Elsevier

Clinical Immunology

Volume 230, September 2021, 108820
Clinical Immunology

Full Length Article
Memory and naïve gamma delta regulatory T-cell gene expression in the first 24-weeks of peanut oral immunotherapy

https://doi.org/10.1016/j.clim.2021.108820Get rights and content

Highlights

  • gdTreg modulation is observed in the 1st 6-weeks of peanut oral immunotherapy, a potential early marker of desensitization.

  • At 24-weeks of POIT, gdTregs upregulate OX40R, GITR, TFGB1, CTLA4, ISG20, CD69, and downregulate IL7R and SELL.

  • gdTreg expression suggests POIT modulates type-2 inflammatory responses.

Abstract

Background

Peanut oral immunotherapy (POIT) has provided desensitization to peanut allergic individuals. Limited immunological evaluation exists during the first 24-weeks of POIT.

Objective

Regulatory T-cells (Tregs) are antigen induced immunosuppressive T-cells important in establishing tolerance. Delineation of early immunologic changes contributing to the development of peanut desensitization would help clarify the mechanism of action in POIT. We performed single-cell RNA sequencing (scRNAseq) on Tregs in pediatric subjects undergoing POIT during the first 24-weeks of therapy to evaluate early immunological changes induced by POIT.

Methods

PBMC samples from peanut allergic subjects between 5 and 12 years of age enrolled in a Phase 1/2a POIT study were collected and analyzed at 0, 6, and 24-weeks after POIT initiation and samples were compared to healthy non-peanut allergic controls. Tregs were enriched from PBMCs and scRNAseq analysis performed. Cell Ranger 3.1.0 (10× Genomics) was utilized to identify cell clusters and differentially expressed genes, and results were analyzed with Seurat suite version 3.0.0.

Results

Gene analysis revealed 10 major clusters corresponding to different cell types observed to change during POIT when compared to the healthy, non-peanut-allergic state. scRNAseq analysis of Tregs revealed strong CD3G expression correlating with gdTregs. scRNAseq analysis of gdTregs revealed dynamic changes occurring within the first 6-weeks of treatment and cell frequencies of naïve and memory gdTregs at 24-weeks of treatment reducing to levels similar to healthy controls. Analysis of transcriptomic cell identity analysis using SingleR showed gene expression in gdTregs similar to healthy control after 24-weeks of POIT treatment. scRNAseq analysis revealed alterations in gene expression for memory and naïve gdTregs during this timeframe. Specifically, expression of OX40R (TNFRSF4), GITR (TNFRSF18), TGFB1, CTLA4, ISG20, CD69 were upregulated in memory gdTregs compared to naive gdTregs by 24-weeks of POIT, while IL7R and SELL were downregulated in memory gdTregs compared to naïve gdTregs.

Conclusions

There are specific expression profiles of peripheral naïve and mature gdTreg cells in peanut allergic patients undergoing POIT in the first 24-weeks of treatment implicating pathways involved in maintenance of immune homeostasis. gdTreg cells may contribute to the tolerogenic effect of POIT within the first 24-weeks of POIT treatment. These findings suggest that gdTregs cells may be an early marker of desensitization in subjects undergoing POIT.

Introduction

Approximately 1–2% of children in the United States are affected by peanut allergies [1]. Current treatment of food allergies includes avoidance and the use of epinephrine to treat allergic reactions. Oral immunotherapy has been FDA approved for the treatment for peanut allergies. Peanut oral immunotherapy (POIT) provides desensitization to peanut allergic individuals. Despite these advancements in food allergy treatments, the POIT mechanisms modulating the adaptive immune system to provide effective desensitization, immune homeostasis, and potentially sustained unresponsiveness have yet to be fully elucidated.

CD4 + FOXP3+ T regulatory cells (Tregs) are known to promote the development of oral tolerance to allergens [2] and maintenance of immune homeostasis. Tregs are important in sustaining oral tolerance and preventing allergic disorders, whereas deficiencies in Tregs have been observed to promote allergic disorders [3]. Patients lacking allergen-specific Tregs have been shown to have an increased incidence of food allergies. In a previous study by Syed et al., it was shown that subjects undergoing POIT had increased allergen-specific Tregs compared to baseline peanut allergic controls that were not on POIT [4]. Additionally, expression of the Treg transcription factor FOXP3 was also increased in subjects who developed sustained unresponsiveness after a period of up to 6-months of treatment avoidance compared to baseline untreated peanut allergic subjects and subjects who did not achieve sustained unresponsiveness following POIT avoidance. Interestingly, when the absolute cell counts of non-allergen specific-Tregs were examined, no significant differences were observed between the untreated and POIT-treated subjects.

