Immunomodulation via FGFR inhibition augments FGFR1 targeting T-cell based antitumor immunotherapy for head and neck squamous cell carcinoma.

Fibroblast growth factor receptor 1 (FGFR1) is overexpressed in multiple types of solid tumors, including head and neck squamous cell carcinoma (HNSCC). Being associated with poor prognosis, FGFR1 is a potential therapeutic target for aggressive tumors. T cell-based cancer immunotherapy has played a central role in novel cancer treatments. However, the potential of antitumor immunotherapy targeting FGFR1 has not been investigated. Here, we showed that FGFR-tyrosine kinase inhibitors (TKIs) augmented antitumor effects of immune checkpoint inhibitors in an HNSCC mouse model and upregulated tumoral MHC class I and MHC class II expression in vivo and in vitro. This upregulation was associated with the mitogen-activated protein kinase signaling pathway, which is a crucial pathway for cancer development through FGFR signaling. Moreover, we identified an FGFR1-derived peptide epitope (FGFR1305-319) that could elicit antigen-reactive and multiple HLA-restricted CD4+ T cell responses. These T cells showed direct cytotoxicity against tumor cells that expressed FGFR1. Notably, FGFR-TKIs augmented antitumor effects of FGFR1-reactive T cells against human HNSCC cells. These results indicate that the combination of FGFR-TKIs with immunotherapy, such as an FGFR1-targeting peptide vaccine or immune checkpoint inhibitor, could be a novel and robust immunologic approach for treating patients with FGFR1-expressing cancer cells.

Introduction

Head and neck squamous cell carcinoma (HNSCC), which affects the 1) oral cavity, 2) nasal cavity and paranasal sinuses, 3) nasopharynx, 4) oropharynx, 5) hypopharynx, and 6) larynx, annually causes an estimated 300,000 deaths worldwide.[1] Despite advances in surgery and chemoradiotherapy, many patients with HNSCC (especially human papillomavirus (HPV)-negative HNSCC) experience recurrence and metastases. The survival rate of HNSCC patients is less than 50%, which has not changed for decades.[2] Although cetuximab – a drug that targets the epidermal growth factor receptor (EGFR) – is clinically approved, its clinical efficacy is limited in advanced HNSCC patients.[3] Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of cancer and demonstrated that cancer immunotherapy to be effective in clinical practice. However, only a small number of patients (about 20%) benefit from ICIs in various cancers, including HNSCC.[4] Therefore, the development of novel cancer immunotherapy for HNSCC patients is warranted.

The fibroblast growth factor receptor (FGFR) family consists of four main receptor-type tyrosine kinases (FGFR1-4) that are associated with tissue restoration, angiogenesis, and oncogenesis.[5] FGFR1 overexpression causes tumor formation through mitogen-activated protein kinase (MAPK), PI3K/AKT, and JAK/STAT signaling[6,7] and poor outcome in HNSCC.[8] Many clinical trials using FGFR inhibitors have been conducted on various cancers and have shown some efficiency and tolerability.[9-11] However, the combined effect of FGFR inhibitors with other therapies is unknown, and studies investigating the immune effects of FGFR1 inhibition are sparse. As the inhibition of other tyrosine kinase receptors, such as EGFR, augments T cell responses,[12] immune-modulation via FGFR1 blockade is a potential approach for immunotherapy.

Increasing the number of tumor-reactive T cells is the key to successful cancer immunotherapy, as there are only a few T cells and they are weak against tumors. While adoptive cell transfer of tumor-reactive T cells can generate many tumor-reactive T cells, it is difficult to translate this therapy into the clinic because of its complexity and associated high cost. Cancer vaccines, created using tumor antigen-targeted synthetic peptides, are among potential therapies for eliciting tumor-reactive T cell responses against solid tumors. Recently, the development of appropriate adjuvants and identification of highly immunogenic antigens has enhanced the antitumor activity of peptide-based cancer vaccines.[13] Although tailor-made peptide vaccines targeting mutation-derived neoantigens have shown potent effects,[14] high costs, and complicated techniques have impeded their acceptance in clinical practice. Thus, peptide vaccines targeting tumor-associated antigens (TAAs), which are expressed in many cancers, are being considered. Identification of effective TAAs and potent adjuvants are required to develop novel and robust cancer immunotherapies.[15]

In this study, we demonstrated that FGFR-tyrosine kinase inhibitors (TKIs) exhibited synergistic activity with ICI in the mouse HNSCC model. FGFR-TKIs amplified the expression of MHC class I and MHC class II in HNSCC cells. The upregulation of MHC class II expression was induced by CIITA, subsequent to inhibition of the FGFR/MAPK pathway. Moreover, we identified a novel FGFR1-derived peptide epitope that could generate antigen-reactive and multiple HLA-DR-restricted CD4+ T cell antitumor responses in healthy donors and HNSCC patients. Notably, we found that FGFR-TKIs augmented the antitumor effects of FGFR-reactive T cells in vitro. Overall, these results suggest that FGFR blockade is a novel and suitable combination approach with T cell-based cancer immunotherapy.

