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The Journal of Immunology, 2004, 173: 5944-5951.
Copyright © 2004 by The American Association of Immunologists

4-1BB and OX40 Act Independently to Facilitate Robust CD8 and CD4 Recall Responses1

Wojciech Dawicki*, Edward M. Bertram2,*, Arlene H. Sharpe{dagger} and Tania H. Watts3,*

* Department of Immunology, University of Toronto, Toronto, Canada; and {dagger} Immunology Research Division, Department of Pathology, Brigham and Women’s Hospital and Department of Pathology, Harvard Medical School, Boston, MA 02115


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Mice deficient in OX40 or 4-1BB costimulatory pathways show defects in T cell recall responses, with predominant effects on CD4 vs CD8 T cells, respectively. However, OX40L can also stimulate CD8 T cells and 4-1BBL can influence CD4 T cells, raising the possibility of redundancy between the two TNFR family costimulators. To test this possibility, we generated mice deficient in both 4-1BBL and OX40L. In an adoptive transfer model, CD4 T cells expressed 4-1BB and OX40 sequentially in response to immunization, with little or no overlap in the timing of their expression. Under the same conditions, CD8 T cells expressed 4-1BB, but no detectable OX40. Thus, in vivo expression of 4-1BB and OX40 can be temporally and spatially segregated. In the absence of OX40L, there were decreased CD4 T cells late in the primary response and no detectable secondary expansion of adoptively transferred CD4 T cells under conditions in which primary expansion was unaffected. The 4-1BBL had a minor effect on the primary response of CD4 T cells in this model, but showed larger effects on the secondary response, although 4-1BBL–/– mice show less impairment in CD4 secondary responses than OX40L–/– mice. The 4-1BBL–/– and double knockout mice were similarly impaired in the CD8 T cell response, whereas OX40L–/– and double knockout mice were similarly impaired in the CD4 T cell response to both protein Ag and influenza virus. Thus, 4-1BB and OX40 act independently and nonredundantly to facilitate robust CD4 and CD8 recall responses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Both 4-1BB (CD137) and OX40 (CD134) are inducible members of the TNFR family involved in costimulation of T cell responses (reviewed in Refs. 1 and 2). In vitro, both CD4 and CD8 T cells up-regulate both 4-1BB and OX40 upon Ag-specific activation (3, 4, 5, 6). However, there is evidence that 4-1BB is expressed to higher levels on CD8 T cells and there is preferential expression of OX40 on CD4 T cells in some models (6, 7). Their ligands, 4-1BBL and OX40L, are predominantly expressed on activated APC such as B cells, dendritic cells, and macrophages (reviewed in Refs. 1 and 2).

In general, OX40 has more prominent effects on CD4 T cells (8) and functions to sustain CD4 T cell survival subsequent to TCR/CD28 signaling (3, 9, 10, 11, 12). Mice lacking OX40 or its ligand show defects in CD4 proliferative responses with little or no effect on CD8 T cells (13, 14, 15, 16). Similarly, mice engineered to constitutively express OX40L show expansion of CD4, but not CD8 T cells (17, 18). Conversely, agonistic Abs against 4-1BB show preferential effects on survival or expansion of CD8 T cells (19, 20, 21, 22), and mice lacking 4-1BB or its ligand show defects in recall CD8 T cell responses (23, 24, 25, 26), with no detectable effects on CD4 T cell responses to lymphocytic choriomeningitis virus or influenza virus (25, 27).

Despite the extensive literature suggesting a predominant role for OX40 on CD4 T cells and for 4-1BB on CD8 T cells, there are exceptions to this segregation of OX40 and 4-1BB function. In graft-vs-host disease models, 4-1BB/4-1BBL can play an equivalent role in MHC-I- or MHC-II-restricted disease (28), although the same is not true for OX40 (29). Furthermore, 4-1BB is required for herpes simplex 1-mediated keratinitis (30), a disease that has been attributed to the Th1 response. In adoptive transfer models, using OVA-specific TCR transgenic T cells, both OX40 and 4-1BB costimulatory pathways can influence CD4 and CD8 responses (6, 31, 32, 33). Thus, OX40 and 4-1BB can clearly influence both T cell subsets, at least in some models.

