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. 2018 Aug 14:9:1861.
doi: 10.3389/fimmu.2018.01861. eCollection 2018.

Memory Inflation Drives Tissue-Resident Memory CD8+ T Cell Maintenance in the Lung After Intranasal Vaccination With Murine Cytomegalovirus

Kaitlyn M Morabito  1   2 , Tracy J Ruckwardt  1 , Erez Bar-Haim  1   3 , Deepika Nair  1 , Syed M Moin  1 , Alec J Redwood  4 , David A Price  5   6 , Barney S Graham  1
Affiliations

Memory Inflation Drives Tissue-Resident Memory CD8+ T Cell Maintenance in the Lung After Intranasal Vaccination With Murine Cytomegalovirus

Kaitlyn M Morabito et al. Front Immunol. .

Abstract

Tissue-resident memory T (TRM) cells provide first-line defense against invading pathogens encountered at barrier sites. In the lungs, TRM cells protect against respiratory infections, but wane more quickly than TRM cells in other tissues. This lack of a sustained TRM population in the lung parenchyma explains, at least in part, why infections with some pathogens, such as influenza virus and respiratory syncytial virus (RSV), recur throughout life. Intranasal (IN) vaccination with a murine cytomegalovirus (MCMV) vector expressing the M protein of RSV (MCMV-M) has been shown to elicit robust populations of CD8+ TRM cells that accumulate over time and mediate early viral clearance. To extend this finding, we compared the inflationary CD8+ T cell population elicited by MCMV-M vaccination with a conventional CD8+ T cell population elicited by an MCMV vector expressing the M2 protein of RSV (MCMV-M2). Vaccination with MCMV-M2 induced a population of M2-specific CD8+ TRM cells that waned rapidly, akin to the M2-specific CD8+ TRM cell population elicited by infection with RSV. In contrast to the natural immunodominance profile, however, coadministration of MCMV-M and MCMV-M2 did not suppress the M-specific CD8+ T cell response, suggesting that progressive expansion was driven by continuous antigen presentation, irrespective of the competitive or regulatory effects of M2-specific CD8+ T cells. Moreover, effective viral clearance mediated by M-specific CD8+ TRM cells was not affected by the coinduction of M2-specific CD8+ T cells. These data show that memory inflation is required for the maintenance of CD8+ TRM cells in the lungs after IN vaccination with MCMV.

Keywords: CD8+ T cells; cytomegalovirus; memory inflation; respiratory syncytial virus; tissue-resident memory; vaccine.

