Human regulatory γδT lymphocytes as novel autoimmunity-protective cells: Lessons from alopecia areata

doi:10.21203/rs.3.rs-5342172/v1

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Human regulatory γδT lymphocytes as novel autoimmunity-protective cells: Lessons from alopecia areata

https://doi.org/10.21203/rs.3.rs-5342172/v1

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Regulatory T cells control autoimmune diseases (AID). Yet, much less is known about the functions of evolutionarily much older Foxp3 + human regulatory γδT cells (γδTregs). Here, we have explored these functions in one of the most common human AID, the hair loss disorder, alopecia areata (AA).

Lesional AA skin showed significantly more γδTreg than non-lesional or healthy skin. Next, we investigated how human γδTregs impact on experimentally induced AA in human scalp skin xenotransplants on SCID/beige mice. PBMC-derived autologous γδTregs were pre-activated with IL-2, IL-15, and zoledronate in vitro and injected intradermally into human scalp xenografts before or after AA induction by autologous CD8 + T cells in vivo. γδTreg not only prevented the development of AA lesions, but also promoted hair regrowth in established AA lesions in the xenotransplants, accompanied by a reduced perifollicular lymphocytic infiltrate and restoration of hair follicle (HF) immune privilege (IP) .

We then co-cultured γδTregs with organ-cultured, stressed (MICA-overexpressing) human scalp HFs in the presence/absence of pathogenic CD8+/NKG2D + T cells that induce HF IP collapse by secreting interferon-g, all under autologous conditions. Under these ex vivo conditions, γδTregs mitigated HF IP collapse induced by CD8 + T cells, primarily through IL-10 and TGF-β1 secretion, enhanced HF keratinocyte proliferation and reduced their apoptosis while preventing premature catagen induction (= AA hallmarks).

These findings in a model human AID introduce human γδTregs as important regulatory lymphocytes that invite novel cell-based therapies in CD8 + T cell-dependent AIDs characterized by IP collapse such as AA.

CD4+/Foxp3 + regulatory T cells (Tregs) have long been appreciated as key immune cells that limit autoimmune responses, maintain immune homeostasis, and promote tissue repair [1–4]. As such, Tregs and their subpopulations have attracted increasing interest in the context of developing cell-based therapies for autoimmune diseases that expand, promote, and therapeutically exploit the potent immunoinhibitory functions of these cells [5–9]. Instead, much less is known about the physiological functions of – evolutionarily much older - regulatory γδT cells (γδTregs) [10, 11]. In particular, the functions of γδTregs in human tissue physiology and pathology remain poorly understood. The current study explores the latter by interrogating γδTregs in the context of one of the most common human autoimmune diseases, the inflammatory hair loss disorder, alopecia areata (AA) [12–14].

This model autoimmune disorder, afflicts growing (anagen) hair follicles (HFs) that have lost their physiological immune privilege (IP) and are being attacked by NKG2D + immune cells, leading to hair loss [12, 13, 15–20]. AA represents a stereotypic hair loss pattern rather than a single disease entity [13, 21–23]:a complete phenocopy of AA can be induced in healthy human scalp skin transplanted onto immunocompromised mice not only by autoreactive CD8 + T cells, but also by other autologous, NKG2D + and interferon-γ (IFNγ)-secreting, presumably non-autoantigen-specific innate/transitional immune cells such as NK cells [24, 25], ILC1-like cells [26], and Vδ1 + γδT cells [27, 28]. Moreover, all of these NKG2D + cell populations are greatly increased and secrete IFNγ around lesional HFs in the skin biopsies of AA patients, often already around non-lesional AA HFs, namely when the proximal HF epithelium overexpresses NKG2D-activating danger signals such as MICA/B, ULBP3, and/or CD1d [23, 25, 29–31]. Even CD8 + T cells can induce AA-like hair loss in mice via autoantigen-independent, but NKG2D-dependent innate mechanisms in mice [32].

Thus, it is important to distinguish at least two pathobiologically distinct variants of AA that share the same characteristic, clinically indistinguishable hair loss pattern [22, 23]: autoantigen-dependent and CD8 + T cell-driven autoimmune AA (AAA), which meets the modified Witebsky criteria of a classical autoimmune disease [33]; and non-autoantigen-specific, CD8 + T cell-independent “non-autoimmune AA” (NAIAA), which may eventually also acquire autoimmune features over time with increasing disease chronicity [13, 22, 23, 34]. Accepting that the human AA hair loss phenotype can be induced by these very different pathomechanisms, it is clinically paramount importance to systematically search for and identify endogenous regulatory mechanisms that are capable of suppressing both AAA and NAIAA and that can both prevent HF IP collapse and restore the HF’s immunoinhibitory milieu, thus protecting it from A relapse [13, 22, 23].

In this context, γδTCs and regulatory T cells (Tregs) are of particular interest. We have previously shown that the former serve as – typically non-autoantigen-specific - “danger recognition” cells in human skin that identify and respond to MICA/B- and/or CD1d-overexpressing, “stressed” HF epithelium and can induce collapse of the HFs physiological IP [20, 35] by secreting IFNγ [14, 27, 28]. Instead, Tregs may be capable of suppressing HF-targeting autoimmunity, namely in the C3H/HeJ mouse model of AA-like hair loss [36–38], even though it has recently been claimed that CD4+/Foxp3 + Tregs play no significant protective role in this mouse model [39] (whose dissimilarities human AA should not be ignored [40]. In mice, Tregs mediate IP in the bulge, the HF’s stem cell niche [41]. Tregs may well execute similar functions also in the human bulb [42, 43], i.e. the critical site of HF immunopathology in AA [13, 22, 42–48], possibly via both antigen- specific and -specific mechanisms [42, 49, 50]. Yet, the verdict on the role of Tregs in human AA is still out, since significant AA-suppressing functions have not yet been documented for Tregs in human AA [44, 51, 52].

