The TNFR family members OX40 and CD27 link viral virulence to protective T cell vaccines in mice (original) (raw)
Virulence correlates with the generation of persistent VACV-specific CD8+ T cell populations. Numerous VACV strains have been isolated, including the prototypical strain used in the laboratory, Western Reserve (WR), as well as others such as Lister (used as a smallpox vaccine in Europe) and the New York City Board of Health strain (NYCBOH) used as a smallpox vaccine in the United States (Dryvax, produced by Wyeth). All viruses are known to differ in expression of several virulence factors, although the exact extent of variation is not fully documented. For example, the VACV WR encodes B18R, which produces an IFN-I–binding protein that neutralizes this cytokine. Lister does not possess this gene, and NYCBOH produces a truncated B18R gene product that is inactive (20). Another difference is the activity targeting the IL-1β–converting enzyme encoded by the B13R gene that is found in WR, but is produced as a truncated protein in NYCBOH and not present in Lister (21). As a direct example of an engineered attenuated vaccine vector, we also used a mutant WR strain in which only the B18R gene was deleted (22), referred to herein as WR-B18R.
When infection was i.p., WR was slowly cleared, with a strong reduction in viral load only obvious between days 14 and 15, whereas NYCBOH and Lister were cleared by day 3 in the spleen and day 7 in the ovaries (Figure 1A). WR-B18R exhibited intermediate characteristics, with reduced titers in the spleen between days 3 and 4, and moderately lower titers compared with WR in the ovaries at day 7. To further demonstrate altered virulence between these strains, mice were exposed to i.n. virus at varying doses (Figure 1B). Whereas even a low dose of 104 PFU WR was not controlled well, resulting in severe weight loss, 106 PFU Lister, NYCBOH, or WR-B18R was not pathogenic, and little to no weight loss was observed. This was not dependent on the background of the host, as similar results were obtained after i.p. or i.n. infection of BALB/c mice (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI42056DS1). Thus, in i.p. and i.n. infection models, the VACVs WR-B18R, Lister, and NYCBOH were highly attenuated compared with the native VACV WR.
Altered virulence of VACV strains. C57BL/6 WT mice were infected i.p. with different strains of VACV (2 × 105 PFU). (A) On the indicated days after infection, ovaries and spleens were removed from individual mice, and VACV titers were determined as described in Methods. (B) WT mice were infected i.n. with 104 PFU WR and 105 or 106 PFU WR-B18R, Lister, and NYCBOH as indicated. Animals were weighed daily. Mean percent of initial body weight is shown. Results are mean (n = 4 per group) from 1 of 3 experiments. *P < 0.05 vs. WR.
To assess induction of CD8+ T cell memory, the immunodominant VACV-reactive CD8+ T cell population was tracked with a tetramer of a peptide of B8R (23–25) or through the production of IFN-γ following stimulation with vaccinia epitopes. At 40 days after i.p. infection, the frequency of memory B8R tetramer–reactive (i.e., B8R+) CD8+ T cells (and CD8+IFN-γ+ T cells; data not shown) in both secondary lymphoid organs and peripheral organs such as the lung was 2- to 4-fold greater in mice infected with WR than in those infected with WR-B18R, Lister, or NYCBOH (Figure 2A). T cell avidity was also assessed by intracellular IFN-γ staining of freshly isolated splenocytes from WR- and Lister-infected mice. At 40 days after infection, memory CD8+ T cells were stimulated for 6 hours with graded concentrations of B8R, and cytokine-producing cells were analyzed by intracellular cytokine staining. As shown in Supplemental Figure 2, the amount of IFN-γ production (as measured by mean fluorescent intensity) on a per-cell basis was almost identical between WR- and Lister-infected groups over a wide range of peptide concentrations, similar to other assessments of IFN-γ (Figure 2, C and D). This suggests that exposure to varying levels of viral antigen primarily affects the frequency of CD8+ T cells elicited rather than the functional activity of individual cells.
