Cytotoxic IFNγ-producing T cells recognizing hair follicles are involved in the pathogenesis of alopecia areata (AA) (Xing et al., 2014Xing L. Dai Z. Jabbari A. Cerise J.E. Higgins C.A. Gong W. et al.Alopecia areata is driven by cytotoxic T lymphocytes and is reversed by JAK inhibition.Nat Med. 2014; 20: 1043-1049Crossref PubMed Scopus (521) Google Scholar), and CD49a/Granzyme B (GrB) expression in lesional T cells correlates with poor treatment responsiveness (Koguchi-Yoshioka et al., 2021Koguchi-Yoshioka H. Watanabe R. Matsumura Y. Okiyama N. Ishitsuka Y. Nakamura Y. et al.The Possible Linkage of Granzyme B-Producing Skin T Cells with the Disease Prognosis of Alopecia Areata.J Invest Dermatol. 2021; 141: 427-9 e10Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar). While B cells contribute to the pathogenesis of autoimmune diseases through multiple effector functions (Chan et al., 1999Chan O.T. Hannum L.G. Haberman A.M. Madaio M.P. Shlomchik M.J. A novel mouse with B cells but lacking serum antibody reveals an antibody-independent role for B cells in murine lupus.J Exp Med. 1999; 189: 1639-1648Crossref PubMed Scopus (598) Google Scholar, Cyster and Allen, 2019Cyster J.G. Allen C.D.C. B Cell Responses: Cell Interaction Dynamics and Decisions.Cell. 2019; 177: 524-540Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, Shlomchik et al., 1994Shlomchik M.J. Madaio M.P. Ni D. Trounstein M. Huszar D. The role of B cells in lpr/lpr-induced autoimmunity.J Exp Med. 1994; 180: 1295-1306Crossref PubMed Scopus (323) Google Scholar) , the involvement of B cells in AA pathogenesis has not been investigated. Although B cells are generally considered as a pathogenic population, accumulating evidences also demonstrate the existence of regulatory B cells (Breg) (Iwata et al., 2011Iwata Y. Matsushita T. Horikawa M. Dilillo D.J. Yanaba K. Venturi G.M. et al.Characterization of a rare IL-10-competent B-cell subset in humans that parallels mouse regulatory B10 cells.Blood. 2011; 117: 530-541Crossref PubMed Scopus (842) Google Scholar, Katz et al., 1974Katz S.I. Parker D. Turk J.L. B-cell suppression of delayed hypersensitivity reactions.Nature. 1974; 251: 550-551Crossref Scopus (244) Google Scholar, Mauri, 2021Mauri C. Novel Frontiers in Regulatory B cells.Immunol Rev. 2021; 299: 5-9Crossref Scopus (7) Google Scholar) which exert their suppressive function via interleukin (IL) -10 secretion. Breg arise from various subpopulations including CD24hiCD38hi immature transitional B cells (Blair et al., 2010Blair P.A. Norena L.Y. Flores-Borja F. Rawlings D.J. Isenberg D.A. Ehrenstein M.R. et al.CD19(+)CD24(hi)CD38(hi) B cells exhibit regulatory capacity in healthy individuals but are functionally impaired in systemic Lupus Erythematosus patients.Immunity. 2010; 32: 129-140Abstract Full Text Full Text PDF PubMed Scopus (1175) Google Scholar, Flores-Borja et al., 2013Flores-Borja F. Bosma A. Ng D. Reddy V. Ehrenstein M.R. Isenberg D.A. et al.CD19+CD24hiCD38hi B cells maintain regulatory T cells while limiting TH1 and TH17 differentiation.Sci Transl Med. 2013; 5: 173ra23Crossref PubMed Scopus (484) Google Scholar), CD24hiCD27+ memory B cells (Iwata et al., 2011Iwata Y. Matsushita T. Horikawa M. Dilillo D.J. Yanaba K. Venturi G.M. et al.Characterization of a rare IL-10-competent B-cell subset in humans that parallels mouse regulatory B10 cells.Blood. 2011; 117: 530-541Crossref PubMed Scopus (842) Google Scholar), and CD27intCD38+ plasmablasts (Matsumoto et al., 2014Matsumoto M. Baba A. Yokota T. Nishikawa H. Ohkawa Y. Kayama H. et al.Interleukin-10-producing plasmablasts exert regulatory function in autoimmune inflammation.Immunity. 