OX40- and 4-1BB-induced signals are each dependent on TNFR-associated factor 2 (34, 35, 36) and contribute to T cell survival by up-regulation of prosurvival members of the Bcl-2 family (10, 37). The possibility of OX40 and 4-1BB expression on both CD4 and CD8 T cells, their similarity in downstream signaling pathways, and their similar function in maintenance of T cell survival suggest that there could be some redundancy in their function in vivo. This possibility is further raised by the finding that blockade of the 4-1BB costimulatory pathway was found to have greater effects on primary CD8 T cell responses than 4-1BBL deficiency (31, 38), raising the possibility of compensatory effects in gene-targeted mice. To evaluate possible redundancy or cooperation between the 4-1BB and OX40 costimulatory pathways, we generated mice lacking both OX40L and 4-1BBL and evaluated the mice for responses to superantigen, influenza virus, as well as to protein Ags in an adoptive transfer model. The results suggest that OX40 and 4-1BB are expressed on different T cells or at different times in vivo, and that they act independently and noncooperatively to promote robust CD4 and CD8 recall responses, respectively.


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

C57BL/6 mice were obtained from Charles River Laboratories (St. Constant, Quebec, Canada). The 4-1BBL–/– mice (23) backcrossed onto the C57BL/6 background (n = 9) were originally obtained from J. Peschon (Amgen, Seattle, WA). OX40L–/– mice (13) (backcrossed onto the C57BL/6 background, n = 8) were crossed with the 4-1BBL–/– mice to generate the double knockout (DKO)4 mice. Genotyping of 4-1BBL locus used the primers 5'-CAC TGA CCG ACC GTG GTA ATG-3', 5'-GAC ATA GCG TTG GCT ACC CGT G-3', and 5'-AGC CCG GTA TCT CTG AGG AG-3'; and of OX40L locus, the primers 5'-AAA CTA TGG AGG TGC AGA-3', 5'-CAG AAG CAA TGT GTC TTG-3', 5'-ATT GAA CAA GAT GGA TTG CAC-3', and 5'-CGT CCA GAT CAT CCT GAT C-3'. OT-I mice (39) were provided by P. Ohashi (Toronto, Ontario, Canada), and OT-II mice (40) were provided by C. Surh (La Jolla, CA). Both transgenic mice express a V{alpha}2+ and V{beta}5.1+ TCR and can be detected in a similar manner. For some experiments, OT-I and OT-II mice were crossed with Thy-1.1 congenic C57BL/6 mice to use as a marker for transferred T cells. Mice were maintained in the University of Toronto animal facilities, and all procedures were approved by the Animal Care Committee, following the guidelines of the Canadian Council on Animal Care.

Adoptive transfer

T cells were purified by negative selection using T Cell Immunocolumns (Cedarlane Laboratories, Hornby, Ontario, Canada) from the spleen and lymph node of either OT-I or OT-II mice. Purified T cells containing 2.5 x 106 CD8+ TCR V{alpha}2+V{beta}5.1+ (OT-I) or CD4+ TCR V{alpha}2+V{beta}5.1+ (OT-II) were injected i.v. into the tail vein of recipient mice. One day later, mice were injected s.c. with 2 mg of OVA (Sigma-Aldrich, St. Louis, MO) and 50 µg of LPS (Sigma-Aldrich). Mice were sacrificed at the times indicated in the figure legends.

Influenza infection

Mice were infected i.p. with 200 hemagglutinating units (HAU) of influenza A HKx31 (H3N2) and sacrificed 7 days (peak of primary response) or 21 days later. Some mice were infected i.p. a second time at day 21 with 200 HAU of a serologically distinct A/PR8/34 (H1N1) strain. The use of the second strain avoids the immunodominant neutralizing Ab response to hemagglutinin and neuraminidase proteins, which would otherwise limit reinfection and limit the secondary CTL response. Mice were sacrificed and spleen cells were harvested and evaluated for responses to the immunodominant CD8 epitope in C57BL/6 mice, nuclear protein (NP) 366–764 (41), using Db/NP366–374 tetramers (42). To enumerate influenza-specific CD8 T effector function, splenocytes were restimulated with NP366–374 peptide and analyzed by intracellular cytokine staining for IFN-{gamma}.

