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The Journal of Immunology, 2006, 177: 4464-4472.
Copyright © 2006 by The American Association of Immunologists, Inc.

Functional Dichotomy between OX40 and 4-1BB in Modulating Effector CD8 T Cell Responses1

Seung-Woo Lee*, Yunji Park{dagger}, Aihua Song*, Hilde Cheroutre{dagger}, Byoung S. Kwon{ddagger} and Michael Croft2,*

* Division of Molecular Immunology and {dagger} Division of Developmental Immunology, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121; and {ddagger} Immunomodulation Research Center, University of Ulsan, Ulsan, Korea


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Members of the TNFR family are thought to deliver costimulatory signals to T cells and modulate their function and survival. In this study, we compare the role of two closely related TNFR family molecules, OX40 and 4-1BB, in generating effector CD8 T cells to Ag delivered by adenovirus. OX40 and 4-1BB were both induced on responding naive CD8 T cells, but 4-1BB exhibited faster and more sustained kinetics than OX40. OX40-deficient CD8 T cells initially expanded normally; however, their accumulation and survival at late times in the primary response was significantly impaired. In contrast, 4-1BB-deficient CD8 T cells displayed hyperresponsiveness, expanding more than wild-type cells. The 4-1BB-deficient CD8 T cells also showed enhanced maturation attributes, whereas OX40-deficient CD8 T cells had multiple defects in the expression of effector cell surface markers, the synthesis of cytokines, and in cytotoxic activity. These results suggest that, in contrast to current ideas, OX40 and 4-1BB can have a clear functional dichotomy in modulating effector CD8 T cell responses. OX40 can positively regulate effector function and late accumulation/survival, whereas 4-1BB can initially operate in a negative manner to limit primary CD8 responses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Costimulatory or coinhibitory molecules are required for driving T cell responses into immunity or tolerance (1). Two major families of membrane proteins have been identified as T cell modulators, the Ig and the TNFR superfamilies (2, 3). Whereas certain TNFR family molecules are involved in inducing apoptosis such as Fas, TNFRI, and TRAILR, others are thought to enhance T cell activation and survival, such as 4-1BB, CD27, CD30, OX40, glucocorticoid-induced TNFR family-related receptor, and herpesvirus entry mediator (HVEM)3 (3, 4). Upon ligation of their respective TNF family ligands, many TNFR family molecules show similar signaling patterns (3, 4). An important question to be resolved then is why T cells express many TNFR molecules that might possess seemingly redundant roles. One hypothesis is that T cells are controlled by sequential interactions between different TNF and TNFR molecules (4). For example, the initial priming and activation of naive T cells could be regulated by constitutively expressed TNFR molecules, whereas activated T cells could receive later acting costimulatory signals from inducible TNFR molecules. Another hypothesis is that each TNFR molecule might have preferential effects on specific types of T cells, or their action might vary depending on the stage of the T cell response when they are ligated (3, 4).

Both 4-1BB and OX40 appear to be closely related in terms of their inducible expression patterns and their apparent signaling capacity, e.g., each can recruit several common TNFR-associated factors (5, 6, 7), activate PI3K-Akt-NF-{kappa}B pathways (8, 9), and up-regulate expression of Bcl-2 family molecules (10, 11). Agonist Abs to 4-1BB and OX40 can enhance CD8 T cell responses against tumors and pathogens (12, 13, 14, 15, 16). Variable data have been reported using gene-deficient mice. Initial data from OX40–/– and OX40 ligand (OX40L)–/– mice suggested that OX40-OX40L is not required for CD8 priming to lymphocytic choriomeningitis virus (LCMV), vesicular stomatitis virus, and influenza virus (17, 18). In contrast, CD8 contact hypersensitivity (18) and alloantigen responses (19) were suppressed, and secondary responses to influenza virus were also impaired (20), in OX40L–/– mice. Moreover, recent studies using OX40-deficient TCR-transgenic CD8 T cells showed that OX40 was required for cell survival during certain primary (21) and secondary CD8 T cell responses (22). The 4-1BB ligand (4-1BBL)–/– mice generated reduced CD8 T cells reactive with LCMV and Listeria at the peak of primary responses (23, 24). However, i.p. infection of influenza virus in 4-1BBL–/– mice showed normal primary CD8 responses, but in this case with impaired memory formation (25, 26, 27). Collectively, these data suggest that OX40-OX40L and 4-1BB-4-1BBL interactions can provide enhancing signals, but their interaction is not always necessary to priming of CD8 T cells.

