Figure 1.
Phenotype of NK1.1+CD3− cells in the SMG.
(A) Naïve NK cells represent a significant population of lymphocytes within the SMG and display reduced levels of KLRG1 and CD27 compared to the spleen and liver. (B) Naïve NK1.1+CD3− cells from the SMG of B6 mice were examined for other NK cell markers.
Figure 2.
SMG NK cells are present in Rag−/− mice and are not thymic or LTi derived.
(A) Organs from naïve Rag−/− mice were examined to confirm the presence of NK cells in the SMG vs. spleen, liver, and blood. NK cells from the SMG were comparably lacking cell surface KLRG1, but expressed increased levels of CD69. (B) Organs from naïve nude mice were examined to confirm the presence of NK cells in the SMG and spleen. (C) NKp46+CD3− cells from the SMG and gut lamina propria of naïve or D14 MCMV infected RORcγt+/GFP reporter mice were examined for GFP expression.
Figure 3.
SMG NK cells become activated and induce the expression of KLRG1 during MCMV infection.
(A) B6 mice were infected with 5×104 pfu/mouse MCMV and sacrificed on D0, D7, 14 or 21 p.i. NK1.1+CD3− cells of the SMG were compared to spleen and liver for expression of KLRG1. (B) The graph represents the percentage of B6 NK cells and CD8+ T cells in the SMG during MCMV infection at D0, 7, 14 and 21. 3–5 experiments of 2–3 mice per group is shown.
Figure 4.
NK cells from the periphery are not recruited to the SMG during MCMV infection.
B6 splenic lymphocytes were depleted of CD19+ and CD5+ cells to enrich for NK cells. Remaining cells were stained with non-reducing amounts of NK1.1 and CD3 and sorted using a FACSAria. NK1.1+CD3− cells were of > 98% purity before i.v. injection into congenic B6 mice at 2×106 cells/mouse. Recipient congenic B6 mice were either infected with 5×104 pfu/mouse MCMV two hours or ten days prior to adoptive transfer. On D7 post-NK cell transfer, mice were analyzed for CD45.2+ NK cell trafficking. The figures represent 1 of 2 separate experiments with 2 mice per group.
Figure 5.
SMG NK cells are hyporesponsive in vivo during MCMV infection.
B6 mice were infected with 5×104 pfu/mouse MCMV and sacrificed on D2, 7, 10 or 14 p.i. IFN-γ production from NK1.1+CD3− cells was determined by intracellular staining. Representative of 5 separate experiments with 2–4 mice pooled per group.
Figure 6.
Poly(I∶C) primed SMG NK cells have impaired IFN-γ and degranulation compared with splenic NK cells.
Lymphoid cells from the SMG and spleens were isolated from 24 hour Poly(I∶C) treated mice. Both organs were processed and incubated for 6 hours with no treatment, crosslinked with anti-NKG2D or anti-Ly49H, or stimulated with IL-12/IL-18. Cells were treated with Monensin and stained for IFN-γ (A) and CD107α (B) and analyzed by flow cytometry. The bar graph shows a compilation of 3 experiments with 9 mice pooled for each experiment. *p = 0.004−0.003 **p = <0.003.
Figure 7.
MCMV activated SMG NK cells have impaired IFN-γ and degranulation compared with splenic NK cells.
Lymphoid cells from the SMG and spleens were isolated from D10 and D2 MCMV infected mice, respectively. Both organs were processed and incubated for 6 hours as described in Figure 6. Cells were treated with Monensin and stained for IFN-γ (A) and CD107α (B) and analyzed by flow cytometry. The bar graph shows a compilation of 4 experiments with 3 mice pooled for each experiment. *p = 0.03−0.003 **p = <0.003.
Figure 8.
T-regs and iNKT cells are absent from naïve and MCMV infected SMG.
(A) Mice expressing a GFP-Foxp3 fusion-protein reporter were infected with MCMV for the time points shown and analyzed for accumulation of Tregs in the spleen, liver, blood and SMG as shown by CD4+GFP+ T cells. Representative of 3 separate experiments with 3–4 mice per group. (B) Splenic, hepatic and SMG leukocytes were isolated from naïve B6 and Jα18 deficient mice. The iNKT cell compartment was analyzed by staining with TCR-β and α-GalCer-loaded CD1d tetramer. Representative of 3 separate experiments with 2–3 mice per group.