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Published in final edited form as: Oral Oncol. 2024 Apr 9;152:106795. doi: 10.1016/j.oraloncology.2024.106795

Enhanced oral versus flank lymph node T cell response parallels anti-PD1 efficacy in head and neck cancer

Michihisa Kono 1,2, Shin Saito 1, Masahiro Rokugo 1, Ann Marie Egloff 1,3, Ravindra Uppaluri 1,3,#
PMCID: PMC11065458  NIHMSID: NIHMS1986937  PMID: 38599127

Abstract

Objectives:

Understanding head and neck tissue specific immune responses is important for elucidating immunotherapy resistance mechanisms to head and neck squamous cell carcinoma (HNSCC). In this study, we aimed to investigate HNSCC-specific immune response differences between oral and subcutaneous flank tumor transplantation in preclinical models.

Materials and Methods:

The MOC1 syngeneic mouse oral carcinoma cell line or versions expressing either the H2Kb-restricted SIINFEKL peptide from ovalbumin (MOC1OVA) or ZsGreen (MOC1ZsGreen) were inoculated into mouse oral mucosa (buccal space) or subcutaneous flank and compared for immune cell kinetics in tumors and tumor-draining lymph nodes (TDLNs) and for anti-PD1 response.

Results:

Compared to subcutaneous flank tumors, orthotopic oral MOC1OVA induced a higher number of OVA-specific T cells, PD1+ or CD69+ activated OVA-specific T cells in both primary tumors and TDLNs. Tumors were also larger in the flank site and CD8 depletion eliminated the difference in tumor weight between the two sites. Oral versus flank SIINFEKL peptide vaccination showed enhanced TDLN lymphocyte response in the former site. Notably, cDC1 from oral TDLN showed enhanced antigen uptake and co-stimulatory marker expression, resulting in elicitation of an increased antigen specific T cell response and increased activated T cells. Parental MOC1 in the oral site showed increased endogenous antigen-reactive T cells in TDLNs and anti-PD1 blockade rejected oral MOC1 tumors but not subcutaneous flank MOC1.

Conclusion:

Collectively, we find distinct immune responses between orthotopic oral and heterotopic subcutaneous models, including priming by cDC1 in TDLN, revealing important implications for head and neck cancer preclinical studies.

Keywords: Head and neck cancer, Oral mucosa, Tumor-draining lymph node, Conventional type1 dendritic cell, Orthotopic model

INTRODUCTION

Immune checkpoint inhibitors (ICIs) provide long-term survival for a limited number of patients with various cancers including head and neck squamous cell carcinoma (HNSCC). HNSCCs, likely due to intratumoral heterogeneity and multiple modes of immune evasion, show a 15-20% immunotherapy response rate in the recurrent and/or metastatic setting. A variety of factors have been reported to predict the therapeutic efficacy of ICIs against HNSCC including T cell infiltration [1, 2] and tumor mutational burden [3] [4]. A deeper understanding of the complex tumor microenvironmental immune response in HNSCCs is needed to improve response rates. In general, the final common pathway for successful immunotherapy is the induction and infiltration of functional antigen-specific T cells into the tumor with anti-PD1 therapies acting on these existing antitumor T cells. Negative influences on development of antigen specific T cell responses include the suppressive microenvironment, T cell exclusionary factors, and tumor intrinsic immune evasion [5]. To overcome HNSCC immunotherapy resistance, a better understanding of tissue specific mechanisms leading to antigen-specific T cell generation is needed.

We previously established novel syngeneic murine oral carcinoma (MOC) cell lines that recapitulate the key driver mutations and heterogeneous immune response of human HNSCC [6-8]. Studies with MOC lines have been reported both with subcutaneous and oral cavity orthotopic approaches [9] [10]. Although oral cavity CD8+ T cell responses have been reported to be superior to flank transplant in an HPV driven HNSCC model [11], mechanistic studies analyzing this finding including in tumor-draining lymph nodes (TDLNs) have not been reported. In this study, we aimed to explore the orthotopic tumor-specific immune response in comparison to the subcutaneous model using MOC cells with a focus on TDLNs. The tumor burden and OVA-specific CD8+ T cell levels in both the tumor and TDLN were compared between OVA-expressing MOC1 (MOC1OVA) within the oral cavity and subcutaneous flank MOC1OVA. Additionally, cDC1 function and costimulatory marker expression in oral TDLNs were compared to subcutaneous TDLNs. Finally, we tested implications of these differences with anti-PD1 therapy. This study highlights specific immunologic response aspects, including antigen presentation in TDLN of the oral cavity site, with implications for preclinical studies, especially those utilizing immune competent models.

