With estimated 880,000 new cases and 450,000 deaths worldwide in 2020, head and neck squamous cell carcinoma (HNSCC) is the sixth most common tumor entity and incidences continue to rise, with a growing proportion of human papillomavirus (HPV)-infection driven tumors over the last decades (1). HNSCC includes tumors located in the oral cavity, oropharynx, hypopharynx, and larynx. Besides some well-known risk factors, such as extensive consumption of tobacco and alcohol, the infection with HPV (primarily genotype 16) causes carcinogenesis in roughly 25% of HNSCC, particularly in the oropharynx (> 70%), hence representing a major leading factor for HNSCC development (2).

Despite the advanced treatment regimen consisting of radical resection and adjuvant high-dose radio(chemo)therapy (R(C)Tx) or definite R(C)Tx in locally advanced HNSCC, the 5-year overall survival (OS) rate of 50-69% still can be improved (3). Moreover, limited prognosis also results from frequent local relapse occurring in up to 60% of HNSCC patients (4). The implementation of immunotherapy approaches, such as anti-programmed cell death protein-1 (anti-PD-1) antibodies, was intended to improve survival outcomes. However, this was noticeable only in 15-30% of HNSCC patients with recurrent or metastatic disease (5, 6). Several studies highlighted that HPV-associated (HPV+) oropharyngeal tumors are generally linked to an improved clinical outcome following R(C)Tx, compared to the HPV-negative (HPV-) ones. Hence, these HPV+ tumors constitute a distinct cancer entity (7, 8), with p16 upregulation being used as their corresponding marker in the 8^th edition of tumor, node, metastasis (TNM) classification, even though the use of p16 overexpression alone as a prognostic and diagnostic marker needs further validation (9). Other parameters, including the combination of HPV-DNA/RNA and p16, hypoxia and hypoxia-induced gene expression, high expression of cancer stem cell markers, tumor volume, solute carrier family 3 member 2 (SLC3A2) and CD44 protein expression, have also been described as prognostic factors for local relapse after R(C)Tx in HNSCC (10). For instance, hypoxia-associated gene signatures or a hypoxic tumor microenvironment (TME) are associated with reduced therapeutic efficacy and poor prognosis (11, 12).

Despite the utility of tumor localization, p16 status, and hypoxia in subgrouping HNSCC patients and predicting clinical outcome, there is still a high variability in treatment response. Therefore, novel biomarkers as well as improved tools to predict therapeutic success and individualize treatment modalities are urgently needed to optimize therapy efficacy, properly adjust therapeutic regimens, and prevent over- or under-treatment with side effects or treatment failure (13).

HNSCC is known to be an immunogenic tumor entity including a high tumor infiltration of myeloid and lymphoid cells (14). However, tumor cells generate an immunosuppressive microenvironment resulting, for instance, in enhanced infiltration of activated regulatory T cells (T_regs) and generation of exhausted T cells (5). Thus, the tumor immune contexture comprising the frequency, spatial distribution, orientation, and functional characteristics of tumor-infiltrating immune cells plays a critical role for the clinical outcome of cancer patients (15, 16). A positive correlation between high densities of tumor-infiltrating T cell subsets, in particular CD8⁺ T cells, and improved prognosis has been reported for various cancer entities, including HNSCC (15, 17). Additionally, dendritic cells (DCs) are known as key players of innate and adaptive immunity (18). DCs are clinically relevant, as demonstrated in various cancer entities (19, 20), and may also significantly impact the outcome of HNSCC patients (21). The function of cancer-infiltrating immune cells is dual and context-dependent, as they can either favor anticancer immunity or promote tumor tolerance. Hence, their role has to be described individually for each cancer entity and immune cell subtype (22). For instance, plasmacytoid DCs (pDCs), described as the main producers of type I interferon upon stimulation, can either participate in antitumor immunity and favorably impact clinical outcome (20), or they may adopt a tolerogenic phenotype and thereby contribute to poor prognosis (23). Other studies investigating the tumor immune architecture revealed that infiltrating T cells also significantly influenced the clinical efficacy of various treatments, including R(C)Tx (24, 25).

