Discordant rearrangement of primary and anamnestic CD8+ T cell responses to influenza A viral epitopes upon exposure to bacterial superantigens: Implications for prophylactic vaccination, heterosubtypic immunity and superinfections

Abstract

Infection with (SAg)-producing bacteria may precede or follow infection with or vaccination against influenza A viruses (IAVs). However, how SAgs alter the breadth of IAV-specific CD8+ T cell (TCD8) responses is unknown. Moreover, whether recall responses mediating heterosubtypic immunity to IAVs are manipulated by SAgs remains unexplored. We employed wild-type (WT) and mutant bacterial SAgs, SAg-sufficient/deficient Staphylococcus aureus strains, and WT, mouse-adapted and reassortant IAV strains in multiple in vivo settings to address the above questions. Contrary to the popular view that SAgs delete or anergize T cells, systemic administration of staphylococcal enterotoxin B (SEB) or Mycoplasma arthritidis mitogen before intraperitoneal IAV immunization enlarged the clonal size of 'select' IAV-specific TCD8 and reshuffled the hierarchical pattern of primary TCD8 responses. This was mechanistically linked to the TCR Vβ makeup of the impacted clones rather than their immunodominance status. Importantly, SAg-expanded TCD8 retained their IFN-γ production and cognate cytolytic capacities. The enhancing effect of SEB on immunodominant TCD8 was also evident in primary responses to vaccination with heat-inactivated and live attenuated IAV strains administered intramuscularly and intranasally, respectively. Interestingly, in prime-boost immunization settings, the outcome of SEB administration depended strictly upon the time point at which this SAg was introduced. Accordingly, SEB injection before priming raised CD127highKLRG1low memory precursor frequencies and augmented the anamnestic responses of SEB-binding TCD8. By comparison, introducing SEB before boosting diminished recall responses to IAV-derived epitopes drastically and indiscriminately. This was accompanied by lower Ki67 and higher Fas, LAG-3 and PD-1 levels consistent with a pro-apoptotic and/or exhausted phenotype. Therefore, SAgs can have contrasting impacts on anti-IAV immunity depending on the naïve/memory status and the TCR composition of exposed TCD8. Finally, local administration of SEB or infection with SEB-producing S. aureus enhanced pulmonary TCD8 responses to IAV. Our findings have clear implications for superinfections and prophylactic vaccination.

Fig 1. SEB administration before intraperitoneal PR8 immunization increases the magnitude of primary T_CD8 responses to select viral epitopes.

(A) BALB/c mice were injected i.p. with PBS or with 50 μg SEB three days before they were immunized i.p. with the PR8 strain of IAV. Seven days later (B,C,E,F,G,H) or at indicated time points (D), splenic (B,C,E,F,G,H) or peripheral blood (D) T_CD8 responses to indicated PR8 epitopes were evaluated by MHC I dextramer staining (B-C), MHC I tetramer staining (D), intracellular staining for IFN-γ (E-F) or in vivo cytotoxicity assays (G-H). Panels B and G illustrate representative contour and histogram plots, respectively. Error bars represent SEM, and *, **, *** and **** denote statistically significant differences with p<0.05, p<0.01, p<0.001 and p<0.0001, respectively, using unpaired, two-tailed Student’s t-tests.

Fig 2. Preferential TCR Vβ usage by T_CD8 clones dictates their enhanced or unaltered responsiveness to PR8 following exposure to bacterial SAgs.

