Background
Alopecia areata (AA) is one of the most common T cell-mediated autoimmune skin diseases, leading to chronic and relapsing hair loss. AA affects both children and adults of all ages and on hairs of all colors, with a prevalence rate at 2% of the overall population without gender predilection [
1]. Clinical evidence supports a high prevalence of comorbid autoimmune conditions among individuals with AA, such as thyroid disease, type 1 diabetes mellitus, inflammatory bowel disease, and systemic lupus erythematosus [
1,
2]. The quality of life in AA patients has been significantly affected by the disappointing outcomes, side effects, and relapses with current conventional therapies, including topical and systematic applications of immunosuppressive regimens (such as corticosteroids and cyclosporine) or immune modulators (e.g., dithranol and diphenylcyclopropenone (DPCP)) [
1,
3]. To overcome these challenges, an innovative and translational technology is necessary to advance the current management of AA. Ideally, this clinical approach should address multiple or all of the underlying causes of autoimmunity in AA. However, similar to all other autoimmune diseases, possible triggers for autoimmunity in AA include genetic, epigenetic, physical, emotional, social, and environmental factors. These complicated factors may act independently or jointly to break down the “immune privilege” of hair follicles through different molecular and cellular mechanisms, resulting in the autoimmune destruction of hair follicles by multiple immune cells, such as CD4
+ and/or CD8
+ T cells and natural killer (NK) cells [
1,
4-
7]. Thus, a comprehensive approach is needed to fundamentally restore the immune privilege of hair follicles and address these multiple immune dysfunctions resulting from a variety of etiological causes.
Hair follicles are normally immune privileged sites, similar to other organ tissue systems, such as the brain, eye, and testis, and they contribute to the regulation of homeostasis through the neuroendocrine-immune network [
8,
9]. Under physiological conditions, the maintenance of the immune privilege status may include the following potential mechanisms: a low expression or absence of major histocompatibility complex (MHC) class I antigens and MHC class I chain-related A (MICA) molecules; a presence of functionally impaired Langerhans cells; and local expression of potent immunosuppressants (for example, transforming growth factor beta 1 (TGF-β1) and α-melanocyte stimulating hormone (MSH)) [
5-
7,
10]. It is well recognized that collapse of hair follicle immune privilege leads to the onset of AA [
7,
11-
14]. Due to the complexity of AA-related autoimmune responses and the similarity with other autoimmune diseases, clinical therapies and trials that only target one or a few components of the autoimmune responses are likely to fail, as has been observed in recent clinical trials for type 1 diabetes [
15-
17]. Successful immune therapies will likely restore the immune balance and peripheral tolerance by a comprehensive modulation within the entire human immune system.
We have previously characterized a novel type of stem cell from human umbilical cord blood, designated a cord blood-derived multipotent stem cell (CB-SC) [
18,
19]. CB-SCs are phenotypically and functionally different from other types of stem cells [
20], including hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs), endothelial progenitor cells (EPCs), and monocyte-derived stem cells [
21,
22]. Preclinical work demonstrated the immune modulation capability of CB-SCs in autoimmune-caused diabetic non-obese (NOD) mice [
23] as well as with autoreactive human T cells from type 1 diabetic patients [
19]. Recently, we reported on the development of the Stem Cell Educator therapy utilizing cultured CB-SCs in clinical trials for both type 1 and type 2 diabetes [
20,
24,
25]. Clinical data demonstrated that a single treatment with the Stem Cell Educator provided lasting reversal of autoimmunity and a rebalance of immune responses that allowed regeneration of islet β cells and improvement of metabolic control in subjects with long-standing type 1 diabetes [
20,
24]. Additionally, a phase 1/2 clinical study demonstrated that Stem Cell Educator therapy can control immune dysfunction and restore the immune balance through the modulation of monocytes/macrophages, leading to a long-lasting improvement of insulin sensitivity and metabolic control in long-standing type 2 diabetic patients [
25]. The combined preclinical and early clinical data [
19,
20,
23-
26] raise the intriguing possibility that the Stem Cell Educator therapy may also be useful in overcoming the autoimmunity involved in AA. Here, we explore the therapeutic potential of Stem Cell Educator therapy in AA subjects.
