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

Abstract

A large gap in our understanding of infant immunity is why natural killer (NK) cell responses are deficient, which makes infants more prone to viral infection. Here we demonstrate that transforming growth factor-β (TGF-β) was responsible for NK cell immaturity during infancy. We found more fully mature NK cells in CD11c(dnR) mice, whose NK cells lack TGF-β receptor (TGF-βR) signaling. Ontogenic maturation of NK cells progressed faster in the absence of TGF-β signaling, which results in the formation of a mature NK cell pool early in life. As a consequence, infant CD11c(dnR) mice efficiently controlled viral infections. These data thus demonstrate an unprecedented role for TGF-β in ontogeny that can explain why NK cell responses are deficient early in life.

Figure 1. TGF-β is a negative regulator of NK cell generation

CD11c^dnR and wild-type mice were injected (i.p.) with 5-FU. Four days later, mice were sacrificed and HSC-enriched bone marrow cells were cultured in the presence of IL-7, SCF, and Flt3L. On day 5, cells were harvested, plated on OP9 stromal cells, and cultured in the presence of IL-15. On day 8, cultures were supplemented with or without TGF-β and outcomes on NK cell production were determined on day 11. (a) FACS plots are from gated CD3⁻cells, and numbers indicate frequency of HSC (c-kit⁺Sca-1⁺), NK cell precursors (CD122⁺NKG2D⁺), immature (NK1.1⁺CD122⁺), and mature (NK1.1⁺DX5⁺) NK cells. (b) Histograms indicate numbers of mature NK cells (CD3⁻NK1.1⁺DX5⁺) from CD11c^dnR (black bar) and wild type (white bar) cultures produced in the absence versus the presence of TGF-β. Data in a,b are representative of two independent experiments with n = 3 mice per experiment.

Figure 2. CD11c^dnR mice produce high numbers of stage F-mNK cells

(a) Histograms indicate frequency and numbers of HSC, pNK, iNK, and mNK cells from the bone marrow of CD11c^dnR (solid bar) versus wild type (open bar) mice. (b) FACS plots are from gated CD3⁻ cells and numbers indicate frequency of mNK cells. Distribution of CD11b versus CD43 is from gated mNK cells and numbers indicate frequency of stage F-mNK cells (CD11b⁺CD43⁺). (c) Graph shows numbers of stage F-mNK cells in the bone marrow of CD11c^dnR (black circle) versus wild type (white circle) mice. *, p < 0.005 (Student’s t-test). (d) FACS plots are from gated mNK cells, and numbers indicate frequency of stage F-mNK cells as identified by CD27⁻CD11b⁺ and CD27⁻CD43⁺ phenotypes. (e) FACS plots show overlay of NK cell subsets in the bone marrow. Upper panels show overlay of CD11b⁺CD43⁺ mNK cells (red dots) versus total mNK cells (blue dots). Lower panels show overlay of CD27⁺CD11b⁻ (blue dots), CD27⁺CD11b⁺ (yellow dots), and CD27⁻CD11b⁺ (red dots) mNK cell subsets. (f) Graphs show numbers of total mNK cells and stage F-mNK cells in the bone marrow versus spleen of CD11c^dnR (red line) versus wild type (blue line) mice. Data in a,b and d,e are representative of three independent experiments with n = 8 and 3 mice, respectively. Results in c,f are representative four independent experiments with n = 11 WT and 13 CD11c^dnR mice.

Figure 3. Massive production of stage F-mNK cells in CD11c^dnR mice is cell autonomous

(a–b) Mixed bone marrow chimeras were constructed with wild type (CD45.2) and CD11c^dnR (CD45.1) donor bone marrow cells provided in the inoculum at 1:1 ratio. Six weeks later, chimeras were sacrificed and donor-derived NK cells were analyzed in the bone marrow, spleen, and liver. (a) FACS plots show the distribution of CD45.2 versus CD45.1 in each organ. NK1.1 versus CD45.1 staining is from gated CD3⁻ cells and numbers indicate frequency of NK cells from each donor. (b) Distribution of CD11b versus CD43 is from gated mNK cells that were gated from wild type (CD45.2⁺) or CD11c^dnR (CD45.1⁺) donor cells. Numbers indicate frequency of stage F-mNK cells produced from each donor. Data in a,b are representative of two independent experiments with n = 3 chimeras per experiment. (c–e) Mixed bone marrow chimeras were constructed with wild type (CD45.2) and CD11c^dnR (CD45.1) donor bone marrow cells provided in the inoculum at 1:1, 2:1, 4:1, and 9:1 ratios. Six weeks later, chimeras were sacrificed and donor-derived NK cells were analyzed in the bone marrow at different stages of development. (c) FACS plots show the distribution of CD45.1 versus CD45.2 in the bone marrow. (d) FACS plots are from gated CD11b⁺CD43⁺ mNK cells and numbers indicate the frequency of CD11c^dnR (CD45.1⁺) origin among stage F-mNK cells. (e) Graph shows the frequency of CD11c^dnR (CD45.1⁺) origin among HSCs, pNK, iNK, and mNK cells at stages D, E, and F. Results in e are from 4:1 chimeras group. Data in c,e are representative of two independent experiments with n = 5 mice per chimeras group.

