Bystander activation of B. pertussis-specific CD4 T_RM cells in vitro

We have previously demonstrated that CD4 T_RM cells accumulate in the lung and nasal tissue of mice during infection with B. pertussis and that these cells persist in the respiratory tissue for at least 9 months and confer protection against rechallenge of convalescent mice. Consistent with these findings, using flow cytometry (gating strategy shown in Fig. S1), we found a high frequency of B. pertussis-specific CD4 T cells in the lungs and nasal tissue of convalescent (>60 days post B. pertussis infection) mice (Fig. 1A), which had a T_RM phenotype (CD44⁺CD69⁺CD103^±) (Fig. S2A). However, IL-17A-producing CD4 T cells were also detected following in vitro stimulation of respiratory mononuclear cells from lung or nasal tissue of convalescent, but not naïve, mice with E. coli-derived LPS in the presence of bone marrow-derived DCs (BMDCs) (Fig. 1A). In contrast, lymph node (LN) CD4 T cells from convalescent mice did not respond to stimulation with LPS and DCs (Fig. 1A). The addition of an anti-IL-12p40 antibody, which targets a common p40 subunit shared by IL-23 and IL-12, significantly reduced IL-17A (Fig. 1B) and moderately reduced IFN-γ (Fig. S2B) production from nasal or lung CD4 T_RM cells following coculture with E. coli LPS in the presence of BMDCs.

We confirmed these findings using FACS-purified nasal- or lung-resident CD4 T cells from convalescent mice. We found substantial IL-17A production by CD4 T cells from nasal tissue and lungs (Fig. 1C) when cocultured with sonicated B. pertussis (sBP) and DCs, confirming the presence of B. pertussis-specific CD4 T cells in the respiratory tissue of convalescent mice. High concentrations of IL-17A were also produced by nasal or lung tissue-resident CD4 T cells from B. pertussis convalescent mice following stimulation with LPS in the presence of DCs (Fig. 1C). Stimulation of nose-resident CD4 T cells with LPS or sBP in the absence of DCs failed to induce IL-17A production (Fig. S2C). Compared with tissue-resident cells, circulating CD4 T cells from the lung produced substantially lower concentrations of IL-17A following stimulation with sBP or LPS in the presence of DCs (Fig. 1C). LPS-induced IL-17A production by tissue-resident CD4 T cells was significantly reduced following the addition of an anti-IL-12p40 neutralizing antibody but not by an anti-MHCII antibody, suggesting that this response is mediated by IL-23/IL-12 signaling and not by TCR engagement with MHC antigen. In contrast, the addition of anti-IL-12p40 or anti-MHCII antibodies reduced sBP-induced IL-17A production in tissue-resident CD4 T cells (Fig. 1C). However, the lack of a statistically significant reduction in the B. pertussis-specific response by either antibody may reflect the fact that B. pertussis-specific IL-17 production results from antigen-APC stimulation, as well as the effects of Th17 cell polarizing cytokines, IL-23 and IL-1β, and blocking one pathway may not significantly inhibit IL-17 production. These data demonstrate that the pathogen-specific T-cell response is mediated by TCR activation and polarizing cytokines, whereas LPS-induced T-cell responses are mediated by cytokines without TCR-MHC interactions.

We next assessed whether B. pertussis-induced CD4 T_RM cells could be activated nonspecifically by the unrelated respiratory pathogen K. pneumoniae. IL-17A-producing CD4 T_RM cells were detected following in vitro stimulation of respiratory mononuclear cells from lung or nasal tissue of convalescent, but not naïve, mice with heat-killed K. pneumoniae (HKKP) in the presence of bone marrow-derived DCs (Fig. 2A). The frequency of IL-17A-producing CD4 T_RM cells was significantly reduced following the addition of an anti-IL-12p40 antibody (Fig. 2A). HKKP also promoted IL-17A production from purified nose-resident CD4 T cells from convalescent mice when cocultured with DCs, and this production was significantly reduced by the addition of anti-IL-12p40 and reduced, although not significantly, by antibodies against IL-12p19 or IL-1β (Fig. 2B). Furthermore, lung tissue-resident, but not circulating, CD4 T cells from convalescent mice produced IL-17A when cultured with HKKP in the presence of DCs, and this was significantly reduced by the addition of antibodies against IL-12p40, IL-23p19 or IL-1β (Fig 2C). Moreover, HKKP induced IL-23, IL-1β, and IL-12p70 production in DCs (Fig 2D). We also demonstrated that bystander activation of T_RM cells was not confined to LPS or gram-negative bacteria; respiratory CD4 T_RM cells from nasal tissue or lungs stimulated with heat-killed Staphylococcus aureus (HKSA) in the presence of DCs produced IL-17A, similar to that induced by HKKP (Fig. S3A-C). Furthermore, purified tissue-resident CD4 T cells from nasal tissue or lungs of B. pertussis convalescent mice produced IL-17A following culture with LPS from E. coli or K. pneumoniae or with monophosphoryl lipid A (MPLA), a derivative of LPS that also binds to TLR4 (Fig. S3D-E). Collectively, these results demonstrate that CD4 T_RM cells induced in the respiratory tract following infection with B. pertussis undergo bystander activation, producing IL-17A in response to LPS or unrelated pathogens in the presence of DCs in vitro, and this appears to be mediated in part by cytokines produced by PAMP-activated innate immune cells, such as DCs.