Discussion
AA is a devastating autoimmune disease that affects patients’ daily lives. Immune dysfunction of AA subjects is complicated, not only localizing in hair follicles, but also having effects outside of hair follicles with the development of other autoimmune diseases. Overcoming the autoimmunity represents one of the key hurdles in the treatment of AA. Systematic applications of immunosuppressive regimens usually yield significant side effects. Localized therapies have been widely utilized in clinic, including intralesional injections of glucocorticoids and the use of topical sensitizers through the induction of contact allergy (for example, dithranol and diphenylcyclopropenone), as well as topical corticosteroids and minoxidil [
1,
3,
39]. To date, although a multitude of therapeutic options exist, neither local treatments nor systematic approaches can provide a cure for AA subjects [
1,
39]. Current clinical proof-of-concept data reveal the safety and efficacy of the Stem Cell Educator therapy approach in the treatment of AA subjects, as demonstrated by clinical outcomes in hair regrowth. This finding opens up a new avenue for AA clinical treatment by using the comprehensive immune modulation induced by Stem Cell Educator therapy. Because this disorder affects children of all hair colors [
1], it is important to highlight that pediatric apheresis presents unique challenges due to children’s low body weight (<40 kg) and height (<140 cm) and the difficulties in vascular access and clinical monitoring. To overcome these technical hurdles and improve the safety in pediatric apheresis, alternative approaches should be considered for the treatment of pediatric AA subjects such as the application of a different apheresis machine with low extracorporeal blood volume, blood priming, and femoral vein catheterization.
AA is characterized as a T cell-mediated autoimmune disease, and CD8
+ T cells seem to dominate the response. They may recognize the MHC class I-restricted melanogenesis-associated autoantigens and/or anagen-associated hair follicle autoantigens and thereby mediate the destruction of hair follicles [
1,
14]. More recently, CD8β
+NKG2D
+ T cells have been characterized as a major player leading to the autoimmune destruction of hair follicles [
27]. Therefore, it is essential to attenuate these effector CD8
+ T cells through the induction of peripheral immune tolerance to these self-antigens. Notably, we found that co-culture with CB-SCs could suppress the proliferation of activated CD8β
+NKG2D
+ T cells and reduce their percentage. Up-regulation of the expression of coinhibitory molecules BTLA and PD-1 on CD8β
+NKG2D
+ T cells may further attenuate their cytotoxic effects. The current study confirmed the expression of BTLA ligand HVEM on CB-SCs. Previous work demonstrated that the strong expression of programmed death-1 ligand (PD-L1) on CB-SCs contributed to the immune modulation of CD8 T cells [
28]. Thus, CB-SCs may directly modulate CD8β
+NKG2D
+ T cells through the PD-1/PD-L1 and BTLA/HVEM pathways.
Additionally, CB-SCs strongly express the autoimmune regulator (Aire) [
24] transcription factor. Aire proteins are usually found in thymic medullary epithelial cells, which play a central role in T cell development and the induction of immune tolerance by mediating ectopic expression of peripheral self-antigens and mediating the deletion of autoreactive T cells [
40,
41]. Knockdown of Aire protein expression resulted in a reduction of PD-L1 expression on CB-SCs. Thus, we hypothesize that, in a way, the Stem Cell Educator therapy may function as “an artificial thymus” that circulates a patient’s blood through a blood cell separator [
20], briefly allows interactions between T cells and other immune cells with CB-SCs
in vitro, induces immune tolerance through the actions of Aire [
24], expression of PD-L1 and HVEM, the release of soluble factors (nitric oxide and TGF-β1), and cell-cell contacting mechanisms [
20,
28], returns the educated autologous lymphocytes to the patient’s circulation, and achieves immune balance and homeostasis in these AA subjects. Of interest, apheresis only withdraws approximately 10-15% of all lymphocytes, and since many pathogenic immune cells likely remain in the hair follicles and the connective tissue sheath which fail to enter into the blood circulation during the apheresis procedure, many autoimmune cells escape direct interaction with the CB-SCs. This would suggest that some of the cells altered by direct encounter with CB-SC can spread the tolerance systemically. Additionally, due to the short life span of most lymphocytes, subjects with severe AA may need additional treatments, perhaps at 3- to 6-month intervals, to improve the efficacy and possibly prevent the relapse of disease. To improve the clinical efficacy of Stem Cell Educator therapy, the sooner treatment with this therapy begins after diagnosis, the higher the chance of rescuing hair follicles and finding a cure for AA.