In HNSCC, the role of the immunological TME is subject of ongoing research. However, there are limited investigations exploring the tumor immune landscape in detail. So far, most studies have focused on detecting the presence of T cells using immunohistochemistry or bulk gene expression profiles of tumor samples (26–29). Several groups reported a positive impact of high densities of CD8⁺ T cells on the survival and relapse of HNSCC patients (5, 27). Nonetheless, these previous studies generally lack a more detailed phenotypic description of infiltrating CD8⁺ T lymphocytes in terms of their phenotype orientation, for instance by assessing the (co-)expression of activating and/or inhibitory markers, their localization relative to the tumor cells, and whether their spatial distribution impacts survival. Moreover, little is known about the infiltration pattern and clinical influence of distinct DC and T-helper (T_H) subsets in HNSCC.

Here, we aimed to explore these unresolved issues by comprehensively investigating the presence and role of several tumor-infiltrating T cell subpopulations, two conventional DC subtypes (cDC1 and cDC2), as well as pDCs in more detail, characterizing their phenotype and spatial distribution in distinct compartments of HNSCC tissue samples by multiplex immunohistochemistry (mIHC). We correlated the densities and proportions of various immune cell subsets with clinical characteristics of the whole HNSCC cohort and different HPV- or hypoxia-associated subcohorts. In addition, we used survival parameters and clinicopathological characteristics of the patient cohort to identify immune cell types with an independent prognostic value. Thereby, we provide a more comprehensive picture of the HNSCC immune architecture supporting the identification of novel prognostic biomarkers for this cancer entity.

The significant influence of the immune landscape on tumor development, progression, as well as on therapeutic outcome, has been described for many solid cancers, including HNSCC. By using conventional hematoxylin and eosin (H&E) whole slide stainings, Ribbat-Idle et al. showed that the immune infiltration pattern is an independent prognostic factor for OS of HNSCC patients, with “cold” tumors being significantly associated with worse clinical outcome in comparison to “hot” and “excluded” tumors (39). Whereas CD8⁺ T cell infiltration in general indicates a positive prognosis in HNSCC, T_H subpopulations, DC subtypes, and CD8⁺ T cells expressing further co-stimulatory/-inhibitory marker proteins lack prognostic evaluation, so far (40, 41).