VACV virulence correlates with magnitude of CD8+ T cell memory. WT mice were infected i.p. (A and C–F) or i.n. (B) with the indicated VACV strains. Splenocytes and lung cells were harvested on day 40 (A), day 60 (B), day 540 (C and D), or days 4, 5, 6, 7, and 8 (E and F) after infection and stained with anti-CD8, -CD44, and -B8R or for intracellular IFN-γ after restimulation with the designated VACV peptides. (A) Representative plots of gated CD8+ T cells staining for CD44 and B8R in spleen and lung. Numbers indicate percent CD44+B8R+ cells after gating on CD8+ T cells. Total numbers of CD8+CD44+B8R+ T cells per organ were determined as described in Methods. *P < 0.05 vs. WR. (B) Number of CD8+IFN-γ+ T cells per lung specific for peptides B8R, A3L, A8R, and B2R. (C and D) Percent CD8+IFN-γ+ T cells per spleen (C) or lung (D) specific for B8R and A8R. (E and F) Total number of CD8+CD44+B8R+ (E) and CD8+IFN-γ+ (F) T cells per spleen at the indicated time points after infection with VACV variants. Results are mean ± SEM (n = 4 per group) from 1 of 2 experiments. *P < 0.05 vs. all other strains or as otherwise denoted.
Furthermore, mice exposed to virus via the i.n. route (Figure 1B) also generated 2- to 5-fold fewer lung-resident memory CD8+ T cells in response to WR-B18R, Lister, or NYCBOH, even when 30-fold more virus was inoculated (Figure 2B). This did not simply apply to the B8R+ population, but was observed when CD8+ T cells were examined reactive with peptides of A3L, A8R, and B2R. Notably, 540 days after i.p. infection, the frequency of persisting memory VACV-reactive CD8+ T cells in both spleen and lung was 3- to 10-fold greater in mice infected with WR compared with those infected with WR-B18R, Lister, or NYCBOH (Figure 2, C and D).
Analysis of the initial effector CD8+ T cell response showed that this difference was likely to be explained by altered molecular regulation during the primary infection, as the size of these populations of VACV-specific T cells correlated to an extent with the size of the memory T cell pools and, again, opposite to the rate of virus clearance (Figure 2, E and F). Therefore, the immune system generates greater numbers of VACV-reactive memory CD8+ T cells with virus that replicates most extensively and is not rapidly cleared, consequently exposing the immune system to viral antigens for extended periods.
Selective use of OX40 to drive VACV-specific CD8+ T cells related to virulence. We then assessed whether the molecular control of CD8+ effector and memory T cells elicited by the attenuated VACV strains paralleled that in response to WR. We recently demonstrated a major role for OX40/OX40L interactions in generating CD8+ T cell memory in response to WR (25) and therefore initially focused on these molecules. OX40 is not constitutively present on T cells, but has to be induced by antigen recognition and inflammatory stimuli; similarly, OX40L is only expressed on APCs such as dendritic cells and B cells after their activation by a variety of stimuli and cytokines (18, 26). Importantly, the absence of OX40 only impaired memory in response to WR, but did not affect the number of memory CD8+ T cells induced by WR-B18R, Lister, or NYCBOH (Figure 3, A and B). In fact, the magnitude of the CD8 response to WR in Ox40–/– mice highly correlated with the response to WR-B18R, Lister, and NYCBOH in WT mice. Therefore, a large cytokine-competent, VACV-reactive memory CD8+ T cell pool only formed when OX40 became active in response to strongly replicating virus. Virus-specific memory T cells did form when OX40 was not brought into play, but their frequency was markedly reduced.
OX40 does not drive CD8+ T cell memory with attenuated viruses or when viral replication is low. (A–G) WT or Ox40–/– mice were infected i.p. with various VACV strains (2 × 105 or 2 × 106 PFU) as indicated. (H) At 1 day after infection with WR, mice were injected i.p. with 200 μg poly:IC. At day 40 (A and B) or day 7 (C, D, F, and G), splenocytes were stained with CD8 plus CD44, CD62L, and B8R or intracellular IFN-γ, and the number of VACV-reactive CD8+ T cells was calculated. (F) Representative plots of gated CD8 cells staining for CD62L and B8R in spleen. Numbers indicate percent CD62L+B8R+ and CD62L–B8R+ cells after gating on CD8+ T cells. Results are mean ± SEM (n = 6 per group). *P < 0.05 vs. WT. Similar results were obtained in 3 separate experiments. (E and H) On day 4 after infection, spleens from the indicated groups were removed from individual mice, and VACV titers were determined as described in Methods. *P < 0.05.
