Assessment of Programmed Cell Death (PD-1) and its Ligand’s (PD-L1) levels in vitiligo

doi:10.21203/rs.3.rs-2882969/v1

Background: Programmed cell death 1 (PD-1) is a cell surface protein that serves as an immune checkpoint in conjunction with its two ligands, PD-L1 and PD-L2. Recently, there has been a lot of interest in the role of the PD-1/PD-L1 pathway in immunoregulation. When PD-1/PD-L1 pathway inhibitors were used in cancer therapy, the observed aggravation of the pre-existing autoimmune disease and the emergence of autoimmune-like symptoms shed light on the potential therapeutic role of increasing PD-L1 expression or activating PD-1 in the fight against diseases with autoimmune pathology, including vitiligo. The present study aimed to assess both PD-1 and PD-L1 levels in vitiligo patients’ marginal and non-lesional biopsies in comparison with normal controls and to correlate them with disease parameters.

Methods: Thirty vitiliginous patients and 30 age and sex-matched controls were included. Full history and clinical examination were done and tissue levels of PD-1 and PD-L1 were measured by ELISA from lesional and non-lesional biopsies.

Results: Levels of tissue PD-1 in marginal biopsies were significantly higher than in non-lesional biopsies (p< .001) and significantly higher than the control PD-l level (p< .001). Non-lesional PD-1 level was also significantly higher than the control PD-l level (p< .001). A statistically significant positive correlation was found between marginal and non-lesional PD-1 levels; (rho=0.792, p< .001). Levels of tissue PD-1 in non-lesional biopsies were significantly higher than in controls (P<0.001) and levels of tissue PD-L1 in non-lesional biopsies were significantly lower than in controls (P<0.001). Non-lesional PD-L1 level was also significantly higher than the control PD-Ll level (p< .001).

Vitiligo

PD-1

PD-L1

Tolerance.

Vitiligo is a chronic skin depigmentation disorder, whose exact pathogenesis has yet to be determined. Many factors interact to cause the disease. The autoimmune (immune imbalance dysregulation) mechanism receives the most attention among these.[1][2]

Immune checkpoints are a class of molecules that regulate over-expressed immune responses to maintain immune homeostasis. They play an important role in peripheral tolerance to melanocyte antigens, and any disruption in these regulatory mechanisms can lead to melanocyte destruction and thus vitiligo.[3]

The inhibitory checkpoint receptor programmed cell death protein (PD-1), is a member of the CD28 inhibitory receptor family. It is expressed inducibly on T cells, B cells, and myeloid cells. It has two ligands, PD-L1 and PD-L2. PD-L1 is expressed on antigen-presenting cells (APCs), tissue, and tumor cells and PD-L2 is expressed mainly on APCs. T cells become inactive when either ligand binds to PD-1. [4]

The PD-1/PD-L1 pathway contributes to central tolerance to self-antigens by negatively selecting autoreactive T cells. Peripherally, upon T cell receptor engagement of naive T cells, PD-1 and PD-L1 are upregulated, either resulting in naive T cell inactivation and inhibition of effector T-cell differentiation and function or, in the presence of TGF-β, can induce the conversion of naive CD4⁺T cells into Foxp3+ induced regulatory T cells (T_reg), which in turn play an important role in peripheral tolerance. [5][6]

The PD-1/PD-L1 signaling pathway is involved in tumor immune escape and disrupting it may activate T cells against cancer cells. [7] The use of PD-1 inhibitors (pembrolizumab and nivolumab) and PD-L1 inhibitors (avelumab, durvalumab, and atezolizumab) inhibitors in cancer therapy resulted in remarkable skin manifestations, with psoriasiform rashes being the most common skin complication. Other skin complications that have been reported include bullous pemphigoid, lichenoid dermatitis, granulomatous skin reactions, and vitiligo.[8]

The development of vitiligo-like lesions (VLL) in patients receiving anti-PD-1/PD-L1 therapy for melanoma highlights the critical role of PD-1/PD-L1 in vitiligo pathogenesis, which could be attributed to a breakdown in tolerance to self-antigens (MART-1, gp100, and tyrosinase-related proteins 1 and 2). [9]

