IFNAR1 Deficiency Impairs Immunostimulatory Properties of Neutrophils in Tumor-Draining Lymph Nodes

Tumor-draining lymph nodes (TDLNs) are the first organs where the metastatic spread of different types of cancer, including head and neck cancer (HNC), occurs and have therefore high prognostic relevance. Moreover, first anti-cancer immune responses have been shown to be initiated in such LNs via tumor-educated myeloid cells. Among myeloid cells present in TDLNs, neutrophils represent a valuable population and considerably participate in the activation of effector lymphocytes there. Tumor-supportive or tumor-inhibiting activity of neutrophils strongly depends on the surrounding microenvironment. Thus, type I interferon (IFN) availability has been shown to prime anti-tumor activity of these cells. In accordance, mice deficient in type I IFNs show elevated tumor growth and metastatic spread, accompanied by the pro-tumoral neutrophil bias. To reveal the mechanism responsible for this phenomenon, we have studied here the influence of defective type I IFN signaling on the immunoregulatory activity of neutrophils in TDLNs. Live imaging of such LNs was performed using two-photon microscopy in a transplantable murine HNC model. CatchupIVM-red and Ifnar1-/- (type I IFN receptor- deficient) CatchupIVM-red mice were used to visualize neutrophils and to assess their interaction with T-cells in vivo. We have evaluated spatiotemporal patterns of neutrophil/T-cell interactions in LNs in the context of type I interferon receptor (IFNAR1) availability in tumor-free and tumor-bearing animals. Moreover, phenotypic and functional analyses were performed to further characterize the mechanisms regulating neutrophil immunoregulatory capacity. We demonstrated that inactive IFNAR1 leads to elevated accumulation of neutrophils in TDLNs. However, these neutrophils show significantly impaired capacity to interact with and to stimulate T-cells. As a result, a significant reduction of contacts between neutrophils and T lymphocytes is observed, with further impairment of T-cell proliferation and activation. This possibly contributes to the enhanced tumor growth in Ifnar1-/- mice. In agreement with this, IFNAR1-independent activation of downstream IFN signaling using IFN-λ improved the immunostimulatory capacity of neutrophils in TDLNs and contributed to the suppression of tumor growth. Our results suggest that functional type I IFN signaling is essential for neutrophil immunostimulatory capacity and that stimulation of this signaling may provide a therapeutic opportunity in head and neck cancer patients.

Tumor-draining lymph nodes (TDLNs) are the first organs where the metastatic spread of different types of cancer, including head and neck cancer (HNC), occurs and have therefore high prognostic relevance. Moreover, first anti-cancer immune responses have been shown to be initiated in such LNs via tumor-educated myeloid cells. Among myeloid cells present in TDLNs, neutrophils represent a valuable population and considerably participate in the activation of effector lymphocytes there. Tumorsupportive or tumor-inhibiting activity of neutrophils strongly depends on the surrounding microenvironment. Thus, type I interferon (IFN) availability has been shown to prime anti-tumor activity of these cells. In accordance, mice deficient in type I IFNs show elevated tumor growth and metastatic spread, accompanied by the pro-tumoral neutrophil bias. To reveal the mechanism responsible for this phenomenon, we have studied here the influence of defective type I IFN signaling on the immunoregulatory activity of neutrophils in TDLNs. Live imaging of such LNs was performed using two-photon microscopy in a transplantable murine HNC model. Catchup IVM-red and Ifnar1 -/-(type I IFN receptor-deficient) Catchup IVM-red mice were used to visualize neutrophils and to assess their interaction with T-cells in vivo. We have evaluated spatiotemporal patterns of neutrophil/T-cell interactions in LNs in the context of type I interferon receptor (IFNAR1) availability in tumor-free and tumor-bearing animals. Moreover, phenotypic and functional analyses were performed to further characterize the mechanisms regulating neutrophil immunoregulatory capacity. We demonstrated that inactive IFNAR1 leads to elevated accumulation of neutrophils in TDLNs. However, these neutrophils show significantly impaired capacity to interact with and to stimulate T-cells. As a result, a significant reduction of contacts between neutrophils and T lymphocytes is observed, with further impairment of T-cell proliferation and activation. This possibly contributes to the enhanced

