Overall magnitude and polarization of Bla g-specific T cell responses
The overall magnitude of CD4+ T cell CR-specific responses is shown inFigure 2a, where for each participant a bar represents the total cytokine response for all the different allergens combined. Each bar is further color coded to show which fraction of the response for each participant is accounted for IL-4 (blue), IFNγ (red), or IL-10 (green). The overall magnitude of baseline responses varied over approximately 2 logs across different subjects in the study cohort (ranging from 21 to 1,198 cells per million of CD4+ T cells).
This large variation between participants was not the result of assay-to-assay variability, as demonstrated by specific controls utilized in the assays. More specifically, in each assay, we also included a PBMC aliquot from an adult volunteer with a moderate degree of cockroach allergy, who underwent apheresis to provide numerous cell aliquots available for use. As shown in Supplementary Figure 1a, limited assay-to-assay variability was observed in repeated assays (n=42) from the lowest to the highest response in the 3.3-fold range, which is far less variable than variability observed within the participant cohort (57-fold). Furthermore, no significant difference was observed in the magnitude of response as a function of the different clinical sites from which the subjects were enrolled (Supplementary Figure 1b ).
Overall, IL-4 responses across the different participants were significantly larger than the IFNγ and IL-10 responses, approximately 2-fold and 4-fold larger, respectively (Figure 2b ). This was expected as all subjects in the study cohort were diagnosed with allergic asthma, and that a Th2 profile is a characteristic feature of allergic responses 39,40. To visualize the nature and degree of polarization as a function of each individual participant, the same data were plotted in an alternative format (Figure 2c ) where, for each participant, the percent of the total response to be ascribed to each of the three cytokines was plotted and grouped according with their polarization profile (>50% of total response). The responses in most participants (n=39/71) were IL-4 polarized (i.e., IL-4 is the dominant cytokine produced), while only 18 and 3 participants were IFNγ and IL-10 polarized, respectively. No clear polarization was noted for an additional 11 participants.
The data in Figure 2a indicate that the most vigorous responses were also the most polarized toward IL-4 production. Further analysis (Figure 2d ) found that the total magnitude (total cytokine response) correlated with Th2 (IL-4) responses (p <0.0001; R=0.59) and inversely correlated with Th1 (IFNγ) and IL-10 producing cells (p= 0.0009; R=-0.38 andp <0.0001; R=-0.53, respectively). These results suggest that stronger responses to CR allergens were more IL-4 polarized, and that weaker responses were associated with IFNγ and/or IL-10 production. Correlation between the different cytokine responses in each individual participant was also analyzed. As shown in Supplementary Figure 2 , IFNγ and IL-10 responses were highly correlated but IL-4 did not correlate with IFNγ or IL-10 responses.
Lastly, in these experiments, we also included a previously described megapool of Bordetella pertussis (BP) epitopes41. Children in our cohort, based on their year of birth, are expected to have been vaccinated with the acellular pertussis (aP) vaccine which is associated with a predominantly Th2 response42,43. As expected, and shown in Supplementary Figure 3a , BP responses in this cohort were Th2 polarized, with a median ratio of IL-4/IFNγ cytokine response of 2.5 and significant participant-to-participant variability. The degree of Th2 polarization observed in the BP responses did not correlate with the degree of polarization observed in the CR responses (Supplementary Figure 3b ), suggesting that there are factors determining the degree of Th2 polarization that are antigen/allergen-specific.