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<front>
<journal-meta>
<journal-id journal-id-type="publisher-id">Front. Cell Dev. Biol.</journal-id>
<journal-title>Frontiers in Cell and Developmental Biology</journal-title>
<abbrev-journal-title abbrev-type="pubmed">Front. Cell Dev. Biol.</abbrev-journal-title>
<issn pub-type="epub">2296-634X</issn>
<publisher>
<publisher-name>Frontiers Media S.A.</publisher-name>
</publisher>
</journal-meta>
<article-meta>
<article-id pub-id-type="publisher-id">799560</article-id>
<article-id pub-id-type="doi">10.3389/fcell.2021.799560</article-id>
<article-categories>
<subj-group subj-group-type="heading">
<subject>Cell and Developmental Biology</subject>
<subj-group>
<subject>Original Research</subject>
</subj-group>
</subj-group>
</article-categories>
<title-group>
<article-title>Dysregulated Peripheral Invariant Natural Killer T&#x20;Cells in Plaque Psoriasis Patients</article-title>
<alt-title alt-title-type="left-running-head">Hu et&#x20;al.</alt-title>
<alt-title alt-title-type="right-running-head">iNKT&#x20;Cell in Psoriasis</alt-title>
</title-group>
<contrib-group>
<contrib contrib-type="author">
<name>
<surname>Hu</surname>
<given-names>Yifan</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
<xref ref-type="aff" rid="aff2">
<sup>2</sup>
</xref>
<xref ref-type="aff" rid="aff3">
<sup>3</sup>
</xref>
<xref ref-type="fn" rid="fn1">
<sup>&#x2020;</sup>
</xref>
<uri xlink:href="https://loop.frontiersin.org/people/1246742/overview"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Chen</surname>
<given-names>Youdong</given-names>
</name>
<xref ref-type="aff" rid="aff2">
<sup>2</sup>
</xref>
<xref ref-type="aff" rid="aff3">
<sup>3</sup>
</xref>
<xref ref-type="fn" rid="fn1">
<sup>&#x2020;</sup>
</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Chen</surname>
<given-names>Zeyu</given-names>
</name>
<xref ref-type="aff" rid="aff2">
<sup>2</sup>
</xref>
<xref ref-type="aff" rid="aff3">
<sup>3</sup>
</xref>
<xref ref-type="fn" rid="fn1">
<sup>&#x2020;</sup>
</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Zhang</surname>
<given-names>Xilin</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
<xref ref-type="aff" rid="aff3">
<sup>3</sup>
</xref>
<uri xlink:href="https://loop.frontiersin.org/people/1155048/overview"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Guo</surname>
<given-names>ChunYuan</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
<xref ref-type="aff" rid="aff3">
<sup>3</sup>
</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Yu</surname>
<given-names>ZengYang</given-names>
</name>
<xref ref-type="aff" rid="aff2">
<sup>2</sup>
</xref>
<xref ref-type="aff" rid="aff3">
<sup>3</sup>
</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Xu</surname>
<given-names>Peng</given-names>
</name>
<xref ref-type="aff" rid="aff2">
<sup>2</sup>
</xref>
<xref ref-type="aff" rid="aff3">
<sup>3</sup>
</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Sun</surname>
<given-names>Lei</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
<xref ref-type="aff" rid="aff3">
<sup>3</sup>
</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Zhou</surname>
<given-names>Xue</given-names>
</name>
<xref ref-type="aff" rid="aff2">
<sup>2</sup>
</xref>
<xref ref-type="aff" rid="aff3">
<sup>3</sup>
</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Gong</surname>
<given-names>Yu</given-names>
</name>
<xref ref-type="aff" rid="aff2">
<sup>2</sup>
</xref>
<xref ref-type="aff" rid="aff3">
<sup>3</sup>
</xref>
</contrib>
<contrib contrib-type="author" corresp="yes">
<name>
<surname>Yu</surname>
<given-names>Qian</given-names>
</name>
<xref ref-type="aff" rid="aff2">
<sup>2</sup>
</xref>
<xref ref-type="aff" rid="aff3">
<sup>3</sup>
</xref>
<xref ref-type="corresp" rid="c001">&#x2a;</xref>
</contrib>
<contrib contrib-type="author" corresp="yes">
<name>
<surname>Shi</surname>
<given-names>Yuling</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
<xref ref-type="aff" rid="aff2">
<sup>2</sup>
</xref>
<xref ref-type="aff" rid="aff3">
<sup>3</sup>
</xref>
<xref ref-type="corresp" rid="c001">&#x2a;</xref>
<uri xlink:href="https://loop.frontiersin.org/people/685895/overview"/>
</contrib>
</contrib-group>
<aff id="aff1">
<sup>1</sup>
<institution>Department of Dermatology, Shanghai Skin Disease Hospital, Tongji University School of Medicine</institution>, <addr-line>Shanghai</addr-line>, <country>China</country>
</aff>
<aff id="aff2">
<sup>2</sup>
<institution>Department of Dermatology, Shanghai Tenth People&#x2019;s Hospital, Tongji University School of Medicine</institution>, <addr-line>Shanghai</addr-line>, <country>China</country>
</aff>
<aff id="aff3">
<sup>3</sup>
<institution>Institute of Psoriasis, Tongji University School of Medicine</institution>, <addr-line>Shanghai</addr-line>, <country>China</country>
</aff>
<author-notes>
<fn fn-type="edited-by">
<p>
<bold>Edited by:</bold> <ext-link ext-link-type="uri" xlink:href="https://loop.frontiersin.org/people/918437/overview">Ji Li</ext-link>, Central South University, China</p>
</fn>
<fn fn-type="edited-by">
<p>
<bold>Reviewed by:</bold> <ext-link ext-link-type="uri" xlink:href="https://loop.frontiersin.org/people/1290728/overview">Zhiqiang Song</ext-link>, Army Medical University, China</p>
<p>
<ext-link ext-link-type="uri" xlink:href="https://loop.frontiersin.org/people/1006387/overview">Danuta Gutowska-Owsiak</ext-link>, University of Gda&#x144;sk and Medical University of Gda&#x144;sk, Poland</p>
</fn>
<corresp id="c001">&#x2a;Correspondence: Qian Yu, <email>yuervictory@163.com</email>; Yuling Shi, <email>shiyuling1973@tongji.edu.cn</email>
</corresp>
<fn fn-type="equal" id="fn1">
<label>
<sup>&#x2020;</sup>
</label>
<p>These authors have contributed equally to this&#x20;work</p>
</fn>
<fn fn-type="other">
<p>This article was submitted to Cell Death and Survival, a section of the journal Frontiers in Cell and Developmental Biology</p>
</fn>
</author-notes>
<pub-date pub-type="epub">
<day>03</day>
<month>02</month>
<year>2022</year>
</pub-date>
<pub-date pub-type="collection">
<year>2021</year>
</pub-date>
<volume>9</volume>
<elocation-id>799560</elocation-id>
<history>
<date date-type="received">
<day>21</day>
<month>10</month>
<year>2021</year>
</date>
<date date-type="accepted">
<day>13</day>
<month>12</month>
<year>2021</year>
</date>
</history>
<permissions>
<copyright-statement>Copyright &#xa9; 2022 Hu, Chen, Chen, Zhang, Guo, Yu, Xu, Sun, Zhou, Gong, Yu and Shi.</copyright-statement>
<copyright-year>2022</copyright-year>
<copyright-holder>Hu, Chen, Chen, Zhang, Guo, Yu, Xu, Sun, Zhou, Gong, Yu and Shi</copyright-holder>
<license xlink:href="http://creativecommons.org/licenses/by/4.0/">
<p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these&#x20;terms.</p>
</license>
</permissions>
<abstract>
<p>
