1 SCC and n=2 control.

[0101] (FIG. 11F-3) A third aspect of quantifying LOX-1 positive PMN-MDSCs by flow cytometry. Graphs represent means n=3 AC, n=1 SCC and n=2 control.

EXAMPLES

[0102]

TABLE-US-00001 TABLE 1 Tumor Samples Tumor Lung tissue Blood Blood Total Total samples 49 94 143 Adenocarcinoma (AC) 13 38 51 Squamous cell carcinoma (SCC) 12 35 47 Large cell carcinoma (LC) 1 1 Other 13 16 Benign 10 4

Example 1

[0103] Cells isolated from NSCLC tumors displayed high viability, however cells isolated from tumors were significantly less viable in comparison to cells isolated from non-tumoral tissue (see FIG. 1A top panel). Squamous cell carcinoma (SCC) contained higher level of epithelial cells (determined by FITC labeled human epithelial antigen and pan-cytokeratin) compared to adenocarcinoma (AC) (see FIG. 1A bottom panel). The infiltration of CD45.sup.+ lymphoid cells was significantly higher in tumor than in non-tumoral tissue (see FIG. 1B). Cells were determined as % of DAPI negative total cell count (see FIG. 1C gating strategy).

Example 2

[0104] The infiltration of all immune cell populations tested was comparable between AC and SCC histological subtypes (see FIG. 2). The immune cell infiltration was higher in tumoral than non-tumoral tissue. When not stated the percentage of immune cell population was determined from viable (DAPI negative) total cell count.

Example 3

[0105] Intratumoral INF-γ producing CD4+ and CD8+ T cells (see FIG. 3 top and bottom panel, respectively) were significantly inhibited in SSC tumors compared to non-tumoral tissue and tumors in AC patients.

Example 4

[0106] Cell suspensions were stimulated with PMA+ ionomycin for 24 h and cytokines were determined by Luminex. There was a significantly higher production of pro-inflammatory cytokines such as GM-CSF, IL-1β, IL-2 and TNF-α in tumors from AC patients over respective non-tumoral tissues (see FIG. 4A and FIG. B). Similarly, GM-CSF and TNF-α production is higher in tumors from AC patients than in tumors from SCC patients. This was observed also for IL-23 (see FIG. 4A and FIG. B). IL-10 was significantly enhanced in tumors from both NSCLC subtypes (see FIGS. 4B). No tumor-associated production of IFN-γ, IL-12p70, IL-13, IL-17, IL-22, IL-9, IL-21, IL-4, IL-6 was observed compared to non-tumoral tissue.

Example 5

[0107] M-MDSC (CD15.sup.−CD14.sup.+CD33.sup.hiHLA-DR.sup.−/lo) are abundant in blood of SCC patients whereas T regulatory cells (CD4.sup.+CD25.sup.+Foxp3.sup.+ CD127.sup.low) are elevated in AC patients (see FIG. 5A). Down-regulation of CD3ζ chain in T cells (see FIG. 5B) and elevated levels of ARG1 are induced in SCC but not in AC patients (see FIG. 5C).

Example 6

[0108] The percentage of the M-MDSC (CD15.sup.−CD14.sup.+CD33.sup.hiHLA.sup.−DR.sup.−/lo) population negatively correlates with the percentage of Tregs (CD4.sup.+CD25.sup.+Foxp3.sup.+CD127.sup.low) in the blood of AC patients but not of SCC patients or age-matched healthy controls (see FIG. 6). These data suggest that CD4.sup.+CD25.sup.+Foxp3.sup.+CD127.sup.low Tregs might represent the major immuno-suppressive population in NSCLC patients with AC.

Example 7: Materials and Methods

[0109]

TABLE-US-00002 TABLE 2 Overview of NSCLC patients Adeno- Squamous cell Age-matched carcinoma carcinoma donors (AC) (SCC) (controls) Sex (male/female) 16/27 34/5 10/7 Age, year (mean ± SD) 63 ± 11 67 ± 9 62 ± 10 Tumor tissue + non- 43 39 — tumoral tissue TMN Stage IA 9 6 — IB 10 9 IIA 5 8 IIB 4 6 IIIA 12 8 IIIB 1 0 IV 2 0 Blood 32 30 17 Plasma 30 30 30

[0110] The experimental protocol is outlined in FIG. 7.

