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. 2004 Oct;78(20):11246-57.
doi: 10.1128/JVI.78.20.11246-11257.2004.

Antioxidant treatment reduces expansion and contraction of antigen-specific CD8+ T cells during primary but not secondary viral infection

Nathan G Laniewski  1 , Jason M Grayson
Affiliations

Antioxidant treatment reduces expansion and contraction of antigen-specific CD8+ T cells during primary but not secondary viral infection

Nathan G Laniewski et al. J Virol. 2004 Oct.

Abstract

During many viral infections, antigen-specific CD8(+) T cells undergo large-scale expansion. After viral clearance, the vast majority of effector CD8(+) T cells undergo apoptosis. Previous studies have implicated reactive oxygen intermediates (ROI) in lymphocyte apoptosis. The purpose of the experiments presented here was to determine the role of ROI in the expansion and contraction of CD8(+) T cells in vivo during a physiological response such as viral infection. Mice were infected with lymphocytic choriomeningitis virus (LCMV) and treated with Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP), a metalloporphyrin-mimetic compound with superoxide dismutase activity, from days 0 to 8 postinfection. At the peak of CD8(+)-T-cell response, on day 8 postinfection, the numbers of antigen-specific cells were 10-fold lower in MnTBAP-treated mice than in control mice. From days 8 to 30, a contraction phase ensued where the numbers of antigen-specific CD8(+) T cells declined 25-fold in vehicle-treated mice compared to a 3.5-fold decrease in MnTBAP-treated mice. Differences in contraction appeared to be due to greater proliferation in drug-treated mice. By day 38, the numbers of antigen-specific CD8(+) memory T cells were equivalent for the two groups. The administration of MnTBAP during secondary viral infection had no effect on the expansion of antigen-specific CD8(+) secondary effector T cells. These data suggest that ROI production is critical for the massive expansion and contraction of antigen-specific CD8(+) T cells during primary, but not secondary, viral infection.