<bold>Background:</bold> Psoriasis is a common immune-mediated skin disease that involves T-cell-mediated immunity. Invariant natural killer T (<italic>i</italic>NKT) cells are a unique lymphocyte subpopulation that share properties and express surface markers of both NK cells and T&#x20;cells. Previous reports indicate that <italic>i</italic>NKT&#x20;cells regulate the development of various inflammatory diseases. IL-17 is a key cytokine in the pathogenesis of psoriasis and a key therapeutic target. Secukinumab is a fully human IgG1&#x3ba; antibody that targets IL-17A, thereby antagonizing the biological effects of IL-17.</p>
<p>
<bold>Objective:</bold> To explore the expression of <italic>i</italic>NKT&#x20;cells in psoriasis patients and the effect of secukinumab on&#x20;them.</p>
<p>
<bold>Methods:</bold> We examined the frequencies of <italic>i</italic>NKT&#x20;cells, Tregs, na&#xef;ve and memory CD4<sup>&#x2b;</sup>and CD8<sup>&#x2b;</sup>T&#x20;cells in the PBMCs as well as their cytokine production in a cohort of 40 patients with moderate-to-severe plaque psoriasis and 40 gender- and age-matched healthy controls. We further collected peripheral blood of another 15 moderate-to-severe plaque psoriasis patients who were treated with secukinumab and evaluated the proportion of <italic>i</italic>NKT&#x20;cells in the PBMCs at baseline and week&#x20;12.</p>
<p>
<bold>Results:</bold> The frequencies of conventional CD4<sup>&#x2b;</sup> T&#x20;cells, CD8<sup>&#x2b;</sup> T&#x20;cells, and Tregs in the PBMCs were comparable between psoriasis patients and healthy controls, but the frequencies of Th17 cells, Tc1 cells and Tc17 cells were increased in psoriasis patients. The frequency of peripheral <italic>i</italic>NKT&#x20;cells and CD69<sup>&#x2b;</sup> <italic>i</italic>NKT&#x20;cells was significantly decreased in psoriasis patients. Both <italic>i</italic>NKT2 cells and <italic>i</italic>NKT17 cells were increased in psoriasis patients, but the ratio of <italic>i</italic>NKT2 cells vs <italic>i</italic>NKT17 cells was significantly reduced in psoriasis patients. After receiving secukinumab, the proportion of <italic>i</italic>NKT&#x20;cells in the PBMCs of patients was increased, while the proportion of <italic>i</italic>NKT17 cells was decreased.</p>
<p>
<bold>Conclusion:</bold> Dysregulated <italic>i</italic>NKT&#x20;cells may be involved in the pathogenesis of psoriasis and secukinumab may play a regulatory role on <italic>i</italic>NKT&#x20;cells.</p>
</abstract>
<kwd-group>
<kwd>psoriasis</kwd>
<kwd>iNKT&#x20;cells</kwd>
<kwd>IFN-&#x3b3;</kwd>
<kwd>IL-4</kwd>
<kwd>IL-17</kwd>
</kwd-group>
<contract-num rid="cn001">81872522 82073429&#x20;81903205 81803120</contract-num>
<contract-sponsor id="cn001">National Natural Science Foundation of China<named-content content-type="fundref-id">10.13039/501100001809</named-content>
</contract-sponsor>
</article-meta>
</front>
<body>
<sec id="s1">
<title>Introduction</title>
<p>Psoriasis is a common inflammatory skin disease that tends to recur frequently and presently has no cure. The incidence of psoriasis varies worldwide, and its prevalence rate in China is 0.47% (<xref ref-type="bibr" rid="B16">Ding et&#x20;al., 2012</xref>). The development of psoriasis primarily involves T-cell-mediated immunity, and the interleukin (IL)-23/T helper 17 (Th17) cell axis plays an essential role in the pathogenesis of psoriasis (<xref ref-type="bibr" rid="B7">Boehncke and Schon, 2015</xref>).</p>
<p>Natural killer T&#x20;cells (NKT&#x20;cells) are a unique lymphocyte subpopulation that shares immune properties and expresses surface markers of both natural killer (NK) cells and T&#x20;cells. Upon activation, NKT&#x20;cells rapidly produce Th1, Th2, and Th17 cytokines (<xref ref-type="bibr" rid="B9">Brennan et&#x20;al., 2013</xref>). In general, NKT&#x20;cells are divided into type I (invariant NKT [<italic>i</italic>NKT]) and type II (non-<italic>i</italic>NKT) cells (<xref ref-type="bibr" rid="B20">Godfrey et&#x20;al., 2004</xref>). <italic>i</italic>NKT&#x20;cells express an invariant TCR&#x3b1; chain consisting of V&#x3b1;14/J&#x3b1;18 paired with a limited range of TCR&#x3b2; chains in mice or V&#x3b1;24/J&#x3b1;18 paired with V&#x3b2;11 in humans (<xref ref-type="bibr" rid="B27">Koseki et&#x20;al., 1991</xref>; <xref ref-type="bibr" rid="B1">Arase et&#x20;al., 1992</xref>; <xref ref-type="bibr" rid="B29">Lantz and Bendelac, 1994</xref>). <italic>i</italic>NKT&#x20;cells demonstrate CD1d restriction, and <italic>&#x3b1;</italic>-GalCer is the first CD1d-presented lipid antigen for <italic>i</italic>NKT&#x20;cells (<xref ref-type="bibr" rid="B24">Kawano et&#x20;al., 1997</xref>). Therefore, immunostaining with <italic>&#x3b1;</italic>-GalCer-loaded CD1d tetramers could be useful for precise identification of <italic>i</italic>NKT&#x20;cells (<xref ref-type="bibr" rid="B24">Kawano et&#x20;al., 1997</xref>).</p>
<p>Based on cytokine production and transcription factor expression, <italic>i</italic>NKT&#x20;cells can be differentiated into at least three subsets: Th1-like <italic>i</italic>NKT&#x20;cells (<italic>i</italic>NKT1) that secrete interferon-&#x3b3; (IFN-&#x3b3;) and express T-bet (<xref ref-type="bibr" rid="B43">Townsend et&#x20;al., 2004</xref>); Th2-like <italic>i</italic>NKT&#x20;cells (<italic>i</italic>NKT2) that produce Interleukin-4 (IL-4) and are dependent on the transcription factors PLZF, GATA3, and IRF4 for development (<xref ref-type="bibr" rid="B25">Kim et&#x20;al., 2006</xref>; <xref ref-type="bibr" rid="B30">Lee et&#x20;al., 2013</xref>); and Th17-like <italic>i</italic>NKT&#x20;cells (iNKT17) that secrete Interleukin-17 (IL-17) and express ROR-&#x3b3;t (<xref ref-type="bibr" rid="B11">Coquet et&#x20;al., 2008</xref>).</p>
<p>
<italic>i</italic>NKT&#x20;cells are a type of key immunoregulatory T&#x20;cell. <italic>i</italic>NKT&#x20;cells have been reported to be involved in the development of various inflammatory diseases. They participate in the control of inflammatory bowel disease, allograft tolerance, and regulation of atopic eczema (<xref ref-type="bibr" rid="B40">Seino et&#x20;al., 2001</xref>; <xref ref-type="bibr" rid="B19">Fuss et&#x20;al., 2004</xref>; <xref ref-type="bibr" rid="B41">Simon et&#x20;al., 2009</xref>; <xref ref-type="bibr" rid="B44">Tsuruyama et&#x20;al., 2012</xref>). Previous research showed that the population of NKT&#x20;cells increases significantly in psoriatic lesions (<xref ref-type="bibr" rid="B8">Bonish et&#x20;al., 2000</xref>; <xref ref-type="bibr" rid="B10">Cameron et&#x20;al., 2002</xref>; <xref ref-type="bibr" rid="B37">Ottaviani et&#x20;al., 2006</xref>; <xref ref-type="bibr" rid="B49">Zhao et&#x20;al., 2008</xref>). In contrast to the accumulation of NKT&#x20;cells in psoriatic plaques, a few studies have documented decreased proportions and compromised immune activities of NKT&#x20;cells in the peripheral blood of psoriasis patients (<xref ref-type="bibr" rid="B46">Van Der Vliet et&#x20;al., 2001</xref>; <xref ref-type="bibr" rid="B26">Koreck et&#x20;al., 2002</xref>; <xref ref-type="bibr" rid="B47">Werner et&#x20;al., 2011</xref>). On the contrary, Langewouters <italic>et&#x20;al.