[0111] Processing of Primary Tumors and Non-Tumoral Tissue from NSCLC Patients

[0112] Tumoral (Tu) and non-tumoral tissue (NTu) were obtained from 82 NSCLC patients undergoing neoadjuvant surgery. The characteristics of NSCLC patients are depicted in Table 2. Tissue samples obtained at the day of surgery were chopped into small pieces and incubated with agitation in RMPI 1640 medium (Gibco) with 100 ng/ml DNAse I and 20 ng/ml Collagenase D (both from Roche) for 45 minutes at 37° C. The cell suspension was then passed through the 100 μm strainer into 50 ml falcon tubes to obtain single cell suspensions. The infiltrated immune cell populations were analyzed by flow cytometry immediately after staining with specific antibodies for 30 min at 4° C. as described below. For further stimulation lymphocytes were counted and seeded into 96-well plates at the concentration of 1×10.sup.6 lymphocytes/nil in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (PAA), 2 mM GlutaMAX I CTS (Gibco) and 100 U/ml penicillin+100 mg/ml streptomycin (Gibco). Cells were stimulated with 50 ng/ml PMA and 10 ng/ml ionomycin (both from Sigma-Aldrich) for 1 h at 37° C. before Brefeldin A (BioLegend, 1000×) was added for 3 h to detect intracellular IFN-γ in CD8.sup.+ and CD4.sup.+ T cells. In some experiments recombinant IL-15 (Peprotech, 13 ng/ml) was added to the cell culture and the proliferating CD8.sup.+ T cells and NK cells were detected by flow cytometry after 3 and 7 days of incubation at 37° C. Cell viability was detected by DAPI (Thermo Fisher Scientific) or by LIVE/DEAD® Fixable Aqua Dead Cell Stain Kit 405 nm excitation (Invitrogen).

[0113] PBMC Isolation and Plasma Collection

[0114] 2-3 tubes of peripheral blood collected in VACUETTE® 9 ml K3 EDTA were obtained from 60 NSCLC patients undergoing neoadjuvant surgery and from 17-30 age-matched volunteers with no history of a malignant disease. 2-4 ml of plasma was collected after centrifugation of the peripheral blood at 3000 rpm for 5 minutes and stored at −80° C. Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Pague gradient centrifugation. PBMCs were counted and seeded into the 96-well plate at the concentration of 1×10.sup.6 PBMCs/ml in complete medium or lysed in RLT buffer for mRNA preservation as described above. Freshly isolated PBMC were analyzed for Tregs and MDSC content by flow cytometry immediately after staining with specific antibodies for 30 min at 4° C. as described below. Some PBMCs were cryopreserved in CryoStor® CS10 (BioLife Solution) in liquid nitrogen before analyses.

[0115] Antibodies Used for Immune Cell Analyses and Staining Protocol

[0116] Epithelial tumor cells—CD45 PE-DyLight594 (Exbio), anti-pan cytokeratin AlexaFluor 488 (eBioscience), anti-human epithelial antigen-FITC (DAKO).

[0117] Dendritic cells: Lin neg (CD3-FITC, CD19-FITC, CD20-FITC, CD56-FITC), CD45-PE-DyLight594, CD11c-APC (all from Exbio), HLA-DR-PE-Cy7 (BD Pharmingen™) Lymphocytes/NK cells: CD3-AlexaFluor 700, CD8-PE-Cy7, CD19-FITC, CD2O-FITC (all from Exbio), CD4-ECD (Beckman Coulter), CD56-PerCP/Cy5.5 (eBioscience). Naive/memory T cells: CD3-AlexaFluor 700, CD8-PE-Cy7, CD45RA-PE, CD45RO-APC, CD62L-FITC (all from Exbio), CCR7-PerCP/Cy5.5 (BioLegend), CD4-ECD (Beckman Coulter).

