The results will be discussed for each paper separately.
Paper I
In Paper I we identified several transcriptomically distinct populations of MFs, DCs and LCs, which will be discussed below.
It is inherently challenging to define a catalogue of cells based on single-cell genomics data. While a cell subset is defined as “A discrete cellular compartment that arises from a distinct progenitor along a unique differentiation programme to carry out a distinct function” (70), this may encompass different transcriptional cell states acquired upon environmental tissue stimuli. However, to assess which of the APC subsets revealed in Paper I represent true subsets, further studies on fate mapping and lineage tracing are required. For simplicity, the term “subsets” will be used for all detected APC populations in the following discussion. To avoid confusion in comparison with future scRNAseq studies, we did not name all DC clusters by number. For instance, the three clusters of DC3s identified were not given separate names (DC4, DC5, etc).
Macrophages
In this analysis, we identified three different subsets of MFs. While most previous studies on oral mucosa MFs have used the M1-M2 classification, we used the terms transient monocyte-like MFs, for reasons described in the introduction.
In healthy human oral mucosa, we identified three transcriptionally distinct MF subsets based on C1Q+, CD14+, CD163+ transcription, and we named them oMF1, oMF2 and oMF3, respectively (Figure 2B in Paper I). oMF1 was enriched for transcripts associated with tissue resident MFs (TRMs) (CD163, C1Qs and LYVE1). oMF3 showed high transcription of calprotectin (heterodimer of S100A8 and -A9) which is associated with transient monocyte-like MFs. oMF2 had an intermediate TRM-phenotype (54). This suggests a model where monocytes are constantly recruited from the blood stream and eventually differentiate into- and contribute to the population of TRMs. Further analysis of sorted MFs from a larger starting material of sorted oMFs are likely to reveal further subsets with phenotypes intermediate of transient monocyte-like MFs and TRMs as reported for the intestine (50, 54). Future trajectory analyses may aid in corroborating this model, and their turnover and replacement kinetics may be assessed using biopsies from bone marrow transplanted patients.
We found that the TRM-like oMF1 expressed high levels of TGFB, IDO and IL10 compared with MF2 and -3. This is in line with the reported “M2-like phenotype” of TRMs in homeostasis. In contrast, the transient monocyte-like oMF3 showed a more proinflammatory “M1”-phenotype with higher relative expression of TNF, IL23 and IL1B (data not shown). In line with this, oMF3 showed to be enriched for GO terms like neutrophil activation and recruitment, MF activation, and cytokine- and chemokine production, in line with their transcriptomic profile. However, oMF3 showed to be enriched for terms associated with wound healing, a classical M2-feature. The oMF2 represented an S100A8/A9lo phenotype indicating some time spent in the tissue, and displayed transcripts of genes involved in host defense, for instance the inflammation-triggering receptor TREM1. In contrast to for oMF3, GO terms associated with antigen presentation were displayed by oMF2, indicating a Th-cell interacting potential of this subset. However, the functional capacities of the different oMF subsets should be corroborated in future studies of sorted oMFs.
Niche-specific imprinting by the local tissue is thought to underpin the phenotype of TRMS in different parts of the body. Our results show several potential ligand-receptor pairs of transient monocyte derived MFs and the intermediate MF subsets with the epithelium, although none of them were selective for MF subsets. This indicates similarity of the niche-imprinting signals that that monocytes and cDCs receive from the oral epithelium. Since oMF1 (cluster 0) was located separately from the other MF subsets, it was not included in the CellPhone analysis in Paper I. Given their main location in the subepithelial niche, future studies should aim at detecting ligand-receptor pairs with oMF subsets and stromal cells like fibroblasts, endothelial and lymphatic cells to in order to reveal potential oral mucosal-specific imprinting interactions. Moreover, micro-anatomical niches within organs can give rise to different phenotypes of TRMs, and it is therefore possible that different anatomical niches of the oral mucosa harbor TRMs with different niche-specific phenotypes (260).