In our study, we investigated the tumor infiltration pattern of several immune cell subpopulations in different tumor compartments. Regarding DC subtypes, predominant pDCs were significantly more abundant in TS than in IET tissue compartment, whereby only minor frequencies of mature and/or activated pDCs (IRF7⁺ pDCs) were identified. In general, HNSCC samples exhibited eightfold higher pDC infiltration in comparison to primary resected colon cancer and pancreatic ductal adenocarcinoma samples in our previous studies using similar BDCA-2 staining procedures (23, 42). Especially in HNSCC, the hypoxic TME seems to inhibit pDC maturation (43). Koucký et al. demonstrated a colocalization of functionally impaired pDCs with T_regs in TS and reported a selective pDC-fostered T_reg expansion (44). When looking at T cell subpopulations in our HNSCC cohort, T_regs represented the major T_H cell subset, with their predominant stromal distribution confirming current literature (45) and may contributing to the mentioned relation of pDCs and T_regs. With respect to further T_H subsets, T_H1 cells were more common than T_H2 cells or T_H17 cells and significantly more frequent in IET than in TS. Although the development of HNSCC is thought to be accompanied by a switch from T_H1- to T_H2-cytokines (46), a CD8⁺-rich TME seems to be linked to a T_H1-dominated immune response (47). Using The Cancer Genome Atlas (TCGA) datasets of HNSCC patients, Lu et al. found an increased level of T_H1 cells in a subcohort with a high abundance of γδT cells, being in turn associated with a longer OS (48). Due to the limited number of marker molecules that can be simultaneously analyzed in one staining panel using our mIHC method, we have chosen CD3 for T cell detection and various major TFs (T-bet, GATA3, RORγT, and FoxP3) for evaluating the proportion of the most common T_H subsets (T_H1 cells, T_H2 cells, T_H17 cells, T_regs). Therefore, rare CD8⁺ T cells (T_C1, T_C2, T_C17) expressing one of these TFs may contribute to the proportion of positively stained TF-expressing CD3⁺ T cells in our study. In this context, T_C1, T_C2, and T_C17 cells have been found in peripheral blood (49) or tumor draining lymph nodes (50) of HNSCC patients by flow cytometry. These studies discriminated the subpopulations based on the cytokine expression profiles without analyzing TF expression. However, CD8⁺ T_C subsets were less frequent than CD4⁺ T_H subsets. In addition to αβT cells, γδT cells expressing the mentioned TFs may also make up a proportion of our positively stained CD3⁺ T cells. IL17-producing γδT cells, termed γδT17, were found in several tumor entities, including HNSCC (51, 52). In further studies, we explored the frequency and phenotype of HNSCC-infiltrating T cells. In comparison to a previous study on soft tissue sarcomas, that utilized a similar T cell staining procedure, HNSCC samples featured an obviously higher T cell infiltration (32). The investigated CD8⁺ T cells showed significantly higher frequencies in TS. This finding is in line with the literature reporting a higher frequencies of stromal T lymphocytes compared to IET-lying ones (53). However, significantly greater proportions of CD8⁺ T cell subpopulations, expressing further co-stimulatory and/or co-inhibitory marker proteins, were present in the IET compartment. In particular, a high expression of the co-inhibitory receptors PD-1 and LAG-3 by CD8⁺ T cells, either alone or in combination, became obvious underlining the described loss of effector function and exhaustion state of tumor-infiltrated CD8⁺ T cells (5).

Following previous approaches, we incorporated several clinicopathological parameters in our analysis that appear important for the progression of HNSCC and treatment response, such as tumor grading, p16 status, and hypoxia, and assessed them in conjunction with immune cell infiltrate patterns. First, we explored the relationship between the immune landscape and tumor grading. While Boucek et al. described a positive association between tumor histological grading and elevated CD8⁺ T cell levels in the blood of HNSCC patients at diagnosis (54), we observed a positive correlation between tumor grading and exhausted CD8⁺ T cells (PD-1⁺LAG-3⁺CD8⁺) within the IET compartment of the Hypox15^low subcohort. This finding is in line with the literature describing a correlation of high LAG-3 expression and high pathological grade (55). Further, a higher grading (G3) was frequently accompanied by a higher proportion of stromal RORγT⁺ T_H17 cells. A high infiltration of T_H17 cells in HNSCC was confirmed by others, hypothesizing on a contribution to cancer progression (21).