We next assessed the direct cytokine stimulation of CD4 T_RM cells. Resident (CD45 i.v.⁻) and circulating (CD45 i.v.⁺) CD4 T cells were purified from the respiratory tissue of convalescent mice and stimulated with combinations of IL-1β, IL-23, IL-18, or IL-12 in the presence or absence of IL-7, which enhances the survival and maintenance of memory T cells [28], and IL-17A and IFN-γ were quantified by ELISA. IL-23 in combination with IL-1β or IL-18 promoted IL-17A production from nose- and lung-resident CD4 T cells, which was enhanced by the addition of IL-7 (Fig. 2E, F). While tissue-resident CD4 T cells responded directly to cytokines, circulating CD4 T cells from convalescent mice failed to produce significant levels of IL-17A following stimulation with combinations of cytokines (Fig. 2E). These findings suggest that B. pertussis infection-induced CD4 T cells that reside in the respiratory tissue parenchyma, but not in the circulation, produce IL-17A in response to homeostatic and T-cell polarizing cytokines independent of TCR engagement.

Bystander activation of B. pertussis-specific CD4 T_RM cells in vivo

We next examined bystander activation of respiratory CD4 T_RM cells in vivo in mice previously infected with B. pertussis. LPS was administered intranasally (i.n.) to convalescent (> 60 days post B. pertussis infection) or naïve mice, and CD4 T_RM cells were assessed in the nasal tissue 4 hours later. Administration of LPS significantly enhanced the number of IL-17A⁺ CD4 T_RM cells in the nasal cavity of convalescent, but not naïve, mice (Fig. 3A). Furthermore, the concentrations of IL-1β, LCN2, and CXCL1, which are driven by IL-17A [29], were significantly higher in the nasal wash of convalescent mice than in that of naïve control mice 4 hours post i.n. administration of LPS (Fig. 3B). In addition, IL23a mRNA expression was significantly enhanced in nasal tissue of convalescent, but not naïve, mice following intranasal administration of LPS (Fig. 3C). Treatment of mice with a neutralizing anti-IL-12p40 antibody one day prior to and during i.n. administration of LPS significantly reduced the number of IL-17A⁺ CD4 T_RM cells in the nasal tissue of convalescent mice (Fig. 3D), suggesting that the nonspecific activation of IL-17A⁺ CD4 T_RM cells is mediated partly by IL-23/IL-12 signaling in vivo.

Intranasal administration of HKKP also enhanced the number of IL-17A⁺ CD4 T_RM cells in the nasal tissue of convalescent but not naïve mice, and this was accompanied by significantly higher concentrations of the antimicrobial peptide (AMP) LCN2 and CXCL1 in the nasal wash (Fig. 3E, F). CXCL1 is involved in neutrophil recruitment, and consistent with this, we found significantly more tissue-infiltrating (CD45 i.v.⁻) neutrophils in the nasal tissue of convalescent mice than in that of naïve control mice following i.n. administration of HKKP (Fig. 3G). These results suggest that CD4 T_RM cells induced during infection with B. pertussis can produce IL-17A nonspecifically in response to LPS or an unrelated pathogen in vivo, and this is accompanied by enhanced chemokine and AMP production and recruitment of neutrophils to the nasal tissue.

