Methods and compositions for treating or preventing inflammatory skin disorders

Abstract

Provided herein is a method for treating or preventing inflammatory skin disorders, such as hidradenitis suppurativa, by administering an agent that inhibits NK cell activity to a subject that has or is at risk of developing inflammatory skin disorders.The agent that inhibits NK cell activity is optionally an agent that blocks the interaction between CD2 and CD58.The agent that blocks the interaction between CD2 and CD58 can be an anti-CD2 agent, such as an anti-CD2 antibody, or an anti-CD58 agent, such as an anti-CD58 antibody.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 342,886, filed May 17, 2022, which is incorporated by reference in its entirety.

[0002] Statement on Federally Sponsored Research This invention was made with Government support under Grants ES026219, 5PO1CA210946, AI149267, and MDO15319 awarded by the National Institutes of Health. The Government has certain rights in this invention. [Background technology]

[0003] Hidradenitis suppurativa (HS) is a chronic debilitating inflammatory skin disease characterized by persistent or recurrent papules, nodules, and abscesses (with or without sinus tracts) in the apocrine gland areas of the body, including the groin and axillae, and under the breasts. The disease is underdiagnosed, but currently has an estimated worldwide prevalence of 1-4%, with a female to male ratio of 3:1. The mean age at onset of the disease is 21.8 years, and individuals of African descent are more commonly affected than those of European descent. The reasons for the early onset, sex, and racial differences in HS are unclear. Clinical features of the disease include painful nodules, foul-smelling discharge, recurrent abscesses, and the development of scarring and fibrosis. Primary affected areas are the groin, axillae, and anal folds. Secondary affected areas are the submammary skin tissue, and areas of skin folds, especially in obese individuals. The clinical features have a dramatic negative impact on quality of life, often contributing to depression and anxiety in patients. Invasive squamous cell carcinoma (SCC) is considered one of the most serious complications of HS with a very high risk of death. HS is classified by Hurley stages I, II, and III based on the severity of the disease and the extent of involvement. Although inflammation is a key feature, major gaps in knowledge include the mechanistic basis of disease onset, progression, overall pathogenesis, and potential for targeted therapy.

[0004] The most common treatments for HS are systemic antibiotics and intralesional corticosteroids to treat infection and attenuate inflammation. TNF-α blockers are the only FDA-approved biologics for the treatment of HS. Anti-IL-17a (secukinumab) therapy has produced favorable clinical responses in the treatment of HS in subjects with refractory disease, and targeting IL-17F and IL-1β for HS is currently in clinical trials, but these therapies provide marginal palliation at best. Furthermore, patients develop resistance to one or more of these therapies and develop refractory HS. Thus, the need for new treatments for HS is essentially unmet. Summary of the Invention

[0005] Provided herein is a method for treating or preventing inflammatory skin disorders such as HS in a subject. The method comprises administering an agent that inhibits NK cell activity to a subject that has or is at risk of developing inflammatory skin disorders. The agent that inhibits NK cell activity is optionally an agent that blocks the interaction between CD2 and CD58. The agent that blocks the interaction between CD2 and CD58 can be an anti-CD2 agent, such as an anti-CD2 antibody, or an anti-CD58 agent, such as an anti-CD58 antibody. The subject with inflammatory skin disorders can have refractory inflammatory skin disorders.

[0006] The methods provided herein optionally further include administering one or more additional therapeutic agents, such as an antibiotic, a steroid, an anti-IL-15 agent, an anti-TNF-α agent, an anti-IL-17a agent, an anti-IL-18 agent, and / or an anti-fibrotic agent.

[0007] This application includes the following figures. The figures are intended to illustrate certain embodiments and / or features of the compositions and methods and to supplement any description(s) of the compositions and methods. These figures are not intended to limit the scope of the compositions and methods unless the written description expressly indicates such examples. [Brief description of the drawings]

