Functional interrogation of lymphocyte subsets in alopecia areata using single-cell RNA sequencing.

Alopecia areata (AA) is among the most prevalent autoimmune diseases, but the development of innovative therapeutic strategies has lagged due to an incomplete understanding of the immunological underpinnings of disease. Here, we performed single-cell RNA sequencing (scRNAseq) of skin-infiltrating immune cells from the graft-induced C3H/HeJ mouse model of AA, coupled with antibody-based depletion to interrogate the functional role of specific cell types in AA in vivo. Since AA is predominantly T cell-mediated, we focused on dissecting lymphocyte function in AA. Both our scRNAseq and functional studies established CD8+ T cells as the primary disease-driving cell type in AA. Only the depletion of CD8+ T cells, but not CD4+ T cells, NK, B, or γδ T cells, was sufficient to prevent and reverse AA. Selective depletion of regulatory T cells (Treg) showed that Treg are protective against AA in C3H/HeJ mice, suggesting that failure of Treg-mediated immunosuppression is not a major disease mechanism in AA. Focused analyses of CD8+ T cells revealed five subsets, whose heterogeneity is defined by an "effectorness gradient" of interrelated transcriptional states that culminate in increased effector function and tissue residency. scRNAseq of human AA skin showed that CD8+ T cells in human AA follow a similar trajectory, underscoring that shared mechanisms drive disease in both murine and human AA. Our study represents a comprehensive, systematic interrogation of lymphocyte heterogeneity in AA and uncovers a novel framework for AA-associated CD8+ T cells with implications for the design of future therapeutics.

Novel therapies for alopecia areata: The era of rational drug development.

Treatments for alopecia areata (AA) have evolved over the decades from broad and nonspecific therapies to those that are now more targeted and rationally selected. This was achieved by means of close cooperation and communication between clinicians and basic scientists, which resulted in the elucidation and understanding of the unique pathophysiology of AA. In this review we discuss this evolution and how novel therapies for AA have changed over the decades, what we have in our current arsenal of drugs for this potentially devastating disease, and what the future holds.

CXCR3 Blockade Inhibits T Cell Migration into the Skin and Prevents Development of Alopecia Areata.

Alopecia areata (AA) is an autoimmune disease of the hair follicle that results in hair loss of varying severity. Recently, we showed that IFN-γ-producing NKG2D(+)CD8(+) T cells actively infiltrate the hair follicle and are responsible for its destruction in C3H/HeJ AA mice. Our transcriptional profiling of human and mouse alopecic skin showed that the IFN pathway is the dominant signaling pathway involved in AA. We showed that IFN-inducible chemokines (CXCL9/10/11) are markedly upregulated in the skin of AA lesions, and further, that the IFN-inducible chemokine receptor, CXCR3, is upregulated on alopecic effector T cells. To demonstrate whether CXCL9/10/11 chemokines were required for development of AA, we treated mice with blocking Abs to CXCR3, which prevented the development of AA in the graft model, inhibiting the accumulation of NKG2D(+)CD8(+) T cells in the skin and cutaneous lymph nodes. These data demonstrate proof of concept that interfering with the Tc1 response in AA via blockade of IFN-inducible chemokines can prevent the onset of AA. CXCR3 blockade could be approached clinically in human AA with either biologic or small-molecule inhibition, the latter being particularly intriguing as a topical therapeutic.

Expression, signaling proficiency, and stimulatory function of the NKG2D lymphocyte receptor in human cancer cells.