INTRODUCTION
In the context of cancer, neutrophils can exhibit both pro-and anti-tumoral properties. Their behavior is determined by signals from tumor cells and the tumor microenvironment (1)(2)(3). In many cancer entities, including head and neck cancer (HNC), high intra-tumoral neutrophil counts have been shown to be associated with a worse patient prognosis (4)(5)(6). Several responsible mechanisms have been identified, including the support of angiogenesis (7,8), the promotion of metastatic spread (9)(10)(11)(12), the regulation of B cell activation (13,14), as well as the regulation of T-cell responses in tumor tissue (15)(16)(17). Neutrophil tumorigenicity is regulated by tumor-derived factors, such as type I interferons (IFNs) (2,8,18,19), transforming growth factor beta (TGFb) (20), or granulocyte-colonystimulating factor (G-CSF) (21). While TGFb and G-CSF have been shown to stimulate pro-tumoral activity of neutrophils, type I IFNs promote anti-tumoral functions of such cells. Of note, type I IFN signaling does not only affect the anti-tumoral differentiation of these cells, but has also been shown to stimulate anticancer immunosurveillance (22,23) as well as negatively affecting the metastatic dissemination of solid tumors via a variety of mechanisms.
Among pathways by which neutrophils affect tumor progression, the regulation of adaptive immune responses has recently been investigated in more detail. In the immediate tumor environment, neutrophils were shown to directly stimulate antitumor responses by presenting antigens to CD4 + T-cells and as well as activating cytotoxic CD8 + T-cells (16,18,24). On the other side of the spectrum, pro-tumoral neutrophils can suppress cytotoxic CD8 + T lymphocytes (25) or secrete chemo-attractants such as CCL17 to attract regulatory T-cells to the tumor microenvironment, which have immunosuppressive attributes and are associated with tumor progression and worse survival (15).
While these neutrophil-related pro-and anti-tumoral mechanisms have mostly been established in the immediate tumor microenvironment, there is growing evidence that neutrophils accumulate in organs of metastasis even before the arrival of tumor cells, and form a "pre-metastatic niche" (26). The formation of the pre-metastatic niche is attributed to the release of factors supporting metastatic seeding of tumor cells. Similar to the immediate tumor microenvironment, type I IFNs were shown to inhibit the pro-tumoral differentiation of neutrophils in the pre-metastatic organs, as shown in a previous study of Wu et al., where mice deficient in interferonalpha/beta receptor (Ifnar1 -/-) developed higher metastasis loads in a mammary and lung carcinoma mouse model (9). This phenomenon depends on the release of pro-metastatic molecules, such as S100A8/9 by neutrophils infiltrating premetastatic lungs.
In HNC, regional metastasis to tumor-draining cervical lymph nodes occurs early during tumor progression and consequently, the lymph node status remains one of the most relevant prognostic factors for HNC patients (27). To date, mechanisms involved in the formation of a pre-metastatic niche in tumor-draining lymph nodes (TDLNs) have not been investigated. Therefore, the goal of this study was to explore the immunoregulatory activity of TDLNs-associated neutrophils prior to the development of nodal metastasis, with a special emphasis on the effects of type I IFN-signaling. A better understanding of the immunoregulatory role of neutrophils in pre-metastatic TDLNs in HNC may well open the door for novel therapeutic approaches.
For the experiments, male littermates between 7-12 weeks were used. All mice used in this study were on the C57BL/6 background. Mice were housed and bred under specific pathogen-free conditions at the animal facility of the University Hospital Essen (Essen, Germany). Animal experiments were performed according to German laws and guidelines of the Federation of European Laboratory Animal Science Associations (FELASA) and were approved by the animal ethics committee.

