CD8+ Effector T Cells
After activation by antigenic and cytokine stimulation by APCs like DCs, naïve CD8
+ T cells differentiate into either memory or cytotoxic effector T cells [
80]. Cytotoxic T cells constitute a subset of T cells with the ability to recognize and kill tumor cells and therefore serve as central players within the anti-tumor response. Indeed many studies report a positive correlation of higher CD8
+ TIL with improved survival in HNSCC patients [
32,
81‐
85]. However, the effect seems to be affected by tumor subsite, relative amount to other tumor infiltrating immune cells, spatial distribution, and HPV status.
In OCSCC, a prognostic benefit of high CD8
+ TIL has been described [
85‐
89,
90•], but the majority of reports show no significant association of high CD8
+ TIL with OS or DSS [
56,
91‐
96]. This lack of consistency might in part be explained by the relative amounts of CD8
+ TILs to other TILs affecting their function. For instance, a high CD8
+/CD4
+ ratio confers superior DSS and DFS [
97], and a low CD8
+/forkhead box protein 3 (FoxP3) ratio confers inferior OS and DFS [
98,
99]. Interestingly, two recent meta-analyses on the role of CD8
+ T cells in OCSCC showed contradictory results. This could be due to the low amount of studies that qualified for meta-analysis, especially by Hadler-Olsen et al. [
100,
101].
In OPSCC, the prognostic role of CD8
+ TILs seems more pronounced with a substantial amount of papers reporting a positive effect on survival of high CD8
+ TIL both stromal and intratumoral [
59,
85,
102‐
108]. HPV-related OPSCC show a significantly higher infiltration of CD8
+ T cells [
12•,
15,
70,
74,
82,
85,
104‐
106]. In fact, it has been reported that HPV-related OPSCC patients with a low CD8
+ TIL count do not show the typical improved survival associated with HPV-related OPSCC [
109]. This could point to a role for the local immune response in the survival of HPV-related OPSCC patients. Not all studies were able to find this association, however [
110]. Although investigated to a lesser extent, high CD8
+ TIL counts have been associated with survival benefit in laryngeal squamous cell carcinoma [
85,
111,
112].
CD4+ T Cells
CD4
+ T cells represent a heterogeneous cell type which can be sub-classified into various subtypes including Th1, Th2, Th9, Th17, follicular helper T cells (Tfh), and Tregs [
113]. This hampers the evaluation of their role in HNSCC as observed effects could be attributable to any of these subtypes, and staining for CD4 alone might not be sufficient. This could explain the ambiguous role of CD4
+ T cells in HNSCC: some papers report a favorable prognostic effect of higher CD4
+ TIL [
12•,
85,
114], while others report the opposite [
88,
115]. However, no significant association between CD4
+ TIL and survival has been reported most frequently [
81,
86,
91,
94,
96,
102]. Although in their meta-analyses, de Ruiter et al. did find a prognostic benefit of higher CD4
+ T cell infiltration in HNSCC, the authors stress to interpret this with caution given the paucity of papers eligible for analyses and a high suspicion of publication bias [
84]. In two recent meta-analyses for OCSCC specifically, Huang et al. report no prognostic value [
100], whereas Hadler-Olsen et al. found data to be insufficient for analyses [
101]. Recently, Cillo et al. [
116•] reported on scRNAseq comparing HPV-related and HPV-unrelated HNSCC, and showed that HPV-related tumors are enriched in the presence of a Tfh cell population when compared with HPV-unrelated tumors. In TCGA data, a Tfh-high profile corresponded with improved survival [
116•]. It must be mentioned that while the majority of HPV-related tumors in this study were OPSCC, the HPV-unrelated tumors were a mix of OCSCC, OPSCC, larynx, and hypopharynx. In all, the role of CD4
+ T cells in HNSCC remains to be clarified. Investigation of CD4
+ T cell subpopulations at various HNSCC subsites specifically is warranted.
Regulatory T Cells
Tregs are a subpopulation of CD4
+ T cells which can be identified by the expression of the transcription factor FoxP3 [
113]. They play a critical role in maintaining host-tolerance and thus preventing auto-immunity, by regulating other immune cells including DCs, NK cells, B-cells, CD4
+ and CD8
+ T cells, but in doing so also facilitate tumor immune escape and tumor progression [
117]. Indeed a large meta-analysis on the prognostic value of FoxP3 showed that high Treg infiltration is associated with inferior OS in several tumor types, but not in HNSCC [
118]. In fact, when considering HNSCC in general, higher CD4
+FoxP3
+ T cell infiltration correlates with better survival [
30,
119,
120]. This does however not seem to apply to all HNSCC subsites as for OCSCC most studies do report lower CD4
+FoxP3
+ TIL counts to be related with improved survival [
121‐
124]. Only one study found an association in the opposite direction [
97], but most, including two meta-analyses, failed to show a significant correlation of FoxP3 with survival in OCSCC [
89,
92,
100,
101,
125].
Echarti et al. recently reported high stromal numbers of Tregs to be correlated with improved survival in HNSCC, and epithelial numbers of Tregs to only gain prognostic importance when stratifying for “inflamed”, “immune excluded”, and “immune desert” tumors based on total number of infiltrating CD8
+ T cells [
126]. This might in fact represent the CD8
+/FoxP3
+ ratio, which confers survival benefit when lower in OCSCC, as published previously by Chen et al. and more recently by Ni et al. [
99,
124]. Interestingly, the intracellular localization of FoxP3 might play a role, as a shift in the subcellular localization of FoxP3 expression from cytoplasmic to nuclear has been described after TCR/CD28 activation [
127,
128]. This indicates that a subgroup of FoxP3
+ T cells might in fact have an effector function. Weed et al. have found that in OCSCC, cytoplasmic localization of FoxP3 was associated with lower risk of recurrence, as opposed to nuclear localization [
129]. In addition, Feng et al. report the spatial relation of FoxP3
+ T cells to CD8
+ T cells to affect OS in OCSCC. In their study using multispectral imaging on 119 HPV-unrelated OCSCC, they showed that a higher density of FoxP3
+ T cells within 30 μm of CD8
+ T cells was significantly associated with worse OS [
90•].
