Ex Vivo Immune Response of Casein-Sensitized Lymphocytes to Bovine Milk Protein Composite Genotypes: Beyond the β-Casein A1/A2 Paradigm

Background: Although the impact of β-casein A1/A2 variants on human health is well-documented, the immunogenic potential of complex milk protein variants, including κ-casein (variants A, B, and E) and β-lactoglobulin (variants A and B) polymorphisms, remains poorly understood. This study aimed to evaluate ex vivo the cellular response of casein-sensitized lymphocytes to digests of milk defined for β-casein, κ-casein and β-lactoglobulin polymorphisms. Methods: BALB/c mice were injected intraperitoneally with casein (CAS group) or PBS (control group), and their lymphocytes were subsequently isolated and stimulated with simulated gastrointestinal digests of milk from cows with the genotypes: REF (A1A2/AB/AB), HM1 (A1A1/AA/AA), HM2 (A2A2/BB/BB), HT1 (A1A1/AE/AB), and HT2 (A1A1/BE/AB). We then analyzed the following: T-cell subpopulations (CD4+CD25+ and CD4+Fosp3+), proliferation, and the secretion (TNF-α, IL-6, IL-10, IL-17A, IL-4) and gene expression (IL-18, IL-1β, TGFβ1) of key cytokines. Results: Lymphocytes from sensitized mice (CAS) showed generally stronger responses to all treatments compared to non-sensitized PBS cells. Of note is the robust pro-inflammatory response of sensitized lymphocytes to HT1 and HT2 digests, as evidenced by a 9- to 10-fold increase in TNF-α and IL-6 secretion compared to the PBS counterparts. This response was uniquely associated with the presence of the κ-casein E variant. Notably, while the population of regulatory lymphocytes (CD4+CD25+ and CD4+Foxp3+) expanded significantly in casein-sensitized lymphocytes in response to all hydrolysates, this expansion was markedly impaired in PBS lymphocytes. Conclusions: Genetic polymorphism of milk proteins, particularly the presence of the κ-casein E variant, significantly modulates the pro-inflammatory potential of milk digests in sensitized lymphocytes. However, while these findings suggest new strategies for reducing milk immunogenicity, further mechanistic research is required to validate the role of the specific composition of protein variants in this phenomenon.