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Immunophenotypic Analysis Reveals Differences in Circulating Immune Cells in the Peripheral Blood of Patients with Segmental and Nonsegmental Vitiligo

  • Marcella Willemsen
    Correspondence
    Correspondence: Marcella Willemsen, Netherlands Institute for Pigment Disorders, Department of Dermatology, Amsterdam University Medical Center, University of Amsterdam, Meibergdreef 9, Amsterdam 1105 AZ, The Netherlands.
    Affiliations
    Netherlands Institute for Pigment Disorders, Department of Dermatology, Amsterdam University Medical Center, University of Amsterdam, Cancer Center Amsterdam, Amsterdam Infection & Immunity Institute, Amsterdam, The Netherlands
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  • Nicoline F. Post
    Affiliations
    Netherlands Institute for Pigment Disorders, Department of Dermatology, Amsterdam University Medical Center, University of Amsterdam, Cancer Center Amsterdam, Amsterdam Infection & Immunity Institute, Amsterdam, The Netherlands
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  • Nathalie O.P. van Uden
    Affiliations
    Netherlands Institute for Pigment Disorders, Department of Dermatology, Amsterdam University Medical Center, University of Amsterdam, Cancer Center Amsterdam, Amsterdam Infection & Immunity Institute, Amsterdam, The Netherlands
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  • Vidhya S. Narayan
    Affiliations
    Netherlands Institute for Pigment Disorders, Department of Dermatology, Amsterdam University Medical Center, University of Amsterdam, Cancer Center Amsterdam, Amsterdam Infection & Immunity Institute, Amsterdam, The Netherlands
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  • Saskia Chielie
    Affiliations
    Netherlands Institute for Pigment Disorders, Department of Dermatology, Amsterdam University Medical Center, University of Amsterdam, Cancer Center Amsterdam, Amsterdam Infection & Immunity Institute, Amsterdam, The Netherlands
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  • E. Helen Kemp
    Affiliations
    Department of Oncology and Metabolism, The Medical School, The University of Sheffield, Sheffield, United Kingdom
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  • Marcel W. Bekkenk
    Affiliations
    Netherlands Institute for Pigment Disorders, Department of Dermatology, Amsterdam University Medical Center, University of Amsterdam, Cancer Center Amsterdam, Amsterdam Infection & Immunity Institute, Amsterdam, The Netherlands
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  • Rosalie M. Luiten
    Affiliations
    Netherlands Institute for Pigment Disorders, Department of Dermatology, Amsterdam University Medical Center, University of Amsterdam, Cancer Center Amsterdam, Amsterdam Infection & Immunity Institute, Amsterdam, The Netherlands
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Open AccessPublished:June 21, 2021DOI:https://doi.org/10.1016/j.jid.2021.05.022
      Accumulating studies have indicated immune-based destruction of melanocytes in both segmental vitiligo (SV) and non-SV (NSV). Whereas SV often occurs unilaterally during childhood and stabilizes after an initial period of activity, the disease course of NSV is usually slowly progressive, with new lesions occurring bilaterally during life. This suggests an involvement of distinct pathophysiology pathways, specifically increased systemic immune activation in patients with NSV but not in patients with SV. This research aimed to identify the differences in immune cells in the blood of patients with SV and NSV through immunophenotyping of circulating cells. Regulatory T cells were unaffected in patients with SV compared with that in healthy controls but decreased in patients with NSV. In patients with NSV, the reduction in regulatory T cells was associated with the presence of other systemic autoimmune comorbidities, which were less present in SV. Similarly, the absence of a melanocyte-specific antibody response in patients with SV suggests less involvement of B-cell immunity in SV. These data show that in contrast to patients with NSV, no increased systemic immunity is found in patients with SV, indicating that SV pathogenesis is associated with a localized cytotoxic reaction targeting epidermal melanocytes.

      Abbreviations:

      cTfh (circulating T follicular helper), NSV (nonsegmental vitiligo), SV (segmental vitiligo), Treg (regulatory T cel), TYR (tyrosinase)

