Skin Infiltration of Pathogenic Migratory and Resident T Cells Is Decreased by Secukinumab Treatment in Psoriasis

IL-17A‒producing T cells play an important role in the pathogenesis of psoriasis. The blockade of IL-17A by mAbs, including secukinumab, dramatically improves the disease. However, their effects on T cells in the patients' skin is not well investigated. Because IL-17A is a secreted protein, secukinumab has no direct effect on IL-17A‒producing cells. However, it is possible that the IL-17A blockade indirectly suppresses its producer cells. Resident memory T (TRM) cells, which are involved in the pathogenesis and the recurrence of psoriasis (Cheuk et al., 2017Cheuk S. Schlums H. Gallais Sérézal I. Martini E. Chiang S.C. Marquardt N. et al.CD49a expression defines tissue-resident CD8+ T cells poised for cytotoxic function in human skin.Immunity. 2017; 46: 287-300Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar, Cheuk et al., 2014Cheuk S. Wikén M. Blomqvist L. Nylén S. Talme T. Ståhle M. et al.Epidermal Th22 and Tc17 cells form a localized disease memory in clinically healed psoriasis.J Immunol. 2014; 192: 3111-3120Crossref PubMed Scopus (227) Google Scholar), may be another target of anti–IL-17A mAbs. Focusing on these issues, we performed a clinical study in 10 patients with psoriasis. The study protocol was approved by Institutional Review Board and written informed consent was obtained from all participants. Patients were treated with secukinumab for 24 weeks. Before the therapy, biopsy specimens were taken from both lesional and nonlesional normal-appearing skin. At week 4 and 24, biopsies were performed again from the areas just in conjunction with the previously biopsied sites. At these timepoints, peripheral blood analysis was also performed. At week 24, nonlesional skin biopsies were taken from the vicinity of the first nonlesional skin in four cases. In each time, a half of the 4-mm punch biopsy specimen was subjected to histologic investigation and the other half was used for ex vivo expansion of skin-infiltrating T cells using IL-2 and anti–CD3 and/or anti‒CD28 mAb-coated microbeads. After two-week expansion, the T cells were analyzed by flow cytometry. The detailed study protocol and methods are described in the Supplementary Materials. The Psoriasis Area and Severity Index scores were decreased more than 90% in all cases by secukinumab treatment, with significant improvement of other clinical scores (Supplementary Figure S1). The changes in the serum parameters and T-cell subsets in the peripheral blood were undetectable after the therapy (Supplementary Figures S2 and S3). Histologic analysis was performed by capturing the whole-biopsy section as digital images using NanoZoomer and its software. The epidermal psoriatic changes were normalized by secukinumab. The number of skin-infiltrating CD3+ T cells of the lesional skin before treatment was significantly higher than that of the nonlesional skin, and they were reduced by the 4-week and 24-week treatment (Supplementary Figure S4). Because the immunohistologic staining study alone was not greatly convincing, we also investigated the T cells expanded from the skin biopsy specimen. As indicated in our previous study, T-cell populations were not substantially altered by the two-week expansion process (Fujiyama et al., 2016Fujiyama T. Ito T. Umayahara T. Ikeya S. Tatsuno K. Funakoshi A. et al.Topical application of a vitamin D3 analogue and corticosteroid to psoriasis plaques decreases skin infiltration of TH17 cells and their ex vivo expansion.J Allergy Clin Immunol. 2016; 138: 517-528.e5Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). There was a clear correlation between the number of expanded T cells and that of histologically enumerated original skin-infiltrating T cells (Figure 1a). The numbers of both CD4+ and CD8+ T cells expanded from the lesional skin before the therapy were significantly more than those of the nonlesional skin and were decreased by the 24-week treatment (Figure 1b). Flow-cytometric analysis with intracellular cytokine staining revealed that both in CD4+ and CD8+ T cells, IL-17A and IFN-γ‒producing T cells expanded from the pretreatment lesional skin were significantly higher in number than those from the nonlesional skin and were decreased at 24 weeks (Figure 1c and d). In patients with moderate to severe psoriasis, IL-17A or associated gene expression was upregulated even in nonlesional skin (Chiricozzi et al., 2016Chiricozzi A. Suárez-Fariñas M. Fuentes-Duculan J. Cueto I. Li K. Tian S. et al.Increased expression of interleukin-17 pathway genes in nonlesional skin of moderate-to-severe psoriasis vulgaris.Br J Dermatol. 