Growing evidence has shown that two factors, namely tumor localization and HPV status, split HNSCC into greatly different subentities, characterized by distinct clinical and therapeutic outcomes (7). Recognition of HPV-related HNSCC as a distinct entity from the HPV-unrelated cases emerged from differences observed in their molecular profiles, tumor development process, clinical presentation, and outcome. This led to a novel staging system for the HPV+ oropharyngeal squamous cell carcinoma in the latest edition of the TNM classification of the Union for International Cancer Control (UICC) (56). The immune contexture, which differs among HNSCC localizations and between HPV+ and HPV- tumors, might represent a pivotal player in shaping these differences. Multiple groups have reported an enhanced infiltration of CD8⁺ T lymphocytes in p16+ HNSCC, that correlated positively with clinical outcome (14, 24, 47, 57, 58). In addition to high CD3⁺ and CD8⁺ T cell densities as well as CD8:T_reg ratio in HPV+ tumors shaping a less immunosuppressive TME as detected in multiple studies (41), we showed that both cDC subtypes (cDC1 and cDC2) had significantly higher frequencies in p16+ vs. p16- tumor samples, primarily in the IET compartment. Similarly, researchers reported a higher infiltration of HPV+ oropharyngeal tumors by a specific CD11c⁺ cDC population compared to the HPV- counterparts (59). Using the TCGA database, Gameiro et al. observed that activated DC transcript levels tend to be higher in HPV+ vs. HPV- tumors, but without reaching significance (60). However, also contradictory results are reported being not able to draw a link between DC frequencies and HPV status or even detecting lower DC amounts in HPV-related tumors (41, 59). Additionally, increased proportions of exhausted T cells (PD-1⁺CD8⁺, PD-1⁺LAG-3⁺CD8⁺), cytotoxic T cells (PD-1⁺GrzB⁺CD8⁺), proliferating T cells (PD-1⁺Ki67⁺CD8⁺), and GATA3⁺ T_H2 cells in TS of patients with p16+ tumors were observed. Similarly to our quantified stainings, HPV-related HNSCC are ascertained to exhibit high expression levels of checkpoint receptors, including PD-1 and LAG-3, underlining a linkage of HPV infection and T cell exhaustion (53, 61, 62). Further, Kansy et al. reported significantly increased fractions of cytotoxic T cells (GrzB⁺CD8⁺) and proliferating T cells (Ki67⁺CD8⁺) in the TS of 27 HPV+ oropharyngeal tumor patients analyzed by multi-color immunofluorescence (63).

Hypoxia is a major factor that shapes the TME and also the response of HNSCC to radiotherapy by regulating i) the survival, proliferation, differentiation, and antitumor capacity of T cells; ii) the survival, migration, antigen presentation, and differentiation of DCs; or iii) the macrophage polarization and tumor development, among others (64, 65). In HNSCC, Kim et al. observed that the level of hypoxia varies across anatomical locations, with tumors in the larynx being less hypoxic than those in the oral cavity, and those in the oropharynx being the most hypoxic ones (66). In our HNSCC cohort, significant lower infiltration of cDC1 cells (IET) and cytotoxic GrzB⁺CD8⁺ T cells (TS) in tumors with hypoxia-associated gene signatures was measurable. According to the literature, hypoxia might diminish the infiltration and activity of DCs as well as T lymphocytes (67, 68) confirming our results. Especially in the p16+ subcohort, additional significant differences were noticeable, as these patients exhibited also a lower proportion of T-bet⁺ T_H1 cells and a concomitantly higher infiltration of T_regs in Hypox15^high tumors being independent of the investigated tumor compartment. It is reported that immunosuppressive immune cells, like T_regs, infiltrate hypoxic regions in HNSCC supporting our results (69).

Besides correlating immune cell infiltrate patterns with clinical parameters, the association of immune cell populations and patient survival was a major research objective of this study. By means of TCGA datasets, Mito et al. showed a lymphocyte gene expression signature that predicts prolonged OS, whereas a myeloid/DC signature indicated lower survival propabilites (5). This suggests that the myeloid compartment favors immune suppression and tumor growth in HNSCC, but its role has not been clearly dissected so far. In our cohort, patients with Hypox15^low HNSCC and a high stromal infiltration of IRF7⁺ pDCs displayed significantly shortened OS and LDMC. The correlation of pDC abundancy and poor prognosis is described for many cancer entities including HNSCC (41, 43). In other studies, pDC infiltration correlated with a high expression of FoxP3 and co-inhibitory markers (PD-1 and LAG-3) setting up an immunosuppressive TME and promoting tumor progression (70, 71). Additionally, positive correlations of especially cDC1 infiltration and, to a lesser extent, cDC2 frequency with favorable LDMC surfaced in our investigation. This fits and adds to the existing knowledge on cDC1s forming clusters with CD8⁺ T cells in the TS compartment of HNSCC patients and, thus, being capable to induce CD8⁺ T cell-mediated antitumoral responses, which in turn correlated with high LDMC in these patients (72). However, we report the positive role of cDC1s in HNSCC by detecting the subset-specific CLEC9A molecule at protein level. Our findings on cDC2s are in line with current literature describing an association of cytokine-producing CD163⁺ cDC2 cells or cDC2, expressing programmed cell death ligand-1 (PD-L1)^high and inducible T cell co-stimulator ligand (ICOSL)^low, with favorable survival (73, 74).

