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. 2007 Apr 15;178(8):5227-36.
doi: 10.4049/jimmunol.178.8.5227.

CD8+ T cell dysfunction and increase in murine gammaherpesvirus latent viral burden in the absence of 4-1BB ligand

Shinichiro Fuse  1 , Sarah Bellfy, Hideo Yagita, Edward J Usherwood
Affiliations

CD8+ T cell dysfunction and increase in murine gammaherpesvirus latent viral burden in the absence of 4-1BB ligand

Shinichiro Fuse et al. J Immunol. .

Abstract

Studies of costimulatory receptors belonging to the TNFR family have revealed their diverse roles in affecting different stages of the T cell response. The 4-1BB ligand (4-1BBL)/4-1BB pathway has emerged as a receptor-ligand pair that impacts not the initial priming, but later phases of the T cell response, such as sustaining clonal expansion and survival, maintaining memory CD8(+) T cells, and supporting secondary expansion upon Ag challenge. Although the role of this costimulatory pathway in CD8(+) T cell responses to acute viral infections has been well-studied, its role in controlling chronic viral infections in vivo is not known to date. Using the murine gammaherpesvirus-68 (MHV-68) model, we show that 4-1BBL-deficient mice lack control of MHV-68 during latency and show significantly increased latent viral loads. In contrast to acute influenza infection, the numbers of MHV-68-specific memory CD8(+) T cells were maintained during latency. However, the virus-specific CD8(+) T cells showed defects in function, including decreased cytolytic function and impaired secondary expansion. Thus, 4-1BBL deficiency significantly affects the function, but not the number, of virus-specific CD8(+) T cells during gammaherpesvirus latency, and its absence results in an increased viral burden. Our study suggests that the 4-1BB costimulatory pathway plays an important role in controlling chronic viral infections.

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Conflict of interest statement

Disclosures

The authors have no financial conflict of interest.

