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