In contrast to studies of OX40-OX40L interaction and those studies of 4-1BBL–/– mice, recent results have suggested that 4-1BB biology is more complex than previously thought, and more complex than OX40 biology. Several studies have shown that agonist Abs to 4-1BB can be highly suppressive for humoral responses (28, 29) and for a number of autoimmune responses (30, 31, 32, 33, 34). Also, other data have now shown enhanced responses in 4-1BB-deficient animals (35, 36). The 4-1BB can be expressed on many kinds of immune cells including CD4+CD25+ regulatory T cells (37), NK cells (38, 39), NKT cells (40), dendritic cells (41, 42), granulocytes (43), and mast cells (44). Thus, various targets of 4-1BB action are possible, including regulatory APC or T cells, but pathogenic T cells have not been ruled out as recipients of suppressive 4-1BB signals, raising the issue of whether 4-1BB can have dual roles on T cells, both positive and negative.

In this study, we have addressed the specific role of OX40 and 4-1BB when expressed on responding naive or primed CD8 T cells. To rule out possible effects from other immune cells, we adoptively transferred OT-I TCR-transgenic CD8 cells from mice deficient in OX40 or 4-1BB into wild-type congenic mice, and then infected them with recombinant defective adenovirus expressing a membrane-bound form of the Ag OVA (Ad-mOVA) that is recognized by the T cells. Significantly, we show that OX40 and 4-1BB regulate effector CD8 T cell responses in a very opposing fashion. OX40 is important to maintain CD8 T cell numbers and functionality at the peak of primary responses, but 4-1BB acts in an inhibitory manner suppressing CD8 T cell expansion and function.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice and virus

OT-I TCR-transgenic mice bred on the C57BL/6 background were used as a source of SIINFEKL peptide-reactive CD8 T cells. OX40–/– and 4-1BB–/– mice were bred with OT-I mice to generate gene-deficient OT-I mice. C57BL/6, B6.PL-Thy1.1 (Thy1.1+), and B6.SJL-Ptprca Pep3b/BoyJ (CD45.1+) mice were purchased from The Jackson Laboratory. Ad-mOVA was purchased from Gene Transfer Vector Core, University of Iowa (Iowa City, IA).

Adoptive transfer of CD8 T cells and virus challenge

Naive CD8 T cells were purified from pooled lymph node (LN) and spleen cells of wild-type and gene-deficient OT-I mice as previously described (45). Briefly, total CD8 T cells were purified first with CD8 T cell isolation kits (Miltenyi Biotec) following the manufacturer’s protocol. Naive CD8 T cells were purified from total CD8 T cells through MACS by incubation with pretitrated biotin labeled anti-CD44 followed by antibiotin microbead. Purified naive CD8 T cells were phenotypically >99% CD8+CD44lowCD62Lhigh. Naive CD8 T cells (1 x 105 cells) were injected i.v. into congeneic B6.PLThy1.1 mice and 2 days after Ad-mOVA (2 x 107 PFU) was injected i.m. into both quadricep muscles. In some cases, purified naive CD8 T cells were labeled with 5 µM CFSE (Molecular Probes) for tracking cell division before adoptive transfer (~1–2 x 106 cells). For secondary responses of CD8 T cells, OT-I cells were stimulated with SIINFEKL peptide for 3 days and then rested with IL-15 (PeproTech) for another 3 days as previously described (22). Primed OT-I cells (5 x 105 cells) were transferred into congenic mice and infected i.m. with Ad-mOVA (2 x 107 PFU).

Flow cytometry analysis

Recipient mice were sacrificed at the indicated times, and the draining LN and spleen were harvested. An aliquot of cells was stained with anti-CD8 PerCP and anti-Thy1.2 FITC (BD Pharmingen). For staining of cell surface markers, the following Abs were used: PE-labeled anti-CD44, anti-CD69, anti-CD127, anti-CD27, anti-CD122, anti-CD62L, anti-CD43 (1B11), anti-CD25, or biotin-labeled anti-OX40 (all from BD Pharmingen), anti-4-1BB (Biolegend) followed by PE-labeled streptavidin (Molecular Probes). Intracellular staining was performed as previously described. Cells were stained with anti-CD8 PerCP and anti-Thy1.2 FITC. Cells then were fixed and permeabilized followed by intracellular staining with anti-TNF PE, anti-IFN-{gamma} allophycocyanin (BD Pharmingen), anti-human granzyme B PE (Caltag Laboratories), anti-mouse Bcl-2 PE, or PE-labeled isotype control Abs (BD Pharmingen).