[0008] [Figure 1] Schematic diagram showing CD2 blockade attenuation of NKT and NK cell activity in HS pathogenesis. NKT and NK cell subpopulations promote tissue destruction and remodeling in HS. CD56bright NKT cell subpopulations expressing IFN-γ, TGF-β, IL-13, IL-10, and / or IL-4 interact with keratinocytes and / or fibroblasts. CD11b+ / -CD56dim NK cells producing IFN-γ interact with keratinocytes. Cognate interactions between CD2 on NKT and NK cell populations and CD58 on keratinocytes and fibroblasts are critical for NKT and NK cell dependent disease processes. [Diagram 2] Analysis of scRNAseq quality control (QC) of six HS samples is shown. The total molecules detected within each sample (nCount-RNA), the total number of genes detected within each sample (nFeature-RNA), and the percentage of mitochondrial genes across the collected samples (percent.MT) are shown before (Figure 2A) and after filtering (Figure 2B). Data from four HS samples met QC and were used for subsequent analysis. [Figure 3A] Transcriptomics analysis to identify NK and NKT cell populations, the major innate immune cells in HS skin. A, UMAP of scRNAseq transcriptomes from normal skin (n=4) and HS skin (n=4). Using Seurat 4.0, 15 clusters representing immune and non-immune cell populations were identified. [Figure 3B] (B) Transcriptomic analysis to identify NK and NKT cell populations, the major innate immune cells in HS skin. (C) Proportions of cell types in each cluster in normal and HS skin. [Figure 3C] (C) Transcriptomic analysis to identify NK and NKT cell populations, which are the major innate immune cells in HS skin. (D) Dot plots showing normalized gene expression in HS for a panel of genes in each cell population. [Figure 3D] A, B, C show transcriptomics analysis to identify NK and NKT cell populations, the major innate immune cells in HS skin. D, D show volcano plots of differentially expressed genes in HS skin tissues (n=8) compared to normal skin tissues (n=6–8). Gene expression was determined using a qRT-PCR OpenArray panel containing 2429 unique target genes. Significantly up- or down-regulated genes are indicated in the top right or top left, respectively (log2FC≧2; P-value≦0.05). [Figure 3E] Figure 1 shows transcriptomics analysis to identify NK and NKT cell populations, the major innate immune cells in HS skin. E is a heat map. miRNA profiles from normal skin (n=6) and HS skin (n=9). Expression was determined using an OpenArray panel containing 754 well-characterized miRNAs. [Figure 3F] Figure 1 shows transcriptomics analysis to identify NK and NKT cell populations, the major innate immune cells in HS skin. F is a volcano plot of miRNAs expressed in normal and HS skin. miRNA profiles from normal skin (n=6) and HS skin (n=9). Expression was determined using an OpenArray panel containing 754 well-characterized miRNAs. Volcano plot (F) shows differentially expressed miRNAs that were up- or downregulated in HS compared to normal skin (upregulated are shown in the top right and downregulated are shown in the top left). Eleven miRNAs with known functions in regulating NK cell differentiation and / or function are labeled in the volcano plot. [Figure 3G](G) Transcriptomics analysis to identify NK and NKT cell populations, which are the major innate immune cells in HS skin. (G) Downstream gene targets of regulated miRNAs in HS with related functions in NKT and / or NK cells. Upregulated gene targets include Let-7, GZMB, PRF1, miR-29a-5p, miR-155, miR-21, miR-29b, Tbx21, miR-126, miR-142-3p, miR-150, miR-200a, and Stat4. Downregulated gene targets include PLZF, miR-27a-5p, miR-181a, CCND1 / CDKN1A / GATA3, PTEN, IRS1, and c-Myb. [Figure 4] We provide annotation of different cell types based on the expression of various cellular markers. A is a dot plot showing the normalized gene expression used to define the different cell populations. B and C show the filtered cell population QC results based on the number of molecules detected in each cell type (nCount-RNA) (B) and the number of genes detected within each cell type (nFeature-RNA) (C). [Diagram 5] The percentages of cell populations in each of the four HS scRNAseq data used in downstream analyses are shown. [Figure 6]Bulk transcriptomics analysis and qRT OpenArray PCR assays are shown. A-D are heatmaps representing bulk transcriptomics analysis of four publicly available datasets (GSE151243, GSE154773, GSE79150, and GSE128637) generated from both microarray and RNA sequencing techniques. Data show comparison between controls (normal (N) / control / non-lesional) and HS (HS or lesional skin). (E) Dot plots showing significantly activated or inhibited canonical pathways in each dataset identified using Ingenuity Pathway Analysis (IPA) (-log(BH p-value ≤ 0.05; z-score > |1|). Enriched pathways include T cell receptor signaling, Il-2 in activated T lymphocytes, and B cell development. (F) Heat map of qRT-PCR OpenArray analysis of four different signaling panels containing 2429 unique target genes in skin samples from normal subjects (n = 6-8) and HS subjects (n = 8). (G) Bar graph showing activated cell signaling pathways in HS compared to normal based on log q-values. [Figure 7] A is a Venn diagram showing the use of HS tissue from patients in different experiments. Overlapping circles represent tissue used in more than one experiment. B is a scatter plot showing relative quantification of 11 miRNAs with known roles in regulating NKT / NK differentiation / maturation / function. Each dot represents tissue from an independent control (left) or HS (right) individual. [Figure 8]Spatial localization of NKT and NK cell populations in HS skin. A-C show immunofluorescence staining of CD3, CD56, and CD2 expressing cells. D-F show immunofluorescence staining of CD2, CD58, and CD56 expressing cells. Images of normal or HS skin tissue are labeled. H&E staining shows "sutured skin" (center) with boxes showing enlarged areas of epidermal / subcutaneous and sinus tract regions in HS. Scale bars in immunofluorescence images are 100 μm. Arrows in immunofluorescence images point to CD2+CD3+CD56bright (NKT cells) and CD2+CD3-CD56dim (mature NK cells) in the epidermal / subcutaneous region and sinus tract (A, C, and D). T cells in immunofluorescence images are CD2+CD3brightCD56-. Immunofluorescence staining in HS sinus tract (D) shows clusters of NKT cells spatially separated from NK cells. B, E, and F show NK (CD2+CD56dim) and NKT (CD2+CD56bright) cells interacting with CD58 (keratinocytes or fibroblasts) expressing cells. B1, C1, C2, E1, F1, and F2 show more detailed magnifications of areas of the cell populations. Data represent HS skin (n=7-16) and normal skin (n=min. 3) sections for immunofluorescence studies. H&E staining was performed on all HS and normal skin sections. [Figure 9] H&E stained micrographs of normal (n=3) and HS skin tissues (n=3). The images show morphological abnormalities of HS skin, such as epidermal hyperplasia, leukocyte infiltration, and tunnels. (Magnification=4x, scale bar=200 μm, merged image is 300 μM). [Figure 10] Photomicrographs of immunofluorescence staining for each individual marker (resolution 4096×4096, magnification=20x, scale bar=100 μm). [Figure 11] Photomicrographs of immunofluorescence staining for each individual marker (resolution 4096×4096, magnification=20x, scale bar=100 μm). [Figure 12]Photomicrographs of immunofluorescence staining of CD8 T cells in HS (resolution 4096 × 4096, magnification = 20x, scale bar = 100 μm). [Figure 13A-F] Shown are photomicrographs of H&E stained skin sections (center) and immunofluorescence staining of CD4 T cells, CD68 and CD20 B cells in HS. A-C show CD4 and CD68 expressing cells in normal and HS skin. B-F show CD20 expressing cells in normal and HS skin. Arrows point to CD20+ cells. [Fig. 13G-H] Shown are photomicrographs of H&E stained skin sections (center) and immunofluorescence staining of CD4 T cells, CD68 and CD20 B cells in HS. G-H are photomicrographs of immunofluorescence staining for each individual marker shown in A-C and D-F. (Resolution 4096 × 4096, magnification = 20x, scale bar = 100 μm). [Figure 14] Photomicrographs of immunofluorescently stained plasma cells in HS for each individual marker (CD2, CD3 and IgG) (resolution 4096×4096, magnification=20x, scale bar=100 μm). [Figure 15] Photomicrographs of immunofluorescence staining of each individual marker (DAPI, CD56, CD3, perforin-1, and CD2) in normal and HS skin samples are shown (resolution 4096×4096, magnification=20x, scale bar=100 μm). [Figure 16] Photomicrographs of immunofluorescence staining of each individual marker (DAPI, CD2, GZMA, CD56, and CD3) in normal and HS skin samples are shown (resolution 4096×4096, magnification=20x, scale bar=100 μm). [Figure 17] Photomicrographs of immunofluorescence staining for each individual marker (DAPI, CD56, CD2, CD11, and GZMB) are shown (resolution 4096×4096, magnification=20x, scale bar=100 μm). [Figure 18]A is a UMAP of NKT and NK cell clusters in HS extracted from the scRNASeq analysis from Figure 3A. B shows the cytotoxicity scores of NKT and NK cells from HS samples. C is a UMAP dimensionality reduction clustering identifying six distinct subclusters of NKT (clusters 0-2), NK (clusters 3 and 5), and MAIT (cluster 4) cells. D shows the expression of NKT and NK-related genes in each of the six subclusters. The analysis shows that populations 0, 1, 2, and 4 express CD3. Population 4 has the gene expression profile of MAIT cells. Populations 3 and 5 have the gene expression profile of NK cells. E shows the percentage of NKT, NK, and MAIT cell populations in the scRNAseq data from each of the four HS patients. [Figure 19] Different NKT and NK cell subclusters from individual HS samples were scored for inflammation (A) and cytotoxicity (B). NK populations had similar inflammation scores, but population 3 (NK) and population 4 (MAIT) had the highest cytotoxicity scores. [Figure 20] Bar graphs of expression of cytokines, chemokines, and growth factors involved in inflammation, recruitment of effector immune cells, and cell proliferation in normal (n=11) (left) and HS (n=13) (right) skin samples. Two-tailed Student's t-test for normal vs. HS (*P≦0.05, **P≦0.01, ***P≦0.001, ****P≦0.0001). [Figure 21] Graph showing cytokine, chemokine, and growth factor levels in normal (n=11) and HS skin samples (n=13). Data represents specimens in which HS was not significantly different from normal. Data is from a multiplex Luminex platform using a 45-Plex Human ProcartaPlex™ Panel 1 (Cat. No. EPX450-12171-901, ThermoFisher, Waltham, MA). [Figure 22]Figure 2 shows that CD2 blockade in organotypic HS skin cultures attenuates proinflammatory and profibrotic signaling pathways. A and B are volcano plots showing changes in gene expression induced by CD2 blockade as determined by TaqMan-based human inflammatory OpenArray panel. The data show that CD2 blockade in HS skin organotypic cultures downregulates the expression of several inflammatory and fibrotic gene markers at the transcriptional level. The same three skin tissues were used for control-IgG and anti-CD2 treatments. [Diagram 23] FIG. 13 is a graph showing cytokine, chemokine, and growth factor stimulation levels in organotypic cultures of HS skin treated with anti-CD2 or IgG. [Figure 24] FIG. 13 is a graph showing cytokine, chemokine, and growth factor stimulation levels in organotypic cultures of HS skin treated with LPS together with anti-CD2 or IgG. [Diagram 25] Figure 3 shows upstream regulators determined by IPA analysis in HS compared to normal OpenArray data from Figure 3D. A shows the top four upstream regulators significantly upregulated in untreated lesional HS skin compared to healthy controls, namely lipopolysaccharide (P≦8.27E-81, z-score=6.550), TNF (P≦6.13E-70, z-score=4.994), IFNG (P≦3.96E-64, z-score=5.459), and TGFB1 (P≦1.12E-60, z-score=3.963). B shows that the upstream regulators TNF (P≦1.70E-32, z-score=-3.721), lipopolysaccharide (P≦1.61E-28; z-score=-4.288), IFNG (P≦1.72E-27; z-score=-2.993) and TGFB1 (P≦3.52E-26, z-score=-2.236) were significantly downregulated after treatment with anti-CD2 in HS skin compared to IgG-treated HS skin in organotypic cultures. [Figure 26] Bar graphs of significantly activated or inhibited signaling pathways using IPA analysis of HS vs. normal skin samples (A) and anti-CD2 vs. control IgG treated HS skin samples (B) are shown. [Figure 27] A summary of IPA analysis of HS skin vs. normal skin samples (A) and anti-CD2 vs. control IgG treated HS skin samples is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] A novel role for innate immune lymphocytes, specifically NKT and NK cells, in the pathogenesis of HS is identified herein. Notably, a striking heterogeneity in NKT and NK cell populations in HS skin was identified, with each subpopulation contributing to non-overlapping aspects of disease pathogenesis, as shown in Figure 1. A common feature of all NKT and NK cell populations was elevated expression of CD2, an adhesion and activation receptor that is integrally involved in the cellular network of HS pathogenesis through its interaction with CD58 on keratinocytes and fibroblasts. Disruption of this interaction using an anti-CD2 blocking antibody attenuated HS pathogenesis-related cytokine / chemokine protein and gene expression. Importantly, the HS tissues studied here were mostly from patients who were refractory to all previous therapies. Thus, the prominent contribution of NK cell populations reflects a novel therapeutic intervention for all stages of HS disease, including HS refractory to current therapies.

