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 new insights into Siglec ligands. Moreover, Siglec-liposomes are in vivo compatible, where we demonstrated that Siglec-7-liposomes bind to the brain vasculature in a mucin-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 a ST3Gal1-dependent and CD43-independent manner. Taken 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 SpyTag. (C) Schematic of site-specific conjugation of SC to DSPE-PEG-Maleimide. (D) SDS-PAGE of SC and DSPE-PEG-SC. (E) Schematic of making SC-liposomes. (F) Binding of V1 and V2 WT Siglec-7- and R124A Siglec-7-liposomes to U937 cells. Data is represented as flow cytometry histograms and median fluorescent intensity (MFI) of Siglec-7 binding. (G) Binding of V2 of WT Siglec-7 and R124A Siglec-7 in different densities on liposomes to U937 cells. Data is 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 pre-complexed with Strep-Tactin (Strep.) and conjugated to liposomes (lipo.) to U937 cells. Data is 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. 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 is presented as flow cytometry histograms and MFI of Siglec binding. The p value for three technical replicates was calculated using an un-paired 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 Treg and Tconv cells isolated from human peripheral blood with Siglec-liposomes. Binding of Siglec-liposomes to (D) naïve and (E) memory Treg and Tconv cells isolated from human peripheral blood. Data is presented as flow cytometry histograms. (F) Binding of Siglec-liposomes to naïve Treg and Tconv cells isolated from human peripheral blood. Data is presented as a heat map. The p value for four (panel B) and five (panel 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 is 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 is presented as a heat map. Binding of (D) Siglec-2/Siglec-7, and (E) Siglec-1/Siglec-2 on the same liposomes to U937 cells. Data is 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. Not significant (ns) 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 is 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 is 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 co-culturing with αCD3/28 beads and Siglec-liposomes. T cell proliferation of human (B) CD4⁺ and (C) CD8⁺ T cells co-cultured with WT and R124A Siglec-7-liposomes. Data is 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 is presented as flow cytometry histograms and proliferation index. Binding of WT and R124A Siglec-7-liposomes to WT and ST3Gal1^−/− T cells. Data is presented as (H) flow cytometry histograms and (I) MFI of Siglec-7 binding. The p value for six (panel B,C) and three (panel D-G) biological replicates was calculated using a paired one-way ANOVA. The p value for three technical replicates was calculated using an unpaired ANOVA. Not significant (ns) p>0.05, **0.01>p≥0.001, ***0.001>p≥0.0001, ****p<0.0001.