Importantly, CD4 + Foxp3Treg cells are not the only regulatory T cells [53–56] since other Foxp3 + cells, namely γδTregs, also exhibit a potent immunoinhibitory phenotype in murine in vivo and human in vitro models [57–62]. Namely in various cancer, pregnancy, allergy and inflammation contexts, regulatory functions of γδTregs have now been documented [57–59, 63–76]. That peripheral γδTregs play a crucial role in maintaining immune balance is also suggested by their declining blood count in association with clinical autoimmune disease manifestations [69, 77, 78]. Yet, their roles in human tissue physiology and pathology remain quite obscure. Elucidating these appears to be important, since Vδ1+/NKG2D+, IFNγ-secreting γδTCs have recently been identified as pathogenic immune cells in AA, namely human NAIAA [27, 28] - a role that had previously been missed in both murine and human AA research. Moreover, human HFs and AA are much more accessible to experimental study than other human tissues and the autoimmune diseases that affect them, in which IP collapse also plays a key role (e.g., autoimmune uveitis [79], multiple sclerosis [80] and diabetes type I [81], and that highly instructive HF organ culture and humanized mouse models are available for preclinically interrogating human AA ex vivo and in vivo [15, 26, 27, 40, 82–85]. Therefore, AA is ideally suited as a model disease for dissecting the functions of γδTregs in human tissue pathology.

On this background, we have asked in the current study:

Do γδTregs, play a protective role in human AA pathobiology? If yes, by which mechanisms? And do autologous human γδTregs isolated from peripheral blood and expanded in vitro deserve consideration as a candidate cell-based therapy in the future management of human AA?

The latter question was further encouraged by our prior demonstration that two other human innate/transitional immune cell populations, i.e., regulatory NK cells [86] and a subset of IL-15-dependent inhibitory NKT cells (iNKT10) [84], are both AA- protective and -therapeutic and can restore a collapsed HF-IP in human scalp skin xenotransplants on immunocompromised mice [25, 26].Studies in cancer immunology have shown that γδTregs can exhibit more potent immunosuppressive activity than CD4 + Tregs [87].Moreover, autologous human γδTregs can be generated relatively easily in vitro from peripheral blood mononuclear cells (PBMCs) in the presence of zoledronate, a potent aminobisphosphonate that expands γδT cells [88, 89] and by co-stimulation with IL-2, IL-15, and TGF-β1 which promotes the development of γδTregs in vitro [90, 91].

In order to critically probe our working hypothesis that γδTregs may be AA protective, we first compared the number and perifollicular distribution of TCRVδ1+/Foxp3 + cells in lesional and non-lesional skin biopsies from AA patients with healthy human control skin. This was followed up in two instructive, mutually complementary preclinical human AA and HF immunobiology research models. In vivo, we used the humanized mouse model of AA, in which autologous PBMCs (pre-activated by high-dose IL-2) are enriched for CD8+/NKG2D + cells and injected intradermally into human skin xenotransplants on SCID/beige mice; this induces a complete phenocopy of human AA lesions in previously healthy human skin [24–26, 84]. Ex vivo, microdissected healthy human scalp HFs, in which IP collapse and restoration can be interrogated, were oran-cultured [27, 82, 92, 93]. These HFs were co-cultured with either autologous peripheral γδTCs or CD8+/NKG2D + cells in the presence or absence of autologous, peripheral blood-derived γδTregs, using our recently described co-culture assay assay [27] (Fig. S1).

Studying human AA as a model disease, we show here that autologous human γδTregs exert both protective and therapeutic effects in AA and deserve consideration as novel cell-based therapeutics in the future management of human autoimmune diseases characterized by IP collapse.

The number of TCRVδ1+/Foxp3 + γδTregs is increased around both lesional and non-lesional hair follicles in AA

Preliminary pilot observations made during our earlier studies that had identified the pathogenic role of Vδ1 + γδT cells in human AA [27, 28] had raised the possibility that the number TCRVδ1+/Foxp3 + cells is increased. Therefore, we first asked whether and where γδTregs are detectable in healthy human scalp skin, compared to lesional and non-lesional scalp biopsies from AA patients.

Triple immunostaining revealed that a few, scattered γδT cells+/Foxp3 + cells can be visualized around and within the epithelial stem cell-rich, keratin 15 + bulge area of HFs in healthy human scalp skin (Fig. 1A), while only extremely few γδT cells /Foxp3 cells could be detected around the proximal hair bulb of healthy anagen VI scalp HFs(Fig. 1B), i.e. the HF compartment that is primarily attacked by an inflammatory cell infiltrate in AA [12]. Surprisingly, the number of these cells was significantly increased around the hair bulb of lesional HFs (Fig. 1B, p < 0.05). Importantly, a significant increase of peribulbar γδT cells/Foxp3 cells was already noticeable around non-lesional HFs of AA patients compared to healthy control HFs (Fig. 1B, p < 0.05), even though this increase was much more pronounced in lesional compared to non-lesional AA HFs (Fig. 1B, p < 0.05). Mirroring the previously reported increase of Vδ1 + T cells in AA [31], the overall number of TCRVδ1+/Foxp3 compared to TCRVδ2/Foxp3 was strikingly increased around both lesional and non-lesional AA HFs (Fig. 1C, D p < 0.01) (we found no significant difference in the number perifollicular TCRVδ2/Foxp3 between any of the three groups) (Fig. 1E).

These clinical data support, but do not prove, the working hypothesis that TCRVδ1+/Foxp3 get involved in some manner in the early stages of human AA pathogenesis, long before hair loss lesions become clinically visible.

