Cytotoxic T-lymphocyte antigen 4 and programmed cell death ligand 1 blockade increases the effectiveness of interleukin-15 immunotherapy in a bovine leukemia model

Abstract

Background and aim: Bovine interleukin 15 (bIL15) is a potential immunotherapy that can block the spread of bovine leukemia virus (BLV). However, immune checkpoints that maintain body homeostasis may reduce their effectiveness. Thus, an analysis of the effectiveness of bIL15 while blocking negative immune regulators is necessary. We aimed to obtain recombinant bIL15 (rbIL15) and determine its percentage using monoclonal antibodies against bovine cytotoxic T-lymphocyte antigen 4 (CTLA-4) and programmed cell death ligand 1 (PD-L1). To achieve this goal, peripheral blood mononuclear cells (PBMCs) from healthy and BLV+ cattle were treated with bIL15 using a CTLA-4- and PD-L1-blocking algorithm.

Materials and methods: The codon-optimized bIL15 gene was synthesized under de novo conditions using polymerase chain reaction (PCR). The synthesized gene was cloned into pET28 and transformed into electrocompetent Escherichia coli BL21 cells; rbIL15 was purified using metal affinity chromatography and analyzed using sodium dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. The expression of the Bcl2, STAT3, and STAT5 genes was studied using qualitative PCR. An enzyme-linked immunosorbent assay (ELISA) was used to analyze interferon (IFN)-γ production by rbIL15-treated mononuclear cells.

Results: Analysis of rbIL15 using SDS-PAGE and western blotting revealed a specific product weighing 24 kDa. The optimal conditions for rbIL15 induction were 0.2 mM isopropyl-β-D-1-galactopyranoside and 37°C. When rbIL15 was added to PBMCs from healthy cattle, the Bcl2, STAT3, and STAT5 genes were expressed. ELISA of the culture medium of rbIL15-treated PBMCs revealed IFN-γ production. When PBMCs from healthy cows were treated with rbIL15, CTLA-4, and PD-L1 blockade together, they did not produce more IFN-γ than the rbIL15 group. Using PBMCs from BLV+ cattle, combination treatment increased IFN-γ production.

Conclusion: The biological activity of rbIL15 is characterized by the induction of transcription factors and the production of IFN-γ. Using rbIL15 with CTLA-4 and PD-L1 blockade in PBMCs from healthy and BLV+ cows led to the production of a transcription factor and cytokine. The results demonstrate the possibility of using this method to improve immunity and immunological memory in patients with chronic viral infections.

Keywords: bovine interleukin-15; bovine leukemia virus; cytotoxic T-lymphocyte antigen 4; monoclonal antibody; programmed cell death ligand 1.

Figure-1

SDS-PAGE and western blot analyses of rbIL15 expression. (a) Total, (b) pellet, (c) super, and (d) western blots. Lane 1: Escherichia coli culture without expression; Lane 2: Protein expression in 2 h; Lane 3: Protein expression in 4 h; Lane 4: Protein expression in 6 h; Lane 5: Protein expression in 12 h; Lane MM: Molecular markers. SDS-PAGE=Sodium dodecyl-sulfate–polyacrylamide gel electrophoresis, rbIL15=Recombinant bovine interleukin 15.

Figure-2

SDS-PAGE optimization and rbIL15 purification. (a) 0.2 mM IPTG, (b) 1 mM IPTG, (c) 37°C, (d) 25°C, and (e) purified rbIL15. Lane 1: Without expression; Lane 2: With expression; Lane MM: Molecular markers. IPTG=Isopropyl-β-D-1-galactopyranoside, SDS-PAGE=Sodium dodecyl-sulfate–polyacrylamide gel electrophoresis, rbIL15=Recombinant bovine interleukin 15.

Figure-3

Tandem mass spectrometry of fragmented rbIL15. rbIL15=Recombinant bovine interleukin 15.

Figure-4

Biological activity of rbIL15. Concentration histograms of (a) IFN-γ and (b) PCR transcription factors of bovine PBMCs cultivated with and without rbIL15. rbIL15=Recombinant bovine interleukin 15, IFN=Interferon, PCR=Polymerase chain reaction, PBMCs=Peripheral blood mononuclear cells.

Figure-5

Effect of combining rbIL15 with CTLA-4 and PD-L1 blockade on healthy cattle PBMCs. (a) Enzyme-linked immunosorbent assay of culture media supernatant to determine IFN-γ concentration. (b and c) Estimated plot of differences between groups. Student’s t-test was used for comparisons. At p < 0.0001, differences were considered significant. rbIL15=Recombinant bovine interleukin 15, IFN=Interferon, PBMCs=Peripheral blood mononuclear cells, CTLA-4=Cytotoxic T-lymphocyte antigen 4, PD-L1=Programed cell death ligand 1.

Figure-6

Effects of bovine BLV+ PBMC activation by combining rbIL15 with CTLA-4 and PD-L1 blockade. (a) Enzyme-linked immunosorbent assay of culture media supernatant to determine IFN-γ concentration, and (b) Estimated differences between groups. Student’s t-test was used for comparisons. At p < 0.0001, differences were considered significant. rbIL15=Recombinant bovine interleukin 15, IFN=Interferon, PBMCs=Peripheral blood mononuclear cells, CTLA-4=Cytotoxic T-lymphocyte antigen 4, PD-L1=Programmed cell death ligand 1, BLV=Bovine leukemia virus.