Regarding an association of T cell subpopulations with clinical outcome, studies resulted in contradictory findings. On the one hand, CD4⁺ T cells were significantly associated with longer OS and disease-specific survival (75), but on the other hand most research did not observe significant correlations of CD4⁺ infiltration and patient prognosis (41, 61). In our p16- subcohort, the infiltration of RORγT⁺ T_H17 cells and FoxP3⁺ T_regs were linked to favorable OS/LDMC and OS, respectively. While T_H17 infiltration was associated with an improved prognosis of patients with HPV+ HNSCC (21), the current literature is balanced between studies reporting positive and negative impacts of T_reg infiltration on survival. Thereby, contradictory reports exist for both different HNSCC locations and for HPV-dependencies (41, 76, 77). Nevertheless, studies agreed on the relation of high CD8⁺ T cell infiltration and a positive impact on HNSCC patients’ survival (61, 78). Likewise, a higher frequency of CD8⁺ T lymphocytes as well as an elevated CD8:T_reg ratio correlated significantly with higher LRC and LDMC in our study. However, regarding the relation of CD8:T_reg ratio and survival properties of HNSCC patients, contradictory studies exist mentioning an adverse association (41, 79), no association or a favorable association (80, 81). A possible explanation for the inconclusive study situation was provided by Echarti et al. (82). In their investigation, the authors found a high T_reg infiltration to diminish OS in “immune desert” and “immune excluded” HNSCC, but a prolonged OS was seen in high T_reg-infiltrated “inflamed” tumors. When assessing CD8⁺ subpopulations and their association with survival parameters of our cohort, cytotoxic GrzB⁺CD8⁺ T cells are unfavorable concerning clinical outcome for patients with Hypox15^high tumors, whereas for patients with Hypox15^low tumors the infiltration of active cytotoxic T cells seems to be linked to good survival. It is known that the hypoxic TME evades immune cell-mediated killing mechanisms by activating autophagy, forming autophagosomes, and neutralizing/degrading cytotoxic mediators, like GrzB (83). Additionally, whole tumor infiltration of PD-1⁺CD8⁺ T cells was associated with prolonged OS in the Hypox15^low subcohort and displaying a similar trend in the whole cohort, too. According to the literature, a high frequency of PD-1⁺CD8⁺ T cells was linked to favorable OS in patients with HPV- HNSCC, whereby the 25^th lowest percentile of these immune cell infiltrate pattern was used for grouping the patients (84).

When evaluating the clinical outcome of HNSCC patients according to p16 status in combination with immune cell infiltrate pattern of pDCs, cDC1, cDC2, CD3⁺, and CD8⁺ T cells, four subgroups can be classified: p16+/immune^high, p16+/immune^low, p16-/immune^high, and p16-/immune^low. In our investigation, especially patients with p16+ HNSCC and high immune cell infiltrate exhibited significantly better clinical outcomes in comparison to both p16- subgroups and tendentially prolonged survival compared to patients with p16+ HNSCC and low immune cell infiltrate. In literature, patients with HPV+ HNSCC and high infiltration of CD8⁺ T cells showed most favorable survival parameters (85) fitting our observations. Similarly, in terms of combined hypoxia/immune phenotype profiles different subgroups might be distinguished: hypoxia^low/immune^high, hypoxia^high/immune^low, hypoxia^low/immune^low, and hypoxia^high/immune^high. The last two phenotypes mentioned are also referred as a mixed or intermediate phenotype. In this regard, different investigators showed that the hypoxia^low/immune^high phenotype was indicative of enhanced survival rates based on hypoxia- and immune response-related gene expression analysis of TCGA datasets or immunohistochemical staining of T cell (CD3) and hypoxia markers (carbonic anhydrase IX) (11, 86–88). In our study, we confirmed the positive association of the hypoxia^low/immune^high phenotype with favorable clinical outcome of HNSCC patients. Though, different from the studies mentioned above, our phenotype clustering based on Hypox15 gene signature to determine the hypoxia status as well as on multiplex immunohistochemical staining combined with semi-automatic quantification of DCs and T cells at protein level to identify the immune cell infiltrate. In sum, favorable survival of patients with p16+ or Hypox15^low HNSCC seems to be particularly associated with a high infiltration of DCs and T cells. Thus, patients with p16+/immune^low and Hypox15^low/immune^low HNSCC might benefit from therapeutically increased immune cell infiltrate.

