Medical use of regulatory T cell activator

Abstract

The present invention provides an activator of CD4+Foxp3+ regulatory T cells for use in treating or preventing an inflammatory epidermal disease in a subject, wherein the disease is characterised by a

Description

Technical Field The present disclosure relates to the prevention and treatment of inflammatory epidermal diseases, including inflammatory autoimmune epidermal diseases such as lichen planus, that are associated with an inflammatory infiltrate comprising cytotoxic CD8+ T cells causing epidermal damage and particularly epidermal cell apoptosis. Background Autoimmunity is an immune response against the body’s own healthy tissue that usually involves T and B lymphocytes and can cause significant diseases that impact a sufferer’s quality of life, and whose treatment can be challenging for medical professionals. These diseases can be classified based on the system(s) or area(s) of the body that is / are affected, with affected systems including, for example, the gastrointestinal system, the musculoskeletal system, the endocrine system, and the cutaneous system. One example of a cutaneous system autoimmune disease is lichen planus (LP). This disease is a relatively uncommon dermatosis, with an incidence estimated between 0.14-1.27% of the general population, and which typically develops in middle-aged adults. The histopathological features of the disease include lesions on the skin, mucous membranes, and the hair follicles and nail apparatus, with these various areas of the body being affected solely, concomitantly, or sequentially (See, e.g., Vicic et al., Int. J. Mol. Sci. 2023, 24(3), 3038). Studies of this disease have shown that it is a T cell-mediated disease in which the pathogenesis seems to be driven by CD4+ Th-cells which release Thl cytokines, leading to recruitment of Langerhans cells and clonal expansion of cytotoxic CD8+ cells which then cause apoptosis of epidermal cells, particularly keratinocytes (Tziotzios et al., J. Am. Acad. Dermatol. (2018), 79(5): pages 807-818; Firth et al., Arch. Dermatol. Res. (2015) 307:333-339). It is also known that CD4+ regulatory T cells are increased in the PBMC of LP patients compared to healthy controls (Domingues et al. J. Transl. Med. (2016) 14:171; Firth et al., Arch. Dermatol. Res. (2015) 307:333-339). However, the reason why these regulatory T cells are unable to control the CD8+ cell proliferation is unknown, and it is suggested that these cells may be dysfunctional (Zhou et al., Inflammation (2016) 39: 1205-1215). Recent clinical approaches to the treatment of this disease include the use of anti-IL-17 antibody secukinumab, the IL-12 / 23-targeting ustekinumab, and the IL-23 inhibitor guselkumab, to block the action of these inflammatory cytokines. In the past, depleting anti-CD4 antibodies have been investigated for the treatment of autoimmune diseases and cancer (e.g., in US 2010 / 015134 Al entitled “Therapy with CD4 binding peptides and radiation”). However, such treatments have not been applied to the treatment of lichen planus and are no longer routinely considered as treatments for autoimmune diseases due to the significant depletion of CD4+ T-cells that they can cause and the resulting side-effects. Non-depleting anti-CD4 antibodies Clenoliximab and TRX1 have previously been suggested for the treatment of many autoimmune disease (e.g., in US 2009 / 0226430 Al entitled “Recombinant anti-CD4 antibodies for Human Therapy”, and in US 2010 / 0021460 Al entitled “Methods of treating autoimmune diseases using CD4 antibodies”). It is currently thought that clenoliximab can mediate in vivo immunomodulatory effects through 1) removal of CD4+ T cells by apoptosis or complement-mediated lysis of mAB-coated CD4+ T cells; 2) receptor modulation caused by internalization or stripping of the CD4 receptor from the cell surface; and 3) inhibition of T cell activation by antagonism of CD4-MHC class II interactions, possible leading to induction of anergy ). Similarly, it is currently thought that TRX1 works by reducing the density of CD4 expressed on T-cell surfaces that can engage in an immune response, thereby decreasing the count of functional CD4+ effector T lymphocytes (https. / / www.creativebioiabs;n^^ To the best of the inventors’ knowledge, these non-depleting anti-CD4 antibodies have not been used for the treatment of lichen planus. Other inflammatory epidermal diseases exist with similar histopathology, involving an inflammatory infiltrate comprising cytotoxic CD8+ cells and apoptosis of epidermal cells. Examples are inflammatory autoimmune epidermal diseases, such as cutaneous or mucosal lupus erythematosus, alloreactive epidermal diseases, such as cutaneous or mucosal graft-versus-host disease, and allergic skin diseases, such as severe cutaneous drug reactions, e.g., Stevens-Johnson syndrome. Further, there are additional autoimmune dermatologic disease where CD8+ T cells attack cells within the epidermis or the cells of skin appendages. For example, alopecia areata is an autoimmune disease associated with attack of the hair follicle by CD8+ T cells, while in certain forms of vitiligo (a condition causing depigmentation of the skin) melanocytes within the epidermis are similarly attacked. There is an on-going need to develop new treatments against these diseases, and particularly those such as lichen planus that have a relatively low prevalence, where research and treatment are restricted. Summary Accordingly, in a first aspect the present invention provides an activator of CD4+Foxp3+ regulatory T cells for use in treating or preventing an inflammatory epidermal disease in a subject, wherein the disease is characterised by an inflammatory infiltrate comprising cytotoxic CD8+ T cells causing epidermal cell apoptosis, apoptosis of melanocytes in the epidermis, or apoptosis of epidermal appendage cells. In a second aspect the present invention provides a method of preparing a medicament suitable for treating a subject suffering from, or for preventing a subject from suffering from, an inflammatory epidermal disease characterised by an inflammatory infiltrate comprising cytotoxic CD8+ T cells causing epidermal cell apoptosis, apoptosis of melanocytes in the epidermis, or apoptosis of epidermal appendage cells, the method comprising activating ex vivo CD4+ Foxp3+ regulatory T cells obtained from the subject with an activator of CD4+ Foxp3+ regulatory T cells. Further, in a third aspect the present invention provides an activated CD4+Foxp3+ regulatory T cell for use in treating or preventing an inflammatory epidermal disease in a