Dutzan et al. have previously presented a characterization of the immune network in human gingiva and buccal mucosa using flow cytometry (9). They reported that the number of leukocytes in buccal mucosa was lower than in gingiva. However, the fraction of the DC-Mac among leukocyte populations appeared to be enriched in buccal mucosa as compared with gingiva. The APC compartment of the buccal mucosa was not characterized further. In healthy gingiva, the majority of APCs were CD14+, including HLADR+CD14+AF+-resident MFs and HLADR+CD14+AF− migratory monocytes. CD14+ cDC3s identified in Paper I may be present within the CD14+AF+ population reported in gingiva by Dutzan et
al., indicating a possible overestimation of the number of MFs. Care must be taken not to include DC3 in this gate in future flow cytometric analyses of oral mucosa.
Dendritic cells
In Paper I, we detected 7 transcriptomically distinct DC-like clusters with one cluster of cDC1-, two clusters of cDC2-, three subsets of cDC3-like cells. In addition, we identified one cluster with a transcriptomic profile compatible with the recently characterized mregDC cell state (47). pDCs were not identified.
cDC1
By scRNAseq analysis we detected a small, but transcriptomically distinct population of XCR1+ Clec9a+ cDC1s. In our flow cytometry analyses in Paper I, we also identified a minor population of CD1cloCD14- cells within the CD45+HLA-DR+ CD207- population, compatible with cDC1s.
This is in line with previous studies from other peripheral tissues (59), and with the study of Dutzan et al. that identified a minor population of CD141+ DC1s in human healthy gingiva (9). scRNAseq is well suited for analyzing rare cell populations (261), and our identification and characterization of this subset in normal buccal mucosa brings novel information about their transcriptomic profile and their potential crosstalk with epithelial subsets
cDC1s exhibit a high capacity for antigen cross-presentation, and may thus play a role in anti-viral defense at this site. Based on previous reports, it was natural to speculate that cDC1s in steady state have a tolerogenic function. However, this was not readily supported by their cytokine profile showing low expression of both pro-and anti-inflammatory cytokines (not shown). However, their suggested tolerogenic functions could lie in stimulation of other cells. In fact, recent data indicate that mucosal cDC1s may provide cross-tolerance to epithelial antigens by induction of regulatory CD8+ T cells (262).
The CADM1:CADM3 interactions we observed between cDC1s and melanocytes make it tempting to speculate that there is a cell-to cell contact between these cells (263). This could be assessed by in situ IHC/IF staining of human oral mucosa biopsies.
Little is known about the role of cDC1s in oral diseases. However, Wang et al. reported that the number of CD141+ Clec9a+ DCs were not significantly increased in OLP lesions, in contrast to CD1c+ DCs and pDC (264). As mentioned above, cDC1s may also provide cross-tolerance to epithelial antigens by induction of regulatory CD8+ T cells (262). Thus, such cells may be crucial to prevent autoreactive inflammation in the oral mucosa, and should be investigated further to understand and possibly treat autoimmune pathology in the oral mucosa.
A recent study suggests that cDC1s increase the density and shape the repertoire of tumor-infiltrating lymphocytes essential for overcoming the PD-1/PDL-1 resistance in tumors (265). Being a major challenge in OSCC treatment, the potential for cDC1s in overcoming PD-1/PDL-1 resistance should be explored in future studies (183).
cDC2
In our scRNAseq analysis we detected two clusters of CD1c+IRF4+Clec10a+FcER1+CD207-CD14- cDC2s.
The total population of cDC2s were larger than that of cDC1s. This is in line with two previous studies of cDC2s in healthy oral mucosa detected by flow cytometry, and with reports of CD1c+ DC2s being the major population of human cDCs in blood and peripheral tissues (9, 59, 264).
The wide range of reported functional capacities for cDC2s, may in part stem from the increasingly accepted heterogeneity within the classical cDC2 compartment (70). Our detection of two discrete cDC2 clusters in the oral mucosa, can serve as a baseline for further studies of oral cDC2 function.
