DISCUSSION
Here we report the direct ex vivo characterization of T cell responses to a panel of eleven previously described CR allergens in a cohort of CR allergic children and adolescents with well-controlled asthma. In particular, we found over 57-fold variation in magnitude of allergen-specific T cell responses. The more vigorous responses were also the most Th2 polarized and while the allergens Bla g 9 and Bla g 5 were most immunodominant, individual subjects exhibited distinctive patterns of allergen dominance. Subjects with higher magnitude of allergen-specific T cell reactivity had lower number of Treg populations. Overall, this study unveils a surprisingly high level of heterogeneity in the pattern of Bla g-specific T cell reactivity, both quantitatively and qualitatively. Since the cohort analyzed is enrolled in a future IT clinical trial, this finding lays the foundation for examining whether this heterogeneity may potentially correlate with IT outcomes.
This study is to the best of our knowledge, the first characterization of ex vivo CR-specific CD4+ T cell responses in CR-sensitized urban and under-resourced children with asthma, and for the majority of the known CR allergens to date, which are also components commonly found in CR extracts used for IT 2,10. Our results also differ from previous attempts of portrayal of CR-specific responses17,19,20, because of the high level of granularity and because the ex vivo approach allowed to characterize responding cells to a minimal gap from bona fide in vivo responses.
We identified a large dynamic range in terms of magnitude of CD4+ T cell responses, despite the narrow age range and similar clinical manifestations of disease among participants. Positive controls included in each assay excluded that this was due to inter-assay variability. Likewise, the variability was not explained by differential responses as a function of the different clinical sites from which each subject was enrolled. A high spread in the magnitude of T cell responses has been previously described for other allergic responses, namely to cat dander, fungus, mite and grass or birch pollen 30,46,47 usingex vivo methodologies similar to our study. It is possible that the ex vivo assay strategy utilized contributed to reveal this heterogeneity, as previous allergy studies in the case of CR16,18,20 or other allergens 24,27utilized in vitro restimulation protocols which might obscure and blunt differences.
We also analyzed the functionality of CD4+ T cell responses, which was dominated by IL-4 production and a Th2 polarization profile. Development of early allergic disease seems to be related to sustained Th2-skewed immunity during childhood 48 and previous studies have also demonstrated that levels of Th2 cytokines such as IL-4, IL-5, and IL-13 are associated with pathogenesis of both allergy and asthma13-15. In our study, Th2 polarization was the dominant phenotype but other patterns were also observed. In particular, while IL-10 responses were rare, a profile of IFNγ production or Th1 polarization was observed in one-fourth of the participants and associated with weaker T cell CR-specific responses. Interestingly, resolution of allergic-related immunopathologies is often also attributed to increase of IFNγ levels rather than reduction of Th2-cytokine production 49, and IL-10 is found to regulate CD4+ Th2 cells during allergic airway inflammation50. Therefore, the close monitoring of both IL-10 and IFNγ cytokines would be of interest in the context of IT and could provide new clues for the association of baseline patterns of polarization or dominant allergens and IT outcomes.
Bla g 9 and Bla g 5 were the most immunodominant allergens. Previous studies have reported Bla g 5 as a dominant T cell allergen10,16,17 but patterns of allergen dominance could be associated with different forms of allergic disease17. These observations were made in adult cohorts, and this raises the possibility that Bla g 9 recognition could be a pattern predominantly associated with childhood CR allergy. This pattern of immunodominance has been observed in subjects from the different clinical sites, irrespective of geographical location, further suggesting that Bla g 5 and Bla g 9 responses to CR are intrinsic to this particular age cohort. Changes in the IgE pattern of reactivity to individual Timothy grass and birch pollen allergens were observed over the course of 20 years for sensitized individuals 51. Future studies could examine whether changes in allergen immunodominance are apparent as a function of age and exposure history.
The pattern of reactivity as a function of the specific subject considered was also heterogeneous with different individuals recognizing either Bla g 9 or Bla g 5 as dominant, with dominant responses to other allergens or no pattern of dominance also observed. Individual immunodominance patterns were observed in adult cohorts16 and for IgE reactivity 52. Different HLA could influence allergen immunodominance. Associations between CR sensitization and HLA class II antigens have been suggested53,54 and consistently shown in food allergies55,56.
In addition, we measured basal levels of non-antigen specific regulatory T cells and observed that T cells inversely correlated with the effector CD4+ T cell responses although it did not reach statistical significance. These results were not unexpected and were consistent with previous observations from Bacher et al. 30. The fact that the population of Treg cells varies amongst participants is also of potential interest, as this will enable monitoring of Treg and addressing whether baseline Treg levels predicts response to IT. A possible outcome is that IT may induce or enhance regulatory T cell function, which can be further tested. Indeed, peanut oral immunotherapy has been shown to increase antigen-induced Treg function57.
Overall, the current study reveals that in a pediatric cohort of CR-sensitized subjects with mild-to-moderate asthma, subjects exhibit substantial heterogeneity in many key immunological parameters associated with CR-specific T cell responses. We hypothesize that this large dynamic range of T cell reactivity may influence outcomes of allergen-specific IT. These findings will also enable future research examining which of these parameters best predict the trajectory of evolution of allergic disease, and responsiveness to therapeutic intervention.