Supplemental Information. Lymphocytes Negatively Regulate NK Cell Activity. via Qa-1b following Viral Infection

Size: px

Start display at page:

Download "Supplemental Information. Lymphocytes Negatively Regulate NK Cell Activity. via Qa-1b following Viral Infection"

Bertha Stevens
6 years ago
Views:

1 Cell Reports, Volume 21 Supplementl Informtion Lymphocytes Negtively Regulte NK Cell Activity vi Q-1b following Virl Infection Hifeng C. Xu, Jun Hung, Aleksndr A. Pndyr, Elisbeth Lng, Yun Zhung, Christine Thö, Jörg Timm, Dieter Häussinger, Mrco Colonn, Hrvey Cntor, Krl S. Lng, nd Philipp A. Lng

2 Figure S1 b c LCMV (log 1 pfu/liver) LCMV (log 1 pfu/lung) Figure S1: High dose infected nimls demotrte elevted IFN-γ production, relted to Figure 1. C6BL/6 were infected with low dose or high dose LCMV WE. Virus titers were mesured from spleen, liver, lung, nd kidney tissue () 1 dy post infection (n=4) (b) 2 dys post infection (n=4) (c) 4 dy post infection (n=4). (Error brs show SEM, p <.1, indictes sttisticlly not significnt between the indicted groups)

3 Figure S2 1 b H2-t2 fo ld in c re s e / WT nive H2-t2 fo ld in c re s e / WT nive nive low high 1 1 N i ve CD19+ CD+CD8+ CD+CD4+ CD-CD19B cell T cell T cell CD11b+ cell 2 d y1 c d Q-1b CD19 CD8 F4/8 Merge Q-1b CD4 F4/8 CD19 nti-cd8 nti-cd4 nti-cd8 nti-cd4 f e Q-1b CD19 CD8 F4/8 Merge Ifnr1-/- y1 d N i ve Ifnr1-/- Figure S2: Q-1b expression is medited by type I interferon on B cells, relted to Figure 2. () CBL/6 mice were infected with low or high dose of LCMV WE. 1 dy fter infection (p.i.) H2-t2 expression levels in sorted CD-CD19+ B cells, CD+CD8+ T cells, CD+CD4+ T cells, nd CD-CD19-CD11b+ cells from spleen tissue were determined (n=-4). (b-d) CD8+ T celldepleted or non-depleted C6BL/6 mice, CD4+ T cell-depleted or non-depleted C6BL/6 mice were infected with high dose LCMV WE. (b) H2-t2 expression levels were mesured in spleen tissue 1 dy fter infection (n=4). (c) CD8+ T cell-depleted or non-depleted C6BL/6 mice nd (d) CD4+ T cell-depleted or non-depleted C6BL/6 mice were infected with high dose LCMV WE. 1 dy p.i., spleen tissue ws nlyzed for Q-1b, CD19, CD8 nd F4/8 expression (n=4). (e-f) (C6BL/6) or Ifnr1-/- nimls were infected with high dose LCMV WE. 1 dy p.i., spleen tissue ws nlyzed for (e) H2-t2 gene expression nd (f) Q-1b, CD19, CD8 nd F4/8 expression (n=6, scle br indictes µm). (Error brs show SEM, p <.1, p <.1, indictes sttisticlly not significnt between the indicted groups) Merge

4 Figure S Nïve Nïve CD69 Sc-1 CD2 CD44 Ly49 CD49b KLRG1 b Nïve Nïve Grnzyme B ILC1 cnk Perforin c Dy2 Dy4 low high low high low high cnk ILC1 ILC2 ILC LTi d cnk ILC1 ILC2 ILC LTi Klrc1 -/- Figure S: Expression of surfce molecules, grnzyme B, nd perforin is not ffected by Q-1b, relted to Figure. (-c) or mice were infected with high dose LCMV WE. () NK or ILC1 surfce molecule expression is shown s indicted in spleen tissue 1 dy p.i. (One representtive FACS blot of n=4- is shown). (b) Gnzyme B nd perforin expression ws determined on splenic cnk cells 1 dy p.i. One representtive FACS blot of n=4- is shown. (c) C6BL/6 mice were infected with low dose or high dose LCMV WE. NKG2 expression were determined on indicted ILC subsets (n=4). (d) or NKG2A deficient nimls were infected with high dose LCMV WE. 1 dy p.i. NKG2 expression were determined on indicted ILC subsets (n=4).

5 Figure S4 b c d IFN- (ng/ml) Time (dy) e LCMV (log 1 pfu/spleen) Time (dy) LCMV (log 1 pfu/lung) * f % tet-gp Blood Effector Effector Memory Centrl Memory % tet-gp Spleen Effector Effector Memory Centrl Memory Figure S4: Norml IFN-I respoe triggers similr erly virus repliction between nd mice, relted to Figure 4. (-e) or mice were infected with high dose of LCMV WE. () IFN-α concentrtio from ser of infected nimls were mesured t indicted time points (n=). (b) Number of conventionl DC (cdc) were determined by CD11c nd MHCII stining in spleen tissue of nd mice t indicted time points post infection (n=). (c) Expression of co-stimultory molecules CD4 (left pnel), CD8 (middle pnel), nd CD86 (right pnel) ws determined on CD11c + MHCII + splenic cdc (n=). (d-e) Virus titers were mesured from spleen, liver, lung, nd kidney tissue (d) 1 dy post infection (n=6) nd (e) 2 dys post infection (n=6). (f) nd mice were infected with high dose LCMV WE. 12 dys post infection, proportion of effector (CD62L - ILR - ), effector memory (CD62L - ILR + ), centrl memory (CD62L + ILR + ) of blood nd splenic gp-specific tetrmer re shown (n=-9). (Error brs show SEM, p <., p <.1, p <.1, indictes sttisticlly not significnt between the indicted groups).