CTL assay

Spleen cells were harvested from infected mice at day 6 and the number of activated OT-I cells (CD8+Thy1.2+CD44high) in individual spleens were normalized by FACS. Target cells were prepared using total spleen cells from CD45.1+ congenic mice that were differentially labeled with CFSE as previously described (46). The cells labeled with CFSEhigh (5 µM) were used as targets and pulsed with SIINFEKL peptide (10 µM), whereas the cells labeled with CFSElow (0.5 µM) were pulsed with OVA323–339 peptide (20 µM) to serve as the internal control. Cells were 1:1 mixed and then cocultured with spleen cells from infected mice for 7 h. Specific cytotoxicity was calculated by determining the ratio of CFSElow/CFSEhigh by FACS. For CTL assay at day 10, total CD8 T cells were purified first with MACS and then incubated with target cells for 18 h.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Overlapping and distinct expression profiles of OX40 and 4-1BB on CD8 T cells during priming

OX40 and 4-1BB have been thought to have similar patterns of expression on activated T cells, largely based on results from in vitro culture systems. Both molecules show clear inducible expression, which starts several hours after naive T cell activation and usually peaks 2–4 days into the response (4). However, expression patterns of both molecules have not been characterized well in vivo. After intranasal challenge with influenza virus, a minor population of T cells infiltrating the lung expressed OX40, but 4-1BB was absent (20). In other studies, CD8 T cells were shown to minimally express OX40 after s.c. injection of high dose OVA with LPS and 4-1BB was expressed only very transiently during the initial 12–36 h (27). In this study, we first compared expression of both molecules on responding naive CD8 cells after i.m. infection with Ad-mOVA in draining LN (Fig. 1A) and spleen (Fig. 1B). To aid visualizing expression throughout the course of cell division, we adoptively transferred a relatively high number of CFSE-labeled OT-I transgenic CD8 T cells (1–2 x 106) into congenic mice.


Figure 1
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  		FIGURE 1. Expression of OX40 and 4-1BB on CD8 T cells during the primary response to adenovirus. Purified naive wild-type or gene-deficient OT-I cells (Thy1.2+) were CFSE labeled (5 µM) and then adoptively transferred (1–2 x 106 cells) i.v. into congenic mice (Thy1.1+). Two days later, Ad-mOVA (2 x 107 PFU) was injected i.m., and then the expression of OX40 and 4-1BB on transferred wild-type OT-I cells (CD8+Thy1.2+) in draining LN (A) and spleen (B) was followed at the indicated times. C, The expression of OX40 and 4-1BB and division profile of transferred gene-deficient OT-I cells in draining LN at day 3. Each line shown represents the cutoff for the isotype control. Data are representative of three experiments.

 
The 4-1BB was detected at day 1 after infection before the CD8 population had divided. The majority of CD8 cells expressed 4-1BB between days 2 and 3, and then expression declined gradually thereafter, with levels being similar regardless of the number of divisions undergone. Interestingly, 4-1BB was long lasting on a significant population of responding CD8 cells and still expressed between days 4 and 7. In contrast, OX40 was largely absent at day 1 after infection and not expressed on activated but undivided cells. Corresponding with presumed Ag load, OX40 was induced more on LN compared with spleen CD8 cells between days 2 and 3. Interestingly, the level of OX40 correlated with the number of cell divisions, being higher in CD8 cells that had undergone more divisions. However, it sharply declined after day 3 and became undetectable on most responding CD8 cells, although a small population of cells (~5%) that had undergone more than eight divisions still expressed low levels of OX40 at late times. In general, OX40–/– and 4-1BB–/– CD8 cells showed wild-type cell division profiles and expressed the reciprocal molecule comparably to wild-type CD8 cells (Fig. 1C), suggesting that each molecule has no apparent role for early cell division and does not cross-regulate the expression of the other. Responsiveness of 4-1BB- and OX40-deficient CD8 cells to PMA/ionomycin or anti-CD3 in APC-free systems in vitro was also comparable to wild-type cells, also implying the absence of developmental defects in the CD8 cells from the knockout animals. Collectively, these results show that OX40 and 4-1BB have very distinct expression patterns on activated CD8 T cells responding to adenovirus. Both molecules can be coexpressed at a particular time during priming, but 4-1BB is expressed earlier than OX40, and 4-1BB is maintained over time, whereas OX40 is more transient.