Autologous human γδTregs can readily be generated in vitro from peripheral blood

We next sought to isolate and enrich autologous peripheral γδTregs (TCRVδ2+/Foxp3+) from the same donors who provided scalp HFs for organ culture or scalp skin for xenotransplantation onto immunocompromised mice. The choice of TCRVδ2+/Foxp3 + was dictated by our inability to isolate a sufficient number of autologous TCRVδ1+/Foxp3 + cells from the skin of these donors (the latter is extremely challenging, since AA patients generally are quite hesitant to permit biopsy-taking, and at best consent to very small biopsies being taken). Though we recently managed to isolate TCRVδ1 + cells from the scalp skin of AA patients for γδT cell-HF co-culture [27], the quantity was insufficient for further purification and expansion of TCRVδ1+/Foxp3. However, peripheral blood TCRVδ1/Foxp3 and TCRVδ2/Foxp3 cells both exhibit immunosuppressive and regulatory effects [57, 61, 62, 88, 94]. These cells are thus suitable for proof-of-principle analyses to functionally explore the regulatory properties of γδTregs. Moreover, in clinical practice, only autologous, easily isolated, peripheral blood-derived γδTregs would be suitable as cell-based AA therapeutics.

In the initial step, we isolated autologous γδT cells from the PBMCs of healthy volunteers who also provided scalp skin samples for ex vivo and in vivo experiments. During this process, PBMCs were stimulated with zoledronate, which selectively activates and expands the γδT cell population [88, 89].Subsequently, these cells were cultured for 12 days in a medium enriched with IL-2, IL-15, and TGF-β1, which facilitates the development of γδTreg from T cells, although, as anticipated from existing literature, the purity of these cultures varied [88, 89] (Fig. S1). Thus, while two of the agents used (zoledronate and IL-2) are common to both γδTreg and γδT cell generation, we attempted to specifically expand γδTreg cells through the addition of IL-15 and TGF-β1.

As confirmed by FACS analysis, this method led to the generation of a viable cell population comprising 46.7 ± 3% of γδTCR cells after PBMCs in vitro pre-treatment. Moreover, a subset of 34.5 ± 12% of γδTCR cells expressed Foxp3, indicating regulatory T cell activities (Fig. 2A) [45, 71, 95–97]. For the in vivo and ex vivo experiments, these cells were then sorted using anti-CD3 and anti-γδTCR antibodies (Fig. S2). Some of the sorted cells were assessed by FACS using surface expressionTCRVδ1 or 2 and for intracellular immunostaining of Foxp3 to determine the amount of γδTreg (Fig. S2). FACS analysis on PBMCs in vitro pre-treated with zoledronate, IL-2, IL-15, and TGF-β1, revealed that 89 ± 5% of the sorted CD3 + γδTCR cells expressing Foxp3 were TCRVδ2+/Foxp3+, while only 0.5% of cells were TCRVδ1+/Foxp3+ (Fig. 2B). In the control group, i.e. PBMCs that had not been exposed to IL-2, IL-15, and TGF-β1, a mere 3.1 ± 1% of sorted CD3 + γδT cells were Foxp3+. This confirms that the CD3 + γδT cells sorted from PBMCs cultured with zoledronate, IL-2, IL-15, and TGF-β1, and used in our in vivo and ex vivo models, predominantly consisted of regulatory γδT cells, with a small proportion of non-regulatory γδT cells (see supplementary information detailing the in vitro pre-treatment, sorting strategy, and the subsequent purity FACS analysis for Foxp3: Fig. S2, Tables S1-3).

In line with the published literature, these data confirm the predominance of Vδ2 + T cells among γδTCs in human PBMCs, ranging between 50 and 95% [69, 98, 99]. We show the combination of IL-2, IL-15, and TGF-β1 stimulation to be effective and sufficient for augmenting the expression of Foxp3 markers on γδTCs as a pragmatic, translationally relevant method for amplifying regulatory TCRVδ2+/Foxp3 + in vitro prior to potential applications in cell-based clinical immunotherapy.

γδTregs promote hair regrowth and restore hair follicle immune privilege in experimentally induced human AA lesions in vivo

Next, we functionally probed our working hypothesis that autologous Vδ2 + γδTregs, generated in the manner described above, may be useful as AA therapeutics. For this, three groups of SCID/beige mice were xenotransplanted with healthy human scalp skin as previously described [24–26, 84] and injected intradermally with autologous immune cells as follows: a negative control group was treated with PBMCs that had been pretreated with Phytohemagglutinin (PHA) alone, which induces broad, non-specific activation of T and B cells (25,26,100); the positive control xenotransplants were injected with CD8+/NKG2D + cells to induce AA lesions; and the test group received both CD8+/NKG2D + cells and thereafter also γδTreg cells, injected 45 days later (Fig. S1). As expected, AA hair loss lesions developed in the xenotransplants within 6 weeks after CD8+/NKG2D + injection in all positive control and test animals, but in none of the negative control mice (Fig. 2C and D).

Fifty days after γδTregs injection in vivo, impressive hair regrowth was observed in all treated human xenotransplants. H&E staining confirmed these macroscopic observations and a normalization of the lesions, as evidenced by improvements in tissue structure and reduction in mononuclear cell infiltrates (Fig. 2E), i.e., a return to the physiological histology seen in healthy anagen VI hair follicles.

Moreover, compared to xenotransplants injected with CD8+/NKG2D + cells, the perifollicular infiltrate was significantly reduced, as indicated by a lower number of CD4+ (p < 0.03) (Fig. 3A), CD8+ (p < 0.03) (Fig. 3B), or IFNγ + immune cells (p < 0.03) (Fig. 3C).

