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Keywords:

  • CD8 T cell;
  • Memory;
  • OX40

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Supporting Information

There is growing evidence that engagement of OX40 (CD134), a member of the TNF receptor superfamily, can directly stimulate antigen-specific CD8+ T cells. It has been shown that CD8+ T cells express OX40 following activation, but the response of antigen-specific CD8+ T cells to OX40 stimulation has not been fully characterized. We utilized an antigen-specific transgenic CD8+ T cell model (OT-I) to determine if OX40 engagement can boost the generation of antigen-specific CD8+ T cell memory. Our results demonstrate that enhanced OX40 costimulation, via an agonist anti-OX40 antibody, increases CD25 and phospho-Akt expression on the antigen-specific CD8+ T cells and significantly increases the generation of long-lived antigen-specific CD8+ memory T cells. The increased numbers of memory CD8+ T cells generated via anti-OX40 treatment still required the presence of CD4+ T cells for their long-term maintenance in vivo. In addition, anti-OX40 costimulation greatly enhanced antigen-specific CD8+ T cell recall responses. These data show that OX40 engagement in vivo increases the number of antigen-specific CD8+ memory T cells surviving after antigen challenge and has implications for the development of more potent vaccines against pathogens and cancer.

Abbreviation:
APC:

allophycocyanin

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Supporting Information

A fundamental feature of protective immunity is the formation of CD8+ T cell memory. Memory CD8+ T cells are generated during the course of an immune response after exposure to antigen. Initially, antigen-specific CD8+ T cells rapidly divide and differentiate into a population of effector cells. Expansion is followed by contraction as antigen levels decrease, leading to the loss of 90–95% of antigen-specific T cells due to intrinsic pressures, in the form of programmed cell death, or a lack of extrinsic survival factors, such as cytokines 14. Generation of antigen-specific memory CD8+ T cells occurs as a result of the protection of T cells from apoptosis during the contraction phase, which allows further differentiation into long-term memory cell populations 5, 6. A critical feature of long-term memory CD8+ T cells is their ability to expand more rapidly and respond more effectively than naive T cells upon antigen rechallenge 7, 8. Several elements are essential for the formation of memory CD8+ T cell populations, including inflammation (“danger signals”), the initial exposure to antigen and costimulatory signals 911.

The costimulatory molecule OX40, a membrane-bound member of the tumor necrosis factor-receptor superfamily, has been widely shown to be expressed by CD4+ T cells as well as CD8+ T cells 12, 13. OX40 has been studied extensively on CD4+ T cells, which express high levels of OX40 after T cell receptor-specific activation. The effects of OX40 engagement on CD4+ T cells include increased migration, enhanced cytokine production by effector cells, overcoming tolerance and increasing the numbers of memory CD4+ T cells 12, 1417. Indeed, it appears that OX40 primarily affects CD4+ T cell function; however, several recent reports have demonstrated that OX40 engagement has a direct effect on antigen-specific CD8+ T cells in vivo and in vitro1821. In these reports, expansion of antigen-specific CD8+ T cells and increased production of IFN-γ were shown to occur as a consequence of OX40 engagement.

In this study, we investigated the effects of OX40 engagement on the generation and maintenance of CD8+ T cell memory to soluble antigen following treatment with anti-OX40 antibody in the CD8+ transgenic ovalbumin (OVA)-specific T cell receptor OT-I adoptive transfer mouse model. Our results demonstrate that OX40 engagement increases the generation of antigen-specific CD8+ memory T cell populations after initial expansion. Although the anti-OX40 treatment led to a significant increase in numbers of memory CD8+ T cells, their long-term maintenance and survival was dependent upon the presence of CD4+ T cells. Additionally, we found potential roles for CD25 and phospho-Akt in expansion and survival, as both were increased after OX40 stimulation in vivo. Finally, anti-OX40 treatment during subsequent rechallenge with antigen produced extremely robust recall responses of antigen-specific memory CD8+ T cells when compared to control mice treated with rat IgG. Thus, OX40-mediated costimulation significantly increased the formation of antigen-specific memory CD8+ T cells and greatly enhanced recall responses.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Supporting Information

Anti-OX40 treatment enhances expansion and survival of antigen-specific CD8+ T cells

