IL-6-inducing whole yeast-based immunotherapy directly controls IL-12-dependent CD8 T-cell responses

Abstract

In current clinical trails, whole yeast-based immunotherapy expressing hepatitis C viral antigens demonstrated statistically significant improvement in end of treatment responses when combined with type I interferon based standard of care, even in standard of care resistant patients. Although preclinical data suggest yeast vaccination, such as type I interferon, facilitates CD8 T-cell immunity, the capacity of yeast to generate immunity in patients resistant to type I interferon calls into question the mechanism(s) underpinning the efficacy of this approach. We show yeast and a Toll-like receptor exclusive agonist, Pam3Cys, differ in CD8 T-cell generation when combined with an agonistic CD40 antibody. Although both yeast and PamCys were largely Toll-like receptor dependent, the primary CD8 response generated by yeast was significantly less than Pam3Cys in wild-type hosts even in a CD4 T-cell-deficient setting. In addition, immunization of IL6 mice with yeast produced a 3-fold to 6-fold increased CD8 response while the Pam3Cys response was unaffected. The yeast but not Pam3Cys-driven CD8 response was inhibited in both wild-type and IL-6 hosts by blocking interleukin (IL)-12. In addition, IL6 mice had increased CD86 expression on their dendritic cells after yeast immunization also inhibited by IL-12 blockade. Collectively, our results indicate the CD8 T-cell response to yeast but not Pam3Cys is influenced by IL-6-mediated control of IL-12 critical for dendritic cell activation. To our knowledge this is the first demonstration that yeast directly influence IL-12-associated CD8 T-cell immunity providing an additional route whereby recombinant yeast may provide efficacy independent of type I interferon.

Figure 1. Addition of αCD40 antibody to the immunization synergistically enhances endogenous CD8 T cell responses

WT mice were immunized intravenously with 100ug of ovalbumin (ova) and either 2×10⁷ heat killed yeast, 50ug of anti-CD40 (αCD40) (FGK4.5), both yeast and anti-CD40, or 25ug of Pam3Cys and anti-CD40. Mice were sacrificed 7 days post immunization and peripheral blood and spleens were taken. Peripheral blood data is shown. A. Percentages shown are live cells that are CD8+, B220−, CD44 high, tetramer+ cells assessed by flow cytometric analysis of peripheral blood from a representative sample. B. The CD8 T cell response to yeast synergy is primarily dependent on MyD88 signaling. Bar graph representing WT (black bars) or MyD88 knock out mice (white bars) antigen specific CD8 T cell response to shown immunizations. Error bars represent the standard deviation and asterisks represent statistically significant values as determined by student’s t-test (*= p<0.05, **= p<0.001, ***= p<.0001). Experiments were performed at least twice with three mice per group.

Figure 2. The magnitude of the CD8 T cell response induced by yeast but not Pam3Cys is negatively influenced by IL-6

A. Immunization analysis of WT or IL6−/−mice was done exactly as described in the legend of figure 1. At least four experiments were performed with 2–4 mice per group per experiment.

Figure 3. CD8 T cell response to yeast is independent of CD4 T cells in both WT and IL6 −/− mice

WT (black bars) or IL6−/− (dark grey bars) mice were depleted of CD4 T cells (WT CD4 depleted –light grey bars, IL6−/− CD4 depleted –white bars) 3 days prior and 4 days post immunization with either Pam3Cys synergy (Pam3Cys-ova-aCD40), yeast synergy (yeast-ova-aCD40), anti-CD40 (ova-aCD40), or nothing (naïve) using the CD4 depleting antibody GK1.5. There were no detectable CD4 T cells following CD4 depletion in the blood or spleen as measured by flow cytometry. Bars represent percentage of SIINFEKL specific class I tetramer in peripheral blood. Error bars and asterisks denote significance as described in the legend of figure 1. Experiments were performed twice with three mice per group.

Figure 4. CD8 T cell response requires IL-12 signaling

A. Mice were immunized and analyzed exactly as described in figure 3. The IL-12p70 blocking antibody C17.8 was administered 18 hours and 2 hours prior to immunization in WT (light grey bars) or IL6−/− (white bars) mice, or not at all in WT (black bars) or IL6−/− (dark grey bars) mice. B. Immunization and analysis of WT (black bars) and p19 knock out mice (IL23) (white bars) was done exactly as described in the legend of figure 1. Statistical analysis was done as described in figure 1. Experiments were done twice with three mice per experimental group.

Figure 5. Dendritic cell activation is rescued by removal of IL-6 and dependent on IL-12

A. Dendritic cell activation marker CD86 is decreased in WT mice in response to y e a s t. W T o r IL6−/− mice were immunized with yeast+ova+aCD40, Pam3Cys+ova+aCD40, or nothing (naïve) 24 hours prior to harvesting splenic DCs. DCs shown are CD11c+. Shown is a representative sample of splenic CD8+ DCs in WT (gray shaded histogram) and IL6−/− (open histogram), or WT naïve (dashed line open histogram) immunized as indicated above each graph. B. Bar graph of data shown in A of WT (black bars) and IL6−/− (white bars) mice. Shown is the geometric mean fluorescence intensity of CD86 for both CD8+ and CD11b+ dendritic cells. C. CD86 is reduced after IL-12 block. Shown is a representative sample of CD8+ DCs in WT and IL6−/− mice were treated with IL-12 blocking antibody (gray shaded histogram) 18 and 2 hours prior to yeast immunization or left untreated (open histogram). Naïve mice are shown with a dashed line. D. Bar graph of data shown in C. CD86 expression was calculated as in A. Statistical analysis was done as described in figure 4. Statistical analysis was done as described in figure 1. Experiments were done twice with three mice per group.

Figure 6. IL-12 is necessary to the dendritic cell

WT TCR transgenic OT-1 CD8 T cells were adoptively transferred into WT (black bars) or IL12Rb2−/− (white bars) mice and immunized with either yeast-ova-aCD40 or ova-aCD40 24 hours post transfer. Total numbers of expanded OT-1 T cells in the spleen were counted and percent expansion based on the WT mouse was calculated. Statistical analysis was done as described in figure 1. This experiment was performed twice with three mice per group.

Figure 7. IL-12 production is increased in CD8+ DCs of IL6−/− mice in response to yeast synergy

WT (black bars) or IL6−/− (white bars) were immunized with either yeast-ova-aCD40 or ova-aCD40. Splenic DCs were harvested 1–2 hours post immunization and stained for intracellular IL-12. DCs are CD11c+, B220−, CD8+, IL-12+. Statistical analysis was done as described in figure 1. This experiment was performed twice with three mice per group.