Materials and methods

Cell lines and mice

HSC2 (human oral SCC; HLA-DR13), HSC3 (human tongue SCC; HLA-DR15), HSC4 (human tongue SCC; HLA-DR1, 4, and 53), and Sa-3 (human gingival SCC; HLA-DR9, 10, and 53) were supplied by the RIKEN BioResource Center (Tsukuba, Ibaraki, Japan). HPC-92Y (human hypopharyngeal SCC; HLA-DR4, 9, and 53) and CA9-22 (human gingival SCC, HLA-DR 8 and 15) were kindly provided by Dr. Syunsuke Yanoma (Yokohama Tsurugamine Hospital, Yokohama, Japan). SAS (human tongue SCC), SCC152 (HPV-positive human tongue SCC), SCC090 (HPV-positive human hypopharynx SCC), and the T-cell leukemia cell line Jurkat was purchased from the American Type Culture Collection (Manassas, VA, USA). UM-SCC-47 (HPV-positive human tongue SCC) was supplied by Merck Millipore (Burlington, MA, USA). MOC1 (tongue SCC derived from C57BL/6 mice) was supplied by Kerafast Inc. (Boston, MA, USA). L cells (mouse fibroblast cell lines) expressing individual human HLA-DR molecules (HLA-DR4 and 53) were kindly provided by Dr. R. Karr (Karr Pharma, St. Louis, MO) and Dr. Sasazuki (Kyushu University, Fukuoka, Japan). C57BL/6 mice (female, 8 to 10 weeks old) were purchased from Charles River Laboratories Japan, Inc. (Yokohama, Japan). All mice were maintained in a reactive pathogen-free facility at the Asahikawa Medical University. The experimental protocol was approved by the Institutional Animal Care and Use Committee of Asahikawa Medical University (#20001).

Flow cytometry

Immune cells from mice were stained with PerCP-conjugated anti-CD4 (GK1.5, BioLegend) mAbs, APC/Cy7-conjugated anti-CD8a (53–6.7) mAbs, and the isotype monoclonal mAb. FITC-conjugated anti-I-A/I-E mAbs (M5/114.15.2, BioLegend) was used for negative gating. After pretreatment with 3 μM FGFR1-TKIs (PD173074; AZD4547; Erdafitinib, Selleck Chemicals), 3 μM mitogen-activated protein kinase (MAPK) inhibitor (MEK inhibitor U0126, Promega), MAPK siRNA (SignalSilence® p44/42 MAPK Erk1/2 siRNA, Cell Signaling Technology), 3 μM STAT3 inhibitor (S3I-201, Selleck Chemicals), or 3 µM PI3K inhibitor (BYL719, Selleck Chemicals) for 48 hr, HLA class I and HLA-DR expression on tumor cell lines was assessed via flow cytometry using anti-HLA class I antibodies (Abs) conjugated with fluorescein isothiocyanate (G46-2, BD Pharmingen) and anti-HLA-DR Abs conjugated with phycoerythrin (TU36, BD Pharmingen). HNSCC cell lines were treated with or without 50 U/ml IFN-γ for 48 hr before the assay. IgG1 (MOPC-21, BioLegend) and IgG2a (MOPC-173; BioLegend) were used as isotype controls. Intracellular IFN-γ staining were performed using Perm/WashTM (BD Pharmingen), Cytofix/CytopermTM (BD Pharmingen), APC-conjugated anti-IFN-γ mAbs (4S.B3, BioLegend), and FITC-conjugated anti-granzyme B mAbs (GB11, BioLegend). Samples were analyzed using the CytoFLEX LX flow cytometer and CytExpert (Beckman Coulter).

Western blotting

The tumor cell line proteins extracted using the MiuteTM Total Protein Extraction Kit (Invent Biotechnologies, Inc.) were subjected to electrophoresis on NuPAGE Bis–Tris gels (Invitrogen, Thermo Fisher Scientific Inc.) and transferred to an Immobilon-P membrane (Merck Millipore). The membrane was incubated with mouse anti-human FGFR1 Abs (M19B2, Novus Biologicals) and mouse anti-human β-actin Abs (C4, Santa Cruz Biotechnology, Santa Cruz, CA) and detected via chemiluminescence using the Amersham ECL Prime Western blotting Detection System (GE Healthcare Life Sciences) and Invitrogen iBright Imaging Systems 1500 (Invitrogen, Thermo Fisher Scientific). Class II transactivator (CIITA) expression was evaluated using mouse anti-CIITA Abs (sc-13556, 7–1 H, Santa Cruz Biotechnology, Santa Cruz, CA). Phosopho-MAPK (pERK1/2) and MAPK(Erk1/2) expression was assessed using rabbit anti-phasopho-p44/42 MAPK (pERK1/2) Ab (Thr202/Tyr204, Cell Signaling Technology) and rabbit anti-p44/42 MAPK(Erk1/2) Ab (137F5, Cell Signaling Technology), respectively. MAPK siRNA was assessed using Lipofectamine ® RNAiMAX Transfection Reagent (Invitrogen, Thermo Fisher Scientific) and OptiMEM I Reduced Serum Medium (Invitrogen, Thermo Fisher Scientific) according to the manufacturer’s instructions (Supplemental Figure S1). Protein expression was analyzed using ImageJ.

Synthetic peptides

HLA-DR-binding epitope sequences of FGFR1 were selected using the computer-based algorithms from SYFPEITHI (http://www.syfpeithi.de/)[16] and Immune Epitope Database Analysis Resource (IEDB, RRID:SCR_013182, https://www.iedb.org/).[17] Amino acids were scored for their likelihood of binding to common HLA-DR molecules. We selected FGFR1305-319 (LPYVQILKTAGVNTT), as it had a high binding score with multiple HLA-DR molecules (DRB1*0101, DRB1*0401, DRB1*0701, DRB1*1101, and DRB1*1501). The FGFR1305-319 peptide was purified by Hokkaido System Science (Sapporo, Japan). Using the same methods, homologous peptides to FGFR1305-319 peptide from FGFR3 and FGFR4 protein were identified and synthesized. The PADRE peptide (aK-Cha- VAAWTLKAAa, where “a” denotes D-alanine and “Cha” denotes L-cyclohexylalanine), which can bind with multiple HLA-DR molecules, was used as a positive control.

