Nasal peanut + CpG immunotherapy enhances peanut-specific IFN-γ in Th2 cells and IL-10 in non-Th2 cells in mice.

To the Editor:

Peanut-specific allergen immunotherapy reduces peanut-specific serum IgE, T helper 2 (Th2) cell cytokines, IL-5 and IL-13, and increases peanut-specific serum IgG, IgG4, mucosal IgA and T regulatory (Treg) cells^{(1, 2)} in hypersensitive individuals. However, it is not well understood how peanut-specific Th2 cells change after immunotherapy. This study aims to evaluate potential mechanisms allergen-specific immunotherapy may use to reduce Th2 responses in peanut-hypersensitive mice.

Utilizing a mouse nasal allergen-specific immunotherapy (NIT) model, we confirmed the beneficial effects of including the toll-like receptor 9 ligand, CpG, as a Th1-enhancing adjuvant in peanut NIT^{(3, 4)} to reduce allergic disease severity. The comparison of CpG-adjuvanted to unadjuvanted peanut-specific NIT was performed in IL-13 lineage-tracing mice to evaluate the contribution of CpG combined with peanut on peanut-specific Th2 cells in hypersensitive mice. This study tested the hypothesis that CpG-adjuvanted peanut NIT transitioned peanut-specific Th2 cells into peanut-specific Th1 and/or Treg cells.

Peanut-hypersensitivity was induced in C57BL/6J mice by gastric gavage with peanut extract (PN) and cholera toxin (CT) (Supporting Figure 1). PN-specific immunity induced by sensitization was similar amongst all peanut-hypersensitive mice since there were no differences in serum and fecal antibodies measured prior to immunotherapy (Supporting Figure 2). After a four-week rest, peanut-hypersensitive mice were nasally treated with saline (placebo), peanut alone (PN), CpG alone (CpG) or peanut + CpG (PN+CpG), three times a week for four weeks. Post-immunotherapy, PN and PN+CpG enhanced PN-specific serum and mucosal antibodies compared to placebo and CpG groups(Figure 1A,B).PN+CpG also increased PN-specific serum IgG2c and fecal IgA compared to PN. NIT was specific to PN responses since CT-specific antibodies were similar among all treatment groups after immunotherapy (Supporting Figure 3).

Figure 1: CpG enhances host responses influenced by peanut-specific immunotherapy and reduce allergen severity.

Peanut-hypersensitive C57BL/6 mice received saline (placebo), PN, CpG or PN+CpG NIT. PN-specific A) serum and B) fecal antibodies reported as geometric mean titers (GMT) were measured after therapy (N=14–41). Systemic anaphylaxis was induced with PN. C) Core body temperature and D) allergy clinical symptom scores were measured for one hour after peanut-exposure (N=5–25) and reported as the mean + standard deviation. Statistical differences in antibodies were determined by one-way ANOVA with Tukey’s multiple comparison (p<0.05). Numbers denote a significant increase compared to corresponding therapy group. Two-way ANOVA determine a significant reduction in disease severity compared to placebo indicated by * (p<0.05).

One-week post-immunotherapy, PN-induced anaphylaxis determined if NIT reduced allergy severity. Body temperature and allergy clinical symptoms were monitored for one hour post-challenge (Figure 1C, D).Unsensitized mice maintained stable body temperature after challenge; however, PN-hypersensitive mice rapidly displayed hypothermia post-PN exposure. Hypersensitive mice treated with PN+CpG began to recover from hypothermia and demonstrate improved allergic symptoms 60 minutes post-challenge compared to placebo-treated mice while neither PN nor CpG treatments were effective. Our model suggests both PN and CpG are required for NIT to reduce PN-induced anaphylaxis severity.

PN-specific T cell cytokines that may suggest disease severity after NIT (Figure 2) were evaluated. PN-induced IL-13, IFN-γ and IL-10 were measured to monitor Th2, Th1 and Treg responses, respectively. Cells from PN-sensitized mice secreted more IL-13 after PN-stimulation compared to unsensitized mice (Figure 2A); but, IL-13 secretion was significantly reduced by PN+CpG compared to placebo, PN and CpG. PN-induced IL-10 (Figure 2B) and IFN-γ (Figure 2C) secretion was increased in PN-sensitized mice compared to unsensitized mice but PN+CpG further enhanced IL-10 and IFN-γ compared to the other immunotherapy groups. Thus, PN+CpG therapy decreases IL-13 and increases IFN-γ and IL-10.

Figure 2: Allergen-specific immunotherapy modifies PN-induced cytokine responses.

Peanut-induced responses were determined by subtracting values in unstimulated cells from peanut-stimulated cells. Significant differences in peanut-induced secreted A) IL-13 B) IL-10 and C) IFN-γ, measured post-immunotherapy was determined by one-way ANOVA with Tukey’s multiple comparisons (p<0.05). Numbers above denote increased cytokine responses compared to corresponding treatment group below (n=9–17). IL-13 lineage-tracing mice revealed changes in peanut-induced cytokine positive cells (D-E) after PN or PN+CpG therapy (n=7–8). CD4+ splenocytes were monitored for IL-13 lineage by TdTomato (Tom) expression, D) IL-10 and E) IFN-γ. Mann-Whitney determined significant differences in peanut-induced cytokine positive cells between PN and PN+CpG-treated mice based on IL-13 expression. *indicates significant increase in cytokines compared to PN alone (p<0.05).

