Regionally compartmentalized resident memory T cells mediate naturally acquired protection against pneumococcal pneumonia

Abstract

As children age, they become less susceptible to the diverse microbes causing pneumonia. These microbes are pathobionts that infect the respiratory tract multiple times during childhood, generating immunological memory. To elucidate mechanisms of such naturally acquired immune protection against pneumonia, we modeled a relevant immunological history in mice by infecting their airways with mismatched serotypes of Streptococcus pneumoniae (pneumococcus). Previous pneumococcal infections provided protection against a heterotypic, highly virulent pneumococcus, as evidenced by reduced bacterial burdens and long-term sterilizing immunity. This protection was diminished by depletion of CD4⁺ cells prior to the final infection. The resolution of previous pneumococcal infections seeded the lungs with CD4⁺ resident memory T (T_RM) cells, which responded to heterotypic pneumococcus stimulation by producing multiple effector cytokines, particularly interleukin (IL)-17A. Following lobar pneumonias, IL-17-producing CD4⁺ T_RM cells were confined to the previously infected lobe, rather than dispersed throughout the lower respiratory tract. Importantly, pneumonia protection also was confined to that immunologically experienced lobe. Thus regionally localized memory cells provide superior local tissue protection to that mediated by systemic or central memory immune defenses. We conclude that respiratory bacterial infections elicit CD4⁺ T_RM cells that fill a local niche to optimize heterotypic protection of the affected tissue, preventing pneumonia.

Figure 1. Repeated respiratory infections establish heterotypic protection against pneumococcal pneumonia

C57BL/6 mice were infected with the indicated pneumococcal serotypes (or saline) one, two, or three times intranasally (i.n.) with doses of 1–3×10⁶ CFU each that were seperated by 1 week. After a 4 week rest period mice were challenged with 1×10⁶ Sp3 CFU i.t. for 24h. Lung (A) and blood (B) bacterial burdens were determined. Significance was determined by one-way ANOVA followed by Dunn’s post hoc test. Dashed line indictaes Sp3 CFU infection input. (C) Mice previously exposed to Sp19F, Sp35B, and Sp23A (i.n.) or saline were infected 4 weeks later with 3×10⁵ Sp3 CFU i.t. and followed for 7 days (Kaplan-Meier curve, n=9–10 per group). Significance was determined by Mantel-Cox test. (D) C57BL/6 mice were given 2 doses of heat-killed Sp19F (1×10⁶ CFU) or saline i.n. and challeged 4 weeks later with 1×10⁶ Sp3 CFU i.t. After 24h lung bacterial burden was assessed. (E) Het Imm (i.n.) or saline controls were infected with 5 ×10⁴ Klebsiella pneumoniae CFU i.t. and lung bacterial burden was assessed after 24h. Significance was determined by Mann-Whitney test. Each individual dot represents a single mouse and horizontal lines in scatter plots represent medians. Two independent experiments were performed in every case. *P < 0.05.

Figure 2. Heterotypic anti-pneumococcal immunity accelerates lung neutrophil recruitment

(A) Representative H&E images of Het Imm and saline control lungs that were collected, fixed with 4% PFA, and paraffin embedded 0 and 24h after an infection with 1×10⁶ Sp3 CFU. Images represent 10X magnification. (B) BALF was collected from Het Imm (i.n.) and saline lungs 7 and 24h following infection with 1×10⁶ Sp3 CFU. Differential cell counts were obtained and data are expressed as total number of neutrophils and macrophages. Bars represent means with standard error of the mean (SEM) displayed. Data were log transformed (Y=Log(Y)) and significance was determined by two-way ANOVA followed by Sidak’s post hoc test (n=8–9 for each group). *P < 0.05 for saline vs Het Imm comparisons. Two independent experiments were performed in every case.

Figure 3. Increased lung-to-neutrophil signaling due to heterotypic immunity

(A–G) Het Imm (i.n.) and saline control mice were infected with 1×10⁶ Sp3 CFU and BALF was collected 7 hours later. Cytokine concentrations were measured in the BALF using a multi-plex Luminex assay. Data are expressed as pg/mL and bars represent means with SEM. Dashed lines indicate the limit of detection for the particular analyte. Significance was determined by Student’s t test (n= 14 per group), *P < 0.05. Three independent experiments were performed.