      Introduction

      Vitiligo is the most common skin depigmenting disorder characterized by white patches resulting from the loss of pigment-producing cells, melanocytes (
      • Bergqvist C.
      • Ezzedine K.
      Vitiligo: a review.
      ). It affects approximately 0.5% of the general population with no apparent differences in rates of occurrence according to sex, skin type, or ethnicity (
      • Boniface K.
      • Seneschal J.
      • Picardo M.
      • Taïeb A.
      Vitiligo: focus on clinical aspects, immunopathogenesis, and therapy.
      ). An international consensus classified vitiligo into two subtypes (
      • Ezzedine K.
      • Lim H.W.
      • Suzuki T.
      • Katayama I.
      • Hamzavi I.
      • Lan C.C.
      • et al.
      Revised classification/nomenclature of vitiligo and related issues: the Vitiligo Global Issues Consensus Conference.
      ). The commonest form, nonsegmental vitiligo (NSV), shows symmetrical depigmentation of the body. Contrary, segmental vitiligo (SV) is less common (±10%) and is characterized by a unilateral distribution. In addition, NSV shows an unpredictable disease course, whereas SV typically stabilizes a few months after onset. Altogether, this suggests that distinct pathophysiology pathways might be involved, which could clarify the differences in clinical presentation and disease course.
      Initially, somatic mosaicism, neurogenic mechanisms, and oxidative stress were suspected to be the underlying cause of SV (
      • Speeckaert R.
      • Lambert J.
      • Bulat V.
      • Belpaire A.
      • Speeckaert M.
      • van Geel N.
      Autoimmunity in segmental vitiligo.
      ). Only recently, immune-mediated pathophysiology of SV has been recognized. Increasing evidence has shown immune-based cytotoxic destruction of melanocytes in SV, with lesional IFN-γ–producing melanocyte antigen‒reactive CD8+ T-cell infiltrates migrating to the basal layer (
      • Attili V.R.
      • Attili S.K.
      Segmental and generalized vitiligo: both forms demonstrate inflammatory histopathological features and clinical mosaicism.
      ;
      • Shin J.
      • Kang H.Y.
      • Kim K.H.
      • Park C.J.
      • Oh S.H.
      • Lee S.C.
      • et al.
      Involvement of T cells in early evolving segmental vitiligo.
      ;
      • van Geel N.A.
      • Mollet I.G.
      • De Schepper S.
      • Tjin E.P.
      • Vermaelen K.
      • Clark R.A.
      • et al.
      First histopathological and immunophenotypic analysis of early dynamic events in a patient with segmental vitiligo associated with halo nevi.
      ). Although NSV is closely associated with other autoimmune disorders, for example, thyroid disease and alopecia areata, systemic autoimmune comorbidities are less common in patients with SV (
      • Dahir A.M.
      • Thomsen S.F.
      Comorbidities in vitiligo: comprehensive review.
      ;
      • Speeckaert R.
      • Lambert J.
      • Bulat V.
      • Belpaire A.
      • Speeckaert M.
      • van Geel N.
      Autoimmunity in segmental vitiligo.
      ). Nevertheless, localized skin inflammation, for example, linear morphea, is repeatedly observed in patients with SV, implying a local inflammatory response (
      • Speeckaert R.
      • Lambert J.
      • Bulat V.
      • Belpaire A.
      • Speeckaert M.
      • van Geel N.
      Autoimmunity in segmental vitiligo.
      ). The comparison between SV and NSV provides a unique setting to study whether antimelanocyte immunity remains localized in SV and spreads systemically in NSV.
      A study on gene expression profiles of patients with SV and NSV and of healthy individuals showed that differentially expressed genes in SV were involved in the adaptive immune response, whereas in NSV, the regulation of the innate immune response and B-cell differentiation and activation was more prominent, implying that SV and NSV may utilize different immune responses and mechanisms for melanocyte destruction (
      • Wang P.
      • Li Y.
      • Nie H.
      • Zhang X.
      • Shao Q.
      • Hou X.
      • et al.
      The changes of gene expression profiling between segmental vitiligo, generalized vitiligo and healthy individual.
      ). Concomitantly, the blood of patients with NSV showed a decrease in regulatory T cells (Tregs) and an increase in unswitched memory B cells compared with healthy control blood, which was related to disease activity (
      • Raam L.
      • Kaleviste E.
      • Šunina M.
      • Vaher H.
      • Saare M.
      • Prans E.
      • et al.
      Lymphoid stress surveillance response contributes to vitiligo pathogenesis.
      ). Considering the positive correlation between switched memory B cells and circulating T follicular helper (cTfh) cells, patients with active NSV show activation of germinal centers and faster B-cell isotype switching (
      • Raam L.
      • Kaleviste E.
      • Šunina M.
      • Vaher H.
      • Saare M.
      • Prans E.
      • et al.
      Lymphoid stress surveillance response contributes to vitiligo pathogenesis.
      ). Despite data showing involvement of B cells and germinal center reactions, the correlation to a humoral response remained unstudied.
      To our knowledge, analysis of circulating immune cells, involvement of a humoral response, and germinal center reactions in SV have not been fully characterized. Whereas associated autoimmune diseases are more common in NSV (
      • Alkhateeb A.
      • Fain P.R.
      • Thody A.
      • Bennett D.C.
      • Spritz R.A.
      Epidemiology of vitiligo and associated autoimmune diseases in Caucasian probands and their families.
      ;
      • Gill L.
      • Zarbo A.
      • Isedeh P.
      • Jacobsen G.
      • Lim H.W.
      • Hamzavi I.
      Comorbid autoimmune diseases in patients with vitiligo: a cross-sectional study.
      ;
      • Hadi A.
      • Wang J.F.
      • Uppal P.
      • Penn L.A.
      • Elbuluk N.
      Comorbid diseases of vitiligo: a 10-year cross-sectional retrospective study of an urban US population.
      ;
      • Spritz R.A.
      • Andersen G.H.
      Genetics of vitiligo.
      ), evidence points to a temporary cytotoxic response targeting melanocytes in SV, suggesting differences in systemic immune cell dysregulation between patients with SV and those with NSV.
      This study aimed to compare the differences in cellular and humoral adaptive immunity and innate immunity in human blood of patients with SV and NSV that can contribute to clinical presentation and disease progression. Our results show no increased systemic immunity in patients with SV, in contrast to that in patients with NSV, and points to localized immune-based cytotoxic destruction of melanocytes.

      Results

       Demographics and clinical characterization of study subjects

      The characteristics of the participants are shown in Table 1. Patients with SV had an average age of 34 years, which is higher than the average age of the general SV population because of the age ≥18 years inclusion criterion. SV had stabilized in our patient cohort, for at least 12 months. To focus our comparative study on the differences between SV and NSV without the interference of active versus stable vitiligo disease activity, we compared stable patients with SV with stable patients with NSV. The majority of included patients with SV had type-2 skin, and only one patient had alopecia areata as autoimmune comorbidity. Patients with NSV had stable disease and an average age of 43 years; the majority of patients had a type-2 or -3 skin type, and 6 of the 22 patients (27.2%) showed autoimmune comorbidities. Patients with SV and NSV did not differ in age of onset, disease duration, and affected body surface area. Healthy controls were comparable with regard to age, sex, and skin type with the patients with vitiligo.
      Table 1Patient Characteristics
      Healthy DonorSVNSVP-Value
      P-value tested with Student’s t-test or Mann‒Whitney test.
      n%IQR/SDn%IQR/SDn%IQR/SD
      Total221222
      Age
       <2529.132514.5
       25–501777.3866.71463.6
       >50313.618.3731.8
       Mean36(23–48)34(22–46)43(29–57)>0.05
      Sex>0.05
       Male627.39751150
       Female1672.73251150
      Skin type
      Skin type according to the Fitzpatrick skin scale.
      >0.05
       Type 10014.5
       Type 21359.1866.71254.5
       Type 3731.9325836.4
       Type 414.518.30
       Type 514.5014.5
       Type 6000
      Comorbidities
       Thyroid disease0313.6
      One patient with NSV showed multiple autoimmune comorbidities (hypothyroidism, DM type 1, and colitis ulcerosa).
       Alopecia Areata18.30
       DM type 10418.2
      One patient with NSV showed multiple autoimmune comorbidities (hypothyroidism, DM type 1, and colitis ulcerosa).
       RA00
       SLE00
       Psoriasis00
       Other
      Other specified, including Addison’s disease, arthritis psoriatica, autoimmune hepatitis, IBD, celiac, CREST, morphea, pernicious anemia, PMR, sarcoidosis, scleroderma, Sjogren’s Syndrome.
      014.5
      One patient with NSV showed multiple autoimmune comorbidities (hypothyroidism, DM type 1, and colitis ulcerosa).
      Vitiligo age of onset (y) mean25±14.833±15.6>0.05
      Disease duration (y) median4(2.3–12.3)8(4–12.5)>0.05
      % Affected body surface area median0.75(0.5–1.9)1(0.9–3.9)>0.05
      Abbreviations: CREST, calcinosis, Raynaud phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia; DM, diabetes mellitus; IBD, inflammatory bowel disease; IQR, interquartile range; NSV, nonsegmental vitiligo; PMR, polymyalgia rheumatica; RA, rheumatoid arthritis; SLE, systemic lupus erythematosus; SV, segmental vitiligo.
      1 P-value tested with Student’s t-test or Mann‒Whitney test.
      2 Skin type according to the Fitzpatrick skin scale.
      3 One patient with NSV showed multiple autoimmune comorbidities (hypothyroidism, DM type 1, and colitis ulcerosa).
      4 Other specified, including Addison’s disease, arthritis psoriatica, autoimmune hepatitis, IBD, celiac, CREST, morphea, pernicious anemia, PMR, sarcoidosis, scleroderma, Sjogren’s Syndrome.