Using the same immunization scheme from previous studies that have shown co-injection of an agonistic anti-OX40 antibody with soluble antigen dramatically enhances the number of antigen-specific memory CD4+ cells, we examined the effects of anti-OX40 on the expansion and survival of OVA-specific TCR-transgenic OT-I CD8+ T cells 12, 17. The transgenic CD8+ T cells used here expressed OX40 between 24 and 72 h after antigen priming (Fig. 1A), which coincided with anti-OX40 treatment. Mice that received adoptively transferred OT-I T cells and were immunized with soluble OVA protein and anti-OX40 exhibited a significant increase in expansion of the transferred OT-I T cells in the spleen, draining lymph nodes and peripheral blood compared to controls (Fig. 1B, C). In addition to an increase in the early expansion of OT-I T cells, anti-OX40 stimulation also increased the long-term survival of these cells. Significantly more OT-I T cells persisted in the peripheral blood, lymph nodes and spleens of anti-OX40-treated mice than control mice for up to 100 days after immunization (Fig. 2A, B). The accumulation of OT-I cells in anti-OX40-treated animals appears to be a result of increased protection from apoptosis, as OT-I CD8+ T cells from animals treated with rat IgG exhibited greater annexin V staining compared to OT-I cells from anti-OX40-treated animals (Fig. 3A). Functionally, surviving OT-I cells from anti-OX40- and rat IgG-treated animals were capable of producing similar levels of INF-γ and TNF-α following in vitro restimulation (data not shown), but anti-OX40 treatment boosted the total number of cytokine-producing cells (Fig. 2C).

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Figure 1. Anti-OX40 increases antigen-specific CD8+ T cell expansion in vivo. (A) CFSE-labeled OT-I T cells (Thy1.1) were adoptively transferred into C57BL/6 recipient mice, which were immunized with OVA s.c. (day 0). Lymph nodes and PBL were harvested at the days indicated and OT-I T cells analyzed for OX40 expression. Graphs depict the percentage of OX40+ cells from one out of four mice from each group. (B) OT-I CD8+ T cells were adoptively transferred into C57BL/6 recipient mice, which were immunized s.c. with OVA and anti-OX40 or rat IgG (day 0). Mice received a second dose of anti-OX40 or rat IgG 1 day after immunization. PBL, draining lymph nodes and spleens were analyzed for OT-I cells (CD8+Vα2+Vβ5+) on days 2, 4 and 6 following immune challenge. (C) Absolute numbers of OT-I cells in the draining lymph node and spleen on day 4.

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Figure 2. Anti-OX40 increases antigen-specific CD8+ T cell survival in vivo. OT-I CD8+ T cells were adoptively transferred into C57BL/6 recipient mice, which were immunized s.c. with OVA and anti-OX40 or rat IgG (day 0). Mice received a second dose of anti-OX40 or rat IgG 1 day after immunization. (A) PBL were analyzed at various times after initial challenge for OT-I cells (CD8+Vα2+Vβ5+). (B) The number of OT-I T cells in the lymph nodes (LN) and spleens was determined 56 days following initial antigen challenge. (C) Anti-OX40 and rat IgG-treated OT-I T cells harvested 35 days after initial priming were analyzed for intracellular expression of IFN-γ and TNF-α following in vitro restimulation (6 h) with SIINFEKL (5 μg/mL). The means of three to ten mice/group ± SEM are shown (*p⩽0.05).

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Figure 3. Phenotype of OT-I CD8+ T cells following anti-OX40 treatment. (A) OT-1 CD8+ T cells from mice that received adoptively transferred T cells and were then immunized with OVA and anti-OX40 or rat IgG were harvested 4 days after immunization and CD8+ T cells purified. Cells were then cultured for 24 h in medium (without restimulation of Ag or cytokines) and OT-1 CD8+ T cells analyzed for annexin V expression. (B) CFSE-labeled OT-I T cells were adoptively transferred into C57BL/6 recipient mice, which were then immunized s.c. with 500 μg soluble OVA on day 0 along with 50 μg anti-OX40 or rat IgG on days 0 and 1. OT-I T cells were harvested from the draining lymph nodes on days 1–4, and the expression of CD25 and CD62L on the donor OT-I T cells (CD8+Thy1.1+) was analyzed by FACS. Graphs depict the percentage of CD25high cells from one out of three or four mice from each group. Data are representative of one out of four independent experiments with similar results. (C) OT-I T cells from mice treated with anti-OX40 or rat IgG were purified and lysates prepared as described in the Materials and methods. The lysates were separated by SDS-PAGE and immunoblotted first with antibodies specific for phospho-Akt (p-Akt) and then, after stripping, with antibodies specific for total Akt. The mean of three to four mice/group ± SEM is shown (*p⩽0.05 and **p⩽0.01).

