Vaccination Peptide Protective Anti-Viral Immunity After 4-1BB ...

4-1BB Costimulation Is Required for Protective Anti-Viral Immunity After Peptide Vaccination1 Joyce T. Tan,* Jason K. Whitmire,† Kaja Murali-Krishna,† Rafi Ahmed,† John D. Altman,† Robert S. Mittler,*† Alessandro Sette,‡ Thomas C. Pearson,2* and Christian P. Larsen2* Peptide vaccination induces T cell activation and cytotoxic T cell development. In an effort to understand what factors can improve immune responses to peptide vaccination, the role of 4-1BB (CD137) costimulation was examined, since 4-1BB has been shown to promote T cell responses in other systems. 4-1BBL-deficient (ⴚ/ⴚ) and wild-type (ⴙ/ⴙ) mice were immunized with a lipidated lymphocytic choriomeningitis virus (LCMV) peptide NP396 – 404. Analysis of peptide-specific responses early after immunization by CTL assay, intracellular IFN-␥ staining, and IFN-␥ enzyme-linked immunospot assay (ELISPOT) indicated that CD8 T cell responses were reduced 3- to 10-fold in the absence of 4-1BB costimulation. Moreover, when agonistic anti-4-1BB Ab was given, CD8 T cell responses in 4-1BBLⴚ/ⴚ mice were augmented to levels similar to those in 4-1BBLⴙ/ⴙ mice. Two months after immunization, 4-1BBLⴙ/ⴙ mice still had epitope-specific cells and were protected against viral challenge, demonstrating that peptide vaccination can induce long-term protection. In fact, 70% of CD8 T cells were specific for the immunizing peptide after viral challenge, demonstrating that strong, epitope-specific CD8 T cell responses are generated after peptide vaccination. In contrast, peptide-immunized 4-1BBLⴚ/ⴚ mice had fewer epitope-specific cells and were impaired in their ability to resolve the infection. These results show that immunization with a single LCMV peptide provides long term protection against LCMV infection and point to costimulatory molecules such as 4-1BB as important components for generating protective immunity after vaccination. The Journal of Immunology, 2000, 164: 2320 –2325.

P

eptide immunization with MHC class I-restricted CTL epitopes is an effective method of vaccination. Peptidebased tumor vaccines given either with IFA or in conjunction with dendritic cells are effective in inducing CTL responses and anti-tumor immunity (1–3). Peptide vaccines are also effective in inducing immunity in many different viral systems, such as Sendai virus and lymphocytic choriomeningitis virus (LCMV)3 (4 –7). Previous studies have shown that lipopeptides, peptides covalently linked to a lipid moiety, are effective in priming CTL responses in mice and in humans (8, 9). In addition, mice that were vaccinated with lipidated H-2Db- or H-2Ld-restricted peptides that consist of a LCMV CTL epitope covalently linked to a Th epitope were protected against LCMV infection (10, 11). In C57BL/6 mice, immunization with other dominant or subdominant H-2Db-restricted LCMV epitopes also induced peptide-specific CTL responses and conferred protection against LCMV challenge (11).

*The Carlos and Marguerite Mason Transplantation Biology Research Center and Department of Surgery, and †Emory Vaccine Center and Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322; and ‡ Epimmune, San Diego, CA 92121 Received for publication October 1, 1999. Accepted for publication December 14, 1999. 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 National Institutes of Health Grants AI40519 and AI44644 (to C.P.L. and T.C.P.) and in part by the Engineering Research Center Program of the National Science Foundation under Award EEC-9731643, The Carols and Margeurite Mason Trust, and National Institutes of Health Grants AI30048 and NS21496 (to R.A.). 2 Address correspondence and reprint requests to Drs. Christian P. Larsen and Thomas C. Pearson, Department of Surgery, Emory University School of Medicine, Room 5105, 1639 Pierce Drive, Atlanta, GA 30322. E-mail address: clarsen@ emory.org 3 Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; 4-1BBL, 4-1BB ligand; ELISPOT, enzyme-linked immunospot assay.

