RIPK3 and Caspase-1/11 Are Necessary for Optimal Antigen-Specific CD8 T Cell Response Elicited by Genetically Modified Listeria monocytogenes

Abstract

Efficient induction of effector and long-term protective antigen-specific CD8⁺ T memory response by vaccination is essential to eliminate malignant and pathogen-infected cells. Intracellular infectious bacteria, including Listeria monocytogenes, have been considered potent vectors to carry multiple therapeutic proteins and generate antigen-specific CD8⁺ T cell responses. Although the role of molecules involved in inflammatory cell death pathways, such as necroptosis (RIPK3-mediated) and pyroptosis (Caspase-1/11-mediated), as effectors of immune response against intracellular bacteria are relatively well understood, their contribution to the adjuvant effect of recombinant bacterial vectors in the context of antigen-specific CD8⁺ T cell response remained obscure. Therefore, we evaluated the impact of RIPK3 and Caspase-1/11 (Casp-1/11) individual and combined deficiencies on the modulation of antigen-specific CD8⁺ T cell response during vaccination of mice with ovalbumin-expressing L. monocytogenes (LM-OVA). We observed that Casp-1/11 but not RIPK3 deficiency negatively impacts the capacity of mice to clear LM-OVA. Importantly, both RIPK3 and Casp-1/11 are necessary for optimal LM-OVA-mediated antigen-specific CD8⁺ T cell response, as measured by in vivo antigen-specific CD8⁺ T cell proliferation, target cell elimination, and cytokine production. Furthermore, Casp-1/11 and Casp-1/11/RIPK3 combined deficiencies restrict the early initiation of antigen-specific CD8⁺ T cell memory response. Taken together, our findings demonstrate that RIPK3 and Casp-1/11 influence the quality of CD8⁺ T cell responses induced by recombinant L. monocytogenes vectors.

Keywords: Casp-1/11; Effector CD8+ T cells; Listeria monocytogenes; RIPK3; ovalbumin.

FIGURE 1

Casp-1/11 but not RIPK3 is necessary for the host ability to clear LM-OVA infection. All the experimental mice were infected for 10³ CFU of LM-OVA for 3 and 7 days, respectively. Spleen size was measured at day 3 and day 7 post-infection. Spleen from each infected mice was harvested and processed individually to make single-cell suspensions. CFU/spleen was determined by plating 10-fold serial dilutions of single-cell suspension from each individual mouse on BHI-Streptomycin agar plates. (A,C) Representative images of the spleen size and (B,D) bacterial burden were measured in infected mice at 3 (A,B) and 7 (C,D) days after LM-OVA infection. Results expressed as the mean of five individual mice per group and are representative of three independent experiments. Statistical analysis was performed by using two-way ANOVA followed by Bonferroni post-tests. ***p < 0.001.

FIGURE 2

Casp-1/11 deficiency impairs in vivo antigen-specific CD8⁺ T cell expansion in response to LM-OVA. (A,B) Frequency and (C) total number of OVA_257–264 (SIINFEKL)-specific CD8⁺ T cells in the spleens of infected mice at 7 days after LM-OVA vaccination, revealed by staining with anti-CD8 antibody and H2-K^b-SIINFEKL dextramer. Results are expressed as (A) selected representative image per group or (B,C) representative bar graphic of three independent experiments showing the mean and standard error of five mice per group. Statistical analysis was performed by using two-way ANOVA followed by Bonferroni post-tests. *p < 0.5, ***p < 0.001.

FIGURE 3

RIPK3 and/or Casp-1/11 deficiencies negatively affects OVA-specific CD8⁺ T cell priming and proliferation in response to LM-OVA. (A) Representative images of OT-I CD8⁺ T cells in vivo proliferation in non-infected and LM-OVA-infected WT, RIPK3^{– /–}, Casp-1/11^{– /–}, and Casp-1/11^{– /–}/RIPK3^{– /–} mice. Frequencies of <1 division and >1 division OT-I CD8⁺ T cell populations in (B) non-infected controls and (C) LM-OVA-infected mice. (B,C) Results are expressed as means of five individual mice per group and are representative of three independent experiments. Statistical analysis was performed by using two-way ANOVA followed by Bonferroni post-tests. **p < 0.01, ***p < 0.001.

FIGURE 4

RIPK3 and/or Casp-1/11 deficiencies negatively affect in vivo antigen-specific CTL activity and cytokine production in response to LM-OVA. (A) In vivo elimination of target cells pulsed with high (10 nM) (CFSE^High), intermediate (0.1 nM) (CTV^High), and low (0.001 nM) (CTV^Low) concentration of OVA_257–264 peptide at 7 days post-infection with LM-OVA. Percentage of CFSE^High, CFSE^Low (non-target cells), CTV^High, and CTV^Low shows the frequency of remaining cells after CTL-mediated target cell elimination. Percentage of live target cells in (B) non-infected and (C) infected mice. (D–G) ELISPOT assay determined the frequency of OVA_257–264 peptide-specific IFN-γ- or TNF-α-producing CD8⁺ T cells. (A) Flow cytometry density plots represent a single mouse from each experimental group. Numbers represent mean percentages of five mice/group. (D,F) ELISPOT images from a single mice/group are representative of five animals/group. (B,C,E,G) Bar graphs depict means and standard deviation of five individual mice per group. Experiments were repeated at least three times with similar results. Statistical analysis was performed by using two-way ANOVA followed by Bonferroni post-tests. *p < 0.05, ***p < 0.001.

FIGURE 5

RIPK3 and/or Casp-1/11 deficiencies reduce antigen-specific CD8⁺ T cells degranulation and intracellular cytokine production in response to LM-OVA. (A) Representative images of frequency of LAMP-1-positive OVA-specific CD8⁺ T cells for LM-OVA-infected and non-infected (control) groups and the (B) frequency variation of LAMP-1⁺-CD8⁺ T cells observed on the LM-OVA-infected group. Immediately, representative image of intracellular H2-K^b-SIINFEKL-specific (C) IFN-γ and (E) TNF-α-producing CD8⁺ T cell expression on control and infected groups. The frequency of these cell populations, H2-K^b-SIINFEKL-specific (D) IFN-γ and (F) TNF-α-producing CD8⁺ T cells, after 7 days of infection with LM-OVA was schematized. Frequencies described in (B,D,F) bar graphs were representative of experiments done in triplicate, and the data depict mean ± SEM of five individual mice per group. Statistical analysis was performed by using two-way ANOVA followed by Bonferroni post-tests. ***p < 0.001.

FIGURE 6

Casp-1/11 and Casp-1/11, RIPK-3 combined deficiencies restrict early OVA-specific CD8⁺ T memory cell response. (A) Surface expression of CD127 and KLRG1 on adoptively transferred CD8⁺ T cells after 7 days of infection was denoted by flow cytometry representative images. Frequency of (B) CD127, (C) KLRG1, and (D) CD127, KLRG1-positive OT-1 CD8⁺ T cells are expressed as means of five individual mice per group and are representative of three independent experiments. Statistical analysis was performed by using two-way ANOVA followed by Bonferroni post-tests. *p < 0.05, **p < 0.01.