Targeting CD8+ T cells prevents psoriasis development
Authors: Paola Di Meglio, Federica Villanova, Alexander A. Navarini, Alessio Mylonas, Isabella Tosi, Frank O. Nestle, Curdin Conrad
Journal: Journal of Allergy and Clinical Immunology (2016)

In psoriasis, intraepidermal T cells are predominantly CD8+ and represent key effector cells. Here, we show that these T cells produce pathogenic IL-17 and that neutralization of CD8+ T cells effectively prevents psoriasis development in vivo. Psoriasis is a common inflammatory skin disease, resulting from the interaction of genetic and environmental triggers, leading to dysregulated immune response of innate and adaptive immune cells.1Di Meglio P. Villanova F. Nestle F.O. Psoriasis.Cold Spring Harb Perspect Med. 2014; 4Crossref PubMed Scopus (188) Google Scholar, 2Lowes M.A. Suarez-Farinas M. Krueger J.G. Immunology of psoriasis.Annu Rev Immunol. 2014; 32: 227-255Crossref PubMed Scopus (973) Google Scholar T lymphocytes infiltrating psoriasis skin lesions play key effector roles by driving disease development and maintenance. Traditionally, CD4+ TH cells producing proinflammatory cytokines, such as IL-17A, IL-22, and IFN-γ, are regarded as the main pathogenic T-cell subpopulation. However, CD8+ T cells, which are present in healthy skin as tissue resident memory T cells (TRM),3Watanabe R. Gehad A. Yang C. Scott L.L. Teague J.E. Schlapbach C. et al.Human skin is protected by four functionally and phenotypically discrete populations of resident and recirculating memory T cells.Sci Transl Med. 2015; 7: 279ra39Crossref PubMed Scopus (356) Google Scholar have been shown to produce a similar profile of proinflammatory cytokines4Hijnen D. Knol E.F. Gent Y.Y. Giovannone B. Beijn S.J. Kupper T.S. et al.CD8(+) T cells in the lesional skin of atopic dermatitis and psoriasis patients are an important source of IFN-gamma, IL-13, IL-17, and IL-22.J Invest Dermatol. 2013; 133: 973-979Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar; they are abundantly present in the psoriatic epidermis and potentially recognize peptide antigens presented on MHC class I molecules, such as HLACw6, which is the strongest psoriasis susceptibility allele.5Tsoi L.C. Spain S.L. Knight J. Ellinghaus E. Stuart P.E. Capon F. et al.Identification of 15 new psoriasis susceptibility loci highlights the role of innate immunity.Nat Genet. 2012; 44: 1341-1348Crossref PubMed Scopus (702) Google Scholar Furthermore, we have previously shown that intraepidermal T cells represent key effector cells in psoriasis development and that impeding the entry of T cells into the epidermis, by blocking α1β1-integrin, prevents the development of psoriasis in the clinically relevant AGR mouse model of psoriasis.6Conrad C. Boyman O. Tonel G. Tun-Kyi A. Laggner U. de Fougerolles A. et al.Alpha1beta1 integrin is crucial for accumulation of epidermal T cells and the development of psoriasis.Nat Med. 2007; 13: 836-842Crossref PubMed Scopus (169) Google Scholar Thus, we set out to explore the pathogenic relevance of CD8+ T cells in psoriasis. We first performed a time course experiment using the AGR mouse model. AGR mice are grafted with noninvolved skin from patients with psoriasis, which spontaneously develops a psoriatic phenotype after 4 to 6 weeks.6Conrad C. Boyman O. Tonel G. Tun-Kyi A. Laggner U. de Fougerolles A. et al.Alpha1beta1 integrin is crucial for accumulation of epidermal T cells and the development of psoriasis.Nat Med. 2007; 13: 836-842Crossref PubMed Scopus (169) Google Scholar Thus, at days 0, 7, 21, and 35, skin transplants were harvested and processed for histological and immunohistochemical assessment as described previously.6Conrad C. Boyman O. Tonel G. Tun-Kyi A. Laggner U. de Fougerolles A. et al.Alpha1beta1 integrin is crucial for accumulation of epidermal T cells and the development of psoriasis.Nat Med. 2007; 13: 836-842Crossref PubMed Scopus (169) Google Scholar In line with earlier findings, while