</italic> found an increase in the number of circulating CD94<sup>&#x2b;</sup>CD161<sup>&#x2b;</sup> NKT&#x20;cells in psoriasis patients (<xref ref-type="bibr" rid="B28">Langewouters et&#x20;al., 2008</xref>). It has been confirmed that human <italic>i</italic>NKT&#x20;cells can produce IL-17 in a pro-inflammatory environment (<xref ref-type="bibr" rid="B34">Moreira-Teixeira et&#x20;al., 2011</xref>). Mars <italic>et&#x20;al.</italic> discovered that <italic>i</italic>NKT&#x20;cells played an important role in limiting the development of the Th17 lineage and provided a natural barrier against Th17 responses in EAE mouse model (<xref ref-type="bibr" rid="B33">Mars et&#x20;al., 2009</xref>). Keunhee&#x2019;s study also showed that <italic>i</italic>NKT&#x20;cells can suppress Th17 cell differentiation (<xref ref-type="bibr" rid="B36">Oh et&#x20;al., 2011</xref>). However, the cell surface markers utilized to identify <italic>i</italic>NKT&#x20;cells in the aforementioned studies, for example, CD3, CD161, and CD94, were not specific to <italic>i</italic>NKT&#x20;cells. In the present study, we used CD1d tetramers, which are exclusive markers of <italic>i</italic>NKT&#x20;cells, to accurately identify <italic>i</italic>NKT&#x20;cells and evaluate their immune functions in psoriasis patients.</p>
<p>Secukinumab is a fully human IgG1&#x3ba; antibody that targets IL-17A, thereby antagonizing the biological effects of the cytokine. In 2015, secukinumab was approved by the European Medicines Evaluation Agency (EMEA) and the U.S. Food and Drug Administration (FDA) for marketing in Europe and the United&#x20;States for the treatment of adult moderate-to-severe plaque psoriasis. We also evaluate the proportion of <italic>i</italic>NKT&#x20;cells in the PBMCs of psoriasis patients treated with secukinumab at baseline and week 12, and analyze whether there is a difference in the proportion of <italic>i</italic>NKT&#x20;cells before and after treatment.</p>
</sec>
<sec sec-type="materials|methods" id="s2">
<title>Materials and Methods</title>
<sec id="s2-1">
<title>Patients</title>
<p>This study was approved by Shanghai Tenth People&#x2019;s Hospital Ethics Committees (IRB approval number: 2013-RES-14). We collected the peripheral blood of 40 moderate-to-severe plaque psoriasis patients and 40 gender- and age-matched healthy controls from December 2017 to December 2019. The disease severity of psoriasis patients was assessed using the psoriasis area and severity index (PASI) score. Patients&#x2019; PASI score were all &#x2265;10 when the blood samples were drawn (<xref ref-type="table" rid="T1">Table&#x20;1</xref>). We also collected peripheral blood of another 15 moderate-to-severe plaque psoriasis patients who were treated with secukinumab before and after the 12&#xa0;weeks of treatment. Another 15 gender- and age-matched healthy controls&#x2019; peripheral blood were also collected. All the participants had no other autoimmune diseases, systemic diseases, malignant tumor or active infections, and had not received systemic therapy for at least 4&#x20;weeks or topical therapy for at least 2&#xa0;weeks. All the procedures were in accordance with the tenets of the Declaration of Helsinki for research involving human subjects. Informed consent was obtained from all the participants, and their clinical information and peripheral blood samples were collected for analysis.</p>
<table-wrap id="T1" position="float">
<label>TABLE 1</label>
<caption>
<p>Details of moderate-to-severe plaque psoriasis patients.</p>
</caption>
<table>
<thead valign="top">
<tr>
<th align="left">Characteristics</th>
<th align="center">Healthy Control (N&#x20;&#x3d;&#x20;40)</th>
<th align="center">Psoriasis Patients (N&#x20;&#x3d;&#x20;40)</th>
</tr>
</thead>
<tbody valign="top">
<tr>
<td align="left">Age, years, mean (SD)</td>
<td align="center">40.1&#x20;&#xb1; 10.05</td>
<td align="center">40.85&#x20;&#xb1; 9.81</td>
</tr>
<tr>
<td align="left">Female sex, n (%)</td>
<td align="center">45</td>
<td align="center">45</td>
</tr>
<tr>
<td align="left">Body mass index</td>
<td align="center">23.87&#x20;&#xb1; 2.44</td>
<td align="center">26.34&#x20;&#xb1; 4.21</td>
</tr>
<tr>
<td align="left">Disease duration, years, mean (SD)</td>
<td align="center">-</td>
<td align="center">16.63&#x20;&#xb1; 9.21</td>
</tr>
<tr>
<td align="left">PASI score, mean (SD)</td>
<td align="center">-</td>
<td align="center">18.88&#x20;&#xb1; 7.63</td>
</tr>
<tr>
<td align="left">PGA score, mean (SD)</td>
<td align="center">-</td>
<td align="center">2.25&#x20;&#xb1; 0.43</td>
</tr>
<tr>
<td align="left">BSA (%), mean (SD)</td>
<td align="center">-</td>
<td align="center">39.53&#x20;&#xb1; 5.59</td>
</tr>
<tr>
<td align="left">DLQI score, mean (SD)</td>
<td align="center">-</td>
<td align="center">11.86&#x20;&#xb1; 7.17</td>
</tr>
</tbody>
</table>
</table-wrap>
</sec>
<sec id="s2-2">
<title>Treatment and Assessments</title>
<p>15 patients received subcutaneous secukinumab 300&#xa0;mg at Week 0, 1, 2, 3, 4. After that, they received subcutaneous secukinumab 300&#xa0;mg every 4&#xa0;weeks for maintenance treatment. During the treatment, patients should not take any other drugs or physical therapy that may affect the evaluation of efficacy, such as calcineurin inhibitors, glucocorticoids, vitamin D3 derivatives, acitretin, methotrexate, cyclosporine, other biological agents, PUVA and NB-UVB. Patients were recommended to use moisturizing cream&#x20;daily.</p>
<p>PASI, PGA and BSA score were used for patients&#x2019; efficacy assessment.</p>
</sec>
<sec id="s2-3">
<title>Isolation of Peripheral Blood and Flow Cytometry</title>
<p>PBMCs were freshly separated from human peripheral blood using Ficoll-Paque Plus (Catalog&#x23; 17-1440-03, GE Healthcare) according to the manufacturer&#x2019;s recommendations. PBMCs were treated <italic>in&#x20;vitro</italic> with Cell Stimulation Cocktail (Catalog&#x23; 00-4970-03, eBioscience) for 5&#xa0;h to detect cytokine secretion. To identify dead cells, the cells were first stained with Fixable Viability Stain 780 (Catalog&#x23; 565,388, BD Biosciences) for 15&#xa0;min at room temperature. Subsequently, the cells were stained for 30&#xa0;min with surface marker antibodies in phosphate-buffered saline containing 2% fetal bovine serum at 4&#xb0;C. For detecting intracytoplasmic cytokines (IC), the cells were fixed with IC Fixation Buffer (Catalog&#x23; 00-8222-49, eBioscience) for 30&#xa0;min at 4&#xb0;C. For analyzing intranuclear transcription factors, the cells were fixed with Fixation/Permeabilization Diluent and Concentrate (Catalog&#x23; 88-8824-00, eBioscience) at 4&#x20;&#xb0;C for 40&#xa0;min. After fixation, the cells were stained with intracellular antibodies in Permeabilization Buffer (Catalog&#x23; 00-8333-56, eBioscience) at 4&#x20;&#xb0;C for 30&#xa0;min.</p>