Tumor Model
The MOPC cells were injected subcutaneously (s.c. 1 x 10 6 in 100 μl PBS) into the flank of C57BL/6 and Ifnar1 -/mice, tumor growth was measured by caliper every 2 to 3 days. Tumor-free animals from the same strains were used as control animals. Tumor volume was calculated as follows: Latest on day 14 (unless stated otherwise) after tumor implantation mice were sacrificed and tissues were harvested under aseptic conditions. Blood was taken by heart puncture with a heparinized syringe and stored on ice. Tumors and inguinal TDLNs were harvested and stored in DMEMc (DMEM, 10% FBS, 1 mM sodium pyruvate and 1% penicillinstreptomycin, all from Gibco, by Thermo Fisher Scientific, Waltham, US) on ice for a maximum 30 minutes before processing.

Histology
On day 14 post tumor cell injection, mice were sacrificed, LNs were extracted and snap-frozen in liquid nitrogen in Tissue-Tek O.C.T. Compound (Sakura Finetek, Japan) containing 5% paraformaldehyde. Afterwards, 7-mm cryosections were prepared, fixed with -20°C cold acetone and stained with a hematoxylin-eosin, based on standard protocol. Microscopy was performed using Olympus BX51 manual upright epifluorescence microscope (Shinjuku, Japan) and tissues were analyzed manually for the presence of metastasis based on the morphology.

Isolation of LN Neutrophils and In Vitro Stimulation of T-Cells With Neutrophils
Neutrophils were isolated from LN single-cell suspensions using flow sorting (FACS Aria III Cell Sorter (BD, Franklin Lakes, US), based on the phenotype (single alive Ly6G + , CD11b + cells) with the purity >95%. T-cells were isolated from single-cell suspension from spleen of naïve WT mice using flow sorting based on the phenotype (single alive CD3 + cells) with the purity >95%. Sorted neutrophils were co-incubated in DMEMc with recombinant murine IFN-l2 (Novus Biologicals, Littleton, US) in the concentration 10 ng per 50 000 cells, during 4h at 37°C in a humidified incubator with 5% CO 2 . After sorting, T-cells were loaded with CFSE (Biolegend, San Diego, US) according to manufacturer recommendation and co-incubated with anti-CD3/28-beads (STEMCELL, Vancouver, Canada) during 72h in the absence or presence of neutrophils (WT and Ifnar1 -/-), isolated from TDLNs (50.000 neutrophils and 50.000 T-cells were used for each condition). On day 3 the cell suspension was incubated for 4h at 37°C with Brefeldin A and Monensin (for IFN-g accumulation) followed by staining of surface and viability markers (30min at 4°C). The intracellular markers [IFN-g and Factin (Phalloidin)] were stained in next step, after fixation and permeabilization with the Fix/Perm Buffer Set according to the manufacturer's instructions (all reagents from Biolegend, San Diego, US).

In Vitro Multiphoton Imaging
Anesthesia and surgeries were performed in accordance with FELASA guidelines. The Catchup IVM-red x C57BL/6 and Catchup IVM-red x Ifnar1 -/mice were placed under ketamine/ xylazine (100 μg/g/10 μg/g in 0.9% NaCl i.p.) anesthesia before surgery. During surgery and following imaging, mice were anesthetized with isoflurane (1,5% mixed with oxygen, 250 ml/ min). The body temperature was kept at ∼36°C using a feedback-controlled heating pad. 30 min prior to the MPmicroscopy, T lymphocytes were fluorescein-labeled by tailvein injection of 100 ml PBS containing 10 mg FITC coupled CD3 antibody (clone 17A2, Biolegend, San Diego, US). Twophoton microscopy of the previously surgically exposed inguinal lymph node was performed using the Leica DM6000 CFS-based M P m i c r o s c o p e w i t h s i m u l t a n e o u s d e t e c t i o n v i a photomultipliers (PMTs) and hybrid reflected-light detectors (HyDs) and an HCX IRAPO L 25×/0.95-NA (numerical aperture) water-immersion objective (Leica, Wetzlar, Germany). Illumination was performed at 960 nm using a Coherent Chameleon Vision II Ti: sapphire laser. Td-Tomatoexpressing neutrophils were detected with a 585/50 filter, and Tcells were detected by FITC signal with a 525/50 filter. The lymph node structures were detected by second harmonic generation (SHG) with a 460/50 filter cube. Rendering threedimensional (3D) stacks and image processing was performed using IMARIS image visualization and analysis software (OXFORD instruments, Belfast, UK). Cells counts were performed in three different regions of interest (ROIs) of 150x150 μm per lymph node. To account cell movement over time, cell counts were performed at the beginning and at the end of the imaging time frame (15 minutes) and then averaged per ROI. Cell interactions were manually counted in ROIs over the duration of 15 minutes. Immediate cell proximity between fluorescently labeled neutrophils and T-cells in a threedimensional plane was counted as a cell-cell interaction.

