Fig 1.
Absence of activating receptor NCR1 does not alter NK cell responses upon LCMV infection.
(A) Total numbers of NK1.1+CD3- lymphocytes in the spleen in naïve mice. Data shown are mean + SEM of n = 6–7 mice; pooled data from 2 experiments. ns, not significant (unpaired two-tailed t-test). (B-G) Analysis of splenic cells of C57BL/6 and NCR1gfp/gfp mice 2 days p. LCMV WE infection. (B) Representative flow cytometry plots pre-gated on CD3- and total number of CD3- DX5+ NK1.1+ lymphocytes are shown. (C) Representative flow cytometry plots pre-gated on CD3- DX5+ NK1.1+ and (D) frequency and total numbers of CD11b+ CD27- are depicted. Data shown are mean + SEM of n = 3 mice representative of 2 independent experiments. ns, not significant; * p<0.05 (unpaired two-tailed t-test). (E-G) Splenocytes were incubated in presence of Brefeldin A for 5h at 37°C and IFNγ and Granzyme B (GrzB) expression was measured. (E) Representative flow cytometry plots pre-gated on CD3- DX5+ NK1.1+ and total numbers of IFNγ (F) and Granzyme B (GrzB) producing cells (G). Data shown are mean + SEM of n = 3 mice representative of 2 independent experiments. ns, not significant (unpaired two-tailed t-test). H) NK cells from day 2 LCMV WE infected mice derived from WT and NCR1gfp/gfp were MACS purified. 20.000 YAC-1 cells were co-cultured with purified NK cells at the indicated ratios of NK to target cells for 5h followed by cytometric analysis. The percentage of apoptotic Annexin V and 7-AAD double positive YAC-1 cells is shown. Data shown are mean + SEM of n = 3 mice representative of 2 independent experiments. ns, not significant (unpaired two-tailed t-test).
Fig 2.
Increase of virus-specific CD8 T cells in absence of NCR1 during acute LCMV infection.
(A) Frequency and total numbers of CD8 T cells in the spleen of naïve mice. Data shown are mean + SEM of n = 3 mice representative of 2 independent experiments. ns, not significant (unpaired two-tailed t test). (B) 1x103 P14 T cells (Ly5.1+) were transferred into WT and NCR1gfp/gfp mice followed by 200 ffu LCMV WE infection. After 7d, organs were harvested and flow cytometric analysis was performed. Number of CD8 T cells in indicated organs is shown. (C) 1x104 P14 T cells (Ly5.1+) were transferred into WT and NCR1gfp/gfp mice followed by 200 ffu LCMV docile infection. After 7d, organs were harvested and flow cytometric analysis was performed. Numbers of endogenous CD8 T cell subsets are shown. Data shown are mean + SEM of n = 5–6 mice pooled from 2 independent experiments. ns, not significant, * p<0.05 (unpaired two-tailed t-test). (D-J) 1x103 P14 T cells (Ly5.1+) were transferred into WT and NCR1gfp/gfp mice followed by 200 ffu LCMV WE infection. After 7d, organs were harvested and flow cytometric analysis was performed. (D) Representative flow cytometry plot pre-gated on CD8 T cells. (E) Representative flow cytometry plot pre-gated on endogenous CD8 T cells in the spleen. (F) Total number of transferred P14 (Ly5.1+), endogenous gp33-tet+ and endogenous np396-tet+ CD8 T cells in the spleen, (G) percentages and (H) total numbers in the lung. (I-J) Splenocytes were incubated in presence of Brefeldin A, gp33 and np396 peptide, respectively, for 6h at 37°C followed by intracellular cytokine staining. (I) Number of IFNγ and TNF producing CD8 T cells after gp33 stimulation is shown. (J) Number of IFNγ and TNF producing CD8 T cells after np396 stimulation is shown. Data shown are mean + SEM of n = 4 mice representative of 2 independent experiments. ns, not significant, * p<0.05,** p<0.01, *** p<0.001 (unpaired two-tailed t-test). (K) 1x103 P14 T cells (Ly5.1+) were transferred into WT and NCR1gfp/gfp mice followed by LCMV WE infection. After 5d, spleens were harvested and viral titers were determined. Data shown are mean ± SEM of n = 6–9 mice pooled from 2 independent experiments. ns, not significant, * p<0.05 (unpaired two-tailed t-test).
Fig 3.
Activated polyclonal CD8 T cells and virus-specific CD8 T cells are directly killed by NK cells via NCR1.