       Patients with NSV with secondary autoimmune comorbidities have less circulating Tregs

      Perturbations in Treg numbers and function in vitiligo remain indistinct. Some studies report systemically reduced Tregs in NSV (
      • Ben Ahmed M.
      • Zaraa I.
      • Rekik R.
      • Elbeldi-Ferchiou A.
      • Kourda N.
      • Belhadj Hmida N.
      • et al.
      Functional defects of peripheral regulatory T lymphocytes in patients with progressive vitiligo.
      ;
      • Dwivedi M.
      • Laddha N.C.
      • Arora P.
      • Marfatia Y.S.
      • Begum R.
      Decreased regulatory T-cells and CD4(+)/CD8(+) ratio correlate with disease onset and progression in patients with generalized vitiligo.
      ;
      • Giri P.S.
      • Dwivedi M.
      • Laddha N.C.
      • Begum R.
      • Bharti A.H.
      Altered expression of nuclear factor of activated T cells, forkhead box P3, and immune-suppressive genes in regulatory T cells of generalized vitiligo patients.
      ;
      • Hegab D.S.
      • Attia M.A.
      Decreased circulating T regulatory cells in Egyptian patients with nonsegmental vitiligo: correlation with disease activity.
      ;
      • Lili Y.
      • Yi W.
      • Ji Y.
      • Yue S.
      • Weimin S.
      • Ming L.
      Global activation of CD8+ cytotoxic T lymphocytes correlates with an impairment in regulatory T cells in patients with generalized vitiligo.
      ;
      • Raam L.
      • Kaleviste E.
      • Šunina M.
      • Vaher H.
      • Saare M.
      • Prans E.
      • et al.
      Lymphoid stress surveillance response contributes to vitiligo pathogenesis.
      ), whereas others show that abundance of Tregs in NSV trends toward an increase (
      • Abdallah M.
      • Saad A.
      Evaluation of circulating CD4+CD25highFoxP3+ T lymphocytes in active non-segmental vitiligo.
      ;
      • Moftah N.H.
      • El-Barbary R.A.
      • Ismail M.A.
      • Ali N.A.
      Effect of narrow band-ultraviolet B on CD4(+) CD25(high) FoxP3(+) T-lymphocytes in the peripheral blood of vitiligo patients.
      ). Moreover, Treg involvement in SV remains unstudied. Therefore, we studied systemic Treg and type-1 Treg numbers and IL-10 production in our cohort of patients with SV and NSV and in healthy donor samples. The gating strategy for Tregs is depicted in Supplementary Figure S1. Circulating Tregs were significantly decreased in the blood of patients with NSV compared with the blood of those with SV and those of healthy controls (Figure 1a). However, IL-10 production by Tregs, measured as IL-10+ cells after intracellular FACS staining, was not affected in these patients (Figure 1a). The level of Tregs in patients with SV did not differ from that in healthy controls (Figure 1a). We verified these findings on the RNA expression level and in an independent patient cohort using the RNA-sequencing dataset from
      • Wang P.
      • Li Y.
      • Nie H.
      • Zhang X.
      • Shao Q.
      • Hou X.
      • et al.
      The changes of gene expression profiling between segmental vitiligo, generalized vitiligo and healthy individual.
      . This dataset contains 20 patients with SV, 20 patients with NSV, and 20 healthy control individuals (within each patient group, 5 patients were pooled into a new sample, giving a total of 4 samples) (
      • Wang P.
      • Li Y.
      • Nie H.
      • Zhang X.
      • Shao Q.
      • Hou X.
      • et al.
      The changes of gene expression profiling between segmental vitiligo, generalized vitiligo and healthy individual.
      ). Gene expression profiles of patients with NSV and SV and of healthy individuals were then analyzed for the presence of a Treg gene expression signature. For this, we made use of a gene signature used to discriminate between Tregs (CD25high) and conventional CD4+ T cells (CD25) (
      • Niedzielska M.
      • Israelsson E.
      • Angermann B.
      • Sidders B.S.
      • Clausen M.
      • Catley M.
      • et al.
      Differential gene expression in human tissue resident regulatory T cells from lung, colon, and blood.
      ). This gene signature comprises 25 genes, of which 21 genes are upregulated and 4 genes are downregulated on Tregs compared with those on conventional CD4+ T cells (Supplementary Table S1). Similar to cellular Treg analysis, patients with NSV showed decreased expression of the Treg signature compared with healthy individuals (P < 0.05) (Figure 1b). Because of the large spread among SV samples, comparing Treg levels in patients with NSV and in those with SV did not reach significance (P = 0.08) (Figure 1b).
      Figure thumbnail gr1
      Figure 1Comparison of circulating Treg subpopulations in patients with vitiligo (a) The percentage of Tregs among CD4+ T cells (left) and IL-10‒producing cells among Tregs (right) in HD (n = 22), patients with SV (n = 12), and patients with NSV (n = 20). (b) Expression of the Treg core gene signature in HD (n = 20), patients with SV (n = 20), and patients with NSV (n = 20) (within each group, five samples were pooled into a new sample). The genes included in this Treg core signature are included in . (c) The percentage of Tregs among CD4+ T cells in patients with NSV without (no Comorb., n = 16) or with comorb. (n = 6). (d) The percentage of Tr1 among CD4+ T cells (left) and IL-10‒producing cells among Tr1 (right) in HD, patients with SV, and patients with NSV. Results are shown as individual dot plots with a line as median and 95% CI for a, c, and d or median and minimum and maximum for b. ANOVA and Student’s t-test are significant as indicated; ∗P < 0.05. CI, confidence interval; Comorb, autoimmune comorbidity; HD, healthy control; NSV, nonsegmental vitiligo; SV, segmental vitiligo; Tr1, type-1 regulatory T cell; Treg, regulatory T cell.
      Because systemic autoimmune comorbidities might influence circulating Treg numbers, we compared Treg levels in patients with NSV with or without comorbidities as indicated in Table 1. Patients with NSV with autoimmune comorbidities showed significantly fewer Tregs than patients with NSV without autoimmune comorbidities (Figure 1c), suggesting that impaired Treg numbers might be the consequence of secondary autoimmune responses and not specific to vitiligo pathogenesis. Treg levels in patients with NSV without autoimmune comorbidities still trended toward a decrease compared with those in healthy controls, but this did not reach significance in our patient cohort size (P = 0.09; data not shown). Nevertheless, gene expression patterns of patients with NSV showed a less pronounced Treg signature, even when autoimmune conditions were absent (the patient cohort of
      • Wang P.
      • Li Y.
      • Nie H.
      • Zhang X.
      • Shao Q.
      • Hou X.
      • et al.
      The changes of gene expression profiling between segmental vitiligo, generalized vitiligo and healthy individual.
      only includes patients with no history of any other autoimmune condition) (Figure 1b).
      In contrast to Tregs, the percentage of type-1 Tregs did not demonstrate significant differences between the studied groups; nonetheless, IL-10‒producing type-1 Tregs were increased in patients with SV (Figure 1d). To conclude, these results further support that Tregs but not type-1 Tregs are negatively affected in (some) patients with NSV, which may facilitate the development of autoimmune comorbidities. In contrast, Tregs remain unaffected in patients with SV, consistent with less systemic autoimmune comorbidities in patients with SV.

