Successful Milk Oral Immunotherapy Promotes Generation of Casein-Specific CD137+ FOXP3+ Regulatory T Cells Detectable in Peripheral Blood

Abstract

Background: Oral immunotherapy (OIT) is an emerging treatment for cow's milk protein (CMP) allergy in children. The mechanisms driving tolerance following OIT are not well understood. Regulatory T cells (T_REG) cells are key inhibitors of allergic responses and promoters of allergen-specific tolerance. In an exploratory study, we sought to detect induction of allergen-specific T_REG in a cohort of subjects undergoing OIT.

Methods: Pediatric patients with a history of allergic reaction to cow's milk and a positive Skin Pick Test (SPT) and/or CMP-specific IgE >0.35 kU, as well as a positive oral challenge to CMP underwent OIT with escalating doses of milk and were followed for up to 6 months. At specific milestones during the dose escalation and maintenance phases, casein-specific CD4⁺ T cells were expanded from patient blood by culturing unfractionated PBMCs with casein in vitro. The CD4⁺ T cell phenotypes were quantified by flow cytometry.

Results: Our culture system induced activated casein-specific FOXP3⁺Helios⁺ T_REG cells and FOXP3^- T_EFF cells, discriminated by expression of CD137 (4-1BB) and CD154 (CD40L) respectively. The frequency of casein-specific T_REG cells increased significantly with escalating doses of milk during OIT while casein-specific T_EFF cell frequencies remained constant. Moreover, expanded casein-specific T_REG cells expressed higher levels of FOXP3 compared to polyclonal T_REG cells, suggesting a more robust T_REG phenotype. The induction of casein-specific T_REG cells increased with successful CMP desensitization and correlated with increased frequencies of casein-specific Th1 cells among OIT subjects. The level of casein-specific T_REG cells negatively correlated with the time required to reach the maintenance phase of desensitization.

Conclusions: Overall, effective CMP-OIT successfully promoted the expansion of casein-specific, functionally-stable FOXP3⁺ T_REG cells while mitigating Th2 responses in children receiving OIT. Our exploratory study proposes that an in vitro T_REG response to casein may correlate with the time to reach maintenance in CMP-OIT.

Keywords: allergy; clinical trial; desensitization; milk immunotherapy; regulatory T cells; tolerance.

Figure 1

Successful OIT patients have increased levels of casein-specific IgG4 and whole milk SPT responses. (A) Typical approach to cow’s milk allergy immunotherapy. (B) SPT wheel size (mm) steadily decreased during CM-OIT in patients successfully achieving desensitization. (C) Casein-specific IgE (kU_A/L) levels in successful OIT patients at baseline (B) did not decrease significantly during the early escalation phase E, late escalation phase L or months after reaching maintenance M. (D) Casein-specific IgG4 (kU_A/L) steadily increased during CM-OIT in patients successfully achieving desensitization. Data is shown from 7 patients with each symbol representing a single patient. Casein-specific IgG4 levels were missing for P5. P-values were determined using a one-way ANOVA with a Dunn’s Multiple Comparison post-test (*p < 0.05, **p < 0.01).

Figure 2

Successful desensitization is characterized by expansion of IFN-γ-producing, but not IL-4-producing T_EFF cells following in vitro restimulation with casein. Representative flow cytometry plots from controls lacking PMA stimulation, early phase and late phase identifying (A), CD4⁺ IFN-γ⁺ T_EFF cells, and (B) CD4⁺ IL-4⁺ T_EFF cells emerging in patient PBMC after a 10 day culture in the presence of casein. (C) Proportions of CD4⁺ IFN-γ⁺ T_EFF cells increased with dose escalation. (D) Proportions of CD4⁺ IL-4⁺ T_EFF cells from culture with casein decreased with dose escalation. (E) Ratios of CD4⁺ IFN-γ⁺ T_EFF to CD4⁺ IL-4⁺ T_EFF from culture with casein increased with dose escalation. Data is shown from 5 patients. P-values were determined using a Wilcoxon Signed Rank non-parametric test.