The occurrence of VLL in patients receiving PD-1/PD-L1 inhibitors for the treatment of cancers other than melanoma demonstrates that this phenomenon is caused by PD-1/PDL-1 blockade and is not caused by or dependent on the underlying melanoma. [10]

Furthermore, in mice with pre-melanosomal protein-1 vitiligo, treatment with a PD-L1 fusion protein reversed the development of depigmentation. By increasing the number of T_reg and suppressing melanocyte-reactive T cells. Due to its importance in melanocyte salvage, PD-L1 fusion protein could be a promising treatment option for vitiligo patients. [11]

This study aimed to assess the level of the PD-1 receptor and its main ligand, PD-L1, in margins of affected and unaffected skin of active nonsegmental vitiligo and compare these levels with normal healthy controls. We also sought to explore patients’ PD-1 and PD-L1 expression in relation to their demographic and clinical parameters, including duration of disease, activity (by VIDA score), and severity (by VASI score). Through this study, we hope to pave the way for new treatment modalities for vitiligo targeting PD-1 pathways.

This study was conducted at the dermatology outpatient clinic, Kasr Al-Ainy Hospital, Faculty of Medicine, Cairo University after approval of the Faculty of Medicine Research Ethical Committee code MD-62-2019.

Written informed consent was obtained from all patients and controls. The study was conducted on 30 patients with active vitiligo and 30 sex and age-matched healthy controls, during the time interval from December 2019 and March 2021. Patients with active vitiligo VIDA 1+ or more were included, as well as all clinical variants of vitiligo except segmental type. Patients who received topical treatment for vitiligo for less than 2 weeks or systemic treatment for vitiligo for less than 4 weeks were excluded, as were PD-1-associated skin immune diseases (e.g., psoriasis, atopy, and bullous pemphigoid) and patients with a present or past history of skin cancer.

All patients were subjected to detailed history taking, which included the onset, course, and duration of the disease, precipitating factors, and the date of the most recent sign of activity (appearance of new lesions or extension of current lesions or recurrence of old lesions). Any previous history of systemic or dermatological diseases. In addition, a drug history (any recent topical or systemic treatment).

Clinical assessment:

all patients were assessed clinically to determine, Type of vitiligo, Signs of activity: confetti-like depigmentation, trichrome pattern, inflammation, Koebnerisation, and Body surface area (BSA) affected were noted for all patients.

Vitiligo activity was scored according to the vitiligo disease activity (VIDA) score[12]. Vitiligo area scoring index (VASI) [13]

The control group included 30 normal individuals presenting to the Plastic Surgery Department, for abdominoplasty. All had no history of any dermatological disease.

Skin biopsy:

One 3 mm punch biopsy was taken from the margin of a vitiliginous patch and another 3 mm punch biopsy was taken from unaffected non-lesional skin at least 10 cm away from the closest vitiligo patch. Biopsies were taken following sterilization with povidone-iodine (Betadine®) and using lidocaine 1% (Xylocaine®) as local infiltration anesthesia.

A 3 mm punch biopsy of normal skin was also taken from the skin of each control during their plastic surgery. The skin biopsies were preserved in a tube containing phosphate-buffered saline and kept frozen at -80 C⁰ for the ELISA study.

PD-1 and PD-L1 were measured using an ELISA kit supplied by Life span biosciences. The optical density (OD value) of each well was determined using a microplate reader set to 450 nm. The OD of an unknown sample was compared to an OD standard curve generated using known antigen concentrations to determine its antigen concentration.

Statistical Methods:

Sample size calculation:

The main outcome variable is the difference in PD-1 and PD-L1 levels in affected vs unaffected skin within patients, to be statistically tested by Wilcoxon signed-rank test. Since this is the first study to be conducted on these parameters in vitiliginous skin, a moderate effect size was assumed as per Cohen’s 1988 criteria, with effect size d=0.5. A statistical power analysis was performed for sample size estimation; with a two-tailed alpha error probability set at 0.05, the projected sample size needed for the aforementioned effect size (d=0.5) is approximately N = 30 vitiligo patients to be able to reject the null hypothesis that this difference is zero with probability (power) 0.8. For reference to normal skin, an equally sized sample of healthy controls was selected. Sample size calculation was done using G*Power 3.1.9.2.