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Anti-OX40 treatment affects the phenotype of activated antigen-specific CD8+ T cells

Given the ability of anti-OX40 treatment to promote enhanced CD8+ T cell expansion following antigen challenge, we next sought to determine whether OX40 engagement affects the early activation phenotype or division rate of OT-I T cells following immunization. CFSE-labeled OT-I T cells (CD25low and CD62Lhigh) were adoptively transferred, and mice were immunized with OVA protein and anti-OX40 or rat IgG. Draining lymph nodes were harvested after antigen priming (days 1, 2, 3 and 4), and the phenotype and cell division rate of OT-I T cells were analyzed. The OT-I T cells from anti-OX40- or rat IgG-treated mice had similar rates of proliferation as measured by dilution of CFSE (Supplemental Fig. 1). Antigen priming and anti-OX40 treatment (as compared to rat IgG) did not significantly increase the expression of CD25 on OT-I T cells early in the response (days 1 and 2); however, anti-OX40 significantly sustained CD25 expression as seen on days 3 and 4 (Fig. 3B). At these later times, the frequency of CD25 expression ranged from ∼20% to 70%. In contrast, downregulation of CD62L, another activation marker, was equivalent in these mice (data not shown).

We next determined whether the activation of Akt, a protein associated with T cell survival, was different between OT-I T cells from mice treated with anti-OX40 or rat IgG. Purified OT-I T cells from the lymph nodes of both anti-OX40- and rat IgG-treated mice 4 days after immunization were subjected to immunoblotting to determine if anti-OX40 had an effect on phospho-Akt expression, as the activated form of Akt is associated with IL-2-mediated T cell survival 22. There were significantly greater levels of phospho-Akt in OT-I T cells from anti-OX40-treated mice (compared to control mice) when phospho-Akt was normalized to the amount of total Akt by NIH Image J software analysis (Fig. 3C). These data suggest that, as observed for CD25 expression, treatment with anti-OX40 most likely sustains the activation of Akt compared to rat IgG.

Anti-OX40 affects the phenotype of antigen-specific CD8+ memory T cells

The generation of memory CD8+ T cells can be followed by analyzing changes in the cell surface phenotype of antigen-specific CD8+ T cells over time. Both CD62L and CD127 (IL-7 receptor) represent useful phenotypic markers to observe memory T cell generation and development 2325. CD127, normally expressed on naive CD8+ T cells, is down-regulated during the effector phase and then re-expressed during contraction. The increased expression of CD127 on CD8+ T cells earmarks these cells as memory cells that can further differentiate into long-lived memory cells 25. As CD8+ T cells transition into long-term memory cells, the increased expression of CD62L, in combination with CD127, identifies a subset of memory cells that can proliferate rapidly following antigen rechallenge 25, 26.

Using CD127 and CD62L to identify CD8+ T memory cells, we examined the phenotype of CD8+ T cells generated following antigen immunization and anti-OX40 treatment. Following adoptive transfer of T cells and immunization as described above, OT-I T cells were analyzed for the expression of CD127 and CD62L at various times after immunization (Table 1). At day 4, the peak of expansion, the frequency of OT-I cells expressing CD127 was <10% in both control and anti-OX40-treated mice. As the population of OT-I T cells contracted, the percentage that expressed CD127 significantly increased in anti-OX40-treated mice as compared to controls (day 12). The frequency of CD127+CD62L+ OT-I T cells continued to increase, compared to the controls, in mice that received anti-OX40 (days 21 and 42). Therefore, it appears that anti-OX40 not only enhances survival of antigen-specific CD8+ T cell populations but also increases the formation of memory cells that co-express CD127 and CD62L.