Figure 4. TGF-β arrests NK cell cycle at stages D/E and limits NK cell transition at stage F

(a–d) CD11c^dnR and wild type mice were injected (i.p.) twice a day with BrdU for 3 days. On day 3, mice were sacrificed and frequency of cycling cells was determined in the bone marrow. (a–b) FACS plots show the distribution of BrdU staining among total mNK cells (a) and gated mNK cells at stages D, E, and F (b). (c) Graph shows the frequency of cycling cells in pNK, iNK, and mNK cells from CD11c^dnR (black circle) versus wild type (white circle) mice. (d) Graph shows the frequency of cycling cells in mNK cells at stages D, E, and F from CD11c^dnR (black circle) versus wild type (white circle) mice. Data in a,d are representative of three independent experiments with n = 2 mice per experiment and results in c,d show all 6 individual mice. (e–f) mNK cells at stages D, E, and F were sorted from the bone marrow. mRNA was isolated and cDNA was subjected to pathway-specific qPCR for analysis of cell cycle genes (e) or SYBR Green qPCR for analysis of transcription factors T-bet, GATA-3, and IRF-2 (f). Data were analyzed using Global Pattern Recognition analytical software (e) or 2-33C3 method (f) and results were expressed as fold of change in CD11c^dnR versus wild type samples. Data in e,f are representative of three independent cell sorting with samples pooled from n = 12 CD11c^dnR and 25 WT mice.

Figure 5. TGF-β imposes constraints on NK cell maturation during ontogeny

NK cells from CD11c^dnR and wild type mice were analyzed in newborns (0–2 days), neonates (7–10 days), infants (17–20 days), and adults (56–84 days). (a) FACS plots show NK1.1 versus DX5 staining on gated CD3⁻ cells, and numbers indicate the frequency of mNK cells as a function of age. (b) Distribution of CD11b versus CD43 is from gated mNK cells and numbers indicate the frequency of stage F-mNK cells as a function of age. (c) Graphs show numbers of total mNK cells and stage F-mNK cells as a function of age in bone marrow and spleen of CD11c^dnR (black circle) versus wild type (white circle) mice. Data in a,b are representative of two independent experiments with n = 4 newborn mice and four independent experiments with n = 8 mice per age group (neonate, infant and adult age). Data in c are representative of four independent experiments with n = 8 mice per age group (neonate, infant and adult age) and results show all 8 individual mice.

Figure 6. Infant CD11c^dnR mice have mature NK cell compartment

(a–b) FACS plots show distribution of CD43 versus NK cell receptors among gated mNK cells (CD3⁻NK1.1⁺DX5⁺), and numbers indicate frequency of terminally mature Ly49C/I⁺, Ly49D⁺, Ly49H⁺, NKG2A⁺, NKG2D⁺, and CD94⁺ mNK cell subsets in CD11c^dnR and wild type mice at infant (17–20 days) and adult (56–84 days) ages. (c) Graphs show the frequency of stage F-mNK cell subsets (CD43⁺Ly49C/I⁺, CD43⁺Ly49D⁺, CD43⁺Ly49H⁺, CD43⁺NKG2A⁺, CD43⁺NKG2D⁺, and CD43⁺CD94⁺) among total mNK cells in CD11c^dnR (black circle) versus wild type (white circle) at infant versus adult ages. Data in a,c are representative of three independent experiments with n = 4 infant and 8 adult mice.

Figure 7. Infant CD11c^dnR mice are protected from MCMV infection

(a) FACS plots are from gated CD3⁻ cells and numbers indicate the frequency of Ly49H⁺ NK cells in the spleen of CD11c^dnR and wild type mice at infant (17–20 days) versus adult (56–84 days) ages. Distribution of CD11b versus CD43 is from gated Ly49H⁺ NK cells and numbers indicate the frequency of stage F-Ly49H⁺ NK cells in the spleen. (b) Graph shows numbers of total Ly49H⁺ NK cells (solid line) versus stage F-Ly49H⁺ NK cells (dashed line) in the spleen of CD11c^dnR (black circle) versus wild type (white circle) mice as a function of age. Data in a,b are representative of three independent experiments with n = 3 infant and 6 adult mice. (c–e) CD11c^dnR and wild type infant mice (17–20 days) under pure C57BL/6 background were infected with MCMV and analyzed 5 days post-infection. (c) Histograms show viral load in spleens and livers from infected CD11c^dnR (black bar) versus wild type (white bar) infant mice. *, p values < 0.0001. (d) Histograms show numbers of Ly49H⁺ NK cells in the spleen before (white bar) and 5 days after (black bar) MCMV infection. (f) On day 5 post-infection, mice were injected (i.p.) with BrdU, and cell proliferation was analyzed in the spleen 3–4 hrs later. FACS plots show the distribution of BrdU staining among gated Ly49H⁺ versus Ly49H⁻ NK cells. Data in c,e are representative of two independent experiments with n = 3 CD11c^dnR and 6 WT infant mice.