The interaction of galectin-8 C-terminal domain with cell surface glycoconjugates modulates membrane elasticity to stimulate antigen uptake and presentation to CD4 T cells.

Galectins constitute a family of soluble lectins with unique capacity to induce macroscale rearrangements upon interacting with cell membrane glycoconjugates. Galectin-8 (Gal-8) is acknowledged for its role in facilitating antigen uptake and processing upon engaging with cell surface glycoconjugates on antigen-presenting cells (APCs). Gal-8 consists of two covalently fused N- and C-terminal carbohydrate recognition domains (N- and C-CRD), each exhibiting distinct glycan specificity. In this study, we utilized single N- and C-CRD recombinant proteins to dissect the nature of Gal-8-glycan interactions during antigen internalization enhancement. Single C-CRD was able to replicate the effect of full-length Gal-8 (FLGal-8) on antigen internalization in BMDCs. Antigen uptake enhancement was diminished in the presence of lactose or when N-glycosylation-deficient macrophages served as APCs, underscoring the significance of glycan recognition. Measurement of the elastic modulus using Atomic Force Microscopy unveiled that FLGal-8- and C-CRD-stimulated macrophages exhibited heightened membrane stiffness compared to untreated cells, providing a plausible mechanism for their involvement in endocytosis. C-CRD proved to be as efficient as FLGal-8 in promoting antigen degradation, suggesting its implication in antigen-processing induction. Lastly, C-CRD was able to replicate FLGal-8-induced antigen presentation in the MHC-II context both in vitro and in vivo. Our findings support the notion that Gal-8 binds through its C-CRD to cell surface N-glycans, thereby altering membrane mechanical forces conducive to soluble antigen endocytosis, processing, and presentation to cognate CD4 T-cells. These findings contribute to a deeper comprehension of Gal-8 and its mechanisms of action, paving the way for the development of more efficacious immunotherapies.

Purification of Recombinant Galectins Expressed in Bacteria.

Galectins are soluble lectins that participate in many physiological and pathological functions. Since they can act extracellularly, the use of the recombinant protein is a recurrent strategy for studying their biological functions. Here, we provide a general protocol for the production of Galectins and their isolated or chimeric domains. We take advantage of their lectin activity and the 6xHis-tag addition for purification, thus obtaining a highly pure and active Galectin to use in both in vitro and in vivo assays. For complete details on the use and execution of this protocol, please refer to Cattaneo et al. (2011), Tribulatti et al. (2012), and Prato et al. (2020).

Galectin-8 Enhances T cell Response by Promotion of Antigen Internalization and Processing.

Galectin-8 (Gal-8) is a mammalian lectin endowed with immunostimulatory ability. In the present work, we demonstrate that Gal-8-glycan interactions on the surface of antigen-presenting cells (APCs) promote antigen binding and internalization, independently from antigen nature. Both Gal-8 and antigen were together internalized and localized in early endosomes. Interestingly, antigen processing by APCs was also accelerated in the presence of Gal-8 as a separate mechanism, distinct from the increased antigen internalization. Moreover, APCs pulsed together with antigen and Gal-8 were able to activate cognate CD4+ T cells more efficiently than those pulsed with antigen alone. This enhanced antigen presentation was still evident in the absence of costimulatory signals and APCs-derived soluble mediators. Therefore, our results provide evidence for as yet unrecognized mechanism by which Gal-8 stimulates the elicitation of the immune response in a lectin-dependent manner, by inducing antigen uptake and processing upon lattice formation at APCs surface.