Fig 2: AsGM1 gradient driven by T. gondii infection captures two distinct ILC1-like subsets(A–J) Analysis of uninfected and T. gondii-infected mice 14 days post-infection. (A) Distribution of CD49a vs. Eomes among splenic Group 1 ILCs (CD45+CD3−NK1.1+NCR1+) from uninfected and T. gondii-infected mice. (B) Frequency and numbers of ILC1s and NK cells among splenic Group 1 ILCs from uninfected and T. gondii-infected mice. (C) MFI of CD200R and Ly6C in splenic ILC1s normalized to splenic NK cells from uninfected and T. gondii-infected mice. (D) Distribution of CD49a vs. Eomes among liver Group 1 ILCs from uninfected and T. gondii-infected mice. Distribution of CD200R vs. CD49b among liver ILC1s from uninfected and T. gondii-infected mice. (E) MFI of CD200R, CD49a, and Ly6C in liver ILC1s normalized to liver NK cells of uninfected and T. gondii-infected mice. (F) AsGM1 expression across liver Group 1 ILC subsets from uninfected and T. gondii-infected mice. (G) Frequency and MFI of AsGM1 in ILC1s, ILC1-like cells, and NK cells in the liver of T. gondii-infected mice. (H) AsGM1 expression in CD45+CD3−NK1.1+ ILCs from the spleen, lung, and liver of uninfected and T. gondii-infected mice. A graded gray bar represents the AsGM1 expression gradient, with dashed histograms indicating isotype control levels. (I) Distribution of NCR1+ and NCR1- ILC subsets among NK1.1+AsGM1int ILCs across spleen, lung, and liver in T. gondii-infected mice. (J) MFI of CD200R, Eomes, CD49b, and CD49a in NK1.1+NCR1−AsGM1int ILCs compared with NK cells and ILC1s in the spleen, lung, and liver of T. gondii-infected mice. A graded gray bar indicates AsGM1 expression. Data represent 2 independent experiments, with n = 6–11 uninfected mice and n = 11–16 infected mice. Statistical analysis was performed using unpaired t-test (B, C, E, and I) and one-way ANOVA (G and J), with significance indicated by ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Error bars represent mean ± s.e.m. (B) and mean ± s.d. (C, E, G, I, and J). Refer to Figures S6–S8 for additional information.

Fig 3: Mapping NK-to-ILC1 continuum in the salivary glands via AsGM1(A) Distribution of CD49a vs. CD49b among Group 1 ILCs (CD45+CD3−NK1.1+) in the salivary glands (SGs) of adult wild-type (WT) and CD11cdnR (TGFβR-resistant NK cells) mice.(B) Expression of Eomes and AsGM1 in CD49a−CD49b+ (NK cells) and CD49a+CD49b+ (SG ILCs) subsets from adult wild-type SGs.(C) MFI of Eomes and AsGM1 expression in SG ILCs relative normalized to NK cells in wild-type SGs.(D) Expression of CD200R, Eomes, CD49a, TRAIL, CD103, and AsGM1 in splenic NK cells before (dotted line) and after IL-15 plus TGFβ treatment (yellow).(E) MFI of AsGM1, CD200R, Eomes, CD103, CD49a, and TRAIL under IL-15 plus TGFβ treatment normalized to untreated NK cell controls.(F) Distribution of CD49a vs. CD49b in SG Group 1 ILCs in adult (2–4 months) and aged (5–12 months) wild-type mice. Gates indicate NK cells (blue), SG ILCs (red), and SG-ILC1-like cells (green).(G) Overlay of NK cells, SG ILCs and SG ILC1-like cells from aged SGs within the distribution of Eomes vs. AsGM1 and Eomes vs. CD200R. Expression of Eomes and AsGM1 is shown in overlayed cell subsets.(H) MFI of Eomes, CD200R, and AsGM1 in SG ILCs and SG ILC1-like cells normalized to NK cells in aged (5–12 months) mice.(I) Age-related frequency shifts in NK cells, SG ILCs, and SG ILC1-like cell subsets.(J) Schematic representation illustrating the stable expression of AsGM1 against the fluctuating expression of CD200R and Eomes in SG Group 1 ILCs, highlighting an age-associated continuum. Data represent 3 independent experiments: n = 11 (A–C), n = 3 (D and E), n = 13 (F–I) for wild-type mice, with n = 5 for CD11cdnR mice (A). Statistical analysis was performed using unpaired t-test (C, H) and one-way ANOVA (E), with significance indicated by ∗∗p < 0.01, ∗∗∗∗p < 0.0001. Error bars represent mean +s.d.

Fig 4: Mapping AsGM1 expression across Group 1 ILCs(A) Distribution of NCR1 vs. AsGM1 in CD3−NK1.1+ ILCs in the spleen, bone marrow (BM), blood, lung, mesenteric lymph nodes (mLN), and liver.(B) Profiles of Eomes, CD200R, CD49b, T-bet, and TRAIL in AsGM1- and AsGM1+ ILCs across tissues.(C) Mean Fluorescence Intensity (MFI) for CD200R, Eomes, CD49b, and T-bet in AsGM1- and AsGM1+ ILCs across tissues.(D) Comparison of activating and inhibitory NK cell markers in AsGM1- and AsGM1+ ILCs in the spleen.(E) Proportion of Group 1 ILCs delineated as CD3−NK1.1+NCR1+Eomes− vs. CD3−NK1.1+NCR1+AsGM1- in the spleen.(F and G) Distribution of AsGM1 and Eomes expression across stages of NK cell development. (F) Gating strategy used to identify pre-NKP (Lin−CD244+CD27+c-kit-IL-7Rα+Flk2−CD122-) and rNKP (Lin−CD244+CD27+c-kit-IL-7Rα+Flk2+CD122-) cells.32 The lineage cocktail includes antibodies against CD19, Ly6D, CD3, NK1.1, and CD11b. (G) Stages within the immature (iNK) and mature (mNK) cell compartments are indicated by phenotypic markers listed. Distribution of AsGM1 versus Eomes expression within each developmental stage, with the proportions of AsGM1intEomes− (Yellow), AsGM1intEomes+ (Green) and AsGM1hiEomes+ (Red) subsets shown for each stage indicated in (F and G). Data represent 2–3 independent experiments: spleen (n = 27), BM (n = 6), blood (n = 5), lung (n = 5), mLN (n = 7), and liver (n = 16). Statistical analysis was performed using unpaired t-tests (C and E) and one-way ANOVA (F and G), with significance indicated by ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Error bars represent mean +s.d. Refer to Figure S3 for additional information. Refer to Figure S3 for additional information.