The stimulatory natural killer group 2 member D (NKG2D) lymphocyte receptor and its tumor-associated ligands are important mediators in the immune surveillance of cancer. With advanced human tumors, however, persistent NKG2D ligand expression may favor tumor progression. We have found that cancer cells themselves express NKG2D in complex with the DNAX-activating protein 10 (DAP10) signaling adaptor. Triggering of NKG2D on ex vivo cancer cells or on tumor lines which express only few receptor complexes activates the oncogenic PI3K-protein kinase B (PKB/AKT)-mammalian target of rapamycin (mTOR) signaling axis and downstream effectors, the ribosomal protein S6 kinase 1 (S6K1) and the translation initiation factor 4E-binding protein 1 (4E-BP1). In addition, as in lymphocytes, NKG2D ligand engagement stimulates phosphorylation of JNK and ERK in MAP kinase cascades. Consistent with these signaling activities, above-threshold expression of NKG2D-DAP10 in a ligand-bearing tumor line increases its bioenergetic metabolism and proliferation, thus suggesting functional similarity between this immunoreceptor and tumor growth factor receptors. This relationship is supported by significant correlations between percentages of cancer cells that are positive for surface NKG2D and criteria of tumor progression. Hence, in a conceptual twist, these results suggest that tumor co-option of NKG2D immunoreceptor expression may complement the presence of its ligands for stimulation of tumor growth.

Disulphide-isomerase-enabled shedding of tumour-associated NKG2D ligands.

Tumour-associated ligands of the activating NKG2D (natural killer group 2, member D; also called KLRK1) receptor-which are induced by genotoxic or cellular stress-trigger activation of natural killer cells and co-stimulation of effector T cells, and may thus promote resistance to cancer. However, many progressing tumours in humans counter this anti-tumour activity by shedding the soluble major histocompatibility complex class-I-related ligand MICA, which induces internalization and degradation of NKG2D and stimulates population expansions of normally rare NKG2D+CD4+ T cells with negative regulatory functions. Here we show that on the surface of tumour cells, MICA associates with endoplasmic reticulum protein 5 (ERp5; also called PDIA6 or P5), which, similar to protein disulphide isomerase, usually assists in the folding of nascent proteins inside cells. Pharmacological inhibition of thioreductase activity and ERp5 gene silencing revealed that cell-surface ERp5 function is required for MICA shedding. ERp5 and membrane-anchored MICA form transitory mixed disulphide complexes from which soluble MICA is released after proteolytic cleavage near the cell membrane. Reduction of the seemingly inaccessible disulphide bond in the membrane-proximal alpha3 domain of MICA must involve a large conformational change that enables proteolytic cleavage. These results uncover a molecular mechanism whereby domain-specific deconstruction regulates MICA protein shedding, thereby promoting tumour immune evasion, and identify surface ERp5 as a strategic target for therapeutic intervention.

NKG2D initiates caspase-mediated CD3zeta degradation and lymphocyte receptor impairments associated with human cancer and autoimmune disease.

Deficiencies of the T cell and NK cell CD3ζ signaling adapter protein in patients with cancer and autoimmune diseases are well documented, but mechanistic explanations are fragmentary. The stimulatory NKG2D receptor on T and NK cells mediates tumor immunity but can also promote local and systemic immune suppression in conditions of persistent NKG2D ligand induction that include cancer and certain autoimmune diseases. In this paper, we provide evidence that establishes a causative link between CD3ζ impairment and chronic NKG2D stimulation due to pathological ligand expression. We describe a mechanism whereby NKG2D signaling in human T and NK cells initiates Fas ligand/Fas-mediated caspase-3/-7 activation and resultant CD3ζ degradation. As a consequence, the functional capacities of the TCR, the low-affinity Fc receptor for IgG, and the NKp30 and NKp46 natural cytotoxicity receptors, which all signal through CD3ζ, are impaired. These findings are extended to ex vivo phenotypes of T and NK cells among tumor-infiltrating lymphocytes and in peripheral blood from patients with juvenile-onset lupus. Collectively, these results indicate that pathological NKG2D ligand expression leads to simultaneous impairment of multiple CD3ζ-dependent receptor functions, thus offering an explanation that may be applicable to CD3ζ deficiencies associated with diverse disease conditions.

Induction of T cell exhaustion by JAK1/3 inhibition in the treatment of alopecia areata.