MATERIALS AND METHODS

Cell lines

Mouse oral squamous cell carcinoma, MOC1 was generated from 7, 12-dimethylbenz(a) anthracene (DMBA)-induced C57BL/6 mouse oral squamous cell cancers as previously described [6] [7]. MOC1OVA was created with a retroviral vector encoding mKate2-SIINFEKL on an MSGV2 backbone (kindly provided by Dr. Clint T. Allen, National Institute of Health) [12]. MOC1ZsGreen was established by transducing MOC1 with a lentiviral construct expressing ZsGreen on pLKO backbone using 293T cells with packaging plasmids and Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific), and ZsGreen-expressing cells were selected with blasticidin. The ZsGreen plasmid was kindly provided by Dr. Gavin Dunn (Massachusetts General Hospital) [13]. These cell lines were cultured with the 2:1 mixture of IMDM (Life technologies) and Hams-F12 nutrient mixture (Thermo Fisher Scientific) with 5% heat inactivated FBS (Sigma Aldrich), 100 U/ml penicillin streptomycin (Thermo Fisher Scientific), 5 μg/ml insulin (Sigma Aldrich), 400 ng/ml hydrocortisone (Sigma Aldrich) and 5 ng/ml EGF (Life technologies). Cell lines were tested for mycoplasma every 6 months.

Animal studies

Female C57BL/6 mice were obtained from Taconic Biosciences. TCR-transgenic C57BL/6-Tg (TcraTcrb)1100Mjb mice (OT-1 mice) were obtained from Jackson Labs and bred in-house. All mice were maintained in the pathogen-free animal facility at Dana-Farber Cancer Institute animal resources facility. All procedures were performed under protocols approved by the institutional animal care and use committee (IACUC) of Dana-Farber Cancer Institute.

Tumor cells (3 × 106) were inoculated into the different mice in the right flank for subcutaneous implantation and in the left buccal mucosa for oral implantation. To accurately compare the growth of these different sites, the tumor weights were measured and compared after harvest. The ipsilateral superficial mandibular LN was defined as the TDLN for buccal mucosa tumor [10] and the ipsilateral inguinal LN for flank tumor [14], and each one LN was harvested and compared. Mice were sacrificed at specified time points or endpoints determined by IACUC guidelines. Tumors were resected, immediately minced with razors in RPMI 1640 (Thermo Fisher Scientific) and then incubated for 30 minutes at 37°C with 100μg/ml collagenase Type IV (Life Technologies). LNs and spleens were resected and gently dissociated using microscope glass slides. Splenocytes were treated with red blood cell lysis buffer (Sigma Aldrich). These cells from the tumor, LN, and spleen were filtered to produce single-cell suspensions.

Flow cytometry and SIINFEKL dextramer staining

To detect live and dead cells, all single-cell suspensions were resuspended in 1:500 Zombie aqua (BioLegend) with PBS (Thermo Fisher Scientific) for 15 minutes at room temperature. After centrifuging, Fc block (BioLegend) with FACS buffer (PBS with 1% FBS and 2mM EDTA) was added to inhibit non-specific antibody binding for 15 minutes at 4°C. Then cells were incubated with fluorophore-conjugated anti-mouse antibodies (Abs) diluted in FACS buffer (1:200) for 20 min at 4°C. Antibody panels are summarized in Table S1. To identify antigen specific CD8+ T cells, PE-conjugated SIINFEKL H-2Kb dextramer (Immudex) was added before surface Ab staining according to the manufacturer's instructions. Cells were washed and centrifuged twice, fixed with paraformaldehyde to stabilize the dye, and assessed by flow cytometry (Miltenyi MACSQuantX or BD LSR Fortessa cytometer) with FlowJo v10.8.1 (BD) software for analysis. ZsGreen+ cells and mKate2+ cells were used as compensation controls. Fluorescence minus one (FMO) controls were used for all samples as negative gating.

ELISA

CD8+ T cells in tumor infiltrating lymphocytes (TILs) were isolated from single-cell suspensions with MACS beads CD8 isolation kits (Miltenyi Biotec). CD8 T cells from TILs (1 × 104) were cocultured with splenocytes (1× 105) from non-tumor bearing wildtype C57BL/6 mice as antigen presenting cells and 1μg/mL SIINFEKL peptides (Thermo Fisher Scientific) in 96-well plates. Lymphocytes from TDLN (5 × 105) were cocultured with peptide. Supernatants were analyzed for mouse IFN-γ by ELISA (Thermo Fisher Scientific) following manufacturer’s instructions.