subject, wherein the disease is characterised by an inflammatory infiltrate comprising cytotoxic CD8+ T cells causing epidermal cell apoptosis, apoptosis of melanocytes in the epidermis, or apoptosis of epidermal appendage cells, wherein the activated CD4+ Foxp3+ regulatory T cell is prepared by activating ex vivo CD4+ Foxp3+ regulatory T cell obtained from the subject with an activator of CD4+ Foxp3+ regulatory T cells. Preferred features of the invention are set out in the dependent claims. In particular, the present inventors have surprisingly found that regulatory T cells from patients suffering from lichen planus are capable of being activated by an activator, and once activated are able to control autoreactive CD8+ T cell proliferation. This provides a previously unknown therapeutic mechanism for treating this disease. In addition, the ability to control CD8+ T cell proliferation in this context as shown by the work in the present application provides a therapeutic mechanism for treating and preventing other inflammatory epidermal diseases or disorders in which CD8+ T cell driven apoptosis of epidermal cells, apoptosis of melanocytes within the epidermis, or apoptosis of epidermal appendage cells occurs. Brief Description of the Drawings To assist understanding of the present disclosure and to show how embodiments may be put into effect, reference is made by way of example to the accompanying drawings in which: Figure 1 shows the amino acid sequences of the heavy chain and light chain of the antibody designated tregalizumab (BT-061). Figures 2A to 2F provide photographs showing the histology of a skin specimen taken from a lichen planus patient, which illustrate the features of the disease. Specifically, Fig, 2A reveals a bandlike lymphocytic infiltrate where cytotoxic CD8+ T cells come into close contact to basal keratinocytes (KC) to induce apoptosis - boxed section is enlarged in Fig, 2B, with apoptotic cells marked with black asterisks). Figs. 2C to 2F show T cell populations in lichen planus (Fig, 2C - CD3 staining; Fig, 2D - CD4 staining; Fig, 2E - CD8 staining; the rectangle marks the detail shown in Figure 2F; Fig, 2F1 - blow-up of Fig. 2E, CD8 stain and Fig2F2 granzyme B (GrB) staining.) The inflammatory infiltrate is rich in CD8+ T cells, which largely express GrB (Fig, 2F2). Note the lining up of GrB+CD8+ cells at the dermal-epidermal junction where they contact basal KC (marked with black arrows - Fig, 2F2). (Magnification: Fig, 2A and 2C to 2E x 200; Fig, 2B and Fig. 2F x 400). Figures 3 A to 3D provide photographs showing the representative immunofluorescence (IF) of a skin specimen taken from a lichen planus patient suffering from cutaneous LP. The specimen was stained for the CD4 T-cell surface marker (Fig, 3A), and Foxp3 (Fig, 3B), a transcription factor specific for regulatory T cells (Tregs). DAPI serves as a nuclear counter stain (Fig, 3C), while Fig, 3D visualizes overlay of the triple staining. One representative out of 10 IF studies is depicted. These results reveal that CD4+ Foxp3+ Tregs are abundantly present within the skin inflammatory infiltrate in LP (Fig, 3B). Figure 4 shows CD4+ Foxp3+ Treg cell numbers are enriched in Lichen planus (LP) biopsy specimen. Tregs are abundantly present in cutaneous LP (LP, n=12) as compared to skin of healthy controls (HC, n=10). Mean average cell numbers of three high-power fields (HPF, 200x) per biopsy were determined separately by two blinded investigators. ****, p<0.0001 Figures 5 A to 5E provide bar charts showing that tregalizumab (BT-061) enhances LP patients’ regulatory T cell (Treg)-mediated inhibition of the Mixed Lymphocyte Reaction (MLR) by controlling the proliferation (Ki-67) of alloreactive CD8+ T cells. Figure 5A depicts dysfunctional Treg function in LP patients (1.7% ± 3.3% (SEM) n=4) in suppressing CD8 proliferation during the MLR as compared to healthy controls (HC, 29.2 ± 2.8% (SEM), n=4). ***, p<0.001. In contrast, Figure 5B reveals that the addition of tregalizumab (BT-061) to graded amounts of Tregs of LP patients strongly reconstituted their suppressive MLR activity. MACS-isolated CD8+ and CD4+ T cells from healthy donors and Tregs as well as APC from LP patients were mixed with or without tregalizumab (BT-061) (Ipg / ml) or tregalizumab (BT-061) solvent (placebo) to address the alloreactive Ki-67 response of CD8+ T cells. Cells were co-cultured for 3 days followed by flow cytometric analysis with the proliferation marker Ki-67. The dashed line represents mean value of the MLR without any supplements. *, p<0.05; **, p<0.01; One-way ANOVA followed by FISHER's Least Significant Difference (LSD) test, n=4. Figure 5C displays the relative inhibition of proliferation shown in Figure 5B. Values represent percentage of inhibition of the mean MLR value without any stimulus (medium). Tregalizumab (BT-061) significantly enhances Treg inhibition of the MLR. **, p<0.01; ***, p<0.001; p<0.0001; ns, non-significant; unpaired t-test (Fig, A), one-way ANOVA followed by FISHER's Least Significant Difference (LSD) test, n=4 (Fig, B and C), SEM, standard error of mean. Figure 5D shows CD4 modulation on regulatory T cells (Tregs) and CD4+ conventional T cells (Tconv) after 3 days of stimulation with tregalizumab (BT-061) or placebo (PL) in the MLR. CD4 modulation is depicted as mean fluorescence intensity (MFI) as compared to medium control (= 100%). Error bars indicate SEM. ****p<0.0001, one-sided ANOVA. Figure 5E shows tregalizumab (BT-061) induced CD 137 expression in regulatory T cells (Tregs) and CD4+ conventional T cells (Tconv) after 3 days of stimulation with tregalizumab (BT-061) in the MLR. CD137 expression is depicted as the mean fluorescence intensity (MFI) difference (A) between tregalizumab (BT-061) and placebo treated MLRs. Error bars indicate SEM. *p<0.05, Mann-Whitney-U-test, n=4. Detailed Description As used herein, singular forms “a”, “an” and “the” include plural reference unless the context clearly dictates otherwise. Accordingly, reference to “an antibody” includes reference to “antibodies”. All technical and scientific terms used herein have the meaning that would be understood by a person of skill in the art to which the invention belongs unless clearly indicated otherwise. As noted above, the present invention is directed to the treatment or prevention of inflammatory epidermal disease in a subject where the disease is associated or characterized by an inflammatory infiltrate (e.g., located in the dermis (dermal infiltrate)) comprising cytotoxic CD8+ T cells, and causing epidermal cell damage, in particular epidermal cell apoptosis, or damage to other