Opposing roles of OX40 and 4-1BB in regulating the accumulation of CD8 T cells

To understand the functional role of OX40 and 4-1BB in priming of CD8 T cells, we adoptively transferred a lower number (1 x 105) of naive (>99% of CD44lowCD62Lhigh) wild-type or gene-deficient OT-I CD8 cells into wild-type congenic recipients and then tracked them after infection with Ad-mOVA. Initial expansion of CD8 cells was seen from day 3, peaking at days 5–7, and then the cells underwent a sharp contraction by day 10 (Fig. 2). Surprisingly, 4-1BB–/– CD8 cells showed clear hyperresponsiveness. The expansion of 4-1BB–/– cells started to outpace that of wild-type cells at day 3 (average number, OT-I cells = 3.58 x 104 vs 4-1BB–/– OT-I cells = 7.23 x 104), and this result was strongly evident by day 5 when ~2-fold more CD8 cells were found (Fig. 2A). Enhanced numbers of 4-1BB-deficient cells were visualized through to day 7; however, there was no statistical difference between wild-type and 4-1BB–/– CD8 cells at this time due to the individual variation among mice. Most likely some CD8 cells lacking 4-1BB already started to undergo clonal contraction while others were still expanding at this time point, thus accounting for the high variability. These results suggest that in the absence of 4-1BB, naive CD8 T cells expand much more efficiently during their primary response.


Figure 2
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  		FIGURE 2. Distinct roles of OX40 and 4-1BB in clonal expansion of CD8 T cells. Wild-type or gene-deficient naive OT-I cells (1 x 105 cells) were transferred into five to seven congenic mice that were immunized as in Fig. 1. The number of activated OT-I cells was calculated at the indicated times by tracking CD8+Thy1.2+CD44high cells. A, The number of wild type or gene-deficient OT-I cells (circle) from an individual mouse are indicated. B, Mean number of OT-I CD8 cells ± SEM over time is graphically represented. Similar results were seen in one separate experiment.

 
In contrast, the expansion of OX40–/– CD8 cells was relatively normal at day 3. There was a slight defect in their accumulation at day 5 but this was not statistically significant (Fig. 2A). However, the number of OX40–/– CD8 cells was greatly reduced at day 7, showing that OX40 has a positive role in maintaining cell numbers relatively late in the primary response. Wild-type CD8 cells underwent strong clonal contraction by day 10, and cells were almost undetectable by flow cytometry after day 14 making it impossible to determine whether the enhanced and reduced responses seen in the absence of 4-1BB and OX40 resulted in sustained differences in T cell frequency once the contraction phase ended. These data indicate that OX40 and 4-1BB have opposing roles in regulating clonal expansion of CD8 T cells in the primary response to adenovirus.

Because we found differences in the accumulation of CD8 cells (Fig. 2), we examined in vivo apoptosis (annexin V) and the expression of the antiapoptotic molecule Bcl-2 by flow cytometry at days 5–7. However, we could not find significant differences in the absence of OX40 or 4-1BB (data not shown).

Altered differentiation of effector CD8 T cells in the absence of OX40 or 4-1BB

When naive CD8 T cells differentiate into fully functional effectors, a number of phenotypic changes occur (47, 48, 49). To determine the role of OX40 and 4-1BB in the differentiation process we characterized effector markers at days 5–7 after infection. At the peak of response, wild-type effector CD8 cells were CD69neg, CD25neg, CD44high, and CD122high (data not shown). We did not find any significant difference in these markers in the absence of OX40 or 4-1BB (data not shown). Additionally, wild-type and OX40/4-1BB-deficient effector cells expressed a comparable level of activation-associated glycoform of murine CD43 (50) and completely down-regulated CD127 (IL-7R) at day 5, with some cells regaining expression at day 7 as previously reported (51) (Fig. 3). These results suggest that OX40–/– and 4-1BB–/– CD8 cells gained a number of characteristics of differentiated effectors. However, we found differences in the expression pattern of CD27 and CD62L, two other differentiation markers (Fig. 3). OX40–/– CD8 cells exhibited less down-regulation of both molecules than wild-type cells at day 5 and day 7. In contrast, 4-1BB–/– CD8 cells exhibited increased kinetics of down-regulation. Based on previous reports showing that highly activated effector CD8 T cells down-regulate CD62L (49, 51), and that CD27 loss is also a characteristic of maturation (52, 53), these results suggested that OX40 was required for full differentiation, whereas 4-1BB acted to limit differentiation at an early time of priming.