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Figures

FIG. 1.
FIG. 1.
Treatment with MnTBAP reduces superoxide levels in CD8+ T cells. Naïve spleen cells (A) and spleen cells from mice that were infected with LCMV Armstrong 5 days earlier and treated with either vehicle (B) or 5 mg of MnTBAP/kg (C) were incubated with HE and then stained with anti-CD8α and CD44 antibodies. The HE levels are plotted in histogram formats, with the MFIs indicated in the upper right corners of the plots. Three to six mice were examined under each condition, and results from a representative mouse are presented in each histogram.
FIG. 2.
FIG. 2.
Treatment with MnTBAP results in increased levels of virus in the spleen during acute infection. C57BL/6 mice were treated with either vehicle or 5 mg of MnTBAP/kg and then infected with LCMV Armstrong. A maintenance dose was administered every 24 h from days 0 to 8. At the given time points, mice were sacrificed, and viral levels in the spleen were determined by plaque assays. The means and standard deviations were calculated and are shown for each time point. Four to six mice were used in each group at each time point. *, significant difference between vehicle- and MnTBAP-treated mice; P ≤ 0.05.
FIG.3.
FIG.3.
Treatment with MnTBAP results in decreased expansion and contraction of CD8+ T cells during primary viral infection. C57BL/6 mice were treated with either the vehicle or 5 mg of MnTBAP/kg and then infected with LCMV Armstrong. A maintenance dose was administered every 24 h. At the indicated time points, mice were sacrificed, the spleen was removed, and cells were stained with anti-CD8α and either anti-CD44 (A), DbGP33-41 (B), DbNP396-404(C), or DbGP276-286 (D). The numbers of activated (A) and antigen-specific CD8+ T cells (B-D) were quantitated, and the averages and standard deviations are shown. To determine the effect of MnTBAP administration on lymphocyte numbers in other tissues, mice were sacrificed on days 8 and 38 postinfection and lymphocytes were isolated from the lung, liver and lymph nodes and stained with anti-CD8α and DbNP396-404 (E). The numbers of NP396-404-specific CD8+ T cells were quantitated, and the average and standard deviation are shown. Six to 10 mice were analyzed at each time point. The gray areas in panels A through D indicate the windows of treatment. *, significant difference between vehicle- and MnTBAP-treated mice; P ≤ 0.05.
FIG.4.
FIG.4.
Decreased production of TNF-α in effector but not memory cells from mice treated with MnTBAP during acute viral infection. C57BL/6 mice were treated with either the vehicle or 5 mg of MnTBAP/kg and then infected with LCMV Armstrong. A maintenance dose was administered every 24 h from days 0 to 8. Mice were sacrificed on days 5 (A), 8 (B), or 38 (C) postinfection, and splenocytes were stimulated with the indicated LCMV epitope. Cells were then stained with anti-CD8α, IFN-γ, and TNF-α. The dot plots are gated on CD8+ T cells, and the number in the plot indicates the percentage of CD8+ T cells that are present in that region. Six to 10 mice were analyzed at each time point, and results for a representative mouse are shown. To determine the effect of MnTBAP on cytokine production (D), splenocytes were isolated from a mouse 8 days postinfection with LCMV Armstrong and were incubated with NP396-404 and increasing concentrations of MnTBAP. The numbers of cytokine producing CD8+ T cells were quantitated, and the average and standard deviation are shown. Six mice were analyzed in two independent experiments.
FIG. 5.
FIG. 5.
Reduced in vitro cytotoxicity of MnTBAP-treated mice is not due to a major defect in per-cell cytolysis. Splenocytes from vehicle- and MnTBAP-treated mice 8 days after infection with LCMV Armstrong were harvested and incubated directly ex vivo (A) at the indicated effector-to-target ratio (E:T) with 51Cr-labeled H-2b targets that were untreated (closed symbols) or infected (open symbols) with LCMV clone 13 in a 5-h 51Cr-release assay. Each sample was assayed in triplicate. To determine per-cell lytic activity (B), splenocytes were stained with anti-CD8α and either DbGP33-41 or DbNP396-404 MHC class I tetramer. The numbers of antigen-specific CD8+ T cells were determined, and 104 cells were incubated with 104 appropriately coated peptide targets. Cell numbers were normalized by the addition of naïve splenocytes. Specific lysis was calculated for each mouse, and the average for each treatment was calculated. The relative lysis was determined by dividing the average for each group by the average for the vehicle group. Three to six mice were used in two independent experiments, and each sample was assayed in triplicate.
FIG. 6.
FIG. 6.
Reduced contraction in MnTBAP-treated mice is due to increased proliferation. C57BL/6 mice were treated with either vehicle or 5 mg of MnTBAP/kg and then infected with LCMV Armstrong. A maintenance dose was administered every 24 h. Direct ex vivo apoptosis on day 8 postinfection (A) was assessed by surface staining splenocytes as described above, followed by a brief incubation with Annexin V and 7AAD and immediate acquisition. Naïve (CD8+CD44low) cells were included as a viability control. The value in each quadrant is the percentage of CD8+DbNP396-404+ cells that fall in the quadrant. The averages and standard deviations for each quadrant from the analysis of vehicle- and MnTBAP-treated mice are plotted (B). Proliferation during the contraction phase (days 8 to 15) was assessed (C) by giving the mice BrdU (0.8 mg/ml) in their drinking water from days 8 to 15 after infection. Splenocytes were isolated on day 15 postinfection and stained with anti-CD8α, DbNP396-404, and anti-BrdU antibody. The plots are gated on CD8+DbNP396-404+ cells, and the numbers on the dot plots indicate the percentages of antigen-specific cells that are BrdU+. The average percentage and standard deviation of CD8+DbNP396-404+BrdU+ cells from vehicle- or MnTBAP-treated mice are plotted (D). Proliferation during the memory phase (days 31 to 38) was assessed (E) by giving the mice BrdU (0.8 mg/ml) in their drinking water from days 31 to 38 after infection. Splenocytes were isolated on day 38 postinfection and stained with anti-CD8α, DbNP396-404, and anti-BrdU antibody. The plots are gated on CD8+DbNP396-404+ cells, and the numbers on the dot plots indicate the percentages of antigen-specific cells that are BrdU+. The average percentage and standard deviation of CD8+DbNP396-404+BrdU+ cells from vehicle- or MnTBAP-treated mice are plotted (F). Three to six mice were examined at each time point, and data from a representative mouse is presented in each dot plot. *, significant difference between vehicle- and MnTBAP-treated mice; P ≤ 0.05.
FIG. 7.
FIG. 7.
The number of antigen-specific CD8+ T cells during secondary LCMV infection is unaffected by MnTBAP treatment. C57BL/6 LCMV Armstrong immune mice were treated with either vehicle or 5 mg of MnTBAP/kg and then infected with LCMV clone 13 (A). A maintenance dose was administered every 24 h. At the indicated time points, mice were sacrificed, the spleen was removed, and cells were stained with anti-CD8α and either anti-CD44 (B), DbGP33-41 (C), DbNP396-404 (D), or DbGP276-286 (E). The numbers of activated (B) and antigen-specific CD8+ T cells (C-E) were quantitated, and the averages and standard deviations are shown. Six to eight mice were analyzed at each time point. *, significant difference between vehicle- and MnTBAP-treated mice; P ≤ 0.05.
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