Abs, tetramers, and flow cytometry

Anti-CD4 (GK1.5) and anti-CD8 (53.6.72) Abs were purified from hybridoma supernatants using protein G-Sepharose and biotinylated using N-hydroxysuccinimidyl-D-biotin. Biotinylated anti-TCR V{beta}5.1,5.2 (MR9-4), anti-CD134 (OX-86), FITC anti-TCR V{beta}5.1,5.2 (MR9-4), PE anti-TCR (V{alpha}2 B20.1), and allophycocyanin anti-IFN-{gamma} (XMG1.2) were purchased from BD Pharmingen (San Diego, CA). Biotinylated and PE anti-CD137 (17B5) and allophycocyanin streptavidin were obtained from eBioscience (San Diego, CA). Allophycocyanin-labeled tetramers containing murine class I MHC H-2Db, {beta}2-microglobulin, and influenza nucleoprotein peptide, NP366–374, were synthesized by National Institute of Allergy and Infectious Disease MHC Tetramer Core Facility (Atlanta, GA). Cells were isolated from spleen and resuspended in staining buffer (PBS/3% FCS/0.2% azide) and 2 µg of anti-FcR (2.4G4) Ab for 10 min. Primary biotinylated Abs were added for 20 min, after which cells were washed twice with staining buffer. Cells were incubated for an additional 20 min with allophycocyanin streptavidin and the fluorescently labeled Ab and/or tetramers. Cells were washed two more times and analyzed using a FACSCalibur and CellQuest software (BD Biosciences, Mountain View, CA). For intracellular IFN-{gamma} analysis, splenocytes were stimulated with 1 µM NP366–374 peptide in the presence of GolgiStop (BD Pharmingen) and stained using BD Cytofix/Cytoperm kit (BD Pharmingen), according to the manufacturer’s protocol.

Cytotoxicity assay

Mice were infected with 200 HAU of influenza A HKx31, as described above. Splenocytes were harvested at various times and restimulated in vitro by the addition of 100 nM H-2Db-restricted peptide NP366–374 to 1-ml cultures containing 5 x 106 spleen cells. On day 5, cells were resuspended to 0.65 ml, and duplicate serial 3-fold dilutions of effectors were performed and assayed for anti-influenza NP-specific CTL activity against 51Cr-labeled EL4 cells pulsed with 50 µM NP366–374 peptide and unpulsed EL4 as control. After 5 h, 70 µl of supernatant was harvested onto 96-well harvest plates (Canberra Packard, Mississauga, Canada) and counted on a Topcount scintillation counter (Canberra Packard). Maximum and spontaneous release was determined from wells that contained 1% SDS or medium alone. The percentage of specific lysis was calculated from the equation: ((experimental 51Cr release – spontaneous 51Cr release)/(maximum 51Cr release – spontaneous 51Cr release)) x 100% = percent specific lysis.

Analysis of IL-2 and IFN-{gamma} production by CD4 T cells

Cells were isolated from spleen, and RBC were lysed with Tris-NH4Cl solution. Splenocytes (5 x 106 cells) were incubated with 250 HAU of heat-killed (30 min at 56°C) influenza virus for 48 and 96 h, and the supernatants were harvested and analyzed for IL-2 and IFN-{gamma}, respectively. IL-2 was detected using the indicator cell line CTLL. Serial dilutions of the culture supernatant were prepared in triplicate and incubated with 104 indicator cells in 100 µl in 96-well plates for 24 h. During the final 6 h, the cells were labeled with [3H]thymidine (Amersham Biosciences, Baie d’Urfe, Canada). Cultures were harvested and analyzed on the TopCount 96-well liquid scintillation counter (Canberra Packard). IFN-{gamma} was detected using ELISA using anti-murine IFN-{gamma} mAbs from BD Pharmingen, according to the manufacturer’s instructions.

Superantigen responses

Mice were injected i.p. with 3 µg of staphylococal enterotoxin A (SEA) (Toxin Technology, Saratoga, FL), and spleens were harvested 2 or 12 days later and analyzed by flow cytometry, as described previously, for TCR V{beta}3 and CD4 or TCR V{beta}3 and CD8 cells.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Comparison of 4-1BB and OX40 expression on CD4 and CD8 T cells

