A mouse model of the LEAP study reveals a role for CTLA-4 in preventing peanut allergy induced by environmental peanut exposure

Abstract

Background: A human study, Learning Early About Peanut Allergy (LEAP), showed that early introduction of peanut products decreases the prevalence of peanut allergy among children. However, the immunologic mechanisms mediating the protective effects of consuming peanut products are not well understood.

Objective: The objective was to develop a mouse model that simulates the LEAP study and investigate the underlying mechanisms for the study observations.

Methods: Adult naive BALB/c mice were fed a commercial peanut butter product (Skippy) or buffer control and concomitantly exposed to peanut flour through the airway or skin to mimic environmental exposure. The animals were analyzed for anaphylactic reaction and by molecular and immunologic approaches.

Results: After exposure to peanut flour through the airway or skin, naive mice developed peanut allergy, as demonstrated by acute and systemic anaphylaxis in response to challenge with peanut extract. Ingestion of Skippy, however, nearly abolished the increase in peanut-specific IgE and IgG and protected animals from developing anaphylaxis. Skippy-fed mice showed reduced numbers of T follicular helper (Tfh) cells and germinal center B cells in their draining lymph nodes, and single-cell RNA sequencing revealed a CD4⁺ T-cell population expressing cytotoxic T lymphocyte-associated protein 4 (CTLA-4) in these animals. Critically, blocking CTLA-4 with antibody increased levels of peanut-specific antibodies and reversed the protective effects of Skippy.

Conclusion: Ingestion of a peanut product protects mice from peanut allergy induced by environmental exposure to peanuts, and the CTLA-4 pathway, which regulates Tfh cell responses, likely plays a pivotal role in this protection.

Keywords: CTLA-4; Follicular T cells; IgE; allergens; allergy; peanuts.

Figure 1.

Oral administration of peanut butter prevents peanut allergy induced by intranasal (i.n.) exposure to peanut flour. (A) Schematic overview of the experimental protocol. (B) Plasma levels of peanut-specific IgE, IgG1, and IgG2a are shown. Serial dilution of plasma was used to determine antibody titers. (C) Plasma levels of Ara h 1-specific IgE are shown. Each dot represents one mouse. (D) After intraperitoneal challenge with peanut extract, changes in rectal temperature and clinical scores were monitored. Data are presented as the mean ± SEM (n=4–6 in each group) and are representative of six experiments. **P<0.01 compared to mice fed control buffer and subjected to i.n. peanut flour exposure or between the groups indicated by horizonal lines. (E) Schematic overview of the experimental protocol for Panels F and G. (F) Plasma levels of peanut-specific IgE and IgG1 are shown. (G) Changes in rectal temperature and clinical scores were monitored. Data are presented as the mean ± SEM (n=6 in each group). *P<0.05 and **P<0.01 compared to mice that did not consume P.B. Bites and subjected to i.n. peanut flour exposure. P.B. Bites, Skippy™ Peanut Butter (P.B.) Bites.

Figure 2.

Oral administration of peanut butter prevents peanut allergy induced by epicutaneous (e.c.) exposure to peanut flour. (A) Schematic overview of the experimental protocol. (B) Plasma levels of peanut-specific IgE, IgG1, and IgG2a are shown. (C) Plasma levels of Ara h 1-specific IgE are shown. Each dot represents one mouse. (D) After intraperitoneal challenge with peanut extract, changes in rectal temperature and clinical scores were monitored. Data are presented as the mean ± SEM (n=4–8 in each group) and are representative of two experiments. *P<0.05, **P<0.01 compared to mice fed control buffer and subjected to i.n. peanut flour exposure or between the groups indicated by a horizonal line.

Figure 3.

Oral administration of peanut butter suppresses T follicular helper (Tfh) cell development in mediastinal lymph nodes (mLNs). (A) Schematic overview of the experimental protocol. (B) Total number of mLN cells. (C) Representative scattergrams from FACS analysis of mLN cells are shown. (D) The proportions and total numbers of Tfh cells. Data are presented as the mean ± SEM and are representative of two experiments. *P<0.05, **P<0.01 between the groups indicated by horizonal lines. (E) Schematic overview of the experimental protocol for Panel F. (F) Cytokine production by mLN cells in vitro. Data are presented as the mean ± SEM. **P<0.01 between the groups indicated by horizonal lines. P.B. Bites, Skippy™ Peanut Butter (P.B.) Bites; n.s., not significant.

Figure 4.

Oral administration of peanut butter does not affect the proportion of Foxp3⁺ T regulatory (Treg) cells. (A) Schematic overview of the experimental protocol. (B) Representative scattergrams from FACS analysis of mediastinal LN (mLN) cells and the proportions of Treg cells are shown. Data are presented as the mean ± SEM in each group and are representative of three experiments. **P<0.01 between the groups indicated by horizonal lines. (C) Schematic overview of the experimental protocol for Panels D and E. (D) Representative scattergrams from FACS analysis of mesenteric LN cells are shown. (E) The proportions and total numbers of Treg cells are shown. Data are presented as the mean ± SEM in each group. P.B. Bites, Skippy™ Peanut Butter (P.B.) Bites.

Figure 5.

The mRNA transcript for T-lymphocyte-associated protein 4 (Ctla4) and transcripts of other Treg cell-related genes are enriched in CD4⁺ T cells from mLNs of peanut-butter fed mice. (A) Schematic overview of the experimental protocol. (B) Unsupervised t-distributed stochastic neighbor embedding (t-SNE) plots of CD4⁺ T cells are shown. (C) t-SNE plots of selected genes are shown. (D) Foxp3^eGFP mice were treated as described in (A), and mRNA transcript for Ctla4 in sorted Foxp3⁻ and Foxp3⁺ cells was analyzed by qRT-PCR. Data are presented as the mean ± SEM in each group. *P<0.05 between the groups indicated by a horizonal line.

Figure 6.

Anti-CTLA-4 treatment promotes development of Tfh and germinal center (GC) B cells in mLNs. (A) Schematic overview of the experimental protocol. (B) Total number of cells in mLNs. (C) Representative scattergrams from FACS analysis of mLN cells are shown. (D) Total numbers of Tfh cells in mLNs. (E) Representative scattergrams from FACS analysis of mLN cells are shown. (F) Total numbers of GC B cells in mLNs. Data are presented as the mean ± SEM (n=4) in each group and are representative of two experiments. *P<0.05 and **P<0.01 between the groups indicated by horizonal lines.

Figure 7.

Anti-CTLA-4 treatment reverses the tolerogenic effects of oral peanut butter. (A) Schematic overview of the experimental protocol. (B) Titers of peanut-specific IgE, IgG1, IgG2a, and IgG2b antibodies in plasma are shown. (C) After intraperitoneal challenge with peanut extract, changes in rectal temperatures and clinical scores were monitored. Data are presented as the mean ± SEM (n=4 in each group) and are representative of two experiments. #P<0.05 and ##P<0.01 compared to mice fed control buffer, treated with isotype control, and subjected to i.n. peanut flour exposure. *P<0.05 and **P<0.01 compared to mice fed peanut butter, treated with isotype control, and subjected to i.n. peanut flour exposure.