       Antibody responses against melanocyte antigens are present only in patients with NSV

      Antibody responses against melanocyte antigens, for example, tyrosinase (TYR) and TRP-2, have been found in the sera of patients with NSV (
      • Kemp E.H.
      • Gavalas N.G.
      • Gawkrodger D.J.
      • Weetman A.P.
      Autoantibody responses to melanocytes in the depigmenting skin disease vitiligo.
      ). However, sera from patients with SV have rarely been tested for the presence of autoantibodies. To test reactivity to melanocyte antigens in patients with SV and NSV, sera were evaluated for antibody reactivity against TYR, TYRP1, TYRP2, PMEL, TYR hydroxylase, MART-1, and MCHR1. Antibodies against selected melanocyte antigens were present in the circulation of a significant proportion of patients with NSV (Figure 2). In total, 8 of 22 (36%) patients with NSV were found to have antimelanocyte antibody responses (Supplementary Table S2). Moreover, three of eight patients showed antibody reactivity to several melanocyte antigens (Supplementary Table S2). Patients with NSV with antibody responses against the antigens mentioned earlier were not significantly different from patients with NSV without autoantibodies regarding age, sex distribution, skin type, presence of secondary autoimmune comorbidities, vitiligo age of onset, disease duration, and percentage of affected body surface area (Supplementary Table S3). Contrary, none of the patients with SV showed an antibody response (Figure 2), indicating that the presence of a humoral immune response to melanocyte antigens is limited to patients with NSV.
      Figure thumbnail gr2
      Figure 2Presence of melanocyte-specific antibody responses in patients with vitiligo. Sera of HD (n = 30), patients with SV (n = 12), and patients with NSV (n = 20) were analyzed in radio-binding assays for the presence of antibodies against TYR, TRP1, TRP2, PMEL, TH, MART-1, and MCHR1. The antibody index for each individual patient is shown and is calculated as c.p.m. immunoprecipitated by tested serum divided by the mean c.p.m. immunoprecipitated by the group of HD sera. Each serum was tested in at least three independent experiments. Next, the mean antibody index was calculated from these values. Patient sera with an antibody index above the upper limit of normal (mean antibody index + 3 × SD of the HD individuals) were regarded as positive for antibody reactivity. c.p.m., count per minute; HD, health control; NSV, nonsegmental vitiligo; SV, segmental vitiligo; TH, tyrosinase hydroxylase; TYR, tyrosinase.

       Patients with SV have fewer circulating antibody-producing plasmablasts

      We subsequently analyzed whether the absence of antibody responses in SV is also reflected in less B-cell activation and plasma-cell differentiation. The gating strategy for B cells is depicted in Supplementary Figure S1. The percentage of total B cells (CD3 CD19+) was comparable between patients with vitiligo and healthy individuals (Figure 3a). Similarly, naive B cells and unswitched and switched memory B cells did not demonstrate significant differences between the studied groups (Figure 3a), implying that B cells seem to mature similarly in patients with vitiligo and in healthy individuals. Similarly, transitional B cells did not differ between patients with SV and NSV nor the patients and the healthy controls (Figure 3b). However, plasmablasts were significantly decreased in blood from patients with SV compared with healthy donor blood (Figure 3c). Concomitantly, plasmablasts showed a trend toward a decrease in patients with SV compared with that in patients with NSV, suggesting that patients with SV have fewer circulating antibody-producing cells.
      Figure thumbnail gr3
      Figure 3Distribution of peripheral B-cell subsets in patients with vitiligo. (a‒c) The percentage of B cells among lymphocytes, naive B cells, unswitched memory B cells, and switched memory B cells among (a) B cells, (b) transitional B cells, and (c) plasmablasts among B cells in HD (n = 22), patients with SV (n = 12), and patients with NSV (n = 20). Results are shown as individual dot plots with means ± SEM. ANOVA test is significant as indicated; ∗P < 0.05. HD, health control; NSV, nonsegmental vitiligo; SV, segmental vitiligo.