[0118] IFN-γ producing T cells: CD3-Alexa 700, CD8-PE-Cy7 (both from Exbio), CD4-ECD (Beckman Coulter), IFN-y-FITC (BD Pharmingen). T regulatory cells in tumors: CD8-PE-Cy7 (Exbio), CD4-ECD (Beckman Coulter), Foxp3-AlexaFluor 488 (eBioscience).

[0119] T regulatory cells in PBMC: CD4-PE-Cy7, CD8-eFluor 450, CD25-PerCP-Cy5.5, CD127-APC, Foxp3-AlexaFluor 488, CD3ζ-PE (all from eBioscience), Ki-67-AlexaFluor 700 (BD Pharmingen).

[0120] MDSC: CD14-BD Horizon V450 (BD Horizon), HLA-DR-Alexa Fluor 700, CD33-PE-Cy7, (BioLegend), CD11b-FITC, CD15-APC (eBioscience)+possibly LOX1-PE (BioLegend).

[0121] Cells were stained extracellularly with the mixture of appropriate antibody in PBS for 30 min at 4° C. For intracellular staining of IFN-γ, Foxp3, CD3 and Ki-67 the cells were fixed for 30 min using Fixation Buffer (eBioscience), permeabilized with Permeabilization Buffer (eBioscience) and stained intracellularly for 30 min at 4° C. Cells were washed with PBS and analyzed by LSRFortessa (BD Biosciences). Data were analyzed with FlowJo software (Tree Star). Flow cytometry data may be expressed as mean fluorescent intensity (MFI).

[0122] Cytokine Production and LOX-1 Plasma Detection

[0123] To determine cytokine production, cell supernatants were harvested 24 h after stimulation with PMA and ionomycin or harvested at day 3 and 7 after stimulation with IL-15 as described above. Cell culture supernatant was stored at −80° C. GM-CSF, IFN-γ, IL-10, IL-12p70, IL-13, IL-17, IL-22, IL-9, IL-1β, IL-2, IL-21, IL-4, IL-23, IL-6, TNFα were determined using Luminex assay (MILLIPLEX™ MAP Human Th17 Magnetic Bead Panel, Merck Millipore) by MagPix (XMAP Technology, Luminex). LOX-1 protein as a marker of PMN-MDSC presence was detected in plasma samples (diluted 1:10 or 1:50) from NSCLC patients by ELISA (RD System).

[0124] Isolation of CD33.sup.+HLADR.sup.−MDSC with Magnetic Microbeads

[0125] CD33.sup.+HLADR.sup.− cells were isolated from the PBMC obtained from leukapheresis of NSCLC patients using MACS microbeads and columns (Miltenyi Biotec). Briefly, thawed PBMC were resuspended in cold MACS buffer and incubated with HLA-DR microbeads (Miltenyi Biotec) for 15 min on ice. Then cells were washed with cold MACS buffer to remove unbound beads and subsequently subjected to depletion of HLA-DR.sup.+ cells on MACS column according to manufacturer's instructions. The negative cell fraction was collected, washed and then incubated with CD33 microbeads. MACS column was used for positive selection of CD33.sup.+HLA-DR.sup.− cells. The purity of the CD33.sup.+ cell population was evaluated by flow cytometry and exceeded 90%.

[0126] Isolation of CD33.sup.+CD14.sup.+HLA-DR.sup.− MDSC by Cell Sorting

[0127] Briefly, PBMC were isolated from leukapheresis of NSCLC patients by using Ficoll Paque and stored at −80° C. Thawed PBMC were resuspended in cold MACS buffer and incubated with CD33 microbeads (Miltenyi Biotec) for 15 min on ice. Then cells were washed with cold MACS buffer to remove unbound beads and subsequently subjected to depletion of CD33 negative cells on MACS column according to manufacturer's instructions. The CD33.sup.+ cell fraction was collected, washed and stained with anti-CD14 and anti-HLA-DR Ab for 20 min at 4° C. Cells were than washed with PBS and the CD33.sup.+CD14.sup.+ HLADR .sup.−/low MDSCs were subsequently sorted using S3e cell sorter (Biorad).