To again determine whether this related to initial priming events during the periods of active virus replication, the CD8+ T cell response at day 7 was assessed. There were approximately 75% fewer primary CD8+B8R+ T cells induced in Ox40–/– mice after WR infection. In contrast, little difference in priming of CD8+ T cells reactive with B8R (or another peptide, A23R; data not shown) was observed in Ox40–/– mice infected with WR-B18R, Lister, or NYCBOH (Figure 3C). To further show that the overall response and use of OX40 was related at least in part to the extent of attenuation and to viral load, mice were infected with 10 times as much virus (Figure 3D). The CD8+ T cell response to WR-B18R was strongly increased, to levels similar to WR, when given at the lower dose; OX40 became relevant and accounted for the enhanced response. The requirement for OX40 observed with a higher inoculum of WR-B18R directly correlated with increased viral titers recovered from the spleen (Figure 3E). A similar trend was also seen with NYCBOH: a 10-fold higher infection dose resulted in slightly elevated CD8+ T cell responses, albeit not to the same extent as WR or WR-B18R, and much of the enhanced response was attributable to OX40.
To extend these findings, we injected poly:IC at the time of WR infection. Treatment with poly:IC is known to lead to elevated levels of cytokines like TNF and IFN-I that can suppress viral replication. Strikingly, poly:IC resulted in only low numbers of VACV-reactive CD8+ T cells being generated, to levels approximating those induced in response to Lister and NYCBOH (Figure 3, F and G). Most importantly, the weaker CD8 expansion seen with WR in the context of poly:IC injection directly correlated with reduced viral titers in the spleen (Figure 3H), and this T cell response was independent of OX40. These data further show that the use of OX40 to drive enhanced CD8+ T cell priming and memory is related to the extent of viral load.
CD28 and CD27 are differentially active, depending on the stage of CD8 response and viral virulence. To determine whether this differential use of a stimulatory receptor applies to other, similar molecules, CD28 and CD27 were examined. CD28 is a costimulatory receptor in the Ig superfamily known to regulate initial activation of T cells in many immune responses in concert with antigen signals (27), and it has variably been reported to be required for CD8+ T cell responses against viruses (28). CD28 is constitutively expressed on T cells and binds B7.1 and B7.2 expressed on many APCs, and CD28-B7.2 interactions play a role in generating pools of VACV-reactive CD8+ T cells after infection with WR (28, 29). CD27 is another TNFR family member that acts independent of CD28 to promote T cell responses. CD27 is also constitutive on all T cells, but CD70, its ligand, is similar to OX40L and only induced once APCs such as dendritic cells or B cells receive certain inflammatory signals (18, 19). CD27/CD70 interactions were previously shown to be active in driving the CD8+ T cell response to WR (30). Furthermore, CD27 and OX40 have previously been shown to cooperate together to regulate T cell memory to influenza virus (31, 32).
When memory was assessed 40 days after VACV i.p. infection, the requirement for CD27 followed the principles demonstrated for OX40: although CD27 contributed substantially to development of the high frequency of CD8+ T cells elicited to WR, it was not active in the weaker responses to Lister and NYCBOH (Figure 4A). This selective use of CD27 was again reflected in the primary effector response at day 8, in that CD27 was not required for the smaller CD8+ T cell populations elicited in response to Lister or NYCBOH, but controlled those induced in response to WR (Figure 4B). There was a small defect in the CD8+ T cell response in Cd27–/– mice infected with the mutant WR-B18R virus, but again, the response in Cd27–/– mice largely followed that in Ox40–/– mice.