Copyright © 2000 by The American Association of Immunologists

To confer protection against viral infection, peptide vaccines must activate significant numbers of CD8 T cells. Signals through costimulatory receptors are important components to the generation of an effective T cell response. In many different experimental systems, the costimulatory molecules CD28 and CD40 are critical for generating strong T cell responses (12–15). In the absence of either one of these costimulatory signals, the ability to generate T cell responses is diminished. The CD28 and CD40 pathways are also important for the induction of T cell responses by several different vaccines. For example, B7- and CD40-dependent responses promote immunity induced by tumor vaccination or DNA vaccines (16 –20). 4-1BB is a costimulatory molecule that is expressed on activated T cells (21, 22), and its ligand, 4-1BBL, is expressed on activated B cells, macrophages, and dendritic cells (23–26). Several studies have shown that signals through 4-1BB induce T cell activation and CD8 T cell survival (23, 26 –33). In addition, 4-1BB costimulation has also been shown to facilitate CTL development to induce clearance of tumors from mice (34 –36). We have previously examined the role of 4-1BB costimulation on the generation of primary T cell responses in the LCMV system (37). Although 4-1BBL⫺/⫺ mice generated effective primary antiviral CD8 T cell responses and were able to eliminate the infection, quantitation of these responses showed that 4-1BBL⫺/⫺ mice generated 2- to 3-fold fewer numbers of LCMV-specific cells compared with 4-1BBL⫹/⫹ mice. In this study the role of 4-1BB costimulation in T cell responses following peptide vaccination was examined by comparing the responses of 4-1BBL⫺/⫺ and 4-1BBL⫹/⫹ mice after immunization with a lipidated MHC class I-restricted LCMV peptide, NP396 – 404, covalently linked to an OVA Th epitope. In the absence of 4-1BB costimulation, CTL generation was reduced 10-fold and the number of Ag-specific CD8 T cells generated was 3- to 10-fold fewer than that in 4-1BBL⫹/⫹ mice. In addition, agonistic anti-4-1BB Abs restored 0022-1767/00/$02.00

The Journal of Immunology peptide-specific CD8 T cell responses to numbers comparable to those in 4-1BBL⫹/⫹ mice, showing that 4-1BB costimulation plays an important role in inducing CD8 T cell responses. Memory CD8 T cell responses were also lower than those in 4-1BBL⫹/⫹ mice. A high percentage of Ag-specific cells was generated after LCMV challenge of immunized 4-1BBL⫹/⫹ mice, whereas immunized 4-1BBL⫺/⫺ mice generated lower percentages of Agspecific cells and were impaired in their ability to eliminate the infection. Together, these studies demonstrate that 4-1BB costimulation is important in the generation of CD8 T cell responses after peptide immunization, and that vaccination with a single peptide epitope along with 4-1BB costimulation can induce long term immunity that is protective against viral challenge.

Materials and Methods Mice and Abs (B6 ⫻ 129)F2 (H-2b) or C57BL/6 (H-2b) were purchased from The Jackson Laboratory (Bar Harbor, ME). (C57BL/6 ⫻ 129)F2 4-1BBL⫺/⫺ mice were provided by Jacques Pechon, Immunex (Seattle, WA) (32). The 3E1 clone of the agonistic anti-4-1BB Ab has been previously described (30). Rat IgG was obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). 4-1BBL⫺/⫺ mice were treated with 200 ␮g of anti-4-1BB Ab or rat IgG on days 0, 2, 4, and 6 after immunization.

NP396 – 404 peptide synthesis and immunization The lipidated MHC class I-restricted LCMV peptide NP396 – 404 consisted of the NP396 – 404 peptide covalently attached to a chicken OVA Th epitope and a lipid moiety (9, 11). The NP396 – 404 peptide binds strongly to the MHC class I molecule Db and is a dominant LCMV CD8 T cell epitope. Mice, 6 –10 wk old, were immunized s.c. at the base of the tail with 10 ␮g of lipidated NP396 – 404 peptide as previously described (11).

Virus Seventy days after NP396 – 404 peptide immunization, mice were challenged with 2 ⫻ 106 PFU of the clone 13 variant of LCMV i.v. (38 – 41). Infectious LCMV in serum was quantitated by plaque assay on Vero cell monolayers as described previously (40).

Tetramer staining Tetramers of H-2Db bound to LCMV peptide NP396 – 404 or GP33– 41 were prepared and used as described previously (42). Spleen cells were stained with allophycocyanin-conjugated H-2Db NP396 – 404 or GP33– 41 tetramers and FITC-conjugated monoclonal anti-mouse CD8 purchased from PharMingen (La Jolla, CA) and analyzed by flow cytometry.

Intracellular staining for IFN-␥ The method for intracellular IFN-␥ staining has been described previously (42). Spleen cells were stimulated in vitro with medium, NP396 – 404, or

FIGURE 2. Intracellular IFN-␥ staining of peptidespecific T cells. Spleen cells from 4-1BBL⫹/⫹ and 4-1BBL⫺/⫺ mice on day 7 postimmunization were stimulated with NP396 – 404 for 5 h in vitro, surface stained with anti-CD8, and intracellularly stained with anti-IFN-␥. A, Representative flow cytometric analysis. Numbers indicate the percentage of CD8 T cells that are IFN-␥ positive. The percentage of IFN-␥-producing CD8 T cells was 5-fold lower in 4-1BBL⫺/⫺ than in 4-1BBL⫹/⫹ mice. B, Absolute number of Ag-specific cells generated in the spleen following immunization. 4-1BBL⫺/⫺ mice generated 3-fold fewer NP396 – 404specific cells than 4-1BBL