2016; 174: 136-145Crossref PubMed Scopus (61) Google Scholar, Matos et al., 2017Matos T.R. O'Malley J.T. Lowry E.L. Hamm D. Kirsch I.R. Robins H.S. et al.Clinically resolved psoriatic lesions contain psoriasis-specific IL-17-producing αβ T cell clones.J Clin Invest. 2017; 127: 4031-4041Crossref PubMed Scopus (151) Google Scholar). Therefore, we also monitored T cells in the nonlesional skin in four cases. The number of IL-17A+ T cells, especially CD8+ cells, was decreased after secukinumab treatment (Figure 1e), suggesting that the IL-17A blockade suppresses IL-17A‒producing T cells in the nonlesional and the lesional skin. To investigate the effect of secukinumab treatment on skin TRM cells or cells expressing its markers, we first performed immunofluorescent staining for CD8 and CD103 (Figure 2a). CD8+CD103+ cells are reported to be an important subset of TRM in psoriasis (Cheuk et al., 2014Cheuk S. Wikén M. Blomqvist L. Nylén S. Talme T. Ståhle M. et al.Epidermal Th22 and Tc17 cells form a localized disease memory in clinically healed psoriasis.J Immunol. 2014; 192: 3111-3120Crossref PubMed Scopus (227) Google Scholar). The number of CD8+CD103+ T cells in the whole skin and in the epidermis was dramatically decreased in the lesional skin, but not in the nonlesional skin, after secukinumab treatment (Figure 2b). Because CD103 expression was not altered during the cultivation (Kurihara et al., 2019Kurihara K. Fujiyama T. Phadungsaksawasdi P. Ito T. Tokura Y. Significance of IL-17A-producing CD8+CD103+ skin resident memory T cells in psoriasis lesion and their possible relationship to clinical course.J Dermatol Sci. 2019; 95: 21-27Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), we also investigated the CD8+CD103+ T cells among the ex vivo expanded T cells. The number of the expanded CD8+CD103+ T cells correlated significantly with the immunohistologically identified number of the original CD8+CD103+ T cells (Figure 2c), indicating that CD8+CD103+ cells were successfully expanded. In the lesional skin, the number of CD8+CD103+ cells, including CD8+CD103+CCR7– and CD8+CD103+CD45RO+ cells, indicative of effector memory properties, was significantly decreased after the treatment. CD8+CD103+CD49a– cells, presumably corresponding to IL-17A‒producing skin TRM cells (Cheuk et al., 2017Cheuk S. Schlums H. Gallais Sérézal I. Martini E. Chiang S.C. Marquardt N. et al.CD49a expression defines tissue-resident CD8+ T cells poised for cytotoxic function in human skin.Immunity. 2017; 46: 287-300Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar), also tended to be decreased (Figure 2d) (Supplementary Figure S5). In the nonlesional skin, the number of CD8+CD103+ T cells was not decreased and moreover, the frequency of CD8+CD103+ T cells was rather increased by the treatment (Figure 2d) (Supplementary Figure S5). It seems that CD103– T cells, namely non-TRM subsets, are more susceptible than TRM cells, and TRM cells are resistant to secukinumab therapy. The mechanism underlying the inhibitory effect of secukinumab on T-cell infiltration remains speculative. Because IL-17A stimulates keratinocytes to produce chemokines, such as chemokine ligand 20 which chemoattracts chemokine receptor 6-expressing Th17 and/or Tc17 cells (Harper et al., 2009Harper E.G. Guo C. Rizzo H. Lillis J.V. Kurtz S.E. Skorcheva I. et al.Th17 cytokines stimulate CCL20 expression in keratinocytes in vitro and in vivo: implications for psoriasis pathogenesis.J Invest Dermatol. 2009; 129: 2175-2183Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar), IL-17A blockade might decrease the chemokine ligand 20 production and down-modulate Th17 and/or Tc17 cell migration to the skin (Girolomoni et al., 2012Girolomoni G. Mrowietz U. Paul C. Psoriasis: rationale for targeting interleukin-17.Br J Dermatol. 