Serum analysis
In all collected blood samples, sIgE to 12 common food allergens (milk, egg, wheat, pea, soy, peanut, fenugreek, hazelnut, celery, cod, salmon and shrimp) were analyzed, as well as sIgE to rx6 (pollens from birch, timothy, mugwort, and mold (cladosporium and alternaria)) and rx7 (mite (d. pteronyssinus), cat dander, horse, dog, and rabbit epithelium) inhalant allergens (summarized in Table 1, serum levels reported in S. Table I). The control subjects (n=8) had no detectable sIgE to any of the allergens tested. The patients reporting adverse reactions to food were assigned to two groups based on the presence or absence of any sIgE to these food and inhalant allergens, hereafter called the IgEpos (n=11) and IgEneg (n=9) allergy groups. This grouping was confirmed by the presence or absence of sIgE also at the time of reporting to The Food Allergy Register (data not shown).
Participant characteristics are summarized in Table 1. The subjects in the control group, the IgEpos, and the IgEneg groups, were of both genders and ranging in age between 28-58, 20-68, and 24-62 years, respectively. Among the patients reporting adverse reactions to food with available clinical data at the time of reporting to the Food Allergy Register, all presented mild to severe symptoms affecting mostly skin, respiratory tract, and/or gastrointestinal tract (Table 1).
Total IgE and IgG4 in the participant’s sera, as well as the IgG4/IgE ratio, did not significantly differ between the groups (Suppl. Table I). There were also no significant differences between the groups for TNF-α, IL-6, IL-8 and MCP-1 in serum (S. Table I), while IL-1b serum levels were below the detection limit for most of the samples (data not shown).
Immunophenotyping of unstimulated cells – using antibody panel 1
Manual biaxial gating (Table 3) assessed the frequencies of traditionally defined immune cell lineages (T cells, B cells, monocytes, NK cells, and DC). No statistically significant differences were detected between the control group and the two allergy groups, nor between the IgEpos and IgEneg group in any of these cell populations (Figure 1).
In the CITRUS analyses, where cells are clustered based on their overall marker similarities, the abundances of cells in the cell clusters generated by clustering on all 33 surface markers (25 phenotyping and 8 functional/activation markers) or by clustering on the 25 phenotyping markers were not statistically significantly different between the three groups.
However, when assessing median marker intensities of the functional/activation markers, indicative for the quantitative marker expression per cell, several differences between the groups were observed. After clustering on the 25 phenotyping markers, the expression of CD371, CD69, CD23, CD25, CD28, and HLA-DR differed for nine different parent clusters (parent cluster denoting the statistically significantly stratifying cluster being highest in hierarchy) and several generations of child clusters (Figure 2A).
Cells in the eight parent clusters were identified as subpopulations of monocytes (Mo#1), Th cells (Th#1, Th#2, Th#3, Th#4), Tc cells (Tc#1, Tc#2), and B cells (B#1). The phenotypes of these subpopulations are presented as marker histograms in Figure 2C.
Monocytes in the Mo#1 cluster were CD28low CD69high HLA-DRhigh CD371high CD23low CD123high. Compared to the control group, this monocyte subpopulation had a statistically significantly increased expression of CD371 in both allergy groups, a statistically significantly increased expression of CD69 in the IgEneg group, and a decreased, although overall low, expression of CD23, reaching statistical significance only in the IgEneg group (Figure 2B). In support, up to five generations of child clusters had similar patterns of statistically significant differences with regard to the expression of CD371 and CD69, and CD23 (Figure 2A, S. Table II).
Th cells in cluster Th#1 were identified as being predominantly of the naïve and central memory type with a high expression of the Th2 cell marker CRTH2, being CD28high CD69low and CD25+. Compared to the controls, these cells had a statistically significantly increased expression of CD25 in both allergy groups, which tended to be stronger in the IgEpos group (Figure 2B). In support, up to four generations of child clusters had a similar pattern regarding CD25 expression (Figure 2A, S. Table II).
Like the monocytes above, Th cells in the clusters Th#2 and Th#3 had a statistically significantly decreased expression of CD23 in both allergy groups compared to the controls, supported by one to three generations of child clusters, although the expression of CD23 was in general very low in these clusters (Figure 2A,B). Both Th#2 and Th#3 cells were expressing CRTH2 and being CD28high CD69low and CD25low/+. Cells in the Th#2 cluster were predominantly of the naïve and central memory type, while cells in the Th#3 cluster were of the naïve type (Figure 2C, S. Table II).
Th cells in the Th#4 cluster were predominantly of the effector memory and effector type being CD28high CD69low CD25+ CD134low CD163low CD371low. Like the Th#1 cells, these Th#4 cells had an increased expression of CD25 in the IgEpos allergy group, being statistically significantly higher than the controls and in the IgEneg allergy group. The IgEpos group additionally had a statistically significantly increased expression of CD28 compared to the controls (Figure 2A, B).
Tc cells in cluster Tc#1 were predominantly of the naïve and effector type being CD28high CD69low HLA-DR+ (Fig 2A, S. Table II). In both allergy groups, the expression of CD28 was statistically significantly increased compared to the control group, supported by two generations of child clusters (Figure 2B).
Tc cells in theTc#2 and B cells in the B#1 clusters both had an increased expression of HLA-DR in the allergy groups compared to the controls, reaching statistical significance only in the IgEpos group (Figure 2B). Tc#2 cells were identified as being predominantly of the naïve and effector type being CD28+ CD69+ HLA-DR+ while the B#1 cells were predominantly of the naïve type being CD28low CD69+ CD25low HLA-DRhigh. Tc#2 cell population had one child cluster and the B#1 cell cluster had up to four generations of child clusters with the same patterns for HLA-DR expression in the three groups (Figure 2A, S. Table II).
