Variability in German Cockroach Extract Composition Greatly Impacts T Cell Potency in Cockroach-Allergic Donors

Abstract

German cockroach extract is used clinically to evaluate allergen-specific sensitization and for subcutaneous allergen-specific immunotherapy, though there are no guidelines for standardization in its manufacture. We performed an immunological evaluation of 12 different cockroach extracts prepared from different sources and their potency to induce allergen-specific T cell reactivity. PBMC from 13 cockroach allergic donors were expanded in vitro with 12 different German cockroach extracts. After culture expansion, cells were re-stimulated with the different extracts and T cell responses were assessed by FluoroSpot (IL-5, IFNγ and IL-10 production). In parallel to the extracts, single allergen peptide pools for allergens from groups 1, 2, 4, 5, and 11 were tested to determine allergen immunodominance. Furthermore, to assess allergy specificity, PBMC from 13 non-allergic donors were also tested with the most potent extract and T cell responses were compared to the allergic cohort. Dramatic variations in T cell reactivity were observed to the different cockroach extract batches. Response magnitudes varied over 3 logs within a single donor. IL-5 production in the allergic cohort was significantly higher compared to the non-allergic cohort (p=0.004). Allergen content determination by ELISA detected much lower concentrations of Bla g 5 compared to Bla g 1 and 2. Mass spectrometric analysis revealed that Bla g 5 was present in similar amounts to Bla g 1 and 2 in extracts made from whole body, whereas it was not detected in extracts made from fecal matter, suggesting that Bla g 5 is not excreted into feces. Different donors exhibit different response patterns to different extracts, potentially dependent on the donor-specific T cell allergen immunodominance pattern and the allergen content of the extract tested. These findings have dramatic implications for the selection of potent extracts used for diagnostic purposes or allergen-specific immunotherapy.

Keywords: German cockroach; T cells; allergen extract; cytokines; respiratory allergy.

Figure 1

Quantification of Bla g 1, 2, and 5 in 12 different German cockroach extracts by mass spectrometry (MS). Bar graphs showing the content of peptides derived from Bla g 1, 2, and 5 as detected by MS in 12 different extracts, including extracts for human use, veterinary use, research use and extracts made from German cockroach fecal matter/frass. Bars indicate median values of triplicate readings and error bars indicate 95% confidence interval. Of note, all 3 graphs have a different y-axis.

Figure 2

Variability in T cell potency and cytokine polarization in 12 different German cockroach extracts. (A) A line graph showing T cell reactivity (sum of IL-5, IFNγ, and IL-10 production) in response to 12 different preparations of German cockroach extract, measured in 5-fold titration steps (0.4, 2, 10, and 50 μg/ml). A dotted line indicates potency of extract to induce a T cell response of 500 spot forming cells (SFC). Geometric means 13 tested donors are shown, with error bars indicated 95% confidence interval (CI). (B–D) Bar graphs showing the IL-5, IFNγ, and IL-10 production. Bars indicate geometric means of 13 donors, with error bars indicating 95% CI. N = 13.

Figure 3

T cell reactivity measured as spot producing cells (SFC) in allergic (black dots) and non-allergic (open circles) individuals in response to extract 7. Bars indicate geometric means, error bars represent 95% confidence intervals. Statistical comparison was performed by Mann-Whitney test, two-tailed. P < 0.05 is considered significant. N = 13 per cohort.

Figure 4

Percent T cell reactivity to Bla g-derived peptide pools. Graphs showing T cell reactivity (sum of IL-5, IFNγ, and IL-10) in individual donors against each Bla g component peptide pool as percent of the sum of all Bla g pools. Donors are grouped based on their predominant T cell reactivity for (A) Bla g 1, (B) Bla g 2, (C) Bla g 5, and (D) Bla g 11. N = 12.

Figure 5

T cell reactivity in three donors in response to two different extracts. Total T cell reactivity (sum of IL-5, IFNγ and IL-10) measured as spot forming cells (SFC) in response to two selected extracts (extract 7 left; extract 12 right) in three individual donors was assessed.

Figure 6

Content of Bla g 1, 2, and 5 and T cell reactivity in response to two selected German cockroach extracts. (A) Content of Bla g 1, 2, and 5 in extract 7 and 12 as measured by mass spectrometry. (B–D) Total T cell reactivity (sum of IL-5, IFNγ and IL-10) against Bla g 1, 2, and 5 -specific peptide pools and extracts 7 and 12 in three different donors, each dominant for either (B) Bla g 1 (donor 1424), (C) Bla g 2 (donor 1398) or (D) Bla g 5 (donor 1437). Bla g pool responses and extract responses are segregated by a dashed line. The left axis applies to the Bla g pool responses (left panel), showing percent of T cell response attributed to each individual Bla g pool. The right axis applies to the extract responses (right panel), showing T cell response magnitude as measured by spot forming cells (SFC).