Table 1. Anti-OX40 treatment increases the frequency of antigen-specific CD8+ T cells expressing a memory phenotypea)
OT-I (% of CD8+)CD127+ (%)CD127+CD62L+ (%)
  1. a) Data represent the mean ± SEM of congenic OT-I CD8+ T cells (CD45.2+) from peripheral blood harvested from animals treated with OVA and anti-OX40 or rat IgG (n=10) and are representative of two independent experiments.b) Different from rat IgG (p⩽0.005)c) Different from rat IgG (p⩽0.0005)

Day 4rat IgG53.47±6.7510.14±1.215.58±1.17
anti-OX4077.03±3.43b)9.25±0.513.93±0.33
Day 12rat IgG6.16±1.1769.60±2.2139.71±3.41
anti-OX4030.52±3.01c)78.80±1.45b)44.66±1.65
Day 21rat IgG1.94±0.2465.59±1.8949.29±1.68
anti-OX4016.01±2.28c)85.78±1.69c)73.16±1.76c)
Day 42rat IgG1.01±0.1573.22±1.5057.55±1.46
anti-OX408.71±1.14c)90.33±0.93c)77.45±1.29c)

CD4+ T cells influence survival of anti-OX40-stimulated antigen-specific CD8+ T cells

CD4+ T cells influence the development of CD8+ T cell responses by providing “help” and also appear to be important in the maintenance of CD8+ T memory cells 2729. In addition, OX40 engagement on CD4+ T cells has been shown to facilitate primary and memory CTL responses 30. Therefore, we sought to assess what role, if any, CD4+ T cells have in the survival of anti-OX40-stimulated antigen-specific CD8+ T cells. We adoptively transferred OT-I T cells into MHC class II-deficient mice before immunization. The initial expansion of OT-I T cells on day 4 was not different; however, the long-term survival of OT-I T cells was significantly impaired 45 days after immunization in MHC class II-deficient animals (Fig. 4). These results suggest that CD4+ T cells are required for the maintenance of antigen-specific CD8+ T cell memory in anti-OX40-treated mice. To corroborate these results from MHC class II-deficient mice, wild-type mice were depleted of CD4+ T cells using a single injection of anti-CD4 prior to adoptive transfer and immunization. Overall, the survival of antigen-specific OT-I T cells in anti-OX40-treated mice depleted of CD4+ T cells was significantly reduced compared to anti-OX40-treated CD4+ T cell intact mice (data not shown).

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Figure 4. Role of CD4+ T cells in anti-OX40-mediated expansion and survival of antigen-specific CD8+ T cells in vivo. OT-I T cells were adoptively transferred into MHC class II-deficient mice (class II ko) or wild-type C57BL/6 (Wt) recipient mice (five mice/group), which were then immunized s.c. with OVA and anti-OX40 or rat IgG (day 0). Mice received a second dose of anti-OX40 or rat IgG 1 day after immunization. PBL were analyzed at various times for OT-I T cells.

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Anti-OX40 enhances memory T cell proliferation upon antigen rechallenge

One of the hallmarks of CD8+ T cell memory is a rapid and vigorous recall response following antigen rechallenge 7, 8. Taking into account the ability of anti-OX40 stimulation to generate robust antigen-specific CD8+ T cell responses when given in combination with primary antigen challenge, we assessed the effect of anti-OX40 on antigen-specific CD8+ T cells in recall responses. Adoptively transferred OT-I T cells in mice primed with OVA and anti-OX40 or rat IgG were allowed 40 days to develop into a memory population, as shown previously (Table 1), and the mice were then rechallenged with OVA and anti-OX40 or rat IgG. The expansion and survival of OT-I T cells was analyzed on various days following secondary immunization. Mice that received anti-OX40 in the primary immunization and were then rechallenged with OVA and anti-OX40 exhibited the most vigorous recall response on day 4 (Fig. 5A). To our surprise, the next most vigorous response was observed in mice that received rat IgG during the primary immunization and were then rechallenged with OVA and anti-OX40. A weaker recall response was observed in mice rechallenged with OVA and rat IgG after receiving anti-OX40 at the time of the primary immunization. As expected, mice that received rat IgG in both the primary and secondary immunization had the weakest recall response, an approximate 9-fold lower frequency of OT-I T cells on day 4 compared to mice treated with anti-OX40 during both immunizations; by day 42 the difference was nearly 30-fold.