<p>To analyze CD4<sup>&#x2b;</sup> T&#x20;cells, CD8<sup>&#x2b;</sup> T&#x20;cells, regulatory T&#x20;cells (Treg), and <italic>i</italic>NKT&#x20;cell frequencies and immunofunctions, PBMCs were stained with the following anti-human antibodies: APC-conjugated <italic>&#x3b1;</italic>-GalCer:CD1d tetramer (NIH tetramer facility, United states), FITC-conjugated anti-CD4 (Catalog&#x23; 11-0048-42, eBioscience), APC-conjugated anti-CD4 (Catalog&#x23; 17-0049-42, eBioscience), PE-conjugated anti-CD4 (Catalog&#x23; 12-0048-42, eBioscience), PE-conjugated anti-CD8 (Catalog&#x23; 12-0086-42, eBioscience), PerCP/Cy5.5-conjugated anti-CD8 (Catalog&#x23; 301,032, eBioscience), PE-conjugated anti-IL-17A (Catalog&#x23; 12-7179-42, eBioscience), APC-conjugated anti-IFN-&#x3b3; (Catalog&#x23; 17-7319-82, eBioscience), PE/cyanine 7 (Cy7)-conjugated anti-IL-4 (Catalog&#x23; 25-7049-82, eBioscience), PE-conjugated anti-CD25 (Catalog&#x23; 12-0259-42, eBioscience), APC-conjugated anti-forkhead box P3 (FOXP3) (Catalog&#x23; 17-4777-42, eBioscience), PE/Cy7-conjugated anti-CD3 (Catalog&#x23; 300,420, BioLegend), FITC-conjugated anti-CD69 (Catalog&#x23; 11-0699-42, eBioscience), FITC-conjugated anti-CD45RA (Catalog&#x23; 11-0458-42, eBioscience), PE/Cy7-conjugated anti-CD45RO (Catalog&#x23; 25-0457-42, eBioscience), FITC-conjugated anti-GATA3 (Catalog&#x23; 53-9966-42, eBioscience), PE-conjugated anti-ROR-&#x3b3;t (Catalog&#x23; 12-6988-82, eBioscience), PerCP/Cy5.5-conjugated anti-T-bet (Catalog&#x23; 644,805, Biolegend), and FITC-conjugated anti-IFN-&#x3b3; (Catalog&#x23; 11-7319-82, eBioscience).</p>
<p>Data were acquired on a FACS Canto II (BD Biosciences) and analyzed using the FlowJo software (Tree Star).</p>
</sec>
<sec id="s2-4">
<title>Statistical Analysis</title>
<p>Data are presented as mean&#x20;&#xb1; SD and shown as dot plots of individual samples. Statistical significance was assessed with a two-tailed paired student&#x2019;s <italic>t</italic>-test. Correlation analysis was performed using the Pearson correlation test. All statistical analyses were performed using the GraphPad Prism software. For all cases, significant differences were considered at <italic>p</italic> values &#x3c;&#x20;0.05.</p>
</sec>
</sec>
<sec sec-type="results" id="s3">
<title>Results</title>
<sec id="s3-1">
<title>Normal Distribution of Circulating CD4<sup>&#x2b;</sup> T&#x20;Cells, CD8<sup>&#x2b;</sup> T&#x20;Cells and Tregs in Psoriasis Patients</title>
<p>First, we examined peripheral conventional T&#x20;cells by flow cytometry. As shown in <xref ref-type="fig" rid="F1">Figures 1A, 1B</xref>, the percentages of CD4<sup>&#x2b;</sup> and CD8<sup>&#x2b;</sup> T&#x20;cells in the PBMCs were comparable between psoriasis patients and healthy controls. There was also no significant difference in the percentages of na&#xef;ve (CD45RA<sup>&#x2b;</sup>) and memory (CD45RO<sup>&#x2b;</sup>) T&#x20;cells within either the CD4<sup>&#x2b;</sup> T&#x20;cell or CD8<sup>&#x2b;</sup> T&#x20;cell subsets between psoriasis patients and healthy controls (<xref ref-type="fig" rid="F1">Figures 1C,D</xref>). We further analyzed T-cell activation based on the expression of CD69. As shown in <xref ref-type="fig" rid="F1">Figures 1E,F</xref>, a higher number of CD69-positive CD4<sup>&#x2b;</sup> T&#x20;cells were found in the psoriasis patients than in the healthy controls. However, no significant differences were observed in the proportion of CD69-positive CD8<sup>&#x2b;</sup> T&#x20;cells between psoriasis patients and healthy controls. Similarly, no significant difference was detected in the proportion of circulating CD4<sup>&#x2b;</sup> CD25<sup>&#x2b;</sup> Foxp3<sup>&#x2b;</sup> Tregs (<xref ref-type="fig" rid="F1">Figure&#x20;1G,H</xref>, gating strategy in <xref ref-type="sec" rid="s11">Supplementary Figure S1</xref>). Thus, our results demonstrated normal distribution of peripheral conventional T&#x20;cells and overactivation of conventional CD4<sup>&#x2b;</sup> T&#x20;cells.</p>
<fig id="F1" position="float">
<label>FIGURE 1</label>
<caption>
<p>Distribution of conventional T&#x20;cells and Tregs in the peripheral blood. <bold>(A)</bold> Flow cytometry analysis of CD4<sup>&#x2b;</sup> and CD8<sup>&#x2b;</sup> T&#x20;cells in PBMCs from moderate-to-severe plaque psoriasis patients and healthy controls. <bold>(B)</bold> Summary plots showing individual results of the frequency of CD4<sup>&#x2b;</sup> and CD8<sup>&#x2b;</sup> T&#x20;cells in moderate-to-severe plaque psoriasis patients versus healthy controls. <bold>(C)</bold> Flow cytometry analysis of memory (CD45RO&#x2b;) and na&#xef;ve (CD45RA&#x2b;) CD4<sup>&#x2b;</sup> and CD8<sup>&#x2b;</sup> T&#x20;cells in PBMCs from moderate-to-severe plaque psoriasis patients and healthy controls. <bold>(D)</bold> Summary plots showing individual results of the frequency of CD45RA&#x2b; and CD45RO &#x2b; CD4<sup>&#x2b;</sup> and CD8<sup>&#x2b;</sup> T&#x20;cells in moderate-to-severe plaque psoriasis patients versus healthy controls. <bold>(E)</bold> Flow cytometry analysis of CD69 expression in CD4<sup>&#x2b;</sup> and CD8<sup>&#x2b;</sup> T&#x20;cells in PBMCs from moderate-to-severe plaque psoriasis patients and healthy controls. <bold>(F)</bold> Summary plots showing individual results of the frequency of CD69&#x20;<sup>&#x2b;</sup> CD4<sup>&#x2b;</sup> T&#x20;cells and CD69&#x20;<sup>&#x2b;</sup> CD8<sup>&#x2b;</sup> T&#x20;cells in moderate-to-severe plaque psoriasis patients versus healthy controls. <bold>(G)</bold> Flow cytometry analysis of CD4<sup>&#x2b;</sup>CD25&#x20;<sup>&#x2b;</sup> Foxp3&#x2b; Tregs in PBMCs from moderate-to-severe plaque psoriasis patients and healthy controls. <bold>(H)</bold> Summary plots showing individual results of the frequency of CD4<sup>&#x2b;</sup>CD25&#x20;<sup>&#x2b;</sup> Foxp3&#x2b; Tregs in psoriasis patients versus healthy controls. Data show mean &#x2b;SEM. <italic>p</italic>-values were determined by paired Student&#x2019;s t-test. ns, no significance, &#x2a;<italic>p</italic>&#x20;&#x3c; 0.05, &#x2a;&#x2a;<italic>p</italic>&#x20;&#x3c; 0.01, &#x2a;&#x2a;&#x2a;<italic>p</italic>&#x20;&#x3c; 0.001 and &#x2a;&#x2a;&#x2a;&#x2a;<italic>p</italic>&#x20;&#x3c; 0.0001.</p>
</caption>
<graphic xlink:href="fcell-09-799560-g001.tif"/>
</fig>
</sec>
<sec id="s3-2">
<title>Increase in Th17 Cells, Tc1 and Tc17 Cells in the PBMCs of Psoriasis Patients</title>