(A) Mice were injected i.p. with PBS or with SEB. Seven days later, NP₁₄₇-, PB2₂₈₉- and HA₅₁₈-specific T_CD8 were identified by ICS for IFN-γ and also surface-stained with 15 mAbs recognizing indicated TCR Vβ regions. The abundance of each Vβ region is expressed as a proportion of all detectable Vβs for each epitope-specific T_CD8 population in individual mice (n = 3 per group). SEB-binding Vβs are shown using different patterns against a red background. (B) A ribbon diagram model of SEB (blue) in complex with mouse TCR Vβ8.3 (gray) and the MHC class II molecule HLA-DR1 (α-chain, red; β-chain, green) was constructed. The cognate peptide displayed within the open groove of HLA-DR1 is shown in black. The insert illustrates the key position of Asn²³ for interactions with indicated residues of the CDR2β region of mouse Vβ8.3. (C) Parallel cohorts of mice (n = 5 per group from two independent experiments) were injected i.p. with PBS, 50 μg of SEB, or 50 μg of SEB_N23A, followed 3 days later by PR8 immunization i.p. After 7 days, T_CD8 responses to indicated epitopes were quantified by ICS for IFN-γ. (D) Mice were injected i.p. with PBS or with 50 ng of MAM before they received, 3 days later, an i.p. inoculum of PR8. On day 7 post-PR8 immunization, epitope-specific T_CD8 were enumerated. Data pooled from two independent experiments are shown. (E) Three days before PR8 inoculation, mice were injected with PBS or with 50 μg of SEA i.p. Seven days after PR8 immunization, epitope-specific T_CD8 were enumerated. Error bars represent SEM. * and ** denote significant differences with p<0.05 and p<0.01, respectively, using unpaired, two-tailed Student’s t-tests (D) or one-way ANOVA followed by Tukey’s post-hoc tests (C).

Fig 3. Systemic pre-exposure to SEB augments the primary NP₁₄₇-specific response following immunization with multiple IAV strains.

Mice were injected i.p. with PBS or SEB followed, 3 days later, by i.p. immunization with WT or recombinant IAV strains expressing the same NP (A). These strains were NT60 (B), HK (C), X31 (D) and J-1 (E). Seven days after IAV inoculation, the absolute numbers of IFN-γ-producing epitope-specific T_CD8 were calculated. Error bars represent SEM. * and ** denote significant differences with p<0.05 and p<0.01, respectively, using unpaired Student’s t-tests.

Fig 4. Pre-exposure to SEB raises the frequency of memory T_CD8 precursors and invigorates recall responses of SEB-binding IAV-specific T_CD8.

(A-B) Mice were injected i.p. with PBS or with 50 μg of SEB before they received i.p. immunization with PR8. On indicated days, members of each cohort were sacrificed for their spleens in which the frequencies of NP₁₄₇-specific (A) and HA₅₁₈-specific (B) T_CD8 were determined by ICS for IFN-γ. Each symbol represents an individual mouse, and data are pooled from two independent experiments yielding similar results. (C) On day 7 post-PR8 immunization, the frequencies of KLRG1⁺CD127^- T_CD8 (short-lived effectors) and KLRG1^-CD127⁺ T_CD8 (memory precursors) were determined after surface staining of splenic cells with mAbs to CD3 and CD8α along with MHC I tetramers detecting NP₁₄₇- or HA₅₁₈-specific cells. (D) Mice were injected with PBS or SEB three days before they were primed with PR8 i.p. Thirty days later, boosting immunization with SEQ12 was conducted using the same injection route. On day 7 post-challenge with SEQ12, the frequencies (E) and absolute numbers (F) of IFN-γ-producing epitope-specific T_CD8 in the spleens were determined. The results depicted in E-F are pooled from two independent experiments. Error bars (A-C and E-F) represent SEM. * and ** denote significant differences with p<0.05 and p<0.01, respectively, using unpaired Student’s t-tests.

Fig 5. Exposure to SEB before boosting immunization upregulates Fas and exhaustion markers on anti-IAV T_CD8 and compromises their anamnestic responses.

(A) Mice were primed with PR8, injected with either PBS or 50 μg of SEB, and then boosted with SEQ12 through the i.p. route according to the schematically depicted schedule. Seven days after SEQ12 inoculation, animals were sacrificed for their spleens in which the frequencies (B) and the absolute numbers (C) of IFN-γ-producing epitope-specific T_CD8 were determined by ICS. (D) NP₁₄₇- and HA₅₁₈-specific T_CD8, which were identified by MHC I tetramers, were co-stained with mAbs against intracellular Ki67 and surface Fas, LAG-3 and PD-1. Error bars represent SEM (n = 4 per group). *, ** and *** denote statistically significant differences with p<0.05, p<0.01 and p<0.001, respectively, using unpaired Student’s t-tests.