Data were coded and entered using the statistical package RStudio (R Core Team, 2017). Data were summarized using mean, standard deviation, median, minimum, and maximum in quantitative data and using frequency (count) and relative frequency (percentage) for categorical data. Comparisons between quantitative variables were done using the non-parametric Mann- Whitney test, for comparison of paired measurements within each patient, the non-parametric Wilcoxon signed-rank test was used. For comparing categorical data, Chi-square test was performed. An exact test was used instead when the expected frequency is less than 5. Correlations between quantitative variables were done using the Spearman correlation coefficient. P-values less than 0.05 were considered statistically significant.

A) Demographic characteristics of the studied groups

1. Patients group:

The patients included 13 males (43.33%) and 17 females (56.67%). Their mean age was 37.3 ± 16. All patients were clinically active with VIDA scores ranging between 2 and 4. The samples were representative of the various extents of active vitiligo, with a mean VASI score of 5.37 ± 2.38, and mean body surface area (BSA) affected 6.39% ± 8.97. (Table 1) shows the demographic and clinical characteristics of the patients’ group.

2-Control group

were also 13 males (43.33%) and 17 females (56.67%) and their ages ranged between 20 and 58 years with a mean of 35.3 ± 18.5. Patient and control groups were matched as regards age and sex. (P-value > 0.05).

Table 1: Demographic and clinical characteristics of the patients

Age (Years)	Range	18 - 70
Age (Years)	Mean± SD	37.3 ± 16.6
Sex N (%)	Males	13 (43.33%)
Sex N (%)	Females	17 (56.67%)
Duration (Years)	Range	0.1 - 35
Duration (Years)	Mean± SD	7.59 ± 8.97
Variant N (%)	Acral	2 (6.7 %)
	Acrofacial	4 (13.3 %)
	Focal	2 (6.7 %)
	Generalized	22 (73.3 %)
Signs of activity N (%)	Spreading	29 (96.7 %)
	Confetti like depigmentation	5 (16.7 %)
	Trichrome	5 (16.7 %)
	Inflammatory	1 (3.3 %)
	Koebner	12 (40.0 %)
VIDA score N (%)	2+	2 (6.7 %)
	3+	7(23.3 %)
	4+	21 (70.0 %)
BSA	Range	0.2 and 36 %
BSA	Mean± SD	6.39 ± 8.97
VASI	Range	0.15 and 32.4
VASI	Mean± SD	5.37 ± 2.38

^{The vitiligo disease activity score (VIDA), body surface area (BSA), Vitiligo area scoring index (VASI)}

B) Level of PD-1 and PD-L1 in patients and control group:

• In healthy controls:

The level of PD-1 (ng/mg protein) ranged between 0.91 and 2.70 with a mean of 1.47 ± 0.499; while the level of PD-L1 (pg/mg protein) ranged between 225and 324, with a mean of 283 ± 27.8.

• In patients:

i. In marginal biopsy:

The level of PD-1 (ng/mg protein) ranged between 3.20 and 10.9 with a mean of 7.89 ± 2.48; while the level of PD-L1 (pg/mg protein) ranged between 102 and 129, with a mean of 115 ± 7.86.

ii. In non-lesional biopsy:

The level of PD-1 (ng/mg protein) ranged between 2.01 and 5.01 with a mean of 3.65 ± 1.11; while the level of PD-L1 (pg/mg protein) ranged between 180 and 324, with a mean of 194 ± 8.12.

C) Comparison of PD-1 and PD-L1 in patients & control groups:

PD-1 levels in study groups

PD-1 levels were significantly higher in marginal biopsies than in non-lesional biopsies and in marginal biopsies than in control biopsies (p<001) (mean difference 4.2 and 6.69 ng/mg protein, respectively). Non-lesional biopsies also had significantly higher PD-1 levels than control biopsies (p<001), with a mean difference of 2.23 ng/mg protein. (Figure 1)