[0010] The major immune cell populations involved in HS to date have been T cells, B cell populations (B cells and plasma cells), neutrophils, and macrophages. Th1 and CD8+ T cell cytokines, IFN-γ and TNF-α, are highly elevated in HS. Similarly, the Th17 signature is prominent in HS, including IL-17A, IL-17F, IL-22, and IL-6. Furthermore, neutrophils are recruited to HS lesions, likely mediated by IL-17, IL-8, and / or infection. Neutrophils form neutrophil extracellular traps (NETs) within HS lesions, promoting in situ immune dysregulation and amplification of the inflammatory cascade through activation of the NLRP3-inflammasome pathway and / or suppression of immune regulatory mechanisms. The inflammatory environment of HS also recruits monocytes and promotes their differentiation into inflammatory M1-like macrophages.

[0011] Recruitment and activation of innate and adaptive immune cells around hair follicles promotes the initiation and progression of HS. However, the nature of the cell populations and the underlying mechanisms remain unclear. Analysis of HS skin and normal skin by scRNAseq revealed that natural killer (NK) cells and CD4+ T cells are the major lymphocyte populations in HS. Confocal scanning revealed that HS, but not normal skin, had elevated numbers of CD56 dim and CD56 bright We have shown that the epidermal and sinusoidal regions of HS contain NK cells, which express high levels of perforin and granzyme A, but not CD3, a population that represents highly cytolytic classical NK cells. dim In contrast, the sinus tract contained primarily NK cells. bright NK cells were abundant. These NK-T cells were juxtaposed with α-SMA expressing fibroblasts, a hallmark of fibrosis. In particular, CD56 dimNK cells expressed high levels of CD2, a receptor that signals increased NK cell cytolytic activity and increased production of IFN-γ. These NK cells associated with keratinocytes that expressed CD58 (LFA-3), a counterreceptor for CD2. Keratinocytes contained significantly elevated levels of IL-15 and IL-18, cytokines induced after CD58 engagement with CD2. HS skin also expressed elevated levels of miR150 and miR155, respectively, microRNAs required for NK cell maturation and cytotoxicity. To test whether CD2 blockade in HS resulted in a reduction of HS-associated chemokines and cytokines, skin from HS patients was cultured in the presence of anti-CD2 blocking Ab or control IgG. CD2 blockade resulted in a significant reduction in inflammatory cytokines (IL-6, IL-15, IL-18, IFN-γ) and chemokines (IL-8, MIP1α, RANTES, IP10). This study showed that different NK cell populations are the main contributors to cytolytic activity as well as fibrosis in HS. Thus, using a multi-omics approach including single cell and bulk transcriptome and regulome to build a deterministic association between multiple biological factors in HS, NK cell populations, CD3+CD56bright NKT cells and classical cytolytic CD56dim NK cells were identified as the major innate lymphoid cells contributing to HS disease. These NKT and NK cells are distributed to different locations within HS skin and contribute to different aspects of HS pathogenesis. Molecularly, NKT and NK cells expressed high levels of the costimulatory / adhesion molecule CD2, whose blockade attenuated the expression profile of inflammatory genes and cytokines in HS. Thus, CD2 blockade is identified as a novel therapeutic approach for the treatment of HS.

[0012] Provided herein are methods for treating or preventing inflammatory skin disorders, such as, but not limited to, HS, cutaneous Crohn's disease, pyoderma gangrenosum, Sweet's syndrome, and psoriasis. The methods include administering an agent that inhibits NK cell activity to a subject having or at risk of developing an inflammatory skin disorder. In some embodiments, the skin disorder is associated with an increase in NK cells and / or an increase in the cytolytic activity of NK cells.

[0013] In some embodiments, the agent that inhibits NK cell activity is an anti-CD2 agent, an anti-IL15 agent, and / or an anti-IL-18 agent. Optionally, the agent that inhibits NK cell activity is an agent that blocks the interaction between CD2 and CD58. In some methods, the agent is an antibody (e.g., an anti-CD2 antibody or an anti-CD58 antibody) or a small molecule. The antibody is optionally a humanized antibody or a portion thereof. By way of example, spurizumab is a humanized monoclonal anti-CD2 IgG1 antibody useful in the methods described herein. Effective fragments of spurizumab or other whole antibodies can be used.

[0014] The agent that inhibits NK cell activity is optionally a small molecule or a blocking peptide that inhibits CD2 signaling or activity.

[0015] In addition to agents that inhibit NK cell activity, also useful in the methods described herein are agents that indirectly inhibit NK cell activity by affecting upstream or downstream functions.

[0016] As used herein, the term antibody includes intact polyclonal or monoclonal antibodies, single chain antibodies, or antibody fragments, such as Fab, Fab', and F(ab')2, scFv, Fv, dsFv diabodies, and Fd fragments, as well as combinations of such antibodies or fragments. Thus, provided herein are intact polyclonal or monoclonal antibodies, single chain antibodies, or antibody fragments, such as Fab, Fab', and F(ab')2, scFv, Fv, dsFv diabodies, and Fd fragments that bind to CD2, CD58, etc. Such antibodies or fragments optionally exhibit the same epitope specificity as spurizumab.

[0017] The term antibody also includes chimeric antibodies (e.g., humanized antibodies) and hybrid antibodies with dual or multiple antigen or epitope specificity. Thus, fragments of antibodies that retain the ability to bind their specific antigen (e.g., CD2m CD58, etc.) are provided. The meaning of antibody or fragments thereof also includes antibody fragments and conjugates of antigen-binding proteins (single chain antibodies).

[0018] As described herein, inhibition of NK cell activity includes any detectable decrease compared to a normal (non-diseased) sample, such as a normal skin sample. Such a decrease can be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to control levels. NK cell activity manifests the cytotoxic capacity of the cells or by stimulating them to produce cytokines. 51 Various methods can be used to evaluate, including but not limited to Cr release assay or flow cytometry-based assay.Various methods are described in the following examples.Therefore, the effective amount of the agent that inhibits NK cell activity can be directly detected by such methods, or the effective amount can be determined based on clinical improvement, such as reducing pain, reducing skin lesions, etc.