Figure 3

FOXP3⁺Helios⁺ is a stringent definition for T_REG cells. PBMC from a representative CMA patient before and after tolerization were stimulated with TT or αCD3 for 4 days before staining for T_REG cells in flow cytometry. (A) Sample flow cytometry plots showing CD25^HighCD127^Low T cells, and (B) FOXP3⁺Helios⁺ T_REG cells both pre-gated on CD4⁺ T cells. (C, D) The proportion of CD4⁺ cells captured by either CD25^HighCD127^Low gating or FOXP3⁺Helios⁺ gating that were exclusive to either CD25^HighCD127^Low or FOXP3⁺Helios⁺ gates were plotted in (C) with the degree of overlap between both populations shown in Euler-diagrams in (D) Cultures were completed in triplicates from a single patient’s PBMC (N=3). P-values were determined using a two-way ANOVA with a Tukey’s post-test (*p < 0.05, **p < 0.01, ***p < 0.001). Bars represent the mean ± s.d.

Figure 4

CD137 and CD154 differentially identify casein-specific T_REG and casein-specific T_EFF cells. Proliferation of CD4⁺ cells was assessed by flow cytometry-based CTV dilution analysis. (A, B) Healthy, non-allergic PBMC was cultured in the presence of casein or TT. (A) Representative flow cytometry plots of FOXP3⁺ T cells depicting CTV dilution in CD4⁺ T cells alongside (B), the quantification (N=3). (C–E) Patient PBMC was cultured in the presence of casein for 10 days before evaluating expanded T cell responses by flow cytometry. (C) Flow cytometric gating strategy using a representative sample identifying proliferative (CTV^-, top panel) and non-proliferative (CTV⁺, bottom panel) T_REG cells (FOXP3⁺Helios⁺) expressing CD137 and proportion of T_EFF (FOXP3^-Helios^-) expressing CD154 from a representative patient. (D) Expression of CD137 was significantly higher in proliferative FOXP3⁺Helios⁺ T_REG cells expanded in patient PBMC (N=3). (E) CD154 expression was significant higher in proliferative FOXP3^-Helios^- T_EFF cells expanded in patient PBMC (N=3). The P-value in B was determined using unpaired t-test. P-values in (C, E) were determined using a Wilcoxon Signed Rank non-parametric test (*p < 0.05). Bars represent the mean ± s.d.

Figure 5

Casein-specific CD137⁺ T_REG cells express higher levels of FOXP3. (A) Representative flow cytometric plots identifying CD137⁺ and CD137^- T_REG (FOXP3⁺ Helios⁺) cells. (B, C) FOXP3 mean fluorescence intensity (MFI) is significantly higher in CD137⁺ T_REG cells than in CD137^- T_REG cells during all phases of CM-OIT (N=3). (D, E) Helios MFI is significantly higher in CD137⁺ T_REG cells than in CD137^- T_REG cells at L and M phases of CM-OIT (N=3). P-values were determined using a Wilcoxon Signed Rank non-parametric test (*P < 0.05). Bars represent the mean ± s.d.

Figure 6

Induction of casein-specific T_REG cells correlated with tolerance, suppressed Th2 responses, and with escalation days to maintenance. (A) Proportion of Helios⁺FOXP3⁺ T_REG cells and (B), proportion of proliferative (CTV^low) Helios⁺FOXP3⁺ T_REG cells from total CD4⁺ T cells expanded in our in vitro culture system with casein do not change significantly during E, L and M phases of CM-OIT. (C) When differentiating T_REG based on CD137 expression, we observe that casein-specific CD137⁺ proliferative T_REG increase during Early, Late and Maintenance phase in successful CM-OIT patients. (D) There was no significant reduction in the proportions of CD154⁺ proliferative T_EFF cells during CM-OIT. (E, G) The induction of CD137⁺ proliferative T_REG correlated with an increase in the CD4⁺IFN-γ⁺ T_EFF cells from culture with casein and the ratio of CD4⁺IFN-γ⁺ T_EFF to CD4⁺IL-4⁺ T_EFF during Early and Late phase. (F) There was also a trend of correlation between CD137⁺ proliferative T_REG and CD4⁺ IL-4⁺ T_EFF cells from culture with casein, although there is a no significance. (H) There is a negative correlation between the proportions of CD137⁺ proliferative T_REG at (E) and escalation days to maintenance. (I, J) There was also a trend of correlation between the proportions of CD137⁺ proliferative T_REG at Late and Maintenance phase. and escalation days to maintenance, albeit no significance. Each symbol represents 1 subject. Of 7 patients, 5 patients from E and L phase are involved in analysis/figure (E–G). Yellow symbols represent data at Early phase Blue symbols represent data at Late phase. Red symbols represent data at Maintenance phase. P-values in (A–D) were determined using a one-way ANOVA with Dunn’s multiple comparisons and in (E–J) with a Pearson correlation (*p < 0.05, n.s, not significant). Bars represent the mean ± s.d.