Involvement of the Programmed Death 1/Programmed Death Ligand 1 Pathway in the Immune Microenvironment of Chronic Periapical Lesions

Abstract

Introduction and aims

This study aimed to determine the pathogenic role and clinicopathological significance of the immune microenvironment including the programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) pathway in chronic periapical lesions.

Methods

A total of 20 chronic periapical lesions consisting of 10 periapical granulomas and 10 periapical cysts were included in this study. Immunohistochemistry was performed for immune cell populations, including PD-L1, PD-1, CD4, CD8, FOXP3, and CD20. Immune cell populations were quantitatively evaluated on digitized slides. The associations between each immune cell population and clinicopathological factors and between immune cell populations were statistically analysed.

Results

Lesion size was positively associated with the density of PD-L1+ macrophages (P < .001, Fisher exact test; r = 0.455, P = .044, Pearson correlation analysis) and CD8+ cytotoxic T cells (P = .020, Fisher exact test; r = 0.471, P = .036, Pearson correlation analysis). No associations were found between immune cell populations and other clinicopathological factors, including age, sex, lesion location, and diagnosis. A moderate positive correlation was observed between the density of PD-L1+ macrophages and CD8+ cytotoxic T cells (r = 0.537, P = .015). The density of PD-1+ cells showed a strong positive correlation with the density of CD4+ helper T cells (r = 0.719, P < .001) and FOXP3+ regulatory T cells (r = 0.784, P < .001).

Conclusion

These findings suggest that cytotoxic T cells are implicated in the progression of chronic periapical lesions, which may be regulated by PD-L1+ macrophages. PD-1 may be involved in helper T cell exhaustion and regulatory T cell activity in chronic periapical lesions.

Clinical relevance

We demonstrated the involvement of PD-L1 and PD-1 in the regulation of T cell immunity in chronic periapical lesions. These findings suggest that activating the PD-1/PD-L1 pathway is a potential therapeutic strategy for chronic periapical lesions.

Introduction

Chronic periapical lesions, mainly consisting of periapical granuloma and periapical cysts, are the result of an inflammatory response to bacterial infection in the root canal. Periapical granuloma, also referred to as apical periodontitis, is defined as a mass of inflamed granulation tissue at the apex of a nonvital tooth. Periapical granuloma may develop into a true epithelium-lined cyst called a periapical cyst. Since radiological features are not definitively diagnostic, histological examination is essential for differentiating between the two types of chronic periapical lesions.¹ Previous studies on the immune microenvironment in chronic periapical lesions have mainly focused on the composition of immune cell populations and their differential distribution by histological type, namely between periapical granuloma and periapical cyst, although conflicting results have been reported.2, 3, 4, 5, 6, 7 In addition, the associations of immune cell populations with clinicopathological factors, such as age, sex, lesion location, and lesion size, remain unexplored in chronic periapical lesions.

Programmed death 1 (PD-1), also known as CD279, is a transmembrane protein expressed on the surface of immune cells, such as T cells. Programmed death ligand 1 (PD-L1), also known as CD274 or B7-H1, is a transmembrane protein mainly expressed on the surface of antigen presenting cells, such as macrophages, and some types of tumor cells.⁸ PD-1, as an immune checkpoint receptor, regulates T cell activation by binding to its immune checkpoint ligands, such as PD-L1.⁹ The immunosuppressive role of the PD-1/PD-L1 pathway has been studied in various cancers, leading to the development of anti-PD-1/PD-L1 antibodies for cancer immunotherapy.¹⁰ In addition, recent studies have also focused on the implications of the PD-1/PD-L1 pathway in the pathogenesis of immune-related diseases, including autoimmune or infectious diseases.^8,11 In the field of dentistry, previous studies on periodontitis have demonstrated the double-edged effects of the PD-1/PD-L1 pathway – an inhibitor of inflammation, reducing periodontal tissue destruction, and an enabler of immune escape of pathogens, promoting persistent infection – and suggested that the PD-1/PD-L1 pathway could be a new avenue for the treatment of periodontitis and its related systemic disorders.¹² However, the involvement of the PD-1/PD-L1 pathway has been scarcely investigated in human periapical lesions despite their high prevalence rate,^5,13,14 with no studies reporting the expression of PD-L1 in lesional tissues.

We hypothesized that the immune microenvironment including the PD-1/PD-L1 pathway is involved in the pathogenesis of chronic periapical lesions. Therefore, this study aimed to determine the clinicopathological significance of the immune microenvironment and the associations between immune cell populations in chronic periapical lesions by using immunohistochemistry.