In vivo assessment of combination therapy with FGFR-TKIs and T-cell based immunotherapy

C57BL/6 mice were intradermally injected with 1 × 106 MOC1 cells. The mice were intraperitoneally administered with PD173074 (20 mg/kg) and anti-PD-1 Ab (200 µg/mouse), three times per week from 18 days after inoculating MOC1 (tumor diameter: 7–8 mm). Tumor growth was monitored every three days by measuring two opposing diameters with a pair of calipers. Tumor volume was calculated as length × width2)/2. Results are presented as mean tumor volume (mm3) with standard deviation (SD). Tumors were harvested on day 43 for assessment of tumor-infiltrating lymphocytes (TILs) and immunohistochemistry. The TILs were disaggregated from tumor tissues using collagenase (1 mg/ml) and gentlMACS (Miltenyi Biotec, Berguch, Germany) according to the manufacturer’s instructions. Surface markers of TILs were assessed via flow cytometry. To assess MHC class I, MHC class II, and PD-L1 expression, formalin-fixed, paraffin-embedded (FFPE) tumor samples were evaluated via immunohistochemistry. Anti-MHC class I (anti-mouse H-2Kb Ab, AF6-88.5, BioLegend), anti-MHC class II (anti-mouse I-A/I-E Ab, M5/114.15.2, BioLegend), and anti-PD-L1 (10 F.9G2, BioLegend) Abs were used as the primary Abs. FFPE specimens were stained using the VENTANA Benchmark GX (Roche Diagnostics).

In vitro generation of FGFR1-reactive CD4

The process used to generate peptide-reactive CD4+ T cell (HTL) lines from healthy donor peripheral blood mononuclear cells (PBMCs), has been previously described in detail.[18] Briefly, dendritic cells (DCs) were induced by stimulating CD14+ cells, isolated using the EasySepTM Human CD14+ Positive Selection Kit (STEMCELL), with GM-CSF (50 ng/ml, PeproTech, Rocky Hill) and IL-4 (1000 IU/ml, PeproTech, Rocky Hill, NJ). HTLs isolated using the EasySepTM Human CD4+ T Cell Isolation Kit (STEMCELL technology) were stimulated by peptide-pulsed autologous DCs for one cycle and γ-irradiated autologous PBMCs for two cycles. HTLs were assessed for production of IFN-γ with FGFR1305-319 peptide stimulation using enzyme-linked immunosorbent assay (ELISA) kits (BD Pharmingen), according to the manufacturer’s instructions, and then compared to the unstimulated control. Microcultures with a significant increase in IFN-γ production after FGFR1305-319 peptide stimulation were subsequently expanded. Finally, FGFR1305-319-reactive HTL lines were isolated by limiting dilution.

Measurement of antigen-reactive responses by FGFR1-reactive CD4+ T cell lines

The measurement methods of targeting antigen-reactive responses by HTLs have been previously described in detail.[18] IFN-γ production in the supernatants co-cultured with FGFR1-reactive HTL lines and autologous PBMCs (1 × 105), L-cells (3 × 104), or FGFR1-expressing HNSCC cell lines (3 × 104) as antigen-presenting cells (APCs) was measured using ELISA kits (BD Pharmingen, San Diego, CA). Production of IL-2, TNF-α, and GM-CSF in these supernatants was evaluated using ELISA kits (BD Pharmingen), according to the manufacturer’s instructions. Tumor cells were treated with 3 μM FGFR1-TKIs for the indicated experiments. To investigate the reactivity with FGFR3- or FGFR4-derived homologous peptides to FGFR1, FGFR1-induced HTL lines were co-cultured with autologous PBMCs (1 × 105) and FGFR3303-317 (TPYVTVLKTAGANTT) or FGFR4313-327 (FPYVQVLKTADINSS) peptides. EGFR875-889 (KVPIKWMALESILHR) was used as a negative control peptide. To enhance HLA-DR expression, HNSCC cell lines were treated with 500 U/ml IFN-γ (PeproTech) for 48 hr before the assay. HLA restriction was assessed using anti-HLA-DR Ab L243 (HB-55, ATCC) and anti-HLA class I Ab W6/32 (HB-95, ATCC). In the indicated experiments, MDM232-46-reactive HTL lines (H40)[19] were used instead of FGFR1-induced HTL lines.

Cytotoxicity assay

Supernatants of cocultured FGFR1-reactive HTL lines with target tumor cell lines were assessed using granzyme B ELISA kits (MABTECH) according to the manufacturer’s instructions. To evaluate the killing activity, target tumor cell lines were labeled using the CellTraceTM CFSE Cell Proliferation Kit (Invitrogen, Thermo Fisher Scientific Inc.). After 6 h of coculturing with various effector/target cell (E:T) ratios of FGFR1-reactive HTL lines, the number of dead tumor cells, labeled using 7-AAD viability staining solution (BioLegend), was quantified via flow cytometry.

FGFR1 peptide-reactive responses by T cells from HNSCC patients

PBMCs from HNSCC patients were co-cultured with FGFR1305-319 peptides in 96-well plates, as described previously.[20] The PADRE peptide (capable of binding to all HLA-DR molecules) was co-cultured with PBMCs as a positive control. Briefly, PBMCs (1 × 105) were stimulated with peptides (10 µg/mL) for 2 cycles every 7 days, and IFN-γ production in the supernatants was measured using ELISA. Anti-HLA-DR Ab L243 (HB-55, ATCC) was applied to confirm that the IFN-γ production is mediated through HLA-DR/peptide/T cell receptor complex. All investigations were approved by the Institutional Ethics Committee of Asahikawa Medical University (#16217), and written informed consent was obtained from all participants.

Statistical analysis

All data were assessed using Student’s t-test or Fisher’s exact test. Statistical significance was set at p < .05.

