J. Öhman, Department of Oral Medicine and Pathology, Institute of Odontology, The Sahlgrenska Academy, University of Gothenburg, Box 450, SE405 30 Gothenburg, Sweden. E-mail: email@example.com
Leukoplakias (LPLs) are lesions in the oral mucosa that may develop into oral squamous cell carcinoma (OSCC). The objective of this study was to assess presence and distribution of dendritic Langerhans cells (LCs) and T cells in patients with LPLs with or without cell dysplasia and in oral squamous cell carcinoma (OSCC). Biopsy specimens from patients with leukoplakias (LPLs) with or without dysplasia and oral squamous cell carcinoma (OSCC) were immunostained with antibodies against CD1a, Langerin, CD3, CD4, CD8 and Ki67, followed by quantitative analysis. Analyses of epithelium and connective tissue revealed a significantly higher number of CD1a + LCs in LPLs with dysplasia compared with LPLs without dysplasia. Presence of Langerin + LCs in epithelium did not differ significantly between LPLs either with or without dysplasia and OSCC. T cells were found in significantly increased numbers in LPLs with dysplasia and OSCC. The number of CD4+ cells did not differ significantly between LPLs with and without dysplasia, but a significant increase was detected when comparing LPLs with dysplasia with OSCC. CD8+ cells were significantly more abundant in OSCC and LPLs with dysplasia compared with LPLs without dysplasia. Proliferating cells (Ki67+) were significantly more abundant in OSCC compared to LPLs with dysplasia. Confocal laser scanning microscopy revealed colocalization of LCs and T cells in LPLs with dysplasia and in OSCC. LCs and T cells are more numerous in tissue compartments with dysplastic epithelial cells and dramatically increase in OSCC. This indicates an ongoing immune response against cells with dysplasia.
Although oral leukoplakias (LPL) have been a challenge to the scientific community for several decades, the cause of LPLs is not fully understood. Genetic alterations in keratinocytes in epithelium are involved , and it is well established that LPLs are lesions that may develop into oral squamous cell carcinoma (OSCC; Fig. 1) [2–5].
The immune system has an important role in protection against cancer, which was shown in the beginning of the 1970s when Noone and co-workers reported that prognosis of OSCC improved when T lymphocytes were present within tumour tissue . The cancer immunosurveillance hypothesis postulates that the immune system can recognize cancer precursor cells and usually destroy them . However, the recently introduced concept of cancer immunoediting is pointing at a dynamic interaction between the immune system and dysplastic cells/tumour cells, with phases of elimination, equilibrium and escape [8, 9].
Dendritic cells (DCs) are potent antigen-presenting cells with a key role in evoking a T cell response . Dendritic Langerhans cells (LCs) are a subset of DCs responsible for immunosurveillance in mucosal linings and skin . Following antigen uptake, DCs migrate to regional lymph nodes and interact with T cells . The interplay between DCs and T cells is crucial in selecting the type and direction of an immune response .
DCs have the capacity to generate effector T cells with the capacity to kill tumour cells . DCs engulf, process and present tumour-associated antigens (TAAs) to naïve or memory T cells in context of major histocompatibility complex (MHC) class I–dependent pathway [11, 13–15]. MHC class I–restricted presentation of TAAs by DCs to CD8-positive T cells generates tumour-specific cytolytic effector T cells that have the capacity to recognize and eradicate tumour cells [16, 17]. Thus, interplay between DCs and T cells is important in mounting an immune response to tumour cells.
Sakakura et al.  have reported that patients with head–neck cancers have a decreased number of DCs in peripheral blood and that the number of DCs is inversely correlated to the number of regulatory T cells (Tregs). It has also been shown that an increased number of Tregs in tumours results in increased tumour growth rate . Conversely, increased numbers of peri- and intratumoral DCs improve prognosis in patients with malignant melanomas .