2012; 167: 717-724Crossref PubMed Scopus (109) Google Scholar). In addition, IL-17A induces the production of various cytokines by various cells in the skin. These overall inflammatory changes may also be suppressed by secukinumab. In summary, the blockade of IL-17A signaling by secukinumab therapy suppresses IL-17A‒producing T-cell infiltration in both lesional and nonlesional skin. A small number of T cells, including TRM cells, remains in the skin even after the 24-week treatment presumably because of their resistant property to the therapy. No data sets were generated or analyzed during the study. Toshiharu Fujiyama: http://orcid.org/0000-0003-3612-8025 Takatsune Umayahara: http://orcid.org/0000-0003-1008-4730 Kazuo Kurihara: http://orcid.org/0000-0003-4964-1549 Takatoshi Shimauchi: http://orcid.org/0000-0002-0369-869X Taisuke Ito: http://orcid.org/0000-0002-9274-7050 Masahiro Aoshima: http://orcid.org/0000-0002-6012-0616 Emiko Otobe: http://orcid.org/0000-0002-4971-865X Hideo Hashizume: http://orcid.org/0000-0002-5809-0040 Hiroaki Yagi: http://orcid.org/0000-0001-9328-8215 Yoshiki Tokura: http://orcid.org/0000-0001-7452-6919 Yoshiki Tokura received honorarium for oral presentation from Novartis. The other authors state no conflict of interest. This study, excluding the resident memory T part, was funded by Novartis. The sponsor played roles in the study design and decision to publish. This study was founded by Novartis (Trial Registration: UMIN000023424). Conceptualization: YT; Data Curation: EO; Investigation: TF, TU, KK, TS, TI, MA, HH, HY; Writing - Original Draft Preparation: TF A total of 10 patients with moderate and severe psoriasis vulgaris, irrespective of gender, aged 20–70 years were recruited from Hamamatsu University Hospital, Shizuoka General Hospital, and Shimada Municipal Hospital (Supplementary Table S1). All patients had been diagnosed with psoriasis vulgaris more than six months before inclusion in the study. All patients had the desire for initiation of secukinumab treatment. Exclusion criteria were as follows: (i) administration of adalimumab, infliximab, and etanercept within three months before initiation of secukinumab; (ii) administration of ustekinumab, alefacept, briakinumab, and efalizumab within six months before initiation of secukinumab; (iii) administration of cyclosporine A, methotrexate, corticosteroid, cyclophosphamide, retinoid, and fumaric acid ester, and treatment with psoralen UVA therapy within four weeks before initiation of secukinumab; (iv) phototherapy with UVA and UVB and topical treatment with reagents possibly affecting psoriatic lesions and symptoms, including corticosteroid, vitamin D3 analogue, tacrolimus, pimecrolimus, retinoid, salicylate petrolatum, salicylate, lactate, tar, uric acid, and α-hydroxy acid (fruit acid), within two weeks before initiation of secukinumab; (v) presence of a condition or abnormality that in the opinion of the investigator would compromise the safety of the patient or the quality of the data; and (vi) pregnancy. It was noted that the use of therapies listed in (i) to (iv) was not allowed until week 24. An open, one-therapeutic arm, single-group structured study was conducted. Patients with plaque psoriasis were treated with secukinumab at 300 mg once a week from baseline to week 4 and then every four weeks thereafter. The primary endpoint was the evaluation of changes in skin-infiltrating T cells before and at week 24 after the initial administration of secukinumab. The major secondary endpoint was the evaluation of changes in peripheral blood T cells and in serum levels of cytokines before and at week 24 after the initial treatment. Other secondary endpoints were the evaluations of changes in skin-infiltrating T cells, in peripheral blood T cells, and in serum levels cytokines before and at week 4 after the initial treatment. For the assessment of clinical severity, Psoriatic Area and Severity Index, BSA, and Dermatology Life Quality Index were monitored before therapy and at week 4 and week 24 during the administration. As a limitation, the final assessment of patient number 3 was delayed by six days. Before the therapy, 4-mm punch biopsy specimens were taken from both lesional skin and nonlesional normal-appearing skin. At week 4 and week 24 after initiation of the therapy, 4-mm punch biopsy specimens were again taken from the same plaque of psoriasis. In addition, peripheral blood samples were collected before therapy and at week 4 and week 24 after the therapy. At week 24, a nonlesional 4-mm punch biopsy specimen was also taken in cases 4 and 8–10. Half of the biopsy specimen was used for histologic investigation and the other half was for ex vivo T-cell expansion study. The skin biopsy specimens were cut into two pieces. A half of the biopsy specimens from the lesions was fixed in 4% formaldehyde and routinely stained with