ELISPOT

Lymphocytes from TDLN (1×105) or CD8 T cells from TILs (1×104) and splenocytes (1×105) as antigen presenting cells (APCs) from untreated C57BL/6 mice were cultured with the endogenous tumor-associated antigen p15E or endogenous neoantigen mutant Yipf1 peptides for 24 hr. The p15E peptide epitope (KSPWFTTL) and Yipf1 peptide epitope (VALATFVTI) were identified using previously described methods and synthesized by Peptide 2.0 ([15] and Saito et al., manuscript in preparation). PMA (2μg/mL, Santa Cruz) and ionomycin (0.1μg/mL, Santa Cruz) were used for positive control. T cell responses were assessed by mouse IFN- γ ELISPOT kit (CTL) according to manufacturer’s instruction. The number of positive spots were counted by the Translational Immunogenomics Lab DFCI Core Facility.

cDC1 co-culture assay

XCR1+ cDC1s were purified from TDLN single cell suspensions using the XCR1+ DC isolation kit MACS beads (Miltenyi Biotec). XCR1+ cDC1s (8× 103) were cocultured with naïve OT-1 CD8+ T cells (1× 105) from OT-1 mouse spleen purified by CD8 isolation kit MACS beads (Miltenyi Biotec) in 96-well plates. After 48hr, the supernatant was analyzed by IFN-γ ELISA.

Vaccination

50μg OVA synthetic long peptide (SMLVLLPDEVSGLEQISIINFEKLTEWTS, Peptide2.0) and 100μg polyI:C (Invitrogen) were injected into oral buccal mucosa or subcutaneous flank of tumor-free mice on day 0 and 3.

Monoclonal antibody treatment

Anti-CD8 (clone YTS169.4), anti-PD-1 (clone RMP1-14), and isotype control (IgG2a, clone 2A3) antibodies were obtained from BioXcell. For CD8 depletion, tumor-bearing mice were injected with 250μg of anti-CD8 or isotype control antibody in PBS intraperitoneally on day −1,6,13. For anti-PD-1 treatment, tumor-bearing mice were injected with 250 μg of anti-PD-1 or isotype control antibody in PBS intraperitoneally on days 10, 12, and 14.

Statistical analysis

For comparisons of two independent groups, Student’s t-test with a two-tailed distribution at 95% confidence was used. Multiple comparisons were performed using ANOVA with Tukey’s multiple comparison adjustment. Statistical analyses were performed by GraphPad Prism 9 software (GraphPad) with *p < .05, **p < .01, ***p < .001, and ns = not significant. All data are shown as mean ± SEM.

RESULTS

Enhanced antitumor immune responses in oral orthotopic versus flank tumors

To compare the immune response to a high-affinity antigen between the oral orthotopic and subcutaneous flank sites, we first enforced expression of the SIINFEKL H2Kb-restricted peptide from ovalbumin (OVA257-264) in the mouse syngeneic oral carcinoma model MOC1. These MOC1OVA cells were inoculated into the buccal mucosa or flank and tumor weights were compared after harvest on days 10 and day 17 after inoculation (Figure 1A). Although there was no difference on day 10, oral tumors showed significantly reduced tumor weights compared to flank tumors on day 17 (Figure 1B). To determine if differential immune responses contributed to this difference, the number of antigen-specific T cells, defined as SIINFEKL-dextramer+ CD8+ T cells, was evaluated by flow cytometry at each time point. More SIINFEKL-dextramer+ CD8+ T cells relative to the tumor weight were present in oral tumors than in flank tumors at both the day 10 and day 17 timepoints (Figure 1C and S1A). Furthermore, PD-1+ SIINFEKL-dextramer+ CD8+ T cells showed increased numbers relative to the tumor weight in oral versus flank tumors (Figure 1D). The percentage of CD69+ activated SIINFEKL-dextramer+ CD8+ T cells were increased in oral tumors on day 17 (Figure 1E). As shown in Figure 1E, IFN-γ production from CD8+ T cells from oral TILs cocultured with SIINFEKL peptide was higher than CD8+ T cells from flank TILs on both day 10 and day 17. Finally, CD8 depletion eliminated the difference between the tumor weights of the two sites, indicating that these differences in response are CD8 T cell-dependent (Figure1G, H, and S1B). Collectively, these results showed that the oral orthotopic site induced an enhanced immune response to high-immunogenicity tumor antigens and produced more functional antigen-specific T cells than the subcutis flank.

Figure 1: Oral tumors elicit enhanced OVA antigen antitumor immune responses compared to flank tumors.

Figure 1:

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A. Experimental schema for B-F. Oral or flank tumors were harvested on day 10 or 17 and analyzed by flow cytometry or culture and ELISA.

B. Tumor weight of oral or flank MOC1OVA on day 10 and 17 (mean ± SEM).

C. SIINFEKL-dextramer+ CD8+ T cells in oral or flank MOC1OVA on day 10 and 17. Left is quantification of the number of SIINFEKL-dextramer+ CD8+ T cells per mg tumor (means ± SEM). Right panels are representative data on day17. The percentage is SIINFEKL-dextramer+ cells in CD8+ T cells.

D. Quantification of the number of PD1+ SIINFEKL-dextramer+ CD8+ T cells per mg tumor in oral or flank MOC1OVA on day 10 and 17.

E. CD69+ dextramer+ CD8+ T cells in oral or flank MOC1OVA on day 17. Left panel is quantification of the percentage of CD69+ cells in SIINFEKL-dextramer+ CD8+ T cells (mean ± SEM). Right panels are representative data (pre-gated on SIINFEKL-dextramer+ CD8+ T cells).