Alopecia areata (AA) is an autoimmune disease caused by T cell-mediated destruction of the hair follicle (HF). Therefore, approaches that effectively disrupt pathogenic T cell responses are predicted to have therapeutic benefit for AA treatment. T cells rely on the duality of T cell receptor (TCR) and gamma chain (γc) cytokine signaling for their development, activation, and peripheral homeostasis. Ifidancitinib is a potent and selective next-generation JAK1/3 inhibitor predicted to disrupt γc cytokine signaling. We found that Ifidancitinib robustly induced hair regrowth in AA-affected C3H/HeJ mice when fed with Ifidancitinib in chow diets. Skin taken from Ifidancitinib-treated mice showed significantly decreased AA-associated inflammation. CD44+CD62L- CD8+ T effector/memory cells, which are associated with the pathogenesis of AA, were significantly decreased in the peripheral lymphoid organs in Ifidancitinib-treated mice. We observed high expression of co-inhibitory receptors PD-1 on effector/memory CD8+ T cells, together with decreased IFN-γ production in Ifidancitinib-treated mice. Furthermore, we found that γc cytokines regulated T cell exhaustion. Taken together, our data indicate that selective induction of T cell exhaustion using a JAK inhibitor may offer a mechanistic explanation for the success of this treatment strategy in the reversal of autoimmune diseases such as AA.

Regulatory network analysis defines unique drug mechanisms of action and facilitates patient-drug matching in alopecia areata clinical trials.

Not all therapeutics are created equal in regards to individual patients. The problem of identifying which compound will work best with which patient is a significant burden across all disease contexts. In the context of autoimmune diseases such as alopecia areata, several formulations of JAK/STAT inhibitors have demonstrated efficacy in clinical trials. All of these compounds demonstrate different rates of response, and here we observed that this coincided with different molecular effects on patients undergoing treatment. Using these data, we have developed a computational model that is capable of predicting which patient-drug pairs have the highest likelihood of response. We achieved this by integrating inferred mechanism of action data and gene regulatory networks derived from an independent patient cohort with baseline patient data prior to beginning treatment.

Selective inhibition of JAK3 signaling is sufficient to reverse alopecia areata.

The Janus kinase/signal transducers and activators of transcription (JAK/STAT) are key intracellular mediators in the signal transduction of many cytokines and growth factors. Common γ chain cytokines and interferon-γ that use the JAK/STAT pathway to induce biological responses have been implicated in the pathogenesis of alopecia areata (AA), a T cell-mediated autoimmune disease of the hair follicle. We previously showed that therapeutic targeting of JAK/STAT pathways using the first-generation JAK1/2 inhibitor, ruxolitinib, and the pan-JAK inhibitor, tofacitinib, was highly effective in the treatment of human AA, as well as prevention and reversal of AA in the C3H/HeJ mouse model. To better define the role of individual JAKs in the pathogenesis of AA, in this study, we tested and compared the efficacy of several next-generation JAK-selective inhibitors in the C3H/HeJ mouse model of AA, using both systemic and topical delivery. We found that JAK1-selective inhibitors as well as JAK3-selective inhibitors robustly induced hair regrowth and decreased AA-associated inflammation, whereas several JAK2-selective inhibitors failed to restore hair growth in treated C3H/HeJ mice with AA. Unlike JAK1, which is broadly expressed in many tissues, JAK3 expression is largely restricted to hematopoietic cells. Our study demonstrates inhibiting JAK3 signaling is sufficient to prevent and reverse disease in the preclinical model of AA.

Blockade of IL-7 signaling suppresses inflammatory responses and reverses alopecia areata in C3H/HeJ mice.

The interleukin-7 (IL-7) signaling pathway plays an important role in regulation of T cell function and survival. We detected overexpression of IL-7 in lesional skin from both humans and C3H/HeJ mice with alopecia areata (AA), a T cell-mediated autoimmune disease of the hair follicle. We found that exogenous IL-7 accelerated the onset of AA by augmenting the expansion of alopecic T cells. Conversely, blockade of IL-7 stopped the progression of AA and reversed early AA in C3H/HeJ mice. Mechanistically, we observed that IL-7Rα blockade substantially reduced the total number of most T cell subsets, but relative sparing of regulatory T cells (Tregs). We postulated that short-term anti-IL-7Rα treatment in combination with a low dose of Treg-tropic cytokines might improve therapeutic efficacy in AA. We demonstrated that short-term IL-7Rα blockade in combination with low doses of Treg-tropic cytokines enhanced therapeutic effects in the treatment of AA, and invite further clinical investigation.