As discussed above, there is evidence that both CD4 and CD8 T cells can express and respond to signals through 4-1BB and OX40. However, the coexpression of 4-1BB and OX40 has not been rigorously tested in vivo. To compare the relative kinetics of 4-1BB and OX40 expression on CD4 and CD8 T cells in vivo, TCR transgenic CD4 or CD8 T cells were transferred into wild-type (WT) mice, and surface phenotype was monitored by flow cytometry at different time points after immunization with Ag plus LPS. The transferred T cells were followed by the expression of the Thy-1.1 marker following transfer into Thy-1.2 congenic recipients. 4-1BB was detected on the transferred CD4 (OT-II) T cells at 12 h through 24 h postimmunization, whereas OX40 was not highly expressed until after 4-1BB levels had dropped, at 48 h (Fig. 1A). The CD8 (OT-I) T cells expressed 4-1BB later, with maximal expression at 24 h, whereas little or no OX40 was detected at any time point (Fig. 1B). Neither OX40 nor 4-1BB was observed on the surface of OT-I or OT-II T cells at 60, 72, or 96 h post-OVA/LPS injection (data not shown). Analysis of Thy-1.1-positive cells for TCR V{alpha}2 and V{beta}5.1 revealed that the majority of the Thy-1.1-positive cells were transgenic T cells (data not shown). Thus, 4-1BB and OX40 are expressed sequentially on CD4 T cells. In contrast, little or no OX40 was detected on CD8 T cells, under conditions in which 4-1BB expression was readily detected. Thus, although OX40 has been detected on CD8 T cells under some circumstances, these results show that, at least under these conditions of antigenic stimulation, CD8 T cells express 4-1BB, but not OX40, and CD4 T cells sequentially express 4-1BB and OX40, with little or no coincident expression.



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  		FIGURE 1. Distinct expression profiles of 4-1BB and OX40 induced by antigenic stimulation in vivo. TCR transgenic CD4 (OT-II Thy-1.1) or CD8 (OT-I Thy-1.1) T cells (2.5 x 106 cells/mouse) were transferred into C57BL/6 Thy-1.2 congenic mice. Twenty-four hours later, mice were injected s.c. with 2 mg of OVA plus 50 µg of LPS. Spleens were harvested at 0, 12, 24, 36, and 48 h post-OVA/LPS injection and stained for Thy-1.1, 4-1BB, and OX40. A, OX40 and 4-1BB expression on OT-II Thy-1.1 CD4 at the indicated time points. B, OX40 and 4-1BB expression on OT-I Thy-1.1 CD8 cells at indicated time points. Data are representative of two and three independent experiments. All plots have been gated on Thy-1.1-positive cells.

 
Superantigen responses

To examine the effect of the combination of 4-1BB and OX40 costimulatory pathways on primary expansion of CD4 and CD8 T cells in vivo, WT, OX40L–/–, 4-1BBL–/–, and DKO mice were injected with SEA, and the expansion of V{beta}3+ T cells was followed over time by flow cytometry. The frequency of CD4 and CD8 V{beta}3+ T cells at the height of the response (day 2) and after contraction of the response (day 12) was indistinguishable between all four groups of mice (Fig. 2). Similar results were obtained when expressed as total cell numbers per spleen (data not shown). The lack of significant differences between the CD4 and CD8 response suggests that neither OX40L, 4-1BBL, nor the combination influences primary T cell expansion in response to superantigen in mice. Because the superantigen response is tested without any additional adjuvant, there may be insufficient induction of TNF family ligands on APC to impact on the response. The intact response to SEA in OX40L–/–4-1BBL–/– mice confirms that T cells develop normally in the absence of the two costimulatory pathways and exhibit normal responsiveness to TCR stimulation in vivo.



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  		FIGURE 2. CD4 and CD8 T cell responses to SEA in WT, 4-1BBL–/–, OX40L–/–, and DKO mice. WT, 4-1BBL–/–, OX40L–/–, and DKO mice were injected i.p. with SEA, and spleens were harvested 2 or 12 days later. A, Splenocytes were stained for TCR V{beta}3 and CD4, and analyzed by flow cytometry. B, Splenocytes were stained for TCR V{beta}3 and CD8, and analyzed by flow cytometry. Data represent mean ± SEM from three to four mice and are representative of two independent experiments.

 
4-1BB/4-1BBL, but not OX40/OX40L interactions are required for the CD8 T cell response to influenza virus

Influenza infection of mice represents a useful model for studying costimulatory requirements of T cells (2). Mice deficient in OX40 show impaired CD4 T cell responses to influenza delivered intranasally (i.n.) with no defects in CD8 T cell responses (14). 4-1BBL–/– mice have decreased CD8 T cell recall responses with no defects in CD4 responses to influenza delivered i.p. (23, 25). Although the i.n. route of immunization, which induces an acute localized infection in the respiratory tract, is the physiological route of infection, we have found that immune responses observed with the less infectious i.p. route of infection are sensitive to costimulation and avoid the complications of inflammation and weight loss associated with the more severe disease observed with i.n. infection. Using this model, we previously determined that 4-1BBL–/– mice showed no difference in initial primary expansion or contraction of the CD8 T cell response, which peaks in the spleen at day 7 postimmunization. However, 4-1BBL–/– mice had decreased CD8 T cell numbers at 3–6 wk after priming and showed decreased secondary responses (25), a defect that is corrected by adding agonistic Abs during the boost, but not the priming phase of the response (43).