[0128] MDSC Suppression Assay

[0129] Purified autologous T cells (50 000 cells per well) were labeled with CFSE, activated using anti-CD3/CD28 expander beads (2.5×10.sup.5 beads per well) and incubated in the presence of different ratios of magnetic beads-purified or sorted MDSC (1:1, 1:2, 1:4 T cell/MDSC ratio). T-cell proliferation was measured as CFSE dilution using flow cytometry on day 6. Suppression is calculated as % of controls=(proliferation of analyzed sample−proliferation of non-proliferating cell)/(proliferation of control−proliferation of non-proliferating cells). The production of IFN-γ and IL-2 in cell culture supernatants was evaluated by ELISA (RD System).

[0130] Isolation of T Regulatory Cells and Suppression Assay

[0131] Thawed PBMC from NSCLC leukapheresis were resuspended in cold PBS and passed through 30 μm strainer to remove cell clumps before isolation. CD25.sup.+CD4.sup.+CD127.sup.low Tregs were isolated using EasySep Human CD4.sup.+CD127.sup.lowCD25.sup.+ regulatory T cell isolation kit (Stemcell Technologies). CD3.sup.+ effector cells were isolated by using EasySep Human T cell enrichment kit (Stemcell Technologies) and stained with CFSE (1 μM, Invitrogen). The purity and CFSE-staining was confirmed by flox cytometry. For suppression assay CFSE-stained CD3.sup.+ T effector cells (50 000 cells per well) were seeded in 96-well plate alone (negative control), activated using anti-CD3/CD28 expander beads (16.7×10.sup.3 Dynabeads per well−ratio 3:1 T cells/beads) only (positive control) or activated with beads and incubated in the presence of different ratios of purified Tregs (2:1, 1:1, 0.25:1, 0.125:1 Tregs/Teff ratio). Cells were incubated in complete RPMI 1640 supplemented with 10% human AB serum (200 μl/well). T cell proliferation was analyzed on day 3 (optimal proliferation of controls—60-80%, at least 3 generations). Suppression is calculated as described above for MDSC suppression assay. The production of IL-2 in cell culture supernatants was evaluated by ELISA (RD System).

[0132] qPCR and Proteomics Analysis

[0133] Total RNA was isolated using an RNeasy Mini Kit (Qiagen). Each sample containing RLT buffer and cell lysate was quickly thawed and processed in accordance with the manufacturer's protocol which included a DNase I digestion step. The RNA concentration and purity were determined using a NanoDrop 2000c (Thermo Scientific), and the RNA integrity was assessed using an Agilent 2000 Bioanalyzer (Agilent). Purified RNA samples were stored at −80° C. until further use. cDNA was synthesized from 100 ng of total RNA using an iScript cDNA Synthesis Kit (BioRad). Expression of ARG1 gene was determined by qPCR on CFX96 Touch™ Real-Time PCR Detection System (BioRad). Each 10 μl reaction contained 5 μl of KAPA PROBE FAST qPCR Master Mix (Kapa Biosystems), 0.5 μl of each forward and reverse primers (500 nM each; TIB Molbiol,), 0.5 μl of TaqMan probe (200 nM; TIB Molbiol), 1.5 μl of RNase-free water and 2 μl of 5× diluted cDNA. Each reaction was done in triplicate. The temperature cycling protocol was following: 3 minutes at 95° C. followed by 45 cycles (95° C. for 15 s and 60° C. for 60 s). The formation of PCR products of the expected lengths was confirmed by agarose gel electrophoresis. The Cq values were determined using CFX Manager software (BioRad) and the relative expressions of the studied genes were calculated with GenEx software (MultiD Analyses) with cut off at 36 cycle. Proteomics analyses were conducted from genes published in NSCLC cancer tumor samples in Cancer Genome Atlas. Immune cell population were analyzed using transcriptome-based computational microenvironment cell populations-counter (MCP-counter) method introduced by Becht et al. (2016) from n=508 AC and n=495 SCC patient' tumor samples.