Figure 3
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  		FIGURE 3. OX40 is required for the down-regulation of effector CD8 T cell markers CD62L and CD27. Mice received naive OT-I cells (1 x 105) and were immunized as in Fig. 1. At 5 (A) and 7 (B) days, the expression of CD27, CD127, CD62L, and CD43 (1B11) were analyzed on gated wild-type and gene-deficient OT-I cells (CD8+Thy1.2+) that were pooled from four mice. Isotype control is shown by shaded histogram. Data are representative of two experiments.

 
Defective functional activity of effector CD8 T cells in the absence of OX40

To address any role of OX40 and 4-1BB in regulating effector function of CD8 T cells, we examined the ability to secrete IFN-{gamma} and TNF. A large proportion of wild-type and 4-1BB–/– CD8 cells rapidly secreted both cytokines by day 6 after infection, as measured by intracellular staining after in vitro stimulation with SIINFEKL peptide (Fig. 4, A and B). However, far fewer OX40–/– CD8 cells were positive for both cytokines. Interestingly at this time, OX40–/– CD8 cells synthesized IFN-{gamma} almost equivalently, but showed a strong defect in the ability to make TNF (Fig. 4B). Effector cytokine production by all CD8 populations diminished over time, but a substantial number of wild-type and 4-1BB–/– CD8 cells still produced both cytokines after restimulation at day 10 (Fig. 4C). Conversely, OX40–/– CD8 cells at this time now showed impaired IFN-{gamma} production as well as TNF production.


Figure 4
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  		FIGURE 4. OX40 regulates cytokine production from effector CD8 T cells. Mice received wild-type and gene-deficient OT-I cells and were immunized as in Fig. 1. Pooled lymphoid cells from groups of four mice were restimulated with SIINFEKL peptide for 6 h, and CD8+Thy1.2+ cells were stained for intracellular IFN-{gamma} and TNF at day 6 (A and B) and day 10 (C). B and C, The population expressing cytokines (circles) compared with an isotype control is shown. Data are representative of three experiments.

 
Lastly, we performed ex vivo direct CTL assays to test the killing activity of surviving CD8 cells. OT-I cells isolated at day 6 showed substantial cytotoxic activities (Fig. 5, A and B). Significantly, even after normalization of OT-I E:T ratios, OX40–/– CD8 cells showed a decreased cytotoxic activity, ~5- (E:T = 1.33:1) to 2-fold (E:T = 4:1) lower than that of wild-type CD8 cells (Fig. 5A). In contrast, 4-1BB–/– CD8 cells showed moderately higher cytotoxic activities. Similar results were found 8 days after infection (data not shown). Correlating with the altered cytotoxic activity of OX40-deficient T cells, we found that the majority of wild-type and 4-1BB–/– CD8 cells expressed high levels of granzyme B at day 6, whereas OX40–/– CD8 cells were strongly impaired in granzyme B expression (Fig. 5C). The level of granzyme B declined by day 8 in all CD8 cells, but there was still a clear defect in OX40–/– CD8 cells (Fig. 5D). Collectively, these results demonstrate that OX40 can control the killing activity of effector CD8 T cells through the regulation of cytotoxic genes such as granzyme B.


Figure 5
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  		FIGURE 5. Opposite roles of OX40 and 4-1BB in cytotoxic activity of effector CD8 T cells. Mice received wild-type and gene-deficient OT-I cells and were immunized as in Fig. 1. A and B, Lymphoid cells were isolated from groups of three to four mice and then normalized for the number of wild-type or gene-deficient OT-I cells after flow staining (CD8+Thy1.2+CD44high). Equivalent numbers of effector cells were incubated with a 1:1 mixture of CFSEhigh-labeled SIINFEKL-loaded target cells and CFSElow-labeled control peptide-loaded cells. After 7 h, cytotoxic activity at different E:T ratios was analyzed as shown in the CFSE histograms (A) and as summarized graphically (B). The values (right corner) in A represent the percentage of specific killing from one representative mouse. Each line in B indicates data from an individual mouse with the thick lines representing th mean ± SEM from three mice. C and D, Lymphoid cells from three to four mice were isolated at day 6 and day 8 and pooled, and stained for intracellular granzyme B in combination with Thy1.2 (C) or Thy1.2 and CD8 (D). The population expressing granzyme B (circles) are compared with an isotype control in C. Similar results were found in two separate experiments.