[0019] The agent that inhibits NK cell activity can be administered to a subject at risk of developing inflammatory skin disorders as described herein. Such subjects include subjects with inflammatory disorders, including gastrointestinal disease (e.g., inflammatory bowel disease such as Crohn's disease), arthritis, acne, diabetes, and metabolic syndrome. Subjects at risk can also include subjects with a family history of inflammatory disease. In addition, chronic smoking and obesity are risk factors for developing inflammatory skin disorders such as HS.

[0020] Provided herein is a method for treating or preventing inflammatory skin disorders in a subject by administering to a subject having or at risk of developing an inflammatory skin disorder an agent that inhibits NK cell activity as described herein and an additional therapeutic agent. The additional therapeutic agent can be selected from the group consisting of biologics (e.g., infliximab and adalimumab), antibiotics, steroids, TNF-α blockers, anti-IL-17a (e.g., secukinumab), hormone supplements, retinoids, metformin, anti-fibrotic agents, and analgesics. Such additional agents can be administered before, after, or simultaneously with treatment with the agent that inhibits NK cell activity. The agent that inhibits NK cell activity is optionally administered in conjunction with the additional therapeutic agent (e.g., as a mixture), separately but simultaneously (e.g., via separate intravenous lines to the same subject), or sequentially (e.g., one of the compounds or agents is given first, followed by the second agent). Thus, the term combination is used to refer to the simultaneous, concurrent, or sequential administration of two or more agents.

[0021] Optionally, the subject to be treated has an inflammatory skin disorder that is refractory to one or more previous treatments for the inflammatory skin disorder, and the previous treatments were not treatments with agents that block the interaction between CD2 and CD58.The treatments to which the subject is refractory include, for example, antibiotics, corticosteroids, TNF-α blockers, anti-IL-17a (e.g., secukinumab) treatments, etc.However, in subjects that are refractory to previous treatments, the agents used in the previous treatments can be used in combination with agents that block the interaction between CD2 and CD58, either simultaneously or sequentially.

[0022] Provided herein is a pharmaceutical composition comprising an agent that inhibits NK cell activity. The pharmaceutical composition can include any compound described herein in a therapeutically effective amount. In some embodiments, the pharmaceutical composition can further include a carrier. Such an effective amount can be easily determined by those skilled in the art as described above. Considerations include the effect of the compound administered, i.e., the agent that inhibits NK cell activity, or the combined effect of the compound with one or more additional active agents when two or more agents are used in or with the pharmaceutical composition. Cell culture assays and animal studies can be used in formulating a range of dosages for use in humans.

[0023] The term carrier refers to a compound, composition, substance, or structure that, when combined with a compound or composition, assists or facilitates the preparation, storage, administration, delivery, efficacy, selectivity, or other features of the compound or composition for its intended use or purpose. For example, the carrier can be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject. Such pharmaceutically acceptable carriers include sterile biocompatible pharmaceutical carriers, including, but not limited to, saline, buffered saline, artificial cerebrospinal fluid, dextrose, and water. Carriers can include any excipients, diluents, fillers, salts, buffers, stabilizers, solubilizers, lipids, stabilizers, or other materials known in the art for use in pharmaceutical formulations.

[0024] Depending on the intended mode of administration, the pharmaceutical composition may be in solid, semi-solid or liquid dosage form, such as tablets, suppositories, pills, capsules, powders, liquids, lotions or suspensions, and may optionally be in unit dosage form suitable for single administration of precise dosage amounts. The composition comprises a therapeutically effective amount of the agent or derivatives thereof described herein in combination with a pharma- ceutically acceptable carrier, and may additionally comprise other agents, pharmaceuticals, carriers, or diluents. Pharmaceutically acceptable means a material that is not biologically or otherwise undesirable, which may be administered to an individual together with the selected agent without causing unacceptable biological effects or interacting in a deleterious manner with other components of the pharmaceutical composition in which it is contained. The composition may also comprise additional agents, such as wetting agents, emulsifying agents, suspending agents, sweetening agents, flavoring agents, or perfuming agents.

[0025] The agents or compositions described herein can be administered in several ways, depending on whether localized (e.g., topical) or systemic treatment is desired and the area to be treated. The compositions are administered via any of several routes of administration, including orally, parenterally, intravenously, intraperitoneally, intracerebroventricularly, intramuscularly, subcutaneously, intracavitary, or transdermally. The pharmaceutical compositions can also be delivered locally to the area requiring treatment, for example, by topical application or local injection. Effective doses for any of the administration methods described herein can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0026] Regardless of the route of administration, the amount of reagent administered or the schedule of administration will vary between individuals based on age, size, weight, condition being treated, method of administration, and severity of the condition. Those skilled in the art will understand that dosage will be optimized by the practitioner, and methods for determining dosage are described, for example, in Remington's Pharmaceutical Science, latest edition. A typical dose of an antibody used alone may range from about 1 μg / kg body weight up to 100 mg / kg body weight or more, preferably 1 μg / kg up to 1 mg / kg per day, depending on the factors mentioned above. An intravenous injection of an antibody or fragment thereof may be, for example, 10 ng to 1 g of the antibody or fragment thereof, preferably 10 ng to 1 mg, depending on the factors mentioned above. For local injection, a typical amount of antibody is in the range of 1 pg to 1 mg. Preferably, the local injection will be at an antibody concentration of 1 to 100 μg / ml, preferably 1 to 20 μg / ml.

[0027] As used herein, the terms treatment, treat or treat refer to a method of reducing one or more symptoms of a disorder or disorder, such as one or more effects of inflammatory skin disorder in a subject. Thus, in the disclosed methods, treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of inflammatory skin disorder. For example, a method for treating HS is considered to be a treatment if one or more symptoms of HS are reduced by 10% in a subject compared to a control. Thus, the reduction can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percentage reduction between 10% and 100% compared to natural or control level. It is understood that treatment does not necessarily refer to a cure or complete removal of a disorder or a symptom of a disorder.

[0028] As used herein, prevent, preventive, or prevention refers to a method of preventing, delaying, avoiding, eliminating, preventing, stopping, or impeding the onset, occurrence, severity, or recurrence of a disease or disorder. For example, the disclosed method is considered to be preventive if there is a reduction or delay in the onset, occurrence, severity, or recurrence of inflammatory skin disorder or one or more symptoms of inflammatory skin disorder in a subject susceptible to inflammatory skin disorder compared to a control subject susceptible to inflammatory skin disorder that has not received the composition described herein. The disclosed method is also considered to be preventive if there is a reduction or delay in the onset, occurrence, severity, or recurrence of inflammatory skin disorder or one or more symptoms of inflammatory skin disorder in a subject susceptible to inflammatory skin disorder after receiving the composition described herein compared to the progression of the subject before receiving treatment. Thus, the reduction or delay in the onset, occurrence, severity, or recurrence of inflammatory skin disorder can be about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount therebetween.

[0029] As used herein, administering or administration refers to the act of introducing, injecting, or otherwise physically delivering an exogenous substance (e.g., an agent that inhibits NK cell activity) to a subject, such as by topical, mucosal, intradermal, intravenous, intramuscular, rectal, oral, subcutaneous delivery, and / or any other method of physical delivery described herein or known in the art. When a disease or a symptom thereof is being treated, administration of the substance typically occurs after the onset of the disease or a symptom thereof. When a disease or a symptom thereof is being prevented, administration of the substance typically occurs before the onset of the disease or a symptom thereof.