PD-L1 levels in study groups

PD-L1 levels in marginal biopsies were significantly lower than both non-lesional PD-l levels and control PD-Ll levels (p<001) (mean difference 79.65 and 171.9 pg/mg protein, respectively). Non-lesional PD-1 level was also found to be significantly lower than the control PD-l level (p< .001). with a mean difference of 92.2 pg /mg protein. (Figure 2)

Patients’ PD-1, PD-L1 in different disease parameters

There was no significant difference in the mean level of PD-1 or PD-L1 in different clinical variants or in patients presenting with different signs of activity, in both marginal and non-lesional biopsies. (p > 0.05). (Table2, Table 3)

Table 2: Comparison of PD-1 in patients and subgroup analysis

PD-1 level (ng/mg protein)		Marginal		Nonlesional	p value
PD-1 level (ng/mg protein)		Mean ±SD		Mean ± SD	p value
In all patients		7.89 ± 2.48		3.65 ± 1.11	p< .001
	Subgroup	Mean ±SD	p value	Mean ± SD	p value
Clinical variant	Acral (N=2)	7.89 ± 3.9669	0.126	3.90 ± 1.4142	0.383
	Acrofacial (N=4)	7.38 ± 1.8360		3.54 ± 1.2750
	Focal (N=2)	9.60 ± 0.1414		4.15 ± 0.0707
	Generalised (N=22)	7.82 ± 2.6374		3.60 ± 1.1603
Signs of activity	Spreading (N=29)	7.98 ± 2.47	0.247	3.68 ± 1.12	0.685
	Confetti-like depigmentation (N=5)	7.52 ± 3.11	0.597	3.31 ± 1.15	0.539
	Trichrome (N=5)	7.2 ± 2.89	0.577	3.68 ± 1.32	1
	Inflammatory (N=1)	3.2	0.118	2.01	0.118
	Koebner (N=12)	8.68 ± 2.4	0.132	3.96 ± 1.19	0.111

^{*p < 0.05 is significant, **p< 0.01= highly significant}

Table 3: Comparison of PD-L1 in patients and subgroup analysis

PD-L1 level (pg/mg protein)		Marginal		Nonlesional	p value
PD-L1 level (pg/mg protein)		Mean ±SD		Mean ± SD	p value
In all patients		115 ± 7.86		194 ± 8.12	p< .001
	Subgroup	Mean ±SD	P value	Mean ± SD	p value
Clinical variant	Acral (N=2)	104.95 ± 3.3234	0.137	202.30 ± 3.5355	0.142
	Acrofacial (N=4)	118.32 ± 11.3388		185.88 ± 5.6630
	Focal (N=2)	116.45 ± 3.1820		194.80 ± 13.4350
	Generalised (N=22)	114.52 ± 7.3484		195.05 ± 7.5498
Signs of activity	Spreading (N=29)	114.77 ± 7.87	0.326	193.93 ± 8.01	0.166
	Confetti-like depigmentation (N=5)	121.88 ± 6.85	0.032*	191.44 ± 7.28	0.404
	Trichrome (N=5)	112.84 ± 6.2	0.697	199.82 ± 6.98	0.107
	Inflammatory (N=1)	119.4	0.418	191.3	0.729
	Koebner (N=12)	114.76 ± 8.69	0.966	194.68 ± 6.35	0.735

^{*p < 0.05 is significant, **p< 0.01= highly significant}

There was no correlation between PD-1 or PD-L1 and the demographic and clinical data of the patients as regards their age, disease duration, BSA affected, VIDA score (representing activity) nor VASI score (representing severity) (Table 4)

Table 4: Correlation between clinical characteristics and PD-1, PD-L1 in patients

	Marginal PD-1		Marginal PD-L1		Nonlesional PD-1		Nonlesional PD-L1
	ρ	p value	ρ	p value	ρ	p value	ρ	p value
Age	- 0.029	0.879	-0.248	0.186	-0.027	0.885	-0.045	0.813
BSA	-0.140	0.460	0.060	0.752	-0.095	0.619	-0.224	0.234
Duration	-0.227	0.228	0.184	0.330	-0.091	0.633	-0.435	0.016

VASI	-0.169	0.373	0.075	0.695	-0.143	0.452	-0.215	0.254

VIDA	-0.286	0.125	-0.090	0.635	-0.176	0.353	0.157	0.409

^{ρ= Spearman’s rho correlation coefficient, *p < 0.05 is significant, **p< 0.01= highly significant}