Interestingly, however, we found that oral mucosa cDC2s expressed low levels of CCR7, indicating low migration, in contrast to what is reported previously. One explanation for this is acquisition of the mregDC state for most activated cDC2s in steady state, as discussed below. Future studies are needed to assess whether classical cDC2 clusters show higher degree of migration upon stimulation of pathogens, or in the case of auto-inflammation.
Peripheral tissue cDC2s are known to express low levels of langerin. Thus, the distinction between cDC2s and LCs in single cell-techniques can be challenging. In Paper I, we found a gradual decrease of langerin among the cDC1c+ DC clusters, and in situ assessment of the cells in tissue sections is needed to confirm the localization of the cDC2 clusters to the subepithelial region. scRNAseq analyses of separate oral epithelium and lamina propria will help draw better conclusions regarding the transcriptomic differences been langerin+ cDC2s and LCs.
DC3
In our scRNAseq analysis we detected three transcriptomically distinct clusters of DC3-like cells in healthy oral mucosa. CD5+CD14+CD163+ DC3s have recently been accepted as a distinct subset, since they have been shown to display a developmental origin distinct from cDC1 and cDC2, which differentiate along a common trajectory. Still, little is known about the functions of DC3s, but they have been reported to expand in various inflammatory settings, and have also shown to have pronounced capability to activate tissue-homing T cells. Since cDC3s were detected within in the classical cDC2 compartment, the mixture of these subsets may explain some of the vast diverse functions reported for cDC2s. However, the DC2 compartment has been reported to be heterogeneous before the identification of the DC3 subset. For instance monocyte-like DC2s reported by Yin et al, likely to represent DC3s, are less active in proliferation assays compared with “DC-like” DC2s and produce mainly Th1 cells (266).
In Paper I, we detected three transcriptomically distinct clusters of DC3-like cells in healthy oral mucosa. This fits with the assumption of Ginhoux et al, of a heterogeneous DC3 compartment, with several cell states (70). More work is needed to fully map the DC3 lineage, and to understand its ontogeny and differentiation program in steady state and during inflammation, both in blood and in peripheral tissues including the oral mucosa. Moreover, the relationship between DC3 and the cDC- and monocytic lineages remains to be assessed. Since DC3 is reported to expand in inflammatory settings, future efforts should be made in order to assess the role of DC3s in different chronic inflammatory oral diseases.
Paper II
Origin of langerin+ cells in NOM
In Paper II, we found that a notable fraction of LCs in the oral epithelium of NOM expressed the proliferation marker Ki-67, indicating that human oral mucosal LCs have the capacity for local self-renewal. However, approximately 50 % of the LCs in NOM expressed CD14, which is compatible with a monocyte origin. This suggests a model in which the oral epithelium harbors a population of self-renewing LCs that are integrated within a dynamic LC pool derived from hematopoietic cells (23), which largely displays a monocyte origin. Different trajectory analysis programs for scRNAseq data exist that enable the study of dynamic changes in gene expression (267). This type of analysis is a natural first step to seek in vivo corroboration of human oLC differentiation. Furthermore, by exploiting the XY
chimerism of cells after bone marrow transplantation in sex-mismatched host and donors, one can assess whether peripheral tissue immune cells are replaced by hematopoietic precursors or self-renew locally in the tissue. For instance, T cells are found to self-renew for years in the human gut (50).
Moreover, studies of LC turnover after limb transplants have shown that skin LCs can self-renew for decades (268). Future studies of bone marrow transplanted individuals could gain valuable insight of human oLC kinetics.
Origin of langerin-expressing cells in oral lichen planus
In the epithelium of OLP, we found a twofold fraction of LCs as compared with NOM. Although the fraction of proliferating LCs was reduced in OLP, the density of Ki-67+ LCs was not different from that seen in NOM. In contrast to NOM, the LCs in OLP lesions were mostly negative for CD14, but a significant fraction expressed the transcription factor IRF4. This indicates that the increased LC numbers in OLP are due to an increased influx of precursor cells with a cDC2 phenotype, that mature to LCs locally. The lack of expression of CD14 on these LCs make it tempting to speculate that these LCs have a oLC1 phenotype. In skin, LC1s are reported to play a more pro-inflammatory potential than the more regulatory LC2s, the latter suggested playing an immunoregulatory counter balance role in psoriatic skin (118). Based on this, it is tempting to hypothesize an influx of oLC1 with a pro-inflammatory role in OLP. A deeper characterization of the oLC subsets in OLP with the use of scRNAseq and flow cytometry, would help to untangle proportions of oLC subsets in OLP epithelium, and provide valuable information of their cytokine profile.
