Single-cell perforin and granzyme expression reveals the anatomical localization of effector CD8+ T cells in influenza virus-infected mice

Abstract

Influenza virus infection activates cytolytic T lymphocytes (CTL) that contribute to viral clearance by releasing perforin and granzymes from cytoplasmic granules. Virus-specific, perforin-dependent CD8(+) CTL were detected in freshly isolated cells from the mouse lung parenchyma but not from the mediastinal lymph nodes (MLN), where they are primed, or from the spleen during primary influenza virus infection. To determine whether this difference was due to the low frequency or incomplete maturation of effector CTL in MLN, we measured expression of perforin, granzymes A, B, and C, and IFN-gamma mRNAs in CD8(+) populations and single cells immediately after isolation from virus-infected mice. Quantitative PCR revealed significant expression of perforin, granzyme A, granzyme B, and IFN-gamma in activated CD8(+) cells from MLN, spleen, and lung parenchyma. Granzyme C expression was not detected. Individual activated or nucleoprotein peptide/class I tetramer-binding CD8(+) cells from the three tissues expressed diverse combinations of perforin, granzyme, and IFN-gamma mRNAs. Although cells from lung expressed granzymes A and B at higher frequency, each of the tissues contained cells that coexpressed perforin with granzymes A and/or B. The main difference between MLN and lung was the elevated frequency of activated CD8(+) T cells in the lung, rather than their perforin/granzyme expression profile. The data suggest that some CTL mature into perforin/granzyme-expressing effector cells in MLN but reach detectable frequencies only when they accumulate in the infected lung.

Figure 1

Phenotype and perforin dependence of cytolytic cells from influenza virus-infected mice. (A) CD8⁺ and CD8⁻ cells from the indicated tissues were purified from 7-day Mem71-infected BALB/c mice and assayed in duplicate in ⁵¹Cr-release assays with Mem71-infected (○), NP_147–155-coated (●), or untreated (▵) P815 target cells. (B) Lung parenchymal cells from 7-day Mem71-infected BALB/c mice were incubated with concanamycin A at 100 ng/ml (▵) or 10 ng/ml (▿) or with (○) or without (●) diluent at the higher dose for 2 h, then assayed in triplicate in ⁵¹Cr-release assays with Mem71-infected or NP_147–155-coated P815 target cells as indicated. The symbols with a broken line (Left) show lytic activity of untreated lung cells against untreated target cells. (C) Unfractionated leukocytes and CD8⁺ cells (24% of the leukocyte population) of naive (CD44^low CD62L^high; 28% of the CD8⁺ population) and activated (CD44^high CD62L^low; 36%) phenotype from lung parenchyma of 7-day Mem71-infected BALB/c mice were assayed in duplicate in ⁵¹Cr-release assays with Mem71-infected (○), NP_147–155-coated (●), or untreated (▴) P815 target cells.

Figure 2

Perforin, granzyme, and IFN-γ expression by resting and activated CD8⁺ cells from uninfected and influenza virus-infected mice. CD8⁺ cells of CD44^low CD62L^high (naive) and CD44^high CD62L^low (activated) phenotype were purified from pooled inguinal, brachial, and axillary LN, spleen, and lung parenchyma of control uninfected BALB/c mice (A) and MLN, spleen, and lung parenchyma of 7-day Mem71-infected BALB/c mice (B) (two to three mice per group). Cells were lysed immediately for determination of CD3ɛ, perforin (Per), granzymes (Gr) A–C, and IFN-γ mRNA levels by QC-PCR. (Left) Numbers of mRNA molecules in 100-cell equivalents; broken lines indicate the threshold of detection. (Right) Average numbers of total, naive, and activated CD8⁺ cells per mouse in each tissue.

Figure 3

Single-cell analysis of perforin, granzyme, and IFN-γ mRNA expression by activated CD8⁺ cells from influenza virus-infected mice. Single CD44^high CD62L^low CD8⁺ cells purified from MLN and lung parenchyma of 7-day Mem71-infected BALB/c mice (n = 4) were lysed immediately for analysis of CD3ɛ, perforin, granzymes A–C, and IFN-γ expression by two-round nested RT-PCR. Detection of a perforin, granzyme, or IFN-γ PCR product is indicated by shading of the boxes. The number of cells with each expression pattern is shown at the right; the frequency expressing each product is shown along the bottom.

Figure 4

Single-cell analysis of perforin, granzyme, and IFN-γ mRNA expression by NP/class I tetramer-binding CD8⁺ cells from influenza virus-infected mice. Single NP_366–374/H-2D^b tetramer-binding and nonbinding CD8⁺ cells purified from MLN, spleen, and lung parenchyma of 8-day HKx31-infected C57BL/6 mice (n = 3) were lysed immediately for analysis of CD3ɛ, perforin, granzymes A–C, and IFN-γ expression by two-round nested RT-PCR. Data are displayed as described in the legend to Fig. 3.

Figure 5

Distribution of the number of mRNA species detected per cell isolated from the MLN and lungs of influenza virus-infected mice. The data in Figs. 3 and 4 were used to determine the percentage of activated (Left) or tetramer-binding (Right) CD8⁺ cells expressing 0–5 of the PCR products for perforin, granzymes A–C, and IFN-γ (gray bars). Percentages of cells expressing perforin with granzymes A and/or B are superimposed in the black bars. In the upper right of each graph is the percentage of cells positive for one or more products.