2014; 41: 1040-1051Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar). Among them, the dysfunction of CD24hiCD38hi Breg are relatively well established not only in systemic autoimmune disorders (Mauri and Menon, 2017Mauri C. Menon M. Human regulatory B cells in health and disease: therapeutic potential.J Clin Invest. 2017; 127: 772-779Crossref PubMed Scopus (241) Google Scholar) but also in cutaneous immune-mediated disorders (Hayashi et al., 2016Hayashi M. Yanaba K. Umezawa Y. Yoshihara Y. Kikuchi S. Ishiuji Y. et al.IL-10-producing regulatory B cells are decreased in patients with psoriasis.J Dermatol Sci. 2016; 81: 93-100Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, Yoshihara et al., 2019Yoshihara Y. Ishiuji Y. Yoshizaki A. Kurita M. Hayashi M. Ishiji T. et al.IL-10-Producing Regulatory B Cells Are Decreased in Patients with Atopic Dermatitis.J Invest Dermatol. 2019; 139: 475-478Abstract Full Text Full Text PDF Scopus (12) Google Scholar). Here, we measured IL-10 production in blood B cells from AA subjects and healthy controls (Ctl), and evaluated the association of IL-10-producing B cells with disease prognosis (details are in Supplementary Materials and Methods). This study is approved by the Institutional Review Board in University of Tsukuba Hospital and Osaka University Hospital. Written informed consent was provided from all participants. By stimulation with lipopolysaccharide and CpG, B cells from AA subjects produced more IL-10 than those from Ctl did (Figure 1a). Among CD24hiCD38hi, CD24loCD38md mature naïve, and CD24hiCD38lo memory fractions, CD24hiCD38hi fraction was the most responsible source of IL-10 both in AA and Ctl, consistent with previous reports (Blair et al., 2010Blair P.A. Norena L.Y. Flores-Borja F. Rawlings D.J. Isenberg D.A. Ehrenstein M.R. et al.CD19(+)CD24(hi)CD38(hi) B cells exhibit regulatory capacity in healthy individuals but are functionally impaired in systemic Lupus Erythematosus patients.Immunity. 2010; 32: 129-140Abstract Full Text Full Text PDF PubMed Scopus (1175) Google Scholar), and IL-10 production from each fraction was significantly higher in AA than in Ctl (Figure 1b). The frequency of B cells and CD24hiCD38hi fraction was comparable between AA and Ctl (Figure S2). AA subjects were then classified into good prognosis (GP) and poor prognosis (PP) groups by the threshold of 50 % hair re-growth area in one year, regardless of treatment modalities. Then, B cells from GP produced significantly more IL-10 compared to those from PP (Figure 1c). IL-10 production from the two fractions other than CD24hiCD38hi was significantly higher in GP than in PP (Figure 1d), implying that IL-10 production from B cells other than the conventional Breg is associated with better prognosis. Considering the central role of IFNγ signaling in AA (Ito et al., 2013Ito T. Hashizume H. Shimauchi T. Funakoshi A. Ito N. Fukamizu H. et al.CXCL10 produced from hair follicles induces Th1 and Tc1 cell infiltration in the acute phase of alopecia areata followed by sustained Tc1 accumulation in the chronic phase.J Dermatol Sci. 2013; 69: 140-147Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, Xing et al., 2014Xing L. Dai Z. Jabbari A. Cerise J.E. Higgins C.A. Gong W. et al.Alopecia areata is driven by cytotoxic T lymphocytes and is reversed by JAK inhibition.Nat Med. 2014; 20: 1043-1049Crossref PubMed Scopus (521) Google Scholar), the effect of B cells on IFNγ-producing activity of T cells was compared among Ctl, GP, and PP. When the peripheral blood mononuclear cells (PBMC) depleted with B cells were cultured, PBMC T cells in the