cells within the epidermis or in epidermal appendages, such as apoptosis of melanocytes in the epidermis or apoptosis of epidermal cell appendage cells. Epidermal appendages are skin associated structures located within the dermal region of the skin, including certain glands and hair follicles. Such epidermal diseases are characterized by (and can be determined based on) histologic examination of specimens taken from disease areas of the body (e.g. biopsy), which show the presence of the cytotoxic CD8+ T cells attacking and causing apoptosis of epithelial cells (e.g. keratinocytes), apoptosis of melanocytes within the epidermis, or apoptosis of cells of epidermal appendages (such as cells of hair follicles). (Apoptotic cells can be identified in samples using stains that bind to DNA, such as Hoechst 33342, which can be used to show the nuclear condensation that is distinctive of apoptotic cells.) The cytotoxic CD8+ T cells can be autoreactive or alloreactive (e.g., as in cutaneous or mucosal graft-versus-host disease after an allogeneic stem-cell transplant) and in some cases may be triggered by an allergic response (e.g., as in severe cutaneous drug reactions). In some examples, histologic examination shows a lymphocytic infiltrate (e.g., a bandlike lymphocytic infiltrate) that reaches the dermal-epidermal junction and evokes a so-called interface dermatitis in which cytotoxic CD8+ T cells come into close contact with the epidermal cells, such as basal keratinocytes, and numerous apoptotic epidermal cells, and particularly apoptotic keratinocytes, are seen. Example histology, taken from a lichen planus patient, are shown in Figures 2A to 2F, and particularly in Figure 2B, where apoptotic cells are marked with black asterisks. The inflammatory epidermal disease may be an autoimmune epidermal disease, an alloreactive epidermal disease, or an allergic skin disease caused e.g., by an adverse reaction to a drug or an infection. In particular, the inflammatory epidermal disease may be selected from lichen planus (including cutaneous, mucosal and follicular), lichen sclerosus (cutaneous and / or mucous membranes (genitals / oral cavity)), cutaneous or mucosal lupus erythematosus (isolated or in the context of systemic lupus erythematosus), dermatomyositis, cutaneous or mucosal graft-versus-host disease, minor or majus erythema exsudativum multiforme (including with mucocutaneous involvement) and in particular caused by infectious (bacterial, viral) antigens, fixed drug eruption, severe cutaneous drug reactions (including Stevens-Johnson syndrome and toxic epidermal necrolysis (also known as Lyell syndrome)), alopecia areata (including alopecia areata localisata (including ophiasis type), alopecia areata diffusa, alopecia areata partialis, alopecia areata totalis, and alopecia areata universalis), non-segmental vitiligo (including vitiligo vulgaris, vitiligo arcofacialis, vitiligo generalisata, and vitiligo universalis), and focal vitiligo. The inflammatory epidermal disease may be selected from lichen planus, lichen sclerosus, cutaneous lupus erythematosus, dermatomyositis, and cutaneous graft-versus-host disease. Preferably the disease is an inflammatory autoimmune skin disease selected from lichen planus, lichen sclerosus, cutaneous lupus erythematosus, and dermatomyositis. More preferably the disease is lichen planus, which is also referred to herein as LP. This covers all the clinically different forms of LP skin / mucosal lesions including oral lichen planus, penile or vulvar lichen planus, lichen planopilaris, lichen unguis, and lichen exanthematicus, which is an eruptive variant of lichen planus. The subject can be any mammal, including a human, non-human primate, or a domesticated mammal such as a cat, dog, or rodent. However, preferably the subject is a human. The subject can be six years or older, preferably sixteen years or older. As noted above, the diseases to be treated are characterised by cytotoxic CD8+ T cells that cause apoptosis of the patient’s keratinocytes. In particular, this occurs despite the presence of regulatory T cells (see Fig. 3A to 3D). See also, for example, Ujiie, H., “Regulatory T cells in autoimmune skin diseases”, Experimental Dermatology, 2019; 28: 642-646. Regulatory T cells (Tregs) are a subpopulation of CD4+ T cells that are known to modulate the immune system, and in particular are known to be important for maintaining tolerance to selfantigens (Sakaguchi et al., Immunological Reviews (2001); 182: 18-32), and in regulating immune homeostasis and inflammation. The family of Tregs consists of two key subsets: (i) naturally arising Tregs (sometimes known as nTregs), which develop in the thymus; and (ii) peripherally induced Tregs (sometimes known as iTregs) which arise in peripheral circulation from conventional T cells. Tregs are generally characterised by the expression of CD4 and CD25 surface biomarkers, and also by the transcription factor Foxp3. Herein regulatory T cells (Tregs) are referred to as CD4+Foxp3+ regulatory T cells (although they can also be considered as CD4+CD25+Foxp3+ regulatory T cells). Functional impairment or low numbers of Tregs are known to be associated with certain autoimmune or allergic disease, and also graft-versus host disease (Rivas et al., J Allergy Clin Immunol. 2016 September; 138(3): 639-652; Ohl and Tenbrock, Eur. J. Immunol. 2015. 45: 344-355; Whangbo et al., Expert Review of Hematology, 2020; 13(2): 141-154.) Their impairment in lichen planus can be postulated from their inability to control the cytotoxic CD8+ cells despite being present in lesional skin in significant numbers. As shown in Figure 5A the Tregs from LP patients are unable to suppress CD8+ cell proliferation during a Mixed-Lymphocyte Reaction (MLR). However, as shown in Figures 5B and 5C, despite possible functional impairment these Tregs are able to be activated with an activation agent (“an activator”) as described herein, and once activated can control CD8+ T cell proliferation. Accordingly, the present invention relates to the medical use of an activator of CD4+Foxp3+ regulatory T cells. In one aspect of the invention the activator is for administration to the subject, i.e., for treatment or prevention of the disease in vivo. In an alternative aspect of the invention CD4+Foxp3+ regulatory T cells can be isolated from a sample taken from the subject to be treated, such as a blood sample, and activated ex vivo, before being transferred back to the subject to treat or prevent the disease. Activators of CD4+Foxp3+ regulatory T cells are known in the art. In particular, the activator may be an antibody or antibody