[0134] Statistical Analysis

[0135] Two-tailed paired t-test or unpaired, non-parametric Mann-Whitney test were applied for data analysis using GraphPad PRISM 6 (San Diego, Calif., USA). The results were considered statistically significant if * p<0.05, ** p<0.01 or *** p<0.001. Data were expressed as mean±SEM.

Example 8: T Cells, B Cells and NK Cell Infiltration is Similar in AC and SCC Tumors, but Myeloid DC (CD11c.SUP.+.HLA-DR.SUP.hi.) are Significantly Decreased in SCC tumors

[0136] Immune cell infiltration is higher in NSCLC tumors than in adjacent non-tumoral tissue. Possible differences in T cell, B cell and NK cell or dendritic cell infiltration between two histologically distinct tumors in adenocarcinoma (AC) and squamous cell carcinoma NSCLC patients (SCC) were analyzed. Single cell suspensions from tumors and non-tumoral tissue obtained from 42 AC and 39 SCC neoadjuvant NSCLC patients (Table 2) were analyzed by flow cytometry. Cell viability was higher in non-tumoral than in tumoral tissue, but tumor cell viability was on average around 85% (see FIGS. 1A top panel). Higher infiltration of CD8.sup.+ and CD4.sup.+ T cells with predominantly memory phenotype, B cells, NK cells and DC in tumors was observed when compared to non-tumoral tissue (see Table 3). Infiltration rates of these immune cell populations between AC and SCC tumors were not statistically different. .

TABLE-US-00003 TABLE 3 Percentage of tumor-infiltrating cell (mean from 43 AC and 39 SCC samples) % Cell type Memory Memory Myeloid CD4.sup.+ T cells CD8.sup.+ T cell dendritic cells CD3.sup.+ CD4.sup.+ (CD45 RO.sup.+ CD8.sup.+ T cells (CD45 RO.sup.+ (CD11c.sup.+ NK cells (CD3.sup.+ (CD3.sup.+ CD4.sup.+ CD4.sup.+ T cells (CD3.sup.+ CD8.sup.+ CD8.sup.+ T cells HLA-DR.sup.hi B cells (CD3.sup.− CD56.sup.+ cells from T cells from from live T cell from from live from live (CD19/20.sup.+ from cells from Tissue live cells) live cells) CD4.sup.+ T cells) live cells) CD4.sup.+ T cells) CD45.sup.+ cells) live cells) live cells) AC Tu 3 1.33 77 1 67 2.4 0.8 0.15 AC NTu 0.03 0.2 65 0.05 40 2 0.1 0.09 SCC Tu 2.9 1.2 70 1 68 1 1 0.14 SCC NTu 0.03 0.2 60 0.2 43 1.8 0.05 0.08

Example 9: Intra-Tumoral T Cells and NK Cells are Functionally More Suppressed in SCC than in AC NSCLC Patients

[0137] Since phenotypic analysis of tumor-infiltrated immune cells offers only quantitative evaluation of immune cell infiltration, tumor and non-tumoral cell suspensions were stimulated with PMA and ionomycin for lh, followed by addition of Brefeldin A for additional 3 h. IFN-γ production in CD8.sup.+ and CD4.sup.+ T cells was subsequently determined by flow cytometry. The gating strategy is shown in FIGS. 8A-1 and 8A-2. The capacity of tumoral and non-tumoral CD8.sup.+ and CD4.sup.+ T cells to produce IFN-γ after non-specific stimulation was similar in T cells from AC and SCC patients (see Table 4). However, the IFN-γ production capacity of SCC tumors CD8.sup.+ and CD4.sup.+ T cells was impaired when compared to T cells coming from adjacent healthy tissue, suggesting that T cells in SCC tumors might be more suppressed in their IFN-y-mediated effector functions than T cells in AC tumors (see Table 4). Default IFN-y production was not observed in T cells in non-stimulated tissues.

