Fig 2: Epithelial CD103+NKp44+lin- cells are the major ILC population in adults. a Frequencies as well as absolute cell numbers per cm2 of epithelial (EP) and lamina propria-derived (LP) NKp44+ NK cells in infant (white circles) and adult (dark circles) intestines (infant NK cell frequencies and absolute counts EP (N = 7) and LP (N = 5), adult NK cell frequencies (N = 9) and absolute counts (N = 8)). b Frequencies of NKp44+ cells within CD103+, CD49a+, or CD69+ NK cells in infants (white circles) and adults (dark circles). NKp44 expression by infant epithelial CD103+ (N = 7), CD49a+ (N = 7), and CD69+ (N = 6) NK cells. NKp44 expression by adult epithelial NK cell subsets (N = 9). Heatmap of median frequencies of NKG2A+, Eomes+, perforin+, granzyme B+, and NKp44+ cells within CD103+, CD49a+, or CD69+ NK cells as well as CD127+ ILCs in infant intestines. c SPADE tree of viable EP CD45+lin- lymphocytes. CD127+ ILCs (yellow outline), NK cells (red outline), including CD127-NKp44+CD103+lin- cells (blue outline) within NK cell population. Expression of NKp44 is shown by color coding in relative intensity. Node sizes represent the size of populations. SPADE analysis was computed by using the same signature parameters as in Fig. 1 (infant samples N = 4, adult samples N = 5, target number of nodes: 200, down sampled events target: 30%). Median frequencies indicated by red lines. Error bars define interquartile ranges between 75th and 25th percentiles. Statistical comparisons are Mann-Whitney U comparisons (a) and Wilcoxon matched-pairs signed rank tests (b). Asterisks represent the following p-values: *p < 0.05 and ***p < 0.001

Fig 3: Infant intestinal NK cells have high NKG2A expression. a viSNE plots of combined flow cytometric data visualizing NKG2A, KIR, and CD57 expression by infant and adult epithelial (EP) and lamina propria-derived (LP) NK cells. Cell density of clusters is shown in first row. The expression of NKG2A, KIR, and CD57 is shown by color coding in relative intensity below. viSNE plots are calculated from concatenated FCS files gated on NK cells (infant samples N = 9, adult samples N = 6, iterations = 7500 perplexity = 100, KL divergence = 2.29). b Frequencies of epithelial and lamina propria-derived infant (white circles) and adult (dark circles) NKG2A+ NK cells and KIR+ NK cells (EP infant samples NKG2A (N = 10), KIR (N = 11), LP infant samples NKG2A (N = 9), KIR (N = 10), EP adult samples NKG2A (N = 9), KIR (N = 8), LP adult samples NKG2A (N = 9), KIR (N = 8)). c Histogram overlay of flow cytometric data showing NKG2A and KIR expression by CD103+ (gray), CD49a+ (dark gray), and CD69+ (black) NK cells from infant and adult intestines. d Frequencies of infant (white circles) and adult (dark circles) intestinal NKG2A+ NK cells and KIR+ NK cells within CD103+, CD49a+, or CD69+ populations. NKG2A expression by infant EP CD103+ (N = 10), CD69+ (N = 10), and CD49a+ (N = 9) NK cells, NKG2A expression by infant LP-derived CD103+ (N = 9), CD69+ (N = 9), and CD49a+ (N = 8) NK cells. NKG2A expression by adult EP and LP-derived NK cells (N = 9). KIR expression by infant EP CD103+ (N = 11), CD69+ (N = 11), and CD49a+ (N = 9) NK cells. KIR expression by infant LP-derived CD103+ (N = 10), CD69+ (N = 10), and CD49a+ (N = 8) NK cells. KIR expression by adult EP (N = 9) and LP-derived (N = 8) NK cells. Median frequencies indicated by red lines. Error bars define interquartile ranges between 75th and 25th percentiles. Statistical comparisons are Mann-Whitney U comparisons (b) and Wilcoxon matched-pairs signed rank tests (d). Asterisks represent the following p-values: *p < 0.05 and **p < 0.01

Fig 4: Infant intestinal NK cells contain high levels of cytotoxic granules. a viSNE plots of combined flow cytometric data visualizing Eomes, perforin, granzyme B, and KIR expression by epithelial (EP) and lamina propria-derived (LP) infant and adult NK cells. Expression of Eomes, KIR, perforin, and granzyme B (GrzB) is shown by color coding in relative intensity. viSNE plots have been calculated from concatenated FCS files gated on NK cells (infant samples N = 7, adult samples N = 5, iterations = 7500 perplexity = 100, KL divergence = 2.62). b Frequencies of perforin+ and GrzB+ NK cells in infants (white circles) and adults (dark circles) (EP infant samples (N = 8), LP infant samples (N = 7), EP adult samples perforin expression (N = 9), GrzB expression (N = 8), LP adult samples perforin expression (N = 9), and GrzB expression (N = 8)). c Frequencies of perforin+ and granzyme B+ cells within CD103+, CD49a+, or CD69+ NK cell populations in infant (white circles) and adult intestines (dark circles) (EP infant samples (N = 8), LP infant samples (N = 7), EP adult samples perforin expression (N = 9), and GrzB expression (N = 8), LP adult samples perforin expression (N = 9), and GrzB expression (N = 8)). d Frequencies of LP-derived CD107a+, IFN-?+, and TNF-a+ NK cells in infant (white circles) and adult intestines (dark circles). Cells were stimulated for 6 h with phorbol 12-myristate 13-acetate (PMA) and ionomycin (infant samples N = 5, adult samples N = 7). e Frequencies of LP-derived CD107a+ cells within CD103+, CD49a+, or CD69+ NK cell populations in infant (white circles) and adult intestines (dark circles) after stimulation with PMA and ionomycin for 6 h (infant samples N = 5, adult samples N = 6). Median frequencies indicated by red lines. Error bars define interquartile ranges between 75th and 25th percentiles. Statistical comparisons are Mann-Whitney U comparisons (b, d) and Wilcoxon matched-pairs signed rank tests (c, e). Asterisks represent the following p-values: *p < 0.05; **p < 0.01; and ***p < 0.001

Fig 5: Intestinal NK cells express tissue-residency markers. a viSNE plots of combined flow cytometric data visualizing CD103, CD49a, and CD69 expression on infant and adult NK cells from epithelium (EP) and lamina propria (LP) intestinal tissues. Cell density of clusters is shown in first row. Expression patterns of tissue-residency markers CD103, CD49a, and CD69 are depicted by color coding in relative intensity in following plots. viSNE plots have been calculated from concatenated FCS files gated on NK cells (infant samples N = 9, adult samples N = 6, iterations = 7500 perplexity = 100, KL divergence = 2.29). b Frequencies of epithelial and lamina propria-derived CD103+, CD49a+, or CD69+ NK cells from infant (white circles) and adult intestines (dark circles) (EP infant samples (N = 10), EP adult samples (N = 13), LP infant samples (CD103 (N = 11), CD69 (N = 9), CD49a (N = 8), LP adult samples (CD103 (N = 13), CD69 (N = 13), CD49a (N = 11)). Median frequencies indicated by red lines. Error bars define interquartile ranges between 75th and 25th percentiles. Statistical comparisons are Mann-Whitney U comparisons. Asterisks represent the following p-values: **p < 0.01 and ***p < 0.001