Treatment With IFN Lambda In Vitro
The MOPC cells were injected subcutaneously (1 x 10 6 s.c. in 100 μl PBS) into the flank of C57BL/6 and Ifnar1 -/mice as described above. For therapy approach, mice were intraperitoneally injected with 8 μg of recombinant murine IFN-l2 (Novus Biologicals, Littleton, US) in 100 μl of 0,9% NaCl solution at days 0, 2, 4, and 7 after tumor setting. Animals injected only with 100 μl of 0,9% NaCl were used as controls. Tumor growth was measured as described above. On the day 9 mice were sacrificed, the right inguinal TDLNs were removed, single-cell suspensions were prepared and flow cytometry was performed as described above.

Treatment With IFN Lambda and In Vitro Multiphoton Imaging
The MOPC cells were injected subcutaneously (1 x 10 6 s.c.) into the flank of Catchup IVM-red x C57BL/6 and Catchup IVM-red x Ifnar1 -/mice. The in vivo multiphoton imaging was performed on day 14 after tumor setting. Treatment with recombinant murine IFN-l2 (20 μg in 100 μg PBS i.p., Novus Biologicals, Littleton, US) was performed twice: 24 hours and 1 hour before the imaging.

Quantitative Real Time Polymerase Chain Reaction
Single cells from TDLNs were collected and stored in RNAlater (Invitrogen) RNA stabilization solution at -20C. The RNA was isolated using Qia Shredder and RNeasy Mini Kit (Qiagen, Hilden, Germany) and the cDNA was produced using the Superscript II Reverse Transcriptase Kit (Invitrogen, Thermo Fisher Scientific, Waltham, MA, US). qRT-PCR was performed at 60°C annealing temperature. As housekeeping gene, Rps9 was used. The mRNA expression was measured using the Luna Universal qPCR Master Mix (New England BioLabs, Ipswich, MA, US). Relative gene expressions were calculated by 2^-DCt formulations. Primer sequences (Rps9, Ifnb1, Tnf, Ccl19, ccl21, Cxcl1, Cxcl2) are available upon request.

Proteome Profiler ™ Array
For analysis of cytokines profiles were used Proteome Profiler Array (Mouse XL Cytokine Array Kit, Cat.N ARY028, R&D Systems, Minneapolis, USA), based on manufacturer guidelines. In short, LN tissue (0.06 g of tissue in 0.2 ml DMEMc) was incubated for 4h at 37°C. Supernatant was collected and incubated overnight at 4°C with antibody-coated spotted membranes from the kit. After incubation, membranes were washed three times, biotin-conjugated antibody mix was added with the following incubation for one hour at room temperature. Next, the membrane was washed three times, and an HRPconjugated antibody was added. Then, membranes were incubated in a chemiluminescence reagent that produced a light signal proportional to the amount of antibody-target analyte complexes formed. Analysis and quantification of signal intensity were done using FIJI (ImageJ) software. Signal intensities were normalized to the control spots intensities on the corresponding membranes.

Statistical Analysis
Statistical analyses were performed using Kruskal-Wallis ANOVA with the Bonferroni correction for multiple comparisons and Mann-Whitney U test for two independent samples. Data are shown as median with interquartile range or as an individual values with median. P<0.05 was considered significant.