<p>Next, we examined the production of IFN-&#x3b3;, IL-4, and IL-17 by conventional T lymphocytes. There was no significant difference in the percentages of Th1 and Th2 cells between psoriasis patients and healthy individuals (<xref ref-type="fig" rid="F2">Figures 2A,B</xref>). Notably, the proportion of Th17 cells was significantly augmented in the PBMCs of psoriasis patients (<xref ref-type="fig" rid="F2">Figures 2A,B</xref>), as reported in previous studies (<xref ref-type="bibr" rid="B18">Furue et&#x20;al., 2019</xref>). Moreover, the proportions of circulating T cytotoxic 1 (Tc1) and Tc17 cells were significantly upregulated in psoriasis patients (<xref ref-type="fig" rid="F2">Figures&#x20;2C,D</xref>).</p>
<fig id="F2" position="float">
<label>FIGURE 2</label>
<caption>
<p>Cytokine-producing T&#x20;cells from peripheral blood. PBMCs isolated from moderate-to-severe plaque psoriasis patients and healthy controls were stimulated with Cell Stimulation Cocktail for 5&#xa0;h. The IFN-&#x3b3;-, IL-4- and IL-17-producing T&#x20;cells were determined by intracellular staining and flow cytometry analysis. <bold>(A)</bold> The proportion of IFN-&#x3b3;&#x2013;,IL-4- and IL-17-producing CD4<sup>&#x2b;</sup> T&#x20;cells in psoriasis patients versus healthy controls. <bold>(B)</bold>Summary plots showing individual results of the frequency of IFN-&#x3b3;&#x2013;,IL-4- and IL-17- producing CD4<sup>&#x2b;</sup> T&#x20;cells in psoriasis patients versus healthy controls. <bold>(C)</bold>The proportion of IFN-&#x3b3;&#x2013;,IL-4- and IL-17-producing CD8<sup>&#x2b;</sup> T&#x20;cells in psoriasis patients versus healthy controls. <bold>(D)</bold>Summary plots showing individual results of the frequency of IFN-&#x3b3;-,IL-4- and IL-17- producing CD8<sup>&#x2b;</sup> T&#x20;cells in psoriasis patients versus healthy controls. Data show mean &#x2b;SEM. <italic>p</italic>-values were determined by paired Student&#x2019;s t-test. ns, no significance, &#x2a;<italic>p</italic>&#x20;&#x3c; 0.05, &#x2a;&#x2a;<italic>p</italic>&#x20;&#x3c; 0.01, &#x2a;&#x2a;&#x2a;<italic>p</italic>&#x20;&#x3c; 0.001 and &#x2a;&#x2a;&#x2a;&#x2a;<italic>p</italic>&#x20;&#x3c; 0.0001.</p>
</caption>
<graphic xlink:href="fcell-09-799560-g002.tif"/>
</fig>
</sec>
<sec id="s3-3">
<title>Decrease in the Frequency of <italic>i</italic>NKT&#x20;Cells in the PBMCs of Psoriasis Patients</title>
<p>
<italic>i</italic>NKT&#x20;cells have been shown to play a crucial role in the development of autoimmune diseases (<xref ref-type="bibr" rid="B5">Bendelac et&#x20;al., 2007</xref>). The <italic>&#x3b1;</italic>-GalCer-loaded CD1d tetramer is the best reagent currently available to accurately distinguish <italic>i</italic>NKT&#x20;cells in terms of specificity and sensitivity (<xref ref-type="bibr" rid="B6">Berzins et&#x20;al., 2011</xref>). As depicted in <xref ref-type="fig" rid="F3">Figures 3A,B</xref>, the proportion of <italic>i</italic>NKT&#x20;cells in the PBMCs from psoriasis patients was lower than that in healthy controls. This indicates that a defect in <italic>i</italic>NKT&#x20;cells might be involved in the development of psoriasis. However, there was no correlation between the proportion of <italic>i</italic>NKT&#x20;cells and PASI score in psoriasis patients (<xref ref-type="sec" rid="s11">Supplementary Figure&#x20;S2</xref>).</p>
<fig id="F3" position="float">
<label>FIGURE 3</label>
<caption>
<p>
<italic>i</italic>NKT&#x20;cells frequency and cell subsets in PBMCs of psoriasis patients. <bold>(A)</bold> Representative FACS dot plots for iNKT&#x20;cells from psoriasis patients and healthy controls. <bold>(B)</bold> Summary plots showing individual results of iNKT&#x20;cell frequency in psoriasis patients versus healthy controls. <bold>(C)</bold> Representative FACS dot plots for iNKT&#x20;cell CD4 expression in psoriasis patients and healthy controls. <bold>(D)</bold> Summary plots showing individual results of the frequency of CD4<sup>&#x2b;</sup> iNKT&#x20;cells in psoriasis patients versus healthy controls. <bold>(E)</bold> Representative histogram and summary plots showing individual results of the MFI for T-bet &#x2b; iNKT&#x20;cells in psoriasis patients and healthy controls. <bold>(F)</bold> Representative histogram and summary plots showing individual results of the MFI for GATA3&#x2b; iNKT&#x20;cells in psoriasis patients and healthy controls. <bold>(G)</bold> Representative histogram and summary plots showing individual results of the MFI for ROR&#x3b3;t &#x2b; iNKT&#x20;cells in psoriasis patients and healthy controls. <bold>(H)</bold> Summary plots showing individual results of GATA3&#x2b; iNKT&#x20;cells/ROR&#x3b3;t &#x2b; iNKT&#x20;cells, ROR&#x3b3;t &#x2b; iNKT&#x20;cells/T-bet &#x2b; iNKT&#x20;cells and GATA3&#x2b; iNKT&#x20;cells//T-bet &#x2b; iNKT&#x20;cells in psoriasis patients versus healthy controls. Data show mean &#x2b;SEM. <italic>p</italic>-values were determined by paired Student&#x2019;s t-test. ns, no significance, &#x2a;<italic>p</italic>&#x20;&#x3c; 0.05, &#x2a;&#x2a;<italic>p</italic>&#x20;&#x3c; 0.01, &#x2a;&#x2a;&#x2a;<italic>p</italic>&#x20;&#x3c; 0.001 and &#x2a;&#x2a;&#x2a;&#x2a;<italic>p</italic>&#x20;&#x3c; 0.0001.</p>
</caption>
<graphic xlink:href="fcell-09-799560-g003.tif"/>
</fig>
</sec>
<sec id="s3-4">
<title>Increase in <italic>i</italic>NKT2 and <italic>i</italic>NKT17 Cell Sublineages in Psoriasis Patients</title>
<p>Human mature <italic>i</italic>NKT&#x20;cells can be divided into functionally distinct CD4<sup>&#x2b;</sup> and CD4<sup>&#x2212;</sup> subsets. CD4<sup>&#x2b;</sup> <italic>i</italic>NKT&#x20;cells produce both Th1 and Th2 cytokines, whereas the CD4<sup>&#x2212;</sup> <italic>i</italic>NKT subset mainly exhibits a Th1 cytokine profile (<xref ref-type="bibr" rid="B21">Gumperz et&#x20;al., 2002</xref>). To investigate whether <italic>i</italic>NKT&#x20;cells from psoriasis patients exhibited phenotypic abnormalities, we first analyzed the proportion of the CD4<sup>&#x2b;</sup> <italic>i</italic>NKT subset in the PBMCs, but no significant differences were detected between psoriasis patients and healthy controls (<xref ref-type="fig" rid="F3">Figures&#x20;3C,D</xref>).</p>
<p>We further analyzed the sublineages of <italic>i</italic>NKT&#x20;cells (<xref ref-type="fig" rid="F3">Figures 3E&#x2013;G</xref>). While the Mean Fluorescence Intensity (MFI) of T-bet<sup>&#x2b;</sup> <italic>i</italic>NKT&#x20;cells (<italic>i</italic>NKT1) remained unaltered, the MFI of GATA3<sup>&#x2b;</sup> <italic>i</italic>NKT&#x20;cells (<italic>i</italic>NKT2) and ROR-&#x3b3;t<sup>&#x2b;</sup> <italic>i</italic>NKT&#x20;cells (<italic>i</italic>NKT17) were significantly increased in psoriasis patients. Moreover, we found that the ratio of GATA3<sup>&#x2b;</sup> <italic>i</italic>NKT&#x20;cells vs ROR-&#x3b3;t<sup>&#x2b;</sup> <italic>i</italic>NKT&#x20;cells decreased and the ratio of ROR-&#x3b3;t<sup>&#x2b;</sup> <italic>i</italic>NKT&#x20;cells vs T-bet<sup>&#x2b;</sup> <italic>i</italic>NKT&#x20;cells increased in psoriasis patients, suggesting that there may be imbalance of <italic>i</italic>NKT&#x20;cells sublineages in psoriasis (<xref ref-type="fig" rid="F3">Figure&#x20;3H</xref>).</p>
