Service Project #8: Cellular Mechanisms of Inflammation, Hemostasis, and Thrombosis

Collaborating Investigator: Mark Ginsberg
Affiliation: Professor of Medicine, University of California San Diego 
Funding Status: NIH PO1HL151433  
Project Period: 08/05/20- 07/31/25

Summary

The goal of SP #8 is to elucidate the role of integrins in modulating T cell signaling. The Ginsberg Lab has uncovered new evidence of cross-talk between interleukin-2 (IL-2) and integrin signaling pathways. An engineered IL-2-based immune-biasing antibody/cytokine fusion protein (immunocytokine, IC) designed in TR&D 3 will be used to interrogate the interplay between cytokine and integrin pathways, furthering the aims of the project.  For SP #8, the NCBIB will push our engineered regulatory T cell (Treg)-biased immunocytokine denoted F5111 IC (TR&D 3) to the Ginsberg Lab to elucidate its effects on both canonical and non-canonical IL-2 signaling networks.

Approach

Aim 1. Production and functional validation of F5111 IC. F5111 IC will be produced recombinantly by the NCBIB using a mammalian cell expression system and purified by protein G chromatography and subsequent size-exclusion chromatography using a fast liquid protein chromatography (FPLC) machine. The purified F5111 IC will be functionally validated through biolayer interferometry-based binding studies against IL-2 and its cognate receptor subunits using an Octet® machine, as well as IL-2 signaling assays on commercial human peripheral blood mononuclear cells.

Specific Aim 2. Evaluation of the effects of F5111 IC on IL-2 canonical versus non-canonical signaling. The Ginsberg Lab will administer F5111 IC from Aim 1 to purified Tregs, as well as to a mixed cellular population (human peripheral blood mononuclear cells) and quantify signaling through canonical IL-2 pathways as well as non-canonical integrin-related pathways. F5111 IC from Aim 1 will also be administered in the murine experimental allergic encephalomyelitis (EAE) model of multiple sclerosis to delineate the signaling mechanisms for IL-2 therapies in this system.

The Ginsberg Lab has discovered that IL-2 signals through a non-canonical chemokine receptor pathway that induces integrin activation in Tregs and promotes their suppressive activity. To explore the effects of Treg-biased IL-2 on activation of canonical versus non-canonical IL-2 signaling, the NCBIB will produce the F5111 IC using a mammalian cell expression system and push this molecule to the Ginsberg Lab to test in cellular assays. The Ginsberg Lab will further characterize our molecule in the murine EAE model of multiple sclerosis to determine whether non-canonical IL-2 signaling mechanisms contribute to the therapeutic efficacy of F5111 IC. Overall, the proposed work will enrich our fundamental understanding of cytokine biology, define the connectivity between cytokine and chemokine pathways, and inform the design of therapeutically relevant interventions to treat autoimmune diseases.