Figure S b c d PD-1 IL-R TIM tet-gp + (#/spleen) 1 1 6 1 1 4 * 1 1 6 1 1 4 IFNγ + CD8 + (#/ liver) 1 6 1 1 4 n.c.gp 2B4 Lg KLRG1 e LCMV (log 1 pfu/spleen) f tet-gp + (#/spleen) 1 1 6 1 1 4 g Nïve Nïve dy4 dy4 h Nïve Nïve Klrc1 -/- dy4 dy4 Klrc1 -/- i 1 6 * 1 6 * 1 1 1 4 1 4 1 n.

() Gp-specific tetrmer ws mesured in the spleen 4 dys post infection (n=4). (b) Gp-specific tetrmer ws mesured in spleen (left) nd liver tissue (right) 8 dys p.i. (n=).

6 Figure S b c d PD-1 IL-R TIM tet-gp + (#/spleen) * IFNγ + CD8 + (#/ liver) n.c.gp 2B4 Lg KLRG1 e LCMV (log 1 pfu/spleen) f tet-gp + (#/spleen) g Nïve Nïve dy4 dy4 h Nïve Nïve Klrc1 -/- dy4 dy4 Klrc1 -/- i 1 6 * 1 6 * n.c.gp Figure S: Absence of Q-1b inhibits T cell immunity, relted to Figure 4. (-f) nd mice were infected with high dose LCMV WE. () Gp-specific tetrmer ws mesured in the spleen 4 dys post infection (n=4). (b) Gp-specific tetrmer ws mesured in spleen (left) nd liver tissue (right) 8 dys p.i. (n=). (c) At dy 8 post infection, splenocytes (left pnel) or liver cells (right pnel) were re-stimulted with the LCMV-specific epitope gp, followed by mesurement of IFN-γ production (n=). (d) One representtive histogrm of surfce molecule expression on dy 8 splenic gp-spefific tetrmer is shown (n=). (e) Virus titers were determined in spleen, liver, lung, nd kidney tissue 8 dys p.i. (n=). (f) Bone mrrow cells from or mice were mixed t 1:1 rtio with bone mrrow cells from Cd8 -/- mice were trferred into lethlly irrdited WT mice. One month lter, these mixed chimeric nimls were infected with 2x1 pfu LCMV WE. 12 dys fter infection gp-specific tetrmer in the spleen (left pnel). Splenocytes were re-stimulted with the LCMV-specific epitope gp, followed by stining for IFN-γ (n=4-, right pnel). (g) nd mice were infected with high dose LCMV WE. 4 dys post infection, expression of NKG2A/C/E ws determined on splenic gp-specific tetrmer (n=4). (h) nd Klrc1 -/- mice were infected with high dose LCMV WE, 4 dys post infection, expression of NKG2A/C/E ws determined on splenic gp-specific tetrmer (n=4). (i) Bone mrrow cells from or Klrc1 -/- mice were mixed t 1:1 rtio with bone mrrow cells from Cd8 -/- mice were trferred into lethlly irrdited WT mice. One month lter, these mixed chimeric nimls were infected with 2x1 LCMV WE. 12 dys fter infection gp-specific tetrmer in the spleen (left pnel). Splenocytes were re-stimulted with the LCMV-specific epitope gp, followed by stining for IFN-γ (n=4-, right pnel) (Error brs show SEM, p <., p <.1, p <.1, indictes sttisticlly not significnt between the indicted groups)

7 Figure S6 nti-nk1.1 b MFI (rbitrry units) 1 PD-1 IL-R TIM 2B4 Lg KLRG1 M F I (rbitr ry un its) 2 12 M F I (rbitr ry un its) 1 M F I (rbitr ry u n its) 2 M F I (rbitr ry un its) 4 2 M F I (rbitr ry u n its) 2 1 nti-nk1.1 nti-nk1.1 Figure S6: NK cell depletion prtilly restores defective T cell immunity, relted to Figure. () CBL/6 mice were treted with or without NK cell depletion ntibody. 2 dys fter tretment, NK cell number ws determined from spleen tissue (n=). (b) NK celldepleted nd non-depleted nd mice were infected with high dose LCMV WE. 12 dys fter infection surfce molecules on splenic gp-specific CD8 + T cells were mesured (n=8-1) (Error brs show SEM, p <.1, indictes sttisticlly not significnt between the indicted groups)

Supplementary Information

Supplementary Information Supplementry Informtion Cutneous immuno-surveillnce nd regultion of inflmmtion y group 2 innte lymphoid cells Ben Roediger, Ryn Kyle, Kwok Ho Yip, Nitl Sumri, Thoms V. Guy, Brin S. Kim, Andrew J. Mitchell,