       Patients with SV have more cTfh2 and cTfh17 cells but no increase in the number of active cTfh cells

      Our data so far indicate that patients with SV have no melanocyte-specific antibody response and a diminished humoral response, illustrated by fewer circulating plasmablasts (Figures 2 and 3c). To verify whether this reduced plasmablast differentiation stems from reduced germinal center help, we analyzed the presence of cTfh cells. Levels of both cTfh cells and active cTfh cells (PD-1+ ICOS+) were unaffected in patients with NSV compared with that in healthy control individuals. Instead, the number of cTfh cells were significantly increased in patients with SV compared with that in patients with NSV (Figure 4a). Similarly, the number of cTfh cells with an active phenotype were increased in patients with SV compared with those in both patients with NSV and healthy controls (P < 0.05) (Figure 4a).
      Figure thumbnail gr4
      Figure 4Comparison of cTfh cell subsets in patients with vitiligo. (a‒c) The percentage of cTfh cells among (a) CD4+ cells and active (PD-1+ ICOS+) cTfh cells, (b) cTfh cell subsets and active cells within CD4+ cells, and (c) cTfh cell subsets in HD (n = 22), patients with SV (n = 12), and patients with NSV (n = 20). (d) The percentage of active cells among cTfh cell subsets in patients with NSV without (no comorb., n = 16) or with Comorb. (n = 6). Results are shown as individual dot plots with means ± SEM. ANOVA and Student’s t-test are significant as indicated; ∗P < 0.05. Comorb., autoimmune comorbidity; cTfh, circulating T follicular helper; HD, health control; NSV, nonsegmental vitiligo; SV, segmental vitiligo.
      On the basis of the expression of CXCR3 and CCR6, cTfh cells can be classified into cTfh1, cTfh2, and cTfh17 cells (Supplementary Figure S1) (
      • Koutsakos M.
      • Nguyen T.H.O.
      • Kedzierska K.
      With a little help from T follicular helper friends: humoral immunity to influenza vaccination.
      ). These cells differ in their ability to provide help to naive and memory B cells, and thus the abundance of specific cTfh cell subsets might be as important as total cTfh cell levels. In blood, cTfh1 cells did not show significant differences between the studied groups (Figure 4b). Contrary, cTfh2 and cTfh17 cells, which are superior IL-21 producers to cTfh1 cells and especially provide help to naive B cells, were significantly increased in patients with SV compared with those in patients with NSV and in healthy controls (P < 0.05) (Figure 4b). More importantly, patients with SV did not show an increase in active cTfh2 and cTfh17 cells (Figure 4c). Concomitantly, active cTfh cell subsets did not differ between patients with NSV and healthy individuals (Figure 4c). Because the provision of B-cell help is limited to activated cTfh cells and the percentage of active cTfh1, cTfh2, and cTfh17 cells do not differ between SV and NSV, it seems that both vitiligo cohorts show normal germinal center help that is not different from that of the healthy controls.
      As for Treg numbers, systemic autoimmune comorbidities might affect cTfh cell levels and activation status. NSV is closely associated with other autoimmune conditions, whereas these are less common in patients with SV (
      • Dahir A.M.
      • Thomsen S.F.
      Comorbidities in vitiligo: comprehensive review.
      ;
      • Speeckaert R.
      • Lambert J.
      • Bulat V.
      • Belpaire A.
      • Speeckaert M.
      • van Geel N.
      Autoimmunity in segmental vitiligo.
      ). These comorbidities may involve humoral responses and increased B-cell help from cTfh cells. Indeed, when patients with NSV were divided into subgroups according to the presence of secondary autoimmune comorbidities, it was evident that systemic autoimmune comorbidities were associated with increased percentages of active cTfh2 cells (P < 0.05) and cTfh17 cells (P = 0.07) but not with cTfh1 cells (Figure 4d). Collectively, our results show increased cTfh2 and cTfh17 cell numbers in SV but no increased germinal center reactions in human vitiligo. Consistent with an increase in activated cTfh2 and cTfh17 cell subsets in type-I diabetes and thyroid disease (
      • Gensous N.
      • Charrier M.
      • Duluc D.
      • Contin-Bordes C.
      • Truchetet M.E.
      • Lazaro E.
      • et al.
      T follicular helper cells in autoimmune disorders.
      ), enhanced cTfh cell activation in patients with NSV with secondary autoimmune comorbidities seems to result from those rather than from skin autoimmunity.

       Patients with vitiligo do not differ from healthy controls with regard to circulating NK cells

      Besides aberrations in the adaptive immune response, innate immunity is suggested to be involved in NSV pathogenesis as well but left unstudied in patients with SV. The gating strategy for NK cells is depicted in Supplementary Figure S1. Systemic NK cell levels were not different in both vitiligo subtypes compared with that in healthy control blood (Figure 5a). Expression of the activating NK cell receptor, NKG2D, on NK cells and cytotoxic CD56dim NK cells was significantly decreased in patients with NSV compared with that in healthy controls but unaffected on cytokine-producing CD56bright NK cells (Figure 5b). However, in the blood of patients with SV, no difference in NKG2D expression was seen compared with that in the blood of healthy controls. Because NKG2D can be expressed by CD8+ T effector memory cells and because an increased expression has been observed in active NSV skin (
      • Jacquemin C.
      • Martins C.
      • Lucchese F.
      • Thiolat D.
      • Taieb A.
      • Seneschal J.
      • et al.
      NKG2D defines a subset of skin effector memory CD8 T cells with proinflammatory functions in vitiligo.
      ), we analyzed NKG2D expression by peripheral CD3+ T cells. No significant differences in the proportion of NKG2D+ CD3+ T cells were seen in the blood of patients with NSV compared with that in the blood of patients with SV and healthy controls (data not shown). Because NK cells were unaffected in patients with SV, innate immunity seems to be less involved in SV pathogenesis.
      Figure thumbnail gr5
      Figure 5Distribution of circulating NK cells in patients with vitiligo. (a) The percentage of NK cells among CD45+ cells, CD56bright cells, and CD56dim cells among NK cells and (b) the percentage of NKG2D+ cells among NK cells, CD56bright cells, and CD56dim cells in HD (n = 22), patients with SV (n = 12), patients with NSV (n = 20). (b). Results are shown as individual dot plots with means ± SEM. ANOVA tests are significant as indicated; ∗P < 0.05. HD, health control; NSV, nonsegmental vitiligo; SV, segmental vitiligo.