Results

Antitumor effects of combination therapy with FGFR-TKIs and ICI in mouse model

To investigate whether the FGFR inhibition can be applied to cancer immunotherapies as an immunomodulator, we evaluated the immunomodulatory effects of FGFR-TKIs in a mouse model of MOC1, a mouse tongue cancer cell line expressing FGFR1 (Figure 1a). MOC1-inoculated mice were treated with combination therapy using an FGFR-TKI (PD173074) and anti-PD-1 Abs as shown in Figure 1b. The combination therapy showed a synergistic antitumor effect and significantly reduced tumor growth (Figure 1c). FGFR inhibitor recruited CD4+ and CD8 + T cells in tumor microenvironment, which was further increased with the combined blockade of FGFR and PD-1 (Figure 1d). Remarkably, immunohistochemistry of tumors treated with PD173074 revealed a significant upregulation of MHC class I and MHC class II (Figure 1e). In vitro analysis also demonstrated that three FGFR-TKIs (PD173074, AZD4547, and erdafitinib) upregulated MHC class I and MHC class II expression on MOC1 cells with or without IFN-γ (figure 1f and g). Moreover, PD-L1 expression in TILs was also increased suggesting that FGFR inhibitor can be a promising adjuvant with PD-1 blockade (Supplemental Figure S2). Because tumors reduce the expression of MHC molecules to escape from immune cells,[21] an immune adjuvant that upregulates expression of MHC molecules on tumor cells is crucial for effective T cell-based cancer immunotherapy. Therefore, FGFR-TKIs might be practical immune adjuvants to combine with T-cell based immunotherapy including ICI via upregulation of MHC expression on tumor cells.

Figure 1.

FGFR1 as an immune adjuvant to combine with ICI in HNSCC mouse models. (a) FGFR1 expression in mouse HNSCC cell lines (MOC1) was examined by Western blotting. (b) Experimental schema. C57BL/6 mice were intradermally injected with MOC1 (1x106). PD173074 (20 mg/kg) and anti-PD-1 Ab (200 μg/mice) was administered 3 times per week from day 18 (tumor size: 7–8 mm). (c) Tumor growth curves. Control (Red), anti-PD-1 Ab monotherapy (Blue), PD173074 monotherapy (Yellow), and combination therapy with PD173074 and anti-PD-1 Ab (Green) (n = 4 or 5 /group). Bars and error bars indicate the mean and SD, respectively (*p < .05, **p < .01, ***<0.001, one-way ANOVA). (d) The mice were sacrificed on day 42, and the percentages of CD4+ T cells and CD8+ T cells in TILs were evaluated with flow cytometry. (e) A representative image of MHC-class I or MHC-class II expression in immunohistochemistry on tumor (Day 42). MHC-class I (central) and MHC-class II (right) was enhanced by PD173074. H&E staining was shown in the left. Scale bars represent 100 µm. (f, g) MHC-class I and MHC-class II expression on MOC1 incubated with 3 μM FGFR-TKIs for 48 hr were evaluated by flow cytometry. MOC1 was treated with or without 50 U/ml IFN-γ for 48 hr before the assay. Red: isotype control, Green: untreated tumor cell lines, Blue: treated with PD173074. Pink: treated with AZD4547. Orange: treated with Erdafitinib. (f) Representative data of flow cytometry. (g) Averages values of mean fluorescence intensity (MFI) by FGFR-TKIs. (*p < .05, **p < .01, ***<0.001, Student’s t test).

FGFR1 blockade upregulates tumor HLA expression through MAPK signaling pathway

Next, we evaluated the effects of FGFR-TKIs as immune mediators in human HNSCC cells in vitro. FGFR1 was expressed in most of the human HNSCC cells tested in this study (Figure 2a), and FGFR-TKIs inhibited the tumor proliferation (Supplemental Figure S3). As well as in the mouse model, three FGFR-TKIs enhanced HLA class I and HLA-DR expression on human FGFR1-expressed tumor cell lines (Figure 2b and c). Since IFN-γ is a potent activator of HLA expression, the increase of HLA expression by FGFR inhibitor was maintained even in the presence of IFN-γ. Because Class II transactivator (CIITA) is a master regulator of MHC Class II gene, we next evaluated the expression of CIITA in tumor cells treated with FGFR inhibitors. CIITA expression was induced by FGFR-TKIs (Figure 2d and e). Since IFN-γ alone could induce CIITA expression (Supplemental Figure 4), FGFR-TKIs further increased the effect of IFN-γ (figure 2f and g).

Figure 2.

The changes of HLA and CIITA expression on HNSCC cell lines by FGFR1-TKIs. (a) FGFR1 expression in human HNSCC cell lines was examined by Western blotting. Jurkat (leukemia cells) was used as a negative control. (b, c) HLA-class I and HLA-DR and expression on HNSCC cell lines incubated with 3 μM FGFR-TKIs for 48 hr were evaluated by flow cytometry. HNSCC cell lines were treated with or without 50 U/ml IFN-γ for 48 hr before the assay. Green: isotype control, Red: untreated tumor cell lines, Blue: treated with PD173074. Pink: treated with AZD4547. Orange: treated with Erdafitinib. (b) Representative data of flow cytometry. (c) Averages values of mean fluorescence intensity (MFI). (d-g) FGFR-TKIs (3 μM) upregulated CIITA expression in HNSCC cell lines. HNSCC cell lines were treated with or without 50 U/ml IFN-γ for 48 hr before the assay. (d) Representative data of Western blotting without IFN-γ. (e) Quantitative analysis of protein expression. (f) Representative data of Western blotting with IFN-γ. (g) Quantitative analysis of protein expression. Each data was representative in the triplicate experiments. Bars and error bars show the mean and SD, respectively. (*p < .05, **p < .01, ***<0.001, Student’s t test).