fragment such as an anti-CD4 antibody or a fragment thereof (e.g. as described in patent application publication no. WO 2011 / 064407), or a protein, such as a gpl20 protein or fragment thereof (e.g. as described in patent application publication no. WO 2008 / 092905), IL-2, or an IL-2 mutein. Some activators that are known to be activators of both regulatory T cells and T effector cells, or that are known to cause undesirable side-effects in vivo, are only suitable for use ex vivo where their action can be effectively targeted to isolated regulatory T cells, e.g. some anti-CD3 antibodies or fragments thereof. Other activators, including the anti-CD4 antibody tregalizumab discussed below (which is also known in the art and referred to herein as BT-061), are capable of selective activation of regulatory T cells. The activator is “capable of activating CD4+Foxp3+ regulatory T cells”. In one example this can be defined as the ability of the activator, e.g. the anti-CD4 antibody or antibody fragment, to activate T cells expressing CD4+ and Foxp3+ in vitro, preferably such that the activated CD4+Foxp3+ regulatory T cells are able to inhibit the production of pro-inflammatory cytokines (e.g. IL-5, IL-4 and / or IL-13, preferably IL-5) in a PBMC culture after TCR stimulation, in particular in a culture containing T effector cells. A testing method for such activity may comprise pre-incubating CD4+Foxp3+ regulatory T cells obtained from a donor (e.g. a healthy human donor) with the antibody or antibody fragment for a period suitable to activate the cells (e.g., 48 - 72 hours), TCR stimulating a T effector cell culture obtained from the same donor, transferring the pre-incubated CD4+Foxp3+ regulatory T cells to the T effector cell culture, and measuring the level of the pro-inflammatory cytokine after 72 hours. The T effector cell culture and / or the CD4+Foxp3+ regulatory T cells may be obtained from a healthy individual, or an individual suffering from a disease according to the present invention as described above. In this assay an activator, e.g., an anti-CD4 antibody or fragment thereof, that is capable of activating CD4+Foxp3+ regulatory T cells will lead to reduction in the amount of the pro-inflammatory cytokine detected as compared to a negative control in which the CD4+Foxp3+ cells have not been pre-incubated with the activator. Preferably the activator being tested leads to the same reduction + / - 25%, more preferably + / -10%, as a positive control with the tregalizumab antibody (BT-061), or an anti-CD4 antibody having the same variable heavy and variable light chain regions as the tregalizumab antibody (BT-061), which is described herein. The ability of the activator to activate CD4+Foxp3+ regulatory T cells can also be determined by methods described in the art, such as those described in WO2011 / 064407. For example, the ability can be assayed by examining the suppressive activity of the CD4+Foxp3+ regulatory T cells after incubation with the activator by co-culturing the CD4+Foxp3+ regulatory T cells with CD4+CD25‘ effector T cells. Activated CD4+Foxp3+ regulatory T cells are able to inhibit proliferation of such effector T cells in effector T cell proliferation assays. The effector T cells can be labelled with CFSE such that any proliferation can be determined. Alternatively, proliferation of effector cells can be determined by [3H] thymidine incorporation. It has also been shown that CDI37+CDI54‘ expression is a universal Treg activation signature ex vivo, which allows the identification and isolation of antigen-activated Tregs (Nowak et al., Frontiers in Immunology, February 2018, Vol. 9, Article 199). Therefore, as a further alternative, activated CD4+Foxp3+ regulatory T cells produced in the assay may also be identified based on CD137+CD154‘ expression. The activator may also be defined as one that does not cause antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). This ability can be determined by methods known in the art. In particular, the anti-CD4 antibody tregalizumab (BT-061) described herein is known not to cause ADCC or CDC (as described in Helling et al., Immunology and Cell Biology (2015) 93: 396-405). Methods of modulating the effector functions of an antibody or antibody fragment, so as to ablate effector functions are known in the art (Saunders, K.O., June 2019, Frontier in Immunology, Vol. 10, Article No. 1296). Antibodies or antibody fragments that are modified such that they do not cause ADCC or CDC are within the scope of the invention. The activator is preferably an antibody, an antibody fragment, or a multispecific or bispecific antibody that binds to the Tregs. A multispecific or bispecific antibody is one which targets at least one other antigen e.g., an antigen of another target cell such as a CD8+ cell, and can be used to assist in bringing the activated Treg cell into contact with the cells on which it has an effect. The antibody may be a human antibody, a humanized antibody, a chimeric antibody, or a fragment thereof. Preferably the antibody is a humanized antibody or a fragment of a humanized antibody. Generally, the antibody further comprises a human constant region (Fc). This constant region can be selected among constant domains from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgGl, IgG2, IgG3 and IgG4. Preferred constant regions are selected among constant domains of IgG, in particular IgGl. The antibodies are preferably IgGl antibodies, and / or the antibody or antibody fragment preferably comprises an Fc portion such that the antibody or antibody fragment is capable of binding to an Fc receptor, preferably FcyRI (i.e. CD64). Most preferably the antibody or antibody fragment comprises the Fc portion of an IgGl antibody. In addition, or alternatively, the antibody or antibody fragment is capable of binding to monocytes via an Fc receptor. The present invention also includes any fragment of the antibody including fragments comprising the V regions thereof. This comprises in particular Fab, Fab', F(ab)'2, Fv and scFv fragments. The activator can be an anti-CD4 antibody or antibody fragment thereof, preferably an antihuman CD4 antibody or fragment thereof. In particular, the anti-CD4 antibody can be the antibody tregalizumab (also known and referred to herein as BT-061) or one based on or derived from this antibody. Such antibodies are nondepleting and are capable of activating regulatory T cells but not effector T cells. Accordingly, preferably the antibody is a humanized anti-CD4 antibody or fragment thereof which is the tregalizumab antibody (BT-061), or a variant or fragment thereof. In