three groups showed the comparable level of IFNγ-producing potency (Figure 1e, f). IFNγ production from CD8 T cells, but not CD4 T cells, of Ctl and PP was augmented by the existence of B cells while this upregulating effect was not observed in GP (Figure 1f). It is thus implied that Ctl B cells mainly exert effector functions on the activation of CD8 T cells, and that the suppressive cascade involving B cells and CD8 T cells, which is to be upregulated in AA, is impaired in PP. Among the subjects whose blood and lesional skin could be analyzed, IL-10 production from blood B cells showed a strong negative correlation with CD49a expression in lesional CD8 T cells isolated by explant culture (Figure 2a). A trend of negative correlation between IFNγ/GrB production in lesional CD8 T cells and IL-10 production in blood B cells was also observed (Figure 2a). FoxP3 expression in CD4 T cells did not show correlation. In immunofluorescence analysis, the density of CD4 and CD8 T cells around hair bulbs was comparable between GP and PP (Figure 2b, c). A few B cells were also observed around hair bulbs, more in GP than in PP, and 43.6 % of the lesional B cells found in all the counted GP specimens were positive for IL-10 (Figure 2b, c). It is thus implied that impaired IL-10 production from blood B cells correlates with cytotoxic phenotype of lesional CD8 T cells, and that the existence of lesional B cells might be associated with recovery from the disease, possibly involving IL-10 signaling. So far as we know, this is the first report to demonstrate the involvement of Breg in AA. Based on the findings in systemic lupus erythematosus where CD24hiCD38hi Breg are impaired (Blair et al., 2010Blair P.A. Norena L.Y. Flores-Borja F. Rawlings D.J. Isenberg D.A. Ehrenstein M.R. et al.CD19(+)CD24(hi)CD38(hi) B cells exhibit regulatory capacity in healthy individuals but are functionally impaired in systemic Lupus Erythematosus patients.Immunity. 2010; 32: 129-140Abstract Full Text Full Text PDF PubMed Scopus (1175) Google Scholar) and IL-10-producing CD24hiCD27+ B cells are increased (Iwata et al., 2011Iwata Y. Matsushita T. Horikawa M. Dilillo D.J. Yanaba K. Venturi G.M. et al.Characterization of a rare IL-10-competent B-cell subset in humans that parallels mouse regulatory B10 cells.Blood. 2011; 117: 530-541Crossref PubMed Scopus (842) Google Scholar), the characteristics of Breg in AA would be therefore the potential of developing multiple IL-10-producing fractions while maintaining the conventional Breg. Our AA cohort includes diverse subjects with different disease background. Information on trichoscopy is insufficient, and our definition of GP and PP is affected by the disease activity at the time of sample collection, with PP group showing a trend of having larger affected area, more broken hairs and less short vellus hairs in trichoscopy, and more frequent pull test positivity (Table S1a, b, Figure S3). PP subjects also frequently received intensive treatments including intravenous corticosteroid pulse therapy (Table S1c), suggesting the skewed treatment decision. The question whether the different reactivity of CD8 T cells to B cells in GP and PP is caused by the nature of CD8 T cells or B cells remains unsolved. The relation of T cell phenotypes with B cell function in blood and lesions should also be further confirmed by larger cohort in the future. In addition, the profile of lesional CD8 T cells can be skewed by the explant culture (Figure S1d). The reproduction of our results using other experimental methods such as single cell RNA-seq analysis would be waited. In summary, various B cell