Induction and exhaustion of lymphocytic choriomeningitis virus-specific cytotoxic T lymphocytes visualized using soluble tetrameric major histocompatibility complex class I-peptide complexes

Abstract

This study describes the construction of soluble major histocompatibility complexes consisting of the mouse class I molecule, H-2Db, chemically biotinylated beta2 microglobulin and a peptide epitope derived from the glycoprotein (GP; amino acids 33-41) of lymphocytic choriomeningitis virus (LCMV). Tetrameric class I complexes, which were produced by mixing the class I complexes with phycoerythrin-labeled neutravidin, permitted direct analysis of virus-specific cytotoxic T lymphocytes (CTLs) by flow cytometry. This technique was validated by (a) staining CD8+ cells in the spleens of transgenic mice that express a T cell receptor (TCR) specific for H-2Db in association with peptide GP33-41, and (b) by staining virus-specific CTLs in the cerebrospinal fluid of C57BL/6 (B6) mice that had been infected intracranially with LCMV-DOCILE. Staining of spleen cells isolated from B6 mice revealed that up to 40% of CD8(+) T cells were GP33 tetramer+ during the initial phase of LCMV infection. In contrast, GP33 tetramers did not stain CD8+ T cells isolated from the spleens of B6 mice that had been infected 2 mo previously with LCMV above the background levels found in naive mice. The fate of virus-specific CTLs was analyzed during the acute phase of infection in mice challenged both intracranially and intravenously with a high or low dose of LCMV-DOCILE. The results of the study show that the outcome of infection by LCMV is determined by antigen load alone. Furthermore, the data indicate that deletion of virus-specific CTLs in the presence of excessive antigen is preceded by TCR downregulation and is dependent upon perforin.

Figure 1

Staining of GP33-specific H-2D^b–restricted TCR transgenic T cells and polyclonal LCMV-specific CTL lines with GP33 tetramers. Spleen cells from a naive B6 mouse and a 318 TCR transgenic mouse were stained with monoclonal antibodies specific for CD8 and the TCR gene segment Vα2 (A and B) or with anti-CD8 and GP33 tetramers (C and D). CD8⁺ CTL lines specific for H-2D^b–restricted LCMV peptide epitopes, GP33–41, NP396–404, and GP276–286 were stained using GP33 tetramers 2 wk after in vitro restimulation with peptide-pulsed spleen cells (E).

Figure 1

Staining of GP33-specific H-2D^b–restricted TCR transgenic T cells and polyclonal LCMV-specific CTL lines with GP33 tetramers. Spleen cells from a naive B6 mouse and a 318 TCR transgenic mouse were stained with monoclonal antibodies specific for CD8 and the TCR gene segment Vα2 (A and B) or with anti-CD8 and GP33 tetramers (C and D). CD8⁺ CTL lines specific for H-2D^b–restricted LCMV peptide epitopes, GP33–41, NP396–404, and GP276–286 were stained using GP33 tetramers 2 wk after in vitro restimulation with peptide-pulsed spleen cells (E).

Figure 2

Staining of GP33-specific D^b-restricted CD8⁺ cells isolated from the CSF of mice infected intracranially with rVVINDG or LCMV-DOCILE. Cells isolated from the CSF of mice infected intracranially with 2 × 10³ PFU of rVVINDG (A) or 30 PFU of LCMV-DOCILE (B) were stained with anti-CD8 antibodies and GP33 tetramers. The histograms show staining of live CD8⁺ cells with GP33 tetramers.

Figure 3

CD8⁺ and GP33-specific D^b-restricted CD8⁺ cells isolated from the spleens of mice infected intravenously with LCMV-DOCILE. Cells isolated from the spleens of mice infected intravenously with low or high dose of LCMV-DOCILE were stained with anti-CD8 antibodies and GP33 tetramers. The graphs describe data collected from histograms generated as described in Fig. 2. Solid bars, the percentage of CD8⁺ cells in the spleens of individual mice at four time points during the acute phase of LCMV infection; open bars, the percentage of CD8⁺ cells that also stained with GP33 tetramers. The results are representative of two independent experiments carried out using groups of two mice. The number of GP33-specific CD8⁺ cells (× 10⁵) are shown in brackets above each bar.

Figure 4

Cytotoxic activity and virus titers in mice infected intravenously with LCMV-DOCILE. LCMV-specific CTL activity was measured using spleen cells from mice that had been infected intravenously with a low (A–D) or high dose (E–H) of LCMV-DOCILE. Spleen cells were tested for lysis of normal MC57 cells (○) or MC57 cells that had either been pulsed with peptide GP33 (▴) or which had been infected with LCMV-DOCILE (•). Virus titers, measured as PFU in the spleen (Spl) and thymus (Thy) of each mouse are shown in the upper right-hand corner of each panel. 200 PFU represents the detection limit of the assay.