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Figure 5. Effect of anti-OX40 on recall responses. (A) OT-I CD8+ T cells were adoptively transferred into Ly5.2 recipient mice (ten mice/group), and the mice were immunized 24 h later s.c. with OVA and anti-OX40 or rat IgG (day 0). Mice received a second dose of anti-OX40 or rat IgG 1 day after immunization. PBL were analyzed 42 days after primary immunization for OT-I cells (CD8+CD45.2+). Mice were then divided into four groups (five mice/group) and immunized a second time with OVA and anti-OX40 or rat IgG. PBL were analyzed 42 days after challenge for OT-I T cells. (B) OT-I T cells were adoptively transferred into C57BL/6 recipient mice (ten mice/group), and the mice were immunized 24 h later s.c. with OVA and anti-OX40 or rat IgG (day 0). Mice received a second dose of anti-OX40 or rat IgG 1 day after immunization. PBL were analyzed 46 days following initial challenge for OT-I T cells (CD8+Vα2+Vβ5+). Mice were then divided into four groups (five mice/group), and 250 μg anti-CD4 (GK1.5) or PBS was injected i.p. to deplete CD4+ T cells. Mice in each group received a second immunization of OVA and anti-OX40 1 day later. PBL were analyzed at various times for OT-I cells (CD8+Vα2+Vβ5+). Anti-CD4 antibody was injected every 10 days i.p. for the duration of the experiment.

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To determine if the antigen-specific CD8+ memory T cells generated during the rechallenge were functional, their ability to produce IFN-γ upon antigen stimulation in vitro was measured. Spleen cells from rechallenged mice were pulsed with SIINFEKL peptide, and IFN-γ production was assessed by intracellular staining. Antigen-specific OT-I T cells from mice primed and rechallenged with OVA and anti-OX40 were analyzed 42 days after rechallenge and produced IFN-γ after in vitro restimulation, suggesting that these memory CD8+ T cells were indeed functional (data not shown).

We further investigated whether the effects of anti-OX40 on CD8+ T cell recall responses were CD4+ T cell-dependent. Recipients of adoptively transferred OT-I T cells were immunized in the primary challenge with soluble OVA and anti-OX40 or rat IgG. Seven weeks after primary immunization, these mice were given a depleting anti-CD4 mAb or control, creating the following four groups: 1) rat IgG/anti-CD4, 2) rat IgG/control, 3) anti-OX40/anti-CD4 and 4) anti-OX40/control. All mice were then immunized with the combination of OVA and anti-OX40 to assess the contribution of OX40-specific “help” in the expansion and survival of antigen-specific CD8+ T cells in the secondary response. The expansion of OT-I T cells in CD4-depleted mice was decreased compared to untreated mice 4 days after secondary challenge (Fig. 5B). Interestingly, the rate of contraction of these responses in CD4-depleted and control animals appeared to be similar. Thus, CD4+ T cells seem to be important in anti-OX40-mediated expansion of OVA-specific OT-I CD8+ T cells during a recall response, and the increase in survival may be dependent on the increased early expansion of CD8+ T cells during the recall response.

We next sought to determine whether anti-OX40 influences the expansion and survival of antigen-specific CD8+ T cells after multiple rounds of immunization. Six to eight weeks following the primary challenge with soluble OVA and anti-OX40, mice were split into two groups and rechallenged with OVA and anti-OX40 or rat IgG. Expansion and contraction of OT-I T cells in mice in these two groups mirrored what was seen previously (Fig. 6, primary challenge vs. Fig. 5A, anti-OX40-primed group). The mice that had received OVA and anti-OX40 were challenged for a third time (secondary challenge) with OVA and anti-OX40, and mice that received OVA and rat IgG in the primary challenge were rechallenged for a second time with OVA and rat IgG. The recall response in the secondary challenge was similar to the primary challenge: anti-OX40 treatment induced a strong initial expansion compared to rat IgG, followed by the establishment of a memory CD8+ T cell population that was nearly twice the level seen in the rat IgG group (Fig. 6). Moreover, multiple antigen rechallenges without anti-OX40 were unable to produce an increase in antigen-specific CD8+ memory T cells.