fractions have the potential of producing IL-10 in the chronic autoinflammatory condition of AA, and the impairment of this potential might be related to the difficulty in controlling the continuous disease pathways. Breg would become an index for evaluating the disease activity and prognosis of AA. Materials and Methods Supplementary Tables Materials and Methods Collection of human samples and the evaluation of treatment efficacy All the protocols in this study were performed in accordance with the Declaration of Helsinki and are approved by the Institutional Review Board in University of Tsukuba Hospital (H28-1) and Osaka University Hospital (20108). Written informed consent was provided from all participants. Blood samples were collected from total 40 AA patients (average 33.4 years old, 16 males and 24 females) who visited University of Tsukuba Hospital or Osaka University Hospital before starting the treatments including Pulse, oral corticosteroid, intralesional corticosteroid injection, or contact immunotherapy. Treatments before the first visit were restricted to topical corticosteroid, oral anti-histamine drugs, and/or ultraviolet therapies. All the patients had started topical corticosteroid therapy before the visit. Except for the contact immunotherapy, the topical corticosteroid was continued. The affected area was graded as S0-S5 according to the alopecia areata investigational assessment guideline (Olsen et al., 1999). The disease prognosis was evaluated 12 months after collecting samples. The patients who achieved 50 % or greater area of hair re-growth were defined as GP and the others as PP, regardless of the treatment modalities. The detailed information on the patients including age, sex, disease duration, baseline clinical observation including disease area (Olsen et al., 1999), trichoscopic findings, the pull test results, and treatment modalities is shown in Supplementary Table S1a-c. Blood samples were also collected from 22 patients at the time of screening test for resecting benign tumors (average 46.5 years old, 8 males and 14 females) and were regarded as Ctl. PBMC were isolated from blood samples by Ficoll density gradient centrifugation (density 1.077; GE Healthcare Bio-Sciences, IL, USA). Four-mm punch biopsied specimens of AA lesions were also collected from total 13 of the 40 subjects at the time of blood collection, and lesional T cells were isolated by 2 weeks of explant culture as described previously (Clark et al., 2006) using Iscove’s Modified Dulbecco’s Medium (Wako, Osaka, Japan) containing 10 % fetal bovine serum, penicillin/streptomycin and 2-mercaptoethanol in the presence of 100 IU/mL of IL-2 (Wako) and 20 ng/mL of IL-15 (Wako). By flow cytometry analysis, the expression of IFNγ and GrB in skin CD8 T cells tended to be upregulated by the explant culture compared to those isolated by 2-hour digestion with 3 mg/mL collagenase III (Worthington Biochemical Co, NJ, USA) and 5 μg/mL deoxyribonuclease (Sigma-Aldrich, MO, USA) (Supplementary Figure S1d) in 3 surgical discards while CD49a expression was maintained. Flow cytometry Monoclonal antibodies directly conjugated with fluorescence and isotype controls were used with optimal concentration (Supplementary Table S2a). Prior to the intracellular cytokine staining, cells were stimulated by lipopolysaccharide (10 μg/mL, MERCK, Darmstadt, Germany) and CpG oligodeoxynucleotides (3 μg/mL, Abeomics, Inc., CA, USA) for 24 hours, and then by phorbol 12-myristate 13-acetate (50 ng/mL, Wako) and ionomycin (750 ng/mL, Wako) for 4 hours. Golgi Plug (BD Biosciences, NJ, USA) was also added in the last 4 hours. Cells