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. 2004 Aug;112(4):559-66.
doi: 10.1111/j.1365-2567.2004.01917.x.

4-1BB and OX40 stimulation enhance CD8 and CD4 T-cell responses to a DNA prime, poxvirus boost vaccine

Michael W Munks  1 , Dan V Mourich, Robert S Mittler, Andrew D Weinberg, Ann B Hill
Affiliations

4-1BB and OX40 stimulation enhance CD8 and CD4 T-cell responses to a DNA prime, poxvirus boost vaccine

Michael W Munks et al. Immunology. 2004 Aug.

Abstract

4-1BB (CD137) is a tumour necrosis factor receptor (TNFR) family member, expressed primarily on CD8 T cells after activation. Signalling through 4-1BB has been reported to enhance CD8 T-cell expansion and to protect activated CD8 T cells from death, resulting in an enlarged memory population. Although stimulating 4-1BB has been shown to significantly improve the immune response to weak immunogens such as tumours, little is known about its effect on the CD8 T-cell response to a powerful viral vector such as vaccinia. To test 4-1BB's ability to improve the murine CD8 T cell response to a DNA prime, poxvirus boost vaccine, similar to those used for human immunodeficiency virus and simian immunodeficiency virus vaccines, we administered 4-1BB agonist antibody at the time of the poxvirus boost. 4-1BB stimulation increased the number of functional memory CD8 T cells by two- to fourfold. However, we saw a similar enhancement at the peak of the response and in the memory phase, thus we found no evidence in the context of virus infection that 4-1BB stimulation could increase the percentage of CD8 T cells that survive the acute activation phase to become memory cells. OX40 (CD134) is an analogous TNFR family member expressed primarily on activated CD4 T cells. OX40 stimulation increased the number of antigen-specific CD4 T cells approximately threefold. Stimulating both 4-1BB and OX40 enhanced the CD8 T-cell response more than 4-1BB alone. Thus stimulating these receptors can improve the response to a powerful virus vector, and may be useful in vaccine development.

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Figures

Figure 1
Figure 1
4-1BB enhances CD8 T-cell activation and memory. BALB/c mice were primed with 100 µg of plasmid pTH.HM twice, 1 week apart. Two weeks later, mice were boosted with 1 × 106 pfu of MVA.HM and received 200 µg of either purified anti-4-1BB mAb or rat IgG i.p. with the boost. At the indicated number of days later, unfractionated spleen cells were cultured directly ex vivo for 6 hr with brefeldin A, in the presence or absence of 1 µg/ml RGPGRAFVTI peptide, then stained for CD8 and intracellular IFN-γ. One representative is shown from each group (a). Each point represents the average percentage (b) and number (c) of antigen-specific CD8 T cells ± SEM (day 6, n = 3 mice per group; day 22, n = 4 mice per group). A second experiment gave similar results.
Figure 2
Figure 2
4-1BB improves long-term CD8 T-cell memory. C57Bl/6 mice were vaccinated with 100 µg of plasmid pOVA twice, two weeks apart. Thirty days later, mice were vaccinated with 2 × 105 pfu of rVV-OVA and received 200 µg of either purified anti-4-1BB mAb or rat IgG i.p. (a) At the indicated number of days later, unfractionated spleen cells were cultured directly ex vivo for 6 hr with brefeldin A, in the presence or absence of 1 µg/ml SIINFEKL peptide, then stained for CD8 and intracellular IFN-γ or (b) TNF-α. (c) The percentage of total spleen cells that were CD8+. (d) OVA-specific CD4 T cells were quantified in parallel to SIINFEKL-specific CD8 T cells. Unfractionated spleen cells were cultured directly ex vivo in the presence or absence of 400 µg/ml whole ovalbumin protein for 10 hr, stimulated for an additional 8 hr in the presence of brefeldin A, then stained for CD4 and intracellular IFN-γ. Data shown are the average ± SEM (day 6, n = 6 mice per group; day 60, n = 10 mice per group). A second experiment gave similar results.
Figure 3
Figure 3
4-1BB and OX40 cooperate to enhance CD8 T and CD4 T cells. C57Bl/6 mice were vaccinated with 100 µg of plasmid pOVA twice, 2 weeks apart. Thirty days later, mice were vaccinated with 2 × 105 pfu of rVV-OVA i.p. At the time of the rVV boost, mice were divided into four groups and received either 200 µg of anti-4-1BB mAb + 200 µg rat IgG, 200 µg of anti-OX40 mAb + 200 µg rat IgG, 200 µg anti-4-1BB mAb + 200 µg anti-OX40 mAb, or 400 µg of rat IgG. Five weeks later, spleen cells were examined for antigen-specific CD4 and CD8 T-cell responses. (a) Unfractionated spleen cells were cultured directly ex vivo in the presence or absence of 400 µg/ml whole ovalbumin protein for 10 hr, stimulated for an additional 8 hr in the presence of brefeldin A, then stained for CD4 and intracellular IFN-γ. OVA-specific CD4 T cells are shown as both a percentage of CD4+ T cells and as the number per spleen. (b) Unfractionated spleen cells were cultured directly ex vivo for 6 hr with brefeldin A, in the presence or absence of 1 µg/ml SIINFEKL peptide, then stained for CD8 and intracellular IFN-γ. SIINFEKL-specific CD8 T cells are shown as both a percentage of CD8+ T cells and total number per spleen. Data shown are the average ± SEM (n = 10 mice per group). A second experiment gave similar results.
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