Assessment of Serum Level of programmed cell death-1 (PD-1) in Patients with Vitiligo

 Background: Among the various checkpoint receptors, cytotoxic T lymphocyte-associated protein-4 (CTLA-4), programmed cell death protein-1 (PD-1) and T-cell immunoglobulin domain and mucin-3(Tim-3) have attracted more attentions and demonstrated promising results in therapeutic approaches in autoimmune diseases as vitilligo.

Objective: To assess the level of serum PD1 and correlate it with the severity of the disease in vitiligo patients, in a trial to clarify its role in vitiligo pathogenesis.

Methods: This study was carried out on 23 patients with vitiligo & 23 healthy controls. They were recruited from the outpatient clinic of Dermatology, Andrology & STDs department, Mansoura University Hospitals.

The following data were obtained for all patients and control group: age, sex, family history, skin phototype, type of vitiligo, and disease duration. Disease severity of patients with vitiligo was determined using the Vitiligo Area Severity Index (VASI).

All participants have done laboratory test for serum PD-1.

Results: Mean PD-1 showed non-statistically significant higher value among cases than control group (12.86 & 12.63, respectively) with (p=0.789). Receiver Operating characteristics curve was used to assess validity of PD-1 in differentiating cases from control group with area under curve was fair (0.595) with the best detected cut off point is 11.05 yielding sensitivity of 82.6% and 34.8% specificity. A non- statistically significant correlation was detected between PD-1 and the following; age, sex, marital status, family history, duration, skin phototype, type of vitiligo and VASI score (p>0.05).

Conclusions: In our study, inspite of small number of patients, it could be concluded that PD-1 seems to be implicated in the pathogenesis of vitiligo. Mean PD- 1 showed non-statistically significant higher value among cases than control group.

Vitiligo is a chronic, acquired, autoimmune pigmentary disorder that affects up to 2% of the population [1]. Various factors are involved in the pathogenesis of vitiligo including genetics, environment, oxidative stress as well as innate and acquired immunity [2]. In tumor immune evasion, immunological checkpoints are crucial. Targeting immunological checkpoints is sufficient to reverse peripheral tolerance and reawaken certain patients'  melanoma-specific cytotoxic T cells, according to research on immunotherapy for the disease. Growing interest in PD-1/PD-L1 as potential targets for vitiligo immunotherapies has been sparked by the similarities between vitiligo(autoimmunity) and melanoma (tumor immunity) [3].

An inhibitory cell surface molecule known as PD-1 or CD279 inhibits T lymphocytes. PD-1 binding on T cells to PD-1 ligands (PD-L1 and PD-L2) on other cell types is necessary for immune suppression. Ligation of PD-1/PD-L1 controls inflammatory reactions and autoimmune disorders by suppressing the activation and activity of autoreactive T cells, preventing their proliferation, and inducing death in these cells [4,5]. In a Pmel-1 vitiligo mouse model, [6] shown that treatment with PD-L1 fusion protein corrected depigmentation by studyingPD-1/PD-L1 signaling as a therapeutic target for vitiligo. The aim of the study: To assess the level of serum PD-1 and correlate it with the severity of the disease in vitiligo patients, in a trial to clarify its role in vitiligo pathogenesis.

A case control study was carried out on 23 vitiligo patients (group A) & 23 matched healthy controls (group B). All participants  were recruited from those attending the outpatient clinic of Dermatology, Andrology& STDs Department, Mansoura University Hospitals, Mansoura, Egypt.

Ethical consideration: The study protocol was approved by Institution Research Board (IRB) of Mansoura Medical College (R.23.01.2042.R1). All individuals in this study provided written informed consent. All levels of the study protected confidentiality and personal privacy. All data were only utilized for research reasons.

Inclusion criteria

Patients with classic clinical features of vitiligo of both sexes who did not receive any systemic treatment for at least 3 months.

Exclusion criteria

Patients on systemic treatment such as immuno suppressive or chemotherapy, pregnant and lactating women, past or current history of smoking, patients with other autoimmune diseases, malignant conditions, inflammatory skin disorders such as psoriasis, dermatitis, asthma and allergy.

All the participants were subjected to:

Thorough History taking: Personal history: age, sex, residence, occupation, family history and past history of systemic illness & previous therapy.

Complete physical examination: General dermatological examination for any associated skin diseases and any signs of systemic diseases.

Vitiligo Area Severity Index(VASI) score:

Disease severity of vitiligo patients was determined using the VASI score [7].

The area of vitiligo in hand units, which was set at 1% per unit, and the degree of depigmentation within each hand unit measured patch (possible values of 0, 10%, 25%, 50%, 75%, 90%, or 100%), were multiplied together to determine the VASI for each body region. The contributions of all body regions were then taken into account to compute the

total body VASI (possible range, 0-100) using the formula below: Wood’s light examination was used to determine any other affected areas.