hematoxylin and eosin for standard histopathology. Deparaffinized specimens were autoclaved in 10 mM citrate buffer (pH: 6.0) for 10 minutes at 120 °C to retrieve the antigenic epitopes and then subjected to CD3, CD4, and CD8 expression analysis by avidin–biotin complex method. Nuclear staining was performed with hematoxylin. For immunofluorescence staining, deparaffinized specimens were autoclaved in 10 mM citrate buffer (pH: 6.0) for 10 minutes at 120 °C, and for blocking, they were treated with 1% BSA for 10 minutes. As primary antibodies, anti-CD8 mouse monoclonal antibody (Dako/Agilent Pathology Solutions, Santa-Clara, CA; 1:100 dilution) and anti-CD103 rabbit polyclonal antibody (Abcam, Cambridge, United Kingdom; 1:100 dilution) were applied for overnight and 2 hours, respectively, at room temperature, followed by secondary antibody staining for each 60 minutes at room temperature (Alexa Fluor 488 anti-rabbit IgG, Biolegend and Alexa Fluor 594 anti-mouse IgG Thermo Fischer Scientific, Walham, MA; 1:100 dilution). The sections were scanned by a digital image scanner, NanoZoomer (Hamamatsu Photonics, Hamamatsu, Japan), and analyzed using its software. The epidermal thickness or thickness of stratum corneum was calculated by measuring the area and the length of epidermal surface line of the whole-tissue image. The histologic enumeration of T cells positive for each marker was performed using the digitally captured whole-section images and expressed as the number per whole section. The other half of the biopsy specimen was used for ex vivo expansion of skin-infiltrating T cells with anti-CD3 and anti-CD28 antibodies and recombinant IL-2 by using our established method (Fujiyama et al., 2016Fujiyama T. Ito T. Umayahara T. Ikeya S. Tatsuno K. Funakoshi A. et al.Topical application of a vitamin D3 analogue and corticosteroid to psoriasis plaques decreases skin infiltration of TH17 cells and their ex vivo expansion.J Allergy Clin Immunol. 2016; 138: 517-528.e5Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, Fujiyama et al., 2014Fujiyama T. Kawakami C. Sugita K. Kubo-Kabashima R. Sawada Y. Hino R. et al.Increased frequencies of Th17 cells in drug eruptions.J Dermatol Sci. 2014; 73: 85-88Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, Hashizume et al., 2010Hashizume H. Hansen A. Poulsen L.K. Thomsen A.R. Takigawa M. Thestrup-Pedersen K. In vitro propagation and dynamics of T cells from skin biopsies by methods using interleukins-2 and -4 or anti-CD3/CD28 antibody-coated microbeads.Acta Derm Venereol. 2010; 90: 468-473Crossref PubMed Scopus (14) Google Scholar). RPMI 1640 medium (Life Technologies, Carlsbad, CA) supplemented with L-glutamine, sodium pyruvate, 2-mercaptoethanol, nonessential amino acids (Life Technologies) and 10% heat-inactivated fetal calf serum or pooled human AB serum was used as culture medium (complete RPMI). Samples were immersed in complete RPMI supplemented with 50 U/ml human recombinant IL-2 (Research and Development Systems) and anti-CD3 and anti-CD28 antibody-conjugated microbeads (Human T-Activator; Dynal, Copenhagen, Denmark). Cells were cultivated in six-well plates for two weeks, and the culture media were changed or added every day to make sure the half of the total volume of media were fresh. We obtained >106 cells per specimen by two-week culture, and all cells were collected and counted. The rest of the expanded cells were stored at –80 °C to analyze cell surface markers of TRM. The expanded cells were examined in their phenotype and intracellular cytokine pattern by flow cytometry. Peripheral blood mononuclear cells will be isolated from heparinized samples and analyzed by surface and cytoplasmic staining for CD3, CD4, CD8, IFN-γ, IL-4, IL17A, and IL-22 and subsequent flow cytometry. FITC-, PE-, PerCP, or APC-conjugated mAbs against CD3, CD4, CD8, CD45RO, CD49a, CD69, and CD103 were purchased from BD PharMingen (San Diego, CA). FITC- or PE-conjugated mAbs against IL-4, IL-17, IL-22, and IFN-γ were from eBioscience (San Diego, CA). PE-conjugated anti-CCR7 was obtained from Research and Development systems (Minneapolis, MN). Aliquots of 106 cells were washed once with phosphate buffered saline (pH: 7.4) with a panel of fluorescence-conjugated mAbs for 15 minutes at room temperature in the dark. Before fluorescent staining, the cell viability >98% was verified by staining 0.4 w/v% Tripan Blue (Fujifilm-Wako, Tokyo, Japan). After washing, the harvested cells