Lung influenza virus-specific memory CD4 T cell location and optimal cytokine production are dependent on interactions with lung antigen-presenting cells

Abstract

Influenza A virus (IAV) infection leads to the formation of mucosal memory CD4 T cells that can protect the host. An in-depth understanding of the signals that shape memory cell development is required for more effective vaccine design. We have examined the formation of memory CD4 T cells in the lung following IAV infection of mice, characterizing changes to the lung landscape and immune cell composition. IAV-specific CD4 T cells were found throughout the lung at both primary and memory time points. These cells were found near lung airways and in close contact with a range of immune cells including macrophages, dendritic cells, and B cells. Interactions between lung IAV-specific CD4 T cells and major histocompatibility complex (MHC)II+ cells during the primary immune response were important in shaping the subsequent memory pool. Treatment with an anti-MHCII blocking antibody increased the proportion of memory CD4 T cells found in lung airways but reduced interferon-γ expression by IAV-specific immunodominant memory CD4 T cells. The immunodominant CD4 T cells expressed higher levels of programmed death ligand 1 (PD1) than other IAV-specific CD4 T cells and PD1+ memory CD4 T cells were located further away from MHCII+ cells than their PD1-low counterparts. This distinction in location was lost in mice treated with anti-MHCII antibodies. These data suggest that sustained antigen presentation in the lung impacts the formation of memory CD4 T cells by regulating their cytokine production and location.

Graphical abstract

Fig. 1

Persistent changes to the lung landscape following IAV infection. C57BL/6 mice were infected intranasally with IAV and lungs taken from naive animals or at 9 or 30 days post-infection. (A) H&E staining on lung sections from naive and IAV-infected mice; all images taken at 10x magnification, scale bar 200 µm. Airways, immune cell clusters (white stars), and areas of damage (black dashed areas) are indicated. Blinded analysis of lung sections was performed using ImageScope software to determine (B) the number of areas of high cell density clusters; (C) average measurement of the cluster area (µm²); (D) average number of cells contained within a cluster; (E) average and (F) individual distance/proximity between clusters and the nearest airway (µm); (G) percentages of damaged lung areas; in (C–F), 1 day 9 and 2 day 30 samples were removed as no clusters were present. In (B–E, and G) each symbol represents a mouse and the line is the mean of the group (B–D) or median (E, G). In (F), each symbol represents a cluster, and the line shows the median of all clusters. Error bars are SEM (B–D) or show the interquartile range (E–G). Data are from two experiments with five or six mice/experiment taken at day 9, and two experiments with six or seven mice/experiment taken at day 30, each slide contained one section of each of the five lung lobes, and data shown are added from across all lobes to provide one value per slide. In (E, F, and G) data are not normally distributed (tested by Shapiro-Wilk) and were analyzed by Mann-Whitney U test, *p < 0.05; ***p < 0.001; ****p < 0.0001. Aw = airways; H&E = hematoxylin and eosin; IAV = influenza A virus; SEM = standard error of the mean.

Fig. 2

Immune cells are found throughout the lung, near airways, and in cell clusters at primary and memory time points following IAV infection. (A) TRACE mice enable identification of responding CD4 T cells via permanent expression of EYFP. Lungs from naive or IAV-infected TRACE mice were analyzed 10 or 40 days post-IAV infection. In (B–C), PCLS were cleared and stained with the indicated antibodies, scale bars are 70 μm (A) or 80 μm (B). In (D), frozen lungs were sectioned and stained with the indicated antibodies to identify IAV-specific CD4 T cells, and MHCII+ CD64, CD11c, or B220+ cells. Data are representative of one image set (B–C) or three (naive), four (B220, CD64), or 6–7 (CD11c) mice per time point from two experiments (D). White arrows indicate EYFP+CD4+ cells in close proximity to CD64, CD11c, or B220+ cells, scale bars are 100 μm. CD = cluster of differentiation; EYFP = enhanced yellow fluorescent protein; IAV = influenza A virus; MHC = major histocompatibility complex; PCLS = precision-cut lung slices; TRACE = T cell Reporter of Activation and Cell Enumeration.

Fig. 3

Lung MHCII+ populations alter across the IAV infection time course. C57BL/6 mice were infected with IAV, and lungs taken at 6 or 30 days post-infection or from naive animals. Following tissue digest, lungs were analyzed by flow cytometry as indicated in Supplementary Fig. 2. In (A), each symbol represents a mouse and the line shows the mean of the group. Data are from two independent experiments per time point with a total of 6–12 per group: naive: three mice/experiment; day 6: six mice/experiment; day 30: three and four mice/experiment. All data were normally distributed (tested by Shapiro-Wilk) and differences between time points assessed by ANOVA followed by a Tukey’s test. In (B), cells were gated on MHCII+ cells that were CD64, CD11c, or B220+, and the proportion of the indicated cell populations determined. Not all data were normally distributed and differences between infection time points and naive samples tested by ANOVA followed by a Dunn’s multiple comparison test. In (A), the horizontal line is the mean, and error bars are SEM. In (B), error bars are SD, and in (A–B) *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. ANOVA = analysis of variance; CD = cluster of differentiation; IAV = influenza A virus; MHC = major histocompatibility complex; SD = standard deviation; SEM = standard error of the mean.

