Innate-like Gene Expression of Lung-Resident Memory CD8+ T Cells during Experimental Human Influenza: A Clinical Study

Abstract

Rationale: Suboptimal vaccine immunogenicity and antigenic mismatch, compounded by poor uptake, means that influenza remains a major global disease. T cells recognizing peptides derived from conserved viral proteins could enhance vaccine-induced cross-strain protection. Objectives: To investigate the kinetics, phenotypes, and function of influenza virus-specific CD8⁺ resident memory T (Trm) cells in the lower airway and infer the molecular pathways associated with their response to infection in vivo. Methods: Healthy volunteers, aged 18-55, were inoculated intranasally with influenza A/California/4/09(H1N1). Blood, upper airway, and (in a subgroup) lower airway samples were obtained throughout infection. Symptoms were assessed by using self-reported diaries, and the nasal viral load was assessed by using quantitative PCR. T-cell responses were analyzed by using a three-color FluoroSpot assay, flow cytometry with MHC I-peptide tetramers, and RNA sequencing, with candidate markers being confirmed by using the immunohistochemistry results for endobronchial biopsy specimens. Measurements and Main Results: After challenge, 57% of participants became infected. Preexisting influenza-specific CD8⁺ T cells in blood correlated strongly with a reduced viral load, which peaked at Day 3. Influenza-specific CD8⁺ T cells in BAL fluid were highly enriched and predominantly expressed the Trm markers CD69 and CD103. Comparison between preinfection CD8⁺ T cells in BAL fluid and blood by using RNA sequencing revealed 3,928 differentially expressed genes, including all major Trm-cell markers. However, gene set enrichment analysis of BAL-fluid CD8⁺ T cells showed primarily innate cell-related pathways and, during infection, included upregulation of innate chemokines (Cxcl1, Cxcl10, and Cxcl16) that were also expressed by CD8⁺ cells in bronchial tissues. Conclusions: CD8⁺ Trm cells in the human lung display innate-like gene and protein expression that demonstrates blurred divisions between innate and adaptive immunity. Clinical study registered with www.clinicaltrials.gov (NCT02755948).

Keywords: CD8+ T cell; controlled human infection; influenza; resident memory.

Figure 1.

Experimental human infection with influenza A/California/4/09(H1N1) virus induces a diversity of clinical outcomes. (A) Forty-two healthy adult volunteers were intranasally inoculated with influenza A/California/4/09(H1N1) and sampled periodically after inoculation. A Consolidated Standards of Reporting Trials diagram is shown. (B) A schematic showing the schedule of sampling. (C) The viral load (VL) of 42 individuals as determined by using M gene RT-PCR findings from nasal lavage. Individual data points and the mean (red) VL are shown. (D) Self-reported total daily symptom scores for infected (red, n = 23) and uninfected (blue, n = 18) individuals. Data are shown as the mean ± SEM. (E) Upper respiratory tract (blue), lower respiratory tract (red), and systemic (green) symptom scores in infected participants. Data are shown as the mean ± SEM. HAI = hemagglutination inhibition.

Figure 2.

Circulating memory CD8⁺ T cells correlate strongly with reduced disease severity in influenza. Influenza-specific CD8⁺ T cells were quantified in blood by using a FluoroSpot assay after stimulation with conserved MHC class I epitopes. (A) When cell numbers allowed, IFN-γ–secreting influenza-specific T cells were measured in peripheral blood mononuclear cells (PBMCs) obtained before inoculation from participants who subsequently became infected (n = 16) or remained uninfected (n = 23). (B) Spearman correlation between preinoculation influenza-specific T cells and the viral load in individuals who subsequently had PCR-positive results (n = 16). (C) Frequency of IFN-γ–secreting T cells stimulated with influenza peptides derived from internal influenza proteins before infection and at the Day 7 and Day 28 time points. T-cell responses to conserved peptide derived from M1 and M2 (matrix), NA (neuraminidase), NP (nucleoprotein), NS2 (nonstructural protein 2), and the RNA polymerase complex (PA, PB1, and PB2) are shown as the median and interquartile range. (D) FluoroSpot images from one representative donor at Day 7 after infection are shown. (E) The proportion of T cells secreting IFN-γ, TNFα, IL-2, or combinations of multiple cytokines in peptide-stimulated PBMCs is shown. Pie chart sections represent the proportion of cells with one, two, or three functions. Individual cytokines and their combinations are denoted by arcs. AUC = area under the curve; ID = identifier; rs = Spearman’s rank correlation coefficient; SFC = spot-forming cell; TNF = tumor necrosis factor.

Figure 3.

