Volume 19, Issue 5, Pages 701-711 (November 2003) GATA-3 Promotes Maturation, IFN-γ Production, and Liver-Specific Homing of NK Cells  Sandrine I Samson, Odile Richard, Manuela Tavian, Thomas Ranson, Christian A.J Vosshenrich, Francesco Colucci, Jan Buer, Frank Grosveld, Isabelle Godin, James P Di Santo  Immunity  Volume 19, Issue 5, Pages 701-711 (November 2003) DOI: 10.1016/S1074-7613(03)00294-2

Figure 1 Lymphoid Reconstitution in HSC Chimeras FL-derived HSC were transferred into alymphoid mice (Rag°γc°) to generate hematopoietic chimeras. (A) Flow cytometric analysis of splenocytes stained for B (CD19+) and T (CD3+) cells. NK cells (NK1.1+ IL2Rβ+) were detected in the CD19−CD3− fraction. (B) Absolute numbers of NK cells (CD3−NK1.1+IL-2Rβ+) and B cells (CD19+) in control C57Bl/6 (black circles, n = 14) and GATA-3° (gray circles, n = 16) chimeras. The mean values are represented by a gray bar. There is a significant decrease in splenic NK cells (p = 0.04) and no difference in B cells in the absence of GATA-3. (C) Expression of IL-2Rβ, 2B4, DX5, and CD2 on splenic CD3−NK1.1+ cells was similar in control C57Bl/6 (shaded histogram) and GATA-3° (bold line) chimeras. Immunity 2003 19, 701-711DOI: (10.1016/S1074-7613(03)00294-2)

Figure 2 Natural Cytotoxicity in the Absence of GATA-3 (A) Freshly isolated splenocytes from control C57Bl/6 (black) and GATA-3° (gray) chimeras showed similar lytic activity against 51Cr-labeled YAC-1 target cells. Indicated numbers of NK cells were added per well as determined by parallel flow cytometric analysis. One representative experiment of four is shown. (B) Control C57Bl/6 (black) and GATA-3° (gray) splenocytes were stimulated for 16 hr with IL-2 alone (circles) or in combination with IL-12 (squares) prior to analysis of lytic activity against YAC-1 target cells. One representative experiment of two is shown. Immunity 2003 19, 701-711DOI: (10.1016/S1074-7613(03)00294-2)

Figure 3 GATA-3° Chimeras Fail to Control Early Listeria Infection Nonreconstituted Rag°γc° mice or CD3ϵ° or GATA-3° chimeras (groups of 4 to 5 mice) were infected intravenously with 2 × 103 colony forming units (CFU) of the L. monocytogenes strain LO29 (Bregenholt et al., 2001). (A) Serum IFN-γ levels were measured 2 days postinfection using a cytokine-specific sandwich ELISA. (B) Bacterial burden in the spleen and liver of infected mice were enumerated 4 days postinfection. One representative experiment of two is shown. Immunity 2003 19, 701-711DOI: (10.1016/S1074-7613(03)00294-2)

Figure 4 GATA-3° NK Cell Are Poor Producers of IFN-γ (A) Polarized murine Th1 and Th2 clones (stimulated with phorbol ester and ionomycin) or splenocytes from control or GATA-3° chimeras (stimulated for 16 hr with a combination of IL-2/12/18) were analyzed for intracellular IFN-γ or IL-13. For NK cells, analysis was performed on electronically gated CD3−NK1.1+ cells. One representative experiment of six is shown. (B) Splenic NK cells from control (in this case, CD3ϵ°) or GATA-3° chimeras were stimulated for 24 hr with IL-2 and IL-12, IL-18, IL-12+18 (2 × 104 cells) or with mAb against CD11b or 2B4 (104 cells) and IFN-γ release measured by ELISA. Significantly more IFN-γ was released in response to IL-12+18 compared to IL-12 alone (p < 0.05). (C) Semiquantitative PCR analysis of T-bet and Hlx expression in sorted splenic NK cells (CD3−NK1.1+IL-2Rβ+) from control or GATA-3° chimeras. Two-fold dilutions of cDNA were tested as well as HPRT levels to control for integrity and quantity of the input cDNA. Immunity 2003 19, 701-711DOI: (10.1016/S1074-7613(03)00294-2)

Figure 5 Peripheral GATA-3° NK Cells Have an Immature Phenotype (A) Expression of CD11b, CD43, and B220 on splenic CD3−IL-2RβNK1.1+ NK cells from CD3ϵ° and GATA-3° chimeras. Percentages of positive cells are indicated. (B) Expression of CD11b and CD43 on bone marrow CD3−IL-2RβNK1.1+ NK cells from CD3ϵ° and GATA-3° chimeras. Percentages of positive cells are indicated. (C) Expression of CD94, Ly49D, Ly49C/I, Ly49G2, and KLRG-1 on splenic CD3−IL-2RβNK1.1+ NK cells from CD3ϵ° and GATA-3° chimeras. Percentages of positive cells are indicated. Immunity 2003 19, 701-711DOI: (10.1016/S1074-7613(03)00294-2)

Figure 6 Selective Deficiency of Liver NK Cells in GATA-3° Chimeras (A) Absolute number of hepatic and bone marrow NK cells in control C57Bl/6 (black circles; n = 14) and GATA-3° (gray circles; n = 16) chimeras. There is a highly significant decrease in liver NK cells (p = 2.3 × 10−5) and a significant increase in BM NK cells (p = 0.002) in the absence of GATA-3. (B) Expression of indicated adhesion molecules on splenic CD3−IL-2RβNK1.1+ NK cells from control C57Bl/6 (shaded histograms) and GATA-3° (bold line) chimeras. (C) Bone marrow from CD3ϵ° and GATA-3° chimeras was labeled with CFSE and injected in Rag°γc° mice. The percentage of CD3ϵ° or GATA-3° NK cells among CFSE+ cells identified in the spleen or liver 15 hr after injection is indicated. Significantly fewer liver NK cells are recovered from GATA-3° BM transfers (p < 0.05). One representative mouse of six (for each genotype) is shown. Immunity 2003 19, 701-711DOI: (10.1016/S1074-7613(03)00294-2)