</sec>
<sec id="s3-5">
<title>Decrease in <italic>i</italic>NKT&#x20;Cell Activation and Increased IL-4- and IL-17-Producing <italic>i</italic>NKT&#x20;Cells in Psoriasis Patients</title>
<p>CD69 has been utilized as a cell-surface marker of <italic>i</italic>NKT&#x20;cell maturation and activation. The percentage of CD69<sup>&#x2b;</sup> <italic>i</italic>NKT&#x20;cells was reduced in the PBMCs of psoriasis patients, suggesting that <italic>i</italic>NKT&#x20;cells are less activated in psoriasis patients competed to healthy controls (<xref ref-type="fig" rid="F4">Figures 4A,B</xref>). In addition, the MFI of IL-4- and IL-17-producing <italic>i</italic>NKT&#x20;cells were significantly increased in psoriasis patients, whereas no significant difference was detected in the MFI of IFN-&#x3b3;-producing <italic>i</italic>NKT&#x20;cells between psoriasis patients and healthy controls (<xref ref-type="fig" rid="F4">Figures 4C&#x2013;E</xref>). This was in accordance with our findings for the <italic>i</italic>NKT&#x20;cell sublineages. Furthermore, there was no significant difference in the ratio of IL-17-producing <italic>i</italic>NKT&#x20;cells vs IFN-&#x3b3;-producing <italic>i</italic>NKT&#x20;cells and the ratio of IL-4-producing <italic>i</italic>NKT&#x20;cells vs IFN-&#x3b3;-producing <italic>i</italic>NKT&#x20;cells between psoriasis patients and healthy controls (<xref ref-type="fig" rid="F4">Figure&#x20;4F</xref>).</p>
<fig id="F4" position="float">
<label>FIGURE 4</label>
<caption>
<p>
<italic>i</italic>NKT&#x20;cells activation status and cytokine production in PBMCs of psoriasis patients. Intracellular IFN-&#x3b3; and IL-4 production of circulating <italic>i</italic>NKT&#x20;cells was analyzed after stimulation with Cell Stimulation Cocktail for 5&#xa0;h. <bold>(A)</bold> Representative FACS dot plots for iNKT&#x20;cell CD69 expression in psoriasis patients and healthy controls. <bold>(B)</bold> Summary plots showing individual results of the frequency of CD69&#x20;<sup>&#x2b;</sup> iNKT&#x20;cells in psoriasis patients versus healthy controls. <bold>(C)</bold> Representative histogram and summary plots showing individual results of the MFI of IFN-&#x3b3;-producing iNKT&#x20;cells in psoriasis patients versus healthy controls. <bold>(D)</bold> Representative histogram and summary plots showing individual results of the MFI of IL-4-producing iNKT&#x20;cells in psoriasis patients versus healthy controls. <bold>(E)</bold> Representative histogram and summary plots showing individual results of the MFI of IL-17-producing iNKT&#x20;cells in psoriasis patients versus healthy controls. <bold>(F)</bold> Summary plots showing individual results of IL-4-producing <italic>i</italic>NKT&#x20;cells/IFN-&#x3b3;-producing <italic>i</italic>NKT&#x20;cells and IL-17-producing <italic>i</italic>NKT&#x20;cells/IFN-&#x3b3;-producing <italic>i</italic>NKT&#x20;cells in psoriasis patients versus healthy controls. Data show mean &#x2b;SEM. <italic>p</italic>-values were determined by paired Student&#x2019;s t-test. ns, no significance, &#x2a;<italic>p</italic>&#x20;&#x3c; 0.05, &#x2a;&#x2a;<italic>p</italic>&#x20;&#x3c; 0.01, &#x2a;&#x2a;&#x2a;<italic>p</italic>&#x20;&#x3c; 0.001 and &#x2a;&#x2a;&#x2a;&#x2a;<italic>p</italic>&#x20;&#x3c; 0.0001.</p>
</caption>
<graphic xlink:href="fcell-09-799560-g004.tif"/>
</fig>
</sec>
<sec id="s3-6">
<title>Increase in the Frequency of <italic>i</italic>NKT&#x20;Cells in Psoriasis Patients After Receiving Secukinumab</title>
<p>Secukinumab is effective in the treatment of moderate and severe plaque psoriasis. Patients&#x2019; characteristics, PASI, PGA and BSA score before and after the treatment are shown in <xref ref-type="table" rid="T2">Table&#x20;2</xref>. We further analyzed the proportion of <italic>i</italic>NKT&#x20;cells in the PBMCs of psoriasis patients before and after they treated with secukinumab, and found an increase of <italic>i</italic>NKT&#x20;cells after the treatment (<xref ref-type="fig" rid="F5">Figures 5A,B</xref>). Moreover, we found a decrease of <italic>i</italic>NKT17 subset in <italic>i</italic>NKT&#x20;cells after the treatment, while <italic>i</italic>NKT1 and <italic>i</italic>NKT2 subset remained no change (<xref ref-type="fig" rid="F5">Figures 5C&#x2013;E</xref>).</p>
<table-wrap id="T2" position="float">
<label>TABLE 2</label>
<caption>
<p>Details of moderate-to-severe plaque psoriasis patients treated with secukinumab.</p>
</caption>
<table>
<thead valign="top">
<tr>
<th align="left">Patient characteristics</th>
<th align="center">Baseline (N&#x20;&#x3d;&#x20;15)</th>
<th align="center">Week 12 (N&#x20;&#x3d;&#x20;15)</th>
</tr>
</thead>
<tbody valign="top">
<tr>
<td align="left">Age, years, mean (SD)</td>
<td align="center">36.53&#x20;&#xb1; 12.08</td>
<td align="center">36.76&#x20;&#xb1; 12.08</td>
</tr>
<tr>
<td align="left">Female sex, n (%)</td>
<td align="center">26.7%</td>
<td align="center">26.7%</td>
</tr>
<tr>
<td align="left">Body mass index</td>
<td align="center">23.7&#x20;&#xb1; 4.59</td>
<td align="center">23.18&#x20;&#xb1; 4.18</td>
</tr>
<tr>
<td align="left">Disease duration, years, mean (SD)</td>
<td align="center">12.73&#x20;&#xb1; 5.52</td>
<td align="center">12.96&#x20;&#xb1; 5.52</td>
</tr>
<tr>
<td align="left">PASI score, mean (SD)</td>
<td align="center">18.81&#x20;&#xb1; 9.78</td>
<td align="center">0.82&#x20;&#xb1; 0.71</td>
</tr>
<tr>
<td align="left">PGA score, mean (SD)</td>
<td align="center">2.27&#x20;&#xb1; 0.44</td>
<td align="center">0.67&#x20;&#xb1; 0.47</td>
</tr>
<tr>
<td align="left">BSA (%), mean (SD)</td>
<td align="center">33.67&#x20;&#xb1; 26.19</td>
<td align="center">2.4&#x20;&#xb1; 2.3</td>
</tr>
<tr>
<td align="left">DLQI score, mean (SD)</td>
<td align="center">14.27&#x20;&#xb1; 5.57</td>
<td align="center">2.93&#x20;&#xb1; 3.49</td>
</tr>
</tbody>
</table>
</table-wrap>
<fig id="F5" position="float">
<label>FIGURE 5</label>
<caption>