      Discussion

      This study provides important insights into the differences between SV and NSV pathogenesis and shows that in contrast to NSV, SV does not involve systemic immune activation. We found that Tregs are less abundant in patients with NSV than in healthy controls but did not differ in patients with SV. Furthermore, a humoral response and germinal center reactions were not observed in patients with SV. This is consistent with fewer autoimmune comorbidities in patients with SV and points to a local autoimmune reaction.
      Previous studies have shown the presence of melanocyte-specific antibodies in some patients with NSV (
      • Kemp E.H.
      • Gavalas N.G.
      • Gawkrodger D.J.
      • Weetman A.P.
      Autoantibody responses to melanocytes in the depigmenting skin disease vitiligo.
      ), whereas this remained largely unstudied in patients with SV. This work shows that the involvement of a humoral response against melanocyte antigens is restricted to patients with NSV. The absence of a melanocyte-specific antibody response is consistent with the observation that there is no systemic immune activation in patients with SV. Although autoantibodies were found in some patients with NSV, we hypothesize that this is an underestimation because we tested only seven common melanocyte autoantibody targets. Indeed, in immunoprecipitation experiments with melanocyte extracts, 100% of patients with NSV and 0% of healthy controls were found to have antimelanocyte antibodies in their sera (
      • Naughton G.K.
      • Eisinger M.
      • Bystryn J.C.
      Detection of antibodies to melanocytes in vitiligo by specific immunoprecipitation.
      ). In addition, incidence and level of autoantibodies have been correlated with disease activity and the extent of the disease, meaning that patients with active vitiligo and patients with 5–10% skin depigmentation are more likely to have circulating antimelanocyte antibodies (
      • Harning R.
      • Cui J.
      • Bystryn J.C.
      Relation between the incidence and level of pigment cell antibodies and disease activity in vitiligo.
      ;
      • Naughton G.K.
      • Reggiardo D.
      • Bystryn J.C.
      Correlation between vitiligo antibodies and extent of depigmentation in vitiligo.
      ).
      Tregs induce anergy in melanocyte-specific T cells in healthy individuals (
      • Maeda Y.
      • Nishikawa H.
      • Sugiyama D.
      • Ha D.
      • Hamaguchi M.
      • Saito T.
      • et al.
      Detection of self-reactive CD8+ T cells with an anergic phenotype in healthy individuals.
      ). More importantly, it is assumed that melanocyte-reactive CD8+ T cells escape anergy by loss of coinhibitory CTLA-4 expression in NSV. Similarly, we show that Treg numbers are decreased in patients with stable NSV, most prominently in patients with secondary autoimmune comorbidities, as hypothesized earlier (
      • Le Poole I.C.
      • Mehrotra S.
      Replenishing regulatory T cells to halt depigmentation in vitiligo.
      ). Even in the absence of autoimmune comorbidities, we found that patients with NSV still show a trend toward a decrease in Treg numbers and signatures. In a previous study, the abundance and activity of circulating Tregs in patients with NSV were shown to be similar to those in healthy controls but reduced in the skin of patients with NSV, explained by the failure of Tregs to home to the skin in vitiligo (
      • Klarquist J.
      • Denman C.J.
      • Hernandez C.
      • Wainwright D.A.
      • Strickland F.M.
      • Overbeck A.
      • et al.
      Reduced skin homing by functional Treg in vitiligo [published correction appears in Pigment Cell Melanoma Res 2010;23:477].
      ). Contrary to our cohort, half of these patients showed progressive disease and were under treatment at the moment of collecting blood samples. Therefore, these results might be explained by different disease activity. This highlights the importance of reporting these patient characteristics in great detail. Because numerous studies have not clearly reported the presence or absence of secondary autoimmune comorbidities or disease activity while studying Tregs in NSV, it is difficult to place results into context because the greatest differences are seen in those with other autoimmunities.
      The involvement of NK cells in vitiligo has been suggested by previous studies. RNA analysis of NSV skin biopsies revealed high expression of genes of the innate immune system, especially NK cells, compared with that in healthy skin, and more NK cells were found in both lesional and nonlesional NSV skin (
      • Yu R.
      • Broady R.
      • Huang Y.
      • Wang Y.
      • Yu J.
      • Gao M.
      • et al.
      Transcriptome analysis reveals markers of aberrantly activated innate immunity in vitiligo lesional and non-lesional skin.
      ). Similarly, NK cells were shown to be significantly increased in the blood of patients with stable NSV compared with that in healthy controls (
      • Tulic M.K.
      • Cavazza E.
      • Cheli Y.
      • Jacquel A.
      • Luci C.
      • Cardot-Leccia N.
      • et al.
      Innate lymphocyte-induced CXCR3B-mediated melanocyte apoptosis is a potential initiator of T-cell autoreactivity in vitiligo.
      ). However, we together with others (
      • Raam L.
      • Kaleviste E.
      • Šunina M.
      • Vaher H.
      • Saare M.
      • Prans E.
      • et al.
      Lymphoid stress surveillance response contributes to vitiligo pathogenesis.
      ) did not detect increased peripheral NK cell levels. Nevertheless, NKG2D expression by NK cells was shown to be significantly decreased in patients with NSV, especially on CD56dim cells, and KLRC4-KLRK1, which encodes NKG2D, was found to be downregulated in the blood of patients with SV (
      • Wang P.
      • Li Y.
      • Nie H.
      • Zhang X.
      • Shao Q.
      • Hou X.
      • et al.
      The changes of gene expression profiling between segmental vitiligo, generalized vitiligo and healthy individual.
      ). In lesional NSV skin, the stress molecules MICA/MICB (ligands for the activating NKG2D receptor) were shown to be expressed in dermal areas but not in nonlesional or healthy skin (
      • Raam L.
      • Kaleviste E.
      • Šunina M.
      • Vaher H.
      • Saare M.
      • Prans E.
      • et al.
      Lymphoid stress surveillance response contributes to vitiligo pathogenesis.
      ). In addition, IFN-γ–producing innate lymphoid cells were shown to initially induce CXCR3B-mediated melanocyte apoptosis (
      • Tulic M.K.
      • Cavazza E.
      • Cheli Y.
      • Jacquel A.
      • Luci C.
      • Cardot-Leccia N.
      • et al.
      Innate lymphocyte-induced CXCR3B-mediated melanocyte apoptosis is a potential initiator of T-cell autoreactivity in vitiligo.
      ). Recently, increased NKG2D expression was found on skin-resident NK cells, NKT cells, and CD8+ effector memory T cells, especially in patients with active disease (
      • Jacquemin C.
      • Martins C.
      • Lucchese F.
      • Thiolat D.
      • Taieb A.
      • Seneschal J.
      • et al.
      NKG2D defines a subset of skin effector memory CD8 T cells with proinflammatory functions in vitiligo.
      ). Contrary to increased NKG2D expression by CD8+ effector memory T cells in the skin of patients with active NSV, no significant difference in the proportion of these cells was seen in the blood of patients with NSV compared with that of healthy controls (
      • Jacquemin C.
      • Martins C.
      • Lucchese F.
      • Thiolat D.
      • Taieb A.
      • Seneschal J.
      • et al.
      NKG2D defines a subset of skin effector memory CD8 T cells with proinflammatory functions in vitiligo.
      ). Therefore, it is suggested that a skin factor (possibly IL-15, IFN-α) is responsible for the promotion of NKG2D expression (
      • Jacquemin C.
      • Martins C.
      • Lucchese F.
      • Thiolat D.
      • Taieb A.
      • Seneschal J.
      • et al.
      NKG2D defines a subset of skin effector memory CD8 T cells with proinflammatory functions in vitiligo.
      ). In addition, NKG2D upregulation in vitiligo occurs in response to insults and stress, which primarily occurs in lesional skin (
      • Plaza-Rojas L.
      • Guevara-Patiño J.A.
      The role of the NKG2D in vitiligo.
      ). Therefore, the involvement of NK cells in NSV and SV pathogenesis remains indistinct but suggests skin-resident NK cells to be involved in the initial initiation of the antimelanocyte autoimmunity during active disease rather than long-lasting systemic NK cell involvement.
      To our knowledge, immunophenotypic analysis of circulating immune cells in the blood of patients with SV, compared with those in patients with NSV and in healthy individuals has not been previously reported in the literature. Our results strengthened the notion that immunity plays an important role in vitiligo pathogenesis. Most importantly, our study highlights the immunological difference between NSV and SV. NSV is characterized by systemic immune activation, decreased Treg levels, and the development of autoimmune comorbidities. In contrast, the absence of systemic immune activation in patients with SV indicates that SV pathogenesis is associated with a localized cytotoxic reaction against epidermal melanocytes.