TABLE-US-00004 TABLE 4 Percentage of IFNγ.sup.+ tumor-infiltrating cells % Cell type IFN-γ.sup.+ cells IFN-γ.sup.+ cells from CD8.sup.+ IFN-γ.sup.+ cells from CD4.sup.+ IFN-γ.sup.+ cells T cells from CD8.sup.+ T cells from CD4.sup.+ Tissue non-stimulated T cells non-stimulated T cells AC Tu 2 24 2 12 AC NTu 0.5 30 3 13 SSC Tu 2 21 1 10 SSC NTU 1.5 35 2 18

[0138] Furthermore, the cytokine production from tissues stimulated for 24 h (see FIGS. 8B) was analyzed by Luminex. The production of GM-CSF, IFN-γ, IL-1β, IL-2, IL-4, IL-23, IL-6 and TNF-α was significantly higher in AC tumors when compared to adjacent non-tumoral tissue. This might be due to a higher lymphocyte (see FIGS. 1B) and epithelial cell (see FIG. 1A bottom panel and Table 5) content in AC tumor tissue than in non-tumoral tissue. However, in SCC tumors the cytokine production, the majority of them being pro-inflammatory immune cytokines, was comparable with non-tumoral tissue, suggesting a higher suppression of pro-inflammatory cytokines after stimulation in SCC tumors when compared to AC as the leucocytes infiltration and the number of epithelial cells in tumors was comparable. There was no cytokine production observed in non-stimulated tissues (see FIG. 8B).

TABLE-US-00005 TABLE 5 Percentage of epithelial cells in tumors % Cell type epithelial cells from live CD45 Tissue negative cells AC Tu 17 AC NTu 4 SSC Tu 17 SSC NTU 0.2

[0139] As PMA and ionomycin represent strong unspecific stimulation of immune cells, IL-15 was used to activate predominantly NK cells and CD8.sup.+ T cells, playing a major role in antitumor immunity. Tumor cell suspensions were incubated alone or with IL-15 for 7 days. The proliferation of CD8.sup.+ T cells and NK cells was analyzed on Day 0, 3 and 7 by Ki67.sup.+ staining followed by flow cytometry. Whereas 68% and 79% of T cells and NK cells, respectively, proliferated on Day 3 of incubation in AC tumors, only 23% and 8% of T cells and NK cells, respectively, proliferated in SCC tumors, confirming a higher immunosuppressive environment in SCC than AC tumors (see Table 6).

TABLE-US-00006 TABLE 6 T cell proliferation % Cell type Ki67.sup.+ cells Ki67.sup.+ cells from CD8.sup.+ from CD3.sup.−CD56.sup.+ Tissue T cells T cells (NK cells) D0 Non Treated SCC 2 0 AC 13 0 D3 Non Treated SCC 0.5 0.5 AC 7 5 +IL-15 SCC 23 8 AC 68 79 D7 Non Treated SCC 1 0.5 AC 8 8 +IL-15 SCC 45 40 AC 62 50

[0140] Immune genes expression analyzed from the TCGA database (The Cancer Genome Atlas, National Cancer Institute and National Human Genome Research Institute, https://cancergenome.nih.gov, see Table 7) show higher expression of antitumor-related immune genes in AC than in SCC which again supports higher immunosuppressive microenvironment in SCC over AC. Interestingly, more antigens is expressed in SCC than AC.