F. Quantification of IFN-γ production level by ELISA (mean ± SEM). CD8+ T cells were purified from oral or flank MOC1OVA, and 1×104CD8+ T cells were cocultured with 1μg/ml SIINFEKL peptide with 1×105 splenocytes from tumor-free mice as APCs for 24 hr. IFN-γ production in the supernatant was measured by ELISA.

G. Experimental schema for G. CD8 depleting antibodies (YTS169.4) were injected intraperitoneally in oral or flank tumor-bearing mice on day-1, 6, 13.

H. Tumor weight of oral or flank MOC1OVA with or without CD8 depletion on day 17 (mean ± SEM).

Figure1A-F are representative of 2 independent experiments.

*p<0.05, **p<0.01, ***p<0.001. Significance was evaluated by unpaired Student’s t-test. Multiple comparisons were performed using ANOVA with Tukey’s multiple comparison adjustment.

Increased OVA-specific T cells were generated in oral TDLN

The general consensus is that tumor antigens are captured by migratory dendric cells (DCs) in the tumor and transported to lymph nodes where priming generates antigen-specific T cells [16]. Thus, we examined for antigen specific responses in TDLNs from oral and flank tumor-bearing mice on days 10 and 17 (Figure 2A). As in the TILs, there were more SIINFEKL-specific T cells in oral TDLNs compared to flank TDLNs on days 10 and 17 (Figure 2B and S2). The number of PD-1+ or CD69+ SIINFEKL-dextramer+ CD8+ T cells was also higher in oral TDLN than in flank TDLN (Figure 2C and D). In addition, the functional tumor antigen response of CD8+ T cells from oral tumor-bearing mice was better in TDLNs (Figure 2E). These data indicated that better priming in oral TDLNs resulted in the increased production of antigen-specific T cells in the oral orthotopic setting, likely leading to enhanced anti-tumor responses.

Figure 2: Increased generation of OVA-specific T cells in oral TDLN.

Figure 2:

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A. Experimental schema for B-E. Oral or flank TDLNs were harvested on day 10 or 17 and analyzed by flow cytometry or in vitro culture and ELISA.

B. SIINFEKL-dextramer+ CD8+ T cells in oral or flank TDLN MOC1OVA on day 10 and 17. Left is quantification of the number of SIINFEKL-dextramer+ CD8+ T cells per TDLN (mean ± SEM). Right panels are representative data on day17. The percentage represents SIINFEKL-dextramer+ cells in CD8+ T cells.

C. Quantification of the number of PD1+ SIINFEKL-dextramer+ CD8+ T cells per TDLN in oral or flank MOC1OVA-bearing mice on day 10 and 17.

D. CD69+ dextramer+ CD8+ T cells in oral or flank TDLN MOC1OVA on day 17. Left is quantification of the percentage of CD69+ cells in SIINFEKL-dextramer+ CD8+ T cells (means ± SEM). Right panels are representative data.

E. Quantification of IFN-γ production level by ELISA (means ± SEM). 5×105 lymphocytes from TDLNs in oral or flank MOC1OVA-bearing mice were cocultured with 1μg/ml SIIFNEKL peptide for 24hr. IFN-γ production of the supernatant were measured by ELISA.

These are representative data from 2 independent experiments.

*p<0.05, **p<0.01, ***p<0.001. Significance was evaluated by unpaired Student’s t-test.

Oral mucosa vaccine induced increased antigen-specific T cells in LNs

To examine whether these distinct responses in the oral versus flank setting were related to the presence of tumor, we next tested response to exogenous antigen delivered as a synthetic long peptide (SLP) vaccine. We compared the response to exogenous antigen between oral and flank TDLN by vaccinating mice with an OVA SLP containing SIINFEKL (SMLVLLPDEVSGLEQISIINFEKLTEWTS) into the buccal mucosa or flank subcutis with polyI:C as adjuvant (Figure 3A). Interestingly, the oral vaccine significantly increased SIINFEKL-dextramer+ CD8+ T cells in the ipsilateral cervical lymph node compared to the draining inguinal lymph node in flank vaccinated mice (Figure 3B). However, this increase was not seen in spleen (Figure 3C), indicating that the oral mucosa SLP vaccine was locally effective in the peripheral draining lymph nodes and ineffective systemically. Furthermore, oral vaccine significantly enhanced CD69+ activated SIINFEKL-dextramer+ CD8+ T cells compared to flank vaccine (Figure 3D). Thus, lymph node DCs in cervical lymph nodes may have superior function of antigen presentation compared to inguinal lymph node DCs.

Figure 3: Oral mucosa induced more exogenous antigen-specific T cells in LNs.