were surface-stained, fixed, permeabilized and stained for intracellular targets using BD Cytofix/Cytoperm (BD Biosciences) according to the manufacturer’s protocol. Dead cells were excluded by use of LIVE/DEAD™ Fixable Dead Cell Stain Kit (Thermo Fisher Scientific, MA, USA). Data collection was performed on Gallios cytofluorometer (Beckman Coulter, CA, USA) or BD FACSCelesta flow cytometer (BD Biosciences), and data were analyzed by Kaluza analysis software (Beckman Coulter). The gating strategies are shown in Supplementary Figure S1a-c. Co-culture B cells were isolated from PBMC by use of human CD20 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and the magnetic cell-sorting system (Miltenyi Biotec) according to the manufacturer’s protocol. The purity of the isolated B cells was over 90 %, and the remained PBMC was also confirmed to contain less than 0.1 % of B cells by flow cytometry (Supplementary Figure S1b). A hundred thousand PBMC depleted with B cells were stimulated by T Cell Activation/Expansion Kit (Miltenyi Biotec) for 48 hours at the ratio that the estimated number of T cells by flow cytometry and the number of beads was 2:1 with or without the presence of 1 × 104 B cells isolated from the same PBMC. Since average 84.9 % (ranging 78.5 % to 92.2 %) of the live PBMC depleted with B cells were T cells, the approximate ratio of T cells and B cells was 8.5:1. IFNγ production from the cultured T cells was analyzed by flow cytometry as described above. Immunofluorescence analyses Formalin-fixed and paraffin-embedded lesional specimens from 13 subjects were sliced into 3 μm thickness on glass slides. Sliced specimens were de-paraffinized and rehydrated, and antigen retrieval was performed using TE buffer (pH 9.0, Agilent, CA, USA). After blocking, sliced specimens were incubated with the antibodies targeting CD4, CD8, CD19, CD20, or IL-10 at 1:200 dilution overnight in 4 °C, followed by the application of the secondary antibodies conjugated with fluorescence at 1:500 dilution for 60 min. The antibodies are listed in Supplementary Table S2b. Mounting Medium with DAPI (Vector Laboratories, CA, USA) was used. The sections were also evaluated by hematoxylin and eosin (H-E) staining (Vector Laboratories). The slides were observed by the fluorescence microscope BZ-700 (Keyence, Osaka, Japan). CD4/CD8/CD20-positive cells were counted per unit area (150μm×150μm area of hair bulbs). The average numbers from 5 sections of each specimen are shown in Figure 2c. The ratio of IL-10-producing B cells was calculated by counting IL-10-positive cells within CD19-positive cells in all the analyzed specimens from GP subjects. Statistical analyses Statistical analyses among two groups were performed by Mann-Whitney test or Wilcoxon matched-pairs signed rank test by use of GraphPad Prism software (GraphPad Software, CA, USA). Kruskal-Wallis test followed by Dunn’s multiple comparisons test was applied to the comparison between three groups. Spearman's rank correlation coefficient was applied for the evaluation of correlation. P < 0.05 was considered as significant; p < 0.05 (*) and p < 0.01 (**).Supplementary Table S1aPatient information. S0∼S5 were defined according to the alopecia areata investigational assessment guideline (Olsen et al., 1999). BH: broken hairs, VH: short vellus hairs, YD: yellow dots, AH: oral anti-histamine drug, CI: contact immunotherapy, IL: intralesional corticosteroid injection, O: oral corticosteroid, P: intravenous corticosteroid pulse therapy, T: topical corticosteroid only, UV: ultraviolet therapy, GP: good prognosis, PP: poor prognosis.AgeSexDisease duration (month)Baseline disease areaTrichoscopyPull testPrior TxTxGP/PPFirst/RecurBlood (Fig 1a-d, 2a, S2)Blood (Fig 1e, f)Skin/blood (Fig 2a)25M2S2BH, YD(+)TP, CIGPFirst〇51F4S1BH, VH(-)TTGPFirst〇CD49a, IFNγ, GrB, FoxP311M5S4BH, VH, YDNAT, UVP, CIPPFirst〇38M14S4YDNAT, UV, AHCIPPRecur〇GrB, FoxP338F2S2BH(+)TCIGPRecur〇CD49a, IFNγ, FoxP329F3S2BH(-)T, UV, AHP, CIPPFirst〇CD49a, IFNγ, FoxP335F9S1VH, YD(-)TCIGPFirst〇CD49a, IFNγ, GrB, FoxP319F2S2BH(+)TP, CIPPRecur〇47M6S1YD(-)T, UVIL, UVGPFirst〇7F5S1BH, VH(+)T, UV, AHCIPPRecur〇36F16S1BH, VH, YD(-)T, AHCIGPFirst〇45F5S2BH, VH, YD(+)TCIPPRecur〇50M36S5YDNAT, UVCI, ILPPRecur〇GrB, FoxP35M2S2BH(+)TCIPPFirst〇23F2S2BH, YD(+)T, UVPGPFirst〇63M4S3BH, VH(+)T, UV, AHP, CIPPRecur〇CD49a, GrB56F4S4BH, VHNAT, AHCIGPRecur〇GrB52F5S2BH, YD(+)T, UVP, OPPFirst〇IFNγ19M3S3BH(+)TPGPFirst〇44F12S2BH, VH, YD(-)T, UVCIGPRecur〇IFNγ13F2S2BH, YD(+)TCIPPFirst〇16M4S3BH, VH, YD(-)T, UVCIPPFirst〇44F1S1VH(-)TTGPFirst〇CD49a, IFNγ, FoxP332M4S3BH, VH, YD(+)T, AHPGPFirst〇CD49a, IFNγ, FoxP312F1S1BH(+)TCIPPRecur〇49F3S3BH, YD(+)T, UV, AHPPPRecur〇10F3S2BH(+)TCIGPFirst〇49M2S2BH(+)TPPPFirst〇CD49a, GrB, FoxP341F4S2BH, YD(+)T, AHPPPUnknown〇47F12S3BH, YD(-)TCIPPUnknown〇22M3S2BH(+)TPPPUnknown〇28M31S2YD(+)T, UV, AHPPPUnknown〇14M4S1BH, VH, YD(-)TTGPUnknown〇38F3S3BH(+)TPGPUnknown〇53M6S3BH, YD(+)T, UVPPPUnknown〇50M144S1BH, VH, YD(-)T, UV, AHCIPPUnknown〇28F5S1VH(-)TCIGPUnknown〇43F1S3BH(+)TPGPUnknown〇25F8S2BH, VH, YD(-)T, UVCIGPUnknown〇28F6S1BH, YD(-)T, UVCIGPUnknown〇 Open table in a new tab Supplementary Table S1bFindings in trichoscopy and pull test in GP and PP. The ratios of the subjects with the existence of BH and VH by trichoscopy and with the positive result by pull test were shown in GP and PP groups.TotalBHVHPull testGP (n = 19)78.9%52.6%44.4%PP (n = 21)85.7%19.0%77.8% Open table in a new tab Supplementary Table S1cTreatment modalities in GP and PP. The ratios of the subjects who received the listed treatment modalities in GP and PP groups. CI: contact immunotherapy, IL: intralesional corticosteroid injection, O: oral corticosteroid, P: intravenous corticosteroid pulse therapy, T: topical corticosteroid only, UV: ultraviolet therapy.TotalCIILOPTUVGP (n = 19)52.6%5.3%0%31.6%15.8%5.3%PP (n = 21)66.7%4.8%4.8%52.4%0%0% Open table in a new tab Supplementary Table S2aAntibodies for flow cytometryAntibodyCloneSpeciesSupplierCD3SK7MouseBioLegendCD4RPA-T4MouseBioLegendCD8aRPA-T8MouseBioLegendCD19HIB19MouseBioLegendCD24ML5MouseBioLegendCD38HB-7MouseBioLegendCD49aSR84MouseBD BiosciencesFoxP3259DMouseBioLegendGrBREA226Human(recombinant)Miltenyi BiotecIFNγ4S.B3MouseBioLegendIL-10JES3-9D7MouseBioLegend Open table in a new tab Supplementary Table S2bAntibodies for immunofluorescenceAntibodyCloneSpeciesSupplierCD44B12MouseAgilentCD8aEP1150YRabbitAbcamCD196OMP31RatInvitrogenCD202H7MouseThermo Fisher ScientificIL-108E4B6MouseProteintechMouse IgG H&L(Alexa Fluor® 488)-GoatAbcamRabbit IgG H&L(Alexa Fluor® 488)-DonkeyAbcamRat IgG H&L(Alexa Fluor® 488)-GoatAbcamMouse IgG H&L(Alexa Fluor® 555)-DonkeyAbcamRabbit IgG H&L(Alexa Fluor® 555)-DonkeyAbcam Open table in a new tab Supplementary Figure S2The frequencies of B cells and CD24hiCD38hi population are comparable between AA and Ctl. Left: the ratio of CD19+ B cells in lymphocytes in 28 AA and 16 Ctl. Right: the ratio of CD24hiCD38hi fraction in B cells in 28 AA and 16 Ctl.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Supplementary Figure S3Clinical background of GP and PP. Left: disease duration of 13 GP 15 PP. Right: affected area of 13 GP and 15 PP.View Large Image Figure ViewerDownload Hi-res image Download (PPT)
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