Activation of NK Cells by an Endocytosed Receptor for Soluble HLA-G

Signaling from endosomes is emerging as a mechanism by which selected receptors provide sustained signals distinct from those generated at the plasma membrane. The activity of natural killer (NK) cells, which are important effectors of innate immunity and regulators of adaptive immunity, is controlled primarily by receptors that are at the cell surface. Here we show that cytokine secretion by resting human NK cells is induced by soluble, but not solid-phase, antibodies to the killer cell immunoglobulin-like receptor (KIR) 2DL4, a receptor for human leukocyte antigen (HLA)-G. KIR2DL4 was constitutively internalized into Rab5-positive compartments via a dynamin-dependent process. Soluble HLA-G was endocytosed into KIR2DL4–containing compartments in NK cells and in 293T cells transfected with KIR2DL4. Chemokine secretion induced by KIR2DL4 transfection into 293T cells occurred only with recombinant forms of KIR2DL4 that trafficked to endosomes. The profile of genes up-regulated by KIR2DL4 engagement on resting NK cells revealed a proinflammatory/proangiogenic response. Soluble HLA-G induced secretion of a similar set of cytokines and chemokines. This unique stimulation of resting NK cells by soluble HLA-G, which is endocytosed by KIR2DL4, implies that NK cells may provide useful functions at sites of HLA-G expression, such as promotion of vascularization in maternal decidua during early pregnancy

CpG Oligodeoxynucleotide and Montanide ISA 51 Adjuvant Combination Enhanced the Protective Efficacy of a Subunit Malaria Vaccine

Unmethylated CpG dinucleotide motifs present in bacterial genomes or synthetic oligodeoxynucleotides (ODNs) serve as strong immunostimulatory agents in mice, monkeys and humans. We determined the adjuvant effect of murine CpG ODN 1826 on the immunogenicity and protective efficacy of the Saccharomyces cerevisiae-expressed 19-kDa C-terminal region of merozoite surface protein 1 (yMSP1(19)) of the murine malaria parasite Plasmodium yoelii. We found that in C57BL/6 mice, following sporozoite challenge, the degree of protective immunity against malaria induced by yMSP1(19) in a formulation of Montanide ISA 51 (ISA) plus CpG ODN 1826 was similar or superior to that conferred by yMSP1(19) emulsified in complete Freund's adjuvant (CFA/incomplete Freund's adjuvant). In total, among mice immunized with yMSP1(19), 22 of 32 (68.7%) with ISA plus CpG 1826, 0 of 4 (0%) with CFA/incomplete Freund’s adjuvant, 0 of 4 (0%) with CpG 1826 mixed with ISA (no yMSP1(19)), and 0 of 11 (0%) with CpG 1826 alone were completely protected against development of erythrocytic stage infection after sporozoite challenge. The adjuvant effect of CpG ODN 1826 was manifested as both significantly improved complete protection from malaria (defined as the absence of detectable erythrocytic form parasites) (P = 0.007, chi square) and reduced parasite burden in infected mice. In vivo depletions of interleukin-12 and gamma interferon cytokines and CD4(+) and CD8(+) T cells in vaccinated mice had no significant effect on immunity. On the other hand, immunoglobulin G (IgG) isotype levels appeared to correlate with protection. Inclusion of CpG ODN 1826 in the yMSP1(19) plus ISA vaccine contributed towards the induction of higher levels of IgG2a and IgG2b (Th1 type) antibodies, suggesting that CpG ODN 1826 caused a shift towards a Th1 type of immune response that could be responsible for the higher degree of protective immunity. Our results indicate that this potent adjuvant formulation should be further evaluated for use in clinical trials of recombinant malarial vaccine candidates

Binding of KIR2DL4-Ig Fusion Proteins to HLA-G–Expressing Cells Is Blocked by Anti-KIR2DL4 and Anti-HLA Class I mAbs

<div><p>(Top) 221-G cells were stained with mAb DX17 (pan–HLA class I mAb) and mAb G233 (HLA-G–specific mAb) (solid lines). Staining with secondary antibody alone is also shown (dotted lines).</p> <p>(Bottom) The 221 and 221-G cells were incubated with 50 μg/ml KIR2DL4-Ig fusion protein in the presence of 20 μg/ml of either isotype-matched control Abs or mAbs specific for class I (DX17), HLA-G (G233), or KIR2DL4 (33). Cells were then stained with goat anti-human IgG1 secondary antibodies and assessed by flow cytometry. The data are expressed as mean fluorescence intensity (MFI).</p></div

KIR2DL4 Resides in Endocytic Compartments

<p>Resting NK cells and 293T-2DL4-gfp cells were fixed, permeabilized, and stained with antibodies against Rab5, EEA-1, and M6PR, followed by Alexa-568–conjugated secondary antibodies. NK cells were further stained with mAb 33 coupled to Alexa-488 to detect KIR2DL4.</p