There was no correlation between level of PD-1 and PD-L1 in controls, marginal nor non-lesional biopsies with Spearman's rho = -0.084, -0.181,0.044 and p-value=0.658,0.337,0.818 respectively

There was a strong positive correlation (rho=0.792) that was highly statistically significant (p< .001) between marginal and non-lesional PD-1 levels in patients, as visualized with the upward trending line of best fit-in but There was no significant correlation between marginal and non-lesional PD-L1 levels in patients (rho=-0.321, p=0.084) (Figure 3)

The observed aggravation of the pre-existing autoimmune disease and the emergence of autoimmune-like symptoms when PD-1/PD-L pathway inhibitors were used in cancer therapy shed light on the potential therapeutic role of increasing PD-L expression or activating PD-1 in the fight against diseases with autoimmune pathology including vitiligo.[14]

PD1 is thought to be a marker of T-cell exhaustion. Patients with active vitiligo had higher levels of circulating PD1⁺ T_regs and CD3⁺CD4⁺PD1⁺ T cells than healthy people, implying that regulatory and effector T cells are depleted in vitiligo.[15]

The present study aims to assess quantitatively the expression of PD-1 receptor and its main ligand, PD-L1, in active nonsegmental vitiligo and compare these levels with normal healthy controls.

Marginal PD-1 level was significantly higher than both non-lesional PD-l level and control PD-l level. Non-lesional PD-1 level was also found to be significantly higher than the control PD-l level. Previous research on PD-1 expression in human vitiliginous skin included an uncontrolled immunohistochemical study on the skin of 30 active vitiligo patients by Awad et al. in 2020. In concordance with our results, they detected PD-1 expression in all marginal biopsies, 83.33% of lesional biopsies, and in 26.67% of non-lesional biopsies. Comparing the number and percentage of PD-1⁺ cells among the three areas revealed a highly significant difference (P < 0.001), with PD-1 expression being highest in marginal biopsies and lowest in non-lesional biopsies [16]

The authors proposed that the higher expression of PD-1 in the marginal skin could be attributed not only to the abundance of the infiltrates there but also to a lower CD4⁺/CD8⁺ T-cells ratio, as a lower CD4⁺/CD8⁺ T-cell ratio is accompanied by more T-cell exhaustion and PD-1 expression. Similarly, Willemsen et al. Detected PD-1 in all perilesional vitiligo skin biopsies from active non-segmental vitiligo patients, with 75% of them presenting in T cells. [17]

Our results confirm the significantly higher PD-1 expression in marginal than non-lesional skin and additionally show that both are significantly higher than PD-1 expression in the skin of normal controls, highlighting the aberrance in PD-1 expression in vitiligo patients. We suggest that the overexpression of PD-1 in vitiligo skin, particularly the margins which contain the densest CD8⁺ T cell infiltration [18][19], is due to PD-1⁺CD8⁺ effector T cells that clonally expand in local chronic inflammations.

In the present study, PD-1 levels in both marginal and non-lesional sites were not related to patient demographics (age and sex) and clinical parameters, including VASI, VIDA, duration, and clinical presentation. These findings match Awad et al., 2020, who also found no correlation between the number and percentage of PD-1⁺ cells and disease parameters including VETI score, VIDA score, duration, activity of the disease, and age of the patients. [16]

The expression of PD‐1 mRNA in CD8+ T cells isolated from the blood of vitiligo patients was positively correlated with the VASI score (p = 0.0552, r = 0.4592) and suggested that this might imply an attempt of the immune system to control the widespread inflammation by up-regulation of inhibitory checkpoint receptors [15]. We didn’t find such a correlation in our study. This discrepancy might be due to the different samples and selection criteria for patients, as Rahimi et al. weren’t exclusively selecting active cases as per the VIDA score. In contrast, in our study, all patients were VIDA≥2. Thus, it is likely that disease activity regardless of extent is the primary driver of PD-1 expression.