<p>Increased frequency of <italic>i</italic>NKT&#x20;cells in psoriasis patients treated with secukinumab. PBMC were isolated from psoriasis patients (n &#x3d; 15) at baseline untreated (Week 0) and 12 Weeks post treatment with secukinumab. <bold>(A)</bold> Representative FACS dot plots for <italic>i</italic>NKT&#x20;cells from psoriasis patients before and after secukinumab treatment versus healthy controls. <bold>(B)</bold> Summary plots showing individual results of <italic>i</italic>NKT&#x20;cell frequency in psoriasis patients before and after secukinumab treatment. <bold>(C)</bold> Summary plots showing individual results of T-bet<sup>&#x2b;</sup> <italic>i</italic>NKT&#x20;cells frequency in psoriasis patients before and after secukinumab treatment. <bold>(D)</bold> Summary plots showing individual results of GATA3<sup>&#x2b;</sup> <italic>i</italic>NKT&#x20;cells frequency in psoriasis patients before and after secukinumab treatment. <bold>(E)</bold> Summary plots showing individual results ROR&#x3b3;t<sup>&#x2b;</sup> <italic>i</italic>NKT&#x20;cells frequency in psoriasis patients before and after secukinumab treatment. Data show mean &#x2b;SEM. <italic>p</italic>-values were determined by paired Student&#x2019;s t-test. ns, no significance, &#x2a;<italic>p</italic>&#x20;&#x3c; 0.05, &#x2a;&#x2a;<italic>p</italic>&#x20;&#x3c; 0.01, &#x2a;&#x2a;&#x2a;<italic>p</italic>&#x20;&#x3c; 0.001 and &#x2a;&#x2a;&#x2a;&#x2a;<italic>p</italic>&#x20;&#x3c; 0.0001.</p>
</caption>
<graphic xlink:href="fcell-09-799560-g005.tif"/>
</fig>
</sec>
</sec>
<sec sec-type="discussion" id="s4">
<title>Discussion</title>
<p>In the present study, we isolated mononuclear cells from peripheral blood and detected the expression of <italic>i</italic>NKT&#x20;cells, conventional T&#x20;cells and their cytokine production, as well as Treg cells by flow cytometry. Our study represents a large-scale systemic analysis of the basic immunophenotypes of psoriasis patients with strictly matched healthy individual controls. We found that there were no differences between psoriasis patients and the healthy controls with regard to the percentages of conventional CD4<sup>&#x2b;</sup> and CD8<sup>&#x2b;</sup> T&#x20;cells, na&#xef;ve, memory CD4<sup>&#x2b;</sup> and CD8<sup>&#x2b;</sup> T&#x20;cells and active CD8<sup>&#x2b;</sup> T&#x20;cells. However, an increase in CD4<sup>&#x2b;</sup> T&#x20;cell activation was found in psoriasis patients. IL-17A is a key cytokine that participates in the pathogenesis of psoriasis. Biologic agents targeting IL-17A or IL-17RA have been demonstrated to have considerable clinical impact in the treatment of psoriasis, and this further proves the vital role of IL-17A in psoriasis (<xref ref-type="bibr" rid="B31">Leonardi et&#x20;al., 2012</xref>; <xref ref-type="bibr" rid="B38">Papp et&#x20;al., 2012</xref>; <xref ref-type="bibr" rid="B45">Van De Kerkhof et&#x20;al., 2016</xref>). Marcel <italic>et&#x20;al.</italic> showed that the proportion of IL-17A-producing CD8<sup>&#x2b;</sup> T&#x20;cells in the blood of psoriasis patients correlates with their PASI score (<xref ref-type="bibr" rid="B42">Teunissen et&#x20;al., 2014</xref>). In our study, we found that the Th17 and Tc17 populations were significantly increased in the PBMCs of psoriasis patients, in accordance with previous studies (<xref ref-type="bibr" rid="B42">Teunissen et&#x20;al., 2014</xref>; <xref ref-type="bibr" rid="B12">Dainichi et&#x20;al., 2018</xref>). We also found that the population of Tc1 cells was significantly increased in psoriasis patients. However, there were no differences in the percentages of Th1, Th2, and Tc2 cells between psoriasis patients and healthy controls. Treg cells are a subset of T&#x20;cells that can suppress the inflammation induced by other T&#x20;cells in autoimmune diseases (<xref ref-type="bibr" rid="B23">Jorn Bovenschen et&#x20;al., 2011</xref>; <xref ref-type="bibr" rid="B4">Barbi et&#x20;al., 2014</xref>; <xref ref-type="bibr" rid="B15">Deng et&#x20;al., 2016</xref>; <xref ref-type="bibr" rid="B32">Ma et&#x20;al., 2018</xref>). Several studies have shown the decreased number and impaired suppressive capacity of Treg cells in autoimmune diseases (<xref ref-type="bibr" rid="B39">Read et&#x20;al., 2000</xref>; <xref ref-type="bibr" rid="B3">Balandina et&#x20;al., 2005</xref>; <xref ref-type="bibr" rid="B50">Zhou et&#x20;al., 2009</xref>; <xref ref-type="bibr" rid="B2">Bailey-Bucktrout et&#x20;al., 2013</xref>; <xref ref-type="bibr" rid="B35">Nakagawa et&#x20;al., 2016</xref>). But research results of Treg cells in peripheral blood of psoriasis varies a lot. Furuhashi found that the number of Treg cells in the PBMCs of severe psoriasis patients with PASI &#x3e;12 decreased significantly, and the number of Treg cells increased after phototherapy (<xref ref-type="bibr" rid="B17">Fabio et&#x20;al., 2018</xref>). Karamehic&#x2019;s study showed that the number of CD4<sup>&#x2b;</sup>CD25<sup>&#x2b;</sup>Treg cells in the PBMCs of psoriasis patients was significantly lower than that of healthy control, but there was no significant correlation with PASI score (<xref ref-type="bibr" rid="B13">Dainichi et&#x20;al., 2019</xref>). In the meantime, the results of multiple studies have shown that the number of Treg cells in the peripheral blood of psoriasis patients is not significantly different from that of healthy control or is more than healthy control (<xref ref-type="bibr" rid="B14">Dantas et&#x20;al., 2016</xref>; <xref ref-type="bibr" rid="B48">Zhang et&#x20;al., 2016</xref>). In our study, we found that the number of CD4<sup>&#x2b;</sup> CD25<sup>&#x2b;</sup> FoxP3<sup>&#x2b;</sup> Treg cells was also not altered in psoriasis patients, but we did not conduct in-depth research on Treg&#x20;cells.</p>
<p>As mentioned earlier, in the present study, we used CD1d-tetramer staining, a specific method for <italic>i</italic>NKT identification, to accurately determine the percentage of <italic>i</italic>NKT&#x20;cells. A significant decrease in peripheral blood <italic>i</italic>NKT&#x20;cells was observed in psoriasis patients. Interestingly, we found that the proportion of <italic>i</italic>NKT&#x20;cells increased in patients treated with IL-17A inhibitor secukinumab. Combined with the increased NKT&#x20;cells in psoriatic lesions reported in previous studies (<xref ref-type="bibr" rid="B8">Bonish et&#x20;al., 2000</xref>; <xref ref-type="bibr" rid="B10">Cameron et&#x20;al., 2002</xref>; <xref ref-type="bibr" rid="B37">Ottaviani et&#x20;al., 2006</xref>; <xref ref-type="bibr" rid="B49">Zhao et&#x20;al., 2008</xref>). We speculated that the <italic>i</italic>NKT&#x20;cells in the PBMCs have accumulated in psoriatic plaques. And when the lesions subside, <italic>i</italic>NKT&#x20;cells may come back to peripheral&#x20;blood.</p>
