Prophylactic Peanut Allergen Ara h 6 Sublingual Immunotherapy Drives Expansion of FoxP3 +Helios- Regulatory T Cells in the Absence of Allergen-Specific IgA

Abstract

Insights into the underlying immunological mechanisms of prophylactic sublingual immunotherapy (SLIT) may support the development of new strategies for improved prevention and treatment of food allergy. Here, we investigated the humoral, regulatory and sublingual tissue immune response to prophylactic SLIT administration of a single purified peanut allergen in Brown Norway (BN) rats. BN rats received daily sublingual administration of peanut allergen Ara h 6 for three weeks. Suppression of sensitisation was evaluated by subsequent intraperitoneal administration of Ara h 6. Ara h 6-specific IgE, IgA, IgG1 and IgG2a-c levels were measured in serum. The frequency of regulatory T (Treg) cells was analysed using flow cytometry. The sublingual tissue response to Ara h 6 was analysed by transcriptional profiling using mRNA-sequencing. Ara h 6 SLIT protected rats from subsequent sensitisation without inducing a detectable humoral immune response (Ara h 6-specific IgE, IgA, IgG1 and IgG2a-c) in serum. SLIT furthermore promoted the relative expansion of induced Helios^- Treg cells within the conventional CD4⁺CD25⁺FoxP3⁺ Treg population in sublingual draining lymph nodes and blood. In conclusion, prophylactic Ara h 6 SLIT drives the relative expansion of induced Helios^- Treg cells in the absence of Ara h 6-specific IgA highlighting a potential novel IgA-independent Treg-related immune response at the sublingual mucosal site.

Keywords: immune response; peanut allergy; prophylaxis; sublingual immunotherapy; tissue response.

FIGURE 1

Sensitisation, clinical reactivity and protein uptake. Animal experimental design (A). Ara h 6‐specific IgE levels in serum (B). Ear swelling test (EST) response to intradermal injections of Ara h 6 (C) or peanut protein extract (PPE) on Day 54 (D). Correlation between EST response to Ara h 6 and Ara h 6‐specific IgE levels (E). Protein uptake measured as Ara h 3 levels in small intestinal compartments (F) and serum (G) following oral gavage of PPE and sacrifice on Day 56. n = 8/group. EPI, epithelium; IP, intraperitoneal; LP, lamina propria; PP, Peyer's patches; SLIT, sublingual immunotherapy. *p < 0.05, **p < 0.01, and ***p < 0.001.

FIGURE 2

Humoral immune responses. Ara h 6‐specific IgA (A), IgG1 (B), IgG2a (C), IgG2b (D), and IgG2c (E) in serum following prophylactic Ara h 6 SLIT (Day 28) and subsequent intraperitoneal immunisations (Day 56). n = 8/group. *p < 0.05, **p < 0.01, and ****p < 0.0001.

FIGURE 3

IgA immune responses. Total IgA in serum (A) and faecal samples (B), and Ara h 6‐specific IgA in faecal samples (C) following prophylactic Ara h 6 SLIT (Day 28) and subsequent intraperitoneal immunisations (Day 56). n = 8/group.

FIGURE 4

Regulatory T cell responses. The frequency of Helios⁻ induced regulatory T cells (iTreg) in sublingual draining lymph nodes (SL‐LN), blood and small intestine compartments following the Ara h 6 SLIT regiment (Day 21) (A) and the subsequent intraperitoneal immunisation regiment (Day 56) (B). n = 8/group. mLN, mesenteric Lymph Nodes; PP, Peyer's patches; SI, small intestine. n = 8/group. *p < 0.05.