Head and neck cancers are a heterogeneous group of tumors that are highly aggressive and collectively represent the sixth most common cancer worldwide.Ninety percent of head and neck cancers are squamous cell carcinomas (HNSCCs).The tumor microenvironment (TME) of HNSCCs consists of many different subsets of cells that infiltrate the tumors and interact with the tumor cells or with each other through various networks. Both innate and adaptive immune cells play a crucial role in mediating immune surveillance and controlling tumor growth. Here, we discuss the different subsets of immune cells and how they contribute to an immunosuppressive TME of HNSCCs. We also briefly summarize recent advances in immunotherapeutic approaches for HNSCC treatment. A better understanding of the multiple factors that play pivotal roles in HNSCC tumorigenesis and tumor progression may help define novel targets to develop more effective immunotherapies for patients with HNSCC.

The BCR recognizes foreign Ags to initiate humoral immunity that needs isotype-switched Abs generated via class switch recombination (CSR); however, stimulating the BCR in the absence of costimulation (e.g., CD40) does not induce CSR; thus, it remains elusive whether and how the BCR induces CSR mechanistically. Autoreactive B cells can maintain anergy via unresponsiveness of their BCRs to self-antigens. However, it remains unknown what molecule(s) restrict BCR signaling strength for licensing BCR-induced CSR and whether deficiency of such molecule(s) disrupts autoreactive B cell anergy and causes B cell-mediated diseases by modulating BCR signaling. In this study, we employ mouse models to show that the BCR's capacity to induce CSR is restrained by B cell-intrinsic checkpoints TRAF3 and TRAF2, whose deletion in B cells enables the BCR to induce CSR in the absence of costimulation. TRAF3 deficiency permits BCRinduced CSR by elevating BCR-proximal signaling intensity. Furthermore, NF-kB2 is required for BCR-induced CSR in TRAF3-deficient B cells but not for CD40-induced or LPS-induced CSR, suggesting that TRAF3 restricts NF-kB2 activation to specifically limit the BCR's ability to induce CSR. TRAF3 deficiency also disrupts autoreactive B cell anergy by elevating calcium influx in response to BCR stimulation, leading to lymphoid organ disorders and autoimmune manifestations. We showed that TRAF3 deficiency-associated autoimmune phenotypes can be rectified by limiting BCR repertoires or attenuating BCR signaling strength. Thus, our studies highlight the importance of TRAF3-mediated restraint on BCR signaling strength for controlling CSR, B cell homeostasis, and B cell-mediated disorders.

BackgroundAntitumor immunity is highly heterogeneous between individuals; however, underlying mechanisms remain elusive, despite their potential to improve personalized cancer immunotherapy. Head and neck squamous cell carcinomas (HNSCCs) vary significantly in immune infiltration and therapeutic responses between patients, demanding a mouse model with appropriate heterogeneity to investigate mechanistic differences.MethodsWe developed a unique HNSCC mouse model to investigate underlying mechanisms of heterogeneous antitumor immunity. This model system may provide a better control for tumor-intrinsic and host-genetic variables, thereby uncovering the contribution of the adaptive immunity to tumor eradication. We employed single-cell T-cell receptor (TCR) sequencing coupled with single-cell RNA sequencing to identify the difference in TCR repertoire of CD8 tumor-infiltrating lymphocytes (TILs) and the unique activation states linked with different TCR clonotypes.ResultsWe discovered that genetically identical wild-type recipient mice responded heterogeneously to the same squamous cell carcinoma tumors orthotopically transplanted into the buccal mucosa. While tumors initially grew in 100% of recipients and most developed aggressive tumors, ~25% of recipients reproducibly eradicated tumors without intervention. Heterogeneous antitumor responses were dependent on CD8 T cells. Consistently, CD8 TILs in regressing tumors were significantly increased and more activated. Single-cell TCR-sequencing revealed that CD8 TILs from both growing and regressing tumors displayed evidence of clonal expansion compared with splenic controls. However, top TCR clonotypes and TCR specificity groups appear to be mutually exclusive between regressing and growing TILs. Furthermore, many TCRα/TCRβ sequences only occur in one recipient. By coupling single-cell transcriptomic analysis with unique TCR clonotypes, we found that top TCR clonotypes clustered in distinct activation states in regressing versus growing TILs. Intriguingly, the few TCR clonotypes shared between regressors and progressors differed greatly in their activation states, suggesting a more dominant influence from tumor microenvironment than TCR itself on T cell activation status.ConclusionsWe reveal that intrinsic differences in the TCR repertoire of TILs and their different transcriptional trajectories may underlie the heterogeneous antitumor immune responses in different hosts. We suggest that antitumor immune responses are highly individualized and different hosts employ different TCR specificities against the same tumors, which may have important implications for developing personalized cancer immunotherapy.

