Role of tumor necrosis factor receptors in regulating CD8 T-cell responses during acute lymphocytic choriomeningitis virus infection

Abstract

The role of tumor necrosis factor (TNF) in regulating various phases of the antiviral T-cell response is incompletely understood. Additionally, despite strong evidence ascribing a role for TNF in protecting against T-cell-dependent autoimmunity, the underlying mechanisms are still obscure. To address these issues, we have investigated the role of tumor necrosis factor receptors (TNFRs) I (p55R) and II (p75R) in regulating CD8 T-cell responses to lymphocytic choriomeningitis virus (LCMV) with wild-type, p55R-deficient (p55(-/-)), p75R-deficient (p75(-/-)), and p55R- and p75R-deficient (DKO) mice. Loss of p55R increased the number of memory CD8 T cells to only one of the two immunodominant epitopes, and p75R deficiency had a minimal impact on the T-cell response to LCMV. Strikingly, deficiency of both p55R and p75R had a more dramatic effect on the LCMV-specific CD8 T-cell response; in the DKO mice, as a sequel to enhanced expansion and a reduction in contraction of CD8 T cells, there was a substantial increase in the number of memory CD8 T cells (specific to the two immunodominant epitopes). While the majority of LCMV-specific memory CD8 T cells in wild-type mice were CD62Lhi CCR7hi (central memory), a major proportion of memory CD8 T cells in DKO mice were CD62Llo CCR7hi. TNFR deficiency did not affect the proliferative renewal of memory CD8 T cells. Taken together, these data suggested that TNFRs p55R and p75R have overlapping roles in downregulating CD8 T-cell responses and establishment of immune homeostasis during an acute viral infection.

FIG. 1.

TNFR-deficient mice generate a normal primary LCMV-specific cytotoxic T-lymphocyte effector response. We infected 6- to 8-week-old C57BL/6 wild-type (+/+), p55R-deficient (p55^−/−), p75R-deficient (p75^−/−), and p55R- and p75R-deficient (DKO) mice with LCMV-Armstrong. (A) Eight days after infection, the MHC class I-restricted cytotoxic activity in the spleens was measured directly ex vivo in a 6-h ⁵¹Cr release assay. Uninfected and LCMV-infected MC57 cells (H-2^b) were used as target cells. Data are the means for three mice per group. (B) On the eighth day after infection, CD8 T cells specific to the cytotoxic T-lymphocyte epitopes NP396-404 and GP33-41 were quantitated with MHC I tetramers. Data are the means for three to seven mice per group ± standard deviation and representative of three independent experiments.

FIG. 2.

Effect of TNFR deficiency on the contraction of LCMV-specific CD8 T cells. Groups of wild-type, p55^−/−, p75^−/−, and DKO mice were infected with LCMV-Armstrong. On the indicated days after infection, splenocytes were stained with anti-CD8, anti-CD44, and MHC class I tetramers. The dot plots are gated on total CD8 T cells, and the numbers are the percentages of epitope-specific CD8 T cells among total splenocytes. The numbers in parentheses are the percentages of LCMV-specific CD8 T cells among total CD8 T cells. Data are representative of three independent experiments.

FIG. 3.

Effect of TNFR deficiency on the proliferation and apoptosis of LCMV-specific CD8 T cells. (A) Groups of wild-type, p55^−/−, p75^−/−, and DKO mice were infected with LCMV-Armstrong and administered BrdU between days 8 and 15 postinfection. On day 15 postinfection, splenocytes were stained with anti-CD8, MHC class I tetramer (NP396 specific), and anti-BrdU antibodies. The number of BrdU-positive LCMV-specific CD8 T cells was determined by flow cytometry. The histograms are gated on tetramer-binding CD8 T cells and the numbers are the percentages of BrdU-positive cells of tetramer-binding CD8 T cells ± standard deviation (data are the means for three to four mice per group). (B and C) Eight days after infection, the number of apoptotic LCMV-specific CD8 T cells in the spleen was quantitated directly ex vivo by staining with anti-CD8, MHC class I tetramers, and annexin V. The percentage of annexin V-binding tetramer-positive CD8 T cells was determined by flow cytometry. The dot plots in panel B are gated on D^b NP396 tetramer-binding CD8 T cells, and the numbers shown are the percentages of apoptotic cells of NP396-specific CD8 T cells. The data in panel C are the means for five to six mice per group ± standard deviation from three independent experiments.

FIG. 4.