Functional assessment of stimulated cells – using antibody panel 2
To assess functional differences between cells from the three groups of participants, intracellular cytokine expression and proliferation (Ki67) were assessed in addition to the main surface phenotyping markers, after cell stimulation with PMA and ionomycin.
When clustering on all 28 markers, statistically significant differences between the groups were identified in a branch of six parent/child cell clusters all being CD3+ T cells co-expressing high levels of TNF-α and IFN-γ (Figure 3A). The abundance of these cells was reduced in both allergy groups compared to the control group, although reaching statistical significance only in the IgEneg group (Figure 3B, S. Table II). The parent cell cluster branched into two child clusters characterized as Th cells predominantly of the effector and effector memory type and Tc cells predominantly of the effector and to a lesser extent of the naïve type.
The Th cells population contained further subpopulations, also expressing IL-2 and/or CD25, whereas the last generation child cluster of the Tc cells contained subpopulations also expressing IL-2 and/or HLA-DR. The phenotype of the Th and Tc parent clusters is presented as marker histograms in Figure 3C.
We further investigated group differences in the median marker intensities of CD69, Ki67, and the cytokines IL-2, IL-4, IL-5, IL-10, IL-13, IL-17A, IL-22, IFN-γ, and TNF-α. CITRUS identified group differences in the expression of TNF-α, IFN-γ, IL-17A, and IL-2. Most striking was the decreased (co)expression of TNF-α in numerous cell clusters in the two allergy groups compared to the controls. The differences in the median marker intensities are visualized for one representative subject of each group in viSNE maps Figure 4A. The phenotype of the cells in these clusters is presented as histograms in Figure 4C.
Cell clusters showing a statistically significantly (in SAM) simultaneous reduction in the expression of TNF-α and IFN-γ were identified as subpopulations of Tc cells (stimulated (s)Tc#1) and NK cells (sNK#1). In the Tc#1 subpopulation, only the reduced expression of IFN-γ in the IgEneg group was reaching statistical significance in the pairwise Kruskal-Wallis test compared to the controls. In the NK#1 cells, the expression of TNF-α was statistically significantly decreased in the IgEneg group compared to the controls and the IgEpos group, whereby the expression of IFN-γ was statistically significantly decreased only in the IgEneg group compared to the IgEpos group (Fig 4B,C, S. Table II). The sNK#1 cell cluster had up to three generations of child clusters and the sTc#1 cell cluster had one child cluster with the same significance pattern regarding TNF-α and IFN-γ expression (data not shown).
A subpopulation identified as Th cells (sTh#1) was significantly differing between allergy groups and the control group in the co-expression of TNF-α and IL-17A. These Th cells were characterized as being predominantly of the naïve type and to a lesser extent of the effector type and showed a tendency of a reduced expression of TNF-α in both allergy groups, although not reaching statistical significance, and an increased expression of IL-17A in the allergy groups, statistically significant only in the IgEpos group (Fig 4B,C, S. Table II). Up to two generations of child clusters had the same pattern regarding TNF-α and IL-17A expression (data not shown).
Two other subpopulations of Th cells (sTh#2, sTh#3) differed in the expression of TNF-α only (Fig 4B). In both clusters, TNF-α was expressed in low levels, but was statistically significantly decreased in the IgEneg group, whereby the IgEpos group had the same tendency. (Figure 4B, C, S. Table II). Cells in the sTh#3 cluster were characterized as being predominantly of the naive and effector type (Figure 4B, C, S. Table II). In support, up to five generations of child clusters of the sTh#2 cluster and two generations of child clusters of the sTh#3 clusters had a similar pattern regarding TNF-α expression (data not shown).
Lastly, one cluster differing in the expression of IL-2 was identified as a population of B cells (sB#1). B cells in that cluster had a decreased expression of IL-2 in the allergy groups, reaching statistical significance only in the IgEneg group (Figure 4B, C, S. Table II).
No group differences in expression of the other cytokines were observed.
After manual gating of CD4+ T, CD8+ T and NK cells according to S. Figure IIA, there was a reduction of TNF-α+ IFN-γ+, IL-2+ cells in both allergy groups, reaching statistical significance only in the IgEneg allergy group (Figure 4D). In Th cells, the group median percentage of TNF-α+ IFN-γ+ IL-2+ cells was 3.42 % in the control group, 0.80 % in the IgEpos group, and 0.14 % in the IgEneg group, while the group median percentage of TNF-α+ IFN-γ+ cells was 14 %, 1.85 %, and 0.71 %, respectively. In Tc cells, the group median percentage of TNF-α+ IFN-γ+ IL-2+ cells was 3.94 % in the control group, 0.39 % in the IgEpos group, and 0.05 % in the IgEneg group, while the group median percentage of TNF-α+ IFN-γ+ cells was 7.96 %, 2.56 %, and 0.77 %, respectively. In NK cells, the group median percentage of TNF-α+ IFN-γ+ IL-2+ cells was 3.34 % in the control group, 0.15 % in the IgEpos group, and 0.04 % in the IgEneg group, while the median percentage of TNF-α+ IFN-γ+ cells was 26.66 %, 0.80 %, and 0.15 %, respectively (Figure 4D). Cell percentages and median marker intensities for each of the cytokines individually are reported in S. Figure II.