Influenza-specific CD8⁺ T cells are preferentially expanded in the human airway during experimental infection. Healthy adult volunteers were experimentally infected with influenza A virus and serially sampled for blood and BAL fluid. (A) The macroscopic appearances of the lower airway were assessed by using bronchoscopy before infection and at Day 7 and Day 28. One representative individual is shown. (B) CD8⁺ T cells in the blood and BAL fluid were costained with anti–Ki-67 and anti-CD38 for analysis by flow cytometry. P values from Wilcoxon matched-pair tests for pair-wise comparisons and from Mann-Whitney tests for unpaired comparisons are shown. (C) Flow cytometry plots are shown for one representative individual. (D) CD8⁺ T cells labeled with the A*01:01-CTELKLSDY tetramer or the A*02:01-GILGFVFTL tetramer were quantified in the blood. Individual data and the medians (red) are shown. P values from Friedman tests with Dunn’s multiple comparison tests are shown. (E) CD8⁺ T cells in blood were labeled with the A*02:01-GILGFVFTL tetramer. Flow cytometry plots are shown for one representative individual. (F) CD8⁺ T cells labeled with the A*01:01-CTELKLSDY tetramer or the A*02:01-GILGFVFTL tetramer were quantified in BAL fluid. Individual data and the medians (red) are shown. (G) CD8⁺ T cells in BAL were labeled with the A*02:01-GILGFVFTL tetramer. Flow cytometry plots are shown for one representative individual. P values from Wilcoxon matched-pair tests are shown.*P < 0.05 and **P < 0.01.

Figure 4.

Influenza infection induces divergent phenotypic changes in the blood and BAL fluid. Whole-blood/peripheral blood mononuclear cells (n = 8) and BAL fluid (n = 4) from influenza-infected individuals were stained with a tetramer, anti-CD3, and CD4 and were costained with (A) Ki-67 and CD38; (B) CD69 and CD103; (C) CD45RA and CCR7 (the median and interquartile range are shown); (D) CD27 and CD28; (E) granzyme B and perforin; and (F) CCR5 and CXCR3 for analysis by flow cytometry. Medians (red) and individual data are shown. P values from Wilcoxon matched-pair tests are shown for pair-wise comparisons of BAL-fluid cells, and P values from Friedman tests with Dunn’s multiple comparison tests are shown for multiple comparisons of blood cells. *P < 0.05 and **P < 0.01. eff = effector; Tcm = central memory T; Tem = eff memory T; Temra = terminally differentiated Tem.

Figure 4.

Influenza infection induces divergent phenotypic changes in the blood and BAL fluid. Whole-blood/peripheral blood mononuclear cells (n = 8) and BAL fluid (n = 4) from influenza-infected individuals were stained with a tetramer, anti-CD3, and CD4 and were costained with (A) Ki-67 and CD38; (B) CD69 and CD103; (C) CD45RA and CCR7 (the median and interquartile range are shown); (D) CD27 and CD28; (E) granzyme B and perforin; and (F) CCR5 and CXCR3 for analysis by flow cytometry. Medians (red) and individual data are shown. P values from Wilcoxon matched-pair tests are shown for pair-wise comparisons of BAL-fluid cells, and P values from Friedman tests with Dunn’s multiple comparison tests are shown for multiple comparisons of blood cells. *P < 0.05 and **P < 0.01. eff = effector; Tcm = central memory T; Tem = eff memory T; Temra = terminally differentiated Tem.

Figure 5.

Resident memory CD8⁺ T cells in the lower airway responding to influenza infection display innate-like signatures. Live CD3⁺CD4⁻CD14⁻CD19⁻CD8⁺ T cells from the blood and BAL fluid of 12 individuals inoculated with influenza A virus at Day 7 before infection and Day 28 after infection were sorted by using a fluorescence-activated cell sorter. (A) Diagrammatic representation of differential gene expression (DEG) comparing compartments and time points. (B) Volcano plot showing DEGs in BAL fluid versus blood CD8⁺ T cells before infection. Red indicates genes upregulated in BAL fluid, and blue indicates genes downregulated in BAL fluid. (C) Preinfection expression of the resident memory T-cell markers CD69, CD103/ITGAE (integrin subunit alpha E), FABP4 (fatty acid binding protein 4), and S1PR1 were compared between BAL fluid and blood CD8⁺ T cells. (D) Functionally annotated gene sets significantly enriched (log₁₀-adjusted P value) with DEGs comparing BAL fluid and blood CD8⁺ T cells were identified by using the tmod R package. The red line denotes the cutoff for significance (false discovery rate < 0.05). (E) Volcano plot demonstrating significant DEGs in BAL-fluid CD8⁺ T cells from five infected individuals at Day 7 after infection compared with before infection. Significantly upregulated DEGs are highlighted in red, significantly downregulated DEGS are highlighted in blue, and significant DEGs are labeled with their gene symbols. (F) Functionally annotated gene sets significantly enriched with DEGs comparing CD8⁺ T cells at each time point with the corresponding preinfection time point in blood and BAL fluid were identified by using the tmod R package. All statistically significant DEGs without Bonferroni-Hochberg correction are included. The darker the red coloration, the more significant the pathway enrichment. DC = dendritic cell; NK = natural killer; Th1 = T-helper cell type 1; TLR = Toll-like receptor.

