Figure legends
Figure 1. Alterations of T cell subsets and the regulatory T cell (Treg) phenotype in neonates between birth and 3 weeks of age.a, The frequency of CD3+, CD4+ and CD8+ cells in neonatal blood samples (n = 17) at birth and at 3 weeks of age, as well as in maternal blood (n = 17) and healthy controls (n = 8). b, The frequency of CD4+ FoxP3+ CD25hi cells in neonatal blood samples (n = 19) at birth and at 3 weeks of age, as well as in maternal blood (n = 19) and healthy controls (n = 13). c, The frequency of CD4+ FoxP3+ CD25hi cells in neonatal blood samples at 3 weeks of age grouped according to the feeding method: exclusively breastfed (n = 9), mixed feeding (n = 5) and formula-fed (n = 5) neonates. d, The expression of selected cell surface markers on Tregs in neonatal blood samples at birth and at 3 weeks of age (n = 19). Horizontal lines represent medians and interquartile ranges. * p < 0.05, ** p < 0.01, *** p < 0.001
Figure 2. Representative dot-plots of intracellular cytokine and cell surface cytotoxic markers following mitogenic stimulation in a neonatal blood sample at birth and at 3 weeks of age as well as in a maternal blood sample. a, Intracellular cytokines were gated within CD4+ cells. b, Cell surface expression of CD107a, a marker of cytotoxic degranulation and the intracellular expression of IFN-γ are shown within CD8+ cells.
Figure 3. Alterations of the pro-inflammatory and cytotoxic immunophenotype in neonates between birth and 3 weeks of age.a, The intracellular frequency and mean fluorescence intensity (MFI) of selected pro-inflammatory cytokines in CD4+ cells in neonatal blood samples at birth and at 3 weeks of age (n = 17). b, The intracellular frequency and MFI of interferon gamma (IFN-γ) in CD4+ cells in neonatal blood samples at 3 weeks of age grouped according to the feeding method: exclusively breastfed (n = 8), mixed feeding (n = 5) and formula-fed (n = 4) neonates. c, The frequency of CD8+ and CD8+ CD107a+ cells and MFI of CD107a in CD8+ cells in neonatal blood samples (n = 17) at birth and at 3 weeks of age, as well as in maternal blood samples (n = 17) and healthy controls (n = 8). Horizontal lines represent medians and interquartile ranges. * p < 0.05, ** p < 0.01, *** p < 0.001
Figure 4. Neonatal T cell response upon maternal antigen stimulation. a, Representative sample of a mixed lymphocyte reaction (MLR) at birth and at 3 weeks of age with negative and positive controls. Positive controls were established with the addition of CD3/CD28 activator beads instead of stimulator cells. Negative controls were established in enriched media only. Samples were incubated for 5 days. b, Percentage of neonatal proliferating T cells (n = 37) at birth and at 3 weeks of age in response to maternal irradiated cells in the CD3+, CD4+ and CD8+ cell subsets. c, Percentage of neonatal proliferating CD3+ cells at birth and at 3 weeks of age in response to maternal irradiated cells grouped according to the feeding method: exclusively breastfed (n = 16), mixed feeding (n = 8) and formula-fed (n = 13) neonates. Horizontal lines represent medians and interquartile ranges. * p < 0.05, ** p < 0.01
Figure 5. Maternal and neonatal T cell response in mixed lymphocyte reactions (MLR) following the depletion of CD25+ cells and pro-inflammatory cytokine production by neonatal T cells in exclusively breastfed neonates in MLR upon maternal antigen stimulation at birth and at 3 weeks of age. a, Representative dot-plots with and without the depletion of CD25+ cells in a neonatal sample at 3 weeks of age, gated within CD3+ cells. CD25+ cells were depleted using magnetic microbead separation. b, Percentage of maternal proliferating T cells (n = 6) in response to neonatal stimulator cells of exclusively breastfed neonates from birth and 3 weeks of age in the CD3+ subset.c, Percentage of neonatal proliferating T cells (n = 6) in response to maternal stimulator cells at birth and at 3 weeks of age in the CD3+ subset of exclusively breastfed neonates. d, The concentration of IFN-γ and TNF-α was found to be lower at 3 weeks of age compared to birth (n = 11). The maximal IFN-γ and TNF-α producing capacity of neonatal T cells was also assessed at birth and at 3 weeks of age by culturing them with CD3/CD28 activator beads. The production of IFN-γ and TNF-α was higher both at birth and at 3 weeks of age compared to the level seen in response to maternal antigens at 3 weeks. Horizontal lines represent medians and interquartile ranges. * p < 0.05, ** p < 0.01, *** p < 0.001
Figure 6. Microbiome analysis of neonatal stool samples at 3 weeks of age in exclusively breastfed (n = 9) and exclusively formula-fed (n = 12) neonates. a, Relative frequency of bacterial phyla in the two cohorts. b, Principal component analysis (PCA) of gut microbiota composition of exclusively breastfed (red) and exclusively formula-fed (blue) neonates at 3 weeks of age determined by bacterial 16S rRNA amplification. Numbers represent the individual study number of each participant. Pooled variables of milk received are represented by the large red circle for breastmilk and the large blue triangle for formula, respectively. c, Association of specific microbial taxa with the feeding method by linear discriminant analysis (LDA) effect size (LEfSe). Red indicates taxa enriched in exclusively breastfed neonates. d, Ranking of gut microbial strains using the Random Forest (RF) method in exclusively breastfed (BM) and exclusively formula-fed neonates at 3 weeks of age.
Figure 7. Integrated analysis of mixed lymphocyte reaction (MLR), flow cytometry and microbiome data of a, exclusively breastfed (n = 9) and b, exclusively formula-fed (n = 5) neonates at 3 weeks of age. Various positive and negative correlations between the studied parameters were revealed. Green represents a positive correlation, whereas red represents a negative correlation. Thicker lines represent stronger correlations. Nodes in blue represent MLR data, nodes in black represent microbiome data, and nodes in purple and orange represent flow cytometry data from the panels with and without mitogenic stimulation, respectively.
Supplementary Figure 1. The intracellular frequency and mean fluorescence intensity (MFI) of selected pro-inflammatory cytokines in CD8+ cells in neonatal blood samples at birth and at 3 weeks of age (n = 17). Horizontal lines represent medians and interquartile ranges. * p < 0.05
Supplementary Figure 2. Maternal T cell response upon neonatal antigen stimulation from birth and 3 weeks of age. a,Percentage of maternal proliferating T cells (n = 37) in the CD3+, CD4+ and CD8+ cell subsets in response to neonatal irradiated cells.b, Percentage of proliferating T cells of a third-party, non-pregnant control individual (n = 6) in the CD3+, CD4+ and CD8+ cell subsets in response to neonatal irradiated cells. Horizontal lines represent medians and interquartile ranges. * p < 0.05