Figure 5

Intracellular cytokine staining of CD8⁺ cells isolated from naive and LCMV-DOCILE–infected B6 mice. Spleen cells isolated from naive mice were stained directly (A) or after stimulation with PMA and ionomycin (B) using anti-CD8 and anti–IFN-γ antibodies. Spleen cells recovered from mice infected 8 d before with a low dose (2 × 10² PFU intravenously) of LCMV-DOCILE were stimulated with PMA and ionomycin and subsequently stained with anti-CD8 antibodies, GP33 tetramers, and anti–IFN-γ antibodies. C represents IFN-γ staining of the gated CD8⁺ cell population, whereas D represents IFN-γ staining of the gated CD8⁺/tetramer⁺ cell population. The percentage of gated cells that express IFN-γ is shown in each panel.

Figure 6

Intracellular cytokine staining of CD8⁺ cells isolated from LCMV-DOCILE–infected B6 mice. Spleen cells recovered from mice that had been infected intravenously with either low- (2 × 10² PFU) or high-dose (2 × 10⁶ PFU) LCMV-DOCILE were stimulated with PMA and ionomycin and subsequently stained with anti-CD8 antibodies, GP33 tetramers, and anti–IFN-γ antibodies. Subsequent FACS^® analysis was carried out as described for Fig. 5. Solid bars, the percentage of CD8⁺/ GP33 tetramer⁺ cells (from the experiment shown in Fig. 3) recovered from the spleens of each mouse; open bars, the percentage of CD8⁺/GP33 tetramer⁺ cells that express IFN-γ.

Figure 7

Staining of GP33-specific CD8⁺ cells isolated from the CSF and spleens of mice infected intracranially with LCMV-DOCILE. Cells isolated from the CSF and spleens of mice infected with low (A, B, E, and F) or high (C, D, G, and H) doses intracranially with LCMV-DOCILE were stained with anti-CD8 antibodies and tetramers. (Left) Staining of live cells with anti-CD8 antibodies; (right) gated CD8⁺ cell population stained with GP33 tetramers. The percentage of CD8⁺ cells (A, C, E, and G) and the percentage of CD8⁺/tetramer⁺ cells (B, D, F, and H) is shown in each panel. The mean fluorescence of splenic GP33 tetramer–stained CD8⁺ T cells is shown in brackets in F and H. The results are representative of two independent experiments carried out using groups of two mice.

Figure 8

Cytotoxic activity and virus titers in mice infected intracranially with LCMV-DOCILE. LCMV-specific CTL activity was measured using spleen cells from mice that had been infected intracranially with a low (A) or high (B) dose of LCMV-DOCILE. Spleen cells were tested for lysis of normal MC57 cells (○), MC57 cells that had either been pulsed with peptide GP33 (▴), and MC57 cells which had been infected with LCMV-DOCILE (•). Virus titers, measured per spleen and brain of each mouse, are also shown.

Figure 9

Intracellular cytokine staining of CD8⁺ cells isolated from the spleens and CSF of mice infected intracranially with a low or high dose of LCMV-DOCILE. Spleen cells and CSF recovered from mice that had been infected intracranially with either low (30 PFU) or high (3 × 10⁴ PFU) dose LCMV-DOCILE were stimulated with PMA and ionomycin and subsequently stained with anti-CD8 antibodies, GP33 tetramers, and anti–IFN-γ antibodies. Subsequent FACS^® analysis was carried out as described for Fig. 5. (Left) CD8⁺ cells that express IFN-γ; (right) CD8⁺ GP33-specific T cells that express IFN-γ. The percentages of CD8⁺ and CD8⁺/tetramer⁺ cells (from the experiment shown in Fig. 7) that express IFN-γ are shown in each panel.

Figure 10

Staining of GP33-specific D^b-restricted CD8⁺ cells isolated from the spleens of B6 and PKOB mice infected intravenously with LCMV-DOCILE. Cells isolated from the spleens of either B6 mice infected with a low dose of LCMV-DOCILE (A and D), PKOB mice infected with a low dose of LCMV-DOCILE (B and E), or B6 mice infected with a high dose of the same virus (C and F) were stained with anti-CD8 antibodies and GP33 tetramers. (Histograms) The gated CD8⁺ cell population stained with GP33 tetramers. The results are representative of two independent experiments carried out using groups of two mice.

Figure 11

Intracellular cytokine staining of CD8⁺ cells isolated from LCMV-infected PKOB mice. Spleen cells recovered from mice that had been infected intravenously with either low-dose (2 × 10² PFU) LCMV-DOCILE were stimulated with PMA and ionomycin and subsequently stained with anti-CD8 antibodies, GP33 tetramers, and anti–IFN-γ antibodies. Subsequent FACS^® analysis was carried out as described for Fig. 5. (Left histograms) the percentage of CD8⁺ cells that express IFN-γ; (right histograms) the percentage of CD8⁺ GP33-specific T cells that express IFN-γ.