An ultrasensitive and modular platform to detect Siglec ligands and control immune cell function

Abstract

Siglecs are immunomodulatory receptors that regulate immune cell function. A fundamental challenge in studying Siglec-ligand interactions is the low affinity of Siglecs for their ligands. Inspired by how nature uses multivalency, we developed Siglec-liposomes as a highly multivalent and versatile platform for detecting Siglec glycan ligands in which recombinant Siglecs were conjugated to liposomes using the SpyCatcher-SpyTag system. Siglec-liposomes offer tunable multivalency and a modular assembly, enabling presentation of different Siglecs on the same liposome. Using Siglec-liposomes, we profiled Siglec ligands on human leukocytes, revealing distinct patterns of Siglec ligands. Moreover, Siglec-liposomes are in vivo compatible, where we demonstrated that Siglec-7-liposomes bind to the brain vasculature in a mucin domain-dependent manner. Given the abundance of Siglec ligands on T cells, we investigated whether Siglec-liposomes modulate T cell function and find that Siglec-7-liposomes increase T cell proliferation in an ST3Gal1-dependent and CD43-independent manner. Together, Siglec-liposomes are a versatile and sensitive tool for detecting Siglec ligands and immunomodulation.

Fig. 1.. Development and validation of Siglec-liposomes.

(A) First-generation Siglec-Fc. (B) V1 and V2 constructs of Siglec-Fc with a genetically encoded ST. (C) Schematic of site-specific conjugation of SC to DSPE-PEG-maleimide. (D) SDS-PAGE of SC and SC-PEG-DSPE. (E) Schematic of making SC-liposomes. (F) Binding of V1 and V2 WT Siglec-7– and R124A Siglec-7–liposomes to U937 cells. Data are represented as flow cytometry histograms and median fluorescent intensity (MFI) of Siglec-7 binding. (G) Binding of V2 WT Siglec-7 and R124A Siglec-7 in different densities on liposomes to U937 cells. Data are represented as flow cytometry histograms and MFI of Siglec-7 binding. Binding of (H) WT and R124A V2 Siglec-7, (I) WT and R116A V2 Siglec-1, and (J) WT and R120A V2 Siglec-2 precomplexed with Strep-Tactin (Strep.) and conjugated to liposomes (lipo.) to U937 cells. Data are presented as flow cytometry histograms and MFI of Siglec binding. The P value for three technical replicates was calculated using an unpaired one-way analysis of variance (ANOVA). ****P < 0.0001.

Fig. 2.. Siglecs expressed in sialic acid–deficient CHO cells show improved binding toward their ligands.

Binding of WT and arginine-mutated of (A) Siglec-7–, (B) Siglec-1–, (C) Siglec-2–, (D) Siglec-3–, (E) Siglec-9–, and (F) Siglec-10–liposomes expressed in CMAS^+/+ and CMAS^−/− CHO cells to U937 cells. Data are presented as flow cytometry histograms and MFI of Siglec binding. The P value for three technical replicates was calculated using an unpaired one-way ANOVA. ****P < 0.0001.

Fig. 3.. Profiling Siglec ligands on primary cells.

(A) Schematic of staining immune cells isolated from human peripheral blood with Siglec-liposomes. (B) Siglec-liposomes binding to B cells, T cells, mature NK cells, monocytes, and neutrophils isolated from PBMCs. (C) Schematic of staining T_reg and T_conv cells isolated from human peripheral blood with Siglec-liposomes. Binding of Siglec-liposomes to (D) naïve and (E) memory T_reg and Tconv cells isolated from human peripheral blood. Data are presented as flow cytometry histograms. (F) Binding of Siglec-liposomes to naïve T_reg and T_conv cells isolated from human peripheral blood. Data are presented as a heatmap. The P value for four (B) and five (F) biological replicates was calculated using a paired one-way ANOVA. *0.05 > P ≥ 0.01, **0.01 > P ≥ 0.001, ***0.001 > P ≥ 0.0001.

Fig. 4.. Siglec-liposomes as a multiplexing platform.

(A) Schematic for multiplexing liposomes with different fluorophores. (B) Binding of single color and multiplexed of AF488–Siglec-1–liposome to U937 cells. Data are presented as flow cytometry histograms and MFI of Siglec binding. (C) Binding of multiplexed and single color of AF488–Siglec-1–, AF555–Siglec-7–, and AF647–Siglec-2–liposomes in different combinations to U937 cells. Data are presented as a heatmap. Binding of (D) Siglec-2/Siglec-7 and (E) Siglec-1/Siglec-2 on the same liposomes to U937 cells. Data are presented as flow cytometry histograms and MFI of Siglec binding. The P value for three technique replicates was calculated using an unpaired one-way ANOVA. ns, not significant; P > 0.05, ****P < 0.0001.

Fig. 5.. In vivo and ex vivo staining of cells and tissues using Siglec-liposomes.

In vivo binding of WT and arginine-mutated of (A) Siglec-7–liposome to CD4⁺ and CD8⁺ T cells and (B) Siglec-2–liposome to B cells isolated from mouse splenocytes. Data are presented as flow cytometry histograms. (C) IF microscopy images from mouse spleen representing in vivo binding of WT Siglec-7–liposomes to T cells. Scale bar, 100 μm. (D) In vivo binding of multiplexed AF488–Siglec-7–liposomes to CD4⁺ and CD8⁺ T cells and AF647–Siglec-2–liposomes to B cells. Data are presented as flow cytometry histograms and MFI of Siglec binding. (E) IF microscopy images of mouse brain tissues representing in vivo binding of Siglec–7–liposomes to blood vessels. Scale bar, 100 μm. IF microscopy images of ex vivo staining of mouse brain tissues with (F) WT Siglec-7–Fc and αCD31, and (G) StcE mucinase enzyme treatment followed by WT Siglec-7–Fc and αCD31. Scale bar, 50 μm. The P value for four biological replicates was calculated using a paired Student’s t test. **0.01 > P ≥ 0.001.

Fig. 6.. Modulation of T cell proliferation by Siglec-7–liposomes.

(A) Schematic of T cell isolation from human blood and coculturing with αCD3/28 beads and Siglec-liposomes. T cell proliferation of human (B) CD4⁺ and (C) CD8⁺ T cells cocultured with WT and R124A Siglec-7–liposomes. Data are presented as flow cytometry histograms and proliferation index. T cell proliferation of (D) WT CD4⁺, (E) WT CD8⁺, (F) ST3Gal1^−/− CD4⁺, and (G) ST3Gal1^−/− CD8⁺ T cells isolated from mouse solenocytes. Data are presented as flow cytometry histograms and proliferation index. Binding of WT and R124A Siglec-7–liposomes to WT and ST3Gal1^−/− T cells. Data are presented as (H) flow cytometry histograms and (I) MFI of Siglec-7 binding. The P value for six [(B) and (C)] and three [(D) to (G)] biological replicates was calculated using a paired one-way ANOVA. The P value for three technical replicates was calculated using an unpaired ANOVA [(H) and (I)]. ns, not significant; P > 0.05, **0.01 > P ≥ 0.001, ***0.001 > P ≥ 0.0001, ****P < 0.0001.