Binding of svH1C to lectin-type receptors.

<p>The buffer in these assays was PBS containing 0.05% Tween-20 (see text for effects of different buffer compositions). The figure shows the amount of streptavidin-peroxidase bound to svH1c that was bound to the receptors. Siglec-1 and CLEC10a contained a C-terminal His tag and were assayed in separate experiments. The other receptors were Fc-chimeras and were included in the same assays. SEM was determined for six assays from four independent experiments. Inhibition by fetuin is shown by the average of single values in two assays in which the glycoprotein was added at 10 μM (red) or 30 μM (green).</p

A Peptide Mimetic of 5-Acetylneuraminic Acid-Galactose Binds with High Avidity to Siglecs and NKG2D

<div><p>We previously identified several peptide sequences that mimicked the terminal sugars of complex glycans. Using plant lectins as analogs of lectin-type cell-surface receptors, a tetravalent form of a peptide with the sequence NPSHPLSG, designated svH1C, bound with high avidity to lectins specific for glycans with terminal 5-acetylneuraminic acid (Neu5Ac)-galactose (Gal)/N-acetylgalactosamine (GalNAc) sequences. In this report, we show by circular dichroism and NMR spectra that svH1C lacks an ordered structure and thus interacts with binding sites from a flexible conformation. The peptide binds with high avidity to several recombinant human siglec receptors that bind preferentially to Neu5Ac(α2,3)Gal, Neu5Ac(α2,6)GalNAc or Neu5Ac(α2,8)Neu5Ac ligands. In addition, the peptide bound the receptor NKG2D, which contains a lectin-like domain that binds Neu5Ac(α2,3)Gal. The peptide bound to these receptors with a K_D in the range of 0.6 to 1 μM. Binding to these receptors was inhibited by the glycoprotein fetuin, which contains multiple glycans that terminate in Neu5Ac(α2,3)Gal or Neu5Ac(α2,6)Gal, and by sialyllactose. Binding of svH1C was not detected with CLEC9a, CLEC10a or DC-SIGN, which are lectin-type receptors specific for other sugars. Incubation of neuraminidase-treated human peripheral blood mononuclear cells with svH1C resulted in binding of the peptide to a subset of the CD14⁺ monocyte population. Tyrosine phosphorylation of siglecs decreased dramatically when peripheral blood mononuclear cells were treated with 100 nM svH1C. Subcutaneous, alternate-day injections of svH1C into mice induced several-fold increases in populations of several types of immune cells in the peritoneal cavity. These results support the conclusion that svH1C mimics Neu5Ac-containing sequences and interacts with cell-surface receptors with avidities sufficient to induce biological responses at low concentrations. The attenuation of inhibitory receptors suggests that svH1C has characteristics of a checkpoint inhibitor.</p></div

svH1C bound to monocytes after digestion of PBMCs with neuraminidase.

<p>(A) Dot plots and histograms of cells in PBMC samples treated 30 min at 37°C with neuraminidase inactivated by heating 20 min in boiling water. (B) Histograms of PBMCs from the same donor as (A) after treatment with active neuraminidase. After enzyme digestion, cells were incubated 30 min on ice with 1 μM biotin-tagged svH1C, washed and incubated 30 min on ice with marker antibodies and streptavidin labeled with PerCP-Cy5.5. Binding to only the monocyte (mono) fraction was significantly increased by treatment with neuraminidase, as shown by the histogram. The upper dot plot in (B) represents the monocyte fraction from (A), whereas the lower dot plot represents the monocyte fraction from (B). The subset of monocytes to which svH1C bound is circled, which accounted for 35% of the total monocyte population. This experiment was performed three times with similar results.</p

Binding of svH1C to NKG2D.

<p>(A) The amount of bound peptide was measured after extensive washing with PBS containing 0.05% Tween-20. Fetuin (5 μM, red; 10 μM, yellow; 30 μM, green) and sialyllactose (12 μM, red; 20 μM, yellow; 40 μM, green) were included as inhibitors. Inhibition is shown by the average of single values from three separate assays. The average 100% value was 2.4 ng conjugate bound. (B) Graphical representation of inhibition of binding of svH1C to NKG2D by fetuin (circles) or sialyllactose (squares) as shown in (A).</p

¹H-¹H 2D TOCSY 400 MHz NMR spectrum of svH1C.

<p>The fingerprint of HN-Hα/aliph svH1C protons on a 16.0204 ppm spectrum was recorded in H2O/D2O (90%/10% v/v, at pH 7.0, T = 298K). The amino acid spin-patterns are indicated with dashed lines. Vertical bracket indicate the HN-Hα & ΗΝ-Ηβ (only for serine residues), while horizontal brackets denotes the amide HN and the lysine side-chain NH groups, which are involved in iso-peptide bonds.</p

Models of monovalent, mimetic sequence of svH1C (colored space-filled structure) docked in the ligand binding site of receptors (yellow).

<p>(A) Siglec-5 (accession no. 2ZG1), predicted binding energy, -47 kJ/mol. (B) NKG2D (accession no. 1MPU), predicted binding energy, -40 kJ/mol.</p

Circular dichroism spectra of svH1C as a function of concentration.

<p>(A) 100 μM peptide in 50 mM borate, pH 9.0; (B) peptide solution in (A) diluted 1:3 with water; (C) peptide solution in (A) diluted 1:9 with water; (D) 100 μM peptide in 50 mM borate adjusted to pH 2.</p

Phosphorylated cell-surface receptors after treatment of human PBMCs with 100 nM svH1C for 5 min.

<p>The cells were collected by centrifugation, lysed and analyzed by the Human Phospho-Immunoreceptor Array (R&D Systems) according to the manufacture’s protocol. This experiment was performed in duplicate two times with similar results.</p

Increases in populations of immune cells in the peritoneal cavity.

<p>svH1C was injected subcutaneously every other day with a dose of 1 nmole/g into C57Bl/6 mice. Peritoneal cells were obtained from 3 animals, pooled, and analyzed by flow cytometry. The bars, in increasing darkness, show populations of specific cell types at day 1, 3 and 5 of treatment, i.e., 24 hours after each injection at day 0, 2 and 4. The markers used to identify cell types are listed across the top of the figure. The total number of each cell type was plotted, with the scale indicated at the top of each cell type. Cells are identified by the usual designations across the bottom of the figure. B_M indicates memory B cells. An asterisk indicates activated populations that expressed CD69.</p

CTLA-4 blockade decreases TGF-β and IL-10 expression by HIV-specific CD8+ T Cells.

<p>PBMC (n = 6) were stimulated with HIV peptides in the presence of anti-CTLA4 (or isotype control), then stained with anti-IFN-γ FITC, anti-TGF-β PE, anti-IL-10 APC, anti-CD3 AmCyan, anti-CD4 PerCP CY5.5, anti-CD8 PE CY7, and analyzed by flow cytomerty. Samples were first gated on the CD3+/CD8+ lymphocyte population then the percent of TGF-β, IL-10, and IFN-γ positive cells were determined. Results were expressed as percent of HIV-specific CD8+ T cells expressing TGF-β, IL-10, or IFN-γ after subtraction of the back ground. (A) Representative plots of HIV-specific CD8+ T cells expressing TGF-β, IL-10, or IFN-γ in the presence or absence of anti-CTLA-4. (B-D) Dashed line represents the cutoff for significant TGF-β (B), IL-10 (C), and IFN-γ (D) expression. Percentages in between brackets are median values. The two dots joined by a line represent the values obtained from the same individual and analysis was performed by paired <i>t-</i>test.</p