Several experimental and clinical studies have addressed immune response in already established tumour diseases (reviewed in ). However, the role of immune response in premalignant disorders is not so well elucidated . Based on the new concept of cancer immunoediting [8, 21], it is possible that the immune system at an early phase of cell dysplasia or early tumorogenesis has the capacity to eliminate cells with DNA damage which have the potential to undergo malignant transformation.
We hypothesize that the immune system senses early dysplastic cell transformation in LPLs and mounts an adequate response to this transformation. Thus, the aim of this study was to compare the presence and distribution of dendritic Langerhans cells and T cells in oral leukoplakias with or without cell dysplasia and relate that to the state in OSCC.
Patients and methods
Patients. Biopsy specimens from a medium-sized group of 35 patients were retrieved from the archives of the Department of Oral Medicine and Pathology with clinical diagnoses of leukoplakia (LPL; n = 23) or squamous cell carcinoma (OSCC; n = 12). Histopathological diagnosis of biopsies of patients with LPLs revealed that eight patients were diagnosed with benign hyperkeratosis (BHK), and 15 patients had hyperkeratosis with dysplasia (HKD). Subsequent clinical diagnoses were set to LPLs either with or without dysplasia. None of the biopsies had signs of fungal infection. Patient characteristics are described in Table 1.
Table 1. Patient characteristics.
Leukoplakias without dysplasia
Leukoplakias with dysplasia
Oral squamous cell carcinomas
No. of patients (female/male)
Age in years: median (range)
Lateral border of the tongue
Floor of the mouth
Degree of dysplasia
Degree of differentiation
The same experienced oral pathologist examined all biopsies from patients with leukoplakias, while biopsies from patients with OSCC were examined and diagnosed by at least two experienced pathologists.
The Ethics Committee at the Sahlgrenska Academy, University of Gothenburg, approved the study, and all patients signed an informed consent before they were included in the study.
Immunohistochemistry. Sections from the paraffin-embedded biopsies were obtained and immune stainings were carried out according to a previous protocol , to describe presence and phenotype of cells in tissue specimens.
Antibodies: The following antibodies to CD1a (Clone 010; Dako A/S, Glostrup, Denmark), CD207/Langerin (12D6 Abcam, Cambridge, UK), CD3 (LN10; Novocastra, Newcastle, UK), CD4 (1F6; Novocastra), CD8 (4B11; Novocastra), Ki67 (MIB-1; Dako A/S) were used.
Immunohistochemistry: Paraffin sections (4 μm) were cut consecutively from each specimen and mounted on electrostatically precharged slides (Superfrost Plus; Menzel-Gläzer, Frankfurt, Germany). The sections were deparaffinized and rehydrated. Retrieval of antigens was carried out with TRIS/EDTA pH 9.0 followed by microwave treatment for 20 min. After cooling at room temperature (RT) and phosphate-buffered saline (PBS) rinsing, immunohistochemistry was performed using a TechMate Horizon Autostainer (DAKO A/S). Sections were incubated with primary antibody for 25 min at RT. Endogenous peroxidase was blocked by ChemMate Peroxidase-Blocking Solution (S 2023; Dako A/S) according to the manufacturer’s instructions. All subsequent washings were performed in ChemMate Buffer kit (K5006; Dako A/S). Secondary antibodies were included in the ChemMate DAKO EnVision Detection Kit, Peroxidase/DAB, Rabbit/Mouse (K5007; Dako A/S), where sections were incubated for 25 min in RT and stainings were developed with peroxidase/diaminobenzidine. Counterstaining was carried out with ChemMate Haematoxylin S2020 (Dako A/S), which was followed by washing and dehydration in ethanol/xylene. Mountex (Histolab AB, Gothenburg, Sweden) was used to permanently mount slides. Sections from tonsils served as positive controls, while omission of primary antibodies served as negative controls.
Quantitative analysis was performed on two sections per biopsy. Digitalized images were obtained from a light microscope (Leitz Wetzler, Leica Microsystems, Wetzlar, Germany) with a Leica DC100 camera (Leica Microsystems) from four to six high-power fields (HPF, ×125). The sections were then analysed with computer software BioPix iQ 2.0 (BioPix, Gothenburg, Sweden). Within the epithelium and connective tissue, positively stained nucleated cells were counted. Results are expressed as the number of positively stained cells/mm2.
