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. 2008 Dec 1;181(11):7969-76.
doi: 10.4049/jimmunol.181.11.7969.

OX40 drives protective vaccinia virus-specific CD8 T cells

Shahram Salek-Ardakani  1 , Magdalini Moutaftsi, Shane Crotty, Alessandro Sette, Michael Croft
Affiliations

OX40 drives protective vaccinia virus-specific CD8 T cells

Shahram Salek-Ardakani et al. J Immunol. .

Abstract

Vaccinia virus (VACV) affords long-lasting protection against variola virus, the agent of smallpox. VACV-reactive CD8 T cells contribute to protection but their molecular control is unknown. We show that the TNFR molecule OX40 (CD134) controls primary VACV-specific CD8 T cell expansion and antiviral cytokine production and dictates development of strong memory to both dominant and subdominant VACV epitopes. Using adoptive transfer of OX40-deficient CD8 TCR-transgenic T cells responding to Ag in the context of VACV infection, we found that this reflects a direct action of OX40 expressed by CD8 T cells. Furthermore, CD8 T cells that can protect against lethal VACV challenge do not develop in mice deficient in OX40. Thus, OX40, which has been found to play little if any role in the generation of CD8 T cells to several viruses, including lymphocytic choriomeningitis virus and influenza, plays a dominant role in shaping the CD8 T cell response to VACV. These data suggest that unique costimulatory pathways might control alternate antiviral CD8 responses, demonstrating the plasticity of the immune response in utilizing different mechanisms to achieve similar ultimate goals.