particular, the tregalizumab antibody (BT-061) has V domains defined by the following polypeptide sequences (and which are described further in the examples below): - H chain V domain: EEQLVESGGGLVKPGGSLRLSCAASGFSFSDCRMYWLRQA PGKGLEWIGVISVKSENYGANYAESVRGRFTISRDDSKNTVYLQMNSLKTEDTAVY YCSAS YYRYDVGAWFAYWGQGTLVTVSS (SEQ ID NO: 3) - L chain V domain: DIVMTQSPDSLAVSLGERATINCRASKSVSTSGYSYIYWYQQ KPGQPPKLLIYL ASILESGVPDRF SGSGSGTDFTLTIS SLQAED VAVYYCQHSRELPWT FG QGTKVEIK (SEQ ID NO: 4). Accordingly, the anti-CD4 antibody or fragment thereof for use in the present invention may comprise a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 3 and a light chain variable domain comprising the amino acid sequence SEQ ID NO: 4. In a preferred embodiment the anti-CD4 antibody comprises a human IgGl constant domain. Variants of this antibody or antibody fragment are also suitable for use in the present invention. Variants include those with V domains defined by polypeptide sequences having at least 85%, preferably at least 90 %, most preferably at least 95% sequence identity with SEQ ID NO: 3 or SEQ ID No: 4. More preferably the antibody or fragment thereof has a heavy chain variable domain with at least 90% sequence identity with SEQ ID NO: 3 and a light chain variable domain with at least 90% sequence identity with SEQ ID NO: 4. Still more preferably the antibody or fragment thereof has a heavy chain variable domain with at least 95% sequence identity with SEQ ID NO: 3 and a light chain variable domain with at least 95% sequence identity with SEQ ID NO: 4. A preferred embodiment is an antibody comprising these variable domain sequences and an IgGl constant domain. In addition or alternatively the antibody or antibody fragment is one which has the CDR sequences of tregalizumab (BT-061) (SEQ ID Nos: 5 to 10). A particularly preferred embodiment is an anti-CD4 antibody or fragment thereof comprising a heavy chain variable domain with at least 95% sequence identity with SEQ ID NO: 3 and which includes the CDR sequences of SEQ ID Nos: 5 to 7, a light chain variable domain with at least 95% sequence identity with SEQ ID NO: 4 and which includes the CDR sequences of SEQ ID Nos: 8 to 10. The manner in which tregalizumab (BT-061) binds to CD4 has been disclosed in Helling et al., Immunology and Cell Biology (2015) 93: 396-405. Further variants of tregalizumab (BT-061) have also been disclosed in WO 2011 / 064407. Accordingly, this information can be used to guide the variation of the sequences of the heavy and light chains. Preferably the variations do not substantially affect the specificity and / or affinity of binding. Alternatively, or in addition, the antibody or antibody fragment is one that binds to an epitope on CD4 comprising amino acids 148-154, 164-168, 185, 187, 189-190 and 192, (as described in Helling et al., Immunology and Cell Biology (2015) 93: 396-405 and in WO2011 / 064407). Methods for determining the ability to bind to a particular epitope are known in the art. The antibody or antibody fragment can also be defined as one that binds to substantially the same epitope as the tregalizumab antibody (BT-061) described herein. Epitope mapping of antibodies can be done by methods that are known in the art (See e.g., Dang et al., “Epitope mapping of monoclonal antibodies: a comprehensive comparison of different technologies”, MARS 2023, vol. 15, no. 1, 2285285). In one aspect the invention provides an anti-CD4 antibody for use in treating or preventing lichen planus in a subject, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2. In a further aspect, the invention provides an anti-CD4 antibody or fragment thereof for use in treating or preventing lichen planus in a subject, wherein the antibody or fragment thereof comprises a heavy chain variable domain with at least 95% sequence identity with SEQ ID NO: 3 and which includes the CDR sequences of SEQ ID Nos: 5 to 7, and a light chain variable domain with at least 95% sequence identity with SEQ ID NO: 4 and which includes the CDR sequences of SEQ ID Nos: 8 to 10. The antibody or antibody fragment referred to herein may be manufactured using encoding polynucleotide sequence. In particular, recombinant DNA constructs can be obtained and introduced in host cells by the well-known techniques of recombinant DNA and genetic engineering. Expression cassettes can be used in which the polynucleotide sequence(s) are linked to appropriate control sequences that allow for regulation of transcription and translation in a chosen host cell. Suitable host cells include mammalian cells such as Sp2 / 0, HeLa, CHO, 3T3, C127, BHK, COS, etc. Accordingly, the antibody or antibody fragment may be one which has been produced in a mouse or a human cell line, and in particular in a cell line mentioned herein. The activator may be comprised in a pharmaceutical composition which further comprises a pharmaceutically acceptable excipient or carrier. The pharmaceutical composition may further comprise a stabilizing agent. The pharmaceutical composition may be suitable for intravenous, subcutaneous, or intramuscular use. In some aspects of the invention the activator is for use by administration to the subject. The antibody or antibody fragment may be administered by any appropriate route, e.g., intravenous, subcutaneous, or intramuscular. Preferably the activator is administered subcutaneously. Depending on the route of administration, the antibody or antibody fragment may be comprised in a vial, a pre-filled syringe, or an autoinjector. The vial may be arranged for use in an autoinjector, i.e., arranged such that it can be fitted or slotted into an autoinjector just prior to use. Suitable doses for the activator can be determined based on established methods. Where the activator is an anti-CD4 antibody or fragment thereof, the antibody or antibody fragment may be administered in a dose of between 10 mg and 200 mg, preferably between 75 mg and 200 mg, more preferably between 100 mg and 200 mg. Doses may also be calculated based on the body weight of the subject, and may be from 1 to 3mg / kg, preferably 1 to 2.5 mg / kg, and most preferably 1.5 to 2 mg / kg. The antibody or antibody fragment may be administered at regular time intervals, e.g., once per week, once every 2 weeks, once every 3 weeks or once every four weeks. As described above, in a further aspect of the invention the activator may be used to activate CD4+Foxp3+ regulatory T cells obtained from the subject ex vivo, before the activated cells are reintroduced back into the subject to treat or prevent the disease. Accordingly, the present invention further provides a method