Confocal laser scanning microscopy. Antibodies: The following primary antibodies to CD1a (Clone 010; Dako A/S), CD3 (SP7; Abcam) and CD8 (SP16; Abcam) were used.
Immunohistochemistry: Sections (15 μm) from paraffin-embedded biopsies from patients with LPLs and OSCC were used and mounted on electrostatically precharged slides (Superfrost Plus; Menzel-Gläzer). The sections were deparaffinized and rehydrated.
Retrieval of antigens for CD1a/CD3 stainings was carried out with incubation in TRIS/EDTA pH 9.0 and for CD1a/CD8 with Sodium Citrate pH 6.0 (CD1a/CD8) in 60 °C overnight. After extensive rinsing in distilled water, sections were incubated in horse serum (2:50) with 1% bovine serum albumin (BSA) in TBS for 30 min at RT. Following incubation with primary antibodies in 4 °C overnight, slides were triple-rinsed in TBS. After this procedure, Alexa Fluor 488 (Invitrogen, Eugene, OR, USA) and Alexa Fluor 647 (Invitrogen) diluted in 1% BSA in TBS were applied. After 60-min incubation in RT, the slides were rinsed.
Sections were then mounted with ProLong Gold antifade reagent with DAPI (Invitrogen) and preserved in darkness until analysis. Omission of primary antibodies served as negative controls. Images (200 × 200 μm) were obtained using a LSM 700 (Carl Zeiss, Jena, Germany) laser scanning microscope using ×25 objectives (oil immersion) and laser wavelengths 365 nm, 488 nm and 647 nm for excitation of the fluorochromes Alexa 488, 647 and DAPI.
Image stacks were scanned with an XYZ resolution of 1024 × 1024 × 8.
Statistical analysis. Analyses of differences between groups were carried out by the Kruskal–Wallis test, followed by the Mann–Whitney U-test as a post hoc test, utilizing the statistical software spss v17 (SPSS Inc., Chicago, IL, USA). A P-value < 0.05 was considered as a significant difference.
CD1a-positive LCs were found foremost in the epithelium, but also in connective tissue. In the epithelium, the DCs form an intraepithelial network, spreading their dendrites wedged in between and in close contact with keratinocytes. In the connective tissue, LCs have a tendency to lose their dendritic appearance and acquire a more irregular shape. In LPLs without dysplasia, LCs are more localized in the suprabasal layer of the epithelium (Fig. 2A) compared to LPLs with dysplasia and OSCC, where LCs are found to be scattered over the entire epithelium (Fig. 2B,C).
There was no significant difference in number of CD1a-positive LCs per area unit when comparing epithelium from patients with LPLs without and with dysplasia and OSCC (Fig. 3A, P =0.238; P =0.067). The connective tissue in LPLs with dysplasia had a significantly higher number of CD1a-positive LCs per area unit compared to connective tissue in LPLs without dysplasia (Fig. 3B; P =0.007). Only scarce, solitary Langerin-positive cells were seen in both epithelium and connective tissue of the three disorders (data not shown). The number of Langerin-positive LCs per area unit did not differ significantly either in epithelium or connective tissue between LPLs with or without dysplasia and OSCC (Fig. 3A,B, P =0.374 and P =0.728).
CD3-positive T cells were present in preferentially stratum basale and stratum spinosum within the epithelium of LPLs either with or without dysplasia. In the connective tissue of LPLs without dysplasia, CD3-positive T cells were sparse, while in LPLs with dysplasia, T cells formed a subepithelial infiltrate (Fig. 2D,E). The number of CD3-positive T cells per area unit was significantly higher in connective tissue and in epithelium of LPLs with dysplasia compared to LPLs without dysplasia (Fig. 4A,B; P =0.002 and P =0.008). In OSCC, CD3-positive cells were present in a quite uniform pattern within tumour tissue (Fig. 2F) and were significantly more abundant in comparison with LPLs with dysplasia (Fig. 4A; P =0.0001).
