CD8 T cell defect of TNF-α and IL-2 in DNAM-1 deficient mice delays clearance in vivo of a persistent virus infection

Abstract

DNAM-1 gene-deficient (-/-) mice take significantly longer to clear an acute and persistent LCMV infection in vivo than DNAM-1 +/+ mice. During acute LCMV priming, at the single cell level, DNAM-1 -/- mice made significantly less cytoplasmic CD8 TNF-α and IL-2 but not IFN-γ than their DNAM-1 +/+ counterparts. Restimulated immune memory CD8 T cells from DNAM-1 -/- and DNAM-1 +/+ mice were equivalent in cytolytic activity against LCMV-infected target cells but DNAM-1 -/- CD8 T cells had significant reductions in TNF-α and IL-2 that were associated on adoptive transfer with the inability to terminate the persistent viral infection.

Fig. 1

LCMV-specific GP 33 CD8 T cells from DNAM-1 −/− and DNAM-1 +/+ mice show equivalent expression of interferon-γ 7, 15, and 30 days following a primary challenge with LCMV ARM 1 × 10⁵ i.p. Panels: Left upper: shows representative FACS data from one of five DNAM-1 −/− or DNAM-1 +/+ mice for generation of interferon-γ for GP 33-specific CD8 T cells harvested from the spleen. The right upper panel shows the mean+1 SD for the group of five to six mice each. No significant difference between DNAM-1 −/− and DNAM-1 +/+ mice occurred. The bottom panel on the left shows representative FACS data for one of five mice for expression of interferon-γ in LCMV-specific GP 61 CD4 T cells at 7, 14, or 30 days following primary LCMV ARM challenge while the lower right panel shows the mean ± 1 SD for the DNAM-1 −/− and DNAM-1 +/+ group. DNAM-1 −/− mice expressed significantly more interferon-γ in primed LCMV-specific GP 61 CD4 T cells at 14 and 30 days when compared to DNAM-1 +/+ mice. At day 7 post-inoculation GP 61 CD4 T cells from DNAM-1 −/− mice expressed more interferon-γ than GP 61 CD4 T cells from DNAM-1 +/+ mice but the difference was not significant (P=0.1). Experiment assaying for interferon-γ in GP 33 CD8 and GP 61 CD4 T cells were repeated twice with similar results. For technical details see (Brooks et al., 2006; Homann et al., 2001; Sullivan et al., 2011).

Fig. 2

Defect in the expression of TNF-α and IL-2 in LCMV-specific GP 33 CD8 T cells in DNAM-1 −/− mice at days 7, 14, and 30 following primary challenge with 1 × 10⁵ PFU of LCMV ARM. Upper panel on the left shows representative FACS data from one of five DNAM-1 −/− mice for expression of TNF-α and IL-2 in the cytosol of LCMV-specific GP 33 CD8 T cells from DNAM-1 −/− and DNAM-1 +/+ mice at day 14 post-LCMV challenge. The lower left panel shows the combined data for DNAM-1 −/− and DNAM-1 +/+ groups (5 mice/group: mean+1 SD). A significant defect (P < 0.005) in TNF-α and IL-2 occurs in LCMV-specific GP 33 CD8 T cells at days 7, 14, and 30 in DNAM-1 −/− mice when compared to DNAM-1 +/+ mice. The right panels show that only at day 14 post-LCMV primary challenge is there a significant difference in heightened expression of TNF-α and IL-2 in GP 61 CD4 T cells in DNAM-1 −/− mice over that seen in GP 61 CD4 T cells from DNAM-1 +/+ mice. Upper panel on the right shows representative FACS data from one of five mice while the lower right panel displays the mean+1 SD of each group of five mice. Experiments performed twice.

Fig. 3

DNAM-1 −/− and DNAM-1 +/+ mice generate robust secondary LCMV-specific CD8 T cell (GP33) and CD4 T cell (GP67) responses but GP 33 CD8 T cells from DNAM-1 −/− mice show a defect in expression of TNF-α and IL-2. DNAM-1 −/− and DNAM-1 +/+ mice received a primary LCMV challenge with 1 × 10⁵ PFU of LCMV ARM i.p. Forty-two days later mice received a secondary challenge of 1 × 10⁶ PFU of LCMV Cl 13 i.v. and spleens were harvested five days later. Lymphocytes obtained from the spleen were stimulated with either peptide GP 33–41 or GP 61–80 as reported (Brooks et al., 2006; Homann et al., 2001). Panel on left shows the significant decrease in TNF-α and IL-2 in DNAM-1 −/− mice compared to DNAM-1 +/+ mice at five days post-secondary response (left lower groups of five mice with mean+1 SD). The left upper panel shows FACS data for expression of interferon-γ from one representative GP 33 CD8 CTL obtained from DNAM-1 −/− and DNAM-1 +/+ mice. There was no defect in expression of interferon-γ in these mice. The panel on the right shows interferon-γ, TNF-α, and IL-2 data for GP 61 CD4 T cells obtained five days after secondary LCMV challenge and shows there is no significant differences in GP 61 CD4 T cells harvested from DNAM-1 −/− compared to DNAM-1 +/+ mice.

Fig. 4

DNAM-1 −/− mice persistently infected with LCMV Cl 13 have a defect in purging virus. Panel A shows the results of adoptive transfer of 2 × 10⁷ immune memory splenocytes obtained from either DNAM-1 −/− or DNAM-1 +/+ mice transferred into DNAM-1 +/+ mice. At time of transfer the host DNAM-1 +/+ mice carried 3 × 10⁵ PFU of LCMV in their sera. By 15 days post-transfer of DNAM-1 +/+ immune memory cells (blue) virus had not cleared from any of the six host mice but viral titer was reduced nearly 2 logs (3 × 10⁵ to 6 × 10³). By day 30 virus was cleared from all mice. In contrast, adoptive transfer of immune memory cells from DNAM-1 −/− mice was reduced less than 1 log (3 × 10⁵ to 9 × 10⁴) at day 15 and virus was still present in 50% of mice by day 62. Similar results with repeated experiment. Panel B shows that failure to clear was not due to allogeneic difference between DNAM-1 −/− and DNAM-1 +/+ mice. Panel C displays that spontaneous clearance of persistent LCMV Cl 13 infection in DNAM-1 −/− mice was significantly delayed when compared to spontaneous clearance in DNAM-1 +/+ mice. For this experiment 10 DNAM-1 −/− and five DNAM-1 +/+ mice were inoculated with LCMV Cl 13 2 × 10⁶ PFU i.v. and displayed equivalent amounts of virus in their sera at day 7. DNAM-1 +/+ mice infected with LCMV Cl 13 spontaneously lowered viral titers and cleared infection significantly better (significance P < 0.005, marked by asterisks) than DNAM-1 −/− mice. Mice serially bled and the amount of virus carried by each group over time is quantified by plaque assay. See (Ahmed and Oldstone, 1988) for details. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)