Human T cells efficiently control RSV infection

Abstract

Respiratory syncytial virus (RSV) infection causes significant morbidity and mortality in infants, immunocompromised individuals, and older individuals. There is an urgent need for effective antivirals and vaccines for high-risk individuals. We used 2 complementary in vivo models to analyze RSV-associated human lung pathology and human immune correlates of protection. RSV infection resulted in widespread human lung epithelial damage, a proinflammatory innate immune response, and elicited a natural adaptive human immune response that conferred protective immunity. We demonstrated a key role for human T cells in controlling RSV infection. Specifically, primed human CD8+ T cells or CD4+ T cells effectively and independently control RSV replication in human lung tissue in the absence of an RSV-specific antibody response. These preclinical data support the development of RSV vaccines, which also elicit effective T cell responses to improve RSV vaccine efficacy.

Keywords: Adaptive immunity; Immunology; Mouse models; T cells; Virology.

Figure 1. Sustained replication of RSV in human lung implants.

(A and B) H&E staining of (A) a naive LoM human lung implant (n = 5 implants analyzed) and (B) a human lung implant from an RSV-infected LoM (n = 12 implants analyzed). Scale bars: 200 μm. Arrows note airways. (C) RSV-RNA expression in the human lung implants of control (2 hours after RSV exposure) and RSV-infected LoM at 4 and 11 days after exposure (n = 5 implants/time point). Crossing point (Cp) indicates the cycle number at which the fluorescence signal of the sample exceeds a background fluorescence value. (D) RSV titers (log₁₀TCID₅₀/mL) in human lung implants of control LoM (2 hours after RSV exposure) and RSV-infected LoM at 4 days and 11 days after RSV exposure (n = 5 implants/time point). Dashed line indicates the assay limit of detection. (E) Number of GFP⁺ cells as determined by flow cytometry in the human lung implants of naive LoM (n = 4 implants) and RSV A2-GFP–infected LoM 4, 7, 11, 14, and 21 days after exposure (n = 4 implants/time point). (F) RSV replication monitored longitudinally in the human lung implants of RSV-Luc infected LoM (n = 6 implants) as measured by bioluminescence (radiance [p sec^–1 cm^–2 sr^–1] represented as total flux) signal. The median (horizontal line), upper and lower quartiles (box ends), and minimum to maximum values (whiskers) are shown. Background luminescence (day 0) is denoted by the dashed line. (G) IHC staining for RSV antigen in the human lung implants of naive LoM (n = 4 implants analyzed) and RSV-infected LoM 4, 7, and 21 days after exposure (positive cells are brown, n = 4 implants analyzed/time point). Scale bars: 200 μm. (C–E) Data are shown as the mean ± SEM; (C and D) a statistical analysis was performed using a 2-tailed Kruskal-Wallis test. P values were adjusted for multiple testing using the Benjamini, Krieger, and Yekutieli FDR method.

Figure 2. RSV infection induces an innate proinflammatory immune response resulting in neutrophil infiltration.

(A) Human IL-1β (left panel), IL-6 (middle panel), and IL-8 (right panel) expression in the human lung implants of control LoM (2 hours after RSV exposure) and RSV-infected LoM at 4 days and 11 days after exposure (n = 5 implants/time point). Crossing point (Cp) indicates the cycle number at which the fluorescence signal of the sample exceeds a background fluorescence value. (B) Flow cytometry gating strategy used to detect neutrophils. (C and D) Levels of mouse neutrophil (%Ly6G⁺ of mouse CD45⁺ cells) in the human lung implants of 2 different human donor cohorts of naive LoM (n = 4 and 6 implants) and RSV-infected LoM 2 days, 4 days, and 7 days after exposure (n = 4 implants/time point/cohort). (E and F) H&E staining of an RSV-infected LoM human lung implant depicting (E) neutrophil accumulation in airway lumen (arrows; scale bars: 50 μm) and (F) neutrophil transmigration (arrows; scale bars: 20 μm). (A, C, and D) Data are shown as mean ± SEM. Statistical significance was determined with a 2-tailed Kruskal-Wallis test. P values were adjusted for multiple testing using the Benjamini, Krieger, and Yekutieli FDR method.

Figure 3. RSV infection is efficiently controlled by the human immune system in BLT-L mice and elicits protective immunity from a second RSV challenge.