Figure 5.

Resident memory CD8⁺ T cells in the lower airway responding to influenza infection display innate-like signatures. Live CD3⁺CD4⁻CD14⁻CD19⁻CD8⁺ T cells from the blood and BAL fluid of 12 individuals inoculated with influenza A virus at Day 7 before infection and Day 28 after infection were sorted by using a fluorescence-activated cell sorter. (A) Diagrammatic representation of differential gene expression (DEG) comparing compartments and time points. (B) Volcano plot showing DEGs in BAL fluid versus blood CD8⁺ T cells before infection. Red indicates genes upregulated in BAL fluid, and blue indicates genes downregulated in BAL fluid. (C) Preinfection expression of the resident memory T-cell markers CD69, CD103/ITGAE (integrin subunit alpha E), FABP4 (fatty acid binding protein 4), and S1PR1 were compared between BAL fluid and blood CD8⁺ T cells. (D) Functionally annotated gene sets significantly enriched (log₁₀-adjusted P value) with DEGs comparing BAL fluid and blood CD8⁺ T cells were identified by using the tmod R package. The red line denotes the cutoff for significance (false discovery rate < 0.05). (E) Volcano plot demonstrating significant DEGs in BAL-fluid CD8⁺ T cells from five infected individuals at Day 7 after infection compared with before infection. Significantly upregulated DEGs are highlighted in red, significantly downregulated DEGS are highlighted in blue, and significant DEGs are labeled with their gene symbols. (F) Functionally annotated gene sets significantly enriched with DEGs comparing CD8⁺ T cells at each time point with the corresponding preinfection time point in blood and BAL fluid were identified by using the tmod R package. All statistically significant DEGs without Bonferroni-Hochberg correction are included. The darker the red coloration, the more significant the pathway enrichment. DC = dendritic cell; NK = natural killer; Th1 = T-helper cell type 1; TLR = Toll-like receptor.

Figure 6.

CD8⁺ cells in the bronchial mucosa during influenza coexpress resident memory T and innate-like markers. Endobronchial biopsy specimens were obtained through bronchoscopy of healthy volunteers (n = 9) inoculated with influenza A virus. (A) Sections obtained from bronchial biopsy specimens of participants with PCR-positive results were stained with anti-CD8 and DAPI. Cells expressing CD8 (green) in the epithelial and subepithelial layers at baseline (left), Day 7 (with a magnified image at Day 7), and Day 28 (right) from one representative individual are shown. (B) Cells expressing CD8 were quantified in the mucosal epithelium and subepithelium in participants with PCR-positive (n = 5) and PCR-negative (n = 4) results before infection and at Day 7 and Day 28 after inoculation. (C) Sections were costained with anti-CD8 (fluorescein isothiocyanate, green), anti-CD103 (cyanine 5 [Cy5], magenta), anti-CD69 (Cy3, red), and DAPI (blue) at Day 7 after infection. Representative images from one individual are shown. (D) Colocalization (yellow) of resident memory T markers (CD69 and CD103) with CD8 observed in the bronchial mucosa is represented in the magnified image. (E) The frequency of CD8⁺ cells expressing markers of tissue residence (CD69, CD103, and FABP4 [fatty acid binding protein 4]), stimulatory molecules (BAFF [B-cell activation factor] and CD14), IFN-stimulated genes (viperin), and chemokines (CCL20, CXCL1, CXCL10, and CXCL16) was quantified in the mucosal epithelium. (F) Colocalization correlation coefficients of each marker with CD8 in the mucosal epithelium were calculated. Biopsy sections (Day 7) were costained with anti-CD8 (fluorescein isothiocyanate, green) and (G) anti-CD14 (Cy5, magenta), (H) anti-CXCL1 (Cy5, magenta), (I) anti-CXCL10 (Cy5, magenta), and (J) anti-CXCL16 (Cy5, magenta) with colocalization shown in yellow. One representative individual is shown. Data are representative of three independent experiments performed on biopsy specimens obtained from five infected individuals (i.e., five biological replicates). Yellow arrows indicate cells with coexpression of CD8⁺ cells with other markers. Scale bars, 20 μm.