CD4-positive cells in LPLs either with or without dysplasia showed a similar distribution pattern as CD3-positive T cells. In the epithelium, some CD4-positive cells displayed a dendritic morphology, which was not the case for cells in connective tissue (Fig. 2J,K).
In LPLs either with or without dysplasia, a subepithelial localization was predominant, while in OSCC, CD4-positive cells were scattered in tumour tissue (Fig. 2L). Increasing numbers of CD4-positive cells were found when examining tissue specimens from LPLs without or with dysplasia and OSCC, respectively. However, there were no significant differences in the number of CD4-positive cells per area unit between LPLs with or without dysplasia in either epithelium or connective tissue (Fig. 4A,B, P =0.636 and P =0.783). In contrast, OSCC showed significantly higher numbers of CD4-positive cells in epithelium in comparison with LPLs with dysplasia (Fig. 4A, P =0.0001).
CD8-positive cells showed the same distribution pattern as CD4-positive cells (Fig. 2M,N,O). No cells with dendritic morphology were spotted. When comparing the epithelial compartments of LPLs without dysplasia with LPLs with dysplasia, the number of CD8-positive T cells did not differ significantly (Fig. 4A; P = 0.076). Interestingly, there were significantly more CD8-positive cells per area unit in the connective tissue in LPLs with dysplasia than in LPLs without dysplasia (Fig. 4B, P =0.042). OSCC tissue specimens contained significantly more CD8-positive cells than the epithelial compartment of LPLs with dysplasia (Fig. 4A; P =0.006).
Proliferating Ki67-positive cells were present in all specimens, although the distribution pattern varied from a suprabasal localization and even staining intensity in LPLs without dysplasia to a scattered distribution pattern and a wide range in staining intensity of positive cells in OSCC (Fig. 1P,Q,R).
Although a clear morphological difference was seen in dysplastic cells with abnormal size and atypical appearance, there were no significant differences in Ki67-positive proliferating cells in tissue specimens from patients with LPLs with or without dysplasia (Fig. 5; P =0.825). When comparing the number of epithelial Ki67-positive cells in tissue specimens from patients with OSCC with specimens from patients with LPLs with dysplasia, there was a significant difference between the groups (Fig. 5, P =0.012).
No significant differences were detected in number of Ki67-positive cells in connective tissue of LPLs with or without dysplasia (data not shown; P =0.325).
Confocal laser scanning microscopy
Spatial interactions of CD1a-positive LCs and T cells were found in all three lesions. In the epithelium of leukoplakias without dysplasia, sole LCs were found nearby CD3-positive T cells, but colocalization was just occasionally detected (Fig. 6A). In contrast, leukoplakias with dysplasia displayed several LC–T cell interactions where colocalization was obvious (Fig. 6B). In OSCC, CD1a-positive LCs were seen in conjunction with CD3-positive T cells in the tumour tissue (Fig. 6C). Colocalization analysis of CD1a-positive LCs and CD8-positive T cells showed a similar pattern. In LPL without dysplasia, CD1a-positive LCs were mainly seen in the interspace between the epithelium and the connective tissue. CD8-positive T cells were seen, but no colocalization with LCs was spotted (Fig. 6D). The number of CD8-positive T cells in connective tissue increased in LPL with dysplasia in comparison with LPL without dysplasia. LCs were seen interacting with CD8-positive T cells at the epithelial-connective tissue junction, but also within the epithelium (Fig. 6E). In OSCC, colocalization of CD1a-positive LCs and CD8-positive T cells could be seen in both tumour tissue and stroma (Fig. 6F).
The key finding of this study is that dendritic LCs and CD8-positive T cells are more abundant in leukoplakias with dysplasia as compared to LPLs without dysplastic cells. Not surprisingly, in OSCC the numbers of LCs and T cells are increased in comparison with the epithelial compartments in LPLs.