[0030] As used herein, the term therapeutically effective amount or effective amount refers to the amount of a compound or composition described herein that is effective to treat disease or disorder when administered to a subject, either in a single dose or in the course of multiple doses.The appropriate dose may depend on various factors, including the specific agent used and whether it is used in combination with other therapeutic agents.Other factors that affect the dose administered to a subject include, for example, the type or severity of the disease.For example, a subject with HS may require administration of a different dosage of a composition comprising an agent that inhibits NK cell activity compared to a subject with psoriasis.

[0031] A therapeutically effective amount is also one in which any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial effects.

[0032] As used in this specification and the appended claims, the singular forms include plural references unless the context clearly dictates otherwise.

[0033] The terms may, may be, can, and can be, and related terms, are intended to convey that the related subject matter is optional (i.e., the subject matter is present in some instances and absent in other instances) and are not a reference to the ability or probability of the subject matter, unless the context clearly indicates otherwise.

[0034] Disclosed are materials, compositions, and components that can be used for, in conjunction with, in preparation for, or are products of the disclosed methods and compositions. These and other materials are disclosed herein, and when combinations, subsets, interactions, groups, etc. of these materials are disclosed, it is understood that each is specifically contemplated and described herein, although specific reference to each of the various individual and aggregate combinations and permutations of these compounds may not be expressly disclosed. For example, if a method is disclosed and contemplated, and multiple modifications that can be made to multiple molecules contained in the method are contemplated, each and every combination and permutation of the method and possible modifications are specifically contemplated, unless specifically indicated to the contrary. Similarly, any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of the present disclosure, including, but not limited to, steps in methods using the disclosed compositions. Thus, if there are a variety of additional steps that may be performed, it is understood that each of these additional steps may be performed with any specific method step or combination of method steps of the disclosed methods, and that each such combination or subset of combinations is to be considered specifically contemplated and disclosed. EXAMPLES

[0035] The following examples reveal a remarkable heterogeneity in NKT and NK cell populations in HS skin, with each subpopulation contributing to non-overlapping aspects of disease pathogenesis, as illustrated in Figure 1. A common feature of all NKT and NK cell populations is the elevated expression of CD2, an adhesion and activation receptor that is integrally involved in the cellular network of HS pathogenesis through its interaction with CD58 on keratinocytes and fibroblasts. Anti-CD2 treatment (referred to as CD2 blockade) disrupts this interaction by blocking CD2-CD58 (LFA-3) binding and attenuating cytokines, chemokines, proteins, and gene expression associated with HS pathogenesis. Thus, CD2 blockade is a very potent suppressor of the immune pathogenesis of HS disease. Importantly, the HS tissues studied here are mostly from patients who were refractory to all previous therapies. Thus, the prominent contribution of the NK cell population reflects a novel therapeutic intervention for such patient populations.

[0036] Example 1: Preparation of single cells Surgically discarded skin tissue from healthy subjects and subjects with HS (Hurley II / III stage) was used. Freshly surgically removed human tissue (3 x 3 cm) was placed in 5 ml of medium 154 (Thermo Fisher, Waltham, MA) supplemented with human keratinocyte growth supplement and further cut into small pieces. 100 μl of freshly prepared Liberase TL (2 mg / mL stock, Roche Diagnostics, Indianapolis, IN) and 700 μl of Tyrode's solution were added to a sterile Petri dish and the tissue was gently minced using a 15 ml centrifuge tube, followed by incubation in a 37°C water bath for 15 min. The suspension mixture was passed through a 70 μm cell strainer (Falcon). Finally, any remaining tissue was also incubated in growth medium supplemented with a solution containing 100 μl of 0.25% trypsin and 700 μl of Tyrode's solution and filtered through a 70 μm cell strainer to the previous cell batch. The cells were then resuspended in 300-500 μl of sterile PBS containing 0.04% BSA in a fresh 1.5 ml Eppendorf tube and centrifuged at 2000 rpm for 5 min. The resuspension and centrifugation steps were repeated before using 7-AAD cells to check cell viability.

[0037] Example 2: Single-cell RNA sequencing analysis and clustering of HS samples To identify immune cell populations associated with HS, we performed single-cell RNA-seq (scRNAseq) using the 10X Genomics protocol. For scRNAseq analysis, normal skin data were obtained from one in-house dataset and three publicly available (GSM4284228, GSM4284235, and GSM4284237) NCBI GEO List datasets (GSE144236). HS skin data were obtained from six HS patients (Table 1), of which four patients met the quality control requirements (Figure 2A and Figure 2B). Data processing included quality control, read alignment, and gene quantification, which were performed using the 10X Genomics Cell Ranger (v.5.0.1) tool (10X Gebnomics, Pleasanton, CA). For normalization, the Seurat (v.4.0) package was used. During quality control, several ribosomal protein genes and overexpressed MALAT1 gene were removed. Doublet Finder package was used to remove doublets / multiple cells and doublets. Harmony package was used for data integration and samples were clustered using Seurat with 25 dimensions and 0.3 cluster resolution for all downstream analyses. Cell populations were annotated using azimuth-based markers and HS scRNAseq cell type identification. (See Gudjonsson JE, Tsoi LC, MaF, Billi AC, van Straalen KR, Vossen A, et al., JCI Insight. 2020;5(19)). The dimensionality reduction algorithm identified 15 clusters including populations of innate and adaptive immune cells, keratinocytes, fibroblasts, endothelial cells, and melanocytes (Figure 3A and Figure 4A-4C). The main lymphoid cell populations identified were T cells, B cells, NKT cells, and NK cells (Figure 3A and Figure 5). The expanded proportion of NKT and NK cells in HS was a novel finding ( Fig. 3B ) and was present in each of the four HS scRNAseq data ( Fig. 5 and Table 2 ). [Table 1]

Table 2

[0038] Several genes associated with lineage and / or function were prominently expressed within immune cell populations from HS samples (Figure 3C and Figure 4A). CD2, a cell surface receptor associated with NKT cells, was highly expressed in NK and T cells in HS (Figure 3C). Cells within the T cell cluster predominantly expressed CD4, rather than CD8A or CD8B, indicating that they were helper T (Th) cells rather than cytotoxic T cells (). Expression of CD8A is also observed within the NK cell cluster, a consistent feature in some NKT cells. These CD8A-expressing NKT cells did not express significant levels of the BATF, ITGAE, or ITGB2 transcription factors, making them unlikely to be misclassified as CD8 T cells. Other genes identified with elevated expression in NKT and NK cells and associated with their enhanced maturation / activation were NCAM1 (CD56), PRF1, TBX21, STAT1, STAT4, PDCD2, GZMA, GZMB, and the cytokine IFNG (Figures 3C and 4A). Genes with decreased expression in HS NKT and NK cells were PRDX2, STAT6, IFNAR1, IFNGR1, and IFNGR2 (Figure 3C). T cells in HS expressed CD4, STAT1, STAT4, RORC, PDCD1, PDCD2, FOXP3, BATF, AHR, and the cytokines IFNG, IL-17A, IL-17F, and IL-21 (Figure 3C). Expression of these genes in T cells is associated with effector (Th1 and Th17) and regulatory (Treg) phenotypes. STAT6, a transcription factor induced and activated by the type 2 cytokine IL-4, was significantly reduced in HST cells, indicating that type 2 immune response lymphocytes (Th2 or ILC2) are underrepresented in HS. PDCD2, AHR, BATF, and STAT1 were increased in a small proportion of HS resident B cells and plasma cells (Figure 3C). CD8 T cells contribute minimally to HS. Monocyte / macrophage cell clusters displayed a proinflammatory gene signature with elevated expression of STAT1, IFNGR1, IFNGR2, and IRF7 ( Fig. 3C ), genes associated with activation of type 1 and type II IFN signaling ( Fig. 3C ).This cluster, together with keratinocytes, has enhanced expression of IL-18, a cytokine that promotes activation and expression of IFN-γ in T and NK cells. Overall, the data indicate that the major lymphocyte populations present in HS skin are NK cells, CD4 T cells, and B lineage cells.