A Subset of TREM2+ Dermal Macrophages Secretes Oncostatin M to Maintain Hair Follicle Stem Cell Quiescence and Inhibit Hair Growth.

Hair growth can be induced from resting mouse hair follicles by topical application of JAK inhibitors, suggesting that JAK-STAT signaling is required for maintaining hair follicle stem cells (HFSCs) in a quiescent state. Here, we show that Oncostatin M (OSM), an IL-6 family cytokine, negatively regulates hair growth by signaling through JAK-STAT5 to maintain HFSC quiescence. Genetic deletion of the OSM receptor or STAT5 can induce premature HFSC activation, suggesting that the resting telogen stage is actively maintained by the hair follicle niche. Single-cell RNA sequencing revealed that the OSM source is not intrinsic to the hair follicle itself and is instead a subset of TREM2+ macrophages that is enriched within the resting follicle and deceases immediately prior to HFSC activation. In vivo inhibition of macrophage function was sufficient to induce HFSC proliferation and hair cycle induction. Together these results clarify how JAK-STAT signaling actively inhibits hair growth.

High-throughput T cell receptor sequencing identifies clonally expanded CD8+ T cell populations in alopecia areata.

Alopecia areata (AA) is an autoimmune disease in which cytotoxic T cells specifically target growing hair follicles. We used high-throughput TCR sequencing in the C3H/HeJ mouse model of AA and in human AA patients to gain insight into pathogenic T cell populations and their dynamics, which revealed clonal CD8+ T cell expansions in lesional skin. In the C3H/HeJ model, we observed interindividual sharing of TCRß chain protein sequences, which strongly supports a model of antigenic drive in AA. The overlap between the lesional TCR repertoire and a population of CD8+NKG2D+ T cells in skin-draining lymph nodes identified this subset as pathogenic effectors. In AA patients, treatment with the oral JAK inhibitor tofacitinib resulted in a decrease in clonally expanded CD8+ T cells in the scalp but also revealed that many expanded lesional T cell clones do not completely disappear from either skin or blood during treatment with tofacitinib, which may explain in part the relapse of disease after stopping treatment.

Pharmacologic inhibition of JAK-STAT signaling promotes hair growth.

Several forms of hair loss in humans are characterized by the inability of hair follicles to enter the growth phase (anagen) of the hair cycle after being arrested in the resting phase (telogen). Current pharmacologic therapies have been largely unsuccessful in targeting pathways that can be selectively modulated to induce entry into anagen. We show that topical treatment of mouse and human skin with small-molecule inhibitors of the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway results in rapid onset of anagen and subsequent hair growth. We show that JAK inhibition regulates the activation of key hair follicle populations such as the hair germ and improves the inductivity of cultured human dermal papilla cells by controlling a molecular signature enriched in intact, fully inductive dermal papillae. Our findings open new avenues for exploration of JAK-STAT inhibition for promotion of hair growth and highlight the role of this pathway in regulating the activation of hair follicle stem cells.

Reversal of Alopecia Areata Following Treatment With the JAK1/2 Inhibitor Baricitinib.

BACKGROUND: Alopecia areata (AA) is an autoimmune disease resulting in hair loss with devastating psychosocial consequences. Despite its high prevalence, there are no FDA-approved treatments for AA. Prior studies have identified a prominent interferon signature in AA, which signals through JAK molecules. METHODS: A patient with AA was enrolled in a clinical trial to examine the efficacy of baricitinib, a JAK1/2 inhibitor, to treat concomitant CANDLE syndrome. In vivo, preclinical studies were conducted using the C3H/HeJ AA mouse model to assess the mechanism of clinical improvement by baricitinib. FINDINGS: The patient exhibited a striking improvement of his AA on baricitinib over several months. In vivo studies using the C3H/HeJ mouse model demonstrated a strong correlation between resolution of the interferon signature and clinical improvement during baricitinib treatment. INTERPRETATION: Baricitinib may be an effective treatment for AA and warrants further investigation in clinical trials.