the proliferation of dermal T cells preceded epidermal changes, the numerical expansion of the epidermal T-cell pool temporally coincided with the onset of the psoriatic phenotype, as shown by the papillomatosis index (Fig 1, A and C, and Conrad et al6Conrad C. Boyman O. Tonel G. Tun-Kyi A. Laggner U. de Fougerolles A. et al.Alpha1beta1 integrin is crucial for accumulation of epidermal T cells and the development of psoriasis.Nat Med. 2007; 13: 836-842Crossref PubMed Scopus (169) Google Scholar). Moreover, the accumulation of epidermal T cells paralleled the increase in proliferating keratinocytes as identified by positive Ki67-staining (Fig 1, B and C). Importantly, in the absence of T-cell expansion upon transplantation, which did not occur in one of the experiments we performed, we did not observe any epidermal pathology, in terms of both papillomatosis and frequency of proliferating keratinocytes (see Fig E1 in this article's Online Repository at www.jacionline.org). Thus, the accumulation of epidermal T cells induces both keratinocyte hyperproliferation and onset of papillomatosis, 2 hallmarks of psoriasis, thereby further confirming the role of intraepidermal T cells as key effectors in psoriasis. In keeping with the classical distribution of CD4+ and CD8+ T cells in human psoriatic lesions, intraepidermal T cells in skin grafts 35 days posttransplant were predominantly CD8+ T cells (see Fig E2 in this article's Online Repository at www.jacionline.org). Psoriatic CD8+ T cells have been previously characterized in terms of their cytokine production; however, little distinction has been made between those residing in the dermis and the epidermis in the absence of post-isolation in vitro culture. Thus, to obtain a faithful functional characterization of psoriatic CD8+ T cells, we isolated T cells from the epidermis and the dermis of psoriasis lesions and performed intracellular cytokine staining and fluorescence-activated cell sorting analyses. Among live CD45+ immune cells, the frequency of CD3+CD8+ T cells was significantly higher in the epidermis than in the dermis (Fig 2, A and B). Interestingly, the frequency of epidermal CD3+CD8+ T cells producing IL17A (Fig 2, C and D) or double-producing both IFN-γ and IL-17A or IL-22 and IL-17A, respectively (Fig 2, E), significantly exceeded that of dermal CD3+CD8+ T cells. No significant difference was found for IFN-γ+, IL-22+, or IL-22+IFN-γ+ T cells between the dermis and the epidermis (see Fig E3 in this article's Online Repository at www.jacionline.org). Thus, the main factor differentiating the epidermal from the dermal CD8+ T-cell population is an active Tc17 phenotype. On the basis of these findings, we sought to determine the in vivo pathogenic relevance of CD8+ T cells infiltrating psoriasis lesions. Therefore, we treated xenotransplanted mice with either 1 mg mAb to human CD8 (M-T807) or the corresponding isotype control mAb on days 0 and 14, or mAb to TNF (infliximab, 1 mg intravenously on days 7 and 21 after transplantation). Isotype control antibody-treated skin grafts developed fully fledged psoriasis over the course of 35 days (Fig 2, F and G). Injection of mAb to CD8 resulted in a significantly reduced papillomatosis index and complete blockade of psoriasis development. The effect was equivalent to that obtained with TNF antagonists, a current benchmark in psoriasis treatment (Fig 2, F and G). CD8+ T cells and their role in psoriasis are currently under the spotlight (see Fig E4 in this article's Online Repository at www.jacionline.org). CD8+ T cells isolated from patients with psoriasis produce psoriasis-relevant cytokines, they are retained in the epidermis as TRM after successful therapy,7Cheuk S. Wiken M. Blomqvist L. Nylen S. Talme T. Stahle 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 and LL-37–specific CD8+ T cells expressing α1β1-integrin, a key molecule for trafficking of T cells into psoriatic epidermis,6Conrad C. Boyman O. Tonel G. Tun-Kyi A. Laggner U. de Fougerolles A. et al.Alpha1beta1 integrin is crucial for accumulation of epidermal T cells and the development of psoriasis.Nat Med. 2007; 13: 836-842Crossref PubMed Scopus (169) Google Scholar have been identified in psoriatic blood.8Lande R. Botti E. Jandus C. Dojcinovic D. Fanelli G. Conrad C. et al.The antimicrobial peptide LL37 is a T-cell autoantigen in psoriasis.Nat Commun. 