To test the combined effects of 4-1BBL and OX40L on influenza responses, DKO, 4-1BBL–/–, OX40L–/–, and WT mice were infected i.p. with influenza A HKx31 and sacrificed at time points representing the peak of primary CD8 T cell expansion (day 7), as well as at day 21, by which time a significant decline in CD8 T cell numbers was previously noted in 4-1BBL–/– mice (25). Splenocytes were analyzed for expansion of NP366–374-specific T cells using Db/NP366–374 tetramers. By day 7, ~6% of CD8 T cells were specific for NP366–374 (Fig. 3A), with no significant difference between the four genotypes of mice. There were no significant differences in the number of splenocytes recovered between the different mice, and, as a result, when the percentages were converted to absolute cell numbers, similar results were obtained (data not shown). Thus, the initial expansion of CD8 T cells is not affected by the lack of 4-1BB or OX40 ligation. Following restimulation with NP366–374 peptide, the number of IFN-{gamma}-producing cells was also similar between the four groups of mice (Fig. 3B). Thus, both the primary expansion and the development of effector function of CD8 T cells are unaffected by the lack of 4-1BB and OX40 costimulation.



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  		FIGURE 3. CD8 T cells require 4-1BBL, but not OX40, for optimum recall response to influenza virus. WT, 4-1BBL–/–, OX40L–/–, and DKO mice were infected i.p. with 200 HAU of influenza A HKx31. Spleens were harvested 7 (A and B) and 21 (C, D, and G) days later or, at day 21, mice were reinfected i.p. with 200 HAU of influenza A PR8 and spleens were harvested at 7 days after secondary challenge (E, F, and H). For enumeration of Ag-specific T cells, splenocytes were isolated, counted, and stained with mAb to CD8, CD62L, and Db/NP366–374 tetramer, and analyzed by flow cytometry (A, C, and E). For analysis of IFN-{gamma}-producing cells, splenocytes were restimulated with NP366–374 peptide in the presence of GolgiStop, stained with mAb to CD8 and IFN-{gamma}, and analyzed by flow cytometry (B, D, and F). Splenocytes from day 21 of the primary response (G) and day 7 of the secondary responses (H) were restimulated with NP366–374 peptide for 5 days, after which time they were incubated with peptide-pulsed 51Cr-labeled EL4 target cells in a standard chromium release assay. CTL data are presented as mean ± SEM of four individual mice per group and are representative of two to three independent experiments.

 
By day 21 postinfection, the numbers of CD8+ tetramer+ T cells in both the DKO and 4-1BBL–/– mice had declined to 0.5% of CD8 T cells, whereas the OX40L–/– and WT mice showed a 2-fold higher frequency of Ag-specific T cells (p < 0.01; Fig. 3C). Similarly, following stimulation with NP366–374 peptide DKO and 4-1BBL–/– splenocytes had 0.4% IFN-{gamma}-producing cells, whereas OX40L–/– and WT mice had significantly more at 0.8% (p < 0.02; Fig. 3D).

After restimulation with peptide for 5 days, the recall CTL activity of T cells primed in DKO and 4-1BBL–/– mice was reduced compared with OX40L–/– and WT animals (Fig. 3G). There were no significant differences between 4-1BBL–/– and DKO, or between OX40L–/– and WT animals with respect to the number of Ag-specific CD8 T cells, their ability to produce IFN-{gamma}, or their ability to kill targets.

To compare the importance of each molecule in the secondary response, mice were rechallenged with a serologically distinct PR8 strain, 21 days after priming. As before, we quantitated the proportion of CD8 T cells that were specific for the NP366–374 peptide, 7 days after reinfection. At this time point, DKO and 4-1BBL–/– animals had 8 and 9% influenza-specific CD8 T cells, whereas the OX40L–/– and WT mice had significantly more cells at 13 and 14%, respectively (p < 0.003; Fig. 3E). We also analyzed the effector function of these T cells by determining their ability to produce IFN-{gamma} in response to NP366–374 peptide. Both DKO and 4-1BBL–/– animals had 9% of CD8 cells producing IFN-{gamma}, and the OX40L–/– and WT mice had significantly more at 15% (p < 0.01; Fig. 3F). The cytolytic capacity also followed the same trend as the tetramer-specific T cell numbers. There were no differences between DKO and 4-1BBL–/– or between OX40L–/– and WT animals, and the animals that were missing 4-1BBL had similarly decreased cytolytic activity regardless of the presence of OX40L (Fig. 3H). These results support the conclusion that 4-1BB/4-1BBL interactions are required for optimal secondary CD8 T cell responses, whereas OX40/OX40L interactions are not. As 4-1BBL and DKO mice were indistinguishable in all parameters measured, there appears to be no compensation between these two TNFR family costimulatory pathways with respect to CD8 T cell responses.