Pathways downstream of FGFR include the MAPK, STAT3, and PI3K signaling pathways, which are crucial for oncogenesis via FGFR signaling. To elucidate the mechanism by which pathway is responsible for FGFR-TKIs-induced HLA expression, tumor cells were treated with MAPK, STAT3, or PI3K inhibitors. Notably, the MAPK inhibitor enhanced HLA class I and HLA-DR expression on tumor cell lines (Figure 3a and b). As a proof of concept, the phosphorylated MAPK was inhibited by FGFR inhibitor (Supplemental Figure S5). The upregulation of HLAs via MAPK inhibition was further confirmed by silencing MAPK gene (Figure 3c and d), whereas STAT3 or PI3K inhibition had no effect on HLA expression in tumors (Figure 3e and f). The expression of HLAs was upregulated with FGFR inhibition even in the presence of IFN- γ, a potent inducer of HLAs (Supplemental Figure S6). MAPK inhibiton also upregulated CIITA expression suggesting that the FGFR1/MAPK pathway might suppresses HLA Class II expression through inhibiting CIITA (Figure 3g and h). These results suggest that FGFR inhibits HLA class I and CIITA/HLA class II expression in tumors through MAPK signaling, and FGFR-TKIs could be immune adjuvants for T-cell-based immunotherapy by upregulating antigen presentation on tumor.

Figure 3.

Upregulation of HLAs and CIITA expression on HNSCC cell lines by MAPK inhibition. (a, b) HLA-DR and HLA-class I expression on HNSCC cell lines incubated with 3 μM MAPK inhibitor and 50 U/ml IFN-γ for 48 hr were evaluated by flow cytometry. Red: isotype control, Green: untreated tumor cell lines, Pink: treated with MAPK inhibitor. (a) Representative data of flow cytometry. (b) Averages values of mean fluorescence intensity (MFI). (c, d) HLA-DR and HLA-class I expression on HNSCC cell lines incubated with siMAPK and 50 U/ml IFN-γ for 48 hr were evaluated by flow cytometry. Red: isotype control, Green: untreated tumor cell lines, Pink: treated with MAPK inhibitor. (c) Representative data of flow cytometry. (d) Averages values of mean fluorescence intensity (MFI). (e, f) HLA-DR and HLA-class I expression on HNSCC cell lines incubated with 3 μM STAT inhibitor or PI3K inhibitor and 50 U/ml IFN-γ for 48 hr were evaluated by flow cytometry. Red: isotype control, green: untreated tumor cell lines, pink: treated with STAT inhibitor. Orange: treated with PI3K inhibitor. (e) Representative data of flow cytometry. (f) Averages values of mean fluorescence intensity (MFI). (g, h) HNSCC cell lines were treated with 3 µM MAPK inhibitor or siMAPK, and class II transactivator (CIITA) expression was examined. (g) Representative data of Western blotting. (h) Quantitative analysis of protein expression. Each data was representative in the triplicate experiments. Bars and error bars show the mean and SD, respectively. (*p < .05, **p < .01, ***<0.001, Student’s t test).

Generation of FGFR1-reactive CD4+ T cell lines

As FGFR-TKIs should be used in FGFR-expressing tumors, it is rational to target FGFR as an immune antigen for combined immunotherapy with FGFR-TKIs. Using computer-based algorithm, we selected peptide FGFR1305-319 (LPYVQILKTAGVNTT) as a potential candidate to elicit CD4+ T cell responses (Supplemental Figure S7). FGFR1305-319 peptide-reactive HTL lines were induced by repeatedly stimulating CD4+ T cells from healthy donors with the FGFR1305-319 peptide. FGFR1305-319 peptide-reactive HTL lines (K1, K2, and K3) released IFN-γ in a peptide dose-dependent manner (Figure 4a). This response was inhibited by anti-HLA-DR Abs but not by anti-HLA class I Abs suggesting that the peptide recognition of HTLs was restricted by HLA class II (Figure 4b). To identify the HLA-DR alleles responsible for the interactions of these HTL lines with the peptide, L-cells transfected with a single HLA-DR allele gene were used as APCs. As shown in Figure 4c, the HTL lines K1 and K3 were restricted to L-cells expressing HLA-DR4, and K2 was restricted to L-cells expressing HLA-DR53. This result suggests that the FGFR1305-319 peptide was capable of inducing T cells that are restricted to multiple HLA-DR molecules. The amino acid sequence of the identified FGFR1 peptide epitope has homologous regions in the FGFR3 and FGFR4 proteins (Figure 4d). Interestingly, FGFR1-reactive T cell lines (K1) recognized homologous FGFR3- and FGFR4-derived peptides (Figure 4e). The response to FGFR3- or FGFR4-derived peptide was inhibited by anti-HLA-DR Abs, and irrelevant EGFR-derived peptide could not activate HTLs suggesting that FGFR1-reactive T cell lines could specifically recognize FGFR family protein (Figure 4f). Collectively, the FGFR1305-319 epitope peptide could be applied as a cancer vaccine against tumors expressing FGFR3 or FGFR4 in addition to FGFR1.

Figure 4.