All members of the study were subjected to measurement of serum level of Programmed Cell Death Protein 1 (sPDCD1/sPD1) ELISA Kit cat no: 201-12-7502. The kit used a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). Add soluble Programmed Cell Death Protein 1 (sPDCD1/sPD1) to monoclonal antibody Enzyme wells pre-coated with Programmed Cell Death Protein- 1 (PD1) monoclonal antibody, incubation. Then, add soluble Programmed Cell Death Protein 1 (PD1) antibodies labeled with biotin and combined with Streptavidin-HRP to form an immune complex; then carry out incubation and washing again to remove the uncombined enzyme. Then add Chromogen Solution A and B, changing the color of the liquid to blue. And at the effect of acid, the color finally becomes yellow. The samples' chroma of color and the concentration of the Programmed Cell Death Protein-1(PD1) were positively correlated.

SPSS software, version 25 (SPSS Inc., PASW statistics for Windows version 25), was used to analyze the data. SPSS Inc., Chicago. Number and percentage were used to describe qualitative data. For non-normally distributed data, the median (minimum and maximum) and mean were used to describe the quantitative data. Following the Shapiro-Wilk test to determine whether the data are regularly distributed, the standard deviation. The acquired results' significance was assessed at the (0.05)level.

Monte Carlo tests, the Fischer exact test, and Chi-Square testing were performed when necessary to compare qualitative data between groups. 

• In cases where the data were not normally distributed, the Mann Whitney U test was employed to compare the two study groups. The strength and direction of a linear link between two ordinal or continuous non-normally distributed variables are assessed using the Spearman's rank-order correlation. 

• Cross tabulation was used to evaluate accuracy and determine the best cutoff predicted values when calculating the validity (sensitivity & specificity) of continuous variables using the Receiver Operating Characteristics Curve (ROC curve).

Both groups were comparable as regard: age, sex, marital status, skin phototype and family history (Table-1). Among studied cases; median disease duration was 2 years ranging from 0.17 to 25 years, 69.6% of patients showed progressive disease course, 21.7% stable and 8.6% regressive course. VASI score distribution was as following; 13% (score -1),4.3% (score 0), 8.7% (score 1), 26.1% (score 2), 13% (score 3) and 34.8% (score 4). 73.9% of cases were vitiligo vulgaris, 21.7 % acrofacial and 4.3% focal vitiligo. 26.1% of patients have previous episodes of pigmentation (Table-2). Mean PD-1 showed a non-statistically significant higher value among cases than control group (12.86 &12.63, respectively) (Table-3).

Receiver Operating characteristics curve was used to assess validity of PD-1 in differentiating cases from control group with area under curve was fair (0.595) with the best detected cut off point was 11.05 yielding sensitivity of 82.6% and 34.8% specificity (Table-4). A non-statistically significant correlation was detected between PD-1 level and the following: age, duration, VASI score (p >0.05), sex (p=0.881), marital status (p=0.324), skin phototype (p=0.791) and family history (p=0.395) (Table-5). A non-statistically significant association was detected between mean PD-1 level and the following: disease duration (p=0.657), type of vitiligo (p=0.858) and previous episodes of pigmentation (p=0.805) (Table-6).

Table 1: Comparison of demographic characteristics between group A (vitiligo patients) and group B (controls).

t: Student t test, FET: Fischer exacttest, χ2: Chi-Square test

Table 2: Disease characteristics of the studied cases.

Table 3: Comparison of PD-1 between studied groups

Table 4: Validity of PD-1 in differentiating between cases and control group.

AUC: Area under curve

Table 5: Correlation between PD-1 & age, duration, VASI score (continuous variables), sex, marital status, skin phototype & family history (qualitative variables) among studied cases.

r: Spearman correlation coefficient, t: Student t test.

Table 6: Relation between PD-1 & disease characteristics (qualitative variables) among studied cases.

t: Student t test, F: One Way ANOVA test

Figure 1: PD-1 distribution among studied groups

Figure 2: ROC Curve for PD-1 in differentiating cases of vitiligo from control group

Our study demonstrated a non-statistically significant higher value of PD-1 among cases than control group (12.86 & 12.63, respectively) with (p=0.789). A non-statistically significant correlations were detected between PD-1 and the following; age, sex, marital status, family history, disease duration, skin phototype, type of vitiligo and VASI score (p >0.05). For patients with metastatic melanoma and many other malignancies, PD-1 targeting monoclonal antibodies are currently among the first-line therapy options [8].  Patients with metastatic melanoma are currently being studied for PD-L1-targeting antibodies, such as atezolizumab [9].