[A pilot study of T cell vaccination in systemic lupus erythematosus].

OBJECTIVE: Autoreactive T cells play a critical role in pathogenesis of systemic lupus erythematosus (SLE). Immunization with inactivated autoreactive T cells (T cell vaccination) may activate the idiotype anti-idiotypic network to deplete specific subsets of autoreactive T cells involved in SLE. We conducted a pilot clinical trial of T cell vaccination to investigate the efficiency and safety of T cell vaccination in treatment of SLE. METHODS: Autoreactive T cell clones were derived from peripheral blood mononuclear cells of 6 SLE patients. After irradiated with 80 Gy gamma radiation, 1 x 10(7) T cells were inoculated subcutaneously at 0, 2, 6, 8 week respectively. The patients were followed up for 20-27 months, and monitored for clinical characteristics and side effects from the vaccination. RESULTS: The clinical manifestations and laboratory abnormalities were improved after inoculation without increasing the dose of corticosteroids and immunosuppressants in most patients. SLEDAI score were decreased remarkably. Proliferative responses against the T cell vaccine were observed in 4/6 patients. No side effect was noticed and CD3+, CD4+ and CD8+ T cell were all in normal ranges after the vaccination and during the follow-up period. CONCLUSION: The results of this pilot study indicate that T cell vaccination is a safe and effective treatment in SLE patients.

Autonomous stimulation of cancer cell plasticity by the human NKG2D lymphocyte receptor coexpressed with its ligands on cancer cells.

The stimulatory NKG2D receptor on lymphocytes promotes tumor immune surveillance by targeting ligands selectively induced on cancer cells. Progressing tumors counteract by employing tactics to disable lymphocyte NKG2D. This negative dynamic is escalated as some human cancer cells co-opt expression of NKG2D, thereby complementing the presence of its ligands for autonomous stimulation of oncogenic signaling. Clinical association data imply relationships between cancer cell NKG2D and metastatic disease. Here we show that NKG2D promotes cancer cell plasticity by induction of phenotypic, molecular, and functional signatures diagnostic of the epithelial-mesenchymal transition, and of stem-like traits via induction of Sox9, a key transcriptional regulator of breast stem cell maintenance. These findings obtained with model breast tumor lines and xenotransplants were recapitulated by ex vivo cancer cells from primary invasive breast carcinomas. Thus, NKG2D may have the capacity to drive high malignancy traits underlying metastatic disease.

Alopecia areata is driven by cytotoxic T lymphocytes and is reversed by JAK inhibition.

Alopecia areata (AA) is a common autoimmune disease resulting from damage of the hair follicle by T cells. The immune pathways required for autoreactive T cell activation in AA are not defined limiting clinical development of rational targeted therapies. Genome-wide association studies (GWAS) implicated ligands for the NKG2D receptor (product of the KLRK1 gene) in disease pathogenesis. Here, we show that cytotoxic CD8(+)NKG2D(+) T cells are both necessary and sufficient for the induction of AA in mouse models of disease. Global transcriptional profiling of mouse and human AA skin revealed gene expression signatures indicative of cytotoxic T cell infiltration, an interferon-γ (IFN-γ) response and upregulation of several γ-chain (γc) cytokines known to promote the activation and survival of IFN-γ-producing CD8(+)NKG2D(+) effector T cells. Therapeutically, antibody-mediated blockade of IFN-γ, interleukin-2 (IL-2) or interleukin-15 receptor ß (IL-15Rß) prevented disease development, reducing the accumulation of CD8(+)NKG2D(+) T cells in the skin and the dermal IFN response in a mouse model of AA. Systemically administered pharmacological inhibitors of Janus kinase (JAK) family protein tyrosine kinases, downstream effectors of the IFN-γ and γc cytokine receptors, eliminated the IFN signature and prevented the development of AA, while topical administration promoted hair regrowth and reversed established disease. Notably, three patients treated with oral ruxolitinib, an inhibitor of JAK1 and JAK2, achieved near-complete hair regrowth within 5 months of treatment, suggesting the potential clinical utility of JAK inhibition in human AA.