 
Enhanced secondary responsiveness of CD8 cells in the absence of 4-1BB

To further address the roles of OX40 and 4-1BB, we analyzed secondary responses of previously primed CD8 T cells. OT-I cells were stimulated in vitro and then rested in IL-15 to generate primed CD8 populations. These were then tracked after adoptive transfer, following an in vivo Ad-mOVA challenge. The majority of CD8 cells expressed OX40 and 4-1BB after 3 days during this secondary response (Fig. 6A). Primed 4-1BB–/– CD8 cells accumulated ~3-fold more than wild-type cells at day 5 after infection, whereas primed OX40–/– CD8 cells showed a defect of expansion (Fig. 6B), largely analogous to the phenotype seen during the primary response of CD8 cells. Consistent with this finding, 4-1BB–/– CD8 cells showed more differentiated phenotypic and functional attributes, such as greater down-regulation of CD62L and increased IFN-{gamma} and granzyme B expression (Fig. 6C). In contrast, no differences were observed between wild-type and OX40–/– CD8 cells. These results additionally illustrate that OX40 and 4-1BB play opposing roles in expansion and reactivity of CD8 cells.


Figure 6
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  		FIGURE 6. Distinct roles of OX40 and 4-1BB in secondary responses of CD8 cells. Mice received in vitro primed wild-type and gene-deficient OT-I cells, and were immunized as in Fig. 1. A, The expression of OX40 and 4-1BB on transferred OT-I cells (CD8+Thy1.2+) in LN at day 3 were analyzed. B, The number of OT-I cells was calculated at day 5 by tracking CD8+Thy1.2+ cells in the spleen. The number of wild-type or gene-deficient OT-I cells from an individual mouse. Bars represent mean number ± SEM of OT-I cells. C, Spleen cells from four mice were pooled at day 5 and stained for CD44, CD62L, and intracellular granzyme B in combination with Thy1.2 and CD8. Intracellular IFN-{gamma} was assessed after restimulation with SIINFEKL peptide for 6 h. Data are representative of two or three experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study we report that there is a functional divergence between OX40 and 4-1BB in modulating CD8 priming and effector CD8 responses to adenovirus-expressed Ag. In line with recent reports, OX40–/– CD8 T cells exhibited a defect in accumulating at high numbers over time, and importantly OX40 positively regulated the effector function of CD8 T cells. Surprisingly, in contrast to OX40, 4-1BB negatively regulated the expansion and maturation of effector CD8 T cells.

There have been inconsistencies reported in the literature regarding a role for OX40 in CD8 T cell responses. Agonist Abs to OX40 have shown clear positive effects on CD8 T cell responses in various models, suggesting CD8 cells can respond directly to OX40 signals (15, 16, 54). However, in such studies the target cell for these Abs has not been clear, and at most, only CD4 cells have been ruled out, still leaving the possibility that OX40 action on non-CD8 cells could indirectly augment CD8 priming. In contrast, our present data with OX40–/– CD8 cells responding to Ag provided by adenovirus, and our recent published data with response to Ag in CFA (21) or Ag expressed by a tumor (22), clearly establish that OX40 can control CD8 priming. However, other reports with OX40- or OX40L-deficient animals conversely have shown no role for these molecules in primary CD8 responses (17, 18, 27), begging the question of why OX40 sometimes can be relevant and sometimes irrelevant to CD8 priming. One factor to account for the discrepancies could be alternate expression patterns of OX40 under different physiological situations. OX40 is probably highly regulated on CD8 cells. OX40 was expressed only very transiently on CD8 cells at days 2–3 after adenoviral infection, in contrast to 4-1BB, which was expressed rapidly and for a long time. This transient OX40 expression correlates with that previously seen in a CFA inflammatory environment and in a tumor environment (P. Bansal-Pakala, A. Song, and M. Croft, unpublished observations). Most likely OX40 induction depends upon the amount of Ag as well as environmental factors such as cytokines and TLR signals made available by local inflammation. For example, in one study CD8 cells failed to express OX40 after injection of OVA in LPS, whereas 4-1BB was expressed (27), correlating with the functional conclusions from these authors that OX40 played no role in CD8 priming. Further studies will be required to understand how OX40 expression is regulated and what inflammatory stimuli control its expression.

In our present, and previous studies, OX40 does not appear to control initial priming events and early responses of CD8 T cells because naive OX40–/– CD8 cells become activated and expand relatively normally. However, they failed to sustain their numbers over time, suggesting that in the absence of OX40 there is a defect in long-term division or survival. These results correspond with previous reports of OX40–/– CD4 and CD8 T cells that showed OX40 suppressed apoptosis through up-regulation of Bcl-2 family proteins (10, 22). In this study, we could not detect any significant differences in the expression of Bcl-2 or in vivo apoptosis between wild-type and OX40–/– OT-I cells, but this may be due to technical difficulties to assess these parameters in vivo (51) rather than reflective of an alternate mechanism of OX40 action.