In the lamina propria of OLP, a massive number of langerin+ APCs was detected. The lack of classical MF markers together with IRF4 expression in the majority of these cells indicated a cDC2-origin. cDC2s are reported to be highly flexible cells, with the capacity to promote a wide range of T cell responses, including Th1, Th17 cells and Tregs (23), and human cDC2s may also provide antigen cross-presentation to CD8 T cells (99). The function of the langerin+ cDC2s in OLP needs to be assessed in future studies.
However, the langerin+ cDC2s in OLP may be critical for Th17 activation, a suggested driver of OLP pathogenesis (144). Still, cDC2s are also reported to activate other T cell subsets, like regulatory T cells, and the possibility of a tolerogenic role, with an attempt to dampen the inflammation, remains.
Importantly, given the heterogeneity within the cDC2 compartment, as discussed for Paper I, previous studies on cDC2 function should be interpreted with caution. Monocyte-like cDC2s (now likely to be classified as DC3s), have been reported to produce mainly Th1 cells in proliferation assays (266), suggesting that the role of cDC2 in Th1 activation may be overestimated. Th1 cells have also been suggested a role in OLP, and it would be interesting to assess whether a fraction of the numerous langerin- APCs expressing classical MF markers like CD14/CD68 and calprotectin are DC3s. Given their reported pronounced capacity to activate tissue-homing T cells, DC3s might also possess activities of relevance for the pathogenesis of OLP (142).
LCs versus langerin+ DC2s
It is demonstrated that dermal langerin+ cDC2s originate independently of epidermal LCs (269, 270).
The fraction of langerin+ DC2s and Langerhans cells in transit is not determined in Paper II.
It is possible that a fraction of lamina propria langerin+ DC2s are Langerhans cells in transit to the regional lymph nodes. However, we consider it unlikely that the massive number of langerin+ DCs in the inflammatory infiltrate of OLP are LCs in transit, especially since the LCs are not depleted from the epithelium. Moreover, it is demonstrated that dermal langerin+ DCs originate and perform their functions independently of LCs (269). Thus, it is tempting to assume that the majority of langerin+ DCs are a result of influx of cDC2s from the blood stream, that upregulate langerin in response to signals locally in the tissue. Santorini et al. reported that the majority of DC-LAMP+ cells in the inflammatory infiltrate of OLP co-expressed CCR7, indicating efflux of DCs from the lamina propria (8). In leukocytes, DC-LAMP is confined to mature dendritic cells (271). Analysis of the expression profile of DC-LAMP for APC clusters from scRNAseq data from Paper I showed that cluster 15 (mregDCs) was the only cluster showing high levels of expression of this marker (Figure 8). This may indicate that the majority of the CCR7+ DC-LAMP+ cells reported by Santorini et al., are mregDCs. However, the fraction of langerin+ cells that coexpress CCR7 should be assessed in future studies.
Figure 8: The expression LAMP 3 in the oAPC clusters revealed with scRNAseq data (Paper I).
Whether the langerin+ cDC2s found in the lamina propria of OLP could be LC precursors remains to be assessed. However, in mouse, oral mucosa LC precursors upregulate both langerin and EPCAM in a TGF-β-dependent manner after translocation into the epithelium (122) making it tempting to assume that langerin+ cDC2s are not more likely to be LC precursors than langerin- cDC2s. However, our CellPhone analysis in Paper II did not reveal the TGFB: ALK5 interaction between epithelial cells and APCs highlighting the possibility that the mechanisms of human oLC differentiation may differ from mice.
