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1 Supporting Information lpek et al /pnas SI Materials and Methods Mice. cell knockout, inos / (Taconic arms), Rag1 /, INγR /, and IL-12p4 / mice (The Jackson Laboratory) were maintained and/or bred under barrier conditions in the ana- arber ancer Institute nimal acility in accordance with institutional and National Institutes of Health guidelines. ell Preparation and ytofluorimetry. Single-cell suspensions were prepared from spleen, skin lymph nodes, lungs, and liver by mechanical disruption. one marrow cells were isolated by flushing media through tibias and femurs. or myeloid cell analysis, spleens were disrupted by forceps teasing and incubated in digestion buffer containing RPMI (Invitrogen),.2 mg/ml collagenase P (Roche),.1 mg/ml Nase I (Sigma), and. mg/ml dispase (Invitrogen) for 4 min at 37. fter R lysis with K solution, cells were suspended in S buffer containing cr-block antibody (2.4G2) and stained with fluorescently labeled monoclonal antibodies (mbs) specific for NK1.1 (PK136), 3ε (14-211), 49b (X), 4 (3-11), 4.1 (2), 44 (1M7), α (3-6.7), 122 (TM-1), (24), NKG2 (7), 11b (M1/7), 27 (LG.31), Ly49H (31), NKp46 (R&), 43 (111), 11c (HL3), 22 (R3-62), 69 (1H.23), P1 (J43), KLRG1 (21), NKG2 (16a11), 4 (m4.7), NM1 (1), Ly49 (4), Ly-49HI///H (1411), Gr1 (R6-), γ-chain/132 (TUGm2), and 3ζ (61.2) or isotype controls. or intracellular staining, splenocytes were plated in 24-well plates at cells/ml in RPMI supplemented with % S and activated with PM ( ng/ml) and ionomycin ( ng/ml) for h, on anti-nk1.1 (PK136; 2 μg/ml) or isotype antibody (2 μg/ml)- coated plates for h with monensin (GolgiStop, iosciences) for the last 4 h, or on anti-3ε ( μg/ml) and anti-2 (2 μg/ ml)-coated plates for 24 h with monensin for the last 1 h. In some experiments, OT-1 splenocytes were transferred i.v. followed by IL-/IL-Rα complex treatment. Splenocytes were stimulated h with SIINKL peptide (2 μg/ml, naspec). fter cell-surface staining with anti-nk1.1, -3ε, -α, 4, or 4.1, cells were fixed and intracellularly stained using ytoix/ytoperm reagents ( iosciences) containing anti-inγ, TNα, or isotype control. egranulation was analyzed using similar conditions in the presence of anti-17a-p or isotype control. Proliferative potential was tested by intracellular staining using anti-k i -67 ( iosciences) or isotype control. LI assay was performed according to manufacturer s protocol (Immunohemistry Technologies). ells were analyzed using S alibur, S ria ( iosciences) and lowjo software (Tree Star). Modifications to IL-/IL-Rα Treatments. Leukocytes were depleted using mbs specific for α (2.43, 2 μg) and 4 (GK1., 3 μg) (ioxell). ntibodies were injected every 3 d intraperitoneally starting 2 d before IL-/IL-Rα complex treatment until the end of the sustained treatments. Leukocyte depletion was verified by cytofluorimetry. TLR3 ligand PolyI: (G Healthcare) was injected intraperitoneally (1 μg in 1 μl of PS per mouse) with the last dose of IL-/IL-Rα complexes during sustained treatments. NK ell ytotoxicity ssays. In vitro cytotoxicity assays were performed by lactate dehydrogenase release (LH) assay (ytotox 96 nonradioactive cytotoxicity assay, Promega) according to manufacturer s protocol. NK cells were enriched in splenocytes by depletion of 3ε + and 19 + cells using biotinylated antibodies and MS (Miltenyi). Proportion of NK cells were determined in each sample by flow cytometry and normalized among three groups to have 2 3% NK cells in each effector population. ssay was performed in 96-well U-bottom plates with Y1 cells with different amounts of NK cell-enriched splenocytes to obtain effector:target ratios indicated. bsorbance was recorded at 49 nm using a Victor 3V plate reader (Perkin-lmer). Percentage of cytotoxicity was calculated as [(xperimental-ffector spontaneous Target spontaneous)/target maximum Target spontaneous) 1]. or in vivo killing, NK cell cytotoxicity against allogeneic target cells was assessed by a carboxyfluorescein succinimidyl ester (S)-based in vivo assay. riefly, syngeneic splenocytes (H-2b) labeled with.2 μm S (S low ) served as the control population, and allogeneic splenocytes (H-2q) labeled with. μm S (S high ) served as the target population. S low and S high cells at a 1:1 ratio (total of cells) were injected i.v. into control mice or IL-/IL-Rα complex-treated mice 2 d after the last dose. The ratio of S low and S high cells in spleen was determined using cytofluorometry 3 h later relative to the ratio in NK1.1-depleted control mice. epletion was verified using anti- 49b staining. Percentage of specific killing was calculated as: [(1 (Ratio sample /Ratio NK1.1 depleted )) 1]. T ell Suppression ssay. Myeloid cells from spleens of mice that received sustained treatment were enriched by depletion of NK1.1 +, 3ε +, 19 + cells using biotinylated antibodies and MS. S-labeled OTIxrag1 / cells (1 1 per well) were cultured alone or in 1:1 ratio with myeloid cells in the presence or absence of SIINKL peptide (1 ng/ml) for 3 d. + cells were analyzed for S dilution by flow cytometry. lpek et al. 1of
