Enhancement of interleukin-2 production by bovine peripheral blood mononuclear cells treated with the combination of anti-programmed death-ligand 1 and cytotoxic T lymphocyte antigen 4 chimeric monoclonal antibodies

Abstract

Our previous studies demonstrate the therapeutic efficacy against bovine diseases of an anti-bovine programmed death-ligand 1 (PD-L1) chimeric antibody. In humans, PD-1 and PD-L1 antibodies are more effective when combined with an antibody targeting cytotoxic T lymphocyte antigen 4 (CTLA-4) and these combination therapies are therefore clinically used. Here we generated an anti-bovine CTLA-4 chimeric antibody (chAb) to enhance the therapeutic efficacy of the PD-L1 antibody. We further analyzed the effects of dual blockade of CTLA-4 and PD-1 pathways on T-cell responses. The established anti-bovine CTLA-4 chAb showed comparable blocking activity on the binding of bovine CTLA-4 to CD80 and CD86 as the anti-bovine CTLA-4 mouse monoclonal antibody. Anti-bovine CTLA-4 chAb also significantly increased IL-2 production from bovine peripheral blood mononuclear cells (PBMCs). Further, the combination of anti-CTLA-4 chAb with anti-PD-L1 chAb significantly upregulated IL-2 production by PBMCs. These results suggest that the combination of antibodies have higher potential to enhance immune responses against pathogens compared with single administration.

Keywords: cattle; chimeric antibody; cytotoxic T lymphocyte antigen 4; interleukin-2; programmed death-ligand 1.

Fig. 1.

The binding and blocking abilities of anti-bovine cytotoxic T lymphocyte antigen 4 (CTLA-4) mAb (4C2-D9). Bovine CTLA-4-EGFP-expressing cells was incubated with anti-bovine CTLA-4 mAbs (4C2-D9 and 4G2-A3) to assess binding and blocking ability. (A) The binding ability of anti-bovine CTLA-4 mAbs was measured using flow cytometry. (B and C) Concentration-dependent blocking effect of the binding of anti-bovine CTLA-4 mAbs (4C2-D9 and 4G2-A3) to CTLA-4/CD80 (B) and CTLA-4/CD86 (C). CTLA-4-EGFP-expressing cells were preincubated with 4C2-D9, 4G2-A3, or a mouse IgG₁ control (1.25, 2.5, 5, 10, and 20 μg/ml). Ig binding was detected using flow cytometry. Each point indicates the mean value of the relative MFI of three independent experiments (relative to the control without antibody; error bar, SEM). Tukey’s test was used for statistical analysis (*P<0.05) between groups treated with the same concentration of 4G2-A3 and mouse IgG₁. ‡P<0.05, between the groups treated with the same concentration of 4C2-D9 and mouse IgG₁. †P<0.05, between the groups with the same concentration of 4C2-D9 and 4G2-A3.

Fig. 2.

The immune activation efficacy of 4C2-D9 alone or combined with the anti-programmed death-ligand 1 (PD-L1) antibody. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy cattle and cultured with (A) 4C2-D9 alone or (B) 4C2-D9 and anti-PD-L1 mAb 4G12 in the presence of Staphylococcus aureus enterotoxin B (SEB). After 3 days, the supernatant was harvested. (A and B) The interferon-γ (IFN-γ) concentration in the supernatant was measured using an ELISA (a, n=14; b, n=17). (C) The interleukin-2 (IL-2) concentration in the supernatant was measured using an ELISA (n=18). The bar indicates the median value of each group. Comparisons between each group were performed using the Wilcoxon matched-pairs test (A) and the Steel-Dwass test (B and C). P<0.05 (*) indicates a significant difference.

Fig. 3.

Establishment of Boch4C2 and assessment of its binding and blocking abilities. (A) Structure of Boch4C2 showing the variable regions of 4C2-D9 (white) and constant regions of bovine IgG₁ and Igλ (black). (B) ExpiCHO-S cells were transfected with a plasmid expressing Boch4C2, and Boch4C2 was purified from the culture supernatant. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses to determine the purity of Boch4C2 were performed under reducing and nonreducing conditions. (C) The binding ability of Boch4C2 was measured using flow cytometry of CTLA-4-EGFP-expressing cells. The binding of Boch4C2 was detected using an anti-bovine IgG secondary antibody. (D and E) The concentration-dependent blocking effect of Boch4C2 and 4C2-D9 on the binding of cytotoxic T lymphocyte antigen 4 (CTLA-4)/CD80 (D) and CTLA-4/CD86. (E) Biotin-conjugated CTLA-4-Ig was preincubated with Boch4C2, 4C2-D9 or a control antibody at the molar ratios as follows: (antibody:CTLA-4-Ig) 0:1, 0.1:1, 0.5:1, 1:1, 2:1, 5:1, and 10:1). Each point indicates the mean value of the relative OD value of three independent experiments (relative to the control without antibody; error bar, SEM). Tukey’s test was used for statistical analysis (*P<0.05, between the groups with Boch4C2 and bovine IgG at same molar ratio. ‡P<0.05, between the groups with different molar ratios of Boch4C2. †P<0.05, between groups with 4C2-D9 and mouse IgG₁ at same molar ratio. §P<0.05, between the groups with different molar ratios of 4C2-D9.

Fig. 4.

Efficacies of immune activation of peripheral blood mononuclear cells (PBMCs) of healthy cattle in response to treatment with Boch4C2 alone or combined with Boch4G12. PBMCs were isolated from healthy cattle and cultured for three days in the presence of Staphylococcus aureus enterotoxin B (SEB). (A and B) 10 μg/ml Boch4C2 or bovine IgG was added to the medium, and the concentrations of interferon-γ (IFN-γ) (A) and interleukin-2 (IL-2) (B) were measured using an ELISA (A, n=12; B, n=18). (C and D) Media contained 10 μg/ml each of Boch4C2 or bovine IgG and 10 μg/ml each of Boch4G12 or bovine IgG. The concentrations of IFN-γ (C) and IL-2 (D) were measured using an ELISA (C, n=13; D, n=18). The bar indicates the median value of each group. The comparison between each group was performed using the Wilcoxon matched-pairs test (B) and the Steel-Dwass test (D). P<0.05 (*) indicates a significant difference.