(A) Bioluminescence (radiance [p sec^–1 cm^–2 sr^–1] represented as total flux) in human lung implants of RSV A2-Luc exposed LoM (blue bars, n = 6 implants) and BLT-L mice (red bars, n = 8 implants). Dashed line indicates background (preexposure) luminescence. (B) RSV antigen (brown) in LoM (n = 4 implants analyzed/time point) and BLT-L mouse (n = 4 implants analyzed/time point) lung implants on days 4, 11, and 21 after exposure. Scale bars: 100 μm. (C) BLT-L mouse lung implants were inoculated with RSV A2-GFP or vehicle (first exposure) and then RSV A2-Luc 21 days later (second exposure). (D) Bioluminescence signal in lung implants of RSV A2-Luc–exposed BLT-L mice that were exposed first to RSV A2-GFP (n = 6 implants; yellow bars) or vehicle (n = 12 implants; gray bars). The median (horizontal line), upper and lower quartiles (box ends), and minimum to maximum values (whiskers) are shown. Dashed line indicates background luminescence (day 0). (E and F) RSV-specific human (E) IgM and (F) IgG plasma levels in naive BLT-L mice (n = 4) and BLT-L mice exposed once (IgM, n = 9; IgG, n = 10) or twice (IgM, n = 11; IgG, n = 9) to RSV. Dashed line indicates seropositivity threshold. (G) RSV neutralization activity of plasma from naive BLT mice (n = 4) and BLT mice exposed once (n = 5) or twice (n = 8) to RSV. Shown is the number of RSV GFP⁺ cells 72 hours after infection representative of 6 replicates. (H) RSV NP_137-145 pentamer-reactive CD8⁺ T cells in the lung implant of a BLT-L mouse exposed twice to RSV. (E–G) Data are shown as mean ± SEM. Two-tailed (A and D) Mann-Whitney U, and (G) Kruskal-Wallis tests were used to determine statistical significance. P values were adjusted for multiple testing using the Benjamini, Krieger, and Yekutieli FDR method.

Figure 4. Human CD8⁺ T cell depletion impairs but does not eliminate the control of RSV replication in vivo.

(A) Experimental diagram to evaluate the effect of CD8⁺ T cell depletion on RSV replication. LoM and BLT-L mice administered placebo or CD8-depleting antibody (3 mg/kg, i.v.) were exposed to RSV-Luc and, 21 days later, rechallenged with RSV-Luc. CD8⁺ T cell depletion was confirmed in blood and tissues (Supplemental Figure 3). (B and C) RSV replication was monitored in the lung implants of LoM (blue boxes, n = 10 implants), placebo-treated BLT-L mice (red boxes, n = 12 implants), and CD8-depleted BLT-L mice (gray boxes, n = 10 implants) following the (B) first and (C) second RSV exposure by measuring the bioluminescence signal (radiance [p sec^–1 cm^–2 sr^–1] represented as total flux). The median (horizontal line), upper and lower quartiles (box ends), and minimum to maximum values (whiskers) are shown. (D) RSV antigen (brown) in the lung implants of LoM, placebo-treated BLT-L mice, and CD8-depleted BLT-L mice 14 days after the second exposure (n = 6 implants analyzed). Scale bars: 100 μm. (E) H&E staining of the lung implants of naive (n = 5 implants analyzed), placebo-treated (n = 4 implants analyzed), or CD8-depleted (n = 8 implants analyzed) BLT-L mice. Scale bars: 100 μm. Arrow indicates fibrin in airway lumen. (F) CD4⁺ and CD8⁺ T cell numbers (left) and activated (CD38⁺HLA-DR⁺) CD4⁺ and CD8⁺ T cell levels (right) in the lung implants of naive BLT-L mice (green; T cell levels, n = 8 implants; activation levels, n = 7 implants) and in placebo-treated (red, n = 10 implants) and CD8-depleted (gray, n = 10 implants) BLT-L mice 14 days after the second RSV exposure. Data are shown as mean ± SEM. Statistical significance was determined with a 2-tailed (B, C, and E) Kruskal-Wallis or (E) Mann-Whitney U test. P values were adjusted for multiple testing using the Benjamini, Krieger, and Yekutieli FDR method.

Figure 5. Adoptively transfered primed autologous human CD4⁺ or CD8⁺ T cells control RSV replication in vivo.

(A) Experimental diagram to evaluate RSV replication in LoM following adoptive transfer of primed autologous CD4⁺ and CD8⁺ T cells. LoM were challenged with RSV-Luc and then, 14 days later, transplanted with autologous CD4⁺ T cells (15 × 10⁶), CD8⁺ T cells (15 × 10⁶), or both CD4⁺ (7.5 × 10⁶) and CD8⁺ (7.5 × 10⁶) T cells isolated from RSV-infected human donor-matched BLT-L mice. RSV-infected LoM that did not receive cells served as a control for RSV replication. (B) Longitudinal analysis of RSV replication in LoM that received no cells (green boxes, n = 8 implants), CD8⁺ T cells (red boxes, n = 8 implants), CD4⁺ T cells (blue boxes, n = 8 implants), or both CD4⁺ and CD8⁺ T cells (purple boxes, n = 8 implants) as measured by bioluminescence signal (radiance [p sec^–1 cm^–2 sr^–1] represented as total flux). Dashed line indicates threshold for bioluminescence detection. The median (horizontal line), upper and lower quartiles (box ends), and minimum to maximum values (whiskers) are shown. Statistical significance was compared with a 2-tailed Kruskal-Wallis test. P values were adjusted for multiple testing using the Benjamini, Krieger, and Yekutieli FDR method. (C) RSV antigen staining (brown) in the human lung implants of RSV-infected LoM 10 weeks after adoptive transfer of no cells, CD8⁺ T cells, CD4⁺ T cells, or both CD4⁺ and CD8⁺ T cells (n = 4 implants analyzed/group). Scale bars: 100 μm.