Inhibiting IL-2 Signaling and the Regulatory T-Cell Pathway Using Computationally Designed Novel Peptides

Regulatory T-cells (T_REG) are the gateway to immune function and typically regulate immune cell activation. Cytokines, including interleukin-2 (IL-2), induce T-cell differentiation and promote a regulatory phenotype. Once activated via the IL-2 receptor (IL-2R), a cascade of events in T-cells initiate signal transducer and activator of transcription 5 (STAT5) and Forkhead box P3 (FOXP3) activation which appear to function as important regulators of this immunologic pathway and promote the development and function of T_REG cells. In non-Hodgkin lymphoma (NHL), we have found that intratumoral T_REG cells are increased in number and suppress immune function. In previous work, we have found that T_REG cells inhibit T-cell proliferation, suppress cytokine production and limit effector cell cytotoxicity. We have also shown that increased serum levels of soluble sIL-2Rα is a prognostic factor in NHL and that sIL-2Rα can bind to IL-2 and promote its signaling thereby increasing T_REG cell numbers. In this study, we developed a strategy to inhibit the binding of IL-2 to sIL-2Rα with the goal of suppressing the induction of FOXP3 and decreasing T_REG cell numbers. To do this, we developed peptides designed to disrupt the interaction between IL2 and sILRα.

In collaboration with CMDBioscience^SM, we developed and analyzed 22 peptide compounds derived by structure-based computational design. Initially, we screened each peptide at increasing concentrations using an ELISA assay to test the inhibition of IL-2/IL-2Rα binding by the solubilized peptide. Candidate peptides were then further tested using upregulation of pSTAT5 and FOXP3 in T-cells measured by flow cytometry as a measure of inhibition of IL-2 signaling. The peptides were developed according to different design hypotheses and grouped into different families; the screening ELISA results indicated 4 promising peptides that inhibited IL2/IL2Rα binding (up to 100% inhibition; max peptide concentration of 100uM). These peptides were then used to determine their effect on STAT5 and FOXP3 expression. A lead candidate peptide consistently reduced the expression of FOXP3 and STAT5 expression compared to cells not exposed to peptide. Use of the peptide to disrupt IL-2 signaling inhibited the development of cells with a T_REG phenotype.

We conclude that structure-based peptide design can be used to identify novel peptide inhibitors that block IL-2/IL-2Rα signaling and inhibit STAT5 and FOXP3 upregulation. These peptides could be used as new therapeutic agents to limit immune suppression by T_REG cells and promote a more effective anti-tumor immune response in NHL.