Abstract
Background
Oral lichen planus (OLP) is a chronic inflammatory disease affecting oral mucosa. Its pathogenesis includes T cell infiltration. T cells may be naïve or in response to antigen stimulation, progress through differentiation stages. The differentiated states of T cells in OLP mucosa have not previously been reported.
Methods
Available OLP microarray gene expression data from Gene Expression Omnibus were analyzed for markers of T cell cytotoxicity. Immunohistochemical studies of T cell subset markers CD4 and CD8 and the T cell differentiation marker killer cell lectin-like receptor G1 (KLRG1) were performed on paraffin embedded formalin fixed oral mucosa biopsy samples from 10 patients with OLP.
Results
Gene expression analysis of OLP oral mucosa samples disclosed increased transcript expression of KLRG1, CD8A, and granzyme K (GZMK). By immunohistochemistry, prominent CD4 + and CD8 + T cell infiltration was seen in all patient samples. KLRG1 + T cells were abundant, constituting a mean of 51% (range 40–65%) of the number of CD8 + T cells. KLRG1 + T cells localized at the epithelium and lamina propria junction, infiltrating both basal and intraepithelial regions and adjacent to both basal and intraepithelial keratinocytes.
Conclusions
OLP oral mucosa T cell infiltration includes KLRG1 + highly differentiated cytotoxic T cells, suggesting continued antigen exposure driving T cells to a highly differentiated phenotype. The known phenotype of these cells, together with microarray detected increases in cytotoxic molecules, suggests that highly differentiated cytotoxic T cells contribute to oral mucosa injury in OLP.
Keywords: Lichen planus, Oral lichen planus, Killer cell lectin-like receptor G1, T-cells, Cytotoxic T-cells, Terminally differentiated T-cells
Introduction
Oral lichen planus (OLP) is a common mucocutaneous disorder that affects 1–2% of the adult population, frequently in women in the sixth decade upwards, and presents as bilateral, fairly symmetric, keratotic striations and erythema of the buccal mucosa, gingiva and ventral tongue [1]. The pathogenesis of oral lichen planus (OLP) includes infiltration of both CD4 + and CD8 + T cells. The subepithelial infiltrate has been reported to include both CD8 + cytotoxic and CD4 + helper T cells, while the intraepithelial lymphocytes have been reported to be mainly CD8 + cytotoxic T cells [2–4]. The intraepithelial CD8 + cytotoxic T cells cluster in areas of basement membrane disruption [5], and can be found adjacent to degenerating keratinocytes [4], suggesting that CD8 + T cells may be engaged in epithelial cell destruction by cytotoxic mechanisms typical of CD8 T cells, such as release of granzymes and direct killing of target cells.
Cytotoxic T cells can be naïve cells, quiescent central memory cells, or become differentiated mature T cells with effector memory and effector phenotypes. Highly differentiated mature cytotoxic T cells express characteristic surface molecules that include KLRG1, which mark those T cells with more potent cytotoxic capacity, including greater production of interferon gamma (IFNg), tumor necrosis factor (TNFa), and granzymes compared with KLRG1-negative T cells [6, 7]. Both classical cytotoxic CD8 + T cells and highly differentiated CD28- CD4 + T cells that are cytotoxic, not helper T cells, express KLRG1 [8]. These CD28- CD4 + T cells constitutively express cytotoxic molecules like granzyme B and perforin. To further understand the state of T cell differentiation present in OLP oral mucosa, we performed immunohistochemistry for KLRG1 and standard T cell subset markers CD3, CD4, and CD8 on OLP oral mucosa biopsy samples.
Materials and methods
The Gene Expression Omnibus database was searched for oral lichen planus expression profiling by microarray datasets, identifying two unique datasets GSE38616 [9] and GSE52130 [10]. These datasets contain oral mucosa gene expression data from patients with OLP (N = 7 in both datasets) and healthy volunteers (N = 7 in both datasets). Fold ratios and non-adjusted p-values (given limited multiple hypothesis testing) of selected cytotoxic signature genes were computed.
Immunohistochemistry was performed on oral mucosa formalin fixed paraffin embedded tissue sections from 10 patients with OLP diagnosed according to consensus criteria [11]. Tissue samples were from discarded specimens that had been obtained for diagnostic purposes and were studied under a medical director approved research protocol. Antibody reagents used were anti-CD3 (Leica, #NCL-L-CD3-565, 1:50), anti-CD4 (Abcam, #ab133616, 1:500), anti-CD8 (LSBio, #LS-B3914, 1:100), anti-KLRG1 (Abcuro, #KLRG1 H-IHC, 1:500, 3.92 ug/ml), and isotype control (Abcam, #ab37415, 3.92 ug/ml). Semiquantitative analysis was performed by two methods: conventional grading of histology using a 0–4 + scale, and by analysis of adjacent CD8 and KLRG1 sections, counting 3 regions in each section for number of CD8 + and number of KLRG1 + cells, and taking the mean of the 3 regions KLRG1+ % of CD8 + cells.
