Analysis of apoptosis of memory T cells and dendritic cells during the early stages of viral infection or exposure to toll-like receptor agonists

Abstract

Profound type I interferon (IFN-I)-dependent attrition of memory CD8 and CD4 T cells occurs early during many infections. It is dramatic at 2 to 4 days following lymphocytic choriomeningitis virus (LCMV) infection of mice and can be elicited by the IFN-inducing Toll receptor agonist poly(I:C). We show that this attrition occurs in many organs, indicating that it is due to T cell loss rather than redistribution. This loss correlated with elevated intracellular staining of T cells ex vivo for activated caspases but with only low levels of ex vivo staining with annexin V, probably due to the rapid clearance of apoptotic cells in vivo. Instead, a high frequency of annexin V-reactive CD8alpha(+) dendritic cells (DCs), which are known to be highly phagocytic, accumulated in the spleen as the memory T cell populations disappeared. After short in vitro incubation, memory phenotype T cells isolated from LCMV-infected mice (day 3) or mice treated with poly(I:C) (12 h) displayed substantial DNA fragmentation, as detected by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, compared to T cells isolated from uninfected mice, indicating a role for apoptosis in the memory T cell attrition. This apoptosis of memory CD8 T cells early during LCMV infection was reduced in mice lacking the proapoptotic molecule Bim. Evidence is presented showing that high levels of T cell attrition, as found in young mice, correlate with reduced immunodomination by cross-reactive memory cells.

FIG. 1.

An increase in CD8α⁺ DCs accounts for the observed increase in annexin V-reactive CD8α⁺ CD44^high cells following poly(I:C) treatment. C57BL/6 mice were inoculated with poly(I:C) i.p. Splenocytes were harvested at 0 h (untreated) and 12 h postinoculation. (A) Percentages of untreated and poly(I:C)-treated CD8α⁺ CD44^high/low, CD8β⁺ CD44^high/low, and CD4⁺ CD44^high/low cells. (B) Percentages of untreated and poly(I:C)-treated annexin V⁺ CD8α⁺ CD44^high, annexin V⁺ CD8β⁺ CD44^high, and annexin V⁺ CD4⁺ CD44^high cells. (C) CD40 expression on untreated or poly(I:C)-treated CD8α⁺ CD44^high and CD8β⁺ CD44^high cells. (D) Annexin V and CD40 expression on untreated or poly(I:C)-treated CD8α⁺ CD44^high cells. (E) Thy1.2, CD3, CD11c, and class II (I-A^B) expression on untreated or poly(I:C)-treated CD40⁺ CD8α⁺ CD44^high (dashed lines) and CD40⁻ CD8α⁺ CD44^high (gray fill) cells. Gates were set upon 7-AAD⁻ cells to exclude dead/necrotic cells. The data are representative of three independent experiments with three mice per group..

FIG. 2.

The poly(I:C)-induced decrease in CD8β⁺ CD44^high T cells correlates with an increase in CD8α⁺ DCs. C57BL/6 mice were inoculated with poly(I:C) i.p. Splenocytes were harvested at 0 (untreated), 3, 6, 9, and 12 h after poly(I:C) inoculation. (A) Percentage of CD8α⁺ DCs at multiple time points following poly(I:C) treatment. (B) Absolute numbers of CD8α⁺ CD11c⁺ DCs at multiple time points following poly(I:C) treatment. *, P < 0.05 relative to untreated mice. Error bars indicate standard deviations. (C) Percentage of CD8β⁺ CD44^high/low T cells at multiple time points following poly(I:C) treatment. (D) Absolute numbers of CD8β⁺ CD44^high T cells at multiple time points following poly(I:C) treatment. Gates were set on 7-AAD⁻ cells. Absolute numbers were based upon percentages obtained via flow cytometry. *, P < 0.05 relative to untreated mice. The data are representative of three independent experiments with three mice per group.

FIG. 3.

Trafficking to other compartments does not account for the loss of CD8β⁺ CD44^high and CD4⁺ CD44^high T cells from the spleen following poly(I:C) treatment. C57BL/6 mice were inoculated with poly(I:C) i.p. Splenocytes, peritoneal exudate cells (PECs), lungs, inguinal lymph nodes (iLN), and peripheral blood lymphocytes (PBLs) were harvested at 0 (untreated) and 12 h postinoculation. (A) Plots display the percentages of untreated and poly(I:C)-treated CD8β⁺ CD44^high/low T cells. (B) Plots display the percentages of untreated and poly(I:C)-treated CD4⁺ CD44^high/low T cells. Gates were set upon 7-AAD⁻ cells. Each plot is representative of three mice per group, and the percentages displayed on each plot are the averages for three mice per group. The data are representative of two independent experiments, each with three mice per group..

FIG. 4.