Accumulation of Neutrophils in Tumors and Tumor-Draining Lymph Nodes Is Elevated in Ifnar1 -/-Mice
Neutrophils have been shown to control the pre-metastatic niche formation, therefore we aimed to investigate the role of TDLNinfiltrating neutrophils in this process with the focus on type I IFNs as regulators of neutrophil activity. First, we evaluated the growth of transplantable MOPC in WT and type I IFN receptor (IFNAR1) knockout (Ifnar1 -/-) mice and observed significantly elevated primary tumor growth in Ifnar1 -/animals ( Figures 1A, B, D, E) compared to WT controls. Moreover, we observed significantly induced IFN-b expression in TDLNs at day 14 post tumor injection ( Figure S1), which could be responsible for the initiation of antitumor adaptive immune responses in WT animals. At this time point, we observed the increased size of TDLNs in Ifnar1 -/animals ( Figures 1C, F), but no visible metastases were present in TDLNs in both mouse strains ( Figure 1G-J). In parallel, elevated neutrophil and T-cell infiltration was observed in Ifnar1 -/animals ( Figures 1K, L and S2).
To underpin the mechanism responsible for elevated neutrophil infiltration of TDLNs in type I IFN deficiency, we analyzed the expression of CCR7 on LN neutrophils, as this receptor is suggested to be involved in the recruitment of neutrophils to T-cell zones (32). We demonstrated a significant increase of CCR7 expression on LN neutrophils in Ifnar1 -/vs WT mice in comparison to tumor-free levels (Figure 2A, Figure   S3) and the elevated fraction of CCR7 + neutrophils in tumorbearing Ifnar1 -/mice in comparison to tumor-free animals estimated by flow cytometry ( Figure S3) (Neutrophils were indicated as single alive CD11b + Ly6G + cells, gating strategy is presented on Figure S4; representative dot plots are presented on Figure S3). These findings are in line with immunofluorescent histological staining, which shows increased amounts of CCR7 + LN neutrophils in tumor situation, especially in Ifnar1 -/mice ( Figures 2B-F, S5). In parallel with increased CCR7 expression on neutrophils, expression of CCR7 on lymphocytes in TDLN was also increased in Ifnar1 -/animals ( Figure S6). At the same time, the expression of other molecules possibly involved in neutrophil homing into lymph nodes, such as CXCR4, CD11b ( Figures S7A, B) and chemotaxis such as CXCL2, TNF-a, CCL21, (Figures S7F-H) was not significantly changed on a gene (Figures S7C, E, G) and protein levels ( Figures S7D, F, H). The expression of CCL19 was changed on the gene level ( Figure  S7I), but not on protein level ( Figure S7J).

Characteristics of Neutrophils Infiltrating TDLNs
Next, we aimed to evaluate changes in the phenotype and functions of TDLN-associated neutrophils related to type I IFN deficiency. We assessed the expression of molecules involved in antigen presentation and T-cell activation on neutrophils and observed a downregulated expression of MHC-I and ICAM-1 on neutrophils derived from Ifnar1 -/animals ( Figures 3A, B, D). At the same time, no differences in the expression of other markers associated with antigen-presenting properties of neutrophils, i.e. MHC-II, CD80 and CD86, were found ( Figures S8A-C).
It was shown that filamentous actin (F-actin) polarization at the immune synapse of antigen-presenting cells maintains synapse stability necessary to induce T-cell activation (33). We performed the phalloidin staining of isolated LN neutrophils to check cytoskeleton organization with flow cytometry, as microscopical evaluation of F-actin in neutrophils is impeded due to the size and complex shape of the nucleus (34). We detected a significantly lower F-actin levels in Ifnar1 -/as compared to WT ( Figures 3C, D), suggesting decreased Factin polarization and therefore impaired immunological synapse stability in IFN I deficiency as a possible reason for impaired interaction between neutrophils and T-cells in TDLNs.
Since MHC-I and ICAM-1 are known to be involved in the formation of immunological synapse between APCs and T-cells, we aimed to visualize the interactions between neutrophils and T-cells in regional LNs of tumor-bearing animals.