      Materials and Methods

       Patient material

      This study was conducted in accordance with the Declaration of Helsinki. All subjects signed written informed consent approved by the Medical Ethics Review Committee of the Amsterdam University Medical Centers (NL 64983.018.18). Peripheral blood samples were obtained from patients with SV (n = 12) or with stable NSV (n = 22) aged ≥18 years who were visiting the outpatient clinic at the Amsterdam University Medical Center (Amsterdam, The Netherlands) according to current vitiligo classification and disease activity scoring (
      • Rodrigues M.
      • Ezzedine K.
      • Hamzavi I.
      • Pandya A.G.
      • Harris J.E.
      Vitiligo Working Group
      New discoveries in the pathogenesis and classification of vitiligo.
      ;
      • van Geel N.
      • Grine L.
      • De Wispelaere P.
      • Mertens D.
      • Prinsen C.A.C.
      • Speeckaert R.
      Clinical visible signs of disease activity in vitiligo: a systematic review and meta-analysis.
      ). Exclusion criterion was disease activity in the past 12 months during standard of care treatment. Similarly, we recruited healthy control subjects (n = 20) aged ≥18 years. The demographic characteristics of patients with vitiligo and healthy controls are represented in Table 1. PBMCs were purified from whole blood by density gradient centrifugation (LymphoPrep, Stemcell Technologies, Vancouver, Canada) and cryopreserved before analysis.

       Antibodies and flow cytometry

      Fluorochrome-conjugated antibodies are specified in Supplementary Table S4. Cell surface staining was performed in FACS buffer (PBS supplemented with 1% BSA and 0.05% sodium azide). Subsequently, cells were fixed in True-Nuclear Fix (BioLegend, San Diego, CA) and stained intranuclear in True-Nuclear Perm Buffer (BioLegend), according to the manufacturer’s instructions. FACS acquisition was performed on a FACSCanto II B (BD Biosciences, Franklin Lakes, NJ) using BD FACSDiva software (BD Biosciences), and data were analyzed using FlowJo software (Tree Star, Ashland, OR).

       Gene expression analysis

      The R2 Genomics Analysis and Visualization platform (http://r2.amc.nl) was used for the analysis of gene expression profiles of patients with NSV and SV and of the healthy individuals (GSE80009) (
      • Wang P.
      • Li Y.
      • Nie H.
      • Zhang X.
      • Shao Q.
      • Hou X.
      • et al.
      The changes of gene expression profiling between segmental vitiligo, generalized vitiligo and healthy individual.
      ).

       Radioligand-binding assays

      Antibodies in serum samples were detected using radioligand-binding assays. Plasmids pcDNA3-TH, pcDNA3_TYR, pcDNA3-PMEL17, pcDNA3-MCHR1, and pcDNA-Melan-A (MART-1) were used according to the manufacturer’s instructions in an in vitro TnT T7-coupled Reticulocyte Lysate System (Promega, Madison, WI) with [35S]-methionine to produce radiolabeled full-length TYR, TRP1, TRP2, PMEL, TYR hydroxylase, MART-1, and MCHR1, respectively. Next, radiolabeled antigens were used in radioligand-binding assays with patient sera (n = 34) and healthy control sera (n = 30) at a 1:100 dilution. The antibody index is calculated as the count per minute immunoprecipitated by tested serum divided by the mean count per minute immunoprecipitated by the group of healthy control sera (
      • Kemp E.H.
      • Waterman E.A.
      • Hawes B.E.
      • O'Neill K.
      • Gottumukkala R.V.
      • Gawkrodger D.J.
      • et al.
      The melanin-concentrating hormone receptor 1, a novel target of autoantibody responses in vitiligo.
      ). Each serum was tested in at least three independent experiments. The mean antibody index was calculated from these values. Patient sera with an antibody index above the upper limit of normal (mean antibody index + 3 × SD of the healthy control individuals) were regarded as positive for antibody reactivity.

       Statistical analysis

      Statistical analysis was performed using GraphPad Prism software (GraphPad Software, San Diego, CA). Comparisons were made with ANOVA analysis, Student’s t-test, or Mann‒Whitney test. Tukey’s multiple comparisons corrections were applied for ANOVA analysis. P-values < 0.05 were considered statistically significant: ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001.

       Data availability statement

      No publicly available datasets were generated during this study. Dataset GSE80009 was used for gene expression analysis.

      ORCIDs

      Nathalie O. P. van Uden: http://orcid.org/0000-0001-9952-1065

      Author Contributions

      Conceptualization: MW, RML; Formal Analysis: MW; Funding Acquisition: RML; Investigation: MW, NFP, NOPVU, VSN, SC, EHK; Methodology: MW, NOPVU, SC, RML; Supervision: MWB, RML; Visualization: MW; Writing - Original Draft Preparation: MW, RML; Writing - Review and Editing: MW, NFP, NOPVU, VSN, SC, EHK, MWB, RML

      Conflict of Interest

      The authors state no conflict of interest.