[0039] Example 3: Collection and analysis of bulk transcriptomics data To study the bulk transcriptome expression profiles altered by HS, four publicly available datasets (GSE151243, GSE154773, GSE79150, and GSE128637) representing both microarray and RNA sequencing technologies from the NCBI GEO database were used. Raw values ​​were log2 normalized for further analysis below. DESeq2 was used to independently identify differentially expressed genes (DEGs) with default parameters (FDR<0.05 and log2FC=|1|). For each HS sample, we identified 1614, 2080, 434, and 359 significantly upregulated genes, and 1354, 1747, 309, and 300 downregulated genes, respectively, compared to the control (Figure 6A-6D). Ingenuity pathway analysis (IPA) revealed that NK cell signaling and other pathways were significantly activated in HS (Figure 6E).

[0040] Example 4: Quantitative PCR open array analysis To validate global differential gene expression from publicly available RNASeq datasets of normal (n = 6–8) and HS (n = 8) skin samples, we used quantitative TaqMan-based real-time qPCR against predesigned gene panels of various regulatory pathways. Of the eight HS tissues analyzed in this assay, four were from the same individuals used for scRNAseq (Figure 7A). Human Inflammation Open Array Panel (Cat. No. 4475389), Human Signaling Panel (Cat. No. 4475392), Human Kinome Panel (Cat. No. 4475388), and Human Stem Cell Open Array Panel (Cat. No. 4475390, Thermo Fisher, USA) were used. Briefly, total RNA from HS and normal skin tissues was isolated using Trizol Reagent (Cat. No. 15596018, Ambion, Thermo Fisher). A total of 2 μg of RNA was reverse transcribed into cDNA using the SuperScript® VILO™ cDNA Synthesis Kit (Cat. No. 11754250, Life Technologies (Thermo Fisher)). Preamplification of cDNA was performed in a total volume of 25 μl containing 12.5 μl of TaqMan preamplification master mix (Cat. No. 4391128), 2.5 μl of custom TaqMan preamplification primer pool (Cat. No. 4441856, ThermoFisher), and 2.5 μl of reverse transcription product. Thermal cycling conditions for preamplification included incubation of the product at 95°C for 10 min, followed by 12 cycles of 95°C, incubation for 15 s, and incubation at 60°C for 4 min. The preamplification product obtained after cycling was incubated at 99.9°C for 10 min, then diluted 20-fold and used in the final dilution reaction according to the manufacturer's instructions. OpenArray chips containing the complete pre-coated primers for the 2429 targets were read on a 12K Flex RT-PCR machine (Thermo Fisher Scientific). Data analysis was performed via Expression suit (v1.3) software available online (Thermo Fisher Scientific) using a global gene normalization method.

[0041] Example 5: qRT-PCR analysis of gene expression From qRT-PCR OpenArrays representing 2429 gene targets, 170 genes were significantly increased (Log2FC≧1; p-value<0.05) and 66 genes were significantly decreased (Log2FC≦-1; p-value<0.05) in HS compared to controls (Figure 3D and Figure 6F). Genes with significantly increased expression in HS compared to controls include CD2, kinase BTK, integrin ITGAX, complement protein C1QA, and several cytokines / chemokines and their receptors, phosphatases, kinases, and other molecules associated with inflammatory signatures (Figure 3D; Figure 6G).

[0042] Example 6: qRT-PCR analysis of microRNAs For microRNA profiling, a quantitative TaqMan-based OpenArray Human MicroRNA Panel (catalog no. 4470187; Thermo Fisher) was used. This panel contains 754 well-characterized human miRNA sequences from the Sanger miRBase v14. All 754 assays have been functionally validated with miRNA artificial templates. Briefly, total RNA was isolated from normal skin (n = 6) and HS skin (n = 9) using the mirVana miRNA isolation kit (catalog no. AM1561) (Figure 3E). TaqMan miRNA reverse transcription kit (catalog no. 4366596, Thermo Fisher Scientific) was used for reverse transcription reaction using Megaplex RT and Preamp pool primers v3 (catalog no. 4444750). Preamplification and amplification thermocycling conditions were used as described in the manufacturer's protocol. miRNA expression was read on a 12K Flex RT-PCR machine and relative quantification was performed by the -ΔΔCt method. Data analysis was performed via Expression suite (v1.3) software available online (Thermo Fisher Scientific) using the global gene normalization method. Bioinformatics analysis of all panels was performed using IPA (Qiagen, Hilden, DE) (version 84978992).

[0043] A total of 36 differentially expressed miRNAs were identified in HS compared to healthy skin, of which 27 miRNAs were significantly upregulated and 9 miRNAs were significantly downregulated (log2FC>|1|, p-value<0.05;) (Figure 3F). Eleven of the 36 differentially expressed miRNAs (miR-150, miR-27a5p, miR-155, miR-21, miR-142-3P, miR-126, miR-29b, Let7, miR-200a) have roles in NK cell differentiation and / or function (Figure 3G and Figure 7B). miRNAs that enhance NKT or NK cell differentiation and / or activity (mir-150, miR-155, miR-21, miR-200a, miR29a-5p, Let7) were increased in expression, whereas miRNAs that regulate NK activity (miR-27a-5p, miR-181a-2-3p) were decreased in HS compared to normal controls. Collectively, the cellular landscape, gene expression profile, and miRNA regulome indicate that NK cell populations are important contributors to the pathogenesis of HS (Figure 3G).

[0044] Example 7: Sample processing and ex vivo culture Clinical specimens were cut into approximately 5 mm × 5 mm pieces of tissue and placed on 0.4 μm transwell filters (Merck Millipore, Burlington, MA) with a thin layer of extracellular matrix (ECM, 90% type 1 collagen (Advanced Biomatrix, Carlsbad, CA) + 10% growth factor reduced Matrigel (Corning, Corning, NY). KBM Gold basal medium (00192151; Lonza, Basel, CH) containing KBM Gold supplement (00192152, Lonza) was added to the transwell filter and the bottom of the well to generate air-liquid (the volume of medium did not cover the top tissue layer) cultures. Anti-human CD2 antibody (10 μg / mL, Cat. No. 300240, Clone: ​​RPA-2.10, Biolegend, San Jose, CA) was added to the bottom of the well to generate air-liquid (the volume of medium did not cover the top tissue layer) cultures. Cells were treated with either IgG antibody (10 μg / mL, Cat# 403502, Clone: ​​QA16A12, Biolegend) or vehicle IgG antibody (10 μg / mL, Cat# 403502, Clone: ​​QA16A12, Biolegend) for 3 days. These treatments were changed daily and at the end of each experiment, a portion of each tissue was fixed for histological processing or snap frozen in liquid nitrogen for RNA and protein analysis. Supernatant culture medium was collected daily and stored at -80°C for cytokine, chemokine, and growth factor profiling.

[0045] Example 8: Histological analysis and confocal immunofluorescence (IF) procedures Hematoxylin and Eosin (H&E) staining: Briefly, skin tissues were fixed in 10% formalin, embedded in paraffin, and sectioned at 5 μm thickness. Skin sections were deparaffinized in xylene, rehydrated, and stained with H&E. Images were acquired using a BZ-X710 bright-field microscope and integrated for Z-stacking and stitching using a BZ-X analyzer (Keyence Corporation, Osaka, JP).