of treating a subject suffering from or preventing a subject suffering from the inflammatory autoimmune skin disease described herein, the method comprising removing a sample comprising CD4+Foxp3+ regulatory T cells from the subject, contacting the CD4+Foxp3+ regulatory T cells with an activator as described herein to activate CD4+Foxp3+ regulatory T cells and administering the activated cells to the subject. Such a method may additionally include an in vitro step of increasing the number of Tregs. This can be done using the expansion strategies described herein (Peters et al., PLoS 2008; 3(9): e3161; Hoffmann et al., Blood 2004; 104(3):895-903). Similarly, the present invention provides activated CD4+Foxp3+ regulatory T cells, which have been activated ex vivo using the activator described herein, for use in treating or preventing the disease described herein in a subject. The CD4+Foxp3+ regulatory T cells are obtained from the subject. Optionally the CD4+Foxp3+ regulatory T cells are expanded prior to reintroduction into the subject as described above. The following are intended as examples only and do not limit the present disclosure. Examples Example 1 - Activation of regulatory T cells in the treatment of lichen planus (LP) patients Antibody The anti-CD4 antibody tregalizumab (BT-061) that is used in the examples is a recombinant, humanized IgGl monoclonal antibody comprised of two heavy chains and two light chains. The amino acid sequence for both heavy and light chains has been predicted from the translation of the nucleotide sequence for the gene of tregalizumab (BT-061) and have been confirmed experimentally. Figure 1 displays the predicted amino acid sequences for the heavy and light chains, as well as the most likely disulphide bond assignment. The heavy chain has SEQ ID NO: 1 and the light chain has SEQ ID NO: 2. The tregalizumab antibody (BT-061) used in the examples has been produced in a mouse myeloma host cell line Sp2 / 0-Agl4 (Sp2 / 0, corresponding to ATCC CRL-1581). Expression of the antibody in Sp2 / 0 is also described in WO 2009 / 124815. The BT-061 antibody has been given the generic name tregalizumab. Mixed Lymphocyte Reaction (MLR) Mixed Lymphocyte Reaction (MLR) is an experimental procedure used to study the immune system's response to foreign cells in vitro. Since in LP autoreactive CD8 cells recognize parts of the skin (the epidermis) as foreign in the context of an autoimmune process, the MLR is suitable as an experimental approach to investigate the ability of a Treg activator to treat such a disease. To set up the MLR, CD8-positive T cells from donor A are mixed with antigen-presenting cells (APC) from donor B and cultivated at 37°C in an incubator. Over the course of 3 days, the cytotoxic CD8 cells recognize the APC as foreign in a nascent immune response and start to proliferate. This proliferative response will be quantified by flow cytometry with the proliferation marker Ki-67. The addition of CD4-positive T lymphocytes (from donor A) to cell culture enables a more physiological approach and ensures measurement of proliferation. The influence of regulatory T cells (Tregs) on the proliferative response of CD8 cells, will be addressed by the admixture of Tregs (from donor B) to the cell cultures. Finally, to address the impact of tregalizumab mAb (BT-061) on the inhibitory capacity of Tregs, tregalizumab (BT-061) will be added to the MLR together with or without Tregs. Detailed experimental setup Anonymized buffy coats from different donors of the Institute for Clinical Transfusion Medicine and Haemotherapy, University Hospital Wurzburg, were used to isolate the required cells. In a typical experimental set-up 5xl04 CD8 cells and 2xl04 CD4 cells from donor A were mixed with 1.5 x 105 APC cells from LP patients (which are thereafter designated as donor B) in a 96-well plate. To address the influence of Tregs on the MLR, 5xl04 Tregs from donor B were co-cultured from the beginning of the MLR. After 3 days, the proliferation of the CD8 cells was determined by flow cytometry using the Ki-67 marker. To analyze the impact of tregalizumab (BT-061) on the MLR in co-culture with Tregs, the mAb was added from the start of cell cultures. Specifically, 5xl07 cells from the buffy coat of healthy donors from donor A and 2-3 x 107 cells from donor B were thawed from the freezer (-80 °C) and cultured in X-Vivo medium (Gibco) overnight in an incubator at 37°C with 5% CO2. The following day, CD8-positive T cells were first isolated from donor A using MACS technology (Miltenyi Biotec, #130-045-201). The initial flow through of the CD8 column was used to purify CD4 cells, from which CD4-positive cells without contaminating Tregs were obtained in a further step using the Tregs isolation kit (Miltenyi Biotec, #130-091-301). In this step, CD25-labelled CD4-positive T cells (Tregs) remain in the MACS column, whereas Treg-depleted CD4-positive cells pass through the column as flow-through. To ensure a high level of Treg-depleted CD4 cells, the procedure was repeated. The CD4 cells were added to the MLR at the above concentration. The Tregs remaining in the column were discarded. APC from donor B, e.g. LP patients, were purified by depletion of CD3 cells using CD3 microbeads (Miltenyi Biotec, #130-050-101) and used in the MLR at the above concentration. Furthermore, Tregs from donor B were isolated using the Tregs isolation kit (Miltenyi Biotec, #130-091-301) according to manufacturer's instructions. Varying concentrations of Tregs, ranging from IxlO2 to IxlO4 cells, were used in the MLR assay. The stock solution (120 mg / ml) of tregalizumab (BT-061) was first diluted 1:120 in X-Vivo medium (Gibco) and then 1:1000 to the final concentration of 1 pg / ml (corresponding to a 1:120,000 dilution of the stock solution). The same dilution was used for the placebo solution containing the dissolvent of tregalizumab (BT-061). After three days, cells were cultured for flow cytometric analysis of proliferation markers for 4 hours with PMA (final concentration 100 ng / ml) and ionomycin (final concentration 1 pg / ml) in the presence of brefeldin A (final concentration 20 pg / ml) in an incubator at 37 °C with 5% CO2. Thereafter, cells were analyzed by flow cytometer (with the markers CD3, CD4, CD8, CD137, CD154, Ki-67, Foxp3, live / dead stain). Results After showing that tregalizumab (BT-061) has the capacity to activate Tregs from healthy donors and inhibit alloreactive proliferation of CD8+ T cells in a MLR (data not shown), we asked if the MLR reaction can be also dampened by tregalizumab (BT-061) in a MLR using Tregs from patients suffering