Generation of FGFR1. (a) FGFR1305-319-reactive CD4+ T cell lines (K1, K2, and K3) were assessed for IFN-γ production in response to irradiated autologous PBMCs as APCs with several concentrations of FGFR1305-319 peptide. (b) HLA restriction analysis of the FGFR1305-319-reactive CD4+ T cell lines. Peptide-reactive responses in the FGFR1305-319-reactive CD4+ T cell lines were evaluated by co-cultured with irradiated autologous PBMCs as APCs in the context of anti-HLA-DR mAb or anti-HLA class I mAb. (c) Assessment of restrictive HLA-DR allele in the FGFR1305-319-reactive CD4+ T cell lines. Each T cells was co-culturing with L-cells expressing individual HLA-DR as APCs. IFN-γ production in the supernatants was assessed by ELISA after co-culturing with APCs for 48 hr. (d) Peptide sequences of FGFR1305-319 and its homologous FGFR family-derived peptide. Underlined letters indicate amino acids that are different from FGFR1305-319 peptide. (e) Evaluation of FGFR1305-319-reactive CD4+ T cell response to the homologous FGFR family-derived peptides. FGFR1305-319-reactive CD4+ T cell lines (K1) were evaluated for IFN-γ production in response to irradiated autologous PBMCs as APCs with FGFR3303-317 peptide or FGFR4 313–327 peptide. (f) HLA restriction analysis of the FGFR1305-319-reactive CD4+ T cell lines to FGFR3 or FGFR4 peptide. FGFR3303-317 or FGFR4313-327 peptide-reactive responses in the FGFR1305-319-reactive CD4+ T cell lines were assessed by co-cultured with irradiated autologous PBMCs as APCs in the context of anti-HLA-DR mAb or anti-HLA class I mAb. EGFR875-889 peptide was used as negative control peptide. Each data was representative in the triplicate experiments. Bars and error bars show the mean and SD, respectively. (*p < .05, **p < .01, ***<0.001, Student’s t test).

Direct tumor recognition and cytotoxic activity by FGFR1305-319-reactive CD4+ T cell lines

To assess whether FGFR1305-319-reactive HTLs could directly recognize tumor cells, HTL lines were co-cultured with FGFR1-expressing HNSCC cell lines. As shown in Figure 5a, FGFR1305-319-reactive HTLs responded to HLA-DR-matched tumor cells but not to HLA-DR-unmatched tumor cells. T cell responses were inhibited with anti-HLA-DR but not with anti-HLA Class I Abs suggesting that T cells react to tumor in the context of HLA-DR (Supplemental Figure S8). Moreover, a FGFR1305-319-reactive HTL line (K1) co-cultured with HLA-DR-matched tumor cells produced granzyme B (Figure 5b). In addition to IFN-γ granzyme B, various Th1 cytokines including IL-2 and TNF-α were produced from FGFR1305-319-reactive HTLs in response to tumor (Supplemental Figure S9 and S10), indicating that FGFR1-induced HTL lines are cytotoxic HTLs.[22] As shown in Figure 5c and d, direct tumor cytotoxicity was observed in these HTLs. These results suggest that the FGFR1305-319 peptide could be a potent antitumor vaccine for generating cytotoxic HTLs that directly kill tumors.

Figure 5.

Direct killing of FGFR1 expressing HNSCC cells by FGFR1. (a) FGFR1305-319-reactive CD4+ T cell lines were co-cultured with HLA-DR matched or unmatched HNSCC cell lines expressing FGFR1 for 48 hr. K1 and K3 were restricted to HLA-DR4, and K2 was restricted to HLA-DR53. The cell lines used were HSC2 (HLA-DR13), HSC3 (HLA-DR15), HSC4 (HLA-DR1,4, and 53), Sa-3 (HLA-DR9, 10, and 53), and HPC-92Y (HLA-DR4, 9, and 53). HNSCC cell lines were treated with 500 U/ml IFN-γ for 48 hr before the assay. IFN-γ production in the supernatants was evaluated by ELISA. (b) Granzyme-B production from FGFR1305-319-reactive CD4+ T cell line (K1: HLA-DR4 restricted) was assessed in supernatants co-cultured with HLA-DR matched or unmatched HNSCC cell lines. (c) Killing activity of FGFR1305-319-reactive CD4+ T cell line (K1) was evaluated by co-culturing with CSFE-labeled HNSCC cell lines for 6 hr with various E: T (Effector: Target cells) ratio, and measuring percentages of CFSE+ 7-AAD+ dead cells with flow cytometry. (d) Representative data of flow cytometry in the killing assay (Effector to target ratio was 20:1). Each data was representative in the triplicate experiments. Bars and error bars show the mean and SD, respectively. (*p < .05, **p < .01, ***<0.001, Student’s t test).

FGFR1305-319-reactive T cells in periphery blood from HNSCC patients

In a clinical setting, the existence of FGFR1-reactive precursor T cells in cancer patients is essential for the translation of cancer peptide vaccines targeting FGFR1. Accordingly, we assessed the presence of FGFR1-reactive T cells via short-term stimulation of PBMCs, isolated from six HNSCC patients, with FGFR1305-319 peptide. PBMCs from untreated HNSCC patients were stimulated with peptides for two cycles every 7 days, and the production of IFN-γ was measured. As shown in Figure 6a and B, T cells from HNSCC patients responded to the FGFR1305-319 peptide, indicating that precursor T cells that can react to the FGFR1 peptide vaccine exist in patients with HNSCC.

Figure 6.

The existence of FGFR1-reactive precursor T cells in HNSCC patients. (a) PBMCs from HNSCC patients were co-cultured with FGFR1305-319 peptides for 2 cycles every one week. T cell response to FGFR305-319 peptide was assessed by measuring IFN-γ production in the supernatants using ELISA. Anti-HLA-DR mAb was used to assess HLA restriction of the T cells. PADRE peptide was used as a positive control. Each data was representative in the triplicate experiments. Bars and error bars show the mean and SD, respectively. (*p < .05, **p < .01, ***<0.001, Student’s t test). (b) The clinical characteristics and peptide-reactivity of the 6 HNSCC patients. <: less than the lower limit of detection.