<p>Human <italic>i</italic>NKT&#x20;cells can be segregated into CD4<sup>&#x2b;</sup> and CD4<sup>&#x2212;</sup> subsets according to their phenotypic and functional characteristics (<xref ref-type="bibr" rid="B21">Gumperz et&#x20;al., 2002</xref>). CD4<sup>&#x2b;</sup> <italic>i</italic>NKT&#x20;cells produce both Th1 and Th2 cytokines, whereas the CD4<sup>&#x2212;</sup> subset exhibits a Th1 cytokine profile. However, we found no difference in CD4<sup>&#x2b;</sup> and CD4<sup>&#x2212;</sup> subsets between psoriasis patients and healthy controls. We also examined the sublineages of <italic>i</italic>NKT&#x20;cells and found that the MFI of GATA3<sup>&#x2b;</sup> <italic>i</italic>NKT&#x20;cells and ROR&#x3b3;t<sup>&#x2b;</sup> <italic>i</italic>NKT&#x20;cells were significantly increased in psoriasis patients. We further analyzed the ratio of GATA3<sup>&#x2b;</sup> <italic>i</italic>NKT&#x20;cells vs ROR-&#x3b3;t<sup>&#x2b;</sup> <italic>i</italic>NKT&#x20;cells and found a decrease in psoriasis patients. Besides, the ratio of ROR-&#x3b3;t<sup>&#x2b;</sup> <italic>i</italic>NKT&#x20;cells vs T-bet<sup>&#x2b;</sup> <italic>i</italic>NKT&#x20;cells increased in psoriasis patients, which indicated that <italic>i</italic>NKT&#x20;cells are more likely to differentiate into ROR-&#x3b3;t<sup>&#x2b;</sup> <italic>i</italic>NKT&#x20;cells in psoriasis. We also found a decrease of <italic>i</italic>NKT17 subset in <italic>i</italic>NKT&#x20;cells after secukinumab treatment in psoriasis patients. This indicated a potential interaction between IL-17 and <italic>i</italic>NKT&#x20;cells. Perhaps the decreased proportion of <italic>i</italic>NKT17 cells might be a counterbalance to the administration of IL-17A agonist. We cannot exclude the possibility that the alternation in <italic>i</italic>NKT&#x20;cells might correlate with disease remission caused by secukinumab treatment. To explore the inner mechanism, we will stimulate <italic>i</italic>NKT&#x20;cells with IL-17 to examine their interactions in our future research.</p>
<p>Although <italic>i</italic>NKT&#x20;cells constitute only a small fraction of lymphocytes, their ability to rapidly secrete large amounts of cytokines, make them an important regulator of the Th1, Th2, and Th17 cytokine balance in immune responses. In our study, we found a decrease in the CD69<sup>&#x2b;</sup> subset in psoriasis patients. This indicated that psoriasis patients may have less activated <italic>i</italic>NKT&#x20;cells than healthy controls. But we also found that <italic>i</italic>NKT&#x20;cells in psoriasis patients secreted higher levels of IL-4 and IL-17, which is consistent with the increase observed in the sublineages of <italic>i</italic>NKT&#x20;cells in psoriasis patients. In our opinion, the reason why <italic>i</italic>NKT&#x20;cells in psoriasis patients secreted higher levels of IL-4 and IL-17 mainly lies in the increase of <italic>i</italic>NKT2 and <italic>i</italic>NKT17 subsets. And the inflammatory environment in psoriasis patients may be the reason for <italic>i</italic>NKT functional lineage differentiation shift. Also, the IFN-&#x3b3; producing <italic>i</italic>NKT1 may express higher CD69 than <italic>i</italic>NKT2 and <italic>i</italic>NKT17. While the proportion of <italic>i</italic>NKT2 and <italic>i</italic>NKT17 augmented in psoriasis patients, the CD69 expression decreased relatively. <italic>i</italic>NKT&#x20;cells anergy may also be a reason for the decrease of CD69<sup>&#x2b;</sup>
<italic>i</italic>NKT&#x20;cell in the PBMCs of psoriasis patients, although it&#x2019;s not the primary mechanism. It has also been reported that Treg cells suppress NKT&#x20;cell tumoricidal function by inducing more CD4<sup>&#x2212;</sup> NKT&#x20;cell anergy and less CD4<sup>&#x2b;</sup> NKT&#x20;cell anergy (<xref ref-type="bibr" rid="B22">Ihara et&#x20;al., 2019</xref>). Therefore, there may be interaction between Treg cells and NKT&#x20;cells in psoriasis, which is also the direction of our further research.</p>
<p>We didn&#x2019;t found difference in the ratio of IL-17-producing <italic>i</italic>NKT&#x20;cells vs IFN-&#x3b3;-producing <italic>i</italic>NKT&#x20;cells and the ratio of IL-4-producing <italic>i</italic>NKT&#x20;cells vs IFN-&#x3b3;-producing <italic>i</italic>NKT&#x20;cells between psoriasis patients and healthy controls, which indicated there were no imbalance between them. But this result still needs to be verified on more samples.</p>
<p>Based on the increase in Th17 and Tc17 levels, lower proportion and level of activation of <italic>i</italic>NKT&#x20;cells, increase in the population of <italic>i</italic>NKT17 cells and higher proportion of <italic>i</italic>NKT&#x20;cells after secukinumab treatment in psoriasis patients, we speculate that dysregulated <italic>i</italic>NKT&#x20;cells may be involved in the pathogenesis of psoriasis.</p>
</sec>
</body>
<back>
<sec id="s5">
<title>Data Availability Statement</title>
<p>The original contributions presented in the study are included in the article/<xref ref-type="sec" rid="s11">Supplementary Material</xref>, further inquiries can be directed to the corresponding authors.</p>
</sec>
<sec id="s6">
<title>Ethics Statement</title>
<p>The studies involving human participants were reviewed and approved by Shanghai Tenth People&#x2019;s Hospital Ethics Committees. The patients/participants provided their written informed consent to participate in this&#x20;study.</p>
</sec>
<sec id="s7">
<title>Author Contributions</title>
<p>YH, YC, ZC, XiZ, CG, ZY, PX, LS, XuZ, YG, and QY conducted the experiments. YS planned the study and evaluated the results. YH, YC, and ZC analyzed the results. YH wrote the paper. YS reviewed and verified the original manuscript. All authors read and approved the final manuscript.</p>
</sec>
<sec id="s8">
<title>Funding</title>
<p>This work was sponsored by grants from the National Natural Science Foundation of China (No. 81872522, 82073429, 81903205, 81803120, 81900612), Innovation Program of Shanghai Municipal Education Commission (No. 2019-01-07-00-07-E00046), the Program of Science and Technology Commission of Shanghai Municipality (No. 18140901800), Excellent Subject Leader Program of Shanghai Municipal Commission of Health and Family Planning (No. 2018BR30), Clinical Research Program of Shanghai Hospital Development Center (No. SHDC2020CR1014B, SHDC12018X06), Shanghai Sailing Program(No. 19YF1438100) Program of Shanghai Academic Research Leader (No. 20XD1403300), and Research Program of Shanghai Skin Disease Hospital (No. 2019KYQD08).</p>
</sec>
<sec sec-type="COI-statement" id="s9">
<title>Conflict of Interest</title>
<p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p>
</sec>
<sec sec-type="disclaimer" id="s10">
<title>Publisher&#x2019;s Note</title>
<p>All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.</p>
</sec>
<sec id="s11">
<title>Supplementary Material</title>
<p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type="uri" xlink:href="https://www.frontiersin.org/articles/10.3389/fcell.2021.799560/full#supplementary-material">https://www.frontiersin.org/articles/10.3389/fcell.2021.799560/full&#x23;supplementary-material</ext-link>
</p>
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<supplementary-material xlink:href="Image1.PDF" id="SM2" mimetype="application/PDF" xmlns:xlink="http://www.w3.org/1999/xlink"/>
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