Tissue-Resident Memory T Cells in the Lungs Protect against Acute Respiratory Syncytial Virus Infection

Abstract

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection in young children. The T cell response plays a critical role in facilitating clearance of an acute RSV infection, and memory T cell responses are vital for protection against secondary RSV exposures. Tissue-resident memory (TRM) T cells have been identified as a subset of memory T cells that reside in nonlymphoid tissues and are critical for providing long-term immunity. There is currently limited information regarding the establishment and longevity of TRM T cell responses elicited following an acute RSV infection as well as their role in protection against repeated RSV infections. In this study, we examined the magnitude, phenotype, and protective capacity of TRM CD4 and CD8 T cells in the lungs of BALB/c mice following an acute RSV infection. TRM CD4 and CD8 T cells were established within the lungs and waned by 149 d following RSV infection. To determine the protective capacity of TRMs, FTY720 administration was used to prevent trafficking of peripheral memory T cells into the lungs prior to challenge of RSV-immune mice, with a recombinant influenza virus expressing either an RSV-derived CD4 or CD8 T cell epitope. We observed enhanced viral clearance in RSV-immune mice, suggesting that TRM CD8 T cells can contribute to protection against a secondary RSV infection. Given the protective capacity of TRMs, future RSV vaccine candidates should focus on the generation of these cell populations within the lung to induce effective immunity against RSV infection.

Figure 1.. RSV-specific CD4 and CD8 T cells localize to the lungs.

BALB/c mice were infected with RSV i.n., and lungs were harvested at days 8, 10, 15, 30, 79, and 149 p.i. a) Frequency of CD4, CD49d⁻CD11a^lo, and CD49d⁺CD11a^hi CD4 T cells in the lung parenchyma after RSV infection. b) Frequency and c) number of virus-specific CD49d⁺CD11a^hi CD4 T cells in the lung parenchyma (IV⁻) and vasculature (IV⁺). d) Frequency of CD8, CD11a^lo, CD11a^hi, and M2₈₂ tetramer positive CD8 T cells in the lung parenchyma after RSV infection. e) Frequency and f) number of IV⁻ and IV⁺ CD11a^hi CD8 T cells in the lung. g) Frequency and h) number of IV⁻ and IV⁺ M2₈₂ tetramer⁺ CD8 T cells in the lung. Data are presented as mean ± SEM from two independent experiments that have been combined (n=3–4 mice per experiment). Statistical analysis was performed with Student’s t test, * p<0.05, ** p<0.01, *** p<0.001.

Figure 2.. CD4 and CD8 T cells numbers wane with time after RSV infection.

BALB/c mice were infected with RSV i.n., and lungs were harvested at days 8, 10, 15, 30, 79, and 149 p.i. Cells were analyzed by flow cytometry and gated on RSV-specific a) TRM CD4 T cells and b) TRM CD8 T cells in the lung parenchyma as shown. Representative staining panels are from day 30 p.i. c) Frequency and d) number of CD69⁺CD103⁻ CD49d⁺CD11a^hi CD4 T cells in the pulmonary parenchyma (IV⁻) and vasculature (IV⁺). e) Number of IV⁻ CD69⁺CD103⁻ CD49d⁺CD11a^hi TRM and total CD49d⁺CD11a^hi CD4 T cells in the lung. f) Frequency and g) number of IV⁻ and IV⁺ CD69⁺CD103⁺ M2₈₂ tetramer⁺ CD8 T cells in the lung. h) Number of CD69⁺CD103⁺ M2₈₂ tetramer⁺ TRM and total M2₈₂ tetramer⁺ CD8 T cells in the lung. Data are presented as mean ± SEM from two independent experiments that have been combined (n=3–4 mice per experiment). Statistical analysis was performed with Student’s t test, * p<0.05, ** p <0.01, *** p<0.001.

Figure 3.. Expression of phenotypic markers by TRM CD4 T cells.

a) Expression of CD62L, CXCR3, and CD122 by CD69⁺CD103⁻ CD49d⁺CD11a^hi CD4 T cells was compared to circulating (IV⁺) CD49d⁺CD11a^hi CD4 T cells. Representative histograms from day 30 p.i. are shown. b) Frequency and c) number of CD62L⁺ cells in the lung. d) Frequency and e) number of CXCR3⁺ cells in the lung. f) Frequency and g) number of CD122⁺ cells in the lung. Data are represented as mean ± SEM from two independent experiments that have been combined (n=3–4 mice per experiment). Statistical analysis was performed with Student’s t test, * p<0.05, ** p<0.01, *** p<0.001.

Figure 4.. Expression of phenotypic markers by CD8 T cells.

a) Expression of CD62L, CXCR3, and CD122 by CD69⁺CD103⁺ M2₈₂ tetramer⁺ CD8 T cells was compared to circulating (IV⁺) M2₈₂ tetramer⁺ CD8 T cells. Representative histograms from day 30 p.i. are shown. b) Frequency and c) number of CD62L⁺ cells in the lung. d) Frequency and e) number of CXCR3⁺ cells in the lung. f) Frequency and g) number of CD122⁺ cells in the lung. Data are presented as mean ± SEM from two independent experiments that have been combined (n=3–4 mice per experiment). Statistical analysis was performed with Student’s t test, * p<0.05, ** p<0.01, *** p<0.001.

Figure 5.. TRM CD4 and CD8 T cells protect against RSV infection.

a) BALB/c mice were infected with RSV i.n. or left uninfected and then challenged with IAV expressing either the CD4 epitope F_51–67 (IAV-F₅₁) or the CD8 epitope M2_82–90 (IAV-M2₈₂) one month later. Mice were treated daily with FTY-720 i.p. for 7 days beginning 3 days prior to recombinant IAV challenge. Four days after IAV challenge, lungs were harvested, and plaque assays were performed. Viral titers of b) IAV-PR8, c) IAV-F₅₁ and d) IAV-M2₈₂ in the lung are shown. The dotted line denotes the limit of detection (LOD) of the assay. Data are presented as mean ± SEM from two independent experiments that have been combined (n=5 mice per experiment). Statistical analysis was performed with Student’s t test, * p<0.05, ** p<0.01, *** p<0.001.