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.

中文翻译：

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花生口服免疫治疗前 24 周的记忆和幼稚 gamma delta 调节性 T 细胞基因表达

背景

花生口服免疫疗法 (POIT) 为花生过敏个体提供了脱敏作用。在 POIT 的前 24 周内存在有限的免疫学评估。

客观的

调节性 T 细胞 (Tregs) 是抗原诱导的免疫抑制性 T 细胞，对建立耐受性很重要。描述有助于花生脱敏发展的早期免疫学变化将有助于阐明 POIT 的作用机制。我们在治疗的前 24 周内对接受 POIT 的儿科受试者的 Treg 进行了单细胞 RNA 测序 (scRNAseq)，以评估 POIT 引起的早期免疫学变化。

方法

在 POIT 开始后 0、6 和 24 周收集并分析来自 5 至 12 岁花生过敏受试者的 PBMC 样本，并将样本与健康的非花生过敏对照进行比较。Tregs 从 PBMC 中富集，并进行了 scRNAseq 分析。使用 Cell Ranger 3.1.0 (10× Genomics) 识别细胞簇和差异表达基因，并使用 Seurat 套件 3.0.0 版分析结果。

结果

基因分析显示，与健康的非花生过敏状态相比，在 POIT 期间观察到的不同细胞类型对应的 10 个主要簇发生了变化。Tregs 的 scRNAseq 分析揭示了与 gdTregs 相关的强 CD3G 表达。gdTregs 的 scRNAseq 分析揭示了在治疗的前 6 周内发生的动态变化，并且在治疗 24 周时幼稚和记忆 gdTregs 的细胞频率降低到与健康对照相似的水平。使用 SingleR 对转录组细胞身份分析的分析显示，在 POIT 治疗 24 周后，gdTregs 中的基因表达与健康对照相似。scRNAseq 分析揭示了在此时间范围内记忆和幼稚 gdTregs 基因表达的变化。具体而言，OX40R (TNFRSF4)、GITR (TNFRSF18)、TGFB1、CTLA4、ISG20、

结论

在接受 POIT 治疗的前 24 周，花生过敏患者的外周幼稚和成熟 gdTreg 细胞有特定的表达谱，这表明参与维持免疫稳态的途径。gdTreg 细胞可能在 POIT 治疗的前 24 周内促成 POIT 的耐受性作用。这些发现表明，gdTregs 细胞可能是 POIT 受试者脱敏的早期标志物。