OX40/OX40L interactions are required for optimal CD4 recall response to influenza

As discussed above, both 4-1BB and OX40 can affect CD4 T cell responses in some models. Therefore, we analyzed the CD4 responses to influenza in the four groups of mice. Splenocytes isolated on day 21 postinfection were incubated with heat-inactivated influenza virus. Heat inactivation renders the virus noninfectious and targets the virus to the MHC class II presentation pathway for activation of CD4 T cell responses. The level of IL-2 and IFN-{gamma} in the supernatants was used as an indicator of activation of influenza-specific CD4 T cells. The absence of 4-1BBL had no significant impact on either IL-2 or IFN-{gamma} production (Fig. 4, A and C). In contrast, OX40L–/– and DKO mice had significantly reduced levels of both IL-2 and IFN-{gamma} compared with the WT and 4-1BBL–/– mice (p < 0.01; Fig. 4, A and C). A similar trend was observed when the mice were rechallenged with the PR8 strain of influenza virus and analyzed 7 days later. Mice deficient in OX40L (OX40L–/– and DKO) have a defect in the CD4 response, as indicated by the significantly lower levels of IL-2 and IFN-{gamma} (Fig. 4, B and D; p < 0.01). Again, the DKO mice do not have a greater reduction in the CD4 response compared with the OX40L–/– animals. Thus, only OX40 contributes to the recall CD4 T cell response to influenza.



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  		FIGURE 4. Analysis of CD4 T cell responses to influenza virus in WT, 4-1BBL–/–, OX40L–/–, and DKO mice. Mice were infected i.p. with 200 HAU of influenza A HKx31 and 21 days later rechallenged with influenza A PR8 strain. Splenocytes from day 21 after priming (A and B) and day 7 after rechallenge (C and D) were restimulated with heat-inactivated influenza virus for 48 h (A and B) and 96 h (C and D), and supernatants were analyzed for IL-2 and IFN-{gamma}, respectively. A and C, IL-2 in the supernatant was determined using a bioassay on CTLL cells. B and D, IFN-{gamma} was determined using ELISA. Data presented are means ± SEM at 1/2 dilution from four to five individual mice representative of two to three independent experiments.

 
Effect of OX40 and 4-1BB costimulation on recall response of adoptively transferred Ag-specific CD4 T cells

One of the shortcomings of the influenza model system is that it is difficult to directly enumerate the responding CD4 T cells early in the primary response. To overcome this shortcoming, an adoptive transfer model with CD4 OT-II transgenic cells was used. T cells were purified from OT-II mice, and each animal received 2.5 x 106 OT-II cells. Twenty-four hours later, mice were injected s.c. with OVA/LPS. One, 3, and 8 days later, splenocytes were stained for TCR V{alpha}2, TCR V{beta}5.1, and CD4, and analyzed by FACS to determine the extent of OT-II T cell expansion. At the peak of the response (day 3), there were reduced numbers of OT-II cells in 4-1BBL–/– and DKO mice, with no differences detected between OX40L–/– and WT, or 4-1BBL–/– and DKO mice (Fig. 5A). These results suggest that OX40/OX40L interactions are dispensable for the primary accumulation of CD4 T cells and that 4-1BB/4-1BBL interactions do not compensate for the loss of OX40L.



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  		FIGURE 5. CD4+TCR transgenic T cell responses in WT, 4-1BBL–/–, OX40L–/–, and DKO mice. TCR transgenic CD4 (OT-II) T cells were transferred into WT, 4-1BBL–/–, OX40L–/–, and DKO mice, and 24 h later, injected with OVA/LPS, as in Fig. 1. A, Spleens were harvested 1, 3, and 8 days post-Ag administration, stained for TCRV{alpha}2, TCRV{beta}5.1, and CD8, and analyzed by flow cytometry. B, To follow a recall response, mice were reinjected 14 days after primary immunization with a second dose of OVA/LPS and OT-II cells enumerated by flow cytometry, as above. Results shown represent the mean ± SEM from three to five mice per group per time point, and are representative of two independent experiments.