Squamous cell carcinoma (SCC) is the second commonest type of skin cancer, and SCCs make up about 90% of head and neck cancers (HNSCCs). HNSCCs harbor two frequent molecular alterations, namely, gain-of-function alterations of phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) and loss-of-function mutations of tumor protein p53 (TP53). However, it remains poorly understood whether HNSCCs harboring different genetic alterations exhibit differential immune tumor microenvironments (TME). It also remains unknown whether PIK3CA hyperactivation and TP53 deletion can lead to SCC development spontaneously. Here, we analyzed the Cancer Genome Atlas (TCGA) datasets of HNSCCs and found that patients with both PIK3CA and TP53 alterations exhibited worse survival, significantly lower CD8 tumor infiltrating lymphocytes (TILs) and higher M0 macrophages than other controls. To better model human tumorigenesis, we deleted TP53 and constitutively activated PIK3CA in mouse keratin-15-expressing stem cells, which leads to the spontaneous development of multilineage tumors including SCCs, termed Keratin-15-p53-PIK3CA (KPPA) tumors. KPPA tumors were heavily infiltrated with myeloid-derived suppressor cells (MDSCs), with a drastically increased ratio of polymorphonuclear-MDSC (PMN-MDSC) versus monocytic-MDSC (M-MDSC). CD8 TILs expressed more PD-1 and reduced their polyfunctionality. Overall, we established a genetic model to mimic human HNSCC pathogenesis, manifested with an immunosuppressive TME, which may help further elucidate immune evasion mechanisms and develop more effective immunotherapies for HNSCCs.

Background While immune checkpoint inhibitors (ICI) were approved for head and neck squamous cell carcinomas (HNSCCs), the response rate remains relatively low. Mechanisms underlying ICI unresponsiveness versus sensitivity are not fully understood. Method To better delineate differential responses to ICI treatment, we employed mouse SCC models, termed KPPA tumors that were caused by deleting p53 and hyperactivating PIK3CA, two most frequently mutated genes in human HNSCCs. We transplanted two KPPA tumor lines (TAb2 versus TCh3) into C57BL/6 recipients and examined the immune tumor microenvironment using flow cytometry. Furthermore, we employed single-cell RNA sequencing to identify the difference in tumor infiltrating lymphocytes (TILs). Results We found that different KPPA tumors exhibited heterogeneous immune profiles pre-existing treatment that dictated their sensitivity or unresponsiveness to anti-PD-L1. Unresponsive TAb2 tumors were highly enriched with functional tumor-associated macrophages (TAMs), especially M2-TAMs. In contrast, sensitive TCh3 tumors contained more CD8 TILs with better effector functions. TAb2 tumor cells drastically expanded F4/80+ TAMs from bone marrow precursors, requiring CSF1 and VEGF. Consistently, a higher combined expression of VEGF-C and CSF1 predicts worse survival in PIK3CAAmp/TP53Mutated HNSCC patients. Unresponsive TAb2 tumors upregulated distinct signaling pathways that correlate with aggressive tumor phenotypes. While anti-PD-L1 did not affect the TME of TAb2 tumors, it significantly increased the number of CD8 TILs in TCh3 tumors. Conclusions We uncovered tumor-intrinsic differences that may underlie the differential responses to ICI by establishing and employing two SCC tumor lines, TAb2 vs. TCh3, both of which harbor TP53 deletion and PIK3CA hyperactivation. Our study indicates the limitation of stratifying cancers according to their genetic alterations and suggests that evaluating HNSCC tumor-intrinsic cues along with immune profiles in the TME may help better predict ICI responses. Our experimental models may provide a platform for pinpointing tumor-intrinsic differences underlying an immunosuppressive TME in HNSCCs and for testing combined immunotherapies targeting either tumor-specific or TAM-specific players to improve ICI efficacy.