TABLE-US-00007 TABLE 7 The expression of T regulatory/T cells and MDSC-related genes in NSCLC patients AC n= SCC n= *p < 0.05 Tregs genes CD25 2.907 2.863 NS FOXP3 2.823 2.792 p = 0.05 CCR7 2.953 2.820 * IL7R 3.055 3.007 * TNF 2.457 2.433 NS IFNG 1.981 1.885 NS IL2 0.644 0.382 * CD5 3.043 2.876 * CD69 3.037 2.896 * CCL22 3.072 2.920 * CCR4 2.547 2.246 * CCL17 2.404 2.077 * CCL5 3.397 3.373 NS CCR5 3.130 2.981 * CXCR3 2.909 2.669 * LAG3 2.941 2.938 NS CTLA4 2.765 2.682 * GITR 2.902 3.116 * ENTPD1 3.475 3.439 * NT5E 3.416 3.137 * IL10 2.233 2.187 NS TGFB1 3.519 3.561 * PD1 2.728 2.619 * TIM3 3.276 3.182 * ICOS 2.652 2.564 * TIGIT 2.899 2.845 * IDO1 3.264 3.217 * MDSC genes IRF8 3.264 3.131 * CEBPB 3.550 3.487 * S100A8 3.124 3.528 * S100A9 3.562 3.731 * RB1 3.428 3.472 * RORA 3.212 3.292 * RORC 3.411 2.623 * CHOP 3.258 3.335 * ARG1 0.583 0.996 * NOS2 2.311 2.713 * CYBB 3.538 3.409 * PDL1 2.781 2.849 * TGFB1 3.519 3.561 * IL4R 3.569 3.516 * GM-CSF 2.164 1.604 * G-CSF 2.028 2.226 * IL13 0.694 0.564 * ILIA 2.054 2.768 * IL10 2.233 2.187 NS TGFB1 3.519 3.561 * Data analysed from TCGA database overexpression (shown in bold)

Example 10: T Regulatory Cells Infiltration is Greater in AC Tumors whereas SSC Tumors are Infiltrated More by Myeloid-Derived Suppressor Cells

[0141] In view of the differences between the immunosuppressive tumor microenvironment of AC and SCC patients two major immunosuppressive immune cell populations—CD4.sup.+CD25.sup.+ Foxp3.sup.+CD127.sup.low Tregs and MDSC, here specifically PMN-MDSC and M-MDSC were analyzed. Fresh tissue samples were analyzed by flow cytometry. The gating strategy is shown in FIG. 9 and was, together with antibody staining for MDSC, adopted from Walter et al. (2012) and Bronte et al. (2016). Both Tregs and MDSC populations were detected in higher numbers in tumors than in non-tumoral tissue in both histological subtypes of NSCLC (see Table 8). However infiltration of Tregs was higher in AC than SCC tumors (see Table 8). Conversely, a higher number of PMN-MDSC and M-MDSC was detected in SCC than in AC tumors (see Table 8). As MDSC cannot be defined solely by phenotypic markers and it is technically challenging to perform suppression assays from intratumoral MDSC, the expression of genes associated with MDSC characterization as described in Bronte et al. (2016) was analyzed (see Table 7). The expression of genes related to Tregs and/or T cell function from TCGA database on (n=508 AC and n=495 SCC patients) was also evaluated. Table 7 shows that most of the higher expression of most genes associated with MDSCs in SCC, a higher expression of genes associated preferentially with Tregs/T cells in AC tumors, confirming the phenotypic observation.

TABLE-US-00008 TABLE 8 Percentage of tumor-infiltrating suppressor cells (mean of 43 AC and 39 SCC samples) % Cell type CD15.sup.− CD14.sup.+ CD4.sup.+ CD25.sup.+ CD14.sup.− CD15.sup.+ CD66.sup.hi Tissue FoxP3.sup.+ T cells CD11b.sup.+ cells HLA-DR.sup.−/low AC Tu 12 0.8 0.002 AC NTu 6 0.5 0.001 SSC Tu 8 5 0.014 SSC NTU 5 0.8 0.001

Example 11: Tregs are More Abundant in Blood of AC Patients whereas the Number of MDSC is Higher in Blood of SCC Patients

