p24 Gag_209–218/HLA-Cw*0102 complexes on T2 cells lead to functional inhibition of primary KIR2DL2(+) NK cells.

<p>(<b>A</b>) Representative histograms of degranulation of KIR2DL2(−) (left panel) and KIR2DL2(+) (right panel) NK cells after co-incubation with peptide-pulsed T2 cells (tinted and black) or without target cells (clear). NK cell degranulation was measured flow cytometrically by surface expression of CD107a. (<b>B</b>) Different levels of NK cell degranulation between KIR2DL2(−) (dark grey) and KIR2DL2(+) (light grey) NK cells after co-incubation with peptide-pulsed T2 cells. Each column represents mean±SEM percentage of CD107a(+) NK cells of 5 of different individuals. Unspecific degranulation of NK cells measured without target cells was deducted from each column.</p

HIV-1 p24 peptide-dependent HLA-Cw*0102 stabilization on T2 cells.

<p>(<b>A</b>) Representative histograms of HLA-Cw*0102 stabilization on T2 cells as determined by flow cytometry. Histograms display HLA-Cw*0102 expression on T2 cells in the presence of control peptides VAP-FA and VAP-DA (black) at a concentration of 40 µg/ml as compared to unloaded T2 cells (grey tinted) and isotype control (clear). (<b>B</b>) HLA-Cw*0102 expression on HIV-1 p24 peptide-pulsed T2 cells. HLA-Cw*0102 expression is illustrated as relative median fluorescence intensity (RFI) as compared to unloaded T2 cells. Each column represents mean±SEM RFI of 5 independent experiments for each HIV-1 p24 OLP. A total of 59 HIV-1 p24 OLPs were analyzed, which previously showed strongest HLA-A/B/C stabilization as determined by an HLA-A/B/C antibody. Of those, 11 peptides with the highest HLA-Cw*0102 expression (>five-fold) were selected for subsequent KIR-Fc binding assays.</p

Ability of p24 Gag_209–218 peptide variants for HLA-Cw*0102 stabilization and KIR2DL2-Fc binding.

<p>(<b>A</b>) Differential expression of HLA-Cw*0102 on T2 cells after co-incubation with peptide variants of HIV-1 p24 Gag_209–218-L (AAEWDRLHPV). Peptide variants differed in length [9 or 10 amino acids (aa)] as well as in sequence (substitution of Leucine in position 7 with various amino acids). HLA-Cw*0102 expression is illustrated as relative median fluorescence intensity (RFI) as compared to unloaded T2 cells. Each column represents mean±SEM RFI of 4 independent experiments for each peptide variant. (<b>B</b>) Relative binding of KIR2DL2-Fc to T2 cells after co-incubation with selected HIV-1 p24 Gag_209–218variants. KIR2DL2-Fc binding is illustrated as RFI as compared to VAP-DA-pulsed T2 cells. Each column represents mean±SEM RFI of 3 independent experiments for each 10 aa variant. (<b>C</b>) Different levels of NK cell degranulation after co-incubation with T2 cells in the presence of different p24 Gag_209–218-L variants. Each column represents mean±SEM percentage of CD107a(+) NK cells of 4 different individuals. Unspecific degranulation of NK cells measured without target cells was deducted from each column. * indicates statistical significance as defined <i>p</i><0.05.</p

Probing of fucose and mannose residues on <i>L</i>. <i>rhamnosus</i> GG pili using AFM.

<p>Fig 1A and 1C depict the adhesion forces and Fig 1B and 1D the rupture length histograms (n = 1024) obtained in buffer, from the interaction between <i>L</i>. <i>rhamnosus</i> GG wild type and fucose- and mannose-binding lectin probes (AAL and HHA resp.). In Fig 1E and 1F the force data for the interaction of a pili-deficient Δ<i>spaCBA</i>::Tc^R mutant (CMPG5357) with the two lectin probes are displayed. Insets show representative retraction force curves.</p

Immunogold labeling reveals colocalization of SpaA and fucose on SpaCBA pili.