Figure 3:

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A. Experimental schema for B-D. OVA synthetic long peptide (50μg, SMLVLLPDEVSGLEQISIINFEKLTEWTS) and polyI:C (100μg) were injected into oral buccal mucosa or subcutaneous flank of tumor-free mice on day 0 and 3. Draining LNs were harvested on day 10 and analyzed by flow cytometry.

B. SIINFEKL-dextramer+ CD8+ T cells in oral or flank vaccinated mice. Left is quantification of the number of SIINFEKL-dextramer+ cells per TDLN (mean ± SEM) and right is representative data.

C. Quantification of the number of SIINFEKL-dextramer+ cells per spleen (mean ± SEM).

D. Quantification of the percentage of CD69+ cells in SIINFEKL-dextramer+ CD8+ T cells in draining LNs of oral or flank vaccinated (mean ± SEM).

These are representative data from 2 independent experiments.

*p<0.05, **p<0.01, ***p<0.001. Significance was evaluated by unpaired Student’s t-test.

Oral cDC1 in TDLN induced higher T cell response

Of the several subsets of DCs, the conventional type 1 DC (cDC1) is the most important subset for antitumor immunity [17]. We next focused on comparing oral mucosa and subcutaneous TDLN cDC1s. To examine the antigen presentation capacity of cDC1 in oral and flank TDLNs, XCR1+ cDC1 were purified from harvested TDLNs and cocultured with CD8+ OT1 T cells (Figure 4A). When CD8+ OT1 T cells were co-cultured with cDC1s from oral TDLNs, we noted increased production of IFN-γ compared to OT1 T cell co-culture with cDC1s from flank TDLNs (Figure 4B). In addition, cDC1s from oral TDLNs induced more CD69+ activated CD8+ OT1 T cells than cDC1s from the flank (Figure 4C). Next, we investigated the capacity of TDLN cDC1s to capture antigens from the oral or flank site. For this analysis, we generated MOC1 expressing ZsGreen, a variant of green fluorescent protein (GFP). DCs containing tumor antigen expressing ZsGreen can be detected as ZsGreen+ cells by flow cytometry [13], allowing evaluation of ZsGreen+ cDC1s in TDLNs (Figure 4D). Oral TDLN had significantly higher numbers and percentages of ZsGreen+ XCR1+ cDC1 than flank TDLN (Figure 4E and S3). In addition to antigen content in DCs, the expression of co-stimulatory factors is crucial for efficient antigen presentation [16]. Oral TDLN had increased CD80, CD86, and CD40 expression in ZsGreen+ XCR1+ cDC1 (Figure 4F). These results indicated that oral mucosa biology allows enhanced antigen delivery to cDC1s that also have enhanced ability to provide co-stimulation.

Figure 4: Oral cDC1 in TDLN had increased functional capacity and induced higher T cell responses.

Figure 4:

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A. Experimental schema for B. XCR1+ cDC1 from TDLN of oral or flank MOC1OVA-bearing mice on day 10 were purified and cocultured with OT-1 CD8+ T cells for 48 hr. The supernatants were analyzed by ELISA and OT1 CD8+ T cells were analyzed by flow cytometry.

B. Quantification of IFN-γ production level in the co-culture supernatant of OT1 CD8+ T cells with oral or flank TDLN XCR1+ cDC1 by ELISA (mean ± SEM).

C. CD69+ OT1 CD8+ T cells cocultured with oral or flank TDLN XCR1+ cDC1. Left is quantification of the percentage of CD69+ cells in SIINFEKL dextramer+ CD8+ T cells (mean ± SEM). Right figures are representative data.

D. Experimental schema for E and F. ZsGreen-expressing MOC1 were inoculated into oral mucosa or flank, and TDLN were harvested on day 10 and analyzed by flow cytometry.

E. The number of ZsGreen+ XCR1+ cDC1 per TDLN in oral or flank MOC1ZsG-bearing mice. Left figure is quantification (mean ± SEM) and right figures are representative data. The percentage is ZsGreen+ cells in XCR1+ cDC1s.

F. Co-stimulatory marker expression of ZsGreen+ XCR1+ cDC1 per TDLN in oral or flank MOC1ZsG bearing mice. Left figures are quantification of the MFI (means ± SEM) and right figures are representative data. (means ± SEM).

These are representative data from 2 independent experiments.

*p<0.05, **p<0.01, ***p<0.001. Significance was evaluated by unpaired Student’s t-test.