Virulence of VACV and stage of CD8+ T cell response determine the selective use of CD28, OX40, and CD27. WT, Cd28–/–, Ox40–/–, or Cd27–/– mice were infected i.p. with VACV variants (2 × 105 PFU) as indicated. At day 40 (A) or day 7 (B), splenocytes were stained with CD8 plus CD44 and B8R, and the number of VACV-reactive CD8+ T cells was calculated. Results are mean ± SEM (n = 6 per group). *P < 0.05 vs. WT. Similar results were obtained in 2 separate experiments. Inset in A shows the same data for WR-B18R, Lister, and NYCBOH with the scale enlarged.
Cd28–/– mice revealed a divergent pattern of usage. CD28 was required for development of primary CD8+ T cells induced to all virus variants when assessed at day 8. In contrast, at day 40, CD28 still contributed significantly to memory CD8+ T cell populations generated in response to WR-B18R, Lister, and NYCBOH, but memory T cell development in response to native WR was unaffected (Figure 4, A and B). The kinetics of development of VACV-reactive CD8+ T cells defined 2 obvious phases: a CD28-dependent phase, regulating primary effector cells, and a CD28-independent phase, beginning at day 12, in which the frequency of CD8+ T cells was equivalent in WT and Cd28–/– mice (Figure 5, A and B). Further demonstrating that this CD28-independent memory generation was driven by the TNFR family molecules, blocking CD70 in Cd28–/– animals from day 5 after infection largely recapitulated the defect seen in the absence of CD27 (Figure 5, C and D). Thus, the strong T cell memory elicited in response to the virulent virus strain was dependent on 2 TNFR family interactions, whereas the weaker memory in response to Lister, NYCBOH, and WR-B18R strains was driven by CD28 and largely independent of the TNFR family molecules.
CD27 signaling is critical for CD28-independent CD8+ T cell responses to WR. (A and B) WT or Cd28–/– mice were infected i.p. with WR (2 × 105 PFU). At the indicated days after infection, splenocytes were stained with CD8 plus CD44 and B8R (A), or intracellular IFN-γ or TNF (B), and the number or percentage of VACV-reactive CD8+ T cells was calculated. (C and D) WT, Cd28–/–, or Cd27–/– mice were infected i.p. with WR and injected i.p. on days 5, 6, 7, and 8 (C) with 150 μg nondepleting anti-CD70 blocking Ab in PBS. At day 13 after infection, splenocytes were harvested and stained for CD8, CD44, and B8R. (D) Representative plots of B8R staining, gating on CD8+ T cells, as well as total number of CD8+CD44+B8R+ T cells per spleen. Numbers within dot plots denote percentage of cells in the respective quadrants/gates. Results are mean ± SEM (n = 4 per group). *P < 0.05. Similar results were obtained in 2 separate experiments.
Immunization with WR, but not with NYCBOH or Lister, protects mice lacking MHC class II against lethal respiratory virus challenge. Next, this alternate use of costimulatory receptors that drove expanded memory was studied in terms of protection against subsequent viral encounter. Because antibody can protect against VACV, we chose a model whereby CD8+ T cell activity can be separated from antibody-mediated protection. After i.n. infection with WR, naive mice exhibit weight loss and die within 6–9 days, but memory CD8+ T cells induced by peptide vaccination can afford protection in this situation (25). MHC class II–deficient (MHCII–/–) mice, which lack CD4+ T cells and cannot mount a VACV-specific neutralizing antibody response, were vaccinated with WR and Lister and challenged 70 days later with a lethal i.n. dose of WR. All mice vaccinated with WR survived the infection and exhibited only 10%–15% weight loss (Figure 6, A and B). Protection was dependent on CD8+ T cells, since their depletion before challenge resulted in 100% mortality. In contrast, no protection was evident in mice vaccinated with Lister or NYCBOH, and all succumbed to the infection with kinetics similar to those of naive mice (Figure 6, A and B, and data not shown). This response paralleled the titers of virus detected in the lungs, which were strongly reduced (100- to 1,000-fold) in mice vaccinated with WR, but not in those vaccinated with Lister (Figure 6C). Consistent with the notion that the frequency of memory T cells dictated protection, far fewer B8R-specific memory CD8+ T cells were present in the spleen and lungs of MHCII–/– mice after immunization with Lister (Figure 6, D and E). This shows that the CD8+ T cell memory elicited because of varying levels of replication, and the differential use of OX40 and CD27, correlated with the degree of protection that could subsequently be provided.