Figure 4. CD4⁺ Th17 cells in lungs with heterotypic immunity during pneumococcal pneumonia

Het Imm (i.n.) and saline control mice were infected with 1×10⁶ Sp3 CFU for 24h. (A) Whole lungs were collected, homogenized and protein was extracted. IFN-γ and IL-17A protein concentrations were measured by ELISA. Data are expressed as pg/lung and bars represent means with SEM. Significance was determined by Student’s t test (n= 10–12 per group). (B) Whole lungs were collected and digested with collagenase to generate single cell suspensions. Cells were stimulated with PMA/Ionomycin for 6h in the presence of a protein transport inhibitor. IL-17A⁺ and IFN-γ⁺ CD4⁺ cells were detected using intracellular cytokine staining (ICS). Representative flow cytometry plots from a Het Imm and saline mouse are displayed. (C) IL-17A⁺ and IFN-γ⁺ CD4⁺ cells were quantified and are displayed as number of cells per lung. Bars represent means with SEM. Significance was determined by Student’s t test (n= 6–7 per group). Two independent experiments were performed in every case. *P < 0.05,

Figure 5. CD4⁺ cells are required for optimal heterotypic protection against pneumococal pneumonia

(A) Het Imm mice (i.t.) were administered either a CD4-depleting GK1.5 antibody 72 and 24h before a pneumonia challenge both intranasally (100ug) and intraperitoneally (500ug), or a control IgG antibody at the same concentrations. Mice were infected with 1×10⁶ Sp3 CFU and whole lungs were collected after 24h of pneumonia to determine lung bacterial burden. (B–C) IL-17A and IFN-γ protein levels were measured in the whole lung homogenates from (A). Data were expressed as pg/lung. Significance was determined by Student’s t test. (D) FTY720 or vehicle control was administered to Het Imm (i.t.) mice at a dose of 1mg/kg 2 days prior and the day of a pneumonia challenge with 1×10⁶ Sp3 CFU. Bacterial burden was assessed in whole lungs after 24h. Significance was determined using a Mann-Whitney test. (E) IL-17A protein levels were measured in the whole lung homogenates from (D). Significance was determined by Student’s t test. Individual dots represent a single mouse and horizontal lines represent medians. Dashed line indicates Sp3 CFU infection input. Bars represent means with SEM. Two independent experiments were performed. * P < 0.05.

Figure 6. Recovery from respiratory infection seeds the lungs with pneumococcus-specific Th17 and Th1 cells

The left lobes of Het Imm (i.t.) and saline control mice were digested with collagenase and CD4⁺ cells were stimulated with killed Sp3-pulsed splenocytes. (A) After 12h of stimulation in the presence of a protein transport inhibitor cells were harvested and CD3⁺ cells were analyzed for the production of IL-17A and IFN-γ by intracellular cytokine staining. Flow cytometry plots show the percentage of IL-17A and IFN-γ production from CD3⁺ cells in 3 groups: pan T cell activator α-CD3/CD28 (left), vehicle-pulsed antigen presenting cells (APCs, middle), and killed pneumococcus-pulsed APCs (APCs-pneumococcus, right). Two independent experiments were performed. (B–C) CD4⁺ cells from Het Imm and saline lungs were stimulated with killed Sp3-pulsed splenocytes for 72h. Supernatants from saline (B) and Het Imm (C) stimulations were collected and cytokine concentrations were measured using a multi-plex luminex assay. In addition to killed pneumococcus three controls are displayed: vehicle-pulsed splenocytes (untim), pan T cell activator α-CD3/CD28, and α-MHC-II blocking antibody added to killed pneumococcus-stimulated samples. IL-22 protein was measured independently using an ELISA. Cytokine concentrations were measured in 3 separate stimulation experiments and are displayed as pg/mL. Bars represent means with SEM. The limit of detection for each assay is indicated (LOD). Data were log transformed (Y=Log(Y)) and significance was assessed by one-way ANOVA followed by Sidak’s post hoc test, *P < 0.05 vs APCs-vehicle (unstim) for each cytokine. Three independent experiments were performed.

