Antibodies, human milk oligosaccharides and T cells in early life immune development: the design, set up and first analysis of the PRIMA human milk cohort

This dissertation forms the foundation of both the clinical and fundamental aspects of the project. It established the cohort used in the clinical study and developed the method for measuring antibodies in breast milk. Additionally, the initial chapters present the first results on antibody and immune cell composition in early milk samples.

A central focus is Human Milk Oligosaccharides (HMOs)—unique, complex sugars found only in human milk. HMOs support infant health and immune development through three main mechanisms:
- Protection against pathogens: They promote beneficial gut bacteria and inhibit harmful microbes.
- Immune modulation: HMOs influence cytokine production, shaping the infant's immune responses.
- Gut barrier reinforcement: They help maintain intestinal integrity, reducing exposure to harmful substances.

The composition of HMOs varies significantly between mothers, largely due to genetic factors. This variation is critical to the diverse roles HMOs play in infant immunity and development.

Chapter 3 explores whether bovine immunoglobulin G (bIgG) can protect against respiratory syncytial virus (RSV). Findings show that bIgG can neutralize RSV and stimulate immune responses in humans, suggesting potential for preventive or therapeutic use, particularly in infants reliant on cow milk-based nutrition.

Chapter 4 outlines the design of the PRIMA cohort study, a multicenter, prospective birth cohort involving 1,000 mother-infant pairs recruited within one month of birth. Breast milk samples are collected regularly over the first six months and analyzed for immune-related components such as pathogen-specific antibodies, T cells, HMOs, and extracellular vesicles (EVs). Parental questionnaires track clinical outcomes biweekly.
The study’s primary outcome is the number of parent-reported respiratory infections requiring medical care. Secondary outcomes include doctor-diagnosed infections and allergies in the first year of life.

A significant contribution of this thesis is the development of a multiplex immunofluorescence assay (MIA) capable of measuring all nine antibody isotypes and subtypes (e.g., IgG1–4, IgA1–2) in small volumes of breast milk and blood. This allows for sensitive and reliable immunological profiling.

In Chapter 6, antibody levels in breast milk, amniotic fluid, and cord blood are compared. All nine antibody types are found in each fluid. Cord blood contains the highest levels overall, except for IgA2, which is most abundant in breast milk. These findings suggest selective maternal antibody transfer pathways, with different fluids playing unique roles in neonatal immunity.

Finally, Chapter 7 explores T cells in breast milk—immune cells previously uncharacterized in this context. Using single-cell RNA sequencing and flow cytometry, the study identifies tissue-adapted T cell populations involved in local immune regulation. These cells likely contribute to the maturation of the infant's immune system, particularly in mucosal tissues such as the gut.