Cytokine/Chemokine Synthesis Induced by Soluble HLA-G in Resting NK Cells

<p>Resting NK cells (5 × 10⁵ cells/well) from three different donors were incubated separately for 48 h with either soluble, control IgM Ab (cIg), soluble anti-KIR2DL4 IgM mAb 36 (anti-2DL4), soluble HLA-G produced in CHO cells (sHLA-G), or beads coated with control anti-HA IgG1 mAb 16B12 (cIg) or with anti-CD16 IgG1 mAb 3G8 (3G8), as indicated. sHLA-G was used together with control IgG2a mAb (cIg) or with anti-HLA-G IgG2a mAb G233 (G233), as indicated. Secretion of the cytokines/chemokines listed on the left is given in pg/ml for each donor separately. Secretion induced by sHLA-G and by Ab-coated beads was compared to that induced by anti-KIR2DL4 mAb for each donor separately and is expressed as a percentage of the anti-KIR2DL4 response. The graphs represent the average ± standard deviation from three experiments.</p

Endocytosis of Soluble HLA-G into 293T-2DL4-gfp Cells Is Blocked by Anti-KIR2DL4 mAb and by Soluble KIR2DL4

<div><p>(A) Recombinant, soluble, sHLA-G at 50 μg/ml or mAb 33 (50 μg/ml) was incubated with 293T-2DL4-gfp cells. sHLA-G was also incubated together with 50 μg/ml mAb 33, 50 μg/ml KIR2DL1-Ig, or 50 μg/ml KIR2DL4-Ig, as indicated on the left. Cells were fixed, permeabilized, and stained with either Alexa-568–conjugated secondary antibodies to detect mAb 33 or anti-HLA-G mAb G233, as indicated. Individual confocal sections are shown.</p> <p>(B) Uptake of sHLA-G into 293T-2DL4-gfp cells correlates with level of KIR2DL4 expression. Red fluorescence intensity of G233 staining and green fluorescence intensity of gfp were quantified in 38 individual cells and plotted on a log scale. A best-fit line was generated by linear regression analysis using EXCEL data analysis software.</p> <p>(C) Ratio of red to green fluorescence was quantified for each loading condition as indicated. Average of 10 cells is shown, and standard deviation is shown as bars.</p></div

Immunolocalization of Endocytosed KIR2DL4 in NK Cells by Electron Microscopy

<div><p>(A) The NK cell lines NKL and YTS-2DL4-gfp were loaded with anti-KIR2DL4 mAb 33 for 120 min. Endocytosed receptor was detected using HRP-conjugated sheep anti-F(ab′)₂ mouse IgG. The HRP reaction product visible as a dark stain identifies the location of endocytosed KIR2DL4. Vesicular structures positive for KIR2DL4 ranged between 250 and 500 nm in size.</p> <p>(B) The NK cell line YTS, stably transfected with KIR2DL4-gfp (YTS-2DL4-gfp), was fixed, permeabilized, and stained with antibody to perforin followed by Alexa-568–conjugated secondary antibodies. Single confocal sections are shown.</p></div

KIR2DL4 Localizes to a Subset of Endosomes Containing Rab5

<p>The 293T cells were transfected with HA-tagged KIR2DL4 and gfp-tagged versions of Rab4, Rab5, Rab7, and Rab11. Forty hours after transfection, cells were fixed and stained with anti-HA mAb, followed by Alexa-568–conjugated secondary antibodies to detect KIR2DL4. Single confocal sections are shown.</p

Internalization of KIR2DL4 Is Dynamin Dependent

<p>The 293T cells transfected with HA-tagged KIR2DL4 together with either wild-type dynamin-gfp (Dynamin Egfp-WT) or a dominant-negative mutant of dynamin (dynamin Egfp-K44A) were loaded with KIR2DL4-specific Cy3-conjugated mAb 33 for 120 min and fixed. Individual confocal sections are shown.</p

Cell Surface Shed and Secreted, Soluble HLA-G Is Endocytosed into KIR2DL4-Containing Vesicles

<div><p>(A) Endocytosis of soluble HLA-G in resting NK cells. The 221 cells and 221 cells transfected with HLA-Cw3 (221-Cw3) were fixed, permeabilized, and stained with mAb F4/326. Resting NK cells were incubated at 37 °C for 120 min with soluble, refolded HLA-C or HLA-G. Cells were then fixed, permeabilized, and stained with reagents to detect HLA-C (F4/326) or HLA-G (G233) as indicated.</p> <p>(B) The NK cell line YTS-2DL4-gfp was loaded at 37 °C for 120 min with refolded HLA-G. Cells were fixed, permeabilized, and stained with mAb G233 to detect co-localization of soluble HLA-G with gfp-tagged KIR2DL4.</p> <p>(C) Recombinant soluble molecules of HLA-G but not HLA-C are endocytosed into 293T-2DL4-gfp cells. Refolded HLA-G and HLA-C were incubated with 293T-2DL4-gfp cells for 2 h. Cells were then fixed, permeabilized, and stained with either mAb G233 (to detect endocytosed HLA-G; upper) or mAb F4/326 (to detect endocytosed HLA-C; middle).</p> <p>(D) The 293T-2DL4-gfp cells were co-cultured with an equal number of 221 cells, 221 cells expressing transmembrane HLA-G (221-G), and 221 cells expressing a soluble isoform of HLA-G (221-sG) for 48 h. Adherent 293T-2DL4-gfp cells were fixed, permeabilized, and stained with mAb G233 followed by Alexa-568–conjugated secondary antibodies prior to acquisition of confocal images. Two 221-G cells are visible in the middle panel.</p></div