Foxp3+ Tregs from Langerhans cell histiocytosis lesions co-express CD56 and have a definitively regulatory capacity

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Highlights

  • Foxp3+ Tregs from Langerhans cell histiocytosis lesions co-express CD56

  • Foxp3+ Tregs from Langerhans cell histiocytosis lesions produce TGF-β

  • Treg enrichment in LCH patients is associated with reduced CD8+CD56+ T cells

Abstract

Langerhans cell histiocytosis (LCH) lesions contain myeloid lineage ‘LCH’ cells. Regulatory T cells (Tregs) are also enriched within lesions, although their role in LCH pathogenesis is unknown. LCH cells are thought to produce the transforming growth factor beta (TGF-β) within lesions, however whether Tregs contribute is unestablished. Using flow cytometry, we analyzed relative frequencies of live Tregs from LCH patients and identified CD56 expression and TGF-β production by lesion Tregs. While CD56+ Tregs were enriched in lesions, overall CD56+ T cells were reduced in the blood from active LCH patients compared to non-active disease patients, and there was a negative correlation between CD8+CD56+ T cells and Tregs. We propose that inducing a Treg phenotype in T cells such as CD56+ T cells may be a mechanism by which LCH cells divert inflammatory T cell responses. Thus, Tregs within LCH lesions are likely an important component in LCH pathogenesis.

Introduction

Langerhans cell histiocytosis (LCH) is a rare disease involving inflammatory lesions, which mostly affect children. Lesions arise in any bodily organ; skin and bone are commonly affected, although liver, spleen or hematological involvement increases patient mortality risk [1]. Lesions are defined by CD1a+/CD207+ myeloid lineage cells (LCH cells), which often have RAS/RAF/MEK/ERK pathway mutations [[2], [3], [4], [5], [6], [7], [8]] and constitutive ERK activation [4]. T cells are amongst other immune cells present in lesions (reviewed in Bechan et al. [9]) and Foxp3+ regulatory T cells (Tregs) in particular are enriched in lesions and also peripheral blood from LCH patients [[10], [11], [12], [13], [14]]. Tregs maintain tolerance in immune environments by competing with other T cells for available interleukin 2 (IL-2) or co-stimulation signals, for example, via expression of cytotoxic lymphocyte-associated antigen 4 (CTLA-4). Tregs also secrete the inhibitory cytokines, transforming growth factor beta (TGF-β) and IL-10 (reviewed in Shevach [15]). Tregs can inhibit anti-tumor immune responses and promote tumor development using these mechanisms (reviewed in Sakaguchi et al. [16] and Chaudhary & Elkord [17]). Tregs in LCH lesions express inducible costimulatory factor (ICOS) and are in close proximity to LCH cells, which express ICOS ligand [11,13]. These observations suggest that specific Treg-LCH cell interactions occur within the lesion environment. Additionally, Allen et al. [14] identified upregulation of CTLA4 in T cells from LCH lesions compared with T cells isolated from LCH donor blood. While the expression was not specifically defined on the Treg population, it is plausible that CTLA-4 is expressed by LCH lesion Tregs, thus they may actively suppress other immune cells. Moreover, Tregs were reported as increased in active LCH (AD) patient blood compared to controls [11], further suggesting a possible role in LCH pathogenesis.

TGF-β and IL-10 are commonly produced by Tregs and were detected within blood [18,19] and lesions [10,11,13,[20], [21], [22]] from AD patients. Additionally, groups demonstrated that LCH cells produced TGF-β [14,21], and TGF-β was a driver of the LCH cell phenotype [18,23,24], while IL-10 was produced largely by macrophages [25] and LCH cells [10] within lesions. Since Tregs are enriched within lesions, and the cytokines they produce are present, it is important to understand whether LCH lesion Tregs are a source of TGF-β and/or IL-10 production in addition to LCH cells.