Interestingly, we discovered a significant strong positive correlation between PD-1 levels in non-lesional biopsies and marginal biopsies. This is the first study to find this link, highlighting the role of PD-1 in active non-segmental vitiligo that is relevant regardless of clinical or demographic factors. Since marginal infiltrates are known to be abundant in CD8⁺ cells and have a lower CD4⁺/CD8⁺ T-cells ratio, we can infer that the increased PD-1 expression detected in our study is also largely due to excessive activation of autoreactive CD8⁺ T cells.

No other human studies have looked specifically at PD-1 expression by skin-resident regulatory or memory T cells in vitiligo.[20] A mouse model of melanoma‐associated vitiligo showed that antigen‐specific CD8⁺ tissue-resident memory cells (TRM) cells within depigmented hair follicles lacked PD‐1 expression.[21] However, in a vitiligo mouse model, autoreactive CD8⁺ TRM cells were found to express PD‐1 [22], which would help explain our observation that the density of PD-1 expression close to lesions was markedly higher than that far away in unaffected skin. Future research should be encouraged to determine whether PD-1 is expressed by TRM cells in vitiligo lesions, as this would pave the way for the development of topical or local PD-1 targeting therapies that could help prevent recurrence.[23] .

Unlike PD-1, PD-L1 expression in skin disease remains largely unstudied. In stark contrast to the PD-1 overexpression, our results demonstrated an opposite pattern of PD-L1 expression, with lesional PD-L1 significantly lower than both non-lesional PD-l level and control PD-Ll level and non-lesional PD-1 level was also found to be significantly lower than control PD-l level.

No relationship was found between PD-L1 levels in either marginal or non-lesional sites and any of the demographic or clinical parameters. Unlike PD-1, the relation between marginal and non-lesional PD-L1 showed a weak downward trend, although statistical significance was not established (rho= -0.321, p=0.084).

Willemsen et al recently published data that supported the findings of the current study by demonstrating that melanocytes and epidermal skin cells from perilesional vitiligo skin lacked PD-L1 expression, with only dermal T cells expressing it. Furthermore, melanocytes derived from non-lesional vitiligo were unable to express PD-L1 upon IFN-γ exposure, unlike other skin cells. [17]

IFN-γ is a critical cytokine in vitiligo pathogenesis, and IFN-γ producing CD8⁺ T cells are abundantly present in lesional vitiligo skin [24]. Therefore, one might expect PD-L1 expression to be upregulated in active vitiligo lesions. However, our results demonstrated that, to the contrary, PD-L1 is under-expressed in margins of active vitiligo lesions compared to unaffected skin. Moreover, in unaffected skin, PD-L1 is also significantly lower than in the skin of normal controls. This suggests that in vitiligo, cytokine-induced PD-L1 expression is dysfunctional and fails to activate the checkpoint mechanism required to stop the autoimmune reaction, leaving melanocytes vulnerable to melanocyte-reactive T-cell attack.

This finding lends support to the theory that vitiligo pathogenesis is aided by abnormal PD-L1 expression. It also explains why active vitiligo patients are more likely than healthy controls to spread the disease to distant sites, as their unaffected skin has lower PD-L1 expression. These sites may be affected later if their PD-L1 levels are insufficient to suppress the incoming PD-1⁺ melanocyte-reactive effector T cells.

Since PD-L1 has a pivotal role in regulating induced T _reg development and function[25], we expect that T_regs fail to develop and are unable to suppress the expanding autoreactive effector T cells.

The lack of PD-L1 expression in vitiligo sheds new light on the mechanisms underlying UV phototherapy. It is believed that UVB induces PD-L1 expression in melanocytes via the stress response signaling pathways HMGB1-Activated IRF3 and NF-κB [26] thus playing a critical role in suppressing local immune responses by inducing the PD-1 checkpoint, which relieves autoimmune inflammation such as vitiligo while also allowing premalignant cells to escape immune surveillance, predisposing to epidermal cancers and melanoma. [27]

We used ELISA to measure PD-1 and PD-L1 expression due to financial constraints. This limited the results to only general quantification, so we cannot determine the anatomical site, nor the cellular participants involved in the skewed expression of PD-1/PD-L1 in vitiligo. Thus, we recommend using flowcytometry or double staining immunohistochemistry to determine whether the increased PD-1 is due to T cytotoxic, T_reg, or T_RM cells and whether the decreased PD-L1 is due to melanocytes, keratinocytes, or dendritic cells.