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Figures

Figure 1
Figure 1. Intact activation but reduced early accumulation of VACV-specific CD8 T cells in OX40-deficient mice
(a) Wt mice were infected i.p with VACV-WR (2 × 105 PFU/mouse). On indicated days post-infection splenocytes were harvested and stained for CD8, CD44, B8R-tetramer, and OX40. Top panel, Percentage of CD44-high expressing B8R-specific CD8 T cells. Bottom panel, Percentage of OX40+ cells gating on CD8+ CD44-high B8R-tetramer positive cells. Quadrant settings based on isotype controls. (b) WT or OX40-deficient (OX40−/−) mice were infected i.p with VACV-WR (2 × 105 PFU/mouse). At day 4, splenocytes were stained with CD8 plus CD44 and B8R-tetramer. Representative plots of tetramer staining, gating on CD8 cells. Percentages of activated B8R-tetramer positive CD8 T cells (CD8+CD44+B8R+) and naïve cells (CD8+CD44−) are indicated. (c) Mice were infected as above. At day 4, CD8 T cell activation was assessed by up-regulation of CD69, CD25, CD43, and down-regulation of CD62L and CD127 on B8R-tetramer positive CD44-high cells (left panel). Naïve (CD44-low B8R-tetramer negative) CD8 T cells were used as controls. Percentages that stained positive for each marker are indicated. Similar results were obtained in 3 separate experiments.
Figure 2
Figure 2. CD8 T cells lacking OX40 are defective in expanding and anti-viral cytokine production after infection with VACV
(a) WT or OX40-deficient (OX40−/−) mice were infected i.p with VACV-WR (2 × 105 PFU/mouse). At day 7, splenocytes were stained for B8R-tetramer, or stimulated with B8R peptide for intracellular IFN-γ and TNF staining. Top, left: Representative plots of B8R-tetramer staining, gating on CD8 T cells. Percentages of CD8+ CD62L-high and CD62L-low B8R-tetramer positive cells are indicated. Top, right: Representative plots for cytokine-staining, gating on CD8+CD62Llow cells. Percentages that stained positive for IFN-γ alone, or TNF and IFN-γ/TNF are indicated. Quadrant settings were based on controls, using infected splenocytes that were not stimulated with peptide, and uninfected splenocytes stimulated with each peptide (data not shown). Bottom: Total numbers of B8R-tetramer positive CD8+CD62L-high and CD62L-low T cells, CD8+IFN-γ+ cells, or CD8+TNF+, and CD8+IFN-γ+TNF+ cells per spleen. Results are mean number ± SEM (n=6 mice/group) from one experiment. *, p < 0.05 (wt mice vs knockout). Similar results were obtained in 3 separate experiments.
Figure 3
Figure 3. OX40 is required for optimal generation of effector CD8 T cells directed against dominant and subdominant VACV epitopes
WT or OX40−/− mice were infected i.p. with VACV-WR (2 × 105 PFU/mouse). On days 4 (a), 7 (b), and 15 (c) post infection IFN-γ-secreting CD8 cells were assessed by intracellular cytokine staining after stimulation with VACV peptides as indicated. Data are either representative plots of IFN-γ staining in gated CD8+CD62Llow T cells, with percent positive indicated, or total numbers ± SEM of CD8+IFN-γ+ T cells per spleen from four individual mice. *, p < 0.05 (WT vs OX40−/−). Similar results were obtained in 3 separate experiments.
Figure 4
Figure 4. Impaired generation of CD8 memory cells to both dominant and subdominant VACV epitopes in OX40-deficient mice
Groups of C57BL/6 wild type or OX40-deficient (OX40−/−) mice were infected i.p (a) or by dermal scarification (b) with VACV-WR (2 × 105 PFU/mouse). At day 40, splenocytes were stimulated with VACV peptides as indicated and CD8 T cell priming assessed by intracellular IFN-γ staining. Top: Representative plots of IFN-γ staining in gated CD8 T cells. Percent positive indicated. Bottom: Total numbers of CD8+IFN-γ+ cells per spleen. Results are mean number ± SEM (n=4 mice/group) from one experiment. *, p < 0.05 (wt mice vs knockout) as determined by Student’s t test. Similar results were obtained in 3 separate experiments.
Figure 5
Figure 5. OX40 is required directly by CD8 T cells responding to VACV infection
CFSE labeled naive WT or OX40−/− OT-I CD8 T cells were adoptively transferred into WT B6 (a-c) or OX40−/− (d) mice. One day later, mice were infected i.p. with recombinant VACV expressing full-length OVA (VACV-OVA; 2 × 106 PFU/mouse) or PBS as indicated. After 8 (a, d) 3 (b) or 30 (c) days, CD8 T cell expansion (a, d), division as measured by CFSE-dilution (b) and memory formation (c) were analyzed by tracking the transgenic TCR. Dot plots: Representative co-staining for Vα2 and Vβ5 after gating on CD8 cells. Percent positive indicated. Bottom: Total numbers of CD8+Vα2+Vβ5+ cells (a-d) or CD8+Vα2+Vβ5+IFN-γ+ cells (d) per spleen. Histogram: cell division of wt and OX40−/− CD8 T cells was analyzed on gated CD8+Vα2+Vβ5+ cells 72 h after infection with VACV-OVA. Results are mean number ± SEM (n=4 mice/group) from one experiment. *, p < 0.05 (wt mice vs knockout) as determined by Student’s t test. Similar results were obtained in 1 additional experiment.
Figure 6
Figure 6. OX40-deficient mice clear primary vaccinia infection with similar kinetics compared with wild type mice
WT or OX40−/− mice were infected i.p. with VACV-WR (2 × 105 PFU/mouse). On indicated days post infection, ovaries were removed and VACV-titers determined as described in methods.
Figure 7
Figure 7. OX40 signals control development of CD8 T cell responses that protect against lethal VACV infection
(a) WT or OX40−/− mice were immunized s.c. at the base of the tail with 10 µg of B8R peptide in IFA. Control groups received adjuvant but no peptide (PBS). 3 weeks post-vaccination, mice were infected i.n. with a lethal dose of VACV-WR (3.5 × 106 PFU/mouse [300 × LD50]). Animals were weighed daily and euthanized if weight loss was greater than 25% body weight. (b) Mean % survival and % of initial body weight from indicated numbers of mice. Mean weight data in some cases were not plotted beyond the point at which mice died and beyond day 7 reflected only mice that survived infection. (c) As indicated, groups of WT mice were depleted of CD4 (αCD4) or CD8 (αCD8) T cells prior to intranasal challenge with VACV. (d-e) % and total numbers of CD8+CD44+ B8R-tetramer+ cells (d) and B8R-reactive IFN-γ producing CD8+CD62L- cells (e) in the lungs before intranasal VACV challenge (d0) and after challenge (d5). Results are mean number ± SEM (n=4 mice/group) from one experiment.
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