Comparative ability of IL-12 and IL-28B to regulate Treg populations and enhance adaptive cellular immunity

Abstract

Improving the potency of immune responses is paramount among issues concerning vaccines against deadly pathogens. IL-28B belongs to the newly described interferon lambda (IFNlambda) family of cytokines, and has not yet been assessed for its potential ability to influence adaptive immune responses or act as a vaccine adjuvant. We compared the ability of plasmid-encoded IL-28B to boost immune responses to a multiclade consensus HIV Gag plasmid during DNA vaccination with that of IL-12. We show here that IL-28B, like IL-12, is capable of robustly enhancing adaptive immunity. Moreover, we describe for the first time how IL-28B reduces regulatory T-cell populations during DNA vaccination, whereas IL-12 increases this cellular subset. We also show that IL-28B, unlike IL-12, is able to increase the percentage of splenic CD8(+) T cells in vaccinated animals, and that these cells are more granular and have higher antigen-specific cytolytic degranulation compared with cells taken from animals that received IL-12 as an adjuvant. Lastly, we report that IL-28B can induce 100% protection from mortality after a lethal influenza challenge. These data suggest that IL-28B is a strong candidate for further studies of vaccine or immunotherapy protocols.

Figure 1

Immunization schedule and plasmid maps. (A) Mice were immunized on day 0 and day 14 with a multiclade HIV Gag construct and with or without adjuvant, followed by electroporation using the CELLECTRA adaptive constant current device after each immunization. On day 21, mice were killed and lymphocytes were isolated and analyzed. (B) Plasmid maps for murine IL-28B and IL-12 constructs.

Figure 2

Expression and secretion of murine IL-12 and murine IL-28B in vitro. (A) Western blotting for murine IL-12p40 and murine IL-28 proteins from HEK 293T cell lysates 48 hours after transfection. Mock-transfected cells received empty pVAX vector. (B) ELISAs show secretion of the active IL-12 p35/p40 heterodimer as well as the IL-28 protein into the supernatants of transfected cells.

Figure 3

HIV Gag-specific IFNγ and IL-4 ELISpots from isolated splenocytes. (A) Effects of cytokine adjuvants on the induction of a Th1 response were measured via the use antigen-specific IFNγ ELISpots performed on isolated splenocytes. ELISpots were performed on splenocytes harvested from mice that received IL-12 as an adjuvant or IL-28B as an adjuvant (n = 4) and IFNγ spot-forming units (SFU) were counted. (B) Effects of cytokine adjuvants on the induction of a Th2 response were measured in the same fashion using IL-4 ELISpots.

Figure 4

HIV Gag-specific IgG in sera from vaccinated animals. Sera from control (pVAX) or immunized animals (n = 4) were assayed for the presence of HIV Gag-specific antibodies via ELISA 1 week after immunization. (A) Total IgG. (B) IgG1. (C) IgG2a.

Figure 5

Differential induction of regulatory T cells and TGFβ secretion during immunization. The presence of regulatory T cells (CD4⁺/CD25^hi/FoxP3⁺) was assayed from isolated splenocytes from all groups (n = 4) via flow cytometry (A). Analysis of flow cytometry shows differences in Treg populations, whereas analysis of cytokine secretion from these cells shows differences in TGFβ release (B). P values reflect comparisons between mice vaccinated with Gag4Y alone with mice vaccinated with Gag4Y plus IL-12 or IL-28B.

Figure 6

Changes in CD8⁺ T-cell populations, granularity, and degranulation. The percentage of CD8⁺ T cells (CD3⁺/CD8⁺) was assessed via flow cytometry in the blood, spleen, and mesenteric lymph nodes (A). Antigen-specific induction of perforin in CD8⁺ T cells was analyzed by flow cytometry via comparison of unstimulated cells (NP) with cells stimulated with HIV Gag peptides (Peptide). Results from a single experiment are shown (B) and averages for all experiments are graphed (C). Antigen-specific cytolytic degranulation was measured via stimulation with peptide in the presence of an antibody to CD107a, followed by analysis using flow cytometry (C). P values reflect comparisons between mice vaccinated with Gag4Y alone with mice vaccinated with Gag4Y plus IL-12 or IL-28B.

Figure 7

Protection from death in a lethal influenza challenge. (A) Mice (n = 8) were immunized on day 0 and day 14 with the influenza NP construct and with or without adjuvant, followed by electroporation using the CELLECTRA adaptive constant current device after each immunization. (B) IFNγ ELISpot responses were measured on day 21. (C) On day 42, mice were challenge intranasally with 10 LD50 A/PR/8/34, an H1N1 influenza strain. Mortality associated with influenza infection was tracked over the course of 14 days.