<p>Immunoelectron microscopy double labeling of <i>L</i>. <i>rhamnosus</i> GG cells (A and B) and the Δ<i>spaCBA</i>::Tc^R mutant (CMPG5357) (C) with SpaA antiserum and the fucose-specific <i>Aleuria aurantia</i> lectin (AAL). Detection of SpaA and AAL was done using 5 nm (white arrows) and 10 nm gold particles (black arrows) respectively. The scale bar represents 500 nm. Original overall pictures are shown as insets of A and B.</p

SpaCBA pili are glycosylated with mannose and fucose.

<p><b>(A) SpaCBA pili are glycosylated on <i>L</i>. <i>rhamnosus</i> GG cells—</b>Cell wall-associated proteins of <i>L</i>. <i>rhamnosus</i> GG wild type (1), the pilus-deficient Δ<i>spaCBA</i>::Tc^R mutant (CMPG5357, 2) and the Δ<i>welE</i>::Tc^R mutant on which the pili are overexposed (CMPG5351, 3), were probed with mannose- and fucose-specific lectins (HHA and AAL resp.). Pili content was visualized by probing with SpaC antiserum (SpaC, black arrow and HMW: high molecular weight pili). Interference of the Msp1 glycoprotein was ruled out (open arrow). Blots and gels were performed in triplicate. (LK = Precision Plus Protein^™ Kaleidoscope^™ Standard, Bio-Rad) <b>(B) Purified pili are glycosylated—</b>SDS-PAGE separated pili (pool A) were stained with PAS glycostain and Sypro^® to visualize their protein content. Pili content was shown by probing of Western blotted samples with SpaC antiserum (HMW: high molecular weight pili). Purified Msp1 (open arrow) was used as a positive control. Representative gels are shown, experiment was carried out in triplicate. (LK = Precision Plus Protein^™ Kaleidoscope^™ Standard, Bio-Rad) <b>(C) SpaCBA pili bind mannose-specific lectins—</b>Purified pili fractions (PRM and pili pool B) were subjected to PAS glycostaining and both Sypro^® and Silver stain to visualize their protein content. Absence of 75 kDa signals on PAS and lectin blots rule out the interference Msp1 (cf. open arrow). Western blotted samples were probed with SpaC antiserum (HMW: high molecular weight pili) and the mannose-specific lectins HHA and GNA, visualizing the pili content of the samples and the presence of mannose, respectively. Representative gels and blots of in triplicate-repeated experiment. (LK = Precision Plus Protein^™ Kaleidoscope^™ Standard, Bio-Rad) <b>(D&E) Mannose- and fucose-specific lectins bind SpaCBA pili—</b>Binding of lectins to plate-coated pili was measured to ELISA. Wells coated with coating buffer served as a negative control. Mannan and Lewis X were coated as a positive control for the mannose-specific HHA (<i>Hippeastrum</i> hybrid, D) and fucose-specific AAL (<i>Aleuria aurantia</i>, E) lectins, respectively. Error bars represent standard deviations of three independent experiments (paired t-test, p < 0.05)</p

Binding of KIR2DL3-Fc and inhibition of primary KIR2DL3+ NK cells is significantly stronger when target cells are pulsed with the T_Gag303V variant peptide.

