Regardless of HPV status, the presence of tumor-infiltrating lymphocytes is associated with more favorable outcomes in HNSCC [6, 7]. Studies in other types of cancers, most notably colon cancer, have followed a similar pattern: patients with high CD3 and CD8 infiltrate are associated with significantly better course of disease than those with low T-cell infiltrates [58].

Adoptive T-cell therapy (ACT) involves harvesting T cells from autologous tumor resection or biopsy, expanding them ex vivo with IL-2, testing for tumor specificity, and then rapidly expanding them for reinfusion into a non-myeloablative lympho-depleted patient. High-dose IL-2 is also administered to support the in vivo expansion of transferred T cells [59, 60]. CD8 T cells often constitute the majority of the tumor-specific T cells in culture; other cells such as CD4 T cells, NK cells, and gamma-delta T cells are also present in culture and have been shown to possess significant antitumor activity [60]. Regulatory T cells are also present in the culture, and depletion of them prior to reinfusion positively correlated with increased response to ACT [61–64].

In metastatic melanoma, tumor-specific T cells can be recovered from 35 to 70 % of patients, and the response in these patients is 50–75 % [65–67]. Notably, 95 % of the complete responders demonstrated a durable response for at least 5 years [67]. The use of ACT for patients with HNSCC has been pioneered at a few centers, with one reporting a remarkable response rate of 43 % in the seven patients studied [68]. Of note, a significant hurdle to adoptive T-cell therapy is the ability to culture and expand tumor-specific T cells from the patients’ autologous tumors. In renal, breast, and colon cancer, the recovery rate is extremely low (0–20 % [69]). Interestingly, in HNSCC the success in obtaining tumor-specific T cells from autologous tumors is around 60 %, a number similar to melanoma [70, 71]. In our center, we have successfully cultured tumor-specific T cells from a similar percentage of patients and have treated a single patient with HNSCC.

Strategies to increase the success of recovering tumor-specific T cells from tumor specimen are currently being studied. Data from our group generated using multispectral fluorescent IHC suggest that the ratio of CD8 effector T cells to FoxP3⁺ Treg is an important biomarker that predicts the success of culturing tumor-specific T cells in melanoma (Feng et al, Manuscript in revision). Our group is applying the same multispectral imaging strategy to characterize the tumor environment in HNSCC (Fig. 9.1). Modulation of the tumor environment either during culture or prior to surgery can potentially enhance this ratio, leading to greater recovery of tumor-specific T cells. Administration of 4-1BB agonist antibody during culture has been shown in patients with melanoma to increase the tumor-specific T-cell function [72], and a study evaluating the effect of neoadjuvant anti-PD-1 therapy on success of culturing tumor-specific T cells is underway.

Cytokines are molecular signaling molecules that allow intercellular communication over a long distance to generate rapid and robust immune responses in a controlled manner. The immune-mediated antitumor activities of a few cytokines have been well characterized in preclinical settings and led to many clinical trials testing the efficacy of interleukins including IL-2, IL-7, IL-12, IL-15, and IL-21, interferons, and GM-CSF. Out of these, high-dose IL-2 was approved for the treatment of metastatic melanoma and renal cell carcinoma [73], and IFN-α was approved for adjuvant therapy for stage 3 melanoma [74].

Interleukin-2 (IL-2) is a member of the IL-2-related family that functions to stimulate T-cell growth through the IL-2 receptor. IL-2 exhibits myriad effects on the immune system in addition to promoting CD4 and CD8 T-cell proliferation and differentiation into effector T cells and memory T cells. IL-2 also expands Tregs, which express high-affinity IL-2 receptors [75, 76]. This acts as a negative feedback mechanism to decrease CD8 T-cell activity through both depletion of IL-2 and inhibition by Tregs.

Systemic high-dose IL-2 plays a key role in the treatment of metastatic melanoma and renal cell carcinoma. Although limited to patients in otherwise good health due to acute toxicities, high-dose IL-2 induces objective clinical response in 15–25 % of patients with metastatic melanoma and renal cell carcinoma, with complete response around 7–12 % [77–80]. Importantly, of the patients achieving complete response, 70–90 % remains disease-free in an ongoing 20-year follow-up [78, 81].

The use of IL-2 in HNSCC has been limited to local-regional or intralesional injection with mixed results. High-dose IL-2 injected local-regionally demonstrated induction of tumor-specific T cells in the draining lymph nodes of patients with HNSCC [82]. However, perilymphatic injection of low-dose, but not high-dose, IL-2 demonstrated temporary regression of HNSCC and two complete responders in lip cancer [83–87]. The mechanism for resistance to IL-2 therapy in HNSCC is unclear. It has been suggested in melanoma that patients who fail to demonstrate significant Treg induction experienced an increased response to therapy [88]. One explanation for these observations is that the presence of abundant regulatory T cells unique to HNSCC could be rapidly expanded with IL-2 and limit the antitumor CD8 response. Combination therapy with systemic high-dose IL-2 and Treg depleting agents, such as cyclophosphamide, may be an avenue to overcome this pathway for the treatment of HNSCC.

Interferon-α (IFN-α): IFN-α is a type I interferon that is involved in the immune response to viral infections. In cancer immunotherapy, administration of IFN-α can activate CD8 T cells as well as the innate immune system, specifically NK cells, DCs, and macrophages [89, 90]. This has led to a heightened antitumor immune response in preclinical models, especially when used in combination therapy with checkpoint blockades [29]. The use of IFN-α clinically has so far been limited to adjuvant setting in high-risk melanoma where it significantly improves disease-free survival; however, the overall survival benefit seems to be limited to a very small subset of patients [74]. Currently IFN-α is being tested in solid tumors in combination with various targeted and immunotherapy agents.

The final part of this chapter will cover in brief biomarkers, a rapidly advancing area for immunotherapy. With increasing numbers of immunotherapy strategies available for the treatment of solid malignancies, including HNSCC, identification of prognostic and predictive biomarkers is paramount to stratify patients and direct therapy. Understanding these biomarkers is an active area of research, with a majority of the attention focusing on the checkpoint blockades such as anti-PD-1. Recent studies have demonstrated that response to anti-PD-1 is heavily dependent on the expression of PD-L1 in the tumor microenvironment as well as the mutational burden of the tumor itself [48, 124, 125]. These findings suggest that the response to anti-PD-1 is hinged on a preexisting antitumor immune response and that anti-PD-1 acts to free the CD8 T cells from inhibition to exert their antitumor activities. A biomarker for anti-CTLA-4 is also being researched; preliminary studies suggest that the response is dependent on FcgRIIIA (CD16)-expressing monocyte-mediated elimination of Tregs [32]. IHC is the most frequently used technique in the identification of these biomarkers. Studies using quantitative IHC have identified CD8 T-cell infiltration as an important prognostic factor in predicting outcome for patients with HNSCC [6, 7]. More recently, multiplex immunohistochemistry has emerged as a more advanced tool for the analysis of the tumor microenvironment, offering a more efficient and comprehensive analysis of the tumor microenvironment for both diagnostic and mechanistic studies [126–128]. This also allows us to explore the relationship between cell types in the peritumoral and intratumoral compartments, quantify the expression of specific biomarkers (Fig. 9.1), and examine their impact on patient prognosis and response to therapy. As research on biomarkers advances, we will hopefully be able to better direct patients, based on their tumor microenvironment and mutational status, to the immunotherapy that potentially offers the best response rate and ultimately a cure for their disease.