Synergistic antitumor effects of FGFR-TKIs with tumor-reactive CD4+ T cells

Finally, we evaluated the antitumor effects of combination therapy with FGFR1-reactive HTLs and FGFR-TKIs. IFN-γ production in FGFR1305-319-reactive HTL lines was augmented by use of FGFR-TKIs (Figure 7a). To determine whether FGFR inhibition functions as an immune adjuvant for FGFR-irrelevant immunotherapy, MDM2-reactive T cells were used as effector cells.[19] As FGFR-TKIs did not alter MDM2 expression in HNSCC cells (Supplemental Figure S11), tumor recognition by MDM232-46-reactive HTLs (H40) was increased by FGFR-TKIs (Figure 7b) suggesting that FGFR blockade can be applied to any T-cell-based immunotherapy. The production of granzyme B and the killing activity of FGFR1305-319-reactive HTL lines were also enhanced by FGFR-TKIs (Figure 7c-e). Based on these findings, FGFR-TKIs and cancer peptide vaccines (such as the FGFR1305-319 peptide), could be a potent combination therapy against FGFR1-expressing tumors.

Figure 7.

Synergistic antitumor effects of FGFR inhibitor with tumor-reactive T cells. (a, b) (a) Responses of FGFR1305-319-reactive CD4+ T cells (K1 and K2) or (b) MDM232-46-reactive CD4+ T cells (H40) to tumor cell lines pretreated by FGFR-TKIs was evaluated by measuring IFN-γ production. HNSCC cell lines were treated with 50 U/ml IFN-γ for 48 hr before the assay. DMSO was used as a negative control. (c) Granzyme B production from FGFR1305-319-reactive CD4+ T cells (K1) against tumor cells pretreated by FGFR-TKIs was evaluated. DMSO was used as a negative control. (d) Killing ability of FGFR1305-319-reactive CD4+ T cells (K1) to tumor cells pretreated by FGFR-TKIs. T cell lines K1 was co-cultured with CSFE-labeled FGFR-TKIs pretreated tumor cells for 6 hr. The percentages of dead cells were measured using 7-AAD staining by flow cytometry. Effector to target ratio was 20:1. Symbols and error bars indicate the mean and SD, respectively. (e) Representative data of flow cytometry. Each data was representative in the triplicate experiments. Bars and error bars show the mean and SD, respectively. (*p < .05, **p < .01, ***<0.001, student’s t test).

Discussion

In this study, we elucidated two aspects of FGFR1 in cancer immunology: as an immune adjuvant and as a target antigen of a peptide vaccine. To the best of our knowledge, there have been no reports elucidating the antitumor T cell response elicited by FGFR1-derived peptides, and only a few studies have investigated the immunomodulatory effects of FGFR1 inhibition. According to the Human Protein Atlas database and previous studies, overexpression of FGFR1 occurs in more than 70% of HNSCC cases.[23,24] The expression of FGFR1 is related to poor prognosis in HNSCC,[25,26] especially in HPV-negative HNSCC, but not in HPV-positive HNSCC.[24,27] Because HPV-negative HNSCC exhibits poor response to standard therapies, developing novel treatments targeting FGFR1 could be a potential approach to treat patients with HNSCC. FGFR1 amplification is associated with poor prognosis in most types of cancer, such as melanoma, lung cancer, pancreatic cancer, and glioblastoma.[28-31] Activation of FGFR1 leads to tumor development through its downstream signaling pathways: the Ras-dependent MAPK, PI3K/AKT, and JAK/STAT pathways.[7] As these networks contribute to aggressive tumor behavior by activating tumor cell proliferation, survival, differentiation, and migration,[32] the development of FGFR1-targeting therapy would be beneficial for patients with aggressive tumors.

Immunomodulatory adjuvants are required for efficient immunotherapy to induce immune cold to hot tumors. Since adjuvants such as Toll-like receptor (TLR) ligands have improved clinical outcomes, chemotherapy and molecular-targeted drugs may also act as immune adjuvants in combined immunotherapy.[13] Few studies have investigated the effects of FGFR-TKIs on tumor immunity. In this study, we showed that FGFR-TKIs upregulated HLA-DR and HLA class I expression in tumor cell lines, such that enhanced HLA-DR expression augmented tumor recognition and killing by FGFR1-reactive HTLs. This effect was also observed in FGFR1-irrelevant antigen-reactive T cells, indicating that FGFR1 blockade can be applied to any tumor vaccine as an immune adjuvant. As FGFR1 blockade alone could directly suppress tumor proliferation and induce cell death, FGFR1 TKIs may damage tumor cells in several ways: 1) direct killing of tumor cells; 2) increased prevalence of dead tumor cells can be a source of tumor antigens in APCs; 3) upregulation of MHC expression in tumor cells, followed by antitumor T-cell activation. Thus, FGFR inhibition has the potential to be used as an adjuvant immunotherapy.

The expression of MHC class II is controlled by CIITA, the expression of which is induced by IFN-γ.[33,34] As FGFR-TKIs upregulated HLA-DR and CIITA expression in the absence of IFN-γ, IFN-γ would be dispensable for CIITA expression with respect to FGFR inhibition. Pannini et al showed that CIITA expression in macrophages is inhibited by TLR2-induced MAPK signaling.[35] We demonstrated that CIITA expression in tumor cells was enhanced by inhibition of MAPK/ERK via small molecular inhibitors or siRNA. As MAPK signaling is a common pathway downstream of various proteins such as FGFR and EGFR, which inhibition upregulates HLA class II,[12,36] MAPK signaling would be a key pathway in tumor-mediated suppressed expression of MHC. Since Dennison et al. have demonstrated that MEK1 knockout tumors upregulate MHC class I expression, CD8 T cell infiltration, and T cell activation,[37] our results further verified this issue not only in mouse models but also in human cells. Given the aforementioned finding, in future studies, it would be appropriate to examine whether other reagents that inhibit the MAPK pathway can change tumor MHC expression and T cell infiltration.