2 treatment nalysis 2 d 2 d 3 d 3 d 2 d Transient treatment #Splenocytes (x1 6 ) NK ε #NK cells (x1 6 ) Lymph nodes #NK cells (x1 4 ) one marrow 4 2 in 4 + gate Liver in 4+ gate Lung ig. S1. nalysis of NK cell expansion upon in vivo stimulation with IL-/IL-Rα complexes. () typical treatment protocol used throughout the study. In sustained treatments, mice received five doses of IL-/IL-Rα complexes every 2 3 d intraperitoneally. In transient treatments, mice received a single dose at the same time as the fifth dose of sustained treatment. Two days later, lymphocytes were analyzed by phenotyping, intracellular cytokine staining, degranulation, and in vivo killing assays. () Total splenocyte numbers in untreated (n = 12) mice or upon transient (n = 12) and sustained (n = 16) treatments. ach circle represents an individual mouse. Lines indicate the average value. () Representative dot plots showing expansion of NK ε (NK) cells in the spleens analyzed by flow cytometry. Numbers indicate percentage of cells in the gates. () Increase in percentage (Left) and number (Right) of splenic NK cells in untreated (n = 12) mice or upon transient (n = 12) and sustained (n = 16) treatments. ach circle represents an individual mouse. Lines indicate the average value. () Percentage and number of NK cells in lymph nodes, percentage of NK cells in bone marrow, and percentage of NK cells within 4 + cells in liver and lungs of untreated (Un) mice or upon transient (Tr) and sustained (Su) treatments (n = 3). Significance: P <., P <.1, P <.1; NS, not significant. ata are means ± S. Spleen one marrow Transient Transient 11b b b b b + KLRG1 + 11b + KRLG1-11b - KLRG1 + 11b - KRLG1 - ig. S2. ccumulation of mature NK cells upon sustained in vivo stimulation with IL-/IL-Rα complexes. ar graphs showing frequency of NK cells in different developmental stages based on 11b and 27 expression (Upper) and 11b and KLRG1 expression (Lower) inspleen() and bone marrow () of untreated mice or upon transient and sustained treatments (n = 6). Statistics only shown for white bars indicating the mature NK population. Significance: P <., P <.1, P <.1. lpek et al. 2of
3 %INγ + NK cells Transient Lymph nodes one marrow Liver Transient anti-nk1.1 stimulation %INγ+ NK cells KLRG1 - KLRG1 + %17a NK1.1 depletion control S 49b ontrol NK1.1-depleted 3.3 S G 1 + PolyI: ig. S3. Impaired effector function of NK cells upon sustained in vivo stimulation with IL-/IL-Rα complexes. () Percentage of splenic INγ + NK cells upon PM/ionomycin stimulation shown (n = 11). () MI of INγ + NK cells upon PM/ionomycin stimulation shown for lymph nodes (n = ), bone marrow (n = 3), and liver (n = 3). White bars, untreated; gray bars, transient; black bars, sustained treatment. () INγ production by KLRG1 (white) and KLRG1 + (black) NK cells in spleen of untreated mice or upon transient and sustained treatments (n = 3). (Left) MI of INγ upon PM/ionomycin stimulation. (Right) Percentage of INγ + NK cells upon anti-nk1.1 stimulation. () egranulation of PM/ionomycin stimulated NK cells from untreated mice or upon transient and sustained treatments (n = ). () Representative histograms showing S + cells in the spleens of mice analyzed for in vivo cytotoxicity assays. Numbers indicate percentage of S low and S high cells. () epletion of NK1.1 + cells using anti-nk1.1 (PK136) antibody for in vivo killing assays. Representative dot plots showing NK cell depletion by 49b staining versus forward scatter (S) in spleen. Numbers indicate percentage. (G) ffect of PolyI: treatment on INγ production by NK cells. MI of INγ + NK cells shown (n = 3). PolyI: was injected during the last dose of sustained treatments. Significance: P <., P <.1, P <.1; NS, not significant. ata are means ± S ontrol depleted ontrol 4 depleted Wild-type cell 2 1 Rag1 #11b + cells (x1 6 ) Gr1 low Gr1 high unstimulated OTI OTI+Myeloid S 2 1 INγR inos IL-12p4 G % %γ-chain