Cyclotides Suppress Human T-Lymphocyte Proliferation by an Interleukin 2-Dependent Mechanism

Abstract

Cyclotides are a diverse and abundant group of ribosomally synthesized plant peptides containing a unique cyclic cystine-knotted topology that confers them with remarkable stability. Kalata B1, a representative member of this family of mini-proteins, has been found to inhibit the proliferation of human peripheral blood mononuclear cells. Analysis of T-cell proliferation upon treatment with chemically synthesized kalata B1 mutants revealed a region comprising inter-cysteine loops 1 and 2 of the cyclotide framework to be important for biological activity. Cytokine signaling analysis using an ‘active’ kalata B1 mutant [T20K], and the reference drug cyclosporin A (CsA) demonstrated that treatment of activated T-lymphocytes with these compounds decreased the expression of the interleukin-2 (IL-2) surface receptor as well as IL-2 cytokine secretion and IL-2 gene expression, whereas the ‘inactive’ kalata B1 mutant [V10K] did not cause any effects. The anti-proliferative activity of [T20K] kalata B1 was antagonized by addition of exogenous IL-2. Furthermore, treatment with [T20K] kalata B1 led to an initial reduction of the effector function, as indicated by the reduced IFN-γ and TNF-α production, but the levels of both cytokines stabilized over time and returned to their normal levels. On the other hand, the degranulation activity remained reduced. This indicated that cyclotides interfere with T-cell polyfunctionality and arrest the proliferation of immune-competent cells through inhibiting IL-2 biology at more than one site. The results open new avenues to utilize native and synthetically-optimized cyclotides for applications in immune-related disorders and as immunosuppressant peptides.

Oral activity of a nature-derived cyclic peptide for the treatment of multiple sclerosis

“…As a preliminary experiment, [T20K]kB1 and [V10K]kB1 were incubated at different concentrations with mouse immune cells ex vivo, which were isolated from spleens of EAE-induced, viz. myelin oligodendrocyte glycoprotein (MOG ; MOG)-immunized mice, to confirm the immunosuppressive effects previously observed in human T cells (13). The proliferation of T cells, measured by flow cytometry, as well as the autocrine acting proliferation-supporting cytokine IL-2 and the T H 1 and T H 17 signature cytokines IFN-γ and IL-17A in the supernatant of MOG-restimulated cells (Fig.…”

“…Extensive screening efforts for the discovery of novel nature-derived drugs has led to the identification of antiproliferative properties of the cyclotide kB1 toward human activated lymphocytes in vitro (13). Recently, structure-activity screening of cyclotides isolated from the plant Oldenlandia affinis (Rubiaceae) established [T20K]kB1 as an active lead compound to inhibit T-cell proliferation and, interestingly, replacement of valine to the positively charged lysine in loop 2 resulted in a loss of activity in vitro (13). In the current work, [T20K]kB1 was synthesized following a modified protocol for the generation of thioester peptides and subsequent cyclization by native chemical ligation ( Fig.…”

“…Antiproliferative effects of the cyclotide kalata B1 and the mutant [T20K]kB1 have been investigated on human mononuclear cells and purified T cells, highlighting the IL-2-specific inhibitory mechanism in vitro (12,13). Release of T H 1 and T H 17 signature cytokines were not only inhibited in vitro when incubated with the cyclotide, but also after ex vivo restimulation of EAE-induced [T20K]kB1-pretreated splenocytes with their natural antigen MOG.…”

“…Recent studies referred to the immunosuppressive effects of the circular peptide kalata B1 (kB1) on activated human T lymphocytes in vitro (12,13). This plant-derived peptide belongs to the family of cyclotides well-known for their cyclic cystineknot topology.…”

“…Cyclotides, in particular [T20K]kB1, inhibit T-cell proliferation by down-regulation of IL-2 release as well as IL-2R/CD25 surface expression (13). The cytokine IL-2 physiologically plays an important role in T-lymphocyte activation and acts as an autocrine factor to stimulate T-cell proliferation (19).…”