To test the hypothesis that CpG-adjuvanted peanut NIT induced the transition of peanut-specific Th2 cells into Th1 and/or Treg cells, CD4+ cells from PN-hypersensitive IL-13-lineage-tracing mice treated with PN or PN+CpG NIT were assessed for peanut-induced IL-10 and IFN-γ. PN+CpG increased PN-induced IL-10+ cells from non-Th2 cells (Tom-) (Figure 2D) and IFN-γ+ from Th2 cells (Tom+) (Figure 2E) compared to PN NIT. There was no difference in IL-13+ cells after PN+CpG and PN NIT. Our data suggest that PN+CpG enhances the number of IL-10+ and IFN-γ+ cells without effecting the number of IL-13+ cells compared to NIT with PN alone.

This study demonstrated enhanced PN-specific serum IgG2c, mucosal IgA, IL-10 and IFN-γ, and decreased IL-13 in mice with improved allergy severity after PN+CpG NIT. PN+CpG increased IFN-γ in CD4+ T cells that once produced IL-13 (i.e., Th2 cells) and IL-10 in non-Th2 cells (never produced IL-13) in IL-13 lineage-tracing mice. These results suggest, for the first time, that CpG-adjuvanted peanut NIT can modulate IL-13-producing Th2 cell cytokine profile to enhance IFN-γ and generate additional IL-10 producing cells, which may reduce anaphylaxis severity.

PN+CpG induced peanut-specific immune responses in PN-hypersenstive mice similar to peanut-allergic individuals who have achieved immunological tolerance, which reduces allergen-specific Th2 responses, after PN immunotherapy^{(1, 2)}. PN NIT enhanced sensitization-induced antibody responses compared to placebo or CpG. However, PN+CpG further boosted PN-specific serum IgG2c and mucosal IgA responses compared to NIT with PN alone and increased peanut-induced IFN-γ and IL-10 while decreasing peanut-induced IL-13 secretion, suggesting a shift from Th2 immunity towards Th1 and Treg immunity. Our results support clinical observations that also report decreased Th2-associated immunity and increased Th1 and/or Treg immunity⁽²⁾.

Changes in secreted IL-13, IL-10 and IFN-γ after PN+CpG immunotherapy led us to investigate the cellular source of these cytokines. We hypothesized that adjuvanted-immunotherapy may change PN-specific T cell cytokines through at least two mechanisms including, 1) enhanced production of Th1 and Treg cytokines in Th2 cells and/or 2) generation of peanut-specific Th1 or Treg cells from naïve precursor T cells that reduce Th2 responses. Th2 cells produce several cytokines including, IL-4, IL-5 and IL-13; however, IL-13 is involved in tissue Th2 responses⁽⁵⁾, and may contribute to allergic symptoms. Our study demonstrates reduced IL-13 secretion with PN+CpG NIT compared to PN NIT but similar numbers of IL-13+ CD4+ cells, which suggests that PN+CpG therapy decreases the amount of IL-13 produced without effecting the number of IL-13+ cells. One mechanism that may reduce IL-13 production is enhanced plasticity in Th2 cells to acquire cytokines that suppress Th2 cytokine responses. IL-13 may be produced by Th1 and Th17 cells⁽⁶⁾; however, low Th1 transcription factor levels are observed in IL-13+/IFN-γ+ cells⁽⁶⁾, suggesting that the IL-13+IFN-γ+ cells are plastic Th2 cells evolving to acquire a Th1 phenotype. We observed increased IFN-γ in CD4+IL-13+ T cells after IT, which may support Th2 cell plasticity. Th2 to Th2+Th1 cell plasticity is reported in mouse effector and memory Th2 cells that produce IFN-γ after in vivo activation with Th1-inducing stimuli⁽⁷⁾. Therefore, it is possible that the addition of CpG to PN immunotherapy formulations increase IFN-γ in Th2 cells to reduce pro-allergy effector cytokines.

PN+CpG may also enhance cytokine responses from non-Th2 cells to suppress IL-13 secretion. Enhanced IL-10 in CD4+IL-13- T cells supports an important role for IL-10 in reducing allergic disease severity, since immunological tolerance to peanut is not achieved in the absence of IL-10⁽⁸⁾. Naïve T cells exposed to PN+CpG may possibly differentiate into IL-10+ effector T cells to suppress allergic Th2 responses. Peanut oral immunotherapy (OIT) reduces IL-4+ cells and increases IFN-γ+ cells in peanut-hypersensitive subjects⁽⁹⁾. Since similar cytokine profiles in specific T cell clones were observed in subjects receiving OIT as placebo therapy, it is unlikely that OIT enhanced Th2 cell plasticity but instead increased non-Th2 cells that minimize the effects of the Th2 cells⁽⁹⁾. While our data also agree with peanut immunotherapy generating non-Th2 cells such as, IL-10+ cells, to reduce allergic Th2 cell effects and desensitize or tolerize the host, it is possible that including potent Th1-enhancing adjuvants such as, CpG, to clinical peanut immunotherapy formulations will both generate new T cells and induce Th2 cell plasticity to, more effectively, reduce allergic disease severity compared to therapy with peanut allergens alone.

Collectively, our study suggests CpG-adjuvanted PN NIT may generate new IL-10+ T cells and induce Th2 cell plasticity to enhance IFN-γ to reduce allergy severity. This information may provide insight towards developing improved peanut allergy immunotherapy formulations.