Anti-PD1 blockade rejected oral but not subcutaneous tumors

Having delineated some of the proximal events that support enhanced immune response in the oral cavity versus flank, we next focused on the antigen specific response in these sites. We examined endogenous and not model antigen specific responses to a p15E derived endogenous retrovirus antigen [15] and also to a MOC1 specific mutated Yipf1 protein derived neoantigen (Saito et al, in preparation). TDLN analysis via ELISPOT demonstrated an enhanced response to the p15e derived antigen and Yipf1 neoantigen in oral versus flank parental MOC1 bearing mice. By contrast, similar endogenous antigen specific responses were seen in the oral or flank MOC1 TIL (Figure 5A, B, C). This finding is consistent with better antigen presentation in the oral TDLN not only for the artificial, highly immunogenic OVA antigen, but also for endogenous natural antigens. Lastly, we compared the response to anti-PD1 blockade in oral and flank MOC1 tumors (Figure 5D), according to a protocol in which flank MOC1 cells were not expected to be rejected [18, 19]. Consistent with the enhanced antigen specific responses in the oral site, anti-PD1 blockade caused rejection of the oral MOC1 cells but not flank MOC1 cells (Figure 5E). Altogether, oral orthotopic tumors displayed superior antigen presentation in the TDLN, resulting in enhanced anti-PD1 therapy outcomes.

Figure 5: Enhanced endogenous antigen specific and anti-PD1 responses in oral versus flank MOC1 tumors.

Figure 5:

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A. Schema for analysis of responses to MOC1 naturally occurring antigens.

B, C. IFN-γ ELISPOT assays in TIL and TDLN. 1×105 lymphocytes in TDLN or 1×104 CD8+ T cells purified from TILs of oral or flank MOC1 were cocultured with p15E or Yipf1 peptide (1μM) for 24 hr and IFN-γ positive spots were measured by ELISPOT assay. Splenocytes from tumor-free mice were used as APCs in TIL culture. B is quantification and C is representative data.

D. Experimental schema for anti-PD1 responses. Oral or flank MOC1-bearing mice were injected intraperitoneally with 250 μg control or anti-PD1 Ab on days 10, 12, and 14.

E. Tumor weight of oral or flank MOC1OVA with or without anti-PD1 treatment.

Figure 5D and E are representative data from 2 independent experiments.

*p<0.05, **p<0.01, ***p<0.001. Significance was evaluated by unpaired Student’s t-test.

DISCUSSION

While ICI responses have altered clinical management in malignant melanoma, kidney, lung, and head and neck cancers, other tumor types such as pancreatic, ovarian and breast cancer have more limited responses [20]. These distinct responses are linked to tumor intrinsic factors such as tumor mutational burden, oncogenic signaling pathways, and the expression of major histocompatibility complex (MHC) [21]. In addition to cancer type, antitumor immunity also depends on the tissue site of the tumor lesion and local immunobiology. Clinical data of melanoma and non-small-cell lung cancer (NSCLC) patients showed that ICI response varied by the metastatic location [22] [23]. In a mouse model of NSCLC, orthotopic versus subcutaneous implantation showed that cDC1s in the lung TDLNs induced reduced functional anti-tumor T cells, resulting in the reduced efficacy of ICI [24] [25]. This was attributed to the interplay with the tissue-specific immune responses and the tumor microenvironment, which significantly influenced the effectiveness of immunotherapy. Thus, dissecting tissue-specific antitumor immune responses is crucial in both clinical and preclinical studies.

We identified significant differences in the immune response between oral orthotopic and subcutaneous tumor implantation. HNSCC arises from mucosal epithelial cells of the oral cavity, nasal cavity and paranasal sinuses, nasopharynx, oropharynx, hypopharynx, and larynx. These mucosal sites in the upper airway and upper gastrointestinal tract are the first sites of contact with antigens from food and air due to constant environmental exposure [26]. The mucosal immune system must prevent the entry of pathogen but maintain immune homeostasis by avoiding excessive inflammation through immune tolerance [26]. Recent studies showed that microbiota can affect the antitumor immunity [27]. Oral microbiota are complex and their crosstalk with the immune cells and the epithelium is critical for the maintaining homeostasis [28]. Therefore, the development of HNSCC is likely impacted in this complex tumor microenvironment with tissue-specific immune responses. The distinctions we have illustrated between oral mucosal and subcutaneous tumors in preclinical models underscores the importance of investigating HNSCC-specific immune responses.

Our HNSCC model showed a differential oral TDLN response and cDC1 function that likely contribute to enhanced antitumor immunity. These data parallel studies with the mEER HPV driven model where transplant into the tongue versus flank showed enhanced CD8+ Granzyme B positive T cell infiltration in tongue tumors [11]. However, a cellular mechanism was not identified for this differential response [11]. The cancer immunity cycle highlights the importance of TDLN and DCs [29]. Capturing tumor antigens, transporting to TDLNs and priming of T cell responses illustrate the central role of DCs for generating tumor antigen-specific T cells. In a HNSCC mouse model, neck dissection was shown to abrogate the antitumor effect of ICIs due to the loss of cDC1, thus reinforcing the significance of TDLNs and cDC1 [10]. For effective priming in TDLN, DC function such as the expression of the co-stimulatory molecules is also crucial. Our data demonstrated that cDC1 in oral TDLN exhibited higher expression of costimulatory markers than subcutaneous TDLN, in addition to oral TDLN having increased numbers of antigen-containing cDC1. The influence of DCs in site specific models and on the immunogenicity has been studied previously, including in models of dermal and subcutaneous injections [30]. Our study illustrates an improved function of cDC1s in the oral tumor microenvironment leading to induction of increased functional antigen-specific T cells [25]. In conclusion, our results highlight and extend the importance of studying both tissue-specific and HNSCC-specific antitumor immunity including the cross talk between tumor and TDLN.