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Figures

Figure 1
Figure 1
Intranasal (IN) vaccination with murine cytomegalovirus (MCMV)-M2 elicits more lung-resident M2-specific CD8+ T cells than intraperitoneal (IP) vaccination. (A–E) Mice were vaccinated with MCMV-M or MCMV-M2 via the IN or IP route. Intravascular staining was used in conjunction with Db/M187–195 and Kd/M282–90 tetramers to quantify epitope-specific CD8+ T cells in the lung tissue and blood after 1 week. (A) Gating strategy used to identify M-specific and M2-specific CD8+ T cells in the tissue and blood of the lungs. (B) Frequency and (C) number of M-specific CD8+ T cells in the tissue and blood of lungs 1 week after MCMV-M vaccination. (D) Frequency and (E) number of M2-specific CD8+ T cells in the tissue and blood of the lungs 1 week after MCMV-M2 vaccination. Bars indicate mean ± SEM (n = 5 mice/group). ****P < 0.0001, **P < 0.01, *P < 0.05 by two-way ANOVA. Data are shown from one experiment and representative of two independent experiments.
Figure 2
Figure 2
The M-specific CD8+ T cell population inflates, whereas the M2-specific CD8+ T cell population contracts, after vaccination with murine cytomegalovirus (MCMV). (A–F) Mice were infected with respiratory syncytial virus (RSV) or vaccinated with MCMV-M or MCMV-M2 alone or a combination of MCMV-M and MCMV-M2 via the intranasal route. Intravascular staining was used in conjunction with Db/M187–195 and Kd/M282–90 tetramers to quantify M-specific (A–C) and M2-specific (D–F) CD8+ T cells in the lung tissue and blood at weeks 1 (W1), 8 (W8), and 16 (W16). Total (A,D) denotes all tetramer+ CD8+ T cells regardless of location. Bars indicate mean ± SEM (n = 5 mice/group). ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05 by two-way ANOVA. Data are shown from one experiment and representative of two independent experiments.
Figure 3
Figure 3
The M2 epitope is preferentially generated by the immunoproteasome. (A,B) Mice were infected with respiratory syncytial virus (RSV) and treated with the immunoproteasome inhibitor ONX-0914 or vehicle control on days 0, 2, 4, and 6 at doses of 2, 6, or 10 mg/kg. Db/M187–195 and Kd/M282–90 tetramers were used to quantify the frequency (A) and number (B) of M-specific and M2-specific CD8+ T cells in the lungs on day 7. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05 by two-way ANOVA. Bars indicate mean ± SEM (n = 5 mice/group). Data are shown from one experiment and representative of two independent experiments.
Figure 4
Figure 4
Intranasal (IN) vaccination with murine cytomegalovirus (MCMV) elicits CD8+ tissue-resident memory T (TRM) cells. (A–E) Mice were infected with respiratory syncytial virus (RSV) or vaccinated with MCMV-M or MCMV-M2 alone or a combination of MCMV-M and MCMV-M2 via the IN route. Intravascular staining was used in conjunction with Db/M187–195 and Kd/M282–90 tetramers to quantify M-specific (A,B,D,F) and M2-specific (A,C,E,G) CD8+ T cells in the lung tissue at weeks 8 (W8) and 16 (W16). (A) Representative flow cytometry plots showing expression of CD69 and CD103 on epitope-specific CD8+ T cells in the lung parenchyma at week 8. (B,D) The number of M-specific CD103+CD69+ TRM cells (B) and CD103−CD69+ TRM cells (D) elicited by infection with RSV or vaccination with MCMV-M alone or together with MCMV-M2. (C,E) The number of M2-specific CD103+CD69+ TRM cells (C) and CD103−CD69+ TRM cells (E) elicited by infection with RSV or vaccination with MCMV-M2 alone or together with MCMV-M. (F,G) Percentage of CD103+ and CD103− M-specific CD69+ TRM cells (F) and M2-specific CD69+ TRM cells (G). ****P < 0.0001 by two-way ANOVA. Bars indicate mean ± SEM (n = 5 mice/group). Data are shown from one experiment and representative of two independent experiments.
Figure 5
Figure 5
Phenotype of M-specific and M2-specific CD8+ T cells elicited by murine cytomegalovirus (MCMV) vaccination. Mice were vaccinated with MCMV-M or MCMV-M2 alone or a combination of MCMV-M and MCMV-M2 via the IN route. Intravascular staining was used in conjunction with Db/M187–195 and Kd/M282–90 tetramers to identify M-specific and M2-specific CD8+ T cells in the blood and tissue of the lungs at week 8. (A) Gating strategy for phenotypic analysis. Populations were defined as follows: CD127+KLRG1−CD62L+ [central memory (CM)]; CD127+KLRG1−CD62L− [effector memory (EM)]; CD127−KLRG1−CD62L− (effector); and KLRG1+CD62L− (KLRG1+ effector). (B) The proportions of CM cells (blue), EM cells (green), effectors (orange), and KLRG1+ effectors (yellow) in the lungs are shown for each specificity. (C) CD44 expression on M-specific, M2-specific, and all CD8+ T cells in the tissue and blood of the lungs. *P ≤ 0.05, **P < 0.01 by permutation test (SPICE). Data are shown from one experiment (n = 5/group) and representative of two independent experiments.
Figure 6
Figure 6
Murine cytomegalovirus (MCMV)-elicited tissue-resident memory T cells expedite viral clearance after infection with respiratory syncytial virus (RSV). (A–D) Mice were vaccinated with MCMV vector, MCMV-M or MCMV-M2 alone, or a combination of MCMV-M and MCMV-M2 via the intranasal route and challenged with RSV at week 16. Viral titers in the lungs were measured by plaque assay on days 3 (A) and 5 (B). ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05 by one-way ANOVA. (C,D) Intravascular staining was used in conjunction with Db/M187–195 and Kd/M282–90 tetramers to quantify M-specific and M2-specific CD8+ T cells in the lungs (C) and the blood (D). ****P < 0.0001, **P < 0.01, *P < 0.05 by two-way ANOVA. Data shown from one experiment and representative of two independent experiments.
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