TGF-β is responsible for NK cell immaturity during ontogeny and increased susceptibility to infection during murine infancy

Jeffrey P. Marcoe, James R. Lim, Keri L. Schaubert, Nassima Fodil-Cornu, Marsel Matka, Alexandra L. McCubbrey, Alexander R. Farr, Silvia M. Vidal

& Yasmina Laouar

Nature Immunology doi:10.1038/ni.2388

Supplementary Figure 1: Phenotypic identification of NK cell developmental pathway in CD11cdnR mice.BM cells were isolated from CD11cdnR and wild type mice and precursor stages were identified along NK cell development. (a) FACS plots show the distribution of c-kit versus Sca-1 on gated lineage negative (Lin-) cells.The cell population Lin-ckit+Sca1+ identifies the hematopoietic stem cells (HSC). (b) FACS plots show thedistribution of DX5 versus NK1.1 among gated CD3-CD122+ cells as indicated by arrows. The earliest stage,pNK, is characterized by the lack of both DX5 and NK 1.1 markers (CD3-CD122+NK1.1-DX5-). The immaturestage, iNK, is distinguished by the expression of NK1.1 but not DX5 (CD3-CD122+NK1.1+DX5-). The maturestage, mNK, is identified by the co-expression of both NK1.1 and DX5 (CD3-CD122+NK1.1+DX5+). (c) FACSplots show the distribution of CD11b versus CD43 among gated mNK cells as indicated by arrows. Gatesidentified by D (CD11b-CD43-), E (CD11b+CD43-) and F (CD11b+CD43+) indicate the three consecutive stages ofNK cell maturation. Frequency and numbers of CD11cdnR versus wild type cells at each developmental stage areindicated in Figure 2. Data in a,c are representative of three independent experiments with n = 8 mice per group.

ckit

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Nature Immunology doi:10.1038/ni.2388

Supplementary Figure 2: Enhanced NK cell maturation in the absence of TGF-βR signaling is demonstrated by acquisition of CD43 and loss of CD27. BM, spleen, and liver were isolated from CD11cdnR and wild type mice and mNKcells were analyzed for expression of CD11b, CD43, and CD27. (a) FACS plots show NK1.1 versus CD3 staining from total cells. (b) FACS plots show the expression of CD122 and DX5 on gated NK1.1+CD3- cells. (c) FACS plots show the distribution of CD11b versus CD43 (left panel) and CD27 versus CD11b (right panel) on gated mNK cells. Numbers indicate the frequency of terminally mature NK cells as identified by expression of CD11b and CD43 (left panel) and loss of CD27 (right panel). Graphs show frequency of CD11b+CD43+and CD27-CD11b+ cells mNK cells from CD11cdnR (black bar) versus wild type (white bar) mice. Data in a,c are representative of three independent experiment with n = 3 mice per group.

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Nature Immunology doi:10.1038/ni.2388

Supplementary Figure 3: Massive production of stage F-mNK cells in CD11cdnR

mice from BALB/c background. CD11cdnR mice and control littermates on pure BALB/c background were examined for terminal maturation of NK cells in the absence versus presence of TGF-βR signaling. FACS plots show DX5 versus TCRαβstaining and numbers indicate frequency of NK cells in the BM. Distribution of CD11b versus CD43 is shown from gated mNK cells and numbers indicate frequency of stage F-mNK cells in CD11cdnR BALB/c mice versus wild type BALB/c littermates. Data are representative of two independent experiments with n = 3 mice per experiment.

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Nature Immunology doi:10.1038/ni.2388

Supplementary Figure 4: NK cells from CD11cdnR mice express NKp46. NK cells from CD11cdnR and wild type mice on C57BL/6 (a, c) and BALB/c (b, d) backgrounds were analyzed for expression of NKp46. FACS plots (a-b) show percentages of NK cells in the spleen as indicated by NK1.1+(or DX5+)TCRαβ-, NKp46+TCRαβ-, and NKp46+NK1.1+(or DX5+) phenotypes. FACS plots (c) and (d) show expression of NKp46 among gated NK1.1+TCRαβ- cells (C57BL/6 ) and DX5+TCRαβ- cells (BALB/c) respectively. Results are from spleen and similar data were obtained from the BM. Data in a,c and d,e are representative of three independent experiments with n = 2 mice per experiment.