IFN I Deficiency as a Reason for Impaired Interaction Between Neutrophils and T-Cells in TDLNs
The Catchup IVM-red mice were used for imaging of neutrophils and their cell/cell interactions in tumor-draining inguinal LNs via two-photon intravital microscopy. To assess the interaction of neutrophils with T Lymphocytes in such LNs in vivo, T Lymphocytes were labeled with FITC-coupled CD3 antibody. We observed that tumor injection led to a significant downregulation of neutrophil/T-cell interactions in TDLNs of Ifnar1 -/mice, but not in WT mice ( Figures 4A-C) ( Supplementary Movies 1, 2). Naïve non-tumor-bearing WT and Ifnar1 -/mice were used as a control. We did not observe any differences in interaction numbers between control groups ( Figure 4C). Of note, to avoid artifacts due to different

Neutrophils Deficient in Type I IFN Signaling Show Decreased Capacity to Activate T-Cells
The observed decrease of neutrophil interactions with T-cells in Ifnar1 -/mice could lead to the impaired activation and proliferation of effector T Lymphocytes. Indeed, we observed significantly reduced activation of T-cells responses in TDLN tissue and isolated cells of Ifnar1 -/mice, estimated by Ki-67 expression ( Figures 4D-J and S9). In line with that, lower IFN-g expression in CD4 + and CD8 + T-cells in TDLNs was observed ( Figures 4K, L) To prove the role of neutrophils in activation of T-cells in vitro, we co-incubated isolated WT vs. Ifnar1 -/-TDLN neutrophils with WT naïve T-cells. Indeed, we observed significantly suppressed T-cell activation after co-incubation with Ifnar1 -/neutrophils, as visualized by reduced IFN-g production by both CD4 + and CD8 + T Lymphocytes ( Figures 4M, N).
Altogether, these results suggest that deficiency in type I IFNs might be responsible for impaired neutrophil/T-cell interactions, resulting in impaired activation of anti-tumor immune responses. This in turn could be responsible for the observed accelerated tumor growth in IFNAR1-deficient mice.

Activation of IFN Signaling Downstream of Non-functional IFNAR1 Rescues T-Cell Stimulatory Capacity of Neutrophils in Ifnar1 -/-Mice In Vitro and In Vivo
Therapeutic strategy aiming to restore type I IFN signaling in neutrophils could enhance their anti-tumoral properties (35). Among others, type III interferons (interferon lambda, IFN-l) are the most functionally similar to type I IFNs and share downstream signaling pathway, although signaling through distinct receptors (36). Therefore, we aimed to evaluate whether IFNAR1 deficiency could be rescued by the activation of IFN signaling downstream of the inactive receptor, using IFNl, hereby potentially activating immunostimulatory properties of Ifnar1 -/-TDLN neutrophils.
We first isolated Ifnar1 -/-TDLN neutrophils and treated them in vitro with IFN-l to evaluate their capacity to stimulate T-cells. Indeed, we observed elevated T-cell activity indicated by their IFN-g production after co-incubation with such treated neutrophils, as compared to untreated Ifnar1 -/cells ( Figures 5A-D). Moreover, treatment with IFN-l increased MHC-I expression by TDLN neutrophils ex vivo ( Figure 5E). Next, we assessed the capacity of IFN-l to rescue cell stimulatory properties of IFNAR1-deficient neutrophils in vivo.
To this end, we treated Ifnar1 -/tumor-bearing mice with IFN-l and monitored neutrophil/T-cell interactions using the in vivo multiphoton imaging, as described above. In agreement with our in vitro data, we observed elevated counts of interactions between neutrophils and T-cells in TDLNs of IFN-l treated mice, compared to untreated animals ( Figure 5F).
Finally, to prove the therapeutical effect of IFN-l on antitumor immune responses and tumor growth in IFNAR1deficient animals in vivo, we set tumors into mice and treated animals with IFN-l as described in methods. Tumor growth was monitored and mice sacrificed at day 9. Indeed, Ifnar1 -/mice treated with IFN-l show significantly decreased tumor growth, as compared to untreated Ifnar1 -/mice ( Figure 5G).
Collectively, these data suggest that inactive IFNAR1 on neutrophils leads to the prominent defect in the neutrophilmediated activation of anti-tumor T-cell responses in TDLNs. IFNAR1-independent rescue of type I IFN signaling, using IFNl, improves the immunostimulatory capacity of neutrophils in TDLNs and contributes to the suppression of tumor growth. Overall, our results suggest that neutrophil-mediated regulation of anti-cancer immune responses in lymph nodes is modulated, at least in part, by type I IFN signaling. Deactivation of type I IFN receptor (IFNAR1) on neutrophils leads to a significant reduction of neutrophil/T-cell interactions accompanied by the decreased activity and proliferation of the T cells, and elevated tumor growth in such mice. Ex vivo analyses confirmed the reduced capacity of such type I IFN-deficient neutrophils to stimulate T-cells, as measured by a significantly suppressed IFN-g production by both CD4 + and CD8 + cells.