Inadequate PD-1/PD-L1 function has been linked to a number of autoimmune disorders, including type 1 diabetes, inflammatory bowel diseases, multiple sclerosis, rheumatoid arthritis, and vitiligo, according to mounting evidence. In diseases with abnormally regulated immune system activity, manipulating PD-1/PD-L1 signaling appearsto have considerable effects [10,11]. [12] assessed the status of regulatory T cells (Tregs), PD1+ Tregs, and levels of fork head box P3(FOXP3), transforming growth factor- β (TGF- β1), cytotoxicT-lymphocyte associated antigen-4(CTLA-4) and genes for chemokine receptors (CCR4, CCR7) and their ligands (CCL21, CCL22) in peripheral blood and in lesional, perilesional, non-lesional sections in active generalized vitiligo [50 patients] and normal skin sections [51 controls]. In comparison to controls, they discovered that patients with vitiligo had a lower percentage of Tregs and a higher percentage of PD1+Tregs. They proposed that elevated PD-1 expression on Tregs andCD4+ T cells may be influenced by autoreactive T cells that express PD-L1 and/or PD-L2, which may cause Treg fatigue and finally apoptosis, resulting in a lack of Tregs in vitiligo patients [12].

Repressing melanocyte-reactive T lymphocytes is the main component of an effective treatment plan for vitiligo [13]. Using Pmel-1 vitiligo mice, [6] evaluated the therapeutic impact of PD-L1 in vivo. For six weeks, 20 g of PD-L1 was injected intraperitoneally three times per week. In adult Pmel-1 mice, treatment with this fusion protein dramatically stopped the progression of depigmentation. They discovered that melanocyte-reactive T cell abundanceis suppressed and that Treg recruitment was considerably boosted by about a two-fold in mouse skin treated with PD-L1 fusion protein. Sadly, these therapeutic effects were not long-lasting [6]. PD-L1 expression can be increased on a variety of cell types in response to proinflammatory cytokines, such as IFN- [14]. Even while lesional vitiligo skin has a large number of IFN-producing CD8+ T cells, cytokine-induced PD-L1 expression may not be occurring or may be insufficient to create a negative feedback loop [3].

[15] investigated the frequency of Tim-3+/PD-1+/CD8+ T cells in 30 patients with vitiligo and 30 sex- and age-matched controls by flow cytometry. They found patients with vitiligo have shown significantly more expression of Tim-3 and PD-1 on their CD8+ T cells compared with controls. They reported a significant positive correlation between the frequency of PD-1+/CD8+, Tim-3+/CD8+, and PD-1+/Tim-3+/CD8+ with disease activity score of patients with vitiligo. They suggested that the inflammatory situation in vitiligo causes excessive activation of CD8+ T cells followed by more expression of PD-1 and Tim-3 to regulate the CD8+T-cell autoreactive response [15].

30 patients with non segmental vitiligo were examined by [16] to determine the pattern of expression of CTLA-4 and PD-1. Skin biopsies from the margins, lesions, and non-lesions were taken. Digital pictures were used to assess the immunohistochemistry expression in the mononuclear inflammatory infiltrates. When compared to non-lesional skin, they discovered that PD-1 and CTLA-4 were significantly expressed in the marginal and lesional infiltrates. Additionally, PD-1 was substantially more expressed at the margins than at the lesions. They suggested that the antimelanocytic, T-cell-mediated immune response in vitiligo takes place at the edge of active lesions. However, there was no comparable noteworthy distinction in CTLA-4expression.

The tissue levels of PD-1 and PD-L1 were examined by [17] from lesional and non-lesional biopsies in 30 patients with active non segmental vitiligo and compared to normal healthy controls. In comparison to both non-lesional and control levels, they discovered that the marginal PD-1 level was considerably higher. Furthermore, it was discovered that the non-lesional PD-1 level was substantially higher than the control level. This draws attention to the abnormal PD-1 expression in people with vitiligo. In comparison to both non-lesional and control levels, PD-L1 levels in marginal biopsies were considerably lower (p 001). Also, noticeably lower than in the skin of normal controls is non-lesional PD-L1. This implies that cytokine-induced PD-L1 expression in vitiligo is defective and fails to trigger the checkpoint mechanism necessary to end the autoimmune reaction, leaving melanocytes open to attack by melanocyte-reactive T cells [17].

In agreement with our study results, [17,16] reported that PD-1 levels were not related to patient demographics (age and sex) and clinical parameters, including VASI, disease duration, activity of the disease and clinical presentation. Our study had limitations, including, small number of patients and lack of tissue study. Also, we did not determine the cellular participants involved in the expression of PD-1/PD-L1 in vitiligo patients.

In our study, in spite of small number of patients, it could be concluded that PD-1 seems to be implicated in the pathogenesis of vitiligo.