The poly(I:C)-induced attrition of CD8β⁺ CD44^high and CD4⁺ CD44^high T cells correlates with an increase in DNA fragmentation. C57BL/6 mice were inoculated with poly(I:C) i.p. Splenocytes were harvested at 0 (untreated) and 12 h after poly(I:C) treatment. DNA fragmentation was assessed via TUNEL staining after a brief in vitro culture of splenocytes for 5 h at either 37°C or 4°C (see Materials and Methods). (A) Plots show the percentage of untreated or poly(I:C)-treated TUNEL⁺ CD8β⁺ CD44^high T cells (37°C and 4°C). (B) Plots show the percentage of untreated or poly(I:C)-treated TUNEL⁺ CD4⁺ CD44^high T cells (37°C and 4°C). Each plot is representative of three mice per group, while the percentages displayed on each plot are the averages for three mice per group. The data are representative of three independent experiments.

FIG. 5.

The poly(I:C)-induced increase in CD8α⁺ DCs may contribute to the rapid clearance of apoptotic cells. A total of 2 × 10⁷ CFSE-labeled wild-type splenocytes (Ly5.1) were adoptively transferred into congenic wild-type recipients (Ly5.2). Splenocytes were harvested at 0 (untreated), 3, 6, 9, and 12 h after poly(I:C) treatment. (A) The capacities of Ly5.2⁺ CD8α⁺ CD11c⁺ and Ly5.2⁺ CD8α⁻ CD11c⁺ DCs to take up Ly5.1⁺ CFSE⁺ cells were correlated with CD40 expression at indicated the time points Doublets were excluded via pulse width, and gates were set upon Ly5.2⁺ CD11c⁺ 7-AAD⁻ cells. (B) The plot shows the percentages of host (Ly5.2) CD8α⁺ and CD8α⁻ CFSE⁺ DC populations following poly(I:C) treatment. Each data point is the average for three mice, and the data are representative of three independent experiments, each with three mice per data point. Error bars indicate standard deviations..

FIG. 6.

The LCMV-induced attrition of CD8β⁺ CD44^high T cells correlates with a substantial increase in DNA fragmentation and caspase 3 activation but not annexin V reactivity. C57BL/6 mice were infection with LCMV-Armstrong i.p. Splenocytes were harvested at 0, 1, 2, 3, and 4 days postinfection. DNA fragmentation was assessed via TUNEL staining after a brief in vitro culture of splenocytes for 5 h at 37°C (see Materials and Methods). (A) Annexin V and CD11c staining on CD8α⁺ CD44^high and CD8β⁺ CD44^high cells at days 0,1, and 2 after LCMV infection. Histogram inset in quadrant 3 show CD40 expression on the annexin V⁺ CD11c⁺ CD8α⁺ CD44^high population (quadrant 2). (B) Absolute CD8β⁺ CD44^high T cell numbers at 0, 1, 2, 3, and 4 days postinfection. Error bars indicate standard deviations. (C) Histogram overlays depicting CD8β⁺ CD44^high annexin V reactivity and caspase 3 activation on days 0, 1, 2, 3, and 4 after LCMV infection. Line color corresponds to day after LCMV infection. (D) Percentages of TUNEL⁺ CD8β⁺ CD44^high T cells isolated from mice at days 0, 1, 2, 3, and 4 postinfection. Absolute numbers were based upon percentages obtained via flow cytometry. The data are representative of three independent experiments with three mice per group..

FIG. 7.

The LCMV-induced apoptosis of CD8β⁺ CD44^high T cells is partially dependent on Bim. C57BL/6 (wild-type) and Bim-deficient (Bim KO) mice were infected with LCMV-Armstrong i.p. Splenocytes were harvested at day 0 (untreated) and 3 days postinfection. DNA fragmentation was assessed via TUNEL staining after a brief in vitro culture of splenocytes for 5 h at 37°C (see Materials and Methods). (A) The plots show the percentages of untreated and LCMV-infected wild-type and Bim KO CD8β⁺ CD44^high T cells. (B) The plots show the percentages of untreated and LCMV-infected wild-type and Bim KO TUNEL⁺ CD8β⁺ CD44^high T cells. Each plot is representative of three mice per group, while the percentages are the averages for three mice per group. The data are representative of three independent experiments..

FIG. 8.

Reduced attrition in aged LCMV-immune mice limits the diversity in the arising T cell response following heterologous Pichinde virus (PV) challenge. Young (6 to 8 months) and aged (18 to 22 months) LCMV-immune mice were prebled to determine NP205-specific CD8⁺ T cell frequencies prior to infection with PV, via an intracellular cytokine assay. Mice were rebled on day 8 after PV infection, and the ratios of the NP205 to the NP38 response were determined via intracellular cytokine assay. (A) Frequencies of NP38-specific CD8⁺ T cells, as revealed by intracellular IFN-γ assay, in young and aged naïve mice. (B) Frequencies of NP38- and NP205-specific CD8⁺ T cells at day 8 after PV infection, as revealed by intracellular IFN-γ assay, in young and old LCMV-immune mice. (C) Ratio of the NP205 to NP38 response for multiple young and old LCMV-immune mice (day 8 after PV infection). The plots in panels A and B show one representative mouse, while panel C shows the NP205/NP38 frequencies for all mice over multiple experiments..