Akhand et al have shown that FGFR inhibitors augment the antitumor effect of anti-PD-1 Abs in breast cancer models by enhancing the intratumoral infiltration of lymphocytes and reducing myeloid suppressor cells.[38] In addition, Palakurthi et al reported that combination therapy with anti-PD-1 Abs and FGFR inhibition led to increased T cell infiltration to support enhanced survival in lung cancer models.[39] Concordant with these findings, our results elucidated that the basis of these synergistic effects of FGFR inhibitors is the upregulation of MHC expression in tumors. As T cells are depleted in microenvironment with FGFR3-expressing tumors,[40] and FGFR inhibition activates T cells in the FGFR2 model,[39] the FGFR family may suppress MHC expression on tumors to evade immune surveillance. Although the antitumor activity of CTLs is outside the scope of this study, upregulation of HLA class I by FGFR1 inhibition may have a positive effect on CTLs. In addition to T cells, inhibition of FGFR in tumor-associated macrophages increases the M1/M2 macrophage ratio.[41] Thus, FGFR inhibition can be applied for immunomodulation, and further studies are required to elucidate the effect of combined immunotherapy with FGFR inhibition in clinical settings (FGFR inhibitor with PD-1 blockade: ClinicalTrials.gov Identifier: NCT05004974).

We identified a novel T cell epitope peptide derived from FGFR1, which elicited antitumor HTL responses. Thus far, CD8+ cytotoxic T lymphocytes (CTLs) have been considered to play central roles in T cell-based immunotherapy. However, tumor cells escape from CTLs through multiple mechanisms, such as downregulation of MHC class I expression.[42] Recent evidence has shown that HTLs are essential for successful tumor clearance.[43] Antigen-reactive HTLs are required to activate CTL and NK cells by producing Th1 cytokines, maturation and activation of macrophages through the CD40L/CD40 pathway, establishes long-lived antitumor memory responses and augments immunosurveillance.[44-46] In addition to their helper function, HTLs exhibit a direct cytotoxic function.[22] Antitumor responses by immunotherapy requires both antigen-reactive CTLs and HTLs, even in tumors that do not express MHC class II molecules.[47] Our in silico sequencing suggested that the FGFR1305-319 peptide is capable of binding to multiple common HLA-DR alleles (DRB1*0101, DRB1*0401, DRB1*0701, DRB1*1101, and DRB1*1501). In this study, we have shown that the FGFR1305-319 peptide could bind to HLA-DR4 and -DR53. This suggests that the FGFR1305-319 peptide could be applied to a large population of patients. Moreover, the FGFR1305-319 peptide possesses a potential HLA-A0201-binding epitope (FGFR1308-315: VQILKTAG). As long peptides containing HTL and CTL epitopes have shown high efficacy in clinical applications,[48] the potential of the FGFR1 peptide to induce both CTLs and HTLs should be considered in the future. In addition, the FGFR1 peptide epitope has amino acid sequences homologous to the FGFR3 and FGFR4 peptide epitopes that may bind to multiple HLA-DRs. Because FGFR1-reactive HTLs can react with FGFR3- and FGFR4-derived peptides, the FGFR1 peptide vaccine can be applied to FGFR family (FGFR1, FGFR3, and FGFR4)-expressing tumors. The disadvantage of targeting TAAs is the risk of damage to normal tissues. However, we have shown that the precursor of FGFR1-reactive T cells were present in healthy donors and cancer patients without autoimmune diseases, suggesting that the risk of autoreactivity is relatively low. Because the migrating TAA-reactive T cells from thymus have moderate- or low-affinity T cell receptors, normal tissues with low FGFR1 expression[24,49] might be ignored from these T-cells. Beside normal tissues, our results showed that the T cells elicited by FGFR1 peptide could recognize and kill tumors with high FGFR1 expression indicating that FGFR1 peptide-based vaccine can induce antitumor T cells without damaging normal tissues. As FGFR inhibitors have been considerably safe in clinical trials,[50] targeting FGFR1 as an immunogen might be a safe and effective approach.

In summary, we demonstrated that FGFR-TKIs augmented antitumor effects of ICI in HNSCC mouse models by upregulating the expression of MHC class I and MHC class II in vitro and in vivo. This upregulation was mediated through the inhibition of the MAPK signaling pathway, but not that of the STAT and PI3K signaling pathways. Furthermore, we identified a novel helper epitope from FGFR1 that could elicit antigen-reactive T cell responses. FGFR1-reactive T cells were restricted to common HLA-DRs, and exhibited direct tumor cell recognition and cytotoxic activity against FGFR1-expressing HNSCC cells. The precursor T cells that react to the FGFR1 epitope peptide were detected in HNSCC patients, suggesting this epitope to be a potential candidate for peptide vaccines. Notably, FGFR-TKIs augmented the antitumor effect of FGFR1-reactive T cells against human HNSCC. These results suggest that FGFR-TKIs are potential immune adjuvants for T-cell-based immunotherapy. Combination therapy with TKIs and cancer vaccines or ICI could be a novel and potent immunotherapeutic approach to treat aggressive cancers with FGFR expression.

Click here for additional data file.

APC	antigen-presenting cell
CIITA	class II transactivator
CTL	CD8+ cytotoxic T lymphocyte
EGFR	epidermal growth factor receptor
E: T	Effector: Target
FGFR	fibroblast growth factor receptor
HNSCC	head and neck squamous cell carcinoma
HTL	CD4+ helper T lymphocyte
ICI	immune checkpoint inhibitor
mAb	monoclonal antibody
MAPK	mitogen-activated protein kinase
MFI	mean fluorescence intensity
PBMC	peripheral blood mononuclear cell
TAA	tumor-associated antigen
TKI	tyrosine kinase inhibitor