Materials and methods

Case selection

Chronic periapical lesions diagnosed as periapical granuloma or periapical cyst were retrieved from the pathology archive of the Department of Oral Pathology, Dankook University Dental Hospital. All diagnoses were confirmed by an experienced oral pathologist (KYO). Cases with insufficient tissue for immunohistochemistry were excluded. Based on the sample size in recent immunohistochemical studies on periapical lesions,^15,16 a total of 20 chronic periapical lesions were included in this study. Clinicopathological data including age, sex, lesion location, and lesion size were collected from medical records. This study was approved by the Institutional Review Board of Dankook University Dental Hospital (approval No. DKUDH IRB 2024-10-006).

Immunohistochemistry

Nondecalcified, formalin-fixed paraffin-embedded tissue samples were selected for each case. To investigate the expression of immune checkpoint molecules (PD-1, PD-L1) and the distribution of T cells (CD4+ helper, CD8+ cytotoxic, FOXP3+ regulatory) and B cells (CD20+), the following six antibodies were used: PD-L1 (clone E1L3N; dilution 1:200; #13684, Cell Signaling Technology), PD-1 (clone NAT105; dilution 1:50; ab52587, Abcam), CD4 (clone 4B12; Ready-to-Use; IR649; Dako), CD8 (clone C8/144B; Ready-to-Use; IR623, Dako), FOXP3 (clone 236A/E7; dilution 1:400; ab20034, Abcam), and CD20 (clone L26; Ready-to-Use; PA0200, Leica Biosystems). Immunohistochemical staining was performed according to the manufacturers’ instructions using a Leica BOND-MAX Autostainer (Leica Biosystems). Tonsil tissue was used as a positive control, and primary antibodies were omitted for negative controls.

Immunohistochemical evaluation

All 120 immunohistochemically stained slides were scanned at 20$\times$ magnification using an Olympus Slideview VS200 slide scanner (Evident Scientific). From each whole slide image, 2 fields with the highest expression were selected at 20$\times$ magnification. Finally, the density (cells/mm²) of each immune cell population was calculated.

Statistical analysis

The chi-square test or Fisher exact test was used to analyse the associations between categorical variables, for which clinicopathological factors, including age and lesion size, and immune cell populations were dichotomized at the median into low and high groups. Pearson correlation analysis was used to analyse the associations between continuous variables. P values <.05 were considered statistically significant. All statistical analyses were performed using IBM SPSS Statistics Version 27.

Results

PD-L1 was mainly expressed in some macrophages (Figure 1A). PD-1 was mainly expressed in a subset of T cells (Figure 1B). CD4 (Figure 1C), CD8 (Figure 1D), and FOXP3 (Figure 1E) were expressed in helper, cytotoxic, and regulatory T cells, respectively. CD20 was expressed in B cells (Figure 1F).

Fig. 1.

Representative images of immunohistochemically stained slides. (A) Membranous PD-L1 staining on a subset of macrophages. (B) Membranous PD-1 staining on a subset of T cells. (C) Membranous CD4 staining on helper T cells. (D) Membranous CD8 staining on cytotoxic T cells. (E) Nuclear FOXP3 staining in regulatory T cells. (F) Membranous CD20 staining on B cells. Some cells showing a typical expression pattern are indicated by arrows.

The size of chronic periapical lesions is positively correlated with PD-L1+ macrophages and CD8+ cytotoxic T cells

The associations between immune cell populations and clinicopathological factors are summarized in Table. Among the clinicopathological factors studied, lesion size showed a significant association with high PD-L1 (P < .001) and CD8 (P = .020) expression. Pearson correlation analysis also showed a moderate positive correlation between lesion size and the density of PD-L1+ macrophages (r = 0.455, P = .044; Figure 2A) and CD8+ cytotoxic T cells (r = 0.471, P = .036; Figure 2B). The other clinicopathological factors, including histological type (periapical granuloma versus periapical cyst), showed no significant relationship with immune cell populations (Table).

Table.

A summary of the associations between immune cell populations and clinicopathological factors in chronic periapical lesions.