Vaccination of MHCII–/– mice with WR, but not NYCBOH or Lister, protects against lethal respiratory virus challenge. MHCII–/– mice were immunized i.p. with VACV variants (2 × 105 PFU). Naive MHCII–/– mice were used as control. 10 weeks after vaccination, mice were infected i.n. with a lethal dose of WR (4.5 × 106 PFU; i.e., 400 × LD50). Some MHCII–/– groups were depleted of CD8+ (αCD8) T cells before i.n. challenge with VACV. Animals were weighed daily and euthanized if weight loss was greater than 30% body weight. Mean percent survival (A) and percent of initial body weight (B) are shown. 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) On day 7 after challenge, tissues from individual mice that survived the infection were collected, and virus titers were determined by plaque assay as described in Methods. Results are mean (n = 4 per group) from 1 experiment. (D and E) Percent and total number of CD8+CD44+B8R+ cells and B8R-reactive, IFN-γ–producing CD8+CD62L– cells in the spleen (D) and lungs (E) of MHCII–/– mice 90 days after immunization with WR or Lister. Results are mean ± SEM (n = 4 per group) from 1 experiment. *P < 0.05 vs. WR.
To formally demonstrate that the frequency of memory CD8+ T cells is a primary determinant of the extent of protection afforded in the absence of CD4+ T cells and antibody, mice were infected with WR; 35 days later, varying numbers of naive (CD8+CD44lo) or VACV-reactive memory (CD8+CD44hiB8R+) T cells were isolated and then transferred directly into the lungs of naive recipients by intratracheal injection. As a positive control, infected mice were bled at the same time, and VACV-immune serum was prepared and transferred i.p. into separate groups of naive mice. All groups were subsequently infected i.n. with a lethal dose of WR. Mice that received naive CD8+ T cells did not survive the infection, whereas 100% were protected by direct intratracheal transfer of 1 × 104 B8R+ T cells (Figure 7A). Lower numbers of VACV-specific CD8+ T cells either protected a fraction of mice or failed to protect at all, with the effect essentially proportional to the number of memory cells transferred. Similarly, the extent of disease as measured by weight loss directly correlated with the number of B8R-reactive memory CD8+ T cells transferred (Figure 7B).
The frequency of CD8+ T cells in the lung prior to challenge directly correlates with the degree of protection against lethal VACV infection. Naive CD8+CD44lo and VACV-reactive memory CD8+CD44hiB8R+ T cells were isolated from WR-infected mice, and varying numbers were instilled into the lungs of naive mice via the trachea. Some groups received 400 μl VACV-immune serum i.p. 1 day after transfer, mice were infected i.n. with a lethal dose of WR (1 × 106 PFU; i.e., 100 × LD50). Animals were weighed daily and euthanized if weight loss was greater than 30% body weight. Mean percent survival (A) and percent of initial body weight (B) are shown. 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. Results are mean (n = 4 per group) from 1 experiment.
Targeting OX40 enhances virus-specific CD8+ T cell responses to attenuated VACVs. We further investigated whether engagement of OX40 would boost the response to an attenuated VACV strain by treating with an agonist antibody after infection. Anti-OX40 enhanced CD8+ T cell priming in response to Lister, with 300%–400% more tetramer- and IFN-γ–producing cells detected regardless of epitope specificity (Figure 8, A and B). To exclude the possibility that the enhanced VACV-specific CD8+ T cell response was dependent on CD4+ T cells, MHCII–/– mice were infected with Lister. Anti-OX40 treatment again strongly enhanced the accumulation of these CD8+ T cells (Figure 8, C and D).