Expression of Foxp3 is synonymous with low CD127 and defines Tregs along with CD3, CD4 and CD25 expression [26,27]. Not all studies investigating Tregs in LCH have used the stringent definition of CD3+CD4+CD25+Foxp3+ or CD127low lymphocytes in concert. Historically, the identification of Tregs inhibited functional studies because staining for Foxp3 requires cell permeabilization. Consequently, the function of Tregs in LCH is not established. Akin to landmark studies [26,27] we replaced anti-Foxp3 with anti-CD127 and used the cell surface phenotype CD3+CD4+CD25+CD127low to identify Tregs, which importantly allows for downstream functional assays on the population. No studies to date have investigated live Tregs from LCH lesions. We comprehensively characterized and quantitated the relative frequency of Tregs from LCH patients, and analyzed their function in order to better understand their role in LCH pathogenesis.

Section snippets

Clinical details of LCH patients

The cohort for this study included 10 male and 7 female patients with LCH ranging from age 2 months to 68 years (Table 1). Bone, skin and lung were the most commonly affected tissues. Other than a single patient, to the best of our knowledge no patients with AD received any treatment with steroids or chemotherapy prior to the collection of specimens. From 7 patients with non-active disease (NAD), 3 received prior treatment. Samples from participants 1 to 13 were analyzed for different factors

Tregs are enriched in AD patients compared to healthy donors

Gating for CD3+CD4+CD25+CD127low lymphocytes [26,27] we identified live Tregs in healthy donor- and LCH patient-derived peripheral blood, and in LCH lesions (Fig. 1A). Akin to previous studies, the proportion of Tregs within CD4+ T cells and total T cells from LCH lesions was significantly increased compared to healthy donor-derived blood (Fig. 1B-C). Furthermore, the proportion of Tregs in the T cell population was significantly increased in AD patient blood compared to healthy donor blood (

Discussion

This study aimed to better define the role of Tregs in LCH pathogenesis by quantitating their relative frequency and testing their function. Functional studies on Tregs in LCH lesions have not been completed previously. We established that the live Treg identification method that substitutes anti-Foxp3 with anti-CD127 is sufficient for detecting Tregs in LCH patients. Our results support previous studies reporting an enrichment of Tregs in LCH patients [[10], [11], [12], [13], [14]], and for

Conclusions

In summary, we highlight that CD56+ T cells collectively may have an important role in the pathogenesis of LCH. Our study primarily established that the alternative Treg identification method that allows for downstream functional studies is appropriate in the context of assaying Tregs from LCH lesions. In addition to our current understanding that Tregs and TGF-β are present within lesions, we showed that Tregs from LCH lesions are biased towards generating TGF-β but do not appear to produce

Ethics approval and consent to participate

This project was approved by Ballarat Health Services and Saint John of God Ballarat Hospital Human Research Ethics Committee (HREC) (HREC/15/BHSSJOG/5 and HREC/10/BHSSJOG/57) and Federation University Australia HREC (A08-100). Written, informed consent was provided by patients, and/or parents of children where appropriate. Buffy coats from healthy donors were obtained from the Australian Red Cross Blood Service.

Availability of data and materials

All data will be available upon request.

Author's contributions

T.G., E.K., J-I.H. and G.K. recruited patients with LCH and provided tissue samples and clinical information. J.M., J.K., S.B. and G.K designed experiments. J.M. performed experiments, analyzed results, prepared figures and wrote the manuscript. G.K. critically revised the manuscript. J.K., E.K., T.G., D.G.P., J-I.H and S.B. reviewed the manuscript. G.K led the investigation.

Funding and acknowledgements

We are thankful for the participation of patients and healthy donors in this study. A portion of LCH patient peripheral blood samples and coded data were supplied by the Children's Cancer Centre Tissue Bank at the Murdoch Children's Research Institute and The Royal Children's Hospital (www.mcri.edu.au/childrenscancercentretissuebank). Establishment and running of the Children's Cancer Centre Tissue Bank is made possible through generous support by Cancer In Kids @ RCH (www.cika.org.au),

Declaration of Competing Interest

The authors declare that they do not have any competing interests.

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