from lichen planus (LP). Therefore, the MLR was set up applying Tregs and APCs from PBMCs of LP patients and CD8+ and CD4+ T cells from healthy donors. The strength of the MLR response was measured via the proliferation of alloreactive CD8+ T cells. Usually this is quantified by the addition of 3H-thymidine to cell cultures, which requires prior treatment of APC with mitomycin C or proliferation-inhibiting irradiation. Since this celldamaging pre-treatment can interfere with the MLR, the proliferative response of CD8+ T cells was determined using Ki-67 expression as evaluated by flow cytometric analysis. Four independent experiments revealed that the mean MLR response translated into a proliferation of 7.5±1.9% (±SEM) of alloreactive CD8+ T cells (Figure 5B) which was not statistically affected by the addition of tregalizumab (BT-061) or tregalizumab (BT-061) solvent (placebo). Of interest, the addition of tregalizumab (BT-061) and 104 Tregs from LP patients to the MLR assay revealed an 78.7% inhibition of the MLR response (1.6±0.4%, ±SEM) while Tregs alone or placebo displayed hardly any effect (7.6±.1.9%, ±SEM or 6.2±1.3%, ±SEM, respectively). The enhanced inhibition of the MLR by tregalizumab (BT-061) was also observed using grading amounts of Tregs, ranging from 103 to 102 (Figure 5B). The relative inhibition of the proliferative CD8 response (where the mean value of the MLR without any stimulus were set to 100%) reflected a 75.5±5.0% (±SEM) suppression of the MLR facilitated by 104 Tregs together with tregalizumab (BT-061) (Figure 5C). In contrast the addition of placebo to MLR-Tregs cultures had no statistically significant effect on MLR inhibition as well as tregalizumab (BT-061) alone. Mean CD4 expression on the T-cells in the MLR was analysed by flow cytometry after the 3 days of stimulation with tregalizumab (BT-061) or placebo (PL). After setting the lymphocyte gate, dead cells were excluded using a dead / live stain. Sequential gating on CD3 and CD8 was then performed. CD4 cells were then selected by excluding CD8 cells. T regulatory cells (Tregs) and CD4+ conventional T cells (Tconv) were separated based on being Foxp3+ or Foxp3‘. The results are shown in Figure 5D, in which CD4 modulation is depicted as mean fluorescence intensity (MFI) as compared to medium control (= 100%). These results show a comparable pronounced CD4 downmodulation induced by tregalizumab (BT-061) in both regulatory T cells (Tregs) and in CD4+ conventional T cells (Tconv). CD137 expression in Treg and Tconv was also analysed by flow cytometry after the 3 days of stimulation in the MLR with tregalizumab (BT-061) or placebo (PL). Gating strategy was performed as in the previous paragraph, followed by sequential gating on Foxp3+ / Foxp3‘ and CD137 / CD154. CD137+ cells were CD154' (data not shown). The results are shown in Figure 5E, in which CD 137 expression is depicted as the MFI difference (A) between BT-061 and placebo treated MLRs. These results show that tregalizumab (BT-061) has induced CD137 expression specifically in Tregs but not in Tconv. (CD137 being known as marker for Treg activation.) Conclusions The MLR model is an accepted model to address alloreactive immune responses. Applying this model, the ex vivo testing of regulatory T cells from patients suffering from the autoimmune skin disease LP revealed that Tregs alone were not capable to suppress the MLR. Strikingly, the addition of the mAb BT-061 to the MLR-Treg-cocultures facilitated a strong Treg-mediated suppression. In contrast, tregalizumab (BT-061) alone or placebo (tregalizumab (BT-061) solvent) did not show a significant effect. Further analysis confirmed that the Treg cells from the MLR had been activated by tregalizumab (BT-061) - CD4 downmodulation had been induced and the cells were CD137+ and CD 154", aknown phenotype of Treg activation (Nowak et al., Frontiers in Immunology, February 2018, Vol. 9, Article 199). The data indicates that the tregalizumab antibody (BT-061) antibody, and T regulatory cell activators in general, can enhance Treg effector functions in LP patients in vivo leading to treatment of the disease. The examples described herein are to be understood as illustrative examples of embodiments of the invention. Further embodiments and examples are envisaged. Any feature described in relation to any one example or embodiment may be used alone or in combination with other features. In addition, any feature described in relation to any one example or embodiment may also be used in combination with one or more features of any other of the examples or embodiments, or any combination of any other of the examples or embodiments. Furthermore, equivalents and modifications not described herein may also be employed within the scope of the invention, which is defined in the claims. All articles and patent publications referred to herein are incorporated by reference in their entirety. Sequences: SEQ ID No: 1 - tregalizumab (BT-061) heavy chain sequence EEQLVESGGG ISVKSENYGA SYYRYDVGAW CLVKDYFPEP GTQTYICNVN PPKPKDTLMI EQYNSTYRW REPQVYTLPP TPPVLDSDGS SPGK LVKPGGSLRL NYAESVRGRF FAYWGQGTLV VTVSWNSGAL HKPSNTKVDK SRTPEVTCW SVLTVLHQDW SRDELTKNQV FFLYSKLTVD SCAASGFSFS TISRDDSKNT TVSSASTKGP TSGVHTFPAV KVEPKSCDKT VDVSHEDPEV LNGKEYKCKV SLTCLVKGFY KSRWQQGNVF DCRMYWLRQA VYLQMNSLKT SVFPLAPSSK LQSSGLYSLS HTCPPCPAPE KFNWYVDGVE SNKALPAPIE PSDIAVEWES SCSVMHEALH PGKGLEWIGV EDTAVYYCSA STSGGTAALG SWTVPSSSL LLGGPSVFLF VHNAKTKPRE KTISKAKGQP NGQPENNYKT NHYTQKSLSL SEQ ID No: 2 - tregalizumab (BT-061) light chain sequence DIVMTQSPDS LAVSLGERAT INCRASKSVS TSGYSYIYWY QQKPGQPPKL LIYLASILES GVPDRFSGSG SGTDFTLTIS SLQAEDVAVY YCQHSRELPW TFGQGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASWCLL NNFYPREAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC SEQ ID No: 3 - tregalizumab (BT-061) heavy chain variable sequence: EEQLVESGGGLVKPGGSLRLSC AASGF SF SDCRMYWLRQAPGKGLEWIGVISVKSEN YGANYAESVRGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCSASYYRYDVGAWFA YWGQGTLVTVSS SEQ ID No: 4 - tregalizumab (BT-061) light chain variable sequence: DIVMTQSPDSLAVSLGERATINCRASKSVSTSGYSYIYWYQQKPGQPPKLLIYLASILE SGVPDRF SGSGSGTDFTLTIS SLQ AED VAVYYCQHSRELPWTFG QGTKVEIK SEQ ID No: 5 - tregalizumab (BT-061) heavy chain CDR1 sequence: DCRMY SEQ ID No: 6 - tregalizumab (BT-061) heavy chain CDR2 sequence: VISVKSENYGANYAESVRG SEQ ID No: 7 - tregalizumab (BT-061) heavy chain CDR3 sequence: SYYRYDVGAWFAY SEQ ID No: 8 - tregalizumab (BT-061) light chain CDR1 sequence: RASKSVSTSGYSYIY SEQ ID No: 9 - tregalizumab (BT-061) light chain CDR2 sequence: LASILES SEQ ID No: 10 - tregalizumab (BT-061) light chain CDR3 sequence: QHSRELPWT

