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BRIEF REPORT Table of Contents  
Ahead of print publication
Circulating follicular T-helper cells in patients with nonsegmental vitiligo


1 Department of Dermatology and Venereology, Faculty of Medicine, Tanta University, Tanta, Egypt
2 Department of Clinical Pathology, Faculty of Medicine, Tanta University, Tanta, Egypt

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Date of Submission08-Nov-2020
Date of Decision15-May-2021
Date of Acceptance08-Aug-2021
Date of Web Publication16-Nov-2021
 

  Abstract 


Vitiligo is a common depigmenting dermatosis with an autoimmune background. Circulating T-follicular helper (Tfh) cells are reported to increase and correlate with disease activity and autoantibody production in several human autoimmune diseases. The potential contribution of Tfh cells in vitiligo pathogenesis has not been well elucidated yet. Hence, this study aimed to evaluate the frequency of circulating Tfh cells in patients with nonsegmental vitiligo (NSV) in comparison to normal healthy subjects to detect their possible role in vitiligo pathogenesis. Venous blood samples were obtained from 25 patients with NSV and 20 age- and sex-matched healthy controls to determine the level of circulating Tfh cells (CD3+ CD4+ CXCR5+ PD-1+ T cell population), by flowcytometry. The frequency of peripheral CXCR5+ PD-1+ lymphocytes which are CD3+ CD4+ was significantly augmented in vitiligo patients than controls. It showed a significant positive correlation with vitiligo disease severity and activity estimated by Vitiligo Area Scoring Index and Vitiligo Disease Activity scores, respectively. These results emphasize on the potential role of Tfh cells in the pathogenesis, extent, and severity of NSV. Antagonizing the autoimmune initiating action of Tfh cells population or targeting their cytokine secretion might offer a helpful future tool for the treatment of patients with NSV particularly in active cases.

Keywords: Autoimmunity, T-follicular helper cells, vitiligo


How to cite this URL:
Hassan GF, Abdel-Dayem MM, Saad MA, Hegab DS. Circulating follicular T-helper cells in patients with nonsegmental vitiligo. Dermatol Sin [Epub ahead of print] [cited 2021 Dec 5]. Available from: https://www.dermsinica.org/preprintarticle.asp?id=330485





  Introduction Top


Vitiligo is the most frequent acquired inflammatory autoimmune depigmenting skin disorder; its prevalence is 0.5%–2.0%. It is caused by the loss of active functioning melanocytes, leading to the development of white macules that can be psychologically devastating and stigmatizing, especially in dark-skinned individuals.[1]

Vitiligo is a multifactorial disease and the autoimmune theory is the most acceptable one due to the prevalence and high frequency of autoantibodies in vitiligo patients in addition to the association with many other autoimmune diseases.[2]

T-follicular helper cells (Tfh) are identified as a specific subset of T-helper cell with unique surface markers as well as receptors, showing increased expression of CXC-chemokine receptor 5 (CXCR5), inducible co-stimulator (ICOS), programmed cell death protein-1 (PD-1), and downregulation of Chemokine receptor 7 (CCR7) besides cytokine production in the high levels of interleukin (IL)-21 and specific transcription factor (Bcl-6) upon activation.[3] Tfh cells play an active role in human autoimmune mechanisms through migration into the B-cell follicles in lymphoid organs and stimulating the cytokine signals with proliferation of B-cells, building a germinal center reaction, leading to antibody-secreting plasma cells and memory cells production.[4]

The mechanism of production of autoantibodies in vitiligo remains unclear. As increased level of IL-21 in vitiligo has been previously reported, the involvement of Tfh cells in vitiligo pathogenesis and disease progression is suggested. Tfh cells and IL-21 not only play a key role in B-cell activation but they are also thought to act as possible players in the immunedysregulation, autoimmunity, and promotion of an increase in CD8+ T-cells that mediate cytotoxic responses against body own tissues.[5] Tfh cells in blood (CXCR5+) are commonly termed circulating Tfh cells. Unfortunately, data clarifying circulating Tfh cell alterations in vitiligo are sparse in the literature. Hence, the aim of this study was to evaluate the frequency of circulating Tfh cells in patients with nonsegmental vitiligo (NSV) in comparison with controls to detect their possible role in vitiligo pathogenesis.


  Methods Top


The study included 25 patients with NSV and 20 normal volunteers (control group). The study was approved by the research ethics committee of the Faculty of Medicine, Tanta University (approval code: 31971 / 12/17) and an informed written consent was signed by each participant. Complete history taking, general examination, dermatological examination to assess extent, severity, and activity of vitiligo according to the Vitiligo Area Scoring Index (VASI),[6] and Vitiligo Disease Activity (VIDA)[7] scores were done.

Patients with segmental vitiligo, those who received systemic treatment or phototherapy in the preceding 3 months before the study, pregnant and lactating females, those with other autoimmune diseases were excluded from the study.

