Initial exploration of a novel fusion protein, IL-4/IL-34/IL-10, which promotes cardiac allograft survival mice through alloregulation

Abstract

Immune mediated graft loss still represents a major risk to transplant recipients. Creative approaches to immunosuppression that exploit the recipient's own alloregulatory mechanisms could reduce the need for pharmacologic immunosuppression and potentially induce immune tolerance. In the process of studying recipient derived myeloid derived suppressor cells (MDSCs), we identified key alloregulatory MDSC mechanisms, mediated by isolatable proteins IL-4, IL-34, and IL-10. We sought to purify these proteins and fuse them for subsequent infusion into transplant recipients as a means of inducing an alloregulatory response. In this introductory investigation, we leveraged molecular engineering technology to create a fusion protein (FP) of three cytokine coding sequences of IL-4, IL-34, and IL-10 and demonstrated their expressions by Western Blot analysis. Following purification, we tested whether FP IL-4/IL-34/IL-10 (FP1) can protect heart transplant allografts. Injection of FP1 significantly prolonged allogeneic cardiac graft survival in a dose-dependent fashion and the increase of graft survival time exceeded survival attributable to IL-34 alone. In vitro, MDSCs cells were expanded by FP1 treatment. However, FP1 did not directly inhibit T cell proliferation in vitro. In conclusion, newly developed FP1 improves the graft survival in cardiac transplantation mouse model. Significant additional work to optimize FP1 or include other novel proteins could supplement current treatment options for transplant patients.

Keywords: MDSC; Transplant; alloregulation; heart transplant; tolerance.

Figure 1.

Generation of IL-4/IL-34/IL-10 fusion protein FP1.

Figure 2.

IL-4/IL-34/IL-10 fusion protein prolonged cardiac allograft survival. (A) 5 mg/Kg of Il-4/IL-34/IL10 fusion protein was intraperitonially injected on day 0 for single dose group (FP1) or on day 0, 3, and 6 for multiple dose group (FP3) post-transplantation. (B) Graft survival was analyzed by abdominal palpation. Survival curves show significant difference between control (No treatment) and FP-treated groups (FP1 or FP3) by log-rank (Mantel-Cox) test, *p < 0.05, Mean survival time (MST, days).

Figure 3.

IL-4/IL-34/IL-10 fusion protein does not alter proliferation capacity of T cells. T cells were obtained and cultured as mentioned in the section of Materials & Methods in the presence/absence of IL-4/IL-34/IL-10 fusion protein (20 ng/ml). Following incubation for 3 days, cells were collected and analyzed for proliferation by flow cytometry of diluted CFSE. (A) The gating strategy for proliferated CD8+/CD4 + cells. (B) Histogram plots show representative flow cytometric data of proliferating cells in CD4 + or CD8 + cells. (C) Bar charts show the frequency of proliferated cells of CD4 + or CD8 + cell. The differences between seven groups were tested by ANOVA and after post Tukey test. T cells were stimulated with CD3 & CD28 antibodies during incubation for 3 days before flow cytometry assay.

Figure 4.

IL-4/IL-34/IL-10 fusion protein promotes in vitro MDSCs expansion. Bone marrow cells were obtained cultured as mentioned in the section of Materials & Methods in the presence/absence of IL-4/IL-34/IL-10 fusion protein (20 ng/ml). Following incubation for 6 days, cells were collected and analyzed for total MDSCs, PMN-MDSCs, and M-MDSCs. (A) The representative histogram images and quantitative analysis of percentage of MDSC subsets induced 6 days after incubation. Data were analyzed as mean value ± SD and student t-test was used to assess the result significance. *p < 0.05, ***p < 0.001, compared with the control group, ns, not significant.