Enhanced CD8+ T cell responses to attenuated VACVs following agonist anti-OX40 treatment. WT (A and B) and MHCII–/– (C–E) mice were infected with 2 × 105 PFU Lister i.p. 1 day later, mice were treated with 150 μg control rat IgG or anti-OX40 (αOX40). 8 days after infection, VACV-specific CD8+ T cells were assessed by tetramer (A and C) or by intracellular IFN-γ staining after stimulation with the indicated VACV peptides (B and D). Data are either representative plots of tetramer staining in gated CD8+ T cells, with percent positive indicated, or total number (mean ± SEM) of CD8+IFN-γ+ T cells per spleen from 4 individual mice. *P < 0.05. Similar results were obtained in 2 separate experiments. (E) MHCII–/– mice were immunized i.p. with WR or Lister (2 × 105 PFU). 1 day later, cohorts of Lister-immunized mice were treated with 150 μg anti-OX40. Naive MHCII–/– mice were used as control. 10 weeks after vaccination, mice were infected i.n. with a lethal dose of WR (4.5 × 106 PFU; i.e., 400 × LD50). Animals were weighed daily and euthanized if weight loss was greater than 25% body weight. Mean percent of initial body weight is shown, with percent survival indicated.
Most importantly, anti-OX40 provided complete protection against death in MHCII–/– mice vaccinated with Lister, which alone was ineffective at preventing lethality (Figure 8E). Notably, the extent of protection when mice were vaccinated with the combination of Lister and anti-OX40 was similar to that when mice were vaccinated with the virulent VACV strain WR. Thus, exogenous OX40 signals given at the time of activation of naive VACV-specific CD8+ T cells promote a large population of CD8+ T cells that have the ability to fully protect against subsequent lethal virus infection.
WR and Lister scarification generates superior protective CD8+ T cell immunity against i.n. viral challenge that is mediated by CD28, OX40, and CD27. Finally, we addressed the findings of a recent study demonstrating that immunization with attenuated VACVs via skin scarification led to significantly higher levels of viral gene expression compared with other routes of immunization (33). Interestingly, this correlated with the development of greater numbers of IFN-γ–producing CD8+ T cells and the level of protection afforded against respiratory VACV challenge. To address whether this phenomenon relates to the use of OX40 and CD27 driving CD8+ T cell memory, we infected mice with WR and Lister by dermal scarification of the tail. Similar to the prior report (33), analysis of viral titers in the tail showed that in contrast to i.p. or i.n. Lister administration, very high levels of replication occurred, only moderately lower than that seen with WR, and Lister was not rapidly cleared (Figure 9A). The CD8+ T cell response to both WR and Lister in WT mice was strongly increased compared with i.p. infection (compare Figure 9, B and C, with Figure 3C); specifically, scarification with Lister resulted in numbers of CD8+B8R+IFN-γ+ T cells similar to those induced with i.p. WR (Figure 9, C and D). Most importantly, and correlating with our prior data and hypotheses, OX40 and CD27, as well as CD28, were then involved in driving this CD8+ T cell response to Lister (Figure 9, C and D). Immunization of MHCII–/– mice with Lister via scarification resulted in complete protection from death against i.n. VACV challenge (Figure 9E), again showing the relationship among viral replication, enhanced priming of CD8+ T cells, and use of these TNFR family costimulatory molecules.
WR and Lister scarification generates superior protective CD8+ T cell immunity against i.n. viral challenge that is mediated by CD28, OX40, and CD27. WT mice were infected by dermal scarification with WR and Lister (2 × 105 PFU). (A) On the indicated days after infection, VACV titers at the site of infection were determined as described in Methods. *P < 0.05. (B–D) 8 days after infection, splenocytes were harvested and stained with anti-CD8, -CD44, and -B8R or for intracellular IFN-γ after restimulation with B8R peptide. (B and C) Representative plots of gated CD8 cells staining for CD44 and B8R in spleen. Numbers indicate percent CD44+B8R+ cells after gating on CD8+ T cells. Total number of CD8+CD44+B8R+ T cells per organ was determined as described in Methods. (D) Percent CD8+IFN-γ+ T cells per spleen specific for B8R peptide. Results are mean ± SEM (n = 4 per group) from 1 of 3 experiments. *P < 0.05. (E) MHCII–/– mice were immunized by dermal scarification with WR and Lister (2 × 105 PFU). Naive MHCII–/– mice were used as control. 10 weeks after vaccination, mice were infected i.n. with a lethal dose of WR (4.5 × 106 PFU; i.e., 400 × LD50). Animals were weighed daily and euthanized if weight loss was greater than 30% body weight. 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.