Quantitative (immuno-)histomorphometry (qIHM) showed the expected prominent increase in the number of TCRVδ2+/Foxp3 + cells in test xenotransplants, compared to the negative and positive control groups (Fig. S3A-C). Given that only a small percentage of the injected γδTregs had been TCRVδ1+/Foxp3 + cells, in contrast to what one can see in lesional and non-lesional HFs of AA patients (Fig. 1B-D), the xenotransplants obviously showed much more TCRVδ2+/Foxp3 + than TCRVδ1+/Foxp3 + cells (Fig. S3A-C).

Crucially, compared to xenotransplants treated only with CD8+/NKG2D + cells, the IP of lesional HFs that had also received a γδTreg injection was at least partially restored, as indicated by upregulation of the potent HF-IP guardians, αMSH and TGF-β1 [27, 35, 82] (p < 0.05 and p < 0.05, respectively) (Fig. 3D and E), and downregulation of HLA-ABC, (p < 0.02) (Fig. 3F) and HLA-DR protein expression in vivo (p < 0.03) (Fig. 3G) to level that was comparable to that of healthy human HFs in negative control xenotransplants. Taken together, this provides the first evidence that autologous human peripheral γδTregs are therapeutic in a model human autoimmune disorder in vivo, namely in the humanized AA mouse model where peripheral γδTregs stimulated hair regrowth and restored HF-IP.

Peripheral γδTregs also protect from AA development by secreting immune privilege guardians

That the number of γδTregs was found to be increased around both lesional and non-lesional HFs in AA patients (Fig. 1C-E) raised the possibility that this might reflect an attempt to protect the HF-IP from collapse early during AA pathogenesis, similar to what has been proposed for the function of Tregs in murine AA [101] and in murine bulge IP [41]. Therefore, we next asked whether peripheral - γδTregs can also prevent or retard the onset of AA and, if yes, if they do so by releasing TGF-β1 and/or IL-10, i.e. recognized potent IP guardians [22, 82, 102]. For this purpose, xenotransplanted SCID/beige mice were treated either with CD8+/NKG2D + cells and γδTreg cells, and simultaneously with TGF-β1- or IL-10-neutralizing antibodies; only with CD8+/NKG2D + cells; or with both CD8+/NKG2D + cells and peripheral γδTreg cells, along with an isotype control (fig. S1).

Fifty days after CD8+/NKG2D + cell injection, the xenotransplants treated only with these cells showed the expected AA-like hair loss. In contrast, co-administration of γδTreg cells effectively prevented the development of hair loss lesions.This preventive effect was abrogated in the presence of TGF-β1 or IL-10 antibodies (Fig. 4A and B). This provides the first demonstration that adequately pre-stimulated peripheral blood γδTreg cells can effectively suppress the development of experimentally induced AA lesions in previously healthy human skin in vivo. Moreover, this shows that this AA-preventive effect is primarily mediated by TGF-β1 and IL-10 secretion by these γδTregs in the vicinity of HFs that are under attack by pathogenic CD8 + T cells.

Immunohistological analysis showed that the xenotransplants treated with CD8+/NKG2D + cells alone or with CD8 + T cells + γδTregs + TGF-β1 or IL-10 neutralizing antibodies displayed characteristic AA features (Fig. 4C), namely the expected peribulbar mononuclear cell infiltrates (Fig. 4C) typically seen in AA (“swarm of bees”) [12, 13]. specifically an increase in CD4+, CD8+, and IFN-γ + cells, along with increased and ectopic HF expression HLA-DR and HLA-ABC, and a decline in the intrafollicular protein expression of the IP guardians, αMSH and TGF-β1 - all indicating HF-IP collapse [27, 35, 82] (Fig. 5A-G). In striking contrast, qIHM revealed that xenotransplants which had been treated with CD8+/NKG2D + γδTreg cells + isotype control retained intact HF structures, showed diminished inflammatory HF infiltrates (Fig. 4C), and maintained normal low-level MHC class Ia expression as well as prominent αMSH and TGF-β1 protein expression (Fig. 5D and F and G). This demonstrates that autologous peripheral Vδ2 + γδTregs also protect from AA development in vivo by secreting IP guardians and underscores the pivotal role of TGF-β1 and IL-10 secretion in these AA-protective effects.

γδTregs suppress CD8+/NKG2D+-induced cytotoxic hair follicle damage ex vivo

We had previously shown that microdissected human anagen scalp HFs temporarily display tissue distress indicators and a considerably weakened HF-IP immediately immediately after the set-up of organ culture and that, in this ‘stressed’, MICA/B-overexpressing state, are vigorously attacked by co-cultured autologous NKG2D + γδT cells and CD8 + T cells in a manner that reproduces all key features of AA-like HF damage ex vivo: HF-IP collapse, premature catagen induction, and HF dystrophy [14]. Therefore, we used this co-culture assay to mechanistically interrogate the HF interactions of CD8+/NKG2D + T cells in the presence or absence of γδTregs under highly controlled ex vivo conditions in serum-free medium, focusing on immunosuppressive activities γδTregs ex vivo.

Cell division analysis revealed a significantly suppressed proliferation of CFSE-labeled CD8+/NKG2D + cells when these were co-cultured with γδTreg cells (p < 0.05) (Fig. 6A), highlighting the potent immunoinhibitory activity of the latter. We refined our ex vivo immunocyte-HF co-culture model (27) to incorporate varying numbers of γδTregs in co-culture with HFs and CD8+/NKG2D + T cells. This enabled us to determine the optimal amount of γδTregs necessary to effectively inhibit the induction of AA hallmark features ex vivo and showed that co-culturing HFs and CD8 + T cells with 500 γδTregs per well was most effective in suppressing the transition of anagen HFs into catagen (Fig. 6B). When γδTregs alone were co-cultured with HFs, as expected, there was no induction of the AA-associated "danger" signals, CD1d and MICA/B (Fig. 7A-D) [27], and the expression levels of αMSH and TGF-β1 (Fig. 7F-G), and hair matrix proliferation and apoptosis were unaltered (Fig. 7E) compared to control HFs.