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Nature Immunology doi:10.1038/ni.2388

Supplementary Figure 5: mNK cells from CD11cdnR origin dominate in mixed BM chimeras. (a) Mixed BMchimeras were constructed with wild type (CD45.2) and CD11cdnR (CD45.1) donor BM cells provided in the inoculum at 1:1 ratio. Six weeks later, chimeras were sacrificed and donor-derived NK cells were analyzed in theBM, spleen, and liver. FACS plots show CD3 versus NK1.1 staining per organ and numbers indicate frequency of NK cells among mixed (CD45.1+ and CD45.2+) donor cells, gated wild type (CD45.2+) and CD11cdnR (CD45.1+) donor cells. Data in a are representative of two independent experiments with n = 3 chimeras per experiment. (b) Mixed BM chimeras were constructed with wild type (CD45.2) and CD11cdnR (CD45.1) donor BM cells provided in the inoculumat 4:1 ratios. Six weeks later, chimeras were sacrificed and donor-derived NK cells were analyzed in the BM at different stages of development. Distribution of CD45.1 marker is shown among pNK (CD3-CD122+NK1.1-DX5-), iNK(CD3-CD122+NK1.1+DX5-), and mNK (CD3-CD122+NK1.1+DX5+) cells including stages D (CD11b-CD43-), E (CD11b+CD43-), and F (CD11b+CD43+). Numbers indicate frequency of cells derived from CD11cdnR donor cells at each developmental stage. Data in b are representative of two independent experiments with n = 5 chimeras.

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Nature Immunology doi:10.1038/ni.2388

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Supplementary Figure 6: TGF-β suppresses T-bet and Gata-3 in NK cells. Splenic NK cells (NK1.1+CD3-) from CD11cdnR and wild type mice were sorted and cultured with IL12 (10 ng/ml) and IL18 (20 ng/ml) in the presence (black bar) versus absence (white bar) of exogenous TGF-β (5 ng/ml). Cells were harvested after 24 hrs of culture and mRNA levels were determined using quantitative real time PCR. Histograms indicate amounts of T-bet, GATA-3, and Eomesodermin (Eomes) mRNA as normalized to amounts of HPRT mRNA. Data are representative of two independent sorting with samples pooled from n= 6 CD11cdnR 10 WT mice.

Nature Immunology doi:10.1038/ni.2388

CD

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Supplementary Figure 7: TGF-β controls acquisition of NK cell maturation but not acquisition of NK cell receptors during ontogeny. (a) At 20 and 56 days of age, CD11cdnR and wild type mice were sacrificed and splenic NKcells were analyzed for expression of NK cell receptors. Graphs show frequency of mNK cell subsets (Ly49C/I+, Ly49D+, Ly49H+, NKG2A+, NKG2D+, and CD94+) among total mNK cells in CD11cdnR (black circle) and wild type(white circle) mice at infant (20 days) versus adult (56 days) ages. (b) FACS plots show the distribution of CD43 versus NK cell receptors among gated mNK cells (CD3-NK1.1+DX5+) in the BM of infant versus adult mice. Resultsare shown as overlay between CD11cdnR (blue dots) and wild type (red dots) cells. Data in a,b are representative of three independent experiments with n = 4 infant and 8 adult mice.

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Nature Immunology doi:10.1038/ni.2388

WT

CD11cdnR

22 ± 2 mg

97 ± 22 mg

Supplementary Figure 8: MCMV infection results in lymphopenia in wild type but not CD11cdnR

Infant mice. CD11cdnR and wild type infant mice (17-20 days) under pure C57BL/6 background were infected with MCMV and analyzed 5 days post-infection. Numbers indicate spleen weight and results are representative of two independent experiments with n = 3 CD11cdnR and 6 WT infected pups.

Nature Immunology doi:10.1038/ni.2388