<p>(A) Representative dot plots of KIR2DL3-Fc staining of 721.221-ICP47-C*03:04 cells pulsed with positive control peptide GAL, negative control peptide GKL, T_Gag303 wild-type peptide, or T_Gag303V variant peptide. Staining was measured by flow cytometry after addition of a secondary PE-conjugated anti-IgG antibody. All cells express GFP, indicating successful transduction of ICP47 and thus optimal blockade of TAP. (B) Quantification of KIR2DL3 binding expressed as percent of cells binding KIR2DL3-Fc. Means of six independent experiments, with error bars representing SD, are shown. Binding of KIR2DL3-Fc was significantly stronger to 721.221-ICP47-C*03:04 cells pulsed with the variant T_Gag303V (YVDRFFK<u>V</u>L) peptide than to those pulsed with wild-type (YVDRFFK<u>T</u>L; <i>p</i> = 0.002) or T_Gag303A (YVDRFFK<u>A</u>L; <i>p</i> = 0.002) peptide. (C and D) Degranulation of primary KIR2DL3+ (C) and KIR2DL3− (D) NK cells measured as CD107a expression after incubation with 721.221-ICP47-C*03:04 cells pulsed with T_Gag303 wild-type and T_Gag303A and T_Gag303V variant peptides. The percentage of CD107a+ NK cells in response to the respective peptide is normalized to the percentage of CD107a+ NK cells for KIR2DL3− and KIR2DL3+ NK cell subsets after co-incubation with target cells pulsed with GKL (GAVDPLLKL) control peptide. 721.221-ICP47-C*03:04 target cells presenting YVDRFFK<u>V</u>L significantly inhibit degranulation of KIR2DL3+ NK cells compared to targets pulsed with YVDRFFK<u>T</u>L (<i>p</i> = 0.0121) or YVDRFFK<u>A</u>L (<i>p</i> = 0.0019). The different peptide variants had no detectable effect on CD107a expression in KIR2DL3− NK cells. All <i>p</i>-values stated are adjusted for multiplicity of testing. Primary NK cells from nine different healthy <i>KIR2DL3+</i> participants were tested. (E) Sensogram of KIR2DL3 dimeric analyte binding to biotinylated HLA-C*03:04/YVDRFFKTL, HLA-C*03:04/YVDRFFKAL, and HLA-C*03:04/YVDRFFKVL monomers on a Streptavidin chip; an empty well served as negative control. The sensogram data are normalized to the amount of respective HLA monomer immobilized on the chip by HC10 antibody. Each fit was obtained using double reference subtraction and is shown as a black line. Affinity of the KIR2DL3/HLA-C*03:04/YVDRFFKVL protein-protein interaction was highest. *<i>p</i> < 0.05; **<i>p</i> < 0.01.</p

KIR footprints in HIV-1 clade C sequence.

<p>^aHIV-1 consensus sequence in 392 study participants.</p><p>^bDominant variant.</p><p>^cConsensus T was also significantly associated with the lack of encoding <i>KIR2DL3/HLA-C*03</i>:<i>04</i> (<i>q</i> = 0.1).</p><p>^dVariant amino acid A at Gag₃₀₃ was significantly associated to an <i>HLA-C*03</i>:<i>04</i> genotype alone (<i>p</i> < 0.001), but not in combination with KIR2DL3.</p><p>KIR footprints in HIV-1 clade C sequence.</p

Structural details of the HLA/peptide/KIR three-way complex.

<p>(A) Overall structure of the HLA/peptide/KIR complex. The peptide is buried in the HLA class I binding grove, while KIR interacts with both helices of HLA class I as well as the C-terminus of the peptide. (B) Comparison of the interactions around the mutated viral residue (top: in fat sticks; bottom: in spheres). The YVDRFFK<u>T</u>L (YTL) wild-type peptide includes a hydrogen bond between the side-chain oxygens of Thr₈ and Asn₈₀, while the YVDRFFK<u>V</u>L (YVL) variant improves the hydrophobic packing against Val₇₆. Interestingly, Asn₈₀ participates in a hydrogen bond expected to confer allotype specificity to KIR2Ds.</p

Equal HLA-C*03:04 stabilization on TAP-blocked 721.221-ICP47-C*03:04 cell line with HIV-1 p24 Gag T_Gag303 wild-type and variant peptides.

<p>(A) Representative histograms of HLA-C*03:04 stabilization with T_Gag303 wild-type and T_Gag303A and T_Gag303V variant peptides compared to addition of no peptide (−). HLA-C*03:04 surface levels were determined by flow cytometry using an anti-pan—HLA class I antibody (clone W6/32). Peptides were added at a saturating concentration of 100 μM. (B) Quantification of HLA-C*03:04 stabilization in the presence of T_Gag303 wild-type and T_Gag303A and T_Gag303V variant peptides, as well as positive endogenous control peptides for HLA-C*03:04 stabilization (GAVDPLLAL and GAVDPLLKL) and a non-HLA-C*03:04-stabilizing influenza-derived control peptide (Flu, ILRGSVAHK). Data represent mean of five experiments with error bars indicating the SD. Relative fluorescence intensity (RFI) was calculated as the gMFI of the sample divided by the gMFI of 721.221-ICP47-C*03:04 cells stained in the absence of peptide.</p