As opposed to our previous studies of OX40 on CD8 cells in adjuvant and tumor systems, we show in response to adenovirus that OX40 can dramatically regulate differentiation and effector function. Again, it is not clear why OX40 has multiple alternate activities depending on the model system, but the type and level of the inflammatory milieu likely dictates the dominance or subdominance of OX40 in these activities. OX40–/– CD8 cells showed defects in down-regulation of the effector markers CD62L and CD27, the secretion of cytokines, and importantly cytotoxic activity. Fully differentiated CD8 T cells are known to down-regulate CD62L and CD27 both of which have been used as markers to distinguish effector or effector memory CD8 T cells (CD62LlowCD27low) after LCMV infection (49). Consistently, when we restimulated OT-I cells in vitro we found that OX40–/– cells were not efficient in producing effector cytokines. OX40–/– CD8 cells showed a more pronounced defect in producing TNF compared with IFN-{gamma}, indicating that the OX40 signal may differentially target this cytokine in fully activated CD8 T cells. Of note, a previous report showed that blocking OX40L with OX40-Ig reduced TNF production from CD8 T cells after influenza virus infection (55), and more recently Ito et al. (56) showed that OX40 signaling selectively promoted TNF secretion in human CD4 T cells in vitro. Thus, the regulation of TNF synthesis by OX40 may be common to CD4 and CD8 T cells in various inflammatory circumstances. As well as TNF production, another striking functional impairment in the absence of OX40 was decreased cytotoxicity. Interestingly, when costaining activation markers and cytokines with granzyme B, we found that wild-type CD8 cells expressing high levels of granzyme B were phenotypically CD62Llow, CD27low, and TNF+ (data not shown), and this group was the phenotype not generated when OX40 was absent. OX40 may then help to program genetic remodeling during the transition from naive to effector cells, resulting in the expression of signature genes central to effector CD8 T cells (57). However, preprimed OX40–/– CD8 cells showed functional characteristics comparable to those of wild-type cells in a secondary response, even though cell accumulation was decreased, possibly suggesting that a role in differentiation only applies during priming of naive CD8 cells.

In contrast to OX40-OX40L, the biology of 4-1BB appears very complex. Ligation of 4-1BB undoubtedly augments the activation, the proliferation, and the survival of CD8 T cells in vitro (11, 58, 59, 60). However, in vivo, agonist anti-4-1BB can lead to completely opposite effects. For example, anti-4-1BB can augment CTL generation and improve immunity against tumors and viruses (12, 13, 14), whereas in other cases it dramatically inhibits immune responses in several autoimmune models (30, 31, 32, 33, 34). One of the reasons might be the promiscuous expression of 4-1BB on many kinds of immune cells that have specific and perhaps opposing roles in various pathophysiological circumstances. For example, it has been suggested that negative effects of anti-4-1BB could be due to promoting apoptosis of CD4 cells (34, 61), and that CD4 and CD8 cells respond differently to 4-1BB ligation (58). Alternatively, anti-4-1BB has been proposed to induce regulatory cells, either in the CD8 lineage (31, 62), or in the dendritic cell lineage (30).

In opposition to some of the experiments with anti-4-1BB, all studies of 4-1BBL–/– mice have either revealed no role for this ligand in initial CD8 priming, or suggested that 4-1BB/4-1BBL interactions play a positive, enhancing role, largely at a late stage of the CD8 T cell response when memory has formed or when memory is beginning to develop (23, 24, 25, 26, 27). Our studies represent the first to specifically address how the lack of 4-1BB on a CD8 cell affects its initial response and clearly show that without 4-1BB, CD8 cells are hyperresponsive to Ag in both primary and secondary responses, at least when provided via adenovirus. We do not believe this hyperresponsiveness is related to the Ag used in this study, or the vector, or the use of OT-I transgenic T cells, in that we have found similar hyperresponsiveness of 4-1BB–/– OT-I cells in mice immunized with OVA in CFA (data not shown), and enhanced CD8 priming to murine CMV in nontransgenic 4-1BB-deficient animals (I. Humphreys and M. Croft, unpublished observations). Moreover, these results agree well with our previous finding that 4-1BB–/– CD4 T cells from OT-II TCR transgenic mice also showed hyperresponsiveness to OVA protein in vivo (36). Thus, we conclude that 4-1BB, in physiological settings, can play a negative or limiting role in both CD4 and CD8 T cell responses, in addition to its apparent positive role, at least during the initial priming of effector T cells. It remains to be determined whether the loss of 4-1BB on CD8 cells affects the generation, maintenance, and responsiveness of long-term memory populations.