Five ml venous peripheral blood sample was withdrawn from patients and controls and testing the sample was done within 24 h from collection by flowcytometry to define circulating Tfh populations which are CXCR5+ PD-1+ within CD3+ CD4+ T-cell population in peripheral blood and to calculate their percentage.[8],[9]

Equipment used

Flowcytometer: FACSCalibur Becton Dickinson (BD Biosciences, Heidelberg, Germany) and FlowJo 7.6 single cell analysis software (Tree Star Inc., Ashland, USA.)

Statistical analysis

Data were fed to the computer and analyzed using IBM SPSS software package version 20.0 (v 16; SPSS Inc., Chicago, IL, USA).


  Results Top


The demographic and clinical data of patients are summarized in [Table 1]. There was no significant difference between patients and controls as regarding gender or age. The frequency of peripheral Tfh (CXCR5+ PD-1+) in vitiligo patients group ranged from 5% to 11.8% with a mean of 7.37 ± 1.24 standard deviation (SD), and in control group, it ranged from 0.98% to 3.50% with a mean of 2.17 ± 0.93 (SD). The frequency of Tfh cells was elevated in NSV patients in comparison with controls [Figure 1], and the difference was statistically significant (P < 0.05).
Table 1: Clinical and flowcytometric data of nonsegmental vitiligo patients' group versus controls

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Figure 1: (a) Forward and side scatters show gated lymphocytes populations, (b) Dot blot shows double positive CD3+ CD4+ T-cells expression.(c and d) Representative dot blot profiles show the frequency of circulating CD185 (CXCR5)+ CD279 (PD-1)+ T-follicular helper cells expression, (c) In healthy control subject, (d) in nonsegmental vitiligo patient.

Click here to view


The frequency of peripheral Tfh in patients' group had no significant correlation with disease duration (r = ‒0.27, P = 0.19). However, it showed a significant positive correlation with vitiligo disease severity and activity estimated by VASI (r = 0.23, P = 0.017) and VIDA scores (r = 0.14, P = 0.003), respectively.


  Discussion Top


Several factors are involved in the pathogenesis of vitiligo as genetic predisposition, environmental triggers, renewal impairment, neural mechanism and most importantly; the role of autoimmunity and silent inflammation, particularly during the progressive episodes of the disease.[2] The autoimmune theory is the leading hypothesis in vitiligo. However, the mechanism of production of autoantibodies remains unclear. Tfh cells imbalance has been incriminated in B-cell activation and antibody production in several autoimmune diseases.[4]

In the present study, there was a statistically significant difference between patients and control group, where vitiligo patients had higher level of Thf cells than control group. These findings could be attributed to the increased levels of several autoantibodies characterizing vitiligo including those directed against melanocyte antigens. As circulating Tfh cells share the same phenotype as germinal center Tfh cells, they are thought to be efficient in stimulating B-cells with the production of autoantibodies in the autoimmune pathogenic mechanism of vitiligo.[10]

A significant correlation was also detected in the current study between circulating CXCR5+/PD-1+ Tfh cells and disease severity and activity estimated by standard VASI and VIDA scores, and these results were in agreement with those of Jacquemin et al.[4] They found that the peripheral Tfh cell frequency was significantly correlated with VIDA score and the percent of affected body surface area.[4] This observation suggested that increased circulating Tfh cell frequency in vitiligo could be associated with increased disease activity and could be potentially used as a disease activity marker due to their role in anti-melanocyte antibody production.

In fact, the presence of several autoantibodies in vitiligo with the recent genetic study that identified two single neucleotide ploymorphism located in CXCR5 of Tfh cells in vitiligo patients make the analysis of the frequency of CXCR5+ Tfh cell subset in human vitiligo blood and correlation of the results with the vitiligo disease clinical findings important to clarify and support the possible role of Tfh cells in the disease pathogenesis.[11]

Tfh cells were suggested to play a central role in the production of autoantibodies in several autoimmune diseases. It was found that circulating Tfh cells identified by CXCR5+ PD-1+ ICOS+ in systemic lupus erythematosus (SLE) patients-as one of common autoimmune diseases associated with vitiligo-were elevated compared to controls. Furthermore, the circulating Tfh cells were significantly correlated with the SLE disease activity index, frequency of circulating plasmablasts, and anti-double-stranded DNA antibody positivity, but not correlated with disease duration. These results showed that the circulating Tfh cells are more specific as an indicator of the disease activity state in SLE.[8]

In a similar context, it was found also that circulating Tfh cells CD4+ CXCR5+ in the peripheral blood of pemphigus patients (another autoimmune and antibody-mediated disease) measured with flowcytometry were significantly elevated.[11] Moreover, no difference in the frequencies of PD-1+ or ICOS+ circulating Tfh cells was detected in pemphigus patients when compared to healthy controls in contrast with bullous pemphigoid in which CXCR5+ PD-1+ ICOS+ cells were significantly elevated among CD4+ T-lymphocytes.[12] These findings show that there are significant differences in specificity of Tfh cells surface markers in correlation with disease activity according to disease type. Furthermore, it was noticed that in both pemphigus and bullous pemphigoid, there was elevated IL-21 in the blood (predominantly produced by Tfh and Th17 cells) and that could play a pivotal effect in T- and B-cells proliferation and antibody production and their levels were significantly reduced after efficient therapy.[12]