DISCUSSION
Previously, we could already show for different tumor entities that the deficiency in type I IFN signaling leads to the elevated tumor growth in Ifnar1 -/mice and changed tumorigenic properties of tumor-associated neutrophils (9). Type I IFN signaling in the immediate tumor microenvironment has also been shown to stimulate anti-cancer immunosurveillance as well as to reduce the metastatic dissemination of carcinomas, which is associated with the progressive tumor growth (22,23). These findings stem from animal models as well as human tissue analysis for different types of solid cancers. In mice, the metastatic spread of mammary or lung carcinoma has been shown to be accelerated in Ifnar1 -/mice (9). Similar effects were demonstrated in human melanoma cells, where an impaired production of type I IFN by cancer cells was associated with bone metastasis formation (38). In colorectal cancer, IFNAR1 was shown to be actively degraded in the tumor tissue by phosphorylation-dependent ubiquitination, which was supported by VEGF (39). This led to the impairment of type I IFN signaling and resulted in the immunosuppression that supported tumor growth (40). Our primary tumor analyses suggest the existence of similar mechanisms in head and neck cancer.
In the light of their prognostic importance for HNC, but also due to their relevance for the initiation of anti-tumor immune responses, we thereafter focused on the analysis of pre-metastatic tumordraining lymph nodes. In particular, we chose to analyze lymph nodes prior to the formation of metastases, which represent an intriguing therapeutic target in HNC patients. Our analyses revealed that TDLN-associated neutrophil counts in tumor-bearing mice were overall significantly elevated, compared to tumor-free animals.
Notably, significantly more neutrophils accumulated in TDLNs of tumor-bearing Ifnar1 -/mice compared to tumor-bearing WT controls. In light of strongly elevated primary tumor growth in Ifnar1 -/mice this phenomenon suggests pro-tumoral bias of such LN neutrophils. As shown for other tumor entities, IFNAR1 deficiency is a well-documented driver of pro-tumoral differentiation of neutrophils (8,41,42), as well as neutrophil migration to organs of metastasis, even prior to metastasis formation (9). To further illuminate the mechanism of neutrophil recruitment, we performed analyses of CCR7 expression on LN neutrophils and observed significant upregulation of this receptor on Ifnar1 -/cells in tumor-bearing animals as compared to tumorfree mice. The role of CCR7 as a receptor facilitating migration of neutrophils to lymph nodes has been previously suggested (32). In parallel with increased CCR7 expression on Ifnar1 -/neutrophils, lymphocytes from Ifnar1 -/-TDLN were also characterized by the elevated CCR7 expression. Such phenomenon can be additionally responsible for increased migration of lymphocytes to TDLN and elevated size of TDLN that is observed in Ifnar1 -/mice. In agreement with our data, activation via IFNAR1 was reported to regulate CCR7 on different cell types (43) In recent years, the immunoregulatory functions of neutrophils in the tumor microenvironment have been described. Neutrophils appear to interact with T-cells in multiple ways, including the presentation of antigens for T-cell activation, as shown in lung cancer model (16). Recruitment of T-cells by neutrophils is known to be facilitated by cytokines, such as TNF-a, Cathepsin G, and neutrophil elastase (15). Our in vivo analyses allowed a detailed estimation of spatiotemporal neutrophil/T-cell interactions in TDLNs. We could demonstrate significantly increased counts of neutrophil/T-cell interactions in tumor-bearing mice, compared to non-tumor-bearing controls. However, tumor-bearing Ifnar1 -/mice show significantly less T-cell interactions per neutrophil in comparison to WT mice. The expression of ICAM-1 expression by neutrophils could be one of the mechanisms involved in this process (18). ICAM-1 is a co-stimulatory