Animal and clinical studies demonstrated that both CD4
+ Th1 cells and CD8
+ T cells are required for the pathogenesis of AA [
1,
4,
42]. Up-regulation of Th1 cytokines in AA subjects, not Th2 cytokines, exacerbates the autoimmune destruction [
31,
43,
44]. Kubo and colleagues reported that there was a positive correlation between the severity of the alopecia and the increase of Th1 cells, inversely proportional to the number of IL-4-producing Th2 cells [
45]. Therefore, the promotion of Th2 immune responses has been proposed to be beneficial for the treatment of AA patients [
1,
4]. The current study demonstrated that Th2 response-associated cytokines such as IL-4, IL-5, and IL-13 in these AA subjects were markedly increased after receiving Stem Cell Educator therapy. Additionally, CD28, one of the major costimulatory molecules contributing to the differentiation of Th2 cells [
33-
36], was up-regulated after Stem Cell Educator therapy. Thus, the combination of CD28 + IL-4 can provide key signals facilitating naïve or memory CD4
+ T cells and giving rise to Th2 cells via the activation of mitogen-activated protein (MAP) kinase and extracellular signal-regulated kinase (ERK) signaling pathways [
36]. Consequently, these Th2 cells and their cytokines may antagonize Th1 cell functions and counterbalance their AA-related autoimmune responses [
32].
Collapse of immune privilege in hair follicles is the major cause of pathogenesis in AA. Due to constitutively low or absent expression of MHC class I antigen in the proximal hair follicle epithelium, hair follicles may initially be attacked by NK cells through NK cell function-activating receptors NKG2D and NKG2C [
5,
46]. Thus, attenuating NK cells is also necessary to reestablish the immune privilege and fundamentally advance the clinical outcomes for the treatment of AA. Notably, current data provide evidence for the up-regulation of TGF-β1 production in peripheral blood mononuclear cells, as well as the formation of a “ring of TGF-β1” in hair follicles of AA subjects after receiving Stem Cell Educator therapy. TGF-β1 can significantly suppress the proliferation and activity of NK cells [
47], in addition to its effects on CD4
+ and CD8
+ T cells [
29]. Thus, TGF-β1 may play a key role in the restoration of immune privilege of hair follicles and the induction of local immune tolerance.
It is also well known that TGF-β1 is a pleiotropic growth factor that plays a key role in the production and remodeling of the extracellular matrix [
48]. Animal studies show that TGF-β1 acts as an essential factor contributing to the regulation of cycling and remodeling of hair follicles via the inhibition of keratinocyte proliferation and induction of apoptosis [
30,
37,
38], as well as one of the key niche factors that regulate melanocyte stem cell immaturity and quiescence in the bulge area of hair follicles [
49]. Previous work demonstrated that the formation of a “ring of TGF-β1” around pancreatic islets may protect the newly regenerated islet β cells against infiltrating lymphocytes and macrophages [
23], providing a safe environment for promotion of regeneration of pancreatic islet β cells in long-standing type 1 diabetic patients [
20,
24]. Thus, the formation of a “ring of TGF-β1” may not only protect hair follicles through the restoration of immune privilege, but may also lead to the activation of epithelial hair follicle stem cells and hair regrowth. Additional molecular and cellular mechanisms underlying the Stem Cell Educator therapy in humans can be further explored by studying easily accessible and abundant hair follicles. Thus, clinical success in AA by the Stem Cell Educator therapy approach may open up new avenues for the treatment of other autoimmune diseases.
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Competing interests
Dr. Zhao (YZ), inventor of this technology, led the clinical study, and has an investment and a fiduciary role in Tianhe Stem Cell Biotechnologies Inc. (licensed this technology from the University of Illinois at Chicago). YeZ, WL, SW, JS, and YuL are employees of Tianhe Stem Cell Biotechnologies Inc. who might have an interest in the submitted work. All other authors (YaL, BY, HW, HL, QL, DZ, YC, JZ, and EG) have no financial interests that may be relevant to the submitted work.
Authors’ contributions
YZ and YaL designed the trial and analyzed the data. YZ drafted the manuscript and obtained the funding. HW and HL collected data. BY, QL, DZ, YC, YeZ, WL, SW, JS, JZ, YuL, and EG provided administrative, technical, or material support. All authors had full access to all the data and take responsibility for the integrity of the data and the accuracy of the data analysis. All authors read and approved the final manuscript.