		PD-L1			PD-1			CD4			CD8			FOXP3			CD20
	n	Low	High	P	Low	High	P	Low	High	P	Low	High	P	Low	High	P	Low	High	P
Diagnosis
PG	10	4	6	.371	5	5	1.000	5	5	1.000	4	6	.371	4	6	.371	4	6	.371
PC	10	6	4		5	5		5	5		6	4		6	4		6	4
Age
$<$35 y	11	5	6	1.000	5	6	1.000	3	8	.070	6	5	1.000	5	6	1.000	4	7	.370
$\ge$35 y	9	5	4		5	4		7	2		4	5		5	4		6	3
Sex
Male	11	4	7	.370	6	5	1.000	7	4	.370	4	7	.370	5	6	1.000	6	5	1.000
Female	9	6	3		4	5		3	6		6	3		5	4		4	5
Jaw
Maxilla	12	5	7	.650	6	6	1.000	4	8	.170	4	8	.170	4	8	.170	5	7	.650
Mandible	8	5	3		4	4		6	2		6	2		6	2		5	3
Teeth
Anterior	12	5	7	.650	8	4	.170	6	6	1.000	4	8	.170	5	7	.650	5	7	.650
Posterior	8	5	3		2	6		4	4		6	2		5	3		5	3
Lesion size
$<$1.2 cm	12	10	2	<.001*	7	5	.650	6	6	1.000	9	3	.020*	8	4	.170	7	5	.650
$\ge$1.2 cm	8	0	8		3	5		4	4		1	7		2	6		5	3

Age and lesion size were dichotomized at the median.

PC, periapical cyst; PG, periapical granuloma.

^⁎Statistically significant.

Fig. 2.

Associations between lesion size and immune cell populations. Lesion size shows a moderate positive correlation with the density of PD-L1+ macrophages (A) and CD8+ cytotoxic T cells (B).

PD-L1+ macrophages are positively correlated with CD8+ cytotoxic T cells in chronic periapical lesions

The associations between the density of PD-L1+ macrophages and T cell populations were investigated using Pearson correlation analysis. As a result, a moderate positive correlation was observed between PD-L1+ macrophages and CD8+ cytotoxic T cells (r = 0.537, P = .015; Figure 3A). Similar spatial distributions were also identified between PD-L1+ macrophages and CD8+ T cells in a tissue context (Figure 3B and C). PD-L1+ macrophages showed no significant relationship with the other T cell populations, including CD4+ helper T cells (r = 0.011, P = .963) and FOXP3+ regulatory T cells (r = 0.058, P = .808).

Fig. 3.

Associations between PD-L1 and CD8. (A) A moderate positive correlation is seen between the density of PD-L1+ macrophages and CD8+ cytotoxic T cells. (B) PD-L1-high, CD8-high case. Note the similar spatial distributions of PD-L1+ cells and CD8+ cells. (C) PD-L1-low, CD8-low case.

PD-1+ cells are strongly positively correlated with CD4+ helper T cells and FOXP3+ regulatory T cells in chronic periapical lesions

The associations between the density of PD-1+ cells and T cell populations were investigated using Pearson correlation analysis. As a result, a strong positive correlation was observed between PD-1+ cells and CD4+ helper T cells (r = 0.719, P < .001; Figure 4A) and between PD-1+ cells and FOXP3+ regulatory T cells (r = 0.784, P < .001; Figure 4B). Similar spatial distributions were also identified between PD-1+ cells, CD8+ T cells, and FOXP3+ T cells in a tissue context (Figure 4C and D). No significant relationship was found between PD-1+ cells and CD8+ cytotoxic T cells (r = 0.097, P = .684).

Fig. 4.

Associations between PD-1 and T cell populations. The density of PD-1+ cells shows a strong positive correlation with the density of CD4+ helper T cells (A) and FOXP3+ regulatory T cells (B). (C) PD-1-high, CD4-high, FOXP3-high case. Note the similar spatial distributions of the immune cell populations. (D) PD-1-low, CD4-low, FOXP3-low case.

Discussion

This study investigated the clinicopathological significance of the immune microenvironment and the associations between immune cell populations in chronic periapical lesions using tissue samples. The results of this study confirmed the hypothesis that the immune microenvironment including the PD-1/PD-L1 pathway is involved in the pathogenesis of chronic periapical lesions.