It is well established that LPLs are lesions that may develop into oral squamous cell carcinoma [2, 3]. The cause of LPLs is not fully understood but genetic alterations in keratinocytes are key events . There are no diagnostic or treatment modalities that can prevent LPLs from transforming into OSCC. At present, there are no methods available to predict which LPLs that will transform into OSCC .
Tumour-infiltrating DCs and T cells have a function in inhibiting tumour progression [18, 23]. The concept of cancer immunoediting has resulted in a view of dynamic interaction between the immune system and dysplastic cells/tumour cells, with phases of elimination, equilibrium and escape . In an early phase of cell dysplasia or early tumour cell formation, the immune system has the capacity to eliminate cells with DNA damage that may result, or already have resulted, in neoplastic transformation . A main approach for the immune system to recognize tumour cells is uptake of TAAs by DCs that via the MHC class I–presenting pathway present tumour peptides to tumour-specific CD8-positive cells [24–26]. Tumour-associated antigen -specific CD8-positive T cells may then recognize and eliminate cancer cells [27, 28]. Another interesting pathway of immune interaction is via tumour-derived exosomes that may both enhance and diminish immune activation . Thus, several ways exist for immune response to combat altered cells, a process where DCs and T cells are key players. Until now most studies have addressed the function of DCs and T cells in already established tumour diseases [29–32]. In contrast, studies designed to delineate the role of these leucocytes in premalignant disorders are sparse. In LPLs, a few studies have addressed the number of DCs [33–35].
In this study, the numbers of dendritic LCs and CD8-positive T cells in LPLs with cell dysplasia are increased in comparison with LPLs without cell dysplasia. Our findings support the results presented by Syafradi and co-workers, who reported that the number of CD8-positive T cells correlated with the grade of dysplasia . Also, Gannot and co-workers found an increase in immune cell infiltration with increasing degree of dysplasia . Possible mechanisms are that LCs within epithelium encounter and engulf TAAs, apoptotic material or tumour-derived exosomes that cause recruitment of DCs.
In established OSCC, Zancope et al.  showed that the number of peritumoral CD8-positive T cells correlated with a lower neoplastic proliferative index. The CD8-positive cells may be cytotoxic T cells that recognize TAAs from malignant or dysplastic cells. However, CD8 molecules are not expressed solely on T cells, as NK cells and plasmocytoid DCs are known to express CD8α molecules . Consequently, it is possible that CD8-positive cells registered in our patient material may in part be NK cells or plasmocytoid DCs. This do not change our interpretation that LPLs with dysplasia cause immune activation to dysplastic cells, as both NK cells and plasmocytoid DCs are known to be of importance in antitumoral defence [40, 41].
CD3-positive T cells in OSCC were found in significantly higher numbers than in LPLs with dysplasia and in LPLs without dysplasia. It is well known that premalignant and malignant diseases mount an immune response, which can be reflected in an influx of T cells . It is also evident from our results that CD3-positive T cells in epithelium gradually increase when comparing non-dysplastic and dysplastic epithelium, and OSCC.
In OSCC, CD4-positive cells were significantly more abundant than in LPLs, which may reflect the influx of lymphocytes to tumours described in numerous cancers . In epithelium of LPLs without or with dysplasia, cells with dendritic morphology were found. As human DCs are known to express CD4 molecules , these DCs most likely are LCs. This means that CD4-positive cell counts are not solely reflecting the number of CD4-positive T cells but also contain CD4-positive LCs.
The increased number of LCs in the submucosa of LPLs with dysplasia may be explained by recruitment of DCs to the region or migration from the epithelium of activated LCs. No signs of fungal or bacterial infiltration that could cause inflammation were noted in our biopsy material. The influx of LCs and T cells observed in this study could be explained by altered self-expression by keratinocytes.