We also propose that the study be expanded to include stable and segmental vitiligo, as well as clinical subgroups of nonsegmental vitiligo and that changes in PD-1 and PD-L1 expression be monitored after active disease stabilization.

Our results suggest that the PD-1/PD-L1 checkpoint seems to be implicated in the loss of peripheral tolerance in human vitiligo, with PD-1 being highly expressed on infiltrating T cells yet insufficiently stimulated due to a lack of local PD-L1 expression. This raises hope that PD-1/PD-L1 checkpoint activation may succeed in stabilizing and reversing depigmentation, whether indirectly by inducing upstream transcription factors such as in UB-UVB therapy or directly using PD-L1 fusion protein therapy as demonstrated in murine vitiligo. At the same time, PD-1/PD-L1 expression on the various cell types in the skin, including T cell subsets (CTL, T_reg, and TRM), melanocytes, dendritic cells, B cells, keratinocytes, and fibroblasts, remain understudied and might help develop a more nuanced understanding of the checkpoint's various functions.

Picardo M, Dell’Anna ML, Ezzedine K, Hamzavi I, Harris JE, Parsad D, et al. Vitiligo. Nature Reviews Disease Primers 2015 1:1. 2015 Jun 4;1(1):1–16.
Sandoval-Cruz M, García-Carrasco M, Sánchez-Porras R, Mendoza-Pinto C, Jiménez-Hernández M, Munguía-Realpozo P, et al. Immunopathogenesis of vitiligo. Autoimmun Rev. 2011 Oct 1;10(12):762–5.
Speeckaert R, van Geel N. Vitiligo: An Update on Pathophysiology and Treatment Options. Am J Clin Dermatol. 2017 Dec 1;18(6):733–44.
Jubel JM, Barbati ZR, Burger C, Wirtz DC, Schildberg FA. The Role of PD-1 in Acute and Chronic Infection. Front Immunol. 2020 Mar 24;11.
Francisco LM, Sage PT, Sharpe AH. The PD-1 pathway in tolerance and autoimmunity. Immunol Rev. 2010 Jul 1;236(1):219–42.
Gianchecchi E, Delfino DV, Fierabracci A. Recent insights into the role of the PD-1/PD-L1 pathway in immunological tolerance and autoimmunity. Autoimmun Rev. 2013 Sep 1;12(11):1091–100.
Jiang X, Wang J, Deng X, Xiong F, Ge J, Xiang B, et al. Role of the tumor microenvironment in PD-L1/PD-1-mediated tumor immune escape. Molecular Cancer 2019 18:1. 2019 Jan 15;18(1):1–17.
Bhardwaj M, Chiu MN, Pilkhwal Sah S. Adverse cutaneous toxicities by PD-1/PD-L1 immune checkpoint inhibitors: pathogenesis, treatment, and surveillance. Cutan Ocul Toxicol. 2022;41(1):73–90.
Hua C, Boussemart L, Mateus C, Routier E, Boutros C, Cazenave H, et al. Association of Vitiligo With Tumor Response in Patients With Metastatic Melanoma Treated With Pembrolizumab. JAMA Dermatol. 2016 Jan 1;152(1):45–51.
Liu RC, Consuegra G, Chou S, Fernandez Peñas P. Vitiligo-like depigmentation in oncology patients treated with immunotherapies for nonmelanoma metastatic cancers. Clin Exp Dermatol. 2019 Aug 1;44(6):643–6.
Miao X, Xu R, Fan B, Chen J, Li X, Mao W, et al. PD-L1 reverses depigmentation in Pmel-1 vitiligo mice by increasing the abundance of Tregs in the skin. Scientific Reports 2018 8:1. 2018 Jan 25;8(1):1–6.
Njoo MD, Das PK, Bos JD, Westerhof W. Association of the Köbner phenomenon with disease activity and therapeutic responsiveness in vitiligo vulgaris. Arch Dermatol. 1999;135(4):407–13.