Heterologous immunity to K. pneumoniae induced by i.n. immunization with a wP vaccine

We examined bystander activation of CD4 T cells induced with a wP vaccine and the possibility that this might mediate heterologous immunity to an unrelated pathogen. We first compared the induction of B. pertussis-specific CD4 T_RM cells and protection against B. pertussis induced by parenteral versus mucosal delivery of the vaccines. Immunization with a wP vaccine by either the i.p. or i.n. routes conferred a high level of protection against lung infection with B. pertussis (Fig. 4A). Immunization with a wP vaccine by the i.p. route conferred protection against infection of the nose but not to the same extent as that induced by the same vaccine delivered by the i.n. route. Following B. pertussis aerosol challenge, the CFU counts in nasal wash and nasal tissue were significantly lower in mice immunized with a wP vaccine by the i.n. compared with the i.p. route (Fig. 4A). This correlated with a significantly higher frequency of B. pertussis-specific IL-17A⁺ and IFN-γ⁺ CD4 T_RM cells in the nasal tissue of i.n. compared with i.p. immunized mice on the day of challenge and 7 days post B. pertussis challenge (Fig. 4B).

Assessment of bystander activation of wP vaccine-induced CD4 T_RM cells revealed that immune cells from the nasal tissue of mice immunized with a wP vaccine by the i.n., but not the i.p., route produced IL-17A when cocultured with HKKP and DCs (Fig. 4C). We also observed a significant increase in the frequency of IFN-γ-producing CD4 T_RM cells following HKKP stimulation of nasal tissue immune cells from mice immunized by either the i.n. or i.p. route (Fig. S4A); however, the frequency of IFN-γ⁺ CD4 T_RM cells was much lower than that of IL-17A⁺ CD4 T_RM cells.

IL-17A production was induced in CD4 T_RM cells from mice immunized with a wP vaccine following in vitro stimulation of nasal mononuclear cells with IL-23 in combination with IL-1β or IL-18 (Fig. 4D). Conversely, IL-17A production was not detected in CD4 T_RM cells from mice immunized with a wP vaccine by the i.p. route or from naïve control mice following in vitro stimulation with Th17-polarizing cytokines (Fig. 4D). A low frequency of IFN-γ-producing CD4 T_RM cells was detected in nasal immune cells following stimulation with the Th1-polarizing cytokines IL-12 and IL-18 (Fig. S4B). Collectively, these results demonstrate that i.n. immunization with the wP vaccine is superior to i.p. route to induce IL-17A-producing CD4 T_RM cells in the nasal mucosa and that CD4 T_RM cells induced following i.n. immunization with a pertussis vaccine can produce IL-17A in response to stimulation with K. pneumoniae or T-cell polarizing cytokines in vitro.

We investigated the possibility that wP vaccine-induced B. pertussis-specific CD4 T_RM cells are also susceptible to bystander activation in vivo and that this might augment protective immunity to K. pneumoniae. Immunization with a wP vaccine by the i.n. route generated IL-17A-seceting CD4 T_RM cells in the nasal cavity, which was significantly augmented following i.n. administration of HKKP (Fig. 5A). IFN-γ-secreting CD4 T_RM cells were also enhanced, but the numbers were low compared with IL-17A-secreting CD4 T_RM cells (Fig. 5A and Fig. S5A). Furthermore, i.n. administration of HKKP promoted a significant increase in the number of tissue-infiltrating (CD45 i.v.⁻) neutrophils and Ly6C⁺CD11b⁺ inflammatory monocytes in the noses of mice that had been immunized with a wP vaccine (Fig. 5B and Fig. S5B). Similarly, i.n. administration of LPS significantly enhanced the number of IL-17A-secreting CD4 T_RM cells in the nasal cavity of mice immunized i.n. with the wP vaccine (Fig. S5C-E), with a corresponding increase in the numbers of inflammatory monocytes and neutrophils infiltrating the nasal tissue (Fig. S5F, G).

Intranasal administration of HKKP enhanced the concentrations of LCN2, CXCL1, and IL-1β in the nasal mucosa of wP-immunized mice, and this effect was significantly reduced following treatment with a neutralizing anti-IL-17A antibody (Fig. 5C). Furthermore, treatment of wP-immunized mice with a neutralizing anti-IL-17A antibody prior to and during administration of HKKP significantly reduced the frequency and moderately reduced the absolute number of tissue-infiltrated neutrophils in the nasal mucosa (Fig. 5D). These results suggest that bystander activation of wP vaccine-induced IL-17A-seceting CD4 T_RM cells by heat-killed K. pneumoniae promotes AMP production and chemokines that recruit neutrophils to the nasal tissue.

