Visualization of granzyme B-expressing CD8 T cells during primary and secondary immune responses to Listeria monocytogenes

Abstract

CD8 T cells contribute to long-term protection against Listeria monocytogenes infection by differentiating into memory T cells. These rapidly respond to antigen or inflammation upon secondary infection. In this study we used CD8 T cells from OT1 mice and CD4 T cells from OT2 mice expressing a fluorescent chimeric granzyme (GZMB-Tom) protein to monitor the primary response to infection with ovalbumin-expressing L. monocytogenes (Lm-OVA). We show that, unlike poorly responding CD4 T cells, CD8 T cells readily proliferated and expressed high levels of GZMB-Tom as early as 2 days after infection. FACS analysis showed GZMB-Tom expression in undivided CD8 T cells, with its level increasing over one to four divisions. OT1 T cells were visualized in the T-cell zone by confocal microscopy. This showed GZMB-Tom-containing granules oriented towards MHCII-positive cells. Twenty hours later, most OT1 T cells had divided but their level of GZMB-Tom expression was reduced. Recently divided OT1 cells failed to express GZMB-Tom. Fourteen hours after secondary infection, GZMB-Tom was re-expressed in memory OT1 T cells responding either to Lm-OVA or L. monocytogenes. Differences in the activation phenotype and in the splenic distribution of OT1 T cells were observed, depending on the challenge. Notably, OTI T cells with polarized granules were only observed after challenge with cognate antigen. This work showed that the GZMB-Tom knock-in mice in which GZMB-Tom faithfully reproduced GZMB expression, provide useful tools to dissect mechanisms leading to the development of anti-bacterial effector and memory CD8 T cells and reactivation of the memory response to cognate antigen or inflammatory signals.

Keywords: cytolytic T lymphocyte; fluorescent granzyme B; immune response to bacteria; memory T cells.

Figure 1

Analysis of the primary immune response to ActA⁻-ovalbumin (OVA) infection using chimeric granzyme B-Tomato protein (GZMB-Tom) -OT1 CD8 T cells and GZMB-Tom-OT2 CD4 T cells. Recipient C57BL/6 (CD45.1) mice were injected with Cell tracker Violet (CTV) -labelled CD8 T cells purified from GZMB-Tom-OT1 (CD45.2) mice and CTV-labelled CD4 T cells purified from GZMB-Tom-OT2 (CD45.2) mice (10⁶ cells for each population). At 40 hr (green) and 60 hr (orange) after ActA⁻-OVA injection (10⁶ bacteria), spleens were collected and analysed by cytometry. Gating was on CD45.2 and CD8-positive (a) or CD45.2 and CD4-positive (b) cell populations. GZMB-Tom fluorescence excited with the 561 nm laser was collected in the G610-20 channel (left), anti-GZMB monoclonal antibody was used to reveal GZMB expression in fixed and permeabilized cells (middle), both being shown as a function of CTV (left, middle) and anti-CD44 expression is shown as a function of GZMB-Tom expression (right). Numbers represent the % positive cells or the mean fluorescence intensity (MFI) of positive cells for the marker on the ordinate. Results from four mice in two different experiments are shown as % divided cells (first set of columns) or % of positive cells for the indicated marker (for the 3 other sets of columns) ± SD for OT1 (c) or OT2 (d) T cells.

Figure 2

Splenic localization of chimeric granzyme B-Tomato protein (GZMB-Tom) -positive OT1 T cells responding to a primary infection with ActA⁻-ovalbumin (OVA). Recipient C57BL/6 (CD45.1) mice were injected with Cell tracker Violet (CTV) -labelled CD8 T cells purified from GZMB-Tom-OT1 (CD45.2) mice (10⁶ cells). At 40 hr (a, c) and 60 hr (b, d) after ActA⁻-OVA injection, spleens were collected. Spleen samples were fixed for 3 hr with Antigenfix (see Materials and methods), embedded in OCT and cut into 8-µm slices for immunohistology. Labelling with anti-CD45.2 (green, a–d), anti-B220 (a, b) and anti-MHCII (blue, c, d) was performed on non-permeabilized slices. GZMB-Tom (red, a–d) was excited with the 561-nm laser of the Zeiss 740 confocal microscope. Confocal analyses used a 40× oil objective. Zoom 0·6×. Tiles (4 × 4 or 2 × 2) of mosaic images are also shown. The scale bars are 100 μm for a and b, 10 μm for c and d. Images are representative of three experiments.

Figure 3

Analysis of the response to ActA⁻-ovalbumin (OVA) or to ActA⁻ bacteria of ActA⁻-OVA-primed chimeric granzyme B-Tomato protein (GZMB-Tom) -OT1 CD8 memory T cells. Recipient CD45.1 mice were injected with 5000 GZMB-Tom-OT1 (CD45.2) CD8 T cells 1 day before infection with ActA⁻-OVA (10⁶ bacteria). Thirty days later, they were either unchallenged (red) or injected with 10⁶ ActA⁻-OVA (green) or ActA⁻ (blue) bacteria and their spleens were collected 14 hr later and analysed by cytometry. With gating on CD45.2 and CD8-positive cells, a dot plot shows expression of GZMB-Tom and CD25 on live cells (a) and an histogram shows interferon-γ (IFN-γ) analysis on fixed and permeabilized cells (b). Representative of two experiments. In (a), numbers represent % cells in a given quadrant for ActA⁻-OVA (green) or ActA⁻ (blue) challenges. Mean fluorescence intensity (MFI) for GZMB-Tom (c) and for CD25 (d) expression is shown within quadrants represented in (a) as GZMB⁺ CD25⁻, GZMB⁺ CD25⁺ and GZMB⁻ CD25⁺, respectively.

Figure 4

Splenic localization of chimeric granzyme B-Tomato protein (GZMB-Tom) -positive OT1 memory T cells responding to a challenge infection with ActA⁻-ovalbumin (OVA) or ActA⁻ bacteria. Spleens of mice described in the legend of Fig.3 were treated for confocal analysis as described for Fig.2. Columns (a–c) show spleen samples from mice 14 hr after challenge with ActA⁻-OVA (a) or ActA⁻ (c) bacteria, or unchallenged (b). Staining for CD45.2 (green) reveals OT1 T cells, and GZMB-Tom (red) is revealed as in Fig.2. The first line shows anti-B220 (Magenta) in a 2 × 2 Tile analysis with a 100-µm scale bar and lines 2–3 show staining for CD11c (blue) with a 100 µm scale bar (line 2). Regions marked 1, 2, 3 are enlarged from column A and regions marked 4, 5, 6 are enlarged from column B and are shown in line 3 (for regions 1 and 4) or in the Supporting information (Fig. S4, line 1; for regions 2, 3, 5, 6). The fourth line shows anti-F4.80 staining (yellow) (crop 1, zoom 0·6), an enlargement in an area of the red pulp (presence of F4.80⁺ macrophages) (see Fig. S4, line 2).