 
Next, we used the adoptive transfer model to examine the contributions of each costimulatory molecule to the CD4 recall response. We transferred OT-II cells as before and administered OVA/LPS. Fourteen days later, a second dose of OVA/LPS was administered and the in vivo recall response was analyzed by flow cytometry. At day 0 of the recall response, which is also day 14 of the primary response, we consistently observed a slight decrease in OT-II T cell numbers in all three knockout animals (Fig. 5B). As the response progresses, it is obvious that OX40L–/– and DKO mice have no detectable secondary response and that the 4-1BBL–/– mice have a delayed and reduced response compared with the WT animals (Fig. 5B). These results also support the conclusion that OX40/OX40L interactions are critical for optimal CD4 T cell responses. This effect appears to be due at least in part to a requirement for OX40 for maintenance of CD4 T cells late in the primary response. The greater defect in recall response suggests that OX40 signals are also important in allowing the CD4 T cells to expand upon rechallenge. In this model, we also observed that the lack of 4-1BBL had a small effect on primary expansion and a somewhat larger effect on secondary CD4 responses. The finding that the CD4 secondary response is completely abrogated in OX40L–/– mice makes it difficult to assess synergy in the secondary response. However, it is clear that 4-1BBL and OX40L do not cooperate in the primary response, and that OX40L has a profound role in the secondary CD4 T cell response under conditions in which the primary response is unchanged.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we show that 4-1BB has a major role in CD8 T cell secondary responses, with more minor effects on CD4 responses. In contrast, OX40L had no detectable effect on primary expansion of CD4 T cells, but had a profound effect on CD4 T cell memory and no detectable effects on CD8 T cells. The 4-1BBL–/– mice were indistinguishable from DKO mice with respect to CD8 T cell responses, and OX40L–/– mice were indistinguishable from DKO mice with respect to CD4 T cell responses. Thus, in both an influenza infection model and a CD4 T cell adoptive transfer model, there was no evidence of cooperation or redundancy between the two costimulatory pathways.

With OVA/LPS immunization, which involves transient activation via a nonreplicating Ag, 4-1BB and OX40 are not extensively coexpressed in vivo (Fig. 1). On CD4 T cells, substantial OX40 expression was only detected after the expression of 4-1BB had returned to baseline, at 48 h. 4-1BB is expressed before the T cells start dividing (31), whereas OX40 is expressed after division has commenced (data not shown) (31). This implies that a T cell could first receive a 4-1BBL signal and then disengage, undergo division, and re-engage an APC. Whether it re-engages the same APC or a different one remains to be determined. The discovery of CD4+CD3– accessory cells that are low in CD80, CD86, and 4-1BBL, but high in OX40L, suggests that OX40 signals can be delivered from a different APC (44). 4-1BB was expressed on CD8 T cells 24 h post-Ag injection, but no OX40 was observed either at the time of 4-1BB expression, or afterward when the cells became CD44high (data not shown) (31). OX40 expression has been documented on in vitro stimulated CD8 T cells (5, 45, 46, 47), but it has not been shown on CD8 T cells directly ex vivo from a mouse. The discrepant results might be due to different levels of antigenic stimulation achieved in the different models, which in turn could impact on the levels of OX40 and its ligand that are induced. Nevertheless, we show that activated CD8 cells express little or no OX40 under conditions that up-regulate 4-1BB, CD69, and CD44. Thus, under conditions of limited antigenic stimulation in vivo, OX40 and 4-1BB are not expressed to a significant extent on the same cells and appear to function independently in the immune response in the spleen. It should be pointed out, however, that under conditions of chronic or prolonged infection or inflammation, there may be more sustained expression of TNFR family costimulatory molecules (30, 48).

CD4 T cell help is required to maintain CD8 T cell numbers at the end of the primary response to influenza, and recall CD8 T cell responses to influenza are reduced in MHC II-deficient animals (49, 50). Therefore, one might have expected OX40 deficiency to impair CD8 responses due to decreased help. CD4 help for CD8 memory responses is important during CD8 T cell priming, but dispensible during the secondary response (51, 52). Because OX40L deficiency impairs secondary, but not primary CD4 T cell expansion, this most likely explains the failure to see an impact of OX40 on help for CD8 T cell responses. The finding that OX40/OX40L appears to have no impact on the primary response of OT-II T cells even in the absence of 4-1BBL further supports the idea that OX40L is not important during T cell primary expansion. Because there was no detectable secondary CD4 T cell response of OT-II T cells in OX40L–/– mice, it was not possible to assess whether there is synergy in the contribution of OX40L and 4-1BBL to CD4 secondary responses. However, it is clear from comparison of the single knockout animals in the same experiment that OX40 costimulation of CD4 recall is quantitatively more important than the effect of 4-1BB on this response.

