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.