(A) Experimental setup of the in vivo killer assay used in B-E. NCR1gfp/gfp were infected with LCMV WE and splenic CD8 T cells were positively MACS sorted and CTV labeled after 7d. Purified CD8 T cells were transferred into day 2 infected B6 and NCR1gfp/gfp mice. After 4 h, spleen and lung tissue was harvested followed by flow cytometric analysis. Recovery of transferred CTV+ CD44+ CD8 T cells in the spleen (B) and in the lung (C). Data shown are mean + SEM of n = 12 mice pooled from 3 independent experiments. ns, not significant, * p<0.05 (unpaired two-tailed t-test). Total numbers of NK1.1+CD3- cells in the spleen (D) and in the lung (E). Data shown are mean + SEM of n = 4 mice representative of 2 independent experiments. ns, not significant, (unpaired two-tailed t-test). (F) Experimental scheme used in G-H. 1x106 P14 T cells (Ly5.1+) were transferred into NCR1gfp/gfp mice followed LCMV WE8.7 and VVG2 co-infection. After 5d, splenic CD8 T cells were MACS sorted and transferred into day 3 infected B6 and NCR1gfp/gfp mice. After 4 hours, spleens were harvested and P14 T cell (Ly5.1+) number was determined by flow cytometric analysis. (G) Recovery of P14 T cells (Ly5.1+) in the spleen. (H) Number of NK1.1+ CD3- cells in the spleen are shown. Data shown are mean + SEM of n = 8 mice pooled from 2 independent experiments. ns, not significant, * p<0.05 (unpaired two-tailed t-test).
Fig 4.
Highly activated and more functional CD8 T cells in absence of NCR1 during chronic infection.
(A) Experimental setup used in B-I. 1x103 P14 T cells (Ly5.1+) were transferred into WT, NCR1gfp/gfp and αNCR1 blocked WT mice followed by LCMV docile infection. After 12d, organs were harvested and analyzed. (B) Total number of endogenous CD8 T cells and (C) frequency of CD44+ CD8 T cells pre-gated on endogenous CD8 T cells in the spleen. (D) Representative flow cytometry plot pre-gated on CD8 T cells. (E) Frequency and total numbers of P14 T cells (Ly5.1+) in the spleen. F-I) Splenocytes were incubated in presence of Brefeldin A, CD107a and gp33 peptide for 6h at 37°C followed by intracellular cytokine staining. (F) Representative flow cytometry plots pre-gated on P14 T cells (Ly5.1+). (G) Frequency and total numbers IFNγ+ CD107a+ P14 T cells (Ly5.1+). (H) Frequency and total numbers of IFNγ+ CD107a+ endogenous CD8 T cells in the spleen. I) Viral load in spleen. Data shown are mean ±SEM of n = 8–9 mice pooled from 3 independent experiments. ns, not significant, * p<0.05, ** p<0.01 (unpaired two-tailed t-test). (J) 1x103 P14 T cells were transferred into WT, NCR1gfp/gfp and αNCR1 blocked WT mice followed by LCMV docile infection. Weight curve normalized to initial body weight. Data shown are mean + SEM of n = 4–7 mice pooled from 2 independent experiments. ns, not significant, * p<0.05 (two way ANOVA followed by Sidak’s multiple comparison test). (K) B6 and NCR1gfp/gfp mice were infected with 1.5x104—2x104 ffu LCMV docile. Spleens were harvested 12 dpi and viral load was determined. Data shown are mean of n = 8 pooled from 2 independent experiments. ns, not significant, * p<0.05, ** p<0.01 (unpaired Mann-Whitney U test).
Fig 5.
Severe disruption of splenic architecture in absence of NCR1 during chronic LCMV infection.
(A-C) 2x104 P14 T cells (Ly5.1+) were transferred into WT and NCR1gfp/gfp mice followed by LCMV WE infection. After 8 days, spleens were harvested and prepared for confocal microscopy and pictures were taken with a 10x objective. (A) Overview of the splenic sections. Region of interest (ROI), dashed line. Scale bar 80 μm (B-C) Ratio of MFI of volume of B cell zones (B220+) and the volume of MFI of T cells found within B cell zone was determined. Shown is the ratio of volume between B cell zone-localized (B) CD3+ cells/ (C) P14 T cells (Ly5.1+) and the volume of the B cell zone, respectively. Data shown are mean + SEM of 61–66 B cell zones derived from 4–5 mice pooled from 2 experiments. ns, not significant, * p<0.05 (unpaired two-tailed t test). (D-F) 2x103 P14 T cells (Ly5.1+) were transferred into WT and NCR1gfp/gfp mice followed by LCMV docile infection. After 8 days, spleens were harvested and prepared for confocal microscopy. (D) Overview of the splenic sections. Region of interest (ROI), dashed line. Scale bar 70 μm (E-F) Ratio of MFI of volume of B cell zones (B220+) and the volume of MFI of T cells found within B cell zone was determined. Shown is the ratio of volume between B cell zone-localized (E) CD3+ cells/ (F) P14 T cells (Ly5.1+) and the volume of the B cell zone, respectively. Data shown mean ±SEM of 54–97 B cell zones from 8–9 mice pooled from 3 experiments * p<0.05, **** p<0.0001 (unpaired two-tailed t-test).