When HFs were co-cultured with both γδTregs and CD8 + T cells, the typically observed premature catagen induction by CD8+/NKG2D + cells [27] was significantly suppressed (Fig. 6B). In addition, γδTregs effectively countered the CD8 + T cell-induced HF-IP collapse, as gauged by the low level of HLA-ABC and HLA-DR protein expression in the HF epithelium of anagen HFs (Fig. 7A and B) [27, 82, 83]. Moreover, co-culture with autologous γδTregs also significantly reduced the AA-associated HF overexpression of “danger” signals like CD1d and MICA/B (Fig. 7C and D). Furthermore, there was a marked increase in the proliferation and reduced apoptosis of hair matrix keratinocytes (Fig. 7E) as well as enhanced expression of the HF IP guardians, α-MSH and TGF-β1 in the outer root sheath (Fig. 7F and G).

These HF co-culture data functionally confirm the potency of γδTregs in suppressing AA-like HF damage caused by CD8+/NKG2D + even under reductionist ex vivo conditions.

γδTregs prevent catagen induction in "stressed" HFs by secreting IL-10 and TGF-β1

Finally, we explored by FACS analysis in this co-culture model whether γδTregs exert the above HF-protective effects primarily by the secretion of immunoinhibitory mediators. Analysis of cells derived from the co-culture of γδTregs with "stressed" HFs showed a significant elevation in intracellular IL-10 and TGF-β1 levels after 6 days of HF co-culture with Vδ2 + γδTregs, compared to CD8+/NKG2D + and γδT cells (Fig. 8A). When HFs were co-cultured with CD8+/NKG2D + alone, the percentage of premature catagen HFs was significantly reduced when IL-10, TGF-β1, or both were added to the culture medium (Fig. 8B).

This is in line with the concept that, if γδTregs secrete IL-10 and/or TGF-β1, as expected from previous work [99, 103, 104], this is likely to exert HF-protective effects. Indeed, ELISA analysis showed a significant increase in TGF-β1 (Fig. 8C) and IL-10 levels (Fig. 8D) in the supernatant of HFs co-cultured with γδTregs and CD8+/NKG2D + T cells compared to controls.

Conversely, there was a significant reduction in the supernatant level of IFNγ the key pathogenic cytokine in AA (105) in HFs that had been co-cultured with both γδTregs and CD8+/NKG2D + T cells (Fig. 8E).

In this combined in vivo and ex vivo study in a model human autoimmune disorder, we introduce human γδ Tregs as regulatory lymphocytes with clinically relevant autoimmunity- and tissue homeostasis-protective functions. Specifically, we provide the first evidence that γδTregs can both protect from AA development and restore hair regrowth in established AA lesions in human skin in vivo. Moreover, we demonstrate that γδTregs maintain and re-establish IP in a human mini-organ (here: the anagen hair bulb of scalp HFs) and protect tissue functionality (here: the growth of hair matrix keratinocytes and anagen maintenance) (Fig. 9).

Human γδTregs thus join skin Tregs [41] as key HF-IP-supporting immune cells in vivo, but – in contrast to the latter – can easily be isolated and expanded from the peripheral blood of affected patients.

We present here a novel cell-based therapeutic strategy in the future personalized medicine management human AA, using autologous, in vitro-pretreated peripheral γδTregs. By employing a rigorous sorting strategy using antibodies against CD3 and γδTCR, and confirming the purity of the γδTreg cells through secondary FACS analysis, we demonstrate that our γδTreg cell preparations consist predominantly of regulatory γδT cells.This strategy also overcomes one of the major shortcomings of AA therapy with JAK inhibitors, namely that the latter seem to fail to effectively and long-lastingly re-establish HF-IP, since therapy discontinuation with these drugs almost invariably is associated with rapid AA relapse [22, 106–109]. Instead, γδTregs effectively both protect and restore human HF-IP (Fig. 9), thus rendering anagen HFs resistant against an immune attack - the most important challenge in the successful long-term management of AA [22, 106].

Moreover, our demonstration of both protective and therapeutic effects of human γδTregs in human scalp skin in vivo and human HFs ex vivo against key AA-associated HF pathology characteristics cast further doubt on how predictive the most frequently used preclinical AA research model, the TLR4-mutant C3H/HeJ mouse, really is for therapeutic interventions in human AA, and how well it mimics the pathobiology of human, rather than represinting a murine variant of AA [40, 110]: recently published data in this mouse model have suggested that “failure of T_reg-mediated immunosuppression is not a major disease mechanism in AA” [40]. Our data show that this verdict does not apply to the role of human γδTregs in human AA. That even non-lesional skin of AA patients already shows an up-regulated number of perifollicular γδTregs may suggest an active attempt of the skin and HF immune system to maintain HF-IP homeostasis. Of course, Lee et al. examined only the most frequently investigated Tregs in mice, whose role in human AA remains unclear [42, 111], but not γδTregs - which might represent the clinically more relevant T cell population with regulatory functions in human AA.

It is tempting to speculate that peripheral blood-derived, autologous γδTregs may also become an effective cell-based therapy in the future management of other autoimmune diseases characterized by IP collapse, such as multiple sclerosis, autoimmune uveitis and orchitis, fetal immune abortion, and diabetes mellitus type I [79–81, 112–114]. That γδTregs can easily be isolated from peripheral blood and expanded in vitro adds to the attractiveness and clinical relevance of this novel strategy of administering personalized medicine cell-based immunotherapy. Unfortunately, none of these other IP collapse-associated diseases is as accessible to preclinical research in clinically relevant human, rather than mouse, models at the in vivo and ex vivo level as AA. The expansion process, involving specific components (zoledronate, IL-2, IL-15, and TGF-β1), significantly increases their numbers, making them viable for therapeutic applications. This recommends AA as a model disease for exploring novel immune intervention strategies under clinically relevant conditions, especially in the context of how to control γδT cell-mediated organ pathology [27, 31] by γδTregs.