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Figure 6. Effect of anti-OX40 on recall responses following multiple antigen challenges. OT-I T cells were adoptively transferred into C57BL/6 recipient mice (n=10), and 24 h later the mice were immunized s.c. with OVA and anti-OX40 (day 0). Mice received a second dose of anti-OX40 1 day after immunization. PBL were analyzed for OT-I T cells (47 days after initial priming). Mice were then divided into two groups (five mice/group) and challenged (primary challenge) with OVA and anti-OX40 (black circles) or rat IgG (white circles). PBL were taken at various times and analyzed for OT-I T cells (CD8+Vα2+Vβ5+). Mice were challenged again (secondary challenge 60 days after the primary challenge): mice previously challenged with antigen and anti-OX40 were injected with OVA and anti-OX40, while mice previously challenged with antigen and rat IgG were injected with OVA and rat IgG. PBL were analyzed at various times for OT-I T cells.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Supporting Information

The generation of immunological memory, central to protective long-term immunity, depends on a number of elements, including antigen engagement of the TCR, inflammation (e.g. danger signals) and T cell costimulatory signals. Anti-OX40 injected in vivo increases the generation and maintenance of CD8+ T cell memory. Indeed, anti-OX40 increased both the overall size of the antigen-specific CD8+ T cell memory population and the frequency of antigen-specific cells that co-express the “central memory” markers CD127 and CD62L 2325. Our data demonstrate that anti-OX40 does not overcome the need for CD4+ T cells to maintain long-term CD8+ T cell memory 3. Finally, we observed that rechallenge of CD8+ memory T cells with antigen in combination with anti-OX40 in vivo leads to dramatic increases in antigen-specific CD8+ T cell expansion.

Examination of differences in the antigen-specific CD8+ T cell population found in the lymph node after priming and anti-OX40 treatment revealed a potential mechanism that may account for some or all of the anti-OX40-mediated survival effects. Anti-OX40 treatment had little effect on the rate of division of antigen-specific T cells; however, as CFSE can only trace approximately eight divisions, it is possible that anti-OX40 treatment allows for additional divisions. We observed anti-OX40 treatment sustained levels of CD25 expression in the lymph nodes. The prolonged increase in CD25 expression on antigen-stimulated CD8+ T cells could account for enhanced survival through accentuated IL-2 signaling. It has been shown that increased IL-2 signaling can enhance the survival of CD8+ T cells mediated through the PI3 kinase/phospho-Akt pathway 22. Indeed, we found increased phosphorylation of Akt in anti-OX40-treated mice compared to controls. Several groups have shown a direct link between IL-2 signaling and phospho-Akt expression, and increased phospho-Akt levels have been shown to be involved in increased survival of T cells 22, 31, suggesting that this pathway may have led to the enhanced anti-OX40-mediated CD8+ T cell survival observed. Alternatively, the increased levels of phospho-Akt may be due to direct effects of OX40 stimulation, as OX40 has been shown to induce and sustain phospho-Akt in CD4+ T cells 32. Sustained elevation of phospho-Akt levels could have effects on Bcl-xL, which may also play a role in OX40-mediated survival of T cells 33, 34.

OX40 costimulation not only had a pronounced impact on the establishment of CD8+ T cell memory following a primary response, it also promoted the production of a vigorous recall response in vivo. We noted two significant effects of OX40 engagement during antigen rechallenge: 1) increased expansion of antigen-specific effector CD8+ T cells and 2) increased survival of antigen-specific CD8+ T cells, likely due to the increased expansion. OX40 engagement during antigen rechallenge induced robust expansion of CD8+ T cells regardless of whether the initial antigen priming included anti-OX40, demonstrating the ability of OX40 costimulation to boost recall responses regardless of the size of the starting memory CD8+ T cell pool. Yet it appears that, other than the increase in cell numbers, OX40 stimulation did not confer any distinct advantages to the resulting memory pool. This is evident as memory T cells from control mice, while lower in number compared to memory cells from anti-OX40-primed animals, expanded to nearly the same extent following anti-OX40 stimulation upon antigen rechallenge. The recall responses boosted by anti-OX40 were found to be partially dependant on CD4+ T cells, which contrasts with another study reporting no effects of CD4+ T cell depletion on recall responses 29. We found that depletion of CD4+ T cells prior to rechallenge with antigen and anti-OX40 led to a significant decrease in the expansion of OT-I CD8+ T cells compared to intact animals, yet the rate of contraction appeared unchanged. Thus, enhanced OX40 stimulation effectively boosts recall responses, and these optimal responses appear to be partially dependent on CD4+ T cells.