Since IL-17A-driven neutrophils play a protective role in host defense against K. pneumoniae [30], we examined the possibility that i.n. immunization with a wP vaccine could confer nonspecific/heterologous immunity to K. pneumoniae. Mice were immunized i.n. with two doses of a wP vaccine at 0 and 4 weeks and challenged with live K. pneumoniae two weeks later. Compared with naïve mice, mice immunized with a wP vaccine had significantly lower bacterial burdens in the nasal tissue at 24 hours and 5 days post K. pneumoniae challenge (Fig. 5E). Intranasal immunization of mice with the wP vaccine induced production of the neutrophil-recruiting chemokine CXCL1 (Fig. 5F) and promoted recruitment of circulating and infiltrating neutrophils to the nasal tissue (Fig. 5G). CXCLI and circulating neutrophils were enhanced following challenge with K. pneumoniae. Treatment of mice with an anti-CD4 antibody before challenge with K. pneumoniae, which depleted CD4 T cells from the nasal cavity (Fig. 5H), reversed the protective effect of a wP vaccine against live K. pneumoniae (Fig. 5I). Depletion of CD4 T cells partially reversed the enhancement of LCN2 following K. pneumoniae challenge in mice immunized with the wP vaccine (Fig. 5J). Taken together, our findings demonstrate that B. pertussis-specific CD4 T_RM cells induced following i.n. immunization with a wP vaccine conferred heterologous immunity against the unrelated respiratory pathogen K. pneumoniae.

Animals

Female C57BL/6 mice were bred and housed in a specific pathogen-free facility in the Comparative Medicine Unit, Trinity College Dublin. Mice were 6–12 weeks old at the initiation of experiments. All animal experiments were carried out in accordance with the guidelines of the Health Products Regulatory Authority (HPRA), the competent authority in Ireland, and approved by the Trinity College Dublin Animal Research Ethics Committee.

Vaccines and immunizations

Mice were immunized twice either i.n. (20 μl/mouse) or i.p. (200 μl/mouse) with 1/20^th of one human dose of a wP vaccine (WHO International Standard 94/532; NIBSC, UK) at 0 and 4 weeks. Mice were challenged 2 weeks later.

Bacterial respiratory challenge

Mice were infected by aerosol challenge with a culture of B. pertussis (Bp338 strain; 1×10⁹ CFU/ml) from a nebulizer (PARI TurboBOY SX) for 10 min as previously described [7]. Mice were considered convalescent >60 days post B. pertussis challenge. Mice were infected i.n. with K. pneumoniae (ATCC strain 43816; 1×10⁶ CFU/mouse in 20 μl). The course of bacterial infection was followed by performing CFU counts on nasal washes, digested nasal tissue or lung homogenates using Bordet-Gengou agar plates for B. pertussis [3] and tryptic soy agar plates for K. pneumoniae[40].

PAMP or antibody treatment of mice

E. coli-derived LPS (LPS-EB Ultrapure from E. coli 0111:B4 strain; Invivogen; 5 μg/mouse) or HKKP (10 μg/mouse) was administered i.n. in 40 μl to mice anesthetized with ketamine/xylazine. HKKP was prepared by heating K. pneumoniae (43816 strain) to 70°C for 30 mins. For IL-12p40 or IL-17A neutralization, mice were injected i.p. with an anti-IL-12p40 antibody (C17.8; BioXcell), an anti-IL-17A antibody (17F3; BioXcell) or IgG2a isotype control antibody 1 day prior (200 μg/mouse) and at the time of (50 μg/mouse) i.n. administration of LPS or HKKP. For depletion of CD4 T cells, mice were injected i.p. with 200 μg of an anti-CD4 antibody (GK1.5; BioXcell) or a corresponding isotype control antibody (Rat IgG2b, κ isotype control; BioXcell) 3 and 1 day before K. pneumoniae challenge.

Detection of respiratory tissue-resident immune cells

To discriminate circulating from tissue-resident lymphocytes by flow cytometry, mice were injected i.v. with 1.5 μg of PE-conjugated anti-CD45 antibody (30-F11; eBioscience) in 200 μl PBS 10 min prior to euthanasia. Circulating lymphocytes are labeled CD45 i.v.⁺, while tissue-resident lymphocytes are protected from i.v. labeling, are therefore identified as CD45 i.v.⁻.