We also addressed the presence of Langerin-expressing LCs. Langerin is a C-type lectin exclusively expressed by LCs . Langerin is predominantly expressed on immature LCs . In all three studied conditions, Langerin-positive LCs were rare, and no significant differences between the three conditions investigated in our study in respect of the number of Langerin-positive LCs were detected. In an earlier study, we reported that the number of Langerin-positive LCs is increased in oral lichen planus lesions in comparison with healthy oral mucosa . Although no direct comparison can be made between these results, it indicates that LCs in LPLs acquire a mature phenotype. Earlier studies have reported that LCs in colorectal carcinomas and parotid tumours have a mature phenotype [45, 46]. This may indicate a functional state of activity when LCs have acquired antigens from dysplastic cells and are migrating to regional lymph nodes. This could be supported by the observation in this study that there was a significant increase in the number of CD1a-positive LCs in submucosal tissue of LPLs with dysplasia in comparison with LPLs without dysplasia.
Autoimmune diseases such as oral lichen planus (OLP) or oral chronic graft versus host disease (GvHD) following allogeneic stem cell transplantation also display an increase in the number of dendritic LCs and T cells in diseased tissue, which we have shown in an earlier study . LCs and T cells are present to a higher extent in OLP and GvHD in comparison with LPLs and OSCC, when comparing with the results found in this study. Another difference is that in OLP and GvHD, CD4-positive T cell subset dominates in comparison with CD8-positive T cell subset, while in the present study CD8-positive T cell subset dominates. A possible explanation may be that cytotoxic CD8-positive T cells are recruited and engaging dysplastic and cancer cells with aberrant MHC class I molecule expression.
Control laser scanning microscopy analyses presented further support to findings from the quantitative analyses. Presence of dysplasia resulted both in an increase in inflammation and also an increase in interaction between dendritic LCs and T cells. This may support the hypothesis that dysplastic cells induce an immune response [7, 48]. In OSCC, the fact that dendritic LCs interspaced tumour tissue, and in some cases showed intense interaction with CD8-positive T cells, gives support to the results presented by Gannot and co-workers who found an increase in immune cell infiltration paralleled by an increasing degree of cell dysplasia, and finally OSCC .
There is prognostic importance of increased cell proliferation in head and neck tumours . In oral LPLs, expression of the proliferation marker Ki67 has been reported to correlate to degree of dysplasia in LPLs , but other groups reports have not confirmed this result . The number of Ki67-positive cells did not differ significantly between LPLs without dysplasia in comparison with lesions with dysplasia in our patients with LPLs. However, a significant increase in proliferating cells was seen when comparing LPLs with dysplasia and OSCC. This is in line with what Torres-Rendon and collaborators have reported . Thus, presence of dysplasia in our patient material does not seem to be correlated to an increased cell division rate in the benign situation, but a correlation appears when there is an established cancer.
LPL with dysplasia probably is an example where immune activation and proliferation of dysplastic cells are in a state of equilibrium, as originally suggested by Dunn et al. . In the clinical setting, LPLs may persist for many years or throughout the patients’ lifetime without malignant transformation . In inflammatory bowel diseases, evidence for immunosurveillance has been presented by Karlsson et al. , who report a correlation between degree of dysplasia in gut epithelium from patients with ulcerative colitis and the number of CD4-positive T cells in draining lymph nodes. In contrast to the latter study, the present study investigates a disorder without underlying inflammatory disease. However, the findings present additional support to the concept of immunosurveillance.
In conclusion, this study presents evidence that dendritic Langerhans cells and T cells respond to cell dysplasia and malignant transformation in oral leukoplakias and carcinomas, which indicates an immune activation to dysplastic epithelial cells and cancer cells. To address immune response in premalignant disorders will be of increasing importance to find strategies to enhance immune activity in not only premalignant lesions but also established malignant tumours.
This study was supported by the Agreement for Doctoral Education, Region Västra Götaland, Sweden, The Assar Gabrielsson Foundation, The Swedish Dental Society and the Gothenburg Dental Society. The authors are obliged to Ms. Christina Eklund, B.Sc. and Ms. Marie Svensson for excellent technical assistance.