Claims

1. An activator of CD4+Foxp3+ regulatory T cells for use in treating or preventing an inflammatory epidermal disease in a subject, wherein the disease is characterised by an inflammatory infiltrate comprising cytotoxic CD8+ T cells causing epidermal cell apoptosis, apoptosis of melanocytes in the epidermis, or apoptosis of epidermal appendage cells.

2. The activator for use according to claim 1, wherein the inflammatory epidermal disease is an autoimmune epidermal disease, an alloreactive epidermal disease, or an allergic skin disease.

3. The activator for use according to claim 1 or claim 2, wherein the epidermal cells are keratinocytes or wherein the epidermal appendage is a hair follicle.

4. The activator for use according to any of the preceding claims, wherein the disease is characterised by interface dermatitis.

5. The activator for use according to any preceding claim, wherein the disease is selected from lichen planus, lichen sclerosus, cutaneous or mucosal lupus erythematosus, dermatomyositis, cutaneous or mucosal graft-versus-host disease, minor erythema exsudativum multiforme, majus erythema exsudativum multiforme, fixed drug eruption, severe cutaneous drug reactions (including Stevens-Johnson syndrome and toxic epidermal necrolysis (Lyell’s syndrome)), alopecia areata, non-segmental vitiligo, and focal vitiligo6. The activator for use according to claim 5, wherein the disease is selected from lichen planus, lichen sclerosus, cutaneous lupus erythematosus, dermatomyositis, and cutaneous graft-versus-host disease.

7. The activator for use according to claim 6, wherein the disease is lichen planus.

8. The activator for use according to any preceding claim, wherein the activator is a protein.

9. The activator for use according to claim 8, wherein the activator is an antibody, an antibody fragment, a bispecific antibody, or a multispecific antibody.

10. The activator for use according to claim 8 or claim 9, wherein the activator is an anti-CD4 antibody or fragment thereof, a gpl20 protein or fragment thereof, IL2 or an IL2 mutein.

11. The activator for use according to claim 10, wherein the anti-CD4 antibody or fragment thereof comprises a variable heavy chain polypeptide sequence having at least 85% sequence identity with SEQ ID NO: 3 and a variable light chain polypeptide sequence having at least 85% sequence identity with SEQ ID NO: 4.

12. The activator for use according to claim 10 or claim 11, wherein the anti-CD4 antibody or fragment thereof comprises a variable heavy chain polypeptide sequence comprising CDR sequences having SEQ ID NOs: 5, 6 and 7, and a variable light chain polypeptide sequence comprising CDR sequences having SEQ ID NOs: 8, 9 and 10.

13. The activator for use according to any of claims 10 to 12, wherein the anti-CD4 antibody or fragment thereof comprises a variable heavy chain polypeptide sequence having SEQ ID NO: 3 and a variable light chain polypeptide sequence having SEQ ID NO: 4.

14. The activator for use according to any of claims 9 to 13, wherein the antibody is an IgG antibody.

15. The activator for use according to any of claims 9 to 14, wherein the antibody is an IgGl antibody.

16. The activator for use according to any of claims 9 to 15, wherein the antibody is an anti-CD4 antibody having a heavy chain sequence of SEQ ID NO: 1 and a light chain sequence of SEQ ID NO: 2.

17. The activator for use according to any preceding claim wherein the activator is comprised in a pharmaceutical composition.

18. The activator for use according to any preceding claim, wherein the subject is a human subject.

19. The activator for use according to any preceding claim, wherein the activator is to be administered to the subject intravenously, intramuscularly, or subcutaneously, and preferably wherein the activator is to be administered subcutaneously.

20. A method of preparing a medicament suitable for treating a subject suffering from, or for preventing a subject from suffering from, an inflammatory epidermal disease characterised by an inflammatory infiltrate comprising cytotoxic CD8+ T cells causing epidermal cell apoptosis, apoptosis of melanocytes in the epidermis, or apoptosis of epidermal appendage cells, the method comprising activating ex vivo CD4+ Foxp3+ regulatory T cells obtained from the subject with an activator of CD4+ Foxp3+ regulatory T cells.

21. The method according to claim 20, wherein the disease is as defined in any of claims 2 to 7.

22. The method according to claim 20 or claim 21, wherein the activator is as defined in any of claims 8 to 17.

23. An activated CD4+Foxp3+ regulatory T cell for use in treating or preventing an inflammatory epidermal disease in a subject, wherein the disease is characterised by an inflammatory infiltrate comprising cytotoxic CD8+ T cells causing epidermal cell apoptosis, apoptosis of melanocytes in the epidermis, or apoptosis of epidermal appendage cells, wherein the activated CD4+ Foxp3+ regulatory T cell is prepared by activating ex vivo CD4+ Foxp3+ regulatory T cell obtained from the subject with an activator of CD4+ Foxp3+ regulatory T cells.

24. The activated CD4+Foxp3+ regulatory T cell for use according to claim 22, wherein the disease is as defined in any of claims 2 to 7.

25. The activated CD4+ Foxp3+ regulatory T cell for use according to claim 24, wherein the activator is as defined in any of claims 8 to 17.

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