Isolation and flow cytometry analysis of cells from nasal tissue and lungs

Nasal tissue and lung immune cells were prepared as described [3]. Cells were incubated with LIVE/DEAD Aqua (1:600) (Invitrogen) and Fc block (αCD16/CD32 FcγRIII) (1:200) (BD Biosciences). Surface markers were stained with fluorochrome-conjugated anti-mouse antibodies and fixed with 2% paraformaldehyde (PFA, ThermoFisher Scientific). To detect intracellular cytokine production directly ex vivo without any additional stimulation, cells were incubated with brefeldin A (5 μg/ml) at 37°C in 5% CO2 during digestion and for an additional hour with brefeldin A (5 μg/ml). To detect B. pertussis-specific cytokine production, mononuclear cells were stimulated for 18–20 hours with sBP (5 μg/mL), anti-CD28 and anti-CD49d (1 μg/mL; BD Biosciences). To detect TCR-independent cytokine production by CD4 T cells, mononuclear cells were stimulated with combinations of recombinant murine IL-1β, IL-23, IL-18, and IL-12 (all at 10 ng/ml) for 20 hours. To detect cytokine production in response to unrelated PAMPs or pathogens, nasal or lung mononuclear cells were cocultured with bone marrow-derived DCs prepared as previously described [41] using recombinant GM-CSF (Peprotech) and stimulated with E. coli-derived LPS (100 ng/ml), HKKP (5 μg/ml) or HKSA (5 μg/ml), which was prepared by heating S. aureus (strain PS80) at 90°C for 45 mins and washing to remove secreted proteins, for 20 hours. Brefeldin A (5 μg/ml) was added for the final 4 hours of culture. For detection of intracellular cytokines, cells were fixed, permeabilized and stained using the eBioscience^TM Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific) according to the manufacturer's instructions. The antibodies for surface and intracellular cytokine staining are listed in Table S1. Flow cytometric analysis was performed on an LSRFortessa, and data were acquired using Diva software (BD Biosciences). Data were analyzed using FlowJo software (Tree Star).

Fluorescence-activated cell sorting of tissue-resident CD4 T cells

Mononuclear cells were isolated and pooled from lungs or nasal tissue as described above. After red blood cell lysis, cells were further purified over a continuous 30% Percoll gradient (GE Healthcare). Target populations (CD4⁺CD3⁺CD8⁻B220⁻TCR δ⁻) were isolated using the BD FACSAria Fusion Cell Sorter. Tissue-resident (CD45 i.v.⁻) or circulating (CD45 i.v.⁺) CD4⁺CD3⁺ T cells (CD8⁻B220⁻γδTCR⁻) were isolated from lungs and nasal tissue and stimulated with combinations of IL-1β, IL-23, IL-18, IL-12, and IL-7 (10 ng/ml). Plate-bound anti-CD3ε (1 μg/ml) and soluble anti-CD28 (1 μg/ml) were used as positive controls. Cells were cultured for 72 hours at 37°C in 5% CO2, and supernatants were removed for quantification of cytokine concentrations by ELISA.

Coculture of isolated CD4 T cells with BMDCs

Isolated nasal or lung CD4 T cells were cocultured with BMDCs (0.2×10⁶ cells/well) and stimulated with E. coli-derived LPS (100 ng/ml), K. pneumoniae-derived LPS (100 ng/ml; strain ATCC 15380; Sigma‒Aldrich), MPLA (PHAD^®) (100 ng/ml; Sigma‒Aldrich), sBp (5 μg/ml), HKKP (5 μg/ml), or medium alone in the presence of anti-IL-12 p40 (C17.8; BioXcell), anti-IL-1β (B122; BD Biosciences), anti-IL-23 p19 (N71-1183; BD Biosciences) (10 μg/ml), anti-MHC class II (2 μg/ml; M5/114.15.2; ThermoFisher Scientific) or appropriate isotype control for 48 hours at 37°C in 5% CO2, and supernatants were removed for quantification of cytokine concentrations by ELISA.

Quantitative Reverse Transcription-PCR

Nasal tissue was collected into RNAlater solution. RNA was subsequently isolated using TRIzol Reagent (Invitrogen) and reverse transcribed into cDNA using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). qRT‒PCR was performed using commercially available Il23a (Mm00518984_m1) primers. qRT‒PCR was performed on a PRISM7500 Sequence Detection System (ABI). The quantity of each gene was determined by normalization to the 18S rRNA (Mm04277571, ABI) internal control.

Statistical analysis

Statistical analysis was performed using Prism 8 (GraphPad Software). Data were analyzed using one- or two-way ANOVA, followed by appropriate multiple comparisons tests or a two-tailed unpaired t test, as appropriate. Data are expressed as the mean with SEM. P values less than 0.05 were considered significant.