Figures

FIGURE 1
FIGURE 1
4-1BB is expressed on virus-specific effector CD8+ T cells. 4-1BB expression on CD8+ T cells specific for the ORF61524–531/Kb and ORF6487–495/Db epitopes was analyzed. Shaded histograms, isotype control; bold lines, anti-4-1BB. Representative plots from the spleen and MLN at indicated time points are shown. Cells are gated on CD8+tetramer+ cells.
FIGURE 2
FIGURE 2
Increased latent viral burden in the absence of 4-1BBL. A, Lungs and spleens were harvested at indicated time points postinfection and were assayed for viral titers by a standard plaque assay using NIH3T3 cells. Each point represents data from an individual animal. B, Total number of splenocytes at day 14 postinfection is graphed. Error bars indicate SD. C–E, The latent viral load was measured in the spleens at day 14 postinfection (C), at 3 mo postinfection (D), or in the lungs 3 mo postinfection (E) by QF-PCR for the ORF50 gene. Each point represents data from an individual animal and horizontal bars indicate mean values. Representative data from two to three independent experiments (A–C) or individual experiments (D and E) are shown.
FIGURE 3
FIGURE 3
The magnitude of the virus-specific CD8+ T cell response is 4-1BBL independent. CD8+ T cells specific for the ORF61524–531/Kb and ORF6487–495/Db epitopes in the peripheral blood (A), MLN (B), and spleen (C and D) were quantified at indicated time points by MHC/tetramer staining. The percentage (A), or the total number (B–D), of CD8+ T cells specific for each epitope are graphed (error bars indicate SD). MLN were pooled for each group. Representative data from two independent experiments with three to four mice per group are shown.
FIGURE 4
FIGURE 4
MHV-68-specific CD8+ T cells maintain their cytokine-secreting ability and phenotype in 4-1BBL-deficient mice. A, Phenotype of ORF61524–531/Kb-specific CD8+ T cells at 86 days postinfection. Representative data plots gated on CD8+tetramer+ cells are shown. Shaded histograms, Isotype control; bold lines, Ab indicated above plots. IFN-γ (B) or TNF-α (C) production by virus-specific CD8+ T cells at days 14 and 85 postinfection was measured by intracellular cytokine staining. B, Representative data plots gated on CD8+ cells are shown. Numbers indicate average percent of CD8+ T cells secreting IFN-γ (±SD). C, Representative data plots gated on CD8+ cells are shown. Numbers indicate percent of IFN-γ-secreting cells that also secreted TNF-α (±SD). For each figure, representative data from two experiments using three to four mice per group are shown.
FIGURE 5
FIGURE 5
Cytotoxicity by virus-specific CD8+ T cells is decreased during latency in 4-1BBL-deficient mice. Cytotoxicity by virus-specific CD8+ T cells was measured 138 days postinfection by an in vivo CTL assay as described in Materials and Methods. Top, Representative data plots from naive B6 (Naive +/+), MHV-68-infected B6 (Infected +/+), or MHV-68-infected 4-1BBL−/− mice (Infected −/−), gated on 7-AAD-negative splenocytes. CTOhigh, No peptide; CFSEhigh, ORF61524–531 pulsed; CFSElow, ORF6487–495 pulsed. Numbers indicate average of total cell numbers in each population (three to four mice per group). Bottom, Specific killing was calculated and graphed. Error bars indicated SD. Representative data from two independent experiments are shown.*, p < 0.05.
FIGURE 6
FIGURE 6
Impaired degranulation by virus-specific CD8+ T cells in the absence of 4-1BBL. A, Degranulation by the virus-specific CD8+ T cells at day 85 postinfection were analyzed as described in Materials and Methods. Representative data plots gated on CD8+IFN-γ+ cells are shown. Numbers indicate average percent positive for CD107a (±SD). Shaded histograms, Isotype control; bold lines, anti-CD107a. B, Granzyme B expression was examined upon restimulation with indicated peptides in vitro for 5 h. Representative data plots gated on CD8+IFN-γ+ cells are shown. Numbers indicate mean fluorescence intensity (±SD). Representative data from two experiments using three to four mice per group are shown.*, p < 0.01.
FIGURE 7
FIGURE 7
MHV-68-specific memory CD8+ T cells are defective in secondary expansion in the absence of 4-1BBL. A, Naive B6 (Naive +/+), MHV-68-infected B6 (Infected +/+), or MHV-68-infected 4-1BBL−/− mice (Infected −/−) were challenged i.p. with 106 PFU of rVV-ORF61 or ORF6 at day 100+ post-MHV-68 infection. Virus-specific CD8+ T cell expansion in the spleen was enumerated at day 5 postchallenge by MHC/peptide tetramer staining. The experimental scheme is indicated above. Plots are gated on CD8+ cells and shown is the average percentage of tetramer+ cells ± SD. **, p < 0.02 (Mann-Whitney U test, two-tailed). B, Expression of 4-1BB on virus-specific CD8+ T cells specific for the indicated epitopes was analyzed at the indicated days after secondary challenge of infected B6 mice with rVV-ORF6. Representative data gated on CD8+tetramer+ cells are shown. C, A total of 2 × 107 splenocytes from MHV-68-infected B6 or 4-1BBL−/− mice 4 mo postinfection were adoptively transferred into naive B6-CD45.1+ recipients. One day posttransfer, mice were challenged with 2.5 × 106 PFU of rVV-ORF6 i.p., and expansion of virus-specific CD8+ T cells was measured by MHC/peptide tetramer staining at 5 days postchallenge. Experimental scheme is shown above, and the percentages of tetramer+CD8+ T cells among the donor population before and after transfer are graphed below. Each dot represents an individual animal and horizontal bars indicate mean value. D, Similar experiments described in C were performed, except that 2 + 107 splenocytes from MHV-68-infected B6-CD45.1 plus 2 mo postinfection were adoptively transferred into naive B6 or 4-1BBL−/− mice recipients. Representative data from two to three independent experiments with three to four mice per group are shown. *, p < 0.05, Student’s t test.
FIGURE 8
FIGURE 8
CD4+ T cell and humoral responses to MHV-68 in 4-1BBL−/− mice. A and B, Virus-specific CD4+ T cell responses were quantified at day 20 (A) or day 154 (B) postinfection by an ELISPOT assays as described in Materials and Methods. Error bars indicate SD. C and D, Virus-neutralizing activity in the serum at 20 (C) or 138 (D) days postinfection was assayed by a virus-neutralizing assay. Error bars indicate SD. For each figure, representative data from two experiments using three to six mice per group are shown.*, p < 0.005, Student’s t test.
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