Figure 7. Lung CD4⁺ T_RM cells and heterotypic pulmonary protection imprint locally

The left (L) and right (R) lobes of uninfected Het Imm (i.t.) and saline control mice were digested separately with collagenase. (A) Representative flow cytometry plots show lung T_RM cells that were identified as CD3⁺/CD4⁺/CD11a^hi/CD69⁺/CD62L^lo/CD44^hi. (B) T_RM cells were quantified in the left (ipsilateral) and right (contralateral) lobes of Het Imm and saline mice. Data are displayed as % CD11a^hiCD69⁺ of CD4⁺ cells. Bars represent means with SEM. Significance was determined by two-way ANOVA followed by Tukey’s post hoc test (n= 4–5 per group, experiment repeated three times). (C) Representative flow cytometry plot of CD44 and CD62L expression on CD11a^hiCD69⁺ lung T_RM cells (red) and lung CD4⁺CD11a^loCD69- cells (blue). (D) Single cells from the left (ipsilateral) and right (contralateral) lobes of Het Imm (i.t.) and saline mice were stimulated with PMA/Ionomycin for 6h in the presence of a protein transport inhibitor. ICS and flow cytometry was performed to monitor cytokine production. Representative flow plots show IL-17A and IFN-γ-producing CD4⁺ cells from Het Imm and saline lungs. (E–F) IL-17A and IFN-γ-producing CD4⁺ cells were quantified in the left (ipsilateral) and right (contralateral) lobes of Het Imm and saline mice. Bars represent means with SEM. Significance was determined by two-way ANOVA followed by Tukey’s post hoc test (n= 5–6 per group). Three independent experiments were performed. (G) Mice on a mixed background were infected twice with saline, intranasal Sp19F (Het Imm i.n.), or intratracheal Sp19F (Het Imm i.t.). After 4 weeks mice from each group were challenged with 1 × 10⁶ Sp3 CFU. Lungs were harvested after 24h and bacterial burden was determined. Individual dots represent a single mouse and horizontal lines represent medians. Dashed line indicates Sp3 CFU infection input. Significance was determined by one-way ANOVA followed by Dunn’s post hoc test. (H) CD4⁺ lymphocytes from the lungs and spleens of uninfected Het Imm were isolated using FACS. Three minutes prior to euthanization intravital cells were fluorescently labeled with anti-CD45-APC antibody via i.v. injection. The cells for transfer were identified as live, i.v. label-negative CD45⁺CD4⁺ cells. Naïve recipient mice were administered saline, lung-derived or spleen-derived CD4⁺ cells (1–4×10⁶ per mouse) from Het Imm mice transferred via i.v. tail vein injection. Three days after the transfer, recipient mice were challenged with 1×10⁶ Sp3 CFU. After 24h, left lung lobes were harvested, mRNA was extracted, and qRT-PCR for IL-17A was performed. Data were displayed as fold induction for each group normalized to uninfected lungs. Significance was determined by one-way ANOVA followed by Tukey’s post hoc test (n=3–5 per group). Bars represent means with SEM. (I) Ipsilateral vs contralateral heterotypic protection model. Het Imm (i.t.) mice received two Sp19F infections in their left lobes. After 4 weeks, half of the mice were challenged with 1×10⁶ Sp3 CFU in their ipsilateral (left) lobe, and the other half challenged in their contralateral (right) lobes. Images represent the instillation (i.t.) of diluted colloidal carbon into C57BL/6 mouse lungs either in the left (L) or right (R) lobes. Lungs were immediately collected and the deposition of the dye was visualized. (J) Naïve C57BL/6 mice were infected with 1×10⁶ Sp3 CFU selectively in either the left or right lobes. Lung bacterial burden was assessed after 24h. (K) The mice described in (I) were challenged with 1×10⁶ Sp3 CFU in the specified location. After 24h whole lungs were collected and bacterial burden was assessed. Individual dots represent a single mouse and horizontal lines represent medians. Dashed line indicates Sp3 CFU infection input. Significance was determined using a Mann-Whitney test. Two independent experiments were performed. * P < 0.05.