[0046] Immunofluorescence confocal analysis: Skin sections were deparaffinized, rehydrated, and then incubated in antigen unmasking solution according to the manufacturer's instructions (Vector Laboratories, Burlingame, CA). Sections were blocked in blocking buffer containing 5% normal goat serum in PBST (PBS + 0.4% triton® X100) for 1 h at 37°C. Sections were then incubated overnight at 4°C with primary antibodies against various proteins in blocking solution. The antibodies and their dilutions used are listed in Tables 3 and 4. Sequential staining was performed to visualize multiple targets in a single sample. After washing with PBST (three times, 10 min each), sections were reincubated with various fluorescently conjugated secondary antibodies (1:200, Invitrogen). Sections were fixed in DAPI containing Vectashield gold antifade medium (catalog number H-1200, Vector Laboratories, Burlingame, CA) and then visualized under a FLUOVIEW FV3000 confocal microscope (Olympus Center Valley, PA) equipped with an FV3000 Galvo scanning unit. Z-projection images were post-processed for noise reduction, 3D image construction, and movie preparation using FV3IS-SW version 2.3.2.169 software provided with the Olympus Fluoview F3000 confocal microscope. [Table 3-1] [Table 3-2] [Table 4-1] [Table 4-2]

[0047] Example 9: Spatial localization of NKT and NK cells in normal and HS skin NKT and NK cells are heterogeneous, with functions determined by tissue and / or organ localization. Immunofluorescence microscopy was used to correlate their localization to different functions in HS pathogenesis. Classical NK cells include CD56bright (immature) and CD56dim (mature and highly cytolytic), whereas NKT cells are CD3+CD56bright. H&E stained sections from normal and HS skin were used to localize cells expressing CD56 (pan NK marker), CD3 (T cells and NKT), CD2 and CD58 (LFA3, a ligand for CD2) from immunofluorescent antibody stained serial sections. Histomorphology of skin from healthy controls revealed clearly defined epithelial and dermal regions (Figure 8A, Figure 8D, and Figure 9). In contrast, HS skin (Hurley late stage II and stage III) showed hyperinflammatory areas with hyperproliferation of the epidermal and dermal compartments (Figures 8B, 8C, 8E, 8F, and 9). Histology showed the presence of sinus tracts projecting deep into the subcutaneous tissue with widespread cellular infiltration of different populations of immune cells (Figure 9). Control skin contained very few T cells (CD3+CD56-) and NKT or NK cells (mainly CD56bright) (Figures 8A, 8B, and 10). In contrast, HS skin was characterized by a high percentage of NK (CD56+), NKT (CD3+CD56+), and T cells (CD3+CD56-) (Figures 8C-8F, and 10). In the epidermal and dermal regions of HS skin, NK cells were mainly CD3-CD56dim, a mature cell phenotype (Figures 8C, 8E, and 10). Near the deep sinus tract and hair follicle regions, the cells were predominantly CD56bright NK cells that co-expressed CD3, a characteristic of NKT cells (Fig. 8D, Fig. 8F, and Fig. 10). A striking feature was the high expression level of CD2 on NKT, NK, and T cells in HS. Expression of CD58, the ligand for CD2, was significantly elevated on epithelial cells in HS skin but not in control skin. Furthermore, these CD58-expressing epithelial cells were juxtaposed to CD2-expressing NK, NKT cells (CD56+), and T cells (CD56-) (Fig. 8B vs. Fig. 8E, and Fig. 8F, and Fig. 11).

[0048] The spatial distribution observed for NKT and NK cell populations was not as clear for T and B cell populations (Figures 12 and 13). Although B cells (CD20+) and plasma cells (IgG+) were observed in HS, they did not show a distinct localization pattern (Figures 13A-H and 14). T cells in the epidermis, dermis and sinus compartments of HS skin were predominantly CD4+, with rare CD8+ cells (Figures 12 and 13). This is consistent with the scRNAseq (Figure 3A) data. In summary, the data indicated that NKT and NK cells were the major lymphocyte populations within HS skin, and they were spatially localized to different regions associated with distinct aspects of disease pathogenesis.

[0049] Example 10: NK and NKT cell populations have distinct roles in the pathogenesis of HS Perforin and granzymes (granzyme A and granzyme B) are proteins required for NKT cell and NK cell-mediated cytotoxic activity of target cells. Normal skin samples contained low frequencies of perforin-1, granzyme A, and granzyme B expressing cells (Figures 15, 16, and 17). In contrast, HS skin had numerous perforin-1, granzyme A, and granzyme B expressing classical NK cells (CD3-CD56dim) within epidermal hair follicles, subcutaneously, and along the border areas of the sinus tract, which may be related to the development of the sinus tract (Figures 15, 16, and 17). NKT and NK cells expressed comparable levels of granzyme A, but NKT cells had apparently lower expression of perforin-1 and granzyme B. Immunohistological staining images showed that NK and NKT cells in HS expressed high levels of CD2, confirming the data from the gene expression analysis above. A significant proportion of CD56dimCD2hi granzyme Bhi cells also expressed high levels of CD11b (MAC1 / ITGAM / CR3) (Figure 17). Overall, the staining patterns reveal heterogeneity in NKT and NK cell populations in different regions of HS skin.

[0050] HS scRNAseq data for NKT and NK cells were extracted from Figure 3A (Figure 18A). Both NKT and NK cells had fairly high cytotoxicity scores based on expression of cytotoxic genes, but NK cells had higher scores (Figure 18B). UMAP clustering revealed six populations based on gene expression profiles, three of which were NKT (CD3+), two were NK (CD3-), and one population had MAIT characteristics (CD3+CD161+) (Figure 18C and Figure 18D). These NK cell populations were present in each of the four HS samples (Figure 18E). Based on gene expression-related function scoring, the NK populations had similar inflammation scores, but the cytotoxicity scores were higher in population 3 (NK) and population 4 (MAIT) (Figure 19).

[0051] Multiple analysis To identify inflammatory cytokine and chemokine responses, ex vivo cultures of HS skin tissues (n=11) or healthy skin tissues (n=13) were exposed to anti-CD2 and multiplex Luminex analysis was performed. Cytokine / Chemokine / Growth Factor 45-Plex Human ProcartaPlex™ Panel 1 (catalog no. EPX450-12171-901, Thermo Fisher) was used. Briefly, tissues were homogenized using cold RIPA buffer (Santa Cruz biotechnologies) containing 2 mM sodium orthovanadate, 1 mM PMSF, and protein cocktail inhibitors (1x; Santa Cruz). Homogenates were centrifuged at 10,000 rpm for 10 min and supernatants were stored at -80°C until assayed. (i) Th1 / Th2 markers: GM-CSF, IFNγ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-12p70, IL-13, IL-18, TNFα (ii) Th9 / Th17 / Th22 / T reg markers: IL-9, IL-10, IL-17A (CTLA-8), IL-21, IL-22, IL-23, IL-27(iii) Inflammatory cytokines: IFNα, IL-1α, IL-1RA, IL-7, IL-15, IL A total of 45 target proteins were evaluated, including (iv) chemokines: eotaxin (CCL11), GROα (CXCL1), IP-10 (CXCL10), MCP-1 (CCL2), MIP-1α (CCL3), MIP-1β (CCL4), RANTES (CCL5), SDF-1α, and (v) growth factors: BDNF, EGF, FGF-2, HGF, NGFβ, PDGF-BB, PIGF-1, SCF, VEGF-A, and VEGF-D. For analysis, 50 μl of conditioned medium was used on a Luminex 200 instrument (Luminex Corporation, USA) as previously described (Kashyap M, Kawamorita N, Tyagi V, Sugino Y, Chancellor M, Yoshimura N, et al., J Urol. 2013;190(2):757-64).