Hamzavi I, Jain H, McLean D, Shapiro J, Zeng H, Lui H. Parametric modeling of narrowband UV-B phototherapy for vitiligo using a novel quantitative tool: the Vitiligo Area Scoring Index. Arch Dermatol. 2004 Jun;140(6):677–83.
Curnock AP, Bossi G, Kumaran J, Bawden LJ, Figueiredo R, Tawar R, et al. Cell-targeted PD-1 agonists that mimic PD-L1 are potent T cell inhibitors. JCI Insight. 2021 Oct 22;6(20).
Rahimi A, Hossein-Nataj H, Hajheydari Z, Aryanian Z, Shayannia A, Ajami A, et al. Expression analysis of PD-1 and Tim-3 immune checkpoint receptors in patients with vitiligo; positive association with disease activity. Exp Dermatol. 2019 Jun 1;28(6):674–81.
Awad SS, Touni AA, Gabril MY. Expression of immune checkpoints in active nonsegmental vitiligo: a pilot study. Int J Dermatol. 2020 Aug 1;59(8):982–8.
Willemsen M, Krebbers G, Tjin EPM, Willemsen KJ, Louis A, Konijn VAL, et al. IFN-γ-induced PD-L1 expression on human melanocytes is impaired in vitiligo. Exp Dermatol. 2022 Apr 1;31(4):556–66.
Luiten RM, Van Den Boorn JG, Konijnenberg D, Dellemijn TAM, Van Der Veen JPW, Bos JD, et al. Autoimmune destruction of skin melanocytes by perilesional T cells from vitiligo patients. Journal of Investigative Dermatology. 2009 Sep 1;129(9):2220–32.
Wu J, Zhou M, Wan Y, Xu A. CD8 + T cells from vitiligo perilesional margins induce autologous melanocyte apoptosis. Mol Med Rep. 2013 Jan 1;7(1):237–41.
Willemsen M, Linkutė R, Luiten RM, Matos TR. Skin-resident memory T cells as a potential new therapeutic target in vitiligo and melanoma. Pigment Cell Melanoma Res. 2019 Sep 1;32(5):612–22.
Malik BT, Byrne KT, Vella JL, Zhang P, Shabaneh TB, Steinberg SM, et al. Resident memory T cells in the skin mediate durable immunity to melanoma. Sci Immunol. 2017 Apr 14;2(10).
Richmond JM, Strassner JP, Rashighi M, Agarwal P, Garg M, Essien KI, et al. Resident Memory and Recirculating Memory T Cells Cooperate to Maintain Disease in a Mouse Model of Vitiligo. Journal of Investigative Dermatology. 2019 Apr 1;139(4):769–78.
Willemsen M, Melief CJM, Bekkenk MW, Luiten RM. Targeting the PD-1/PD-L1 Axis in Human Vitiligo. Front Immunol. 2020 Nov 6;11.
Yang L, Wei Y, Sun Y, Shi W, Yang J, Zhu L, et al. Interferon-gamma inhibits melanogenesis and induces apoptosis in melanocytes: A pivotal role of CD8 + cytotoxic T lymphocytes in vitiligo. Acta Derm Venereol. 2015;95(6):664–70.
Francisco LM, Salinas VH, Brown KE, Vanguri VK, Freeman GJ, Kuchroo VK, et al. PD-L1 regulates the development, maintenance, and function of induced regulatory T cells. Journal of Experimental Medicine. 2009 Dec 21;206(13):3015–29.
Wang W, Chapman NM, Zhang B, Li M, Fan M, Nicholas Laribee R, et al. Upregulation of PD-L1 via HMGB1-activated IRF3 and NF-κB contributes to UV radiation-induced immune suppression. Cancer Res. 2019 Jun 6;79(11):2909.
Wang W, Wu ZH. Immune checkpoint molecules: “new” kids on the block of skin photoimmunology. Genes Dis. 2019 Jan 1;8(1):1–5.

No competing interests reported.

Assessment of Programmed Cell Death (PD-1) and its Ligand’s (PD-L1) levels in vitiligo

Status:

Version 1

Abstract

Figures

Introduction

Patients and methods

Results

Discussion

Conclusions

References

Additional Declarations

Status:

Version 1