Two studies have suggested a potential role for 4-1BB and OX40 in responses to superantigen. In particular, agonistic anti-OX40 Ab when combined with LPS and superantigen allows maintenance of an expanded CD4 T cell population (9). Similarly, agonistic anti-4-1BB Abs can result in increased expansion and survival of T cells following superantigen administration in mice, with greater effects on CD8 T cells (21). However, in the present study, we found no evidence of a role for endogenous OX40L or 4-1BBL in primary expansion in response to superantigen.

The lack of impact of the OX40L deficiency on the CD8 response to influenza is consistent with previous findings by Kopf et al. (14), who used the i.n. model of infection to show a similar defect in CD4 T cell responses, as we observe with the i.p. model. Our finding that DKO mice have a similar defect as 4-1BBL–/– animals confirms that OX40L–/– is dispensable for the CD8 response to influenza, and that this is not merely due to compensation by 4-1BB/4-1BBL.

Three studies have shown that CD8 TCR transgenic OT-I T cells respond to OX40 costimulation in vivo (6, 32, 33). In most of the experiments, anti-OX40 was used to enhance signals through OX40, which most likely amplifies effects of this signaling pathway over that seen when endogenous ligand is removed. However, Bansal-Pakala et al. (32) observed defects in OX40–/– OT-I T cell expansion when mice were immunized with peptide in CFA in the absence of added anti-OX40 Ab. Thus, as observed in vitro, it appears that if a sufficiently strong antigenic signal is given, then CD8 T cells can up-regulate and respond to OX40 stimulation.

In terms of CD4 responses, OX40/OX40L interactions are required for the establishment of a robust CD4 memory pool that can vigorously respond to rechallenge with influenza virus or protein Ag. The adoptive transfer model also reveals a dependence on 4-1BBL for the CD4 recall response, but it is relatively minor compared with the requirement for OX40. The mice that lacked OX40L (OX40L–/– and DKO) had a completely absent secondary expansion of OT II cells, whereas the 4-1BBL–/– showed delayed and reduced expansion of OT-II cells upon rechallenge. Furthermore, the influenza model failed to reveal any defects in CD4 T cell responses in the absence of 4-1BBL.

Some studies have shown quantitative defects in Ab responses in the absence of OX40 (16, 44). However, most studies have shown that OX40 and 4-1BB costimulatory interactions are dispensible for humoral immunity (13, 14, 15, 25). Consistent with these findings, we observed no defect in influenza-specific Ab production or class switch to IgG1 or IgG2a in any of the four genotypes of mice (data not shown). The lack of effect of removing OX40L on the primary CD4 or Ab responses is not due to compensation by 4-1BB because the DKO animals do not show a defect in the expansion of transferred CD4, in sAg-activated CD4 T cells, or Ab production, further supporting the idea of distinct functions for the two costimulatory receptors.

In conclusion, our results show that 4-1BB and OX40 can be expressed separately on T cells in vivo, with OX40 restricted to CD4 T cells and its expression delayed compared with 4-1BB. By comparing 4-1BB/4-1BBL and OX40/OX40L in the same model, the data clearly show distinct roles for the two costimulatory pathways in the development of CD8 and CD4 recall responses, with little or no effect on primary expansion of T cells. Analysis of mice deficient in both costimulatory pathways revealed that the two costimulatory pathways function independently and nonredundantly to facilitate robust CD8 and CD4 T cell responses.


   Acknowledgments
 
We thank Eugene Ravkov of the National Institute of Allergy and Infectious Diseases tetramer facilty for production of Db/NP tetramers.


   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 research was supported by a grant from the Canadian Institutes of Health Research (to T.H.W.). Back

2 Current address: Division of Immunology and Genetics, The John Curtin School of Medical Research, The Australian National University, P.O. Box 334 Mills Road, Canberra, Australia 2601. Back

3 Address correspondence and reprint requests to Dr. Tania H. Watts, Department of Immunology, University of Toronto, 1 King’s College Circle, Toronto, Ontario, M5S 1A8. E-mail address: tania.watts{at}utoronto.ca Back

4 Abbreviations used in this paper: DKO, double knockout; HAU, hemagglutinating unit; i.n., intranasal; NP, nuclear protein; SEA, staphylococcal enterotoxin A; WT, wild type. Back

Received for publication July 9, 2004. Accepted for publication August 17, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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