Cryopreservation of tissue-engineered epithelial sheets in trehalose.

Tissue-engineered epidermal membranes are useful for clinical wound healing. To facilitate these products in the clinic, optimized storage methods need to be developed. We studied the efficiency of extracellular trehalose at various concentrations for cryopreserving human tissue-engineered epidermal membranes compared with that of dimethyl-sulfoxide (DMSO) used by most organ banks for cryopreserving skin grafts and artificial skin substitutes. Keratinocyte (KC) viability, proliferation and marker expression following cryopreservation in trehalose were examined with similar results to those using DMSO. Trehalose concentration (0.4m) was optimized according to the described cellular activities following cryopreservation. Artificial epidermal substitutes were then cryopreserved in trehalose at the optimized concentration. Cell viability, growth factor secretion and wound healing properties of cryopreserved artificial epidermal substitutes using nude mice were examined and compared with those of DMSO cryopreservation. Cryopreservation with trehalose enhanced human KC viability in suspension and artificial skin substitutes. In addition, trehalose cryopreservation provided fast recovery of EGF and TGF-ß1 secretion by KCs after thawing. When transplanted into nude mice, trehalose-cryopreserved artificial skin repaired skin defects in a similar manner to that of a non-cryopreserved control. Moreover, trehalose-cryopreserved artificial skin resulted in engraftment and wound closure that was significantly enhanced compared with that of DMSO-cryopreserved epidermal membranes. The results indicate that the use of trehalose improves cryopreservation of tissue-engineered epithelial sheets.

Normally occurring NKG2D+CD4+ T cells are immunosuppressive and inversely correlated with disease activity in juvenile-onset lupus.

The NKG2D receptor stimulates natural killer cell and T cell responses upon engagement of ligands associated with malignancies and certain autoimmune diseases. However, conditions of persistent NKG2D ligand expression can lead to immunosuppression. In cancer patients, tumor expression and shedding of the MHC class I-related chain A (MICA) ligand of NKG2D drives proliferative expansions of NKG2D(+)CD4(+) T cells that produce interleukin-10 (IL-10) and transforming growth factor-beta, as well as Fas ligand, which inhibits bystander T cell proliferation in vitro. Here, we show that increased frequencies of functionally equivalent NKG2D(+)CD4(+) T cells are inversely correlated with disease activity in juvenile-onset systemic lupus erythematosus (SLE), suggesting that these T cells may have regulatory effects. The NKG2D(+)CD4(+) T cells correspond to a normally occurring small CD4 T cell subset that is autoreactive, primed to produce IL-10, and clearly distinct from proinflammatory and cytolytic CD4 T cells with cytokine-induced NKG2D expression that occur in rheumatoid arthritis and Crohn's disease. As classical regulatory T cell functions are typically impaired in SLE, it may be clinically significant that the immunosuppressive NKG2D(+)CD4(+) T cells appear functionally uncompromised in this disease.

Fas-ligand-mediated paracrine T cell regulation by the receptor NKG2D in tumor immunity.

Tumor-associated ligands of the activating NKG2D receptor can effectively stimulate T cell responses at early but not late stages of tumor growth. In late-stage human tumor settings, we observed MIC-driven proliferation of NKG2D(+)CD4(+) T cells that produced the cytokine Fas ligand (FasL) as a result of NKG2D costimulation but were themselves protected from Fas-mediated growth arrest. In contrast, FasL suppressed proliferation of T cells in vitro that did not receive NKG2D costimulation. Similar observations with normal peripheral blood NKG2D(+)CD8(+) T cells demonstrated unrecognized NKG2D-mediated immune functions, whereby FasL release promotes tumor cell death and NKG2D costimulation prolongs T cell survival. These effects, beneficial in conditions of limited NKG2D ligand expression, may be counterweighed when massive expression and shedding of MIC occurs, such as in some late-stage tumors, that causes sustained NKG2D costimulation and population expansion of immunosuppressive T cells.