[0142] As Tregs infiltration was higher in AC and MDSC infiltration higher in SCC tumors, the presence of these cells was also analyzed in blood of NSCLC patients. The immunosuppressive population was quantitatively measured by flow cytometry in PBMCs isolated from NSCLC patients and age-matched donors with no history of malignant disease (see Table 2, FIG. 5A). PBMCs wear also functionally tested by suppression assays (see FIG. 11). The gating strategy is shown in FIG. 10A. Tregs (CD4.sup.+CD25.sup.+Foxp3.sup.+CD127.sup.low), PMN-MDSC (CD14.sup.−CD15.sup.+CD11b.sup.+) and M-MDSC (CD15.sup.−CD14.sup.+CD33.sup.hiHLA-Dr.sup.−/low) were analyzed from frozen (see FIG. 5A and Table 9) and from freshly isolated PBMCs (see Table 10). Tregs were more abundant in the blood of AC patients than in SCC patients and age-matched controls. Moreover, Tregs detection was not affected by cryopreservation. Tregs suppression assays further showed that Tregs from both histological subtypes were similarly competent to decrease CD8.sup.+ and CD4.sup.+ T cell proliferation (see FIG. 11A). This suggests only quantitative differences in Tregs in blood between AC and SCC patients. Interestingly, the number of Tregs correlated negatively with both MDSC populations only in blood of AC patients (see FIG. 6 for M-MDSC and 10B, 10C for PMN-MDSC).

TABLE-US-00009 TABLE 9 Percentage of MDSC in PBMC from frozen samples % Cell type CD14.sup.− CD15.sup.+ CD15.sup.− CD14.sup.+ CD11b.sup.+ cells CD66.sup.hi HLA- from DR.sup.−/low from cryopreserved cryopreserved Tissue PBMC PBMC Control 7 0.05 AC 3 0.05 SSC 6.5 0.18

[0143] To the contrary to Tregs, the numbers of both types of MDSC are affected by cryopreservation. However, analysis of fresh PBMC samples showed more M-MDSC cells in the blood of SCC than AC patients and age-matched controls (see Table 10). The number of PMN-MDSC was comparable between AC and SCC patients and was higher than in age-matched controls. As PMN-MDSC cannot be distinguished from neutrophils with no suppressive function we analyzed LOX1 expression on these cells by flow cytometry (see FIG. 11F-1, 11F-2 and 11F-3). The presence of LOX-1 positive PMN-MDSC was largely increased in SCC patients, compared to AC patients and healthy controls. Furthermore, LOX-1 protein associated with the presence of PMN-MDSC was significantly increased in plasma from SCC patients in comparison to AC patients and healthy age-matched controls (see FIG. 11E). Similarly, expression of ARG1, an effector molecule predominantly expressed in PMN-MDSCs, was increased in SCC patients (FIG. 5C). Immunosuppressive action of MDSCs was shown to inhibit CD3ζ expression in T cells. Indeed, CD3ζ chain expression was only inhibited in CD8.sup.+ and CD4.sup.+ T cells in SCC patients when compared to AC patients or age-matched controls (FIG. 5B and FIG. 11 D).

TABLE-US-00010 TABLE 10 Percentage of Treg cells and MDSC in PBMC from fresh samples % Cell type CD4.sup.+ CD25.sup.+ CD14.sup.− CD15.sup.+ CD15.sup.− CD14.sup.+ FoxP3.sup.+ T cells CD11b.sup.+ cells CD66.sup.hi HLA- from live fresh from live fresh DR.sup.−/low from live Tissue PBMC PBMC fresh PBMC Control 0.8 13 0.2 AC 1.3 32 0.2 SSC 0.5 31 0.8

[0144] To prove that MDSCs found in SCC patients might be more suppressive than MDSCs found in AC patients MDSC suppression assays were performed (see FIG. 11A-C). M-MDSC were purified either with magnetic beads (CD33.sup.+ HLA.sup.−DR.sup.−/low) (see FIG. 11B) or by cell sorting (CD33.sup.+CD14.sup.+HLA-DR.sup.−/low) (see FIG. 11C) and mixed with stimulated CFSE-labeled autologous T cells. Interestingly, M-MDSCs from both histological subtypes inhibited CD8.sup.+ and CD4.sup.+ T cell proliferation to the same extend. However, only M-MDSCs from SCC patients decreased substantially IL-2 and IFN-γ production from stimulated T cells in comparison to M-MDSCs from AC patients (see FIG. 11A-B). These results show that M-MDSCs are not only increased in the blood of SCC patients but also exhibit higher suppressive activity on T cells than M-MDSC in blood from AC patients.

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