were resuspended in phosphate buffered saline and subjected to flow cytometry. More than 5 × 104 cells per sample were analyzed on a FACSCanto2 (BD). Results were analyzed using a FlowJo software (TreeStar, Ashland, OR). The gating process was shown in Supplementary Figure S6. For the intracellular cytokine staining assay, 106 cells/well in 24-well plate were incubated in complete RPMI containing 10–8 M phorbol 12-myristate 13-acetate (Sigma-Aldrich St Louis, MO), 10–6 M calcium ionophore (Sigma-Aldrich) and 1 μl/ml of BerGolgi stop (BD) for 6 hours at 37 °C. The cells were harvested and stained with fluorescent-tagged mAbs against CD3 and CD8 followed by cytokine staining using Cytofix/Cytoperm Plus Kit with GolgiStop (BD) according to the manufacturer's protocols. Because CD4 expression is downregulated during treatment with phorbol 12-myristate 13-acetate and calcium ionophore, we estimated CD4+ T cells as CD3+ CD8– T cell population. The study protocol was approved by the Institutional Review Board (Clinical Research Review Board of Hamamatsu University School of Medicine) and then subsequently approved by the Institutional Review Boards of Shimada Municipal Hospital and Shizuoka General Hospital. These studies were performed according to the Declaration of Helsinki and Ethical Guidelines for Medical and Health Research Involving Human Subjects (Ministry of Education, Culture, Sports, Science and Technology and the Ministry of Health, Labour and Welfare in Japan). Written informed consents were obtained from all participants. In addition, the studies to characterize ex vivo expanded TRM cells and to conduct immunohistopathologic analysis of TRM cells were separately approved by the Clinical Research Review Board of Hamamatsu University School of Medicine. The results from Kolmogolov-Smirnov test indicated that our datasets did not meet normality, and thus, nonparametric tests were applied. We applied Friedman test, and when there were significant differences, post hoc pairwise multiple comparison (Dunn-Bonferroni test) were performed by SPSS software (IBM, version 21). For comparison of nonpaired samples, Mann–Whitney U-test was applied. For the correlation analysis, we applied Spearman's correlation. P < 0.05 was considered statistically significant.Supplementary Figure S2Peripheral blood parameters in 10 patients before and at four and 24 weeks after secukinumab treatment. ESR, erythrocyte sedimentation rate; LDH, lactate dehydrogenase; VEGF, vascular endothelial growth factor; WBC, white blood cell.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Supplementary Figure S3The numbers and the frequencies of cytokine-bearing peripheral blood T cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Supplementary Figure S4Histologic findings in skin specimens before and at four and 24 weeks after secukinumab treatment. Digitally captured whole-section images of 4-mm punch section were analyzed. The mean thickness of epidermis and stratum corneum and the number of CD3+ T cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Supplementary Figure S5Representative dot plots of CD103 (left panels) and the frequency among CD8+ cells and number of CD103+CD45RO+, CD103+CCR7–, and CD103+CD49–cells (right panels).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Supplementary Figure S6Gating strategy. Small debris and doublets were eliminated and CD3+ and CD8+/– cells were gated. As a limitation of the study, we did not perform dead-cell exclusion simultaneously with the other staining; however, the gated cells contained less than 1% of dead cells. FSC-A, forward scatter area; FSC-H, forward scatter height; FSC-W; forward scatter width; SSC-A, side scatter area; SSC-H, side scatter height; SSC-W, side scatter width.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Supplementary Table S1Patients Enrolled in This StudyCaseAgeSexHospitalPASILesional Skin BiopsyNonlesional Skin Biopsy164MHUH25.4HipShoulder227FHUH22.9HipBack367MSGH21BackBack439MHUH19.7ThighThigh552FHUH47ThighThigh655FHUH9.2KneeThigh750MHUH18.4ThighThigh866MSMH13KneeKnee945MSGH29.4ThighAbdomen1063MSGH38.2BackBackAbbreviations: HUH, Hamamatsu University Hospital; PASI; Psoriasis Area and Severity Index; SGH, Shizuoka General Hospital; SMH, Shimada Municipal Hospital. Open table in a new tab Abbreviations: HUH, Hamamatsu University Hospital; PASI; Psoriasis Area and Severity Index; SGH, Shizuoka General Hospital; SMH, Shimada Municipal Hospital.