Supplementary Material

1

Supplemental figure S1A Gating strategy for TILs.

Supplemental figure S1B Representative data showing efficacy of CD8+ T cell depletion in anti-CD8 antibody treated mice.

Supplemental figure S2 Gating strategy for lymph nodes.

Supplemental figure S3 Gating strategy for DCs.

2

Table S1 Antibodies used in this study

Highlights:

  • Oral tumors induced enhanced antigen specific T cell responses compared to the flank.

  • cDC1s in oral TDLNs are highly functional and elicit a robust T cell response.

  • anti-PD1 treatment induces rejection of oral but not flank tumors.

Acknowledgements

We thank all members of the Uppaluri lab and Dr. Gavin P. Dunn for thoughtful discussions. We thank Clint Allen for the mKATE2-SIINFEKL plasmid and Dr. Gavin P. Dunn for the ZsGreen plasmid. Work in Uppaluri lab is supported by (NIH/NCI/NIDCR U01DE029188 and NIH/NIDCR R01DE027736). The schematic diagrams were created with BioRender under the publication license.

Footnotes

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Disclosures

RU serves on Merck, Regeneron and Daichi-Sankyo advisory boards. The MOC models developed by RU have been filed with the Washington University Office of Technology Management and are licensed for distribution by Kerafast.