Paper III
In Paper III, we found a trend for a favorable 5-year disease-specific survival for patients with high KLF4 expression as compared with those with low expression. In line with previous studies, cases of OTSCC with a marked/moderate inflammation in the tumor stroma had a significantly better 5-year disease-specific survival as compared with patients with slight or no inflammation (201). Here, the 5-year disease-specific survival rate was 68 %. However, the combination of high levels of KLF4 expression and high inflammation score identified a subgroup of patients with a significantly better 5-year disease-specific survival as compared with the rest of the OTSCC patient group. Here, 88 % of the patients were alive 5 years after diagnosis, compared with 68% for the separate inclusion of inflammation.
Furthermore, this combination was found to be an independent prognostic marker in contrast to clinical stage, T-stage, and N-stage in a Cox multivariate analysis of 5-year disease-specific survival. This highlights that combining several biomarkers and other variables can provide more accurate estimates of prognosis.
In two external KLF4 mRNA datasets (The Cancer Genome Atlas and The Genotype-Tissue Expression Portal), lower KLF4 mRNA expression was found in OSCC and head and neck squamous cell carcinomas (HNSCC) than in control oral epithelium. These data support our findings of low KLF4 expression in several OTSCC.
This suggests a model where KLF4, expressed in normal oral keratinocytes, is downregulated in OTSCC.
In support of KLF4 expression normal oral epithelial cells, as reported by others (217) ,we found an intermediate expression of KLF4 epithelial cells compared with other main cell types when analyzing the scRNAseq data from Paper I (Figure 9).
Figure 9. Expression of KLF4 in scRNAseq data of the human oral mucosa (data from Paper I). The numbers on the x-axis refer to cluster identity. Ep, Epithelium; End, Endothelium; Imm, Immune cells; SM, Smooth muscle cells; Fib, Fibroblasts.
In Paper III, we found that, following a 24 h exposure to IFN-γ, nuclear expression of KLF4 was increased in intensity and quantity in two different OSCC cell lines. This is in line with a previous study identifying KLF4 as a downstream target of IFN- γ in colon carcinoma cells, and that KLF4 mRNA expression was increased in such cells after stimulation with IFN-γ in vitro (272).
IFN-γ have been reported to inhibit cell growth and induce apoptosis in tumor cells. In addition, IFN-γ may also inhibit angiogenesis in tumor tissue and stimulate activity of M1 pro-inflammatory MFs to overcome tumor progression. IFN-γ is therefore considered to be a potentially useful in adjuvant immunotherapy for different types of cancer (273). However, IFN-γ can also have pro-tumorigenic actions, for instance by stimulating the synthesis of immune checkpoint inhibitory molecules and indoleamine-2,3-dioxygenase (IDO) (274-276). Thus, IFN-γ may orchestrate both pro-tumorigenic and antitumor immunity, likely in a context-dependent manner. Our results point to KLF4 as a potential contributor in this context in OTSCC.
The possible role of IFN-γ on KLF4 expression in OTSCC tumor cells in vivo, remains to be assessed.
Moreover, the molecular mechanisms of IFN-γ-induced KLF4 expression in OTSCC, the sources of IFN-
γ in OTSCCs as well as the prognostic role of this cytokine, independently and in combination with KLF4 need to be examined in order to better understand this interplay in OTSCC.
In this study, we did not explore the composition of the inflammatory infiltrate, and thus, which subsets of immune cells that are present in the OTSCC tumor-associated infiltrates remains to be assessed. Based on evaluation of cell morphology in IHC sections, the majority of immune cells appeared to be lymphocytes. This observation fits with the reports that tumor-associated lymphocytes often are associated with better prognosis (201, 202, 277). However, different subsets of lymphocytes exist, and Foxp3+ regulatory T cells (Tregs) can have pro-tumorigenic actions, partly by suppressing the actions of cytotoxic lymphocytes (278). Thus, it would have been interesting to assess the fraction of different lymphocyte subsets in the infiltrates of OTSCC and the possible role of different subset dominance in OTSCC prognostication. Moreover, future studies should aim at exploring the communication network between tumor cells and immune cells, as well as other cells in the TME.
CellPhone analysis could show a valuable map of possible interactions that can be explored by functional studies.