While there is substantial research focusing on the cytotoxic effector functions of γδT cells, including in AA [31, 115–117], our study is in line with and underscores previous work that has highlighted potential therapeutic applications of γδTregs. For instance, Ren et al. have shown that Lactobacillus acidipiscis-induced γδTregs, can mitigate experimental autoimmune encephalomyelitis in mice by suppressing the development of pathogenic Th1 and Th17 cells [11]. Furthermore, γδTregs have been effective in mitigating the onset of graft-versus-host disease (GVHD) in mice, underscoring their potential in a variety of immunotherapeutic applications [68, 105, 118–120]. It is important to recognize inherent limitations of our pilot study. For example, while our ex vivo model does not replicate physiological HF innervation, vascularization, and multi-level interactions with circulating immune cells, in our full-thickness human scalp skin xenotransplants on SCID/beige mice, the presence of mouse proteins may and the absence of circulating human immune cells may confound the results. Also, given the need to operate under strictly autologous conditions, only a low number of human skin and PBMC donors, xenotransplanted mice, and organ-cultured HFs could be studied, thus warranting independent confirmation of our findings in additional donors, HFs, and mice, and in both sexes, different ethnicities, and a wider range of age groups.

With these limitations in mind, mechanistically, our study suggests that γδTregs exert their AA-protective and –therapeutic activities mainly by secreting the potent HF-IP guardians, IL-10 and TGF-β1 [117, 121]. This is in line with the demonstration that γδTregs can effectively suppress the proliferation of cytotoxic T lymphocytes via IL-10 and TGF-β1 [122]. We also show that γδTregs reduce IFNγ secretion by HF-pathogenic CD8 + T cells ex vivo. Given that TGF-β1 is a physiological catagen inducer both in murine and human HFs [123–126], our findings suggest a context-dependent modulation of human HF cycling: in the inflammatory microenvironment of AA, the increased secretion of TGF-β1 by γδTregs may primarily counteract the well-documented, potent catagen-promoting effects of IFNγ [89], the key pathogenic cytokine in AA. Moreover, promoting telogen effluvium via enhanced, TGF-β1-mediated catagen induction may be the price a growing (anagen) HF pays for protecting its IP and for warding off IFNγ-induced HF cytotoxicity [27, 127].

In summary, that autologous, peripheral blood-derived γδTregs can both prevent and treat a model human autoimmune disorder in vivo respectively ex vivo, demonstrates the effectiveness of human γδTregs in restoring IP and tissue homeostasis and invites a novel cell-based treatment strategies that utilize these regulatory T cells, namely in in the management of AA. Furthermore, it is worth critically exploring whether autologous cell-based therapy with human γδTregs could also be effective in preventing and treating other autoimmune diseases (AIDs) where immune privilege collapse is a key factor in their pathogenesis. Such diseases could include multiple sclerosis, type I diabetes, autoimmune uveitis, and immune abortion, among others.

Patients, tissue and blood samples

For the in situ analyses we used archival paraffin-embedded biopsy specimens of human AA scalp skin lesions from the Department of Pathology, Rambam Medical Center (4 females, 12–35 years, mean age 20.5 ± 9.5; 3 males, 6–38 years, mean age 18 ± 12). AA was diagnosed both clinically and by histopathology, and none of the enrolled patients showed clinical evidence or had a personal history of other AA-associated autoimmune diseases [3].In addition, 5 non-lesional AA biopsies were obtained from Rambam Health Care Campus (4 females, 28–41 years, mean age 37 ± 7; 1 males, 48 years). For the in vivo and ex vivo analyses, clinically healthy frontotemporal scalp skin specimens and peripheral blood were obtained from 14 healthy volunteers (9 females, aged 49–74 years, mean age 54 ± 9; 4 males, aged 27–47 years, mean age 34 ± 7) undergoing cosmetic facelift surgery. The scalp skin specimens were collected for three primary purposes: transplantation onto SCID mice, human HF culture and preparation of paraffin blocks for histological and immunohistochemical staining. The study was approved by the institutional review board of the Rambam Health Care Campus (RMB-0182-14).

PBMC isolation and culture

PBMCs were isolated from heparinized whole blood from healthy donors by Lymphoprep density gradient centrifugation. Cells were frozen for further assays (in a freezing solution: 70% FBS, 20% RPMI1640 and 10% DMSO) or cultured at a concentration of 3x10⁶ cells/ml in 24 wells plate with medium (RPMI 1640, 10% human AB serum, 1% Penicillin-Streptomycin antibiotics, 2mML-glutamine. To induce the expansion of γδT cells, various agents, including zoledronate, were added to the culture. Additionally, the presence of IL-2, IL-15, and TGF-β1 [65, 66] was utilized to promote the development of γδT regulatory T cells (Table S1). On days 3 and 5, half of the supernatant volume was discarded and replaced with fresh medium containing cytokines (without zoledronate). After 7 days cells were sorted by FACSAria and cultured together with the HFs or used for different assays (fig. S2).

FACS sorting

Stimulated PBMCs (Table S1) were collected, centrifuged at 300g for 5min and washed with PBS containing 1% BSA and 2%penicillin-streptomycin antibiotic mixture. For the staining of the surface antigens, cells were incubated with mAbs for 30min at 4^◦C (Table S2) according to the manufacturer’s instructions.

The cells were sorted by FACSAria as follows:

CD8 + NKG2D + cells

anti-CD3(APC/FITC), CD8(Pe/cy7, 300913), anti-NKG2D (VioBright FITC, 130-104-888).