REFERENCES

  • [1].Herbst RS, Soria JC, Kowanetz M, Fine GD, Hamid O, Gordon MS, et al. Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients. Nature. 2014;515:563–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [2].Tumeh PC, Harview CL, Yearley JH, Shintaku IP, Taylor EJ, Robert L, et al. PD-1 blockade induces responses by inhibiting adaptive immune resistance. Nature. 2014;515:568–71. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [3].Cristescu R, Mogg R, Ayers M, Albright A, Murphy E, Yearley J, et al. Pan-tumor genomic biomarkers for PD-1 checkpoint blockade-based immunotherapy. Science. 2018;362. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [4].Pfister DG, Haddad RI, Worden FP, Weiss J, Mehra R, Chow LQM, et al. Biomarkers predictive of response to pembrolizumab in head and neck cancer. Cancer Med. 2023;12:6603–14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [5].Galon J, Bruni D. Approaches to treat immune hot, altered and cold tumours with combination immunotherapies. Nat Rev Drug Discov. 2019;18:197–218. [DOI] [PubMed] [Google Scholar]
  • [6].Judd NP, Winkler AE, Murillo-Sauca O, Brotman JJ, Law JH, Lewis JS Jr., et al. ERK1/2 regulation of CD44 modulates oral cancer aggressiveness. Cancer Res. 2012;72:365–74. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [7].Onken MD, Winkler AE, Kanchi KL, Chalivendra V, Law JH, Rickert CG, et al. A surprising cross-species conservation in the genomic landscape of mouse and human oral cancer identifies a transcriptional signature predicting metastatic disease. Clin Cancer Res. 2014;20:2873–84. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [8].Kono M, Saito S, Egloff AM, Allen CT, Uppaluri R. The mouse oral carcinoma (MOC) model: A 10-year retrospective on model development and head and neck cancer investigations. Oral Oncol. 2022;132:106012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [9].Judd NP, Allen CT, Winkler AE, Uppaluri R. Comparative analysis of tumor-infiltrating lymphocytes in a syngeneic mouse model of oral cancer. Otolaryngol Head Neck Surg. 2012;147:493–500. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [10].Saddawi-Konefka R, O'Farrell A, Faraji F, Clubb L, Allevato MM, Jensen SM, et al. Lymphatic-preserving treatment sequencing with immune checkpoint inhibition unleashes cDC1-dependent antitumor immunity in HNSCC. Nat Commun. 2022;13:4298. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [11].Dorta-Estremera S, Hegde VL, Slay RB, Sun R, Yanamandra AV, Nicholas C, et al. Targeting interferon signaling and CTLA-4 enhance the therapeutic efficacy of anti-PD-1 immunotherapy in preclinical model of HPV(+) oral cancer. J Immunother Cancer. 2019;7:252. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [12].Sun L, Moore E, Berman R, Clavijo PE, Saleh A, Chen Z, et al. WEE1 kinase inhibition reverses G2/M cell cycle checkpoint activation to sensitize cancer cells to immunotherapy. Oncoimmunology. 2018;7:e1488359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [13].Bowman-Kirigin JA, Desai R, Saunders BT, Wang AZ, Schaettler MO, Liu CJ, et al. The Conventional Dendritic Cell 1 Subset Primes CD8+ T Cells and Traffics Tumor Antigen to Drive Antitumor Immunity in the Brain. Cancer Immunol Res. 2023;11:20–37. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [14].Zhao X, Kassaye B, Wangmo D, Lou E, Subramanian S. Chemotherapy but Not the Tumor Draining Lymph Nodes Determine the Immunotherapy Response in Secondary Tumors. iScience. 2020;23:101056. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [15].Shibata H, Xu N, Saito S, Zhou L, Ozgenc I, Webb J, et al. Integrating CD4(+) T cell help for therapeutic cancer vaccination in a preclinical head and neck cancer model. Oncoimmunology. 2021;10:1958589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [16].Zagorulya M, Spranger S. Once upon a prime: DCs shape cancer immunity. Trends Cancer. 2023;9:172–84. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [17].Broz ML, Binnewies M, Boldajipour B, Nelson AE, Pollack JL, Erle DJ, et al. Dissecting the tumor myeloid compartment reveals rare activating antigen-presenting cells critical for T cell immunity. Cancer Cell. 2014;26:638–52. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [18].Kansal V, Burnham AJ, Kinney BLC, Saba NF, Paulos C, Lesinski GB, et al. Statin drugs enhance responses to immune checkpoint blockade in head and neck cancer models. J Immunother Cancer. 2023;11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [19].Sun L, Clavijo PE, Robbins Y, Patel P, Friedman J, Greene S, et al. Inhibiting myeloid-derived suppressor cell trafficking enhances T cell immunotherapy. JCI Insight. 2019;4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [20].Yarchoan M, Hopkins A, Jaffee EM. Tumor Mutational Burden and Response Rate to PD-1 Inhibition. N Engl J Med. 2017;377:2500–1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [21].Kalbasi A, Ribas A. Tumour-intrinsic resistance to immune checkpoint blockade. Nat Rev Immunol. 2020;20:25–39. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [22].Lee JHJ, Lyle M, Menzies AM, Chan MMK, Lo S, Clements A, et al. Metastasis-specific patterns of response and progression with anti-PD-1 treatment in metastatic melanoma. Pigment Cell Melanoma Res. 2018;31:404–10. [DOI] [PubMed] [Google Scholar]
  • [23].Nishino M, Ramaiya NH, Chambers ES, Adeni AE, Hatabu H, Janne PA, et al. Immune-related response assessment during PD-1 inhibitor therapy in advanced non-small-cell lung cancer patients. J Immunother Cancer. 2016;4:84. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [24].Horton BL, Morgan DM, Momin N, Zagorulya M, Torres-Mejia E, Bhandarkar V, et al. Lack of CD8(+) T cell effector differentiation during priming mediates checkpoint blockade resistance in non-small cell lung cancer. Sci Immunol. 2021;6:eabi8800. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [25].Zagorulya M, Yim L, Morgan DM, Edwards A, Torres-Mejia E, Momin N, et al. Tissue-specific abundance of interferon-gamma drives regulatory T cells to restrain DC1-mediated priming of cytotoxic T cells against lung cancer. Immunity. 2023;56:386–405 e10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [26].Moutsopoulos NM, Konkel JE. Tissue-Specific Immunity at the Oral Mucosal Barrier. Trends Immunol. 2018;39:276–87. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [27].Choi Y, Lichterman JN, Coughlin LA, Poulides N, Li W, Del Valle P, et al. Immune checkpoint blockade induces gut microbiota translocation that augments extraintestinal antitumor immunity. Sci Immunol. 2023;8:eabo2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [28].Pandiyan P, Conti HR, Zheng L, Peterson AC, Mathern DR, Hernandez-Santos N, et al. CD4(+)CD25(+)Foxp3(+) regulatory T cells promote Th17 cells in vitro and enhance host resistance in mouse Candida albicans Th17 cell infection model. Immunity. 2011;34:422–34. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [29].Chen DS, Mellman I. Oncology meets immunology: the cancer-immunity cycle. Immunity. 2013;39:1–10. [DOI] [PubMed] [Google Scholar]
  • [30].Joncker NT, Bettini S, Boulet D, Guiraud M, Guerder S. The site of tumor development determines immunogenicity via temporal mobilization of antigen-laden dendritic cells in draining lymph nodes. Eur J Immunol. 2016;46:609–18. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1

Supplemental figure S1A Gating strategy for TILs.

Supplemental figure S1B Representative data showing efficacy of CD8+ T cell depletion in anti-CD8 antibody treated mice.

Supplemental figure S2 Gating strategy for lymph nodes.

Supplemental figure S3 Gating strategy for DCs.

NIHMS1986937-supplement-1.pdf (639.1KB, pdf)
2

Table S1 Antibodies used in this study

NIHMS1986937-supplement-2.xlsx (10KB, xlsx)

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