2014; 5: 5621Crossref PubMed Scopus (334) Google Scholar The preferential anatomical location in the epidermis makes CD8+ T cells ideally located to engage in a pathogenic cross talk with keratinocytes (Fig E4); a recent mouse model of psoriasiform murine inflammation relying on keratinocyte genetic abnormalities identified CD8+ T cells as critical players.9Gunderson A.J. Mohammed J. Horvath F.J. Podolsky M.A. Anderson C.R. Glick A.B. CD8(+) T cells mediate RAS-induced psoriasis-like skin inflammation through IFN-gamma.J Invest Dermatol. 2013; 133: 955-963Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar In addition, we show that the accumulation of epidermal T cells, which mainly reflect CD8+ T cells, correlates with the onset of keratinocyte hyperproliferation and papillomatosis, 2 characteristic features of psoriasis. Epidermal CD8+ T cells display highly pathogenic features, and the significantly increased frequency of those producing IL-17A, alone or in combination with IL-22 and IFN-γ, makes them a reasonable primary source for this pivotal cytokine, whose clinical targeting is proving highly successful.10Langley R.G. Elewski B.E. Lebwohl M. Reich K. Griffiths C.E. Papp K. et al.Secukinumab in plaque psoriasis–results of two phase 3 trials.N Engl J Med. 2014; 371: 326-338Crossref PubMed Scopus (1428) Google Scholar Finally, we show that blockade of CD8+ T cells via a neutralizing mAb prevents the development of psoriasis in a clinically relevant xenotransplantation mouse model, thus uncovering a critical role for them in driving pathology. These findings may provide the basis for the design of new strategies targeting CD8+ T cells for the treatment of psoriasis. We gratefully acknowledge the participation of patients with psoriasis attending St John's Institute of Dermatology Clinic and the University Hospital of Zurich. We thank H. Sreeneebus at St John's Institute of Dermatology for skin biopsy collection and K. Reimann at Beth Israel Deaconess Medical Center for providing the M-T807 CD8-depleting antibody. Animal studies were approved by the Kantonale Veterinaeramt of Zurich. Human studies, conducted according to the Declaration of Helsinki, were approved by the institutional review boards of the University Hospital of Zurich and Guy's and St Thomas' Hospital and informed patient consents were obtained. Xenotransplantation of noninvolved psoriatic skin, obtained from 3 patients with psoriasis, was performed as previously described using AGR129 mice, which are deficient in type I (A) and type II (G) IFN receptors in addition to being Rag2−/− (R). After 4 to 6 weeks, these skin grafts spontaneously develop a psoriatic phenotype including thickening of the epidermis (acanthosis), elongation of the rete ridges (papillomatosis), and increased numbers of dermal and epidermal T cells, closely reflecting the pathology of patient samples.E1Conrad C. Boyman O. Tonel G. Tun-Kyi A. Laggner U. de Fougerolles A. et al.Alpha1beta1 integrin is crucial for accumulation of epidermal T cells and the development of psoriasis.Nat Med. 2007; 13: 836-842Crossref PubMed Scopus (210) Google Scholar For fluorescence-activated cell sorting analyses of skin T cells, we obtained 4-mm full-thickness skin biopsies from 5 patients with psoriasis, incubated them in 0.5 mol/L EDTA at 37°C for 3 hours to separate the epidermis and the dermis, and then digested them separately in 0.8 mg/mL collagenase type IV in RPMI + 10%FCS + 1%Pen/Strep (cRPMI) at 4°C overnight. EDTA treatment does not affect the expression of cell