In a previous study, rheumatoid arthritis (RA) patients were found to have more CD4+ PD-1+ CXCR5+ Tfh cells in peripheral blood compared with healthy controls and the percentage of these cells correlated positively with the values of erythrocyte sedimentation rate, rheumatoid factor, C-reactive protein, and anti-cyclic citrullinated peptide antibodies. Higher mRNA expression of Bcl-6 and lower Blimp-1 mRNA expression were observed in patients with RA compared to healthy controls, and the expression level of IL-21 was higher in RA patients. The authors concluded that activated Tfh cells in the peripheral blood may be responsible for the development of RA.[9]


  Conclusion Top


The noticeable increased frequencies of Tfh cells in NSV patients and their strong correlation with disease severity and activity suggest a potential role of this cell population in the pathogenesis, extent as well as activity of the disease. Further studies on this cell population may give new insights for future investigations to check the state of vitiligo activity. Furthermore, new treatment strategies targeting Tfh cells or their cytokines might offer helpful therapeutic tools for treatment of vitiligo.

Study limitations

There are two major limitations in this study that could be addressed in future research. First, the study did not focus on studying the Tfh cell subsets or their cytokine secretion profile in studied subjects (where Tfh1 cells are considered as nonhelping to B cells, while Tfh2 and Tfh17 display a stronger functional capacity to help B cells differentiation). Second, the present study did not investigate the serum level of autoantibodies of relevance in vitiligo, such as anti-melanocyte antibodies. Investigating theses autoantibodies and correlating them with Tfh cell or their cytokines population could be beneficial for understanding vitiligo pathogenesis.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Ezzedine K, Eleftheriadou V, Whitton M, van Geel N. Vitiligo. Lancet 2015;386:74-84.  Back to cited text no. 1
    
2.
Dahir AM, Thomsen SF. Comorbidities in vitiligo: Comprehensive review. Inter J Dermatol 2018;57:1157-64.  Back to cited text no. 2
    
3.
Gensous N, Charrier M, Duluc D, Contin-Bordes C, Truchetet ME, Lazaro E, et al. T follicular helper cells in autoimmune disorders. Front Immunol 2018;9:1637.  Back to cited text no. 3
    
4.
Jacquemin C, Taieb A, Boniface K, Seneschal J; FHU ACRONIM. Imbalance of peripheral follicular helper T lymphocyte subsets in active vitiligo. Pigment Cell Melanoma Res 2019;32:588-92.  Back to cited text no. 4
    
5.
Zhou L, Shi YL, Li K, Hamzavi I, Gao TW, Huggins RH, et al. Increased circulating Th17 cells and elevated serum levels of TGF-beta and IL-21 are correlated with human non-segmental vitiligo development. Pigment Cell Melanoma Res 2015;28:324-9.  Back to cited text no. 5
    
6.
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;140:677-83.  Back to cited text no. 6
    
7.
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;135:407-13.  Back to cited text no. 7
    
8.
Choi JY, Ho JH, Pasoto SG, Bunin V, Kim ST, Carrasco S, et al. Circulating follicular helper-like T cells in systemic lupus erythematosus: Association with disease activity. Arthritis Rheumatol 2015;67:988-99.  Back to cited text no. 8
    
9.
Cao G, Chi S, Wang X, Sun J, Zhang Y. CD4+CXCR5+PD-1+T follicular Helper cells play a pivotal role in the development of rheumatoid arthritis. Med Sci Monit 2019;25:3032-40.  Back to cited text no. 9
    
10.
Morita R, Schmitt N, Bentebibel SE, Ranganathan R, Bourdery L, Zurawski G, et al. Human blood CXCR5(+) CD4(+) T cells are counterparts of T follicular cells and contain specific subsets that differentially support antibody secretion. Immunity 2011;34:108-21.  Back to cited text no. 10
    
11.
Boniface K, Seneschal J, Picardo M, Taïeb A. Vitiligo: Focus on clinical aspects, immunopathogenesis, and therapy. Clin Rev Allergy Immunol 2018;54:52-67.  Back to cited text no. 11
    
12.
Hennerici T, Pollmann R, Schmidt T, Seipelt M, Tackenberg B, Möbs C, et al. Increased frequency of T follicular helper cells and elevated interleukin-27 plasma levels in patients with pemphigus. PLoS One 2016;11:e0148919.  Back to cited text no. 12
    

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Correspondence Address:
Doaa Salah Hegab,
Department of Dermatology and Venereology, Faculty of Medicine, Tanta University Hospitals, El Geish Street, Tanta 31111
Egypt
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ds.ds_35_21



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