γδTreg cells

anti-CD3(APC/FITC), anti-γδTCR(PE/APC).

γδT cells

anti-CD3(APC/FITC), anti-γδTCR(PE/APC).

PHA cultured cells

anti-CD3(APC/FITC).

The sorted cells were collected to a tube with RPMI 1640 medium enriched with 20% HS, 1% penicillin-streptomycin antibiotic mixture, 2mML glutamine. Following the cells were centrifuged, suspended, counted and 1) stained with TCRVδ2 and Foxp3 for confirming purity of Tregs, 2) co-cultured with HFs, and 3) injected in xenotransplants.

Flow Cytometry and CFSE Analysis

Cultured cells were collected, centrifuged at 300g for 5min and washed with PBS containing 1% BSA. For the staining of surface antigens, cells were incubated with mAbs for 30min at 4^◦C. For the intracellular staining a fixation and permeabilization step was required and performed according to the manufacturer's instructions. Cells were incubated with the intracellular Abs for 30min at 4◦C (Table S3). To ensure accuracy and avoid false-positive results or data inconsistencies due to fluorescent spillover, we performed compensation using single-stain controls for each fluorophore. Compensation beads (Compbeads) were incubated with each mAb for 25 miutes. Cells were read by FACSFortessa Flow Cytometer and further analyzed using FlowJo. 1xBD Perm/Wash buffer 1xBD Perm/Wash buffer (BD Cytofix/CytopermTM Fixation/Permeabilization Kit). Compensation was done by using CompBeads (BDTM Biosciences, Cat# 552843) [128]. CFSE cell division tracker kit was purchased from Biolegend. The green fluorescence intensity of CFSE within each cell was measured by flow cytometry, and data were calculated using Flowjo software. The proliferation index measures the fold increase in cell number during the course of the experiment. It was calculated by dividing the number of original cells present at the start of the experiment by the number of cell divisions.

Enzyme-Linked Immunosorbent Assay (ELISA)

FACS-sorted cells were cultured in a 96-well plate at a density of 10x10⁴ cells per well. The cells were incubated for 48 hours, both with and without HFs. After the incubation period, the culture medium was collected and aliquoted. These aliquots were then stored at -80°C for future analyses. Quantification of the cytokines secreted into the media was done by using a sandwich ELISA kit according to the manufacturer’s instructions as follows: IFNγ (ELISA deluxe max kit, Biolegend Inc.), TGF-β1 (DuoSet ELISA kit, R&D System Inc.) and IL-10 (DuoSet ELISA kit, R&D System Inc.).

Human HF organ culture

HFs were isolated from the scalp skin tissue after 6 days at tissue culture. Anagen VI HFs were microdissected as previously described (91). Each HF was placed in a single well, in 96-wells plate, with 100µl culture medium (William's medium plus 1% penicillin-streptomycin antibiotic mixture, 2mML-glutamine, 0.1% hydrocortisone and 0.1% insulin). After 24h, i.e. at a time when organ-cultured HFs are maximally stressed, overexpress ‘danger’ signals like MICA/B and display a compromised HF-IP [14], 100 µL of sorted cells - including PHA-cultured PBMCs, CD8+/NKG2D + cells, γδTregs, and γδT cells - were added to the wells with the HFs (RPMI 1640, 1% penicillin-streptomycin antibiotic mixture, 2mML glutamine). After 6 days, the HFs were photodocumented and embedded in optimal cutting temperature (OCT) matrix for immunohistochemistry.

Immunofluorescence microscopy for the ex vivo study

The HFs were embedded in a cryomold and covered with OCT for a few minutes to release air bubbles. Then the cryomold was placed in a bath on dry ice covered with methanol until solidification. The cryoblocks were transferred to an open tube which were also placed in the bath for a few minutes to permit methanol residues to evaporate and stored at -80⁰C until cryosectioning (5µm). Cryosections were carefully placed on glass slides and allowed to dehydrate for 5 minutes at RT. Following this initial dehydration step, the slides were stored at -80°C until they were ready for immunofluorescence microscopy.Prior to staining, the cryosections underwent a second dehydration step, lasting 40 minutes. Post-dehydration, the HF cryosections were fixed by incubating them in acetone for 10 minutes at -18°C. Slides were dehydrated for 20 minutes and transferred to distilled water. Sections were blocked with goat serum for 30 minutes to minimize nonspecific binding and were incubated with the relevant primary Ab overnight (Table S4). Slides were incubated with an appropriate biotinylated secondary Ab (FITC-conjugated goat anti-mouse Ab, Rhodamine-conjugated goat anti-mouse IgG Ab or Alexa Fluor 488-conjugated goat anti-rabbit IgG Ab) for 30 minutes after three washes with PBS, and counterstained by DAPI (Thermo Fisher Scientific). Staining was visualized using a confocal Microscope - Zeiss LSM 700.

Histochemistry, immunohistology and qIHM

Five-micrometer paraffin sections of 7 lesional, 5 non-lesional AA biopsies and 14 healthy human scalp skin xenotransplants, each derived from a distinct donor, were processed for histochemistry or immunohistochemistry. The following primary antibodies were used: anti-TCRVδ1 (Invitrogene), anti-TCRVδ2 (Invitrogene-), anti-Foxp3 (Abcam), anti-CD8 (Cell Marque), anti-CD4 (DAKO), anti-HLA-A,B,C (Abcam), anti-HLA-DR (Abcam), anti- IFNγ (Abcam), anti- α-MSH (LSBio) and anti-TGF-β1 (Abcam) (Table S4) [24, 71]. The last four abs were assessed as standard HF IP read-out parameters [22, 27, 82].