Results
Transcriptome gene expression data analysis from GSE38616 and GSE52130 of OLP compared to healthy oral mucosa (Table 1) demonstrated directionally increased expression of transcripts indicative of multiple types of T cells (CD3D) and B cells (CD20). There were generally no increase in markers with relative specificity for NK cells (NCR1), mast cells/basophils (FCER1), myeloid dendritic cells (CD1c), plasma cells (SDC1/CD138), and plasmacytoid dendritic cells (CLEC4C/BDCA2). Genes associated with mucosal associated invariant T cells (SLC4A10, WNT11, LTK, and FLT4) [12], were not increased, with the exception of KLRB1 (CD161), a marker of specialized populations of mucosal invariant T cells (MAIT) cells, Th17 lineage cells, and NK cells.
Table 1.
Fold-ratio of OLP compared to normal mucosa for selected genes in datasets GSE38616 and GSE52130. Generally, increases in expression of T cell genes and markers of T cell cytotoxicity are present
| Gene | GSE38616 | GSE52130 |
|---|---|---|
| CD3D | 2.08 | 1.97 |
| CD20 | 4.07 | 1.01 |
| NCR1 | 0.99 | 1.01 |
| FCER1 | 0.71 | 0.81 |
| CD1C | 1.10 | 1.01 |
| SDC1/CD138 | 0.88 | 1.00 |
| CLEC4C/BDCA2 | 1.28 | 1.13 |
| SLC4A10 | 0.96 | 0.99 |
| WNT11 | 0.88 | 1.01 |
| LTK | 0.87 | 1.04 |
| FLT4 | 1.07 | 1.05 |
| KLRB1 | 1.85 | 1.27 |
| KLRG1 | 2.28 | 1.69 |
| CXCL9 | 4.50 | 5.75 |
| CXCL10 | 3.52 | 3.45 |
| CXCL11 | 4.66 | 1.50 |
| CD8A | 1.68 | 1.55 |
| GZMA | 1.80 | 1.22 |
| GZMB | 1.33 | 2.81 |
| GZMH | 1.02 | 0.72 |
| GZMK | 3.61 | 1.74 |
| PRF1 | 1.21 | 1.12 |
| IFNG | 1.55 | 1.37 |
Multiple markers of T cell cytotoxicity were increased in both OLP datasets (Table 1; Fig. 1). For examples, in GSE38616, increases in interferon gamma (IFNG) and IFNG-inducible chemokines CXCL9, CXCL10, and CXCL11 were present. KLRG1 expression was 2.3 fold increased (p = 0.03). KLRG1 expression was highly correlated with the cytotoxic T cell marker CD8A (p = 0.0004, R = 0.96) and granzyme K GZMK (p = 0.002, R = 0.94). Similar findings were present in GSE52130.
Fig. 1.
Elevated cytotoxicity marker and KLRG1 gene expression in oral lichen planus oral mucosa. Analysis of oral mucosa biopsy samples gene expression in Gene Expression Omnibus datasets (A) GSE38616 and (B) GSE52130. Increased expression of KLRG1, multiple markers of T cell cytotoxicity (CD8A, granzymes, perforin), interferon gamma (IFNG) driven chemokine expression (CXCL9, CXCL10, CXCL11), including elevated KLRG1, and KLRG1 correlation with markers of T cell cytotoxicity CD8A and granzyme K (GZMK)
In immunohistochemical studies, all samples had infiltrating CD3 T cells, both CD4 and CD8, and KLRG1 + cells. Semi-quantitative grading of KLRG1 infiltration varied from mild (1+) to dense (4+). KLRG1 + cells constituted a mean of 51% (range 40–65%) of CD8 + T cells (Table 2). KLRG1 + cells colocalized with areas of CD8 T cell infiltration in lymphocytic dense infiltrates at the epithelium and lamina propria junction (Fig. 2). Both CD8 T cells and KLRG1 + T cells also localized within the intraepithelial regions, both at the basal layers and more superficially, and often adjacent to keratinocytes, though KLRG1 + T cells were less frequently present in intraepithelial regions than CD8 + T cells (Fig. 3).
Table 2.