Moreover, the infiltration of CLEC9A⁺ cDC1 and CD8⁺ T cells emerged as independent prognostic markers for improved clinical outcome besides clinicopathological parameters, like T stage, p16 status, and tumor localization, as shown in the multivariate Cox regression and our developed ICIS. By using a TCGA dataset of 497 HNSCC tissue samples, Liu et al. identified a significant association of CLEC9A gene and OS (univariate hazard ratio [HR]: 0.876, multivariate HR: 0.906) (89). Moreover, Furgiuele et al. reported CD8⁺ density to be an independent prognostic marker and an deduced ICIS, comprising tumor infiltration of T lymphocytes (CD8⁺, T_regs) and macrophages (CD68⁺), improved HNSCC patients’ prognosis (90). Therefore, in the context of the still ongoing implementation of an ICIS for HNSCC (90, 91), cDC1 and CD8⁺ T cells are promising candidates for an ICIS to predict the clinical outcome of HNSCC patients.

In our study, we comprehensively investigated HNSCC infiltration patterns of several DC subtypes and T cell subpopulations, with respect to their phenotype and spatial distribution. The stromal tumor compartment is highly infiltrated by pDCs and T lymphocytes, with T_regs and exhausted CD8⁺ T cells being the predominant phenotypes and shaping an immunosuppressive tumor immune architecture. HPV-associated tumors showed significantly higher infiltration of several investigated immune cell populations, whereby tumors with hypoxia-associated gene signatures exhibited reduced infiltration. Furthermore, our study contributes to the identification of novel prognostic biomarkers for HNSCC, as especially cDC1 and CD8⁺ T cells were independent prognostic factors for clinical outcome and potentially contribute to the evaluation of a novel and more precise ICIS. These parameters may help to further stratify patient subgroups in need of treatment escalation. If confirmed in a validation cohort, combined treatment approaches with enhancing tumor perfusion (using hyperthermia, irradiation, potentially particle therapy) and immune checkpoint inhibitor therapy in high-risk tumors may be subject of future investigations. In patients with HNSCC receiving primary R(C)Tx, pretherapeutic diagnostic approaches with functional imaging such as ¹⁸F-fluoromisonidazol positron emission tomography (¹⁸F-FMISO PET) to detect hypoxic tumor areas and combining these findings with an ICIS based on the analysis of biopsy material could add prognostic information and lead to further individualized treatment strategies. This might improve the design of future radiochemo(immuno)therapy.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding authors.

The studies involving humans were approved by Ethics Committee of Technische Universität Dresden. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.

JK: Conceptualization, Writing – original draft, Writing – review & editing, Data curation, Formal analysis, Investigation, Methodology, Resources. IP: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review & editing. RR: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review & editing, Visualization. AR: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Visualization, Writing – original draft, Writing – review & editing. SL: Data curation, Formal analysis, Investigation, Methodology, Visualization, Writing – original draft, Writing – review & editing. IB: Data curation, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review & editing. LR: Data curation, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review & editing. AL: Writing – original draft, Writing – review & editing, Funding acquisition, Resources. RW: Writing – original draft, Writing – review & editing, Funding acquisition. MK: Funding acquisition, Writing – original draft, Writing – review & editing, Resources. MS: Funding acquisition, Writing – original draft, Writing – review & editing, Conceptualization.