Fig. 4

IAV-specific CD4 T cells are maintained in lung airways at memory time points. TRACE mice were infected with IAV, and lungs taken 10 or 40 days post-infection. Lung sections were stained with the indicated antibodies to identify MHCII+ cells and IAV-specific CD4 T cells. In (A and H), images are representative of 6–7 mice (day 10: three mice from two experiments; day 40: three or four mice/experiment). In (A), green and white arrows show EYFP+CD4+ cells near or further away from airways, respectively. In (H), arrows indicate CD11c+MHCII+ cells next to EYFP+CD4+ cells and boxes indicate cells more than  20 μm away from CD4+EYFP+ cells. In A and H scale bars are  10 μm. In (B–G) and (I–N), each symbol represents a mouse from the same experiments and the line shows the mean of the group, error bars are SEM (C–G and I, K–N) or median and interquartile range, (B, J). In (B and J), data are not normally distributed (tested by Shapiro-Wilk), and difference tested between time points tested by a Mann-Whitney U test, data in (I, K, and L) were normally distributed and differences tested by t tests. In all: *p < 0.05; **p < 0.01; ***p < 0.001. CD = cluster of differentiation; EYFP = enhanced yellow fluorescent protein; IAV = influenza A virus; MHC = major histocompatibility complex; SEM = standard error of the mean; TRACE = T cell Reporter of Activation and Cell Enumeration

Fig. 5

Immunodominant but not polyclonal IAV-specific memory CD4 T cells have a reduced IFNγ response following anti-MHCII treatment. C57BL/6 (A, C, E) or TRACE (B, D, F) mice were infected with IAV on day 0 and given control IgG or anti-MHCII intranasally on days 6 and 12. On day 40, IAV-specific CD4 T cells were detected ex vivo using IA^b/NP_311-325 tetramers (C) or following restimulation with NP_311-325 peptide (E) or co-culture with IAV-Antigen treated bmDCs (D, F). In (G–L), TRACE mice were infected with IAV and CD4+EYFP+ cells and MHCII tetramer+ examined following 10 or 40 days. In (A–B), cells are gated on CD4+ cells as shown in Supplementary Fig. 7 and the numbers are the percent of cells in the indicated gate, in (K–L) cells are gated shown in Supplementary Fig. 8. In (A, C, E) data are from two experiments with 3/4 mice/experiment and colored symbols indicate the two experiments; in (B, D and F) data are from five experiments with the following numbers of mice/group: IgG, 3, 2, 5, 5, 5; anti-MHCII: 4, 4, 4, 4, 5. In (G–L), mice are from two experiments with: day 10: four or seven mice/group; day 40: four or eight mice/group. Symbols show the mean of the group and error bars are SEM. In (E), data are not normally distributed (tested by Shapiro-Wilk), and difference tested by Mann-Whitney U test, the Y axis is set at the level of detection. In (G), data are not normally distributed and tested via a Mann-Whitney U test. In (H–L), data are normally distributed, and differences tested using a t test in (H–J) and paired t tests between cell types in (K–L). In all graphs, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. bmDC = bone marrow-derived dendritic cell; CD = cluster of differentiation; EYFP = enhanced yellow fluorescent protein; IAV = influenza A virus; IFN = interferon; Ig = immunoglobulin; MHC = major histocompatibility complex; NP = nucleoprotein; SEM = standard error of the mean; TRACE = T cell Reporter of Activation and Cell Enumeration.

Fig. 6

Anti-MHCII treatment alters the location of IAV-specific CD4 T cells. TRACE mice were infected with IAV on day 0 and on days 6 and 12 given control IgG or anti-MHCII intranasally. On day 40, lungs were frozen and tissue sections stained with the indicated antibodies. Data are combined from two independent experiments: IgG: 12 mice:6/experiment; anti-MHCII: eight mice 4/experiment. In (A), white and green arrows indicate EYFP+CD4+ cells and EYFP+CD4+PD1+ cells respectively; scale bar is 100 μm. In (B–G), each symbol represents a mouse, the line shows the mean of the group, and error bars are SEM. For each mouse 2–5 sections were analyzed. In (B, D, E and F) data are not normally distributed (tested by Shapiro-Wilk) and differences tested by Mann-Whitney U test. In (C, G and H), data are normally distributed and differences tested by t test (C, G) or Paired t test (H). In (H), four anti-MHCII mice are removed as no EYFP+CD4+ PD1+ cells were found in the analyzed sections. CD = cluster of differentiation; EYFP = enhanced yellow fluorescent protein; IAV = influenza A virus; Ig = immunoglobulin; MHC = major histocompatibility complex; PD1 = programmed death ligand 1; SEM = standard error of the mean; TRACE = T cell Reporter of Activation and Cell Enumeration