surface markers, such as CD4, in contrast to the widely used dispase treatment (data not shown). Subsequently, digested tissue was stimulated with phorbol 12-myristate 13-acetate (50 ng/mL) and ionomicin (1 μg/mL) in the presence of brefeldin A (3 μM) and monensin (3 μM) in cRPMI at 37°C for 5 hours. Dead cells were excluded from the analysis by staining with Live Dead Yellow (Life Technologies, Carlsbad, Calif). Cells were stained for surface markers, fixed and permeabilized, and stained for intracellular cytokines. The following antibodies were used: anti-CD3 APC (SK7, BD Biosciences, Franklin Lakes, NJ), anti-CD4 BV650 (SK3, BD Biosciences), anti-CD8 PE-Texas Red (3B5, Invitrogen, Carlsbad, Calif), anti-CD45 V500 (HI30, BD Biosciences), anti–IL-17A V450 (N49-653, BD Biosciences), anti–IL-22 PerCP-eFluor710 (22URTI, eBiosciences, San Diego, Calif), and anti–IFN-γ A700 (B27, BD Biosciences).Fig E2As in classical human psoriasis, intraepidermal T cells in the AGR mouse model represent mostly CD8+ T cells. Microscopic view of representative CD3, CD4, and CD8 immunostaining of nonlesional psoriatic skin upon development of fully fledged psoriasis, 35 days after engraftment onto AGR mice. Arrowheads depict intraepidermal CD8+ T cells, and arrows depict dermal CD4+ T cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3No differences in IL-22 and IFN-γ production between epidermal and dermal CD8+ T cells. Functional characterization of epidermal and dermal CD3+ CD8+ T cells isolated from the epidermis and the dermis of 5 patients with psoriasis. Percentages of IL-22+, IFN-γ+, and IL-22+ IFN-γ+ CD8+ T cells, as obtained by intracellular cytokine staining upon phorbol 12-myristate 13-acetate/ionomycin stimulation, show no differences between dermal and epidermal CD8+ T cells. Each line connects epidermal and dermal CD8+ T cells from the same patient.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E4The role of CD8+ T cells in psoriasis immunopathogenesis. Environmental triggers (eg, skin injury, known as Koebner phenomenon) induce the expression of LL37 by keratinocytes, which forms complexes with self-DNA/RNA released by dying cells. These complexes activate skin-infiltrating plasmacytoid dendritic cells (pDCs) to produce IFN-α, which in turn—together with LL37-RNA complexes—promotes maturation and activation of conventional dendritic cells producing IL-23. This leads to expansion and activation of autoreactive CD8+ T cells, as well as CD4+ T cells, in the dermis. Although CD4+ T cells remain principally within the dermis, activated CD8+ T cells acquire expression of very late antigen (VLA)-1 and migrate into the epidermis. Subsequently, potentially upon recognition of autoantigens on keratinocytes via MHC-I, intraepidermal CD8+ T cells release IL-17, which is critically involved in psoriatic inflammation and its pathogenesis.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table E1Cellular and histologic changes over time during psoriasis developmentExperiment depicted in Fig 1Day 0Day 7Day 21Day 35Dermal T cells3.22 (0.71)16.08 (3.51)22.30 (8.66)6.65 (2.11)Epidermal T cells0.23 (0.08)0.33 (0.24)10.50 (1.62)16.32 (1.68)Papillomatosis index1.507 (0.06)1.598 (0.04)2.529 (0.28)2.978 (0.32)Ki-6716.67 (3.83)11.67 (1.70)56.39 (9.91)72.78 (29.51)Mean and SEM values of experimental data depicted in Fig 1, A and B. Mean (± SEM) values of dermal and epidermal T cells, papillomatosis index, and Ki-67 positive keratinocytes in skin samples upon xenotransplantation in the AGR mouse model at indicated time points. Values correspond to the data depicted in Fig 1, A and B. Open table in a new tab Mean and SEM values of experimental data depicted in Fig 1, A and B. Mean (± SEM) values of dermal and epidermal T cells, papillomatosis index, and Ki-67 positive keratinocytes in skin samples upon xenotransplantation in the AGR mouse model at indicated time points. Values correspond to the data depicted in Fig 1, A and B.