Memory phenotype CD8 T cells in TNFR-deficient mice. (A and B) Groups of wild-type, p55^−/−, p75^−/−, and DKO mice were infected with LCMV-Armstrong. Two hundred and ten days after infection, splenocytes were stained with anti-CD8 and anti-CD44 antibodies. The dot plots in panel A are gated on total splenocytes, and the numbers are the percentages of CD44^hi CD8 T cells among total CD8 T cells. Data in panel B are the means for three mice per group ± standard deviation; data shown is representative of six independent experiments. (C) Groups of uninfected wild-type, p55^−/−, p75^−/−, and DKO mice (age matched with mice used in experiments in panels A and B) were sacrificed, and splenocytes were stained with anti-CD8, anti-CD44, anti-CD122, and anti-Ly-6C antibodies. The percentages of CD44^hi, CD122^hi, and Ly-6C^hi cells among CD8 T cells were determined by flow cytometry. The data are the means for two to three mice per group ± standard deviation.

FIG.5.

CD8 T-cell memory in TNFR-deficient mice. Between days 120 and 480 after infection with LCMV-Armstrong, the number of LCMV-specific memory CD8 T cells in the spleens of wild-type, p55^−/−, p75^−/−, and DKO mice was determined by staining with anti-CD8 antibodies, MHC class I tetramers (loaded with NP396 or GP33 peptides), and anti-CD44 antibodies. The data in panel A were obtained on day 480 after infection. Dot plots are gated on total CD8 T cells and show staining for CD44 and the indicated MHC class I tetramers. The numbers are the percentages of tetramer-binding CD8 T cells among total splenocytes. Note the enhanced frequencies of NP396-404-specific memory CD8 T cells in p55^−/− and DKO mice compared to wild-type mice. A notable enhancement in the frequencies of GP33-41-specific memory CD8 T cells is seen in DKO mice. Panel B shows the total number of LCMV-specific memory CD8 T cells in the spleens of wild-type, p55^−/−, p75^−/−, and DKO mice in several independent experiments (experiment 1, day 120 postinfection; experiment 2, day 210 postinfection; experiment 3, day 300 postinfection; experiment 4, day 480 postinfection). Panel C shows the total number of naïve (CD44^lo) and activated/memory (CD44^hi) CD8 T cells in the spleens of wild-type, p55^−/−, p75^−/−, and DKO mice in various experiments. Data in panels B and C are the means for 3 to 10 mice per group for each experiment ± standard deviation. Please note the log scale in the graph.

FIG. 6.

LCMV-specific memory CD8 T cells in the nonlymphoid organs of TNFR-deficient mice. Fifteen months after LCMV infection, mononuclear cells were isolated from the livers of wild-type, p55^−/−, p75^−/−, and DKO mice. Mononuclear cells isolated from the livers were stained with anti-CD8 antibodies and MHC class I tetramers. The dot plots are gated on viable mononuclear cells based on forward and side scatter and show staining for CD8 and MHC class I tetramers (loaded with NP396-404 peptide). The numbers are mean percentages of tetramer-binding CD8 T cells among mononuclear cells ± standard deviation; numbers in parentheses are percentages of tetramer-binding CD8 T cells among total CD8 T cells in a representative mouse. The data are the means for three to four mice per group and representative of two independent experiments.

FIG. 7.

Expression of CD62L and CCR7 on LCMV-specific memory CD8 T cells in TNFR-deficient mice. Three hundred and eighty days after infection with LCMV, splenocytes were stained with anti-CD8, anti-CD62L, MHC class I tetramers (specific to NP396 and GP33 epitopes), and CCL19-Fc (ligand for CCR7). The dot plots are gated on tetramer-binding CD8 T cells. The numbers in each quadrant represent percentages of cells (± standard deviation) of a particular phenotype among NP396-specific CD8 T cells (upper panel) or GP33-specific CD8 T cells (lower panel). Data are the means for two to three mice per group and representative of two independent experiments.

FIG. 8.

Proliferative renewal of LCMV-specific memory CD8 T cells in TNFR-deficient mice. Groups of wild-type, p55^−/−, p75^−/−, and DKO mice were infected with LCMV-Armstrong. One hundred and twenty days after infection, LCMV-immune mice were administered BrdU in drinking water for 8 days. After 8 days of BrdU treatment, mice were sacrificed and splenocytes were stained with anti-CD8 antibodies, MHC class I tetramers (specific to NP396-404), and anti-BrdU antibodies. The histograms showing BrdU staining are gated on tetramer-binding CD8 T cells. The bold and broken lines represent staining of tetramer-binding CD8 T cells for BrdU in BrdU-treated and untreated LCMV-immune mice, respectively. The numbers are the percentages of NP396-404-specific memory CD8 T cells that incorporated BrdU in the 8-day period, and the data are the means for four mice per group± standard deviation from two independent experiments.