Skew in T cell receptor usage with polyclonal expansion in lesions of oral lichen planus without hepatitis C virus infection

Abstract

Oral lichen planus (OLP) is a refractory disorder of the oral mucosa. Its predominant symptoms are pain and haphalgesia that impair the quality of life of patients. OLP develops via a T cell-mediated immune process. Here, we examined the characteristics of the infiltrating T cells in terms of the T cell receptor (TCR) repertoires, T cell clonality, T cell phenotypes and cytokine production profiles. TCR repertoire analyses and CDR3 size spectratyping were performed using peripheral blood mononuclear cells (PBMCs) and tissue specimens of OLP biopsies from 12 patients. The cytokine expression profiles and T cell phenotypes were measured by real-time quantitative polymerase chain reaction. We observed that there were skewed TCR repertoires in the tissue samples (TCRVA8-1, VA22-1, VB2-1, VB3-1 and VB5-1) and PBMCs (TCRVA8-1, VB2-1, VB3-1 and VB5-1) from OLP patients. Furthermore, the CDR3 distributions in the skewed TCR subfamilies exhibited polyclonal patterns. We observed increases in CD4(+) T lymphocytes, interleukin (IL)-5, tumour necrosis factor (TNF)-alpha and human leucocyte antigen D-related in the OLP tissue specimens. Taken together, the present results suggest that T cells bearing these TCRs are involved in the pathogenesis of OLP, and that IL-5 and TNF-alpha may participate in its inflammatory process.

Fig. 1

Mean percentage frequencies of TCRVA and TCRVB in normal oral mucosa (NOM) tissue specimens from six healthy volunteers. The expressions of the different T cell receptor (TCR) repertoires in individual NOM tissue specimens are shown as mean ± standard deviation (a). TCRVA and TCRVB gene expression profiles in the NOM tissue specimens of healthy volunteers (b).

Fig. 2

The T cell receptor (TCR) repertoires in oral lichen planus (OLP) tissue specimens are skewed in comparison with those in healthy peripheral blood mononuclear cells (PBMCs). There are significant increases in TCRVA8-1 and VA22-1 (a) as well as VB2-1, VB3-1 and VB5-1 (b) in OLP tissue specimens compared with healthy PBMCs (*P < 0·05). There are no significant increases in VA8-1, VA22-1, VB2-1, VB3-1 and VB5-1 in normal oral mucosa (NOM) tissue specimens compared with healthy PBMCs (c). All data are shown as mean ± standard deviation.

Fig. 3

The T cell receptor (TCR) repertoires in oral lichen planus (OLP) peripheral blood mononuclear cells (PBMCs) are skewed in comparison with those in healthy PBMCs. There are significant increases in T cells bearing TCRVA8 (a) as well as VB2, VB3 and VB5 (b) in OLP PBMCs compared with healthy PBMCs (*P < 0·05).

Fig. 4

Polyclonal expansion of T cell receptors (TCRs) within the oral mucosa of patients with oral lichen planus (OLP). Because the TCR repertoire analysis revealed skewed numbers of T cells bearing TCRVA8-1, VA22-1, VB2-1, VB3-1 and VB5-1, CDR3 size spectratyping was performed for all samples. In NOM tissue specimens, the CDR3 size distributions show monoclonal or oligoclonal patterns. In contrast, the CDR3 spectratyping patterns for OLP tissue specimens exhibit polyclonal or oligoclonal patterns.

Fig. 5

Histopathological findings for normal oral mucosa (NOM) and oral lichen planus (OLP) tissue specimens. In NOM tissue specimens, lymphocytes are habitually present. In OLP tissue specimens, band-like infiltrates of lymphocytes in the superficial lamina propria and disruption of the basement membrane are detected. The insets show higher magnification images of the boxed regions. Magnifications: ×20 and ×200.

Fig. 6

Immunohistochemical staining for CD3 (a), CD4 (b), CD8 (c), CD20 (d) and human leucocyte antigen D-related (HLA-DR) (e) using commercially available monoclonal antibodies following the ENVISION technique with 3,3-diaminobenzidinetetrahydrochloride as the indicator agent. (f) Negative control. CD3⁺, CD4⁺ and CD8⁺ T lymphocytes are detected among the infiltrating cells toward the basal membrane (a–c). Normal oral mucosa (NOM) tissue specimens contain low numbers of CD3⁺, CD4⁺ and CD8⁺ T lymphocytes (a–c). Expression of CD20 is detected in both OLP and NOM tissue specimens (d). HLA-DR expression is increased significantly in OLP tissue specimens (e). Magnification: ×200.

Fig. 7

Comparison of the expression levels of T cell phenotypes between normal oral mucosa (NOM) (n = 6) and oral lichen planus (OLP) (n = 12) tissue specimens (a) or healthy (n = 9) and OLP (n = 12) peripheral blood mononuclear cells (PBMCs) (b). The mRNA expression levels of CD4⁺ and CD8⁺ T cells were measured by real-time quantitative polymerase chain reaction (PCR). Each dot indicates a single sample. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression was used as an internal control. The expression levels of CD8⁺ T cells are higher than CD4⁺ T cells in NOM and OLP tissue specimens (*P < 0·001). The CD4/CD8 ratio is higher in OLP tissue specimens than in NOM tissue specimens (**P < 0·0001).

Fig. 8

Comparison of the expression levels of several cytokines between normal oral mucosa (NOM) (n = 6) and oral lichen planus (OLP) (n = 12) tissue specimens (a) or healthy (n = 9) and OLP (n = 12) peripheral blood mononuclear cells (PBMCs) (b). The mRNA expression levels of interleukin (IL)-2, IL-4, IL-5 and tumour necrosis factor (TNF)-α were measured by real-time quantitative polymerase chain reaction (PCR). Each dot indicates a single sample. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression was used as an internal control. The mRNA levels expressed by the ratio of the amount of the each cytokine-specific cDNA to the amount of GAPDH-specific cDNA were compared between NOM and OLP tissue specimens or healthy and OLP PBMCs. (a) The expression levels of IL-5 and TNF-α are higher in OLP tissue specimens than in NOM tissue specimens. The IL-2 expression levels are lower in OLP tissue specimens than in NOM tissue specimens. (b) The IL-5 expression levels are higher in OLP PBMCs than in healthy PBMCs.