ndln NK Cells (x10 3 ) Days post-infection (A/PR/8) *** *** *** Liver NK Cells (x10 4 ) Days post-infection (MCMV)

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1 A mln NK Cells(x ) 6 1 * ndln NK Cells (x ) ns C Lung NK Cells(x ) * D LN NK Cells (x ) Days post-infection (A/PR/8) * * E Liver NK Cells (x ) 1 7 Days post-infection (A/PR/8) * * * 1 7 Days post-infection (A/PR/8) Days post-infection (MCMV) Days post-infection (MCMV) Supplementary Figure 1. Trafficking of NK cells to priming and target tissues throughout the course of viral infection. (A-E) Time course of absolute numbers of NK cells (CD-NK1.1+) in the mln) (A), ndln) (), lung (C), draining lymph nodes (DLN) (D), and liver (E), of C7L/6 at different time points post-apr8 (A-C) or post-mcmv (D,E) infection. Control (white-filled bars) mice were mock-infected by receiving either μl of PS (APR8 control) i.n. or. ml of RPMI (MCMV control) i.p., whereas infected mice (blackfilled bars) were either infected with PFU APR8 in μl of PS i.n. or x PFU MCMV in. ml of RPMI i.p. n= or mice per group representative of - experiments. One-way ANOVA with Tukey post-test used to compare groups. * p<., p<.1, * p<.1.

2 A CD7-CD11b- CD7+CD11b- CD7+CD11b+ CD7-CD11b+ * 1 / / / / CD7-CD11b- CD7+CD11b- CD7+CD11b+ CD7-CD11b+ * * NK Cell subsets (%) Control mln (H b ) A/PR/8 # of NK Cells (x ) * * * * Control A/PR/8 Supplementary Figure. Trafficking of NK cells to priming and target tissues throughout the course of viral infection. (A) Frequency of immature (CD7-CD11b-, CD7+CD11b-) and mature (CD7+CD11b+, CD7-CD11b+) NK cells in the mediastinal lymph nodes (mln) of C7L/6 (Hb) mice on day post-apr8 infection. () Absolute number of immature and mature unlicensed and licensed NK cell subsets in the mln of C7L/6 (Hb) mice on day post-apr8 infection. n= mice per group representative of - experiments. Two-way ANOVA with Tukey post-test used to compare groups. p<.1, *p<.1.

3 K K K K 1K 1K K K Singlets 8. K FSC-H K 1K K K Lymphocytes 78. K Ly9C/I 7.1 K K K FSC-A 1K K CD K FSC-A NK Cells.87 K NK1.1 Ly9G 9.1 K Ly9C/I A K K K 1K 1K 1K K K K K K K.6 Ly9C/I Ly9G Ly9A Ly9A Ly9G Ly9A.1 K. 6.9 Ly9G oolean Exclusion Gating Ly9 Negative Single positive Ly9s Ly9A-C/I-G.7 Double positive Ly9s Ly9A-C/I+G17. K K Triple positive Ly9s Ly9A-C/I+G+ 1.9 Ly9A+C/I+G+. 1K Ly9C/I Ly9C/I Ly9A+C/I-G. K Ly9C/I K Ly9C/I Ly9G K Ly9G Ly9G Ly9A+C/I-G % K 1K Ly9A Ly9A-C/I-G+.8 K Ly9G 6.9% Ly9A+C/I+G.67 K 1K - Ly9C/I+G+A+ (.%) Ly9C/I+G+A- (1.9%) Ly9C/I+G-A+ (.67%) Ly9C/I+G-A- (17.%) K Ly9A K Ly9C/I-G+A+ (9.6%) Ly9C/I-G+A- (.8%) Ly9C/I-G-A+ (.%) Ly9C/I-G-A- (.7%) K Ly9A K - Ly9G Receptor inds to Ly9C/I Hb Ly9G Hd Ly9A Hd C7L/6 (Hb).D (Hd) Ly9G Receptor inds to C7L/6 (Hb).D (Hd) Ly9C/I+G+A+ Hb, Hd Ly9C/I+G+A- H, H d Ly9C/I+G-A+ H, H d Ly9C/I+G-ALy9C/I-G+A+ b H b H d Ly9C/I-G+A- Hd Ly9C/I-G-A+ H Ly9C/I-G-A- b - d Supplementary Figure. Comparison between boolean gating and conventional gating to determine the phenotype of NK cell subsets. (A) Representative gating strategy comparing boolean gating (red square) and conventional gating (grey square) to identify Ly9-receptor subsets by staining for Ly9A, -G, and C/I on splenocytes from C7L/6 mice. oolean gating (red square) allows for the simultaneous identification of Ly9-negative, single-positive, double-positive, and triple-positive Ly9-expressing NK cell subsets, whereas conventional gating allows for pairwise comparisons of Ly9 receptors. oolean gating allows for the identification and quanitification of CD-NK1.1+Ly9C/I+G+A+,

4 Supplementary Figure continued. Ly9C/I+G+A-, Ly9C/I+G-A+, Ly9C/I+G-A-, Ly9C/I-G+A+, Ly9C/I-G+A-, Ly9C/I-G-A+, and Ly9C/I-G-A- NK cell subsets, represented as percentages of each subset among total NK cells in the pie chart (bottom). () Classification of licensed and unlicensed NK cell subsets in C7L/6 (Hb) and.d (Hd) mice based on the ability of specified Ly9 receptors to bind MHC class I molecules using either conventional gating (grey square) or oolean gating (red square).