The dramatic effects of anti-OX40 costimulation on recall responses reported here do not fully mirror the effects of OX40/OX40L in other models. Our data appear to be in conflict with data from several studies that showed either a minor effect or no effect at all on CD8+ T cell recall responses in OX40L-deficient mice 35, 36. However, other evidence shows a direct effect of OX40 on CD8+ T cells in these responses using OX40-deficient CD8+ T cells 20, 34. Differences in these studies may be due in part to the model, antigen or immunization scheme. Another potential explanation for the different results in studies using OX40L-deficient mice and anti-OX40 could involve CD4+CD25+ T regulatory cells (Treg), which have been shown to effectively suppress recall responses 37, 38. It has been shown that Treg-mediated suppression of immune responses can be abrogated by anti-OX40 stimulation 39, 40. Thus, anti-OX40 may release CD8+ recall responses from Treg suppression due to engagement of OX40 throughout the animal.

Our results have implications for the development of more potent viral vaccines and anti-cancer therapies that aim to boost both primary and memory immune responses. Optimal vaccine strategies must accomplish the following: 1) generate large numbers of effector cells after primary and subsequent antigen challenges, 2) produce memory cells with high proliferative/functional potential and, 3) for tumor-specific responses, overcome tolerance. For viral vaccines, anti-OX40 costimulation could boost T cell responses by increasing effector and memory T cell populations during chronic and acute phase of infections. We have shown that anti-OX40 enhances CD8+ and CD4+ T cell responses, both of which are required to produce an optimal response during chronic infection 41, 42. Most effective tumor vaccines require both CD4+ and CD8+ responses, and because many tumor antigens are self-antigens, the vaccines must also overcome the barrier of self-tolerance. Costimulation may be the key to overcoming this hurdle, as tumor vaccines that increase costimulation or boost costimulatory molecule expression on DC have been extremely effective 43, 44. Thus, enhanced OX40 costimulation could tip the balance from tolerance to activation. A second practical application of anti-OX40 costimulation would be the boosting of naturally occurring anti-tumor immunity. In a number of mouse tumor models, anti-OX40 treatment alone has been shown to induce tumor eradication, potentially acting upon both the CD4+ and CD8+ T cell arms of adaptive immunity 45. Furthermore, OX40 expression has been observed on CD8+ T cells found in the tumor infiltrate, making these cells potential targets for anti-OX40 activation 46. Therefore, anti-OX40 costimulation may improve vaccine strategies as well as anti-tumor therapies in the future.

In conclusion, we have identified a potential new role for OX40 in CD8+ T cell responses, increasing the formation and survival of antigen-specific memory CD8+ T cells. In addition, OX40 costimulation of recall responses induced vigorous expansion of antigen-specific CD8+ T cells following a secondary challenge that, in turn, led to a large increase in the establishment of long-term memory. Since the formation of long-term CD8+ T cell memory is a critical component of immunity against a host of pathogens and tumors, our results have the potential to enhance the efficacy of vaccines and cancer immunotherapies.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Supporting Information

Mice

Female C57BL/6 and Ly5.2 mice (4- to 6-wks-old) were purchased from the Charles River Laboratory (Boston, MA) and used at 6–10 wks of age. MHC class II-deficient animals (Abb, H2-Ab1) were purchased from Taconic (Germantown, NY). OT-I (Thy1.1) were bred and maintained at the EARCI animal facility.