Fig 6. Exposure to SEB after PR8 immunization augments IAV-specific T_CD8 responses.

(A) Mice were inoculated with PR8 and then injected with PBS or SEB (50 μg) i.p. Seven days after the SEB challenge (or after PBS injection), animals were euthanized, and PR8-specific T_CD8 were enumerated by ICS for IFN-γ in each spleen. Data pooled from two independent experiments yielding similar results are shown in B-C. In separate experiments, CFSE^low naïve splenocytes displaying NP₁₁₈ (control target cells) and CFSE^high naïve splenocytes pulsed with NP₁₄₇ (cognate target cells) were mixed in equal numbers and injected i.v. into PR8-inoculated mice that had subsequently received PBS or SEB. Mice were sacrificed after 1, 2 or 4 hours for their spleens in which target cells were identified based on their differential CFSE intensities. (D) Representative histograms from a 1-hour in vivo killing assay are depicted. (E) In vivo cytotoxicity at indicated time points is averaged and shown for 3 mice/group/time point. Error bars represent SEM. *, **, *** and **** denote differences with p<0.05, p<0.01, p<0.001 and p<0.0001, respectively, using unpaired Student’s t-tests.

Fig 7. Intraperitoneal administration of SEB before sublethal PR8 infection enhances the splenic, but not the pulmonary, NP₁₄₇-specific response.

(A) Indicated numbers of BALB/c mice received no treatment or injected with PBS or SEB i.p. followed, 3 days later, by intranasal instillation of PBS or a 25-μL inoculum of infectious allantoic fluid approximating 0.3 MLD₅₀ of PR8. Weight loss was monitored for 10 days. (B) On day 4 post-infection, viral titres in the lungs and BAL were determined using a modified TCID₅₀ assay detailed in Materials and Methods. Each symbol represents an individual mouse. (C) Ten days after infection, lungs, spleens and BAL fluid were collected from separate cohorts from 5 independent experiments. Epitope-specific T_CD8 were enumerated in each spleen and also in pooled lung and BAL samples by ICS for IFN-γ. Error bars represent SEM. * and ** denote significant differences with p<0.05 and p<0.01, respectively, using one-way ANOVA (A) or an unpaired Student’s t-test (C).

Fig 8. Intranasal instillation of SEB before PR8 does not worsen infection-induced weight loss but increases the pulmonary response to NP₁₄₇.

(A) BALB/c mice were injected i.n. with 50 ng of SEB in 25 μL PBS (n = 7) or with PBS alone (n = 6) three days before they were sublethally infected with PR8. Animals were monitored daily after PR8 infection, and their weight loss was recorded (B). On day 10 post-infection, the percentages of NP₁₄₇-specific T_CD8 were determined in the lungs of PBS- and SEB-pretreated mice by MHC I tetramer staining (C). Representative contour plots are depicted, and mean ± SEM values are indicated. ** denotes a significant difference between PBS- and SEB-treated mice with p<0.01 using an unpaired Student’s t-test.

Fig 9. Respiratory infection with an SEB deletion mutant of S. aureus before PR8 results in weaker NP₁₄₇-specific T_CD8 responses.

(A) COL and COL Δseb were cultured in half-BHI medium, and their growth rates at 37°C were monitored. OD₆₀₀ readings were used to generate a growth curve for each strain. Mice were then given PBS, 1 × 10⁸ CFUs of COL or 1 × 10⁸ CFUs of COL Δseb i.n. three days before they were infected sublethally with PR8. On day 4 post-PR8 infection, mice (n = 3/group) were sacrificed for their lungs and BAL fluid in which infectious PR8 titres were determined by TCID₅₀ assays. Each symbol represents an individual mouse (B). Separate cohorts of mice were monitored for weight loss until day 10 post-PR8 infection (C), at which point animals were sacrificed for their spleens and lungs in which NP₁₄₇-specific T_CD8 were enumerated by MHC I tetramer staining or ICS for IFN-γ (C). Mean ± SEM values are depicted for 11 mice per group pooled from three independent experiments yielding similar results. Statistical comparisons between COL- and COL Δseb-infected mice were carried out using an unpaired Student’s t-test, and * denotes a significant difference with p<0.05.