ligand expressed on APCs that was demonstrated to stabilize cell-cell interactions and to facilitate antigen presentation to lymphocytes (44,45). Reduced expression of MHC-I of the neutrophil surface could also lead to impaired contacts with T-cells (46). The actin cytoskeleton plays also a crucial role in the formation and maintenance of the immunological synapse between antigen-presenting cell and T-cell (47). Interestingly, we observed a reduction of F-actin signal in Ifnar1 -/neutrophils, which could indicate the decreased capacity of these cells to form immunological synapses with T-cells.
Lower counts of neutrophil/T-cell interactions in Ifnar1 -/mice were accompanied by the reduced proliferation of T-cells in LN of such mice. Moreover, stimulation of WT T-cells with Ifnar1 -/neutrophils resulted in decreased activity and proliferation of these T-cells, as evaluated by down-regulated Ki67-staining and decreased IFN-g expression. Altogether, these results suggest that IFN-deficiency does not only reduce the number of interactions between neutrophils and T-cells, but in addition, it leads to less efficient activation of T-cells. These findings are in agreement with previous data showing the supporting role of IFNs in the activity and antigen presentation capacity of APCs, including monocytes and dendritic cells (DCs) (48,49).
With regard to a potential therapeutic approach, we attempted a rescue experiment using the systemic application of IFN-l to Ifnar1 -/mice. Interferon IFN-l, the member of type III IFNs, signals via distinct receptor complex, but activates common Janus kinase (JAK) and signal transducer and activator of transcription (STAT) pathways, similar to IFN-a/b. Moreover, type III IFNs share many biological activities with type I IFNs (36). Beside direct effects on cancer cells and T lymphocytes (50), recent studies demonstrated the capacity of IFN-l to promote NK-cell mediated innate immunity in solid cancer models (51,52). In addition, IFN-l has recently been suggested to play a role in neutrophil stimulation during inflammatory processes (53). Importantly, we observed that IFN-l treatment of tumor-bearing Ifnar1 -/mice led to a significant increase of neutrophil/T-cell interactions in TDLNs of these mice, to the levels observed in WT mice. This was accompanied by significantly reduced tumor growth in such IFN-l−treated mice. Treatment of isolated LN-associated neutrophils with IFN-l in vitro rescued the capacity of these cells to activate T-cells and possibly to stimulate T-cell proliferation. Such therapeutic intervention might be useful not only to suppress primary tumor growth, but also to prevent the formation of metastases, which would be of particular interest in the treatment of HNC (27). Nevertheless, more research is needed to evaluate this possibility. While potential adverse effects of a systemic administration of type I IFNs have been well documented (54), targeted approaches may be more promising and have been successfully applied in animal models (55). For HNC applications, recently reported synergistic effects of type I IFN and checkpoint inhibitors, which have been approved for clinical use in the past years, may be of particular value (56).
Among other mechanisms of tumor-driven immunosuppression, the downregulation of IFNAR1 plays a special role, decreasing the effectiveness of anti-tumor innate and adaptive immune responses, which enables tumor growth and permits the formation of premetastatic niches, minimizing the efficiency of anti-cancer type I IFN therapy. Optimization of therapeutical strategies rescuing the type I IFN signaling, e.g. with IFN-l, improves the immunostimulatory properties of neutrophils and thus restores the anti-cancer activity of lymphocytes, providing an attractive therapeutical option for cancer treatment.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
The animal study was reviewed and approved by Federation of European Laboratory Animal Science Associations (FELASA).