Sequencing identifies specific milk protein causing eosinophilic esophagitis
Key takeaways:
- The patient had a clinically established eosinophilic esophagitis milk allergy.
- Conventional T cells that expressed eoeTCR-4 proliferated when researchers stimulated them with recombinant beta-casein.
Gene sequencing enabled researchers to identify the specific milk protein that triggered a patient’s eosinophilic esophagitis, according to a study published in The Journal of Allergy and Clinical Immunology.
This is the first time that an EoE trigger has been identified at the molecular level, David A. Hill, MD, PhD, attending physician with the division of allergy and immunology, Children’s Hospital of Philadelphia, and colleagues wrote.
“Unlike its more common cousin, anaphylactic food allergy — which causes immediate symptoms of hives, vomiting, and swelling — EoE is a chronic food allergy where the symptoms of chest and abdominal pain develop over months and years,” Hill told Healio.
As a result, Hill continued, diagnosing EoE and identifying the foods that cause it can be challenging.
“To develop the next generation of diagnostics and therapeutics for EoE, we need to know the specific parts of the immune system that are recognizing and responding to food allergens,” Hill said. “We also need to know what specific parts of the foods that we are eating are causing the disease.”
The researchers collected blood from a patient with clinically established EoE milk allergy and used milk protein to stimulate its mononuclear cells. Next, they used single-cell sequencing, which is “an innovative and state-of-the-art technique,” Hill said.
“This approach allows us to sequence the genetic material of single cells — in this case, the T cells that were responding to milk proteins,” Hill said. “Using this approach, we were able to identify the genetic sequence of the T-cell receptor that was recognizing milk in this patient.”
A subset of memory T-helper (Th) cells experienced clonal expansion and unique T-cell receptors were enriched because of the stimulation. Also, a pathogenic effector Th2 (peTh2) gene signature was upregulated in several clonotypes in the 20 T-cell receptor pairs that were most milk-expanded.
Calling them the most unique from one another among the peTh2 cell-high clonotypes, the researchers chose eoeTCR-4, eoeTCR-6, and eoeTCR-7 for functional testing because they would maximize the likelihood of identifying a milk-specific T-cell receptor.
The researchers then transduced the T-cell receptors into sort-purified CD25-CD4+ conventional T cells they acquired from a subject who was tolerant of milk and who shared or approximated all the patient’s human leukocyte antigen (HLA) DR and DQ alleles.
Also, the researchers tested the transduced conventional T cells for proliferation when cultured with dendritic cells that were protein-pulsed, autologous and derived from monocytes.
The conventional T cells that expressed eoeTCR-6 and eoeTCR-7 did not react to the milk proteins. Also, the conventional T cells that expressed eoeTCR-4 proliferated when the researchers stimulated them with recombinant beta-casein, but they did not proliferate when they were stimulated with other proteins.
Using a peptide library including amino acids 59 to 224 and the beta-casein-binding site predictions for the class II alleles that the patient and the control subject shared, the researchers determined that this proliferation was caused by peptide 1, comprising amino acids 59 to 78, and not by the other peptides.
Testing involving three more control subjects with the same class II alleles as the patient and first control showed that eoeTCR-4 is specific to peptide 1 and restricted to HLA-DRB1*07:01, establishing that eoeTCR-4 is specific to milk.
An examination of the RNA/TCR sequencing data further found the highest proportion of milk-expanded clones and most of the cells that expressed eoeTCR-4 in cluster 5 as well as increased IFN-alpha and IFN-gamma response genes in clusters 3, 5, and 8.
Additionally, genes related to activation, cytotoxicity, exhaustion, and regulation had elevated expression in the eoeTCR-4+ clones.
Based on this and other testing, the researchers said that eoeTCR-4 probably is a private T-cell receptor or rare enough that a larger number of patients with EoE milk allergy and the HLA-DRB1*07:01 allele would be needed to detect it. Plus, they continued, patients with EoE might or might not share the eoeTCR-4 antigen.
“Based on our knowledge of anaphylactic food allergy, we know that certain parts of foods tend to be more allergenic and therefore more likely to cause food allergy,” Hill said.
Consequently, he continued, the number of allergenic peptides in food proteins that primarily are responsible for causing EoE probably is limited.
“Nevertheless, a single EoE patient is likely allergic to multiple peptide components of the same food, and different EoE patients will be more or less allergic to different food peptides,” Hill said. “We need to expand this line of research to more patients to understand the full complement of molecules that mediate EoE milk allergy.”
Also, Hill said, similar studies should be performed involving patients with EoE caused by other foods, such as soy, wheat and egg. Although these data would aid in diagnosing EoE, he continued, applications for treatment remain on the horizon.
“Cell-specific treatments for EoE are a little further off than diagnostics, but the first step in being able to modify or delete a food-specific T cell is knowing the molecular identity of that cell,” Hill said.
“We are actively moving this line of research towards the development of novel clinical tests that will aid in the identification of EoE-causal foods and the development of test-directed personalized elimination diets,” he said.
Reference:
- Children’s Hospital of Philadelphia researchers lead first study to identify molecular basis of food allergy. https://www.chop.edu/news/childrens-hospital-philadelphia-researchers-lead-first-study-identify-molecular-basis-food. Published Feb. 4, 2025. Accessed Feb. 6, 2025.