5 A 6% (±%) Unl A/PR/8 mln (H d ) Ly9C/I+G+A+ (1.%) Ly9C/I+G+A- (9.%) Ly9C/I+G-A+ (.1%) Ly9C/I-G+A+ (.%) Ly9C/I-G+A- (16.8%) Ly9C/I-G-A+ (.9%) Ly9C/I+G-A- (1.9%) Ly9C/I-G-A- (.%) % (±%) NK Cells (x ) Control A/PR/8 mln (H d ) * C 7% (±%) Unl A/PR/8 Lung (H d ) Ly9C/I+G+A+ (1.7%) Ly9C/I+G+A- (1.7%) Ly9C/I+G-A+ (6.%) Ly9C/I-G+A+ (7.%) Ly9C/I-G+A- (.%) Ly9C/I-G-A+ (11.%) Ly9C/I+G-A- (1.7%) Ly9C/I-G-A- (.%) 6% (±%) D NK Cells (x ) 1 Control A/PR/8 Lung (H d ) * * Supplementary Figure..D NK subsets show similar licensing distribution patterns following viral infection. (A) Frequency or () absolute number of licensed (CD-NK1.1+Ly9C/I+G+A+, Ly9C/I+G+A-, Ly9C/I+G-A+, Ly9C/I-G+A+, Ly9C/I-G+A-, or Ly9C/I-G-A+; shades of red) and unlicensed (CD-NK1.1+Ly9C/I+G-A-, or Ly9C/I-G-A-; shades of blue) NK cell subsets in the mediastinal lymph nodes (mln) of.d (Hd) mice on day post-apr8 infection. (C) Frequency or (D) absolute number of licensed (shades of red) and unlicensed (shades of blue) NK cell subsets in the lung of.d (Hd) mice on day post-apr8 infection. n= mice per group representative of - experiments. (,D) Two-way ANOVA with Tukey post-test used to compare groups.

6 Lung CD1 D Liver C Lymph Nodes CD CD CD A CD1 CD1 E F Ly9C/I Ly9I H Ly9A Ly9G Ly9G I Ly9A Ly9A - Ly9G Ly9G Ly9A Ly9I Ly9G G Ly9I Ly9G Ly9G Supplementary Figure. Verification of successful depletion of specific NK populations with in vivo monoclonal antibody depletion in C7L/6 mice. (A-C) non-depleted migg (left) and NK1.1 (PK16) (right) depleted C7L/6 mice had lungs (A), livers (), and lymph nodes (C) stained for CD and CD1. (D-F) Mice were either administered mouse/rat IgG (top) or given either anti-ly9c/i (E6) (middle) or anti-ly9g (D11) (bottom) and lungs (D), livers (E), and lymph nodes (F) were removed and stained for CD-NK1.1+ and Ly9G (clone Cwy) vs Ly9I (clone YLI-9). (G-I) Mice were either administered rat IgG (top) or given anti-ly9a (YE1/) (bottom) and lungs (G), livers (H), and lymph nodes (I) were removed and stained for CD-NK1.1+ and Ly9G (clone D11) vs Ly9A (clone YE1/8..6). µg of rigg, anti-nk1.1, anti-ly9g, anti-ly9c/i, or anti-ly9a in. ml of PS i.p. was given days prior to infections. Flow plots are representative of mice from separate experiments.

7 A Liver CD1 CD1 Lymph Nodes CD1 E D C CD CD CD Lung F Ly9C/I Ly9I Ly9I Ly9I Ly9G Ly9G Ly9G Ly9G Supplementary Figure 6: Verification of successful depletion of specific NK populations with in vivo monoclonal antibody depletion in.d mice. (A-C) non-depleted migg (left) and NK1.1 (PK16) (right) depleted.d mice had lungs (A), livers (), and lymph nodes (C) stained for CD and CD1. (D-F) Mice were either administered mouse/rat IgG (top) or given either anti-ly9c/i (E6) (middle) or anti-ly9g (D11) (bottom) and lungs (D), livers (E), and lymph nodes (F) were removed and stained for CD-NK1.1+ and Ly9G (clone Cwy) vs Ly9I (clone YLI-9). µg of rigg, anti-nk1.1, anti-ly9g, or anti-ly9c/i in. ml of PS I.P. was given days prior to infections. Flow plots are representative of mice from separate experiments.

8 A Ly9C/I+ Ly9C/I+ Ly9A+ Ly9A+ C D GM-CSF+ NK Cells (x ) Ly9G+ * * mln (H d ) - A/PR8 IFNγ+ NK Cells (x ) 1 Ly9G+ * Lung (H d ) - A/PR8 Ratio of GM-CSF to IFNγ.6... * % of GM-CSF+ NK Cells 6 1 NKGA+ ns NKGA- Supplementary Figure 7..D and human NK subsets display cytokine profiles consistent with licensing. (A) Absolute number of NK cells that are GM-CSF+ and either CD-NK1.1+Ly9G+, Ly9A+, or Ly9C/I+ at day after APR8 infection in the mediastinal lymph nodes (mln) of.d mice. () Absolute number of NK cells that are IFN-γ+ and either CD-NK1.1+Ly9G+, Ly9A+, or Ly9C/I+ at day after APR8 infection in the lung of.d. (C) Aggregate ratio of GM-CSF to IFN-γ production by unlicensed and licensed NK cells. (D) Percentage of NKGA+ or NKGA- among either licensed or unlicensed NK cell populations that are GM-CSF+ after 16 hours of PMA/Ionomycin stimulation. Murine data: n= mice per group representative of - experiments. One-way ANOVA with Tukey posttest used to compare groups. Human data: Experiments were performed in duplicates; 1- aggregated single-positive NK cell subsets per unlicensed or licensed group. Five distinct individuals were used to obtain NK cells from PMCs. * p<., p<.1, * p<.1.

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