In OPSCC, a positive correlation of higher CD4
+FoxP3
+ T cell infiltration with improved survival is reported [
59,
85,
120,
130,
131], but a significant association with poor survival has also been described [
132]. When considering HPV, it seems that papers reporting significantly higher CD4
+FoxP3
+ infiltration in HPV-related OPSCC [
103,
130,
131,
133] are in balance with reports that do not find such relation [
104,
106,
120,
134,
135]. In their study, Ward et al. reported higher absolute counts of FoxP3
+ T cells in HPV-related OPSCC and correlated this to improved survival [
109]. However, the authors state the observed absolute increase could merely be a reflection of higher overall T cell infiltration in HPV-related OPSCC, since relative amounts of FoxP3 did not differ significantly. One other study described such correlation [
131]. In their meta-analysis, de Ruiter et al. were unable to analyze HPV-related tumors due to insufficient data [
84].
B Lymphocytes
The composition and prognostic role of B-cells in the TME of HNSCC is relatively understudied. As part of the adaptive immune system, B lymphocytes play an important role in the immune response following the onset of malignant tumors [
136]. Mature B cells can produce antibodies which could bind to tumor cells and induce ADCC by NK cells or Fc-receptor mediated phagocytosis by macrophages, they can also act as APCs or can directly interact with CD4
+ T cells (through CD40/CD40L) or CD8
+ T cells (through CD27/CD70), thereby, providing help to the anti-tumor immune response [
137]. In addition to these anti-tumor features, regulatory B-cells (Bregs) are thought to stimulate tumor growth by negatively interacting with other immune cells as well as with tumor cells. It is assumed that Bregs inhibit NK cell- and cytotoxic T cell–mediated tumor immunity, suppress the differentiation of Th1/Th17 cells, and promote the alteration of T-helper cells and macrophages towards the Th2/M2 types. Moreover, Bregs might disable the cellular immune response against the tumor by promoting the conversion of CD4
+/CD25
− T lymphocytes to FoxP3
+ Tregs, thereby, enabling tumor growth and the development of metastasis. Bregs secrete IL-6, IL-10, IL-35, and TGF-β, all known for their suppressive effect on the immune response [
138].
HPV-related HNSCC were shown to contain increased percentages of tumor-infiltrating CD19
+/CD20
+ B lymphocytes compared with HPV-unrelated HNSCC and non-cancerous mucosa [
17•,
70,
74,
139•,
140,
141]. When performing RNA-sequencing, it was even shown that phenotypic differences exist between B lymphocytes in HPV-related and HPV-unrelated HNSCC [
140]. Hladíková et al. found that in HPV-related OPSCCs, the B cell population within the TME seems to be represented mainly by memory B lymphocytes with an activated, antigen-experienced phenotype, characterized by high expression of CD27, no expression of IgD and low expression of IgM [
141]. When they subsequently divided the HPV-related OPSCCs into two groups based on the amount of B cell infiltrate (B
low (B cell proportions < 0.5% of total cells) versus B
hi tumors), they noticed that in the B
hi group the tumor-infiltrating B cells displayed significantly higher levels of activation markers like HLA-ABC, HLA-DR, CD86, and CD40 when compared with the B
lo group, corresponding with an activated phenotype. Moreover, B
hi OPSCCs possessed higher proportions of proliferating Ki-67
+ B cells than the B
low counterparts [
141].
The prognostic impact of B lymphocytes in the TME of HNSCC remains somewhat ambiguous. While most studies report a favorable effect of B lymphocytes on HNSCC patient survival [
17,
141‐
143], some studies fail to find any effect on survival [
70] or even claim that certain subsets of B cells, particularly Bregs, may contribute to an immunosuppressive, cancer promoting microenvironment [
144]. Distel et al. demonstrated that the prognostic impact of CD20
+ B cells may be dependent on the type of treatment and stage of disease [
145]. In their study, they investigated 115 patients with oro- or hypopharyngeal squamous cell carcinoma by dividing them in two groups: a low-risk group (
n = 62) of early stage disease treated with primary surgery and postoperative radiotherapy, and a high-risk group (
n = 53) of inoperable, advanced stage disease treated with definitive chemoradiotherapy. Immunohistochemistry for CD3, CD4, CD8, CD20, CD68, FoxP3, and granzyme B was performed on all pretreatment biopsies and percentages of the various subsets of TIL were correlated with survival. Surprisingly, it was observed that in the low-risk group higher numbers of CD20
+ B cells were consistently associated with improved locoregional tumor control (
p = 0.02), while it was negatively associated with prognosis in the high-risk group (
p = 0.04). Of note, B cells have the ability to form tertiary lymphoid structures (TLS) when they aggregate in a network of follicular DCs surrounded by T cells and high-endothelial venules [
146]. In OCSCC, TLS are more frequently seen in stage I–II than in stage III–IV disease, and the presence of TLS is associated with improved survival [
147]. In their scRNAseq study, Cillo et al. reported an increased presence of germinal center B cells in HPV-related versus HPV-unrelated HNSCC, corresponding with the presence of TLS [
116•].