Immunity to Respiratory Infection Is Reinforced Through Early Proliferation of Lymphoid TRM Cells and Prompt Arrival of Effector CD8 T Cells in the Lungs

Abstract

Cross-protection between serologically distinct strains of influenza A virus (IAV) is mediated by memory CD8 T cells that recognize epitopes from conserved viral proteins. Early viral control begins with activation of tissue-resident memory CD8 T cells (T_RM) cells at the site of viral replication. These CD8 T cells do not act in isolation, as protection against disseminated infection is reinforced by multiple waves of effector cells (T_EFF) that enter the lungs with different kinetics. To define how a protective CTL response evolves, we compared the functional properties of antiviral CD8 T cells in the respiratory tract and local lymphoid tissues. When analyzed 30 dpi, large numbers of antiviral CD8 T cells in the lungs and mediastinal lymph nodes (MLNs) expressed canonical markers of T_RM cells (CD69 and/or CD103). The check point inhibitor PD-1 was also highly expressed on NP-specific CD8 T cells in the lungs, while the ratios of CD8 T cells expressing CD69 and CD103 varied according to antigen specificity. We next used in vitro experiments to identify conditions that induce a canonical T_RM phenotype and found that that naïve and newly activated CD8 T cells maintain CD103 expression during culture with transforming growth factor-beta (TGFβ), while central memory CD8 T cells (T_CM) do not express CD103 under similar conditions. In vivo experiments showed that the distribution of antiviral CTLs in the MLN changed when immune mice were treated with reagents that block interactions with PD-L1. Importantly, the lymphoid T_RM cells were poised for early proliferation upon reinfection with a different strain of IAV and defenses in the lungs were augmented by a transient increase in numbers of T_EFF cells at the site of infection. As the interval between infections increased, lymphoid T_RM cells were replaced with T_CM cells which proliferated with delayed kinetics and contributed to an exaggerated inflammatory response in the lungs.

Keywords: cytotoxic T cell; immune regulation; immunological “memory”; respiratory infection; viral immunity.

Figure 1

The phenotypes of T_RM cells vary according to antigen-specificity. Antiviral CTLs were recovered from the lungs and MLN 35 dpi with WSN-OVA_I. Antigen experienced CTLs were identified using high CD11a and CD44 expression. (A) Contour plots show antigen-experienced CTLs analyzed with MHCI tetramers. The tetramer⁺ CTLs were analyzed for CD103, CD69, and PD-1 expression. Percentages of cells in each quadrant are means ± SD (n = 5/group). Gates were set using non-CD8 T cells. (B–E) Bar graphs show total numbers of tetramer⁺ CTLs, plotted using means ± SD (n = 5/group). Shading shows (B,D) ratios of cells expressing CD103 and/or PD-1, (C,E) ratios of cells expressing CD103 and/or CD69. (F) The numbers of CTLs in each quadrant were compared for the NP and PA epitopes. P-values were calculated using Student's t-tests.

Figure 2

T_CM do not upregulate CD103 during culture with TGFβ. (A) C57BL/6 mice were infected systemically (i.v.) with LM-OVA. CD62L-positive CD8 T cells in the spleen were analyzed 32 dpi. The contour plots indicate gates that were used for analyses. Histograms (top row) show CD103 expression on naïve CD8 T cells, while T_CM cells do not express CD103 (gray shading). Lower panels show CD103 expression naïve OTI cells from the spleens of uninfected mice (bottom row, dashed line). Naïve OTI cells do not express CD103 after ablation of the TGFβ receptor (bottom row, gray shading). (B) Naïve OTI cells were transferred to B6 mice 48 h before infection with LM-OVA. After 3 months, T_CM cells were sorted from the spleens using CD45.1 expression. Purified CD8 T cells (naïve and T_CM) were stimulated with plate-bound anti-CD3/CD28 and rIL-2 (20 U/ml) for 48 h. Live cells were transferred to new wells (no antigen) and cultured for 48 h with rIL-2 plus/minus TGFβ (10 ng/ml). (C) Naïve OTI cells were cultured with plate-bound anti-CD3/CD28 and rIL-2. In addition, some wells were supplemented with SB-431542 (10 μM) to avoid stimulation with serum-derived TGFβ. After 48 h, activated OTI cells were analyzed for CD103/CD69 expression (top panels). Additional cells were cultured for an additional 48 h with rIL-2 and no antigen stimulation (lower panels). (D) Naïve OTI cells were labeled with CFSE-dye and transferred to mice that were previously (30 d) infected with WSN-OVA_I. The MLNs were recovered 5 days after transfer and lymphocytes were cultured for 72 h with rIL-2 plus/minus TGFβ. Three experiments gave similar results.

Figure 3

The distribution of lymphoid T_RM cells changes during PD-1L blockade. Naïve OTI cells were transferred to B6 mice 48 h before infection with WSN-OVA_I. At 30 and 32 dpi, antibodies that block interactions with PD-L1 (or isotype control) were administered by IV injection (250 μg). After 35 days sections of fixed MLN were stained with antibodies that are specific for CD45.1 (green); CD31 (yellow); CD11c (blue); B220 (red), and LYVE-1 (magenta). Z-stacks were recorded at 10X and 20X normal magnification. (A–D) MLNs analyzed after treatment with control antibodies. (E,F) MLNs analyzed after treatment with antibodies that block interactions with PD-L1. The inset boxes (dashed lines) mark the locations of enlarged images shown in (A-2,C-2). (D,G) The Imaris software colocalization function was used to detect contacts (white) between OTI cells (green) and LYVE⁺ vessels (Magenta). (H) Numbers of OTI cells per 10 micron Z-stack (*P = 0.0107).

Figure 4

Infection-induced pathology is not substantially altered by PD-L1 blockade. C57BL/6 mice were infected with WSN-OVA_I and treated twice with (A–C) antibodies to PD-L1 or (D–F) isotype control. Sections of fixed lung tissue were stained 35 dpi using hematoxylin and eosin. Images were recorded at 20X normal magnification. Arrows indicate Bronchioles (black) and blood vessels (white).

Figure 5

Early proliferation by lymphoid T_RM cells corresponds with increased numbers of anti-viral CTLs in the lungs. C57Bl/6 mice were infected with WSN-OVA_I and challenged with X31-OVA. Secondary infections were administered between 30 and 35 dpi (ER), or 120–160 dpi (LR). On the days indicated, each mouse received a single dose of BrdU (given by IP injection) and antiviral CTLs were analyzed 4 h later. (A) On day 3 post recall (D3pr), the MLNs were analyzed for antigen-experienced CTLs using high CD11a and CD44 expression. The contour plots show frequencies of tetramer⁺ cells. Histograms show BrdU incorporation within the tetramer gates. Percentages are means ± SD (n = 5/group). (B–E) The bar graphs show total numbers of Tetramer⁺ CTLs, including BrdU⁺ cells (hatched shading). Bars are means ± SD (n = 5/group). The numbers of cells that incorporated BrdU after ER and LR were compared using unpaired T-tests. NS, P > 0.05; *P < 0.05; **P < 0.01; ****P < 0.0001.

Figure 6

Sections of fixed lung tissue were stained with H&E 4 dpi. Representative images from 5 mice are shown. (A,B) Lungs after early recall (C,D) Lungs after late recall. Arrows indicate Bronchioles (black) and blood vessels (white).