What then might explain an augmented T cell response when 4-1BB cannot be expressed? It is possible that in addition to the generation of positive signals through 4-1BB, there might be situations in which 4-1BB can transmit negative signals to a CD8 cell. This possibility would go along with the suggestion from autoimmune and graft-vs-host disease studies, in which anti-4-1BB is suppressive, that chronic 4-1BB signals may lead to apoptosis (activation-induced cell death) of pathogenic T cells (34, 61). This idea is feasible because we observed stable 4-1BB expression throughout the course of the primary adenovirus response. However, we failed to detect differences in apoptosis between wild-type and 4-1BB-deficient CD8 cells. Alternatively, a rate-limiting signal from 4-1BB might be due to promoting the development of regulatory CD8 cells that are generated at the same time as effector CD8 cells. Thus, it is possible that some naive CD8 cells naturally take an alternate differentiation pathway enhanced by 4-1BB signals, and become regulatory. In the absence of 4-1BB, these cells would not then be generated, leading to enhanced effector CD8 responsiveness. Additional experiments will be needed to definitively conclude whether this mechanism is operative, but our phenotypic studies did not reveal any evidence of the presence/absence of potential regulatory populations. Lastly, another possibility stems from recent observations made with the TNFR family molecule, HVEM. HVEM has been thought to be costimulatory by interacting with LIGHT (63). However, HVEM–/– T cells were found hyperresponsive to stimuli, and HVEM–/– mice exhibited enhanced T cell-directed autoimmune hepatitis (64), somewhat similar to the phenotype we described previously with 4-1BB-deficient CD4 cells and in our study with 4-1BB–/– CD8 T cells. HVEM has now been shown to also bind the ITIM-containing B and T lymphocyte attenuator (BTLA) molecule that is expressed on both activated T cells and APC, and can transmit a negative signal, at least in part by recruiting the phosphatase Src homology protein-2 (65, 66, 67). Thus, HVEM interaction with LIGHT or BTLA resembles the interaction of B7-1-B7-2 with CD28 or CTLA4 (68), indicating that one molecule can bind partners driving either a stimulatory or inhibitory phenotype (69). It is an intriguing idea that the hyperresponsiveness of 4-1BB–/– T cells might be explained through 4-1BB possessing another ligand in addition to 4-1BBL that might provide a negative or modulatory influence in trans to other T cells or to APC, perhaps similar to BTLA. The fact that 4-1BB knockout and 4-1BBL knockout mice do not appear to phenocopy each other makes this possibility appealing.

In summary, we provide novel data to demonstrate that OX40 and 4-1BB physiologically operate in an opposing manner during initial priming of CD8 effector T cells. This study suggests the hypothesis that several TNFR family molecules, originally thought to play overlapping and potentially redundant roles, actually form an intricately balanced network. Some molecules might function only in a positive manner (e.g., OX40, CD27), whereas others might function positively or negatively (e.g., 4-1BB, HVEM), depending on either the stage of the T cell response or the availability of multiple opposing ligands, with their overall action being to fine-tune immune reactivity.


   Acknowledgments
 
We thank Mary Cheng and Xiaohong Tang for technical assistance and Marta L. Fraga for helpful discussion. This manuscript is no. 744 from the La Jolla Institute for Allergy and Immunology (San Diego, CA).


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Grant AI42944 from the National Institutes of Health (to M.C.) and by Grant R01 EY013325 from the National Institutes of Health, by the Science Research Center Fund from the Korea Science & Engineering Foundation, and by Grant KRF-2005-084-E00001 from the Korea Research Foundation (to B.S.K.). Back

2 Address correspondence and reprint requests to Dr. Michael Croft, Division of Molecular Immunology, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121. E-mail address: mick{at}liai.org Back

3 Abbreviations used in this paper: HVEM, herpesvirus entry mediator; OX40L, OX40 ligand; LCMV, lymphocytic choriomeningitis virus; 4-1BBL, 4-1BB ligand; Ad-mOVA, recombinant defective type 5 adenovirus expressing membrane-bound OVA; LN, lymph node; BTLA, B and T lymphocyte attenuator. Back

Received for publication January 13, 2006. Accepted for publication June 28, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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