Hematoxylin and eosin (H&E) staining was performed on cryo- or paraffin sections as previously described [26, 86].

The immunoreactivity patterns were assessed in standardized, well-defined reference areas by qIHM by experienced, blinded observers, following our standard protocols for evaluating human HF immunology read-outs (84) counting at least three reference areas each on three non-consecutive sections, presented randomly to the blinded observer(s). Specifically, immunoreactive cells around and within the HFs were counted in an area of 0.66 mm².

For HLA-A,B,C, HLA-DR, MICA/B, CD1d, α-MSH and TGF-β1 (Table S4) image analysis was performed using Image J software. Protein expression was measured by calculating the percentage of staining coverage within the analyzed area.

TUNEL analysis

Apoptotic cells were evaluated using a commercial TUNEL kit (Roche) with antidigoxigenin fluorescein labeling and according to manufacturer’s protocol. Ki-67 (Invitrogen) was visualized using Alexa Flour 594-conjugated goat anti-mouse (Jackson, 115-585-062). Sections were counterstained by DAPI (Thermo Fisher Scientific). Staining was visualized using a confocal Microscope - Zeiss LSM 700. Quantification was performed as previously described [26].

Humanized AA mouse model

In the current study, 23 female SCID/beige mice (C.B-17/IcrHsd-scid-bg) (Harlan Laboratories Ltd., Jerusalem, Israel) were used at 2–3 months of age and were housed in the pathogen-free animal facility of the Rappaport Faculty of Medicine, Technion – Israel Institute of Technology. Animal care and research protocols were in accordance with institutional guidelines and were approved by the Institutional Committee on Animal Use (17-08-115-IL).

For the humanized AA mouse model [24–26, 84, 85, 129], full-thickness biopsies were taken from four healthy female donors (49–62 years, mean age 57 ± 6 undergoing plastic surgery on the scalp. Biopsies obtained from each donor were dissected into quadratic skin fragments, each measuring 3 mm in both vertical and horizontal dimensions, resulting in a total surface area of 9 mm² per fragment. Three of these fragments were orthotopically grafted into the subcutaneous layer of each SCID/beige mouse, as previously described [24–26]. Seven days after surgery, mice were treated with Minoxi-5 (hair regrowth treatment for men containing 5% Minoxidil active ingredient) by spreading it on the grafts twice a day until optimal expedited hair growth was seen after ca. two months.

Two sets of in vivo experiments were conducted. In the first set, with a therapeutic focus, nine mice were randomly divided into three groups:

1. Group 1 (Control group): Comprised of 3 mice injected with PHA cultured PBMCs, serving as a negative control.
2. Group 2: Consisted of 3 mice injected with CD8+/NKG2D + cells to induce AA lesions.
3. Group 3: Included 3 mice initially injected with CD8+/NKG2D + cells to induce AA lesions. Fifty days post CD8+/NKG2D + cell injection, these mice received a single injection of γδTreg cells (6X10⁵ cells/xenograft).

All mice were sacrificed ninety-five days following the initial CD8+/NKG2D + cell injection to assess the therapeutic impact of γδTreg cell treatment.

In the second set of experiments, designed to evaluate preventive effects, the xenotransplants of 14 mice were injected with CD8+/NKG2D + cells to induce AA lesions. On the same day, 10 of these mice also received an injection of γδTregs (6X10⁵ cells/xenograft) and were further divided into treatment subgroups:

1. Three mice received intradermal injections of TGF-β1 neutralizing antibodies (50 ng, daily).
2. Three mice were treated with intradermal injections of IL-10 neutralizing antibodies (50 ng, daily).
3. Four mice were injected with an isotype control.

The remaining 4 mice in the cohort of 14 did not receive any additional treatment post CD8+/NKG2D + cell injection. All mice were sacrificed fifty days after the initial injections to evaluate the preventive efficacy of the various treatments.

Statistical analysis

Data are presented as the mean ± standard error of mean (SEM) or fold change of mean ± SEM; p values of < 0.05 were regarded as significant. Gaussian distribution of the data was analyzed using Shapiro-Wilk test. Significant differences were analyzed using unpaired Student′s t-test (comparison between one set of data) or One Way ANOVA (comparison between multiple sets of data) for parametric data, or Mann–Whitney test (comparison between one set of data and sham or vehicle) for nonparametric data or Kruskal–Wallis test, and Dunn's test (comparison between multiple sets of data).

Funding

This study was supported in part by the Technion Research and Development Foundation

(TRDF) to A.G.

Author Contributions

Conceptualization: AG, RP

Methodology: AG

Investigation: AK, NG

Visualization: AK, MB, RK, AZ, NS

Supervision: AG

Writing—original draft: AG

Writing—review & editing: AG, RP,MB

Conflict of Interest

The authors state no conflict of interest. For the record, AG performs contract preclinical hair research for competing industry clients on AA; MB is the CEO of Monasterium, a contract research organization that also tests hair growth-modulatory agents; RP is CEO of CUTANEON, a company that also develops hair therapeutics. None of the authors is involved in commercializing gdTregs-based cell therapy.

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Human regulatory γδT lymphocytes as novel autoimmunity-protective cells: Lessons from alopecia areata

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Version 1

Abstract

Figures

INTRODUCTION

RESULTS

Peripheral γδTregs also protect from AA development by secreting immune privilege guardians

γδTregs prevent catagen induction in "stressed" HFs by secreting IL-10 and TGF-β1

DISCUSSION

MATERIALS AND METHODS

Patients, tissue and blood samples

PBMC isolation and culture

FACS sorting

Flow Cytometry and CFSE Analysis

Enzyme-Linked Immunosorbent Assay (ELISA)

Human HF organ culture

Histochemistry, immunohistology and qIHM

TUNEL analysis

Humanized AA mouse model

Statistical analysis

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1