Identification of cow milk epitopes to characterize and quantify disease-specific T cells in allergic children.

BACKGROUND

Cow milk (CM) allergy is the most prevalent food allergy in young children in the United States and Great Britain. Current diagnostic tests are either unreliable (IgE test and skin prick test) or resource-intensive with risks (food challenges).

OBJECTIVE

We sought to determine whether allergen-specific T cells in CM-allergic (CMA) patients have a distinct quality and/or quantity that could potentially be used as a diagnostic marker.

METHODS

Using PBMCs from 147 food-allergic pediatric subjects, we mapped T-cell responses to a set of reactive epitopes in CM that we compiled in a peptide pool. This pool induced cytokine responses in in vitro cultured cells distinguishing subjects with CMA from subjects without CMA. We further used the pool to isolate and characterize antigen-specific CD4 memory T cells using flow cytometry and single-cell RNA/TCR sequencing assays.

RESULTS

We detected significant changes in the transcriptional program and clonality of CM antigen-specific (CM+) T cells elicited by the pool in subjects with CMA versus subjects without CMA ex vivo. CM+ T cells from subjects with CMA had increased percentages of FOXP3 cells over FOXP3 cells. FOXP3 cells are often equated with regulatory T cells that have suppressive activity, but CM+ FOXP3 cells from subjects with CMA showed significant expression of interferon-responsive genes and dysregulated chemokine receptor expression compared with subjects without CMA, suggesting that these are not conventional regulatory T cells. The CM+ FOXP3 cells were also more clonally expanded than the FOXP3 population. We were further able to use surface markers (CD25, CD127, and CCR7) in combination with our peptide pool stimulation to quantify these CM+ FOXP3 cells by a simple flow-cytometry assay. We show increased percentages of CM+ CD127CD25 cells from subjects with CMA in an independent cohort, which could be used for diagnostic purposes. Looking specifically for T2 cells normally associated with allergic diseases, we found a small population of clonally expanded CM+ cells that were significantly increased in subjects with CMA and that had high expression of T2 cytokines and pathogenic T2/T follicular helper markers.

CONCLUSIONS

Overall, these findings suggest that there are several differences in the phenotypes of CM+ T cells with CM allergy and that the increase in CM+ FOXP3 cells is a potential diagnostic marker of an allergic state. Such markers have promising applications in monitoring natural disease outgrowth and/or the efficacy of immunotherapy that will need to be validated in future studies.