      Supplementary Materials

      Figure thumbnail fx1
      Supplementary Figure S1Gating strategy for lymphocyte subpopulations. Tregs, cTfh cells, B cells, and NK cells were analyzed in four separate flow cytometry panels. In each panel, lymphocytes were selected as CD45+ cells (upper row). Tregs were considered to be CD25+ FoxP3+ cells from CD4+ cells, and Tr1 were gated as CD49b+ Lag-3+ cells from CD4+ cells (second row). In both Tregs and Tr1, IL-10‒producing cells were studied. cTfh cells were gated as CXCR5+ cells from CD4+ cells (third row). On the basis of the expression of CCR6 and CXCR3, we identified cTfh1 cells (CCR6 CXCR3+), cTfh2 cells (CCR6 CXCR3), and cTfh17 cells (CCR6+ CXCR3). In all cTfh subsets, we studied the activation status by the markers PD-1 and ICOS. B cells were identified as CD19+ CD3 cells from CD45+ cells (fourth row). B cells were then classified into naive B cells (IgD+ CD27), usm B cells (IgD+ CD27+), and sm B cells (IgD CD27+). Within the switched memory B-cell population, we analyzed the immunoglobulin subtype by staining for IgM. From B cells, we could also identify trans B cells (CD24+ CD38+) and plasmablasts (CD24 CD38+). Finally, NK cells were considered to be CD3 CD56+ cells (bottom row). Within the NK cell population, we separated CD56bright and CD56dim NK cells. Finally, we analyzed the expression of NKG2D on these cells. cTfh, circulating T follicular helper; FSC-A, forward scatter area; FSC-H, forward scatter height; FSC-W; forward scatter width; sm, switched memory; SSC-A, side scatter area; SSC-H, side scatter height; SSC-W, side scatter width; Tr1, type1 regulatory T cell; trans, transitional; Treg, regulatory T cell, usm, unswitched memory.
      Supplementary Table S1Differentially Expressed Genes between CD25high Tregs and Conventional CD25 CD4+ T Cells
      GeneUpregulated/Downregulated on CD25high Tregs
      FOXP3Upregulated
      IKZF2Upregulated
      IL2RAUpregulated
      CTLA4Upregulated
      TIGITUpregulated
      TNFRSF18 (GITR)Upregulated
      TNFRSF4 (OX40)Upregulated
      LAG3Upregulated
      HAVCR2 (TIM-3)Upregulated
      LRRC32 (GARP)Upregulated
      ICOSUpregulated
      IL10Upregulated
      EBI3 (IL35B)Upregulated
      IL1RL1 (ST2)Upregulated
      BATFUpregulated
      LAYNUpregulated
      CSF2RBUpregulated
      TRIB1Upregulated
      ENTPD1 (CD39)Upregulated
      UTS2Upregulated
      RTKN2Upregulated
      IL7RDownregulated
      ENC1Downregulated
      NKG7Downregulated
      CD40LGDownregulated
      Abbreviations: Treg, regulatory T cell.
      Supplementary Table S2Antibody Indexes for Sera from Antibody-Positive Patients with NSV
      PatientTYRTRP1TRP2PMELTHMART-1MCHR1
      Patient 191.11.020.691.063.460.980.99
      Patient 215.893.914.090.920.921.101.09
      Patient 233.952.852.821.151.090.990.94
      Patient 241.021.100.882.941.061.000.91
      Patient 297.885.715.480.981.020.881.02
      Patient 300.830.851.091.014.291.070.94
      Patient 331.061.160.965.120.860.910.98
      Patient 401.040.910.933.741.141.051.11
      Abbreviations: TH, tyrosinase hydroxylase; TYR, tyrosinase.
      Supplementary Table S3Patient Characteristics
      CharacteristicsAntibody-Positive NSVAntibody-Negative NSV
      n%IQR/SDn%IQR/SD
      Total814
      Age, y
       <2517
       25–50675857
       >50225536
       Mean46(29–63)42(30–54)
      Gender
       Male225964
       Female675536
      Skin type
      Skin type according to the Fitzpatrick skin scale.
       Type 1017
       Type 2562.5750
       Type 3337.5536
       Type 400
       Type 5017
       Type 600
      Comorbidities112.5536
      Vitiligo age of onset (y), mean32±13.934±19.1
      Disease duration (y), median7(4–11.3)8(4.5–20)
      % Affected body surface area median1(1–4)1.5(0.5–4.6)
      Abbreviations: IQR, interquartile range; NSV, nonsegmental vitiligo.
      1 Skin type according to the Fitzpatrick skin scale.
      Supplementary Table S4Used Antibodies
      MarkerCloneFluorochromeCompanyCatalog No.
      CD452D1BV510BioLegend368526
      CD3SK7FITCBioLegend344804
      CD56HCD56BV421BioLegend318327
      CD163G8PE-Cy7BioLegend302015
      Granzyme BQA16A02APC/Fire 750BioLegend372210
      CD94DX22PEBioLegend305506
      NKG2D1D11APCBioLegend320808
      NKp44P44-8PerCP-Cy5.5BioLegend325113
      CD27O323PE-Cy7BioLegend302838
      IgDIA6-2PEBioLegend348204
      CD19HIB19APCBioLegend302212
      CD24ML5PerCP-Cy5.5BioLegend311116
      CD38HIT2BV421BioLegend303526
      IgMMHM-88APC/Fire 750BioLegend314546
      CXCR5J252LD4PEBioLegend356904
      CXCR3G025H7PerCPBioLegend353740
      CCR7G043H7BV421BioLegend353208
      CCR6G034E3APCBioLegend353416
      ICOSC398.4AAPC/Fire 750BioLegend313536
      CD49bP1E6-C5APCBioLegend359310
      CD25BC96APC/Fire 750BioLegend302642
      CD4SK3FITCBioLegend344604
      Lag-311C3C65PerCPBioLegend369312
      CD127A019D5PEBioLegend351304
      IL-10JES3-9D7PE-Cy7BioLegend501420
      FOXP3206DBV421BioLegend320124
      PD-1MIH4PE-Cy7eBioscience25-9969-42
      Abbreviations: APC, allophycocyanin; No., number.

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