[0052] Expression of cytokines IFN-γ, IL-1β, IL-4, IL-6, IL-8, IL-12p70, IL-15, IL-17a, IL-18, IL-22, IL-27, and TNF-α was significantly elevated in HS compared to controls (Figure 20). Along with TNF-α, IFN-γ was the major effector cytokine expressed by NK and NKT cells. IL-1β induces cytokine / chemokine production by keratinocytes, IL-8 (CXCL8), which plays a key function in neutrophil recruitment, both of which are significantly enhanced in HS. Keratinocytes are the primary expressers of IL-15 and IL-18, which were highly elevated in HS skin. These cytokines not only enhance the cytotoxic activity of NK cells but also induce the expression of CD2, thus playing a role in NKT cells and classical NK cell-mediated HS pathogenesis. IL-12 is elevated in HS and, importantly, promotes NK cell survival and expansion independent of IL-15 and IL-18. Levels of IFN-α, IL-1α, IL-2, IL-7, IL-9, IL-31, and TNF-β were similar in HS and control skin (Figure 21). IL-13 was elevated in HS skin, but its levels were not statistically different from controls (Figure 21). Several chemokines and growth factors were elevated in HS skin compared to controls. These include CCL2 (MCP1), CCL3 (MIP-1α), CCL4 (MIP-1β), CCL5 (Rantes), CCL11 (eotaxin), CXCL1 (GROα / KC), CXCL9, and CXCL10 (IP10), CXCL12 (SDF-1), NGFβ, EGF, FGF-2, HGF, and PIGF1 (Figure 20). Growth factors BDNF, PDGF-BB, PIGF-1, VEGF, and SCF were present in HS skin, but they did not change significantly from controls (Figure 21). A complex interplay of cytokines, chemokines, and growth factors produced by NK cell populations, keratinocytes, and HS cell populations perpetuates the pathogenic mechanism of the disease. Results indicate that upregulation of CD2 in all NK cell populations and T cells is a key feature of HS.These data indicate that CD2 blockade in HS can reverse the expression of disease-associated cytokines, chemokines, and growth factors as well as gene expression signatures.

[0053] Testing CD2 blockade in organotypic cultures HS skin organotypic cultures (Goliwas KF, Kashyap MP, Khan J, Sinha R, Weng Z, Oak ASW, et al., Inflammation. 2022;45(3):1388-401) were treated with either anti-CD2 mAb vs. IgG (control) or anti-CD2 mAb vs. IgG with LPS. Cultures treated with anti-CD2 mAb vs. IgG alone showed changes in gene expression as measured by inflammatory gene expression arrays. Genes related to NK and T cell activation and effector function were downregulated after treatment with anti-CD2 (Figure 22A and Figure 23). Anti-CD2 also reversed pathogenic cytokine expression patterns and gene expression signatures in LPS-treated HS skin cultures. LPS is a bacterial component known to induce a variety of innate immune responses. Specifically, anti-CD2 treatment led to a decrease in the expression of LPS-induced cytokines / chemokines / growth factors and gene signatures associated with TLR2 / 4 signaling, including IL-10, IL-15, IL-18, FGF-2, IRAK2, MAPK, ILFR, and others (Figure 22B and Figure 24).

[0054] Pathway enrichment analysis Canonical pathway analysis for DEGs was performed using Ingenuity Pathway Analysis (IPA) (Kramer A, Green J, Pollard J, Jr., and Tugendreich S., Bioinformatics. 2014;30(4):523-30) with significant fold change and test statistics. Additional functional enrichment analysis was performed by enrichR (Chen EY, Tan CM, Kou Y, Duan Q, Wang Z, Meirelles GV, et al., BMC Bioinformatics. 2013;14:128) and metascape (Zhou Y, Zhou B, Pache L, Chang M, Khodabakhshi AH, Tanaseichuk O, et al., Nat Commun. 2019;10(1):1523) pathway analysis platforms with standard significance (q-value < 0.05). Disease enrichment analysis was performed via enrichR with standard significance (q-value < 0.05).

[0055] To determine which signaling pathways are downregulated in HS by CD2 blockade, IPA analysis was applied using the qRT-PCR HS skin vs. normal skin array data (Figure 3D) and anti-CD2 vs. IgG array data. The top four upstream regulatory drivers in HS were lipopolysaccharide (P≦8.27E-81, z-score=6.550), TNF (P≦6.13E-70, z-score=4.994), IFNG (P≦3.96E-64, z-score=5.459), and TGFB1 (P≦1.12E-60, z-score=3.963) (Figure 25A). These were also the top four regulatory drivers downregulated by anti-CD2 treatment: TNF (P≦1.70E-32, z-score=-3.721), lipopolysaccharide (P≦1.61E-28; z-score=-4.288), IFNG (P≦1.72E-27; z-score=-2.993), and TGFB1 (P≦3.52E-26, z-score=-2.236) (FIG. 25B). The majority of signaling pathways upregulated in HS versus normal skin (FIGS. 26A and 27A) were downregulated by CD2 blockade in organotypic cultures (FIGS. 26B and 27B).

[0056] Visualization Single-cell RNAseq analysis plots (clusters, heatmaps, dot plots, cell-cell communication) were generated via R (v4.0.3) statistical software. Violin plots, volcano plots, and network centrality comparison violin plots of all experimental validations were drawn using GraphPad Prism.

[0057] statistical analysis To identify marker genes within single-cell RNA-seq clusters, a non-parametric Wilcox test was used with default parameters. Standard cutoffs (FDR ≤ 0.05; log2fc ≥ |1|) were used to identify DEGs in bulk transcriptome datasets. Activated and inhibited canonical pathway analysis was determined by parameters (z-score ≥ |1|; BH p-value ≤ 0.05). The significance of experimentally validated DEGs was (Student's T-test (p-value) ≤ 0.05 and log2fc ≥ |1|). Functional enrichment of genes / proteins was performed with significance (-log10 (q-value > 1.3). Network power-law distribution cutoff was significant (R2 > 0.7; Student t-test p-value ≤ 0.05). Pairwise network centrality distribution comparison significance was achieved by (Welch's t-test ≤ 0.05). Correlation between two network centralities was calculated by R (v4.0.3). The top 5% nodes with high centrality were selected as important protein candidates. The cutoff for proteins lying between the inner layers was within the 90th percentile of the shell. Centrality enrichment significance was tested by hypergeometric test p-value ≤ 0.05. Standard cutoffs (p-value ≤ 0.05; log2fc ≥ |1|) were used to identify DEMs in the miRNAome dataset.

Claims

1. A composition for treating or preventing an inflammatory skin disorder in a subject having or at risk of developing such a disorder, comprising a drug that inhibits NK cell activity.

2. The composition according to claim 1, wherein the inflammatory skin disorder is hidradenitis suppurativa (HS).

3. The composition according to claim 1 or 2, wherein the agent that inhibits NK cell activity blocks the interaction between CD2 and CD58.

4. The composition according to claim 3, wherein the agent that blocks the interaction between CD2 and CD58 is an anti-CD2 agent.

5. The composition according to claim 4, wherein the anti-CD2 agent is an anti-CD2 antibody.

6. The composition according to claim 5, wherein the anti-CD2 antibody is cyprizumab.

7. The composition according to claim 3, wherein the agent that blocks the interaction between CD2 and CD58 is an anti-CD58 agent.

8. The composition according to claim 7, wherein the anti-CD58 agent is an anti-CD58 antibody.

9. The composition according to claim 1, characterized in that the composition is administered in combination with an anti-IL-15 agent, an anti-IL-18 agent, and / or an antifibrotic agent.

10. The composition according to claim 1, wherein the inflammatory skin disorder of the subject is refractory to one or more prior treatments for the inflammatory skin disorder, and the prior treatments were not agents that block the interaction between CD2 and CD58.

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