Semiquantitative grading of T cell infiltration in oral lichen planus oral mucosa biopsy samples by immunohistochemistry. T cell subset intensity and area of infiltration graded 0 (no cells) to 4+ (widespread and dense infiltration). The mean % of CD8 + T cells that were KLRG1 + from counting 3 regions of each section ranged from 40–65%
| Case | CD3 | CD4 | CD8 | KLRG1 | Isotype | KLRG1+ % of CD8+ |
|---|---|---|---|---|---|---|
| 9369 | 3+ | 3+ | 2+ | 2+ | 0 | 60% |
| 9511 | 2+ | 2+ | 3+ | 2+ | 0 | 55% |
| 9626 | 4+ | 3+ | 2+ | 2+ | 0 | 51% |
| 9714 | 4+ | 4+ | 4+ | 1+ | 0 | 45% |
| 9748 | 3+ | 2+ | 3+ | 2+ | 0 | 41% |
| 9844 | 4+ | 3+ | 3+ | 2+ | 0 | 52% |
| 9915 | 4+ | 2+ | 4+ | 4+ | 0 | 58% |
| 10,115 | 4+ | 3+ | 4+ | 3+ | 0 | 65% |
| 10,139 | 1+ | 1+ | 1+ | 1+ | 0 | 46% |
| 10,396 | 4+ | 3+ | 3+ | 2+ | 0 | 40% |
Fig. 2.
CD8 and KLRG1 + T cell infiltration in oral lichen planus oral mucosa. Oral mucosa biopsy sample immunohistochemistry shown for patient 9915 (A-C, low magnification; D-F high magnification), patient 10,115 (G-I), patient 9369 (J-L), and patient 9748 (M-O). Dense lymphocytic infiltrates at the epithelium and lamina propria junction show abundant CD8 + and KLRG1 + T cells
Fig. 3.
Basal layer and intraepithelial CD8 and KLRG1 + T cell infiltration. Oral mucosa biopsy immunohistochemistry shows intraepithelial, including the basal cell layer, infiltration with CD8 + and KLRG1 + T cells adjacent to keratinocytes. Arrows track specific cells across increasing magnification (left to right in all rows). (A-G) Patient 10,115, with CD8 (A-C) and KLRG1 (D-F) immunohistochemistry. (G-J) Patient 9844, with KLRG1 immunohistochemistry
Discussion
OLP is a chronic inflammatory condition affecting oral mucous membranes resulting in a range of clinical manifestations from no symptoms to food sensitivity to significant pain. Multiple arms of the immune system may be involved. The pathogenesis of OLP is incompletely understood and includes cytotoxic T-cell infiltration of the basal lamina zone resulting in basal keratinocyte apoptosis and basal lamina destruction [13, 14]. In cutaneous lichen planus, in vitro studies have demonstrated clones of cytotoxic CD8 + T cells, but not CD4 + T cells, directed against autologous epidermal keratinocytes [15]. Specialized forms of cytotoxic T cells, such as MAIT cells, have also been implicated in the pathogenesis of OLP [13]. Other elements of the immune system are also involved and their involvement may be T cell dependent, such as mast cells, speculated to be recruited and degranulated by T cell secreted chemokines [16].
Here, we further characterized the developmental stage of infiltrating cytotoxic T cells. Cytotoxic T cells are generally identified by their expression of CD8 though some CD4 + T cells, particular those that have lost CD28 expression, are also cytotoxic cells [8]. Cytotoxic T cells differentiate through characteristic stages of maturity, and the most highly differentiated cells have the greatest cytotoxic capacity to injure tissue. These mature highly differentiated T cells can be identified by expression of KLRG1.
In particular, we have identified KLRG1 + cells infiltrating oral mucosa at the epithelium and lamina propria junction and within the intraepithelial layer, including adjacent to basal layer cells. Based on the known properties of human KLRG1 + cells from normal and diseased blood and tissue samples [6, 7], as high producers of cytokines and granzymes, these cells can be hypothesized to be involved in the destruction of tissue elements in OLP.
Acknowledgements
Dr. Sook-Bin Woo, DMD, MMSc provided tissue samples for this study from the Center for Oral Pathology, StrataDx, Lexington, MA.
Funding
Abcuro, Inc. provided funding for immunohistochemistry studies performed by Histowiz, Inc.
Data Availability
No further availability is provided.
Code Availability
N/A.
Authors/ contributions: All authors contributed to manuscript writing. SAG, DSF, and SBW were involved in conception. FL performed the immunohistochemical studies.
Declarations
Conflicts of interest/competing interests
SAG is a founder and consultant to Abcuro, Inc.; DSF is a consultant to Abcuro, Inc.
Ethics approval
Not applicable.
Consent to participate
Not applicable.
Consent for publication
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Footnotes
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Associated Data
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Data Availability Statement
No further availability is provided.
N/A.
Authors/ contributions: All authors contributed to manuscript writing. SAG, DSF, and SBW were involved in conception. FL performed the immunohistochemical studies.