Tregs have always been important in the maintenance of immune homeostasis. Similar to Tregs, gamma delta Tcells (gdTcells) have also gained attention given their ability to regulate and maintain immune homeostasis. Unlike Tregs and other conventional Tcells, gdTcells do not require antigen processing or presentation via a major histocompatibility complex (MHC) [5]. gdTcells serve as antigen presenting cells which is an important and innate feature, thus serving as frontline protectors against unconventional antigens, such as stress molecules and environmental allergens [6,7]. gdTcells are predominantly found in the mucosal layers, such as in the intestinal epithelium, and maintain tissue integrity when faced with stress factors [8]. Early preliminary studies have shown the regulatory role of gdTcells in the modulation of oral tolerance and development of allergic disease [9,10]. However, studies examining the role of gdTcells in food immunotherapy trials are limited, and more investigation is needed given the prevalence of gdTcells in the intestinal mucosa.

Desensitization to peanut during POIT begins as early as the first 24-weeks of therapy, however limited immunological evaluation has been performed during this timeframe. We analyze Treg, and more specifically, gdTreg subpopulations through single-cell RNA sequencing (scRNAseq) in pediatric subjects undergoing POIT from samples at baseline, 6-weeks, and 24-weeks after starting POIT to further explore the immunological and genetic changes occurring due to treatment. Here we delineate early immunological and gene expression changes during peanut desensitization.

Section snippets

Study design and protocol

We conducted a single-center, open-label Phase 1/2a POIT trial at Texas Children's Hospital. The study was performed in accordance with the Declaration of Helsinki which was approved by the Baylor College of Medicine Institutional Review Board (H-26819). All patients and parents provided written and informed assent and consent, respectively. This trial was registered under ClinicalTrials.org (NCT 02203799). Patients between the ages of 5–16 years of age with a clinical history of an

Treg analysis reveals 10 major clusters corresponding to distinct cell types

To evaluate the enriched Tregs and to identify whether other populations were included, the resulting cells-by-gene matrix was processed using dimensionality reduction and was visualized after uniform manifold approximation and projection (UMAP). Using an unsupervised approach, ten major clusters corresponding to different cell types were identified (Fig. 1). These ten clusters were annotated according to the expression of cell marker genes and corresponded to Treg cells, monocytes, NK cells,

Discussion

Our study utilized scRNAseq to explore the diversity of the peripheral blood Treg populations in subjects undergoing POIT in the first 24-weeks of therapy. scRNAseq provides an in-depth avenue to detect how treatments such as POIT can affect the transcriptome for individual immune cellular subpopulations. This data demonstrates dynamic changes in Tregs and more specifically gdTregs, suggesting a role gdTregs may have in the development of successful desensitization and potentially oral

Conclusions

Together, the transcriptomic findings observed in the gdTregs clusters provide information to suggest the maturation of gdTregs in the development of successful POIT desensitization, as well as modulating immune homeostasis in peanut allergy and during POIT. Our findings show that naïve and memory gdTreg subpopulations with Th2 modulatory gene expression are present in POIT treated samples, and that these populations dynamically change as early as the first 6-weeks of POIT treatment. Overall,

Funding

This work was supported by the Texas Children's Hospital Pediatric Pilot Grant; the American Association for Immunologist Travel for Techniques Grant; and the generous support of the Wareing Family Fund and Scurlock Foundation. KR and CC were partially supported by, CPRIT RP200504, NIH/NIEHS P42 ES027725, and NIH/NIEHS P30 ES030285.

Acknowledgements

This work was performed at the Single Cell Genomics Core at BCM partially supported by NIH shared instrument grants (S10OD018033, S10OD023469, S10OD025240), P30EY002520 and CPRIT RP200504 to Rui Chen. We would like to acknowledge the Texas Children's Hospital William T. Shearer Center for Human Immunobiology for their generous support for this research. We would like to thank Dr. Alexandre Carisey and Ms. Rebecca Kairis from the Texas Children's Hospital William T. Shearer Center for Human

References (28)

  • B. Moser et al.

    gdT-APCs: a novel tool for immunotherapy?

    Cell. Mol. Life Sci.

    (2011)
  • R. Zheng et al.

    The role of gdTcells in allergic disease

    J Immunol Res

    (2014)
  • D. Kabelitz et al.

    Antigen recognition by human gamma delta T lymphocytes

    Int. Arch. Allergy Immunol.

    (2000)
  • C.P. Frossard

    Gut T cell receptor-gd(+) intraepithelial lymphocytes are activated selectively by cholera toxin to break oral tolerance in mice

    Clin. Exp. Immunol.

    (2015)
  • Cited by (9)

    • Multi-omics profiling approach in food allergy

      2023, World Allergy Organization Journal
    • Immunotherapy: State-of-the-art review of therapies and theratypes

      2022, Journal of Allergy and Clinical Immunology
      Citation Excerpt :

      After 24 weeks of peanut oral immunotherapy (OIT), naive and memory γδTreg cells stabilized to a similar level as in healthy controls. Transcriptomic studies in memory γδTreg cells showed that anti-inflammatory genes were upregulated and proinflammatory genes were downregulated.4 These cells might be an early marker of desensitization in subjects undergoing peanut OIT.

    View all citing articles on Scopus
    View full text