Unlike previous studies on chronic periapical lesions, which mainly compared immune cell counts between different histological types (eg, periapical granuloma versus periapical cyst),2, 3, 4, 5, 6, 7 the analysis performed in this study was extended to other factors, including age, sex, lesion location, and lesion size. As a result, lesion size showed a positive correlation with PD-L1+ macrophages and CD8+ cytotoxic T cells, although the other clinicopathological factors, including histological type, were not significantly associated with the immune cell populations studied. Chronic periapical lesions vary in size from a barely detectable size to several centimetres in diameter.¹ In a previous study analysing inflammatory cells isolated from human periapical lesions by immunocytochemistry, the proportion of CD8+ T cells was higher in large lesions than in small lesions, while no difference was found in the proportion of CD4+ T cells.¹⁷ Similarly, a positive correlation between lesion size and the density of CD8+ cytotoxic T cells was found at the tissue level in this study by assessing the density of different immune cells using immunohistochemistry. Periapical lesions occur as a defensive reaction to bacterial infection in the root canal and bacterial toxins spread to the periapical area.¹ In the early stages, neutrophils and macrophages play a major role in bacterial clearance.¹⁸ If left untreated, however, this defensive reaction may become less effective with continuous bacterial invasion, and as the lesions progress, chronic inflammatory cells, including lymphocytes and plasma cells, begin to predominate.¹ Among these inflammatory cells, CD8+ cytotoxic T cells, which have the ability to kill infected cells and extracellular bacteria,¹⁹ appear to be primarily involved in the progression of chronic periapical lesions based on this and the previous studies.¹⁷

The role of PD-L1 in chronic periapical lesions has rarely been investigated. In a previous study evaluating leukocytes isolated from human chronic apical periodontitis by flow cytometry, PD-L1 expression was higher in leukocytes from lesions than in those from healthy gingival tissues, and among leukocytes, macrophages showed higher PD-L1 expression than other immune cells, suggesting that PD-L1 is an important immune checkpoint.¹³ Another study using rat models showed a time-dependent increase in PD-L1 expression in chronic apical periodontitis, suggesting that PD-L1 is upregulated according to disease progression.²⁰ In this study, similar to these previous findings, a positive correlation was observed between lesion size and the density of PD-L1+ macrophages at the tissue level in human chronic periapical lesions. Additionally, this study demonstrated that PD-L1+ macrophages are positively correlated with CD8+ cytotoxic T cells. Together, the results of this study suggest the inhibitory role of PD-L1 in cytotoxic T cell-mediated inflammation in chronic periapical lesions.

The increased expression of PD-1 has been reported in chronic apical periodontitis,^13,20 and its expression on T cells has also been visualized in a previous study, suggesting that T cell exhaustion characterized by PD-1 might contribute to the restriction of the inflammatory response in periapical lesions.⁵ T cell exhaustion is a state of T cell dysfunction caused by long-term exposure to antigen with high expression of coinhibitory molecules, such as PD-1, TIM-3, LAG-3, and CTLA-4, on the cell surface, leading to immunosuppression.⁸ Therefore, a strong positive correlation between the density of PD-1+ cells and CD4+ helper T cells observed in this study suggests that chronic exposure of helper T cells to bacterial antigens may induce T cell exhaustion mediated by PD-1 expression in chronic periapical lesions. In addition to helper T cells, FOXP3+ regulatory T cells also showed a strong positive correlation with PD-1 expression in this study. The implications of regulatory T cells in the control of periapical lesions have been demonstrated in previous studies.^3,7 Regulatory T cells modulate the immune system using their suppressive cell membrane molecules, such as PD-1, CTLA-4, CD39, and CD73.²¹ Taken together, it can be inferred that regulatory T cell activation is associated with PD-1 in chronic periapical lesions.

This study presents novel findings on the close link between the PD-1/PD-L1 pathway and T cell immunity in chronic periapical lesions using human tissue samples, possibly leading to the application of the immune checkpoint pathway in the clinical setting. Recent literature has presented new therapeutic approaches based on human and animal studies in periapical lesions.²² In the same context, this study suggests that activating the PD-1/PD-L1 pathway, which may limit T cell-mediated tissue damage at the site of infection, is a potential therapeutic strategy for chronic periapical lesions. However, further studies are needed to determine the opposite effects of immunosuppression on bacterial immune evasion and subsequent outcomes. Furthermore, since this study is limited by its relatively small sample size, large cohort studies are warranted to validate the role of the immune microenvironment in the progression of chronic periapical lesions. Further investigations using an expanded panel of immune cell markers may also be of value in elucidating the detailed mechanism of immune regulation in these lesions.

Conclusion

The results of this study suggest that cytotoxic T cells are implicated in the progression of chronic periapical lesions, which may be regulated by PD-L1+ macrophages. PD-1 may be involved in helper T cell exhaustion and regulatory T cell activity in chronic periapical lesions.

Conflict of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article.