Adoptive transfer and immunization

Spleens from OT-I mice were harvested and processed by crushing between two frosted glass microscope slides, and red blood cells were lysed with ACK (Gibco, Carlsbad, CA). The percentage of OT-I T cells, identified by Vα2/Vβ5 co-expression, was determined by FACS prior to adoptive transfer using Vα2-FITC, Vβ5-PE and CD8-PE CyChrome antibodies (BD Biosciences Pharmingen, San Diego, CA). In some cases OT-I mice were depleted of CD4+ cells by i.p. injection of anti-CD4 (GK1.5) 24 h prior to harvest. A total of 3 × 106 Vα2/Vβ5 cells were adoptively transferred i.v. into C57BL/6 or Ly5.2 recipients. One or two days later, mice were immunized s.c. with 500 μg OVA (Sigma, St. Louis, MO) and 50 μg anti-OX40 (OX86) or rat IgG (Sigma). The following day mice were given a second injection of anti-OX40 or rat IgG. In the rechallenge experiments, mice were immunized on the stated days with a single s.c. dose of OVA and anti-OX40 or rat IgG.

FACS analysis of cells from peripheral blood, lymph nodes and spleens

Mice were bled via the tail vein into 50 μL heparin, and 750 μL RPMI was added. The blood was underlayed with 500 μL Lympholyte M (Cedarlane Laboratories, Hornby, Ontario, Canada) and centrifuged. The cells at the interface were collected and washed with FACS buffer (1% FBS, 0.1% sodium azide in PBS). Spleens and lymph nodes were processed as previously described and stained with the following antibodies: FITC-Vα2, PE-Vβ5, allophycocyanin (APC)-CD62L, APC-Cy7-CD8, CyChrome-CD8, FITC-CD45.1, PE-Thy1.1, APC-CD25 or biotinylated-CD25, biotinylated-CD127 (BD Biosciences Pharmingen), PE-Cy5-CD127 (eBioscience, San Diego, CA) and biotinylated-OX40. Harvested samples, isotype controls and single-stain controls were run on aFACScalibur (Becton Dickinson, Franklin Lakes, NJ) or a CyAn flow cytometer (Dako-Cytomation, Ft. Collins, CO).

Intracellular cytokine staining

Splenic T cells were obtained as described above and stimulated for 6 h in vitro with 2.5 or 5 μg/mL SIINFEKL peptide in RPMI containing 10% fetal bovine serum and 1 mg/mL brefeldin A (BD Biosciences Pharmingen). The cells were harvested and stained with FITC-CD45.2 and PE-Cy5-CD8. Cells were permeabilized with CytoPerm/PermWash buffers and stained with allophycocyanin (APC)-IFN-γ and PE-TNF-α (BD Biosciences Pharmingen).

Immunoblot analysis

Lymph nodes were isolated 4 days after immunization, and cells were incubated with biotinylated anti-CD45.1. OT-I T cells were purified (>90%) with anti-biotin microbeads using an AutoMacs cell sorter (Miltenyi, Germany). Lysates were prepared, run on polyacrylamide gels (1.5 × 106 cell equivalents/lane) and transferred onto nitrocellulose membranes. Antibodies for p-Akt and Akt (Cell Signaling, Beverly, MA) were used to detect the proteins. ImageJ1.32j software (NIH, Bethesda, MD) was used to analyze the phospho-Akt/Akt data.

Determination of apoptosis

Lymph nodes from mice that received adoptively transferred T cells, followed by immunization, were harvested 4 days after immunization, and the CD8+ cells were purified (93–96%) by MACS beads as previously described. A total of 3 × 105 CD8+ cells were plated into wells of round-bottom 96-well plates in 100 μL media and were incubated overnight at 37°C. Cells were harvested 24 h later and stained according to the manufacturer's instructions with biotin-Vα2, FITC-Vβ5, CyChrome-CD8 and PE-annexin V (BD Biosciences Pharmingen).

CFSE labeling of OT-I CD8+ T cells

A total of 2 μL of a 5 mM solution of 5,6-carboxy-fluorescein-succinimidyl-ester (CFSE; Molecular Probes, Eugene, OR) in DMSO (Sigma) was added per 5 × 107 cells/mL in HBSS and incubated for 10 min at 37°C. Cells were then washed prior to injection.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Supporting Information

We would like to Drs. Walter Urba and Edwin Walker for their critical review of this manuscript and Dr. Michael Gough for his expertise in immunoblot technology. This work was supported by NIH grant R01-CA102577-04, Army Prostate Cancer Grant #DAMD 17-03-1-00B and the MJ Murdock Charitable Trust.

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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Supporting Information

Supporting information for this article is available on the WWW under http://www.wiley-vch.de/contents/jc_2040/2007/36428_s.pdf or from the author.

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