γ-chain MI H ζ-chain MI ig. S4. ffect of different immune cells and immunomodulatory molecules on NK cell function sustained in vivo stimulation with IL-/IL-Rα complexes. () ffect of (Left) and 4 (Right) T cell depletion on NK cell function upon sustained in vivo stimulation with IL-/IL-Rα complexes. MI of INγ + NK cells upon PM/ionomycin stimulation shown (n = 3). () ffect of cells on NK cell function assessed in wild-type () or cell-knockout () mice upon sustained in vivo stimulation with IL-/IL-Rα complexes. MI of INγ + NK cells upon PM/ionomycin stimulation shown (n = 3). () MI of INγ + NK cells in IL-/IL-Rα treated or untreated Rag1 mice upon PM/ionomycin stimulation (n = 3). () Number of 11b + Gr1 low (white bars) or Gr1 high (black bars) cells in spleens upon IL-/IL- Rα treatments (n = 9). () Representative histograms showing OV-specific OTI T cell proliferation in the presence of SIINKL peptide and myeloid cells enriched from spleens of mice that received sustained in vivo stimulation with IL-/IL-Rα complexes. Numbers indicate percentage of divided cells. () ffect of INγ (Left), inos (enter) and IL-12p4 (Right) on NK cell function upon sustained in vivo stimulation with IL-/IL-Rα complexes. MI of INγ + NK cells upon PM/ ionomycin stimulation shown (n = 3). (G) ar graphs show percentage of (Left) and common γ-chain + (enter) NK cells, and MI of common γ-chain + NK cells (Right) (n =3 ). (H) MI of 3ζ-chain + NK cells (n = 3). Significance: P <., P <.1; NS, not significant. ata are means ± S. lpek et al. 3of
4 % T cells # T cells (x1 6 ) %44hi Transient P KLRG %69+ %P1+ %KLRG %INγ + T cells %17a+ T cells Transient anti-3ε/2 stimulation INγ + INγ - %INγ+ OTI cells Peptide stimulation ig. S. xpansion and activation of T cells upon in vivo stimulation with IL-/IL-Rα complexes. () Increase in percentage (Left) and number (Right) of splenic T cells in untreated (n = 12) mice or upon transient (n = 12) and sustained (n = 16) in vivo stimulation with IL-/IL-Rα complexes. ach circle represents an individual mouse. Lines indicate the average value. () 44 expression by splenic T cells. (Left) Representative histograms showing 44 expression. Numbers indicate %44 high cells. lack line, 44; gray-filled line, isotype control. (Right) Percentage of T cells with 44 high phenotype in untreated mice or upon transient and sustained treatments (n = ). () xpression of activation markers on T cells from spleen. (Left) Representative histograms showing 69, P1, and KLRG1 expression on T cells. Numbers indicate percentage. (Right) Percentage of 69 + (n = 3), P1 + (n = ), and KLRG1 + (n = 3) T cells in spleens of untreated mice or upon transient and sustained treatments. () Percentage (Left) and MI (Right) of INγ + T cells upon PM/ionomycin stimulation. (n = 21), transient (n = 21), sustained (n = 2). ach circle represents an individual mouse. Lines indicate the average value. () INγ production by and degranulation of T cells stimulated on anti-3ε/2 coated plates in the presence of anti-17a-p or isotype control (not shown) antibody. Percentage of 17a + INγ (black) and INγ + (white) T cells from untreated mice (n = 6) or upon transient (n =7) and sustained (n = 6) treatments shown. () Transferred OTI T cells were stimulated with SIINKL peptide and percentage of INγ + OTI cells was determined (n = 3). Significance: P <., P <.1, P <.1; NS, not significant. ata are means ± S. IL-/IL-Rα NK cells T cells Mouse IL- + Murine IL-Rα %INγ+ cells Human IL- + Murine IL-Rα %INγ+ cells ig. S6. omparison of the effect of mouse and human IL- in IL-/IL-Rα complexes on INγ production by NK and T cells. IL-/IL-Rα complexes were prepared using mouse (2 μg) or human (. μg) IL- with murine IL-Rα (12 μg and 3 μg, respectively). Representative bar graphs showing INγ production by NK and T cells in untreated mice or upon transient or sustained treatments (n = 3). Significance: P <., P <.1, P <.1; NS, not significant. ata are means ± S. lpek et al. 4of
5 Table S1. Lowered activation status and altered balance of activating and inhibitory receptors on NK cells upon sustained in vivo stimulation with IL-/IL-Rα complexes Marker Parameter analyzed (% or MI) Sample size 22 % ± ± ± c MI 6 2. ± ± ± MI ± ± ±.2 43 MI ± ± ± % ± ± ± 6.1 NKG2 MI 22. ± ± ± 6.2 NKp46 MI 3 16 ± ± ±. 24 MI 7.4 ± ± ± 1 4 MI ± ± ±.6 NM1 % ± ± 1.. ± 2.9 Ly-49H % 6.6 ±.1 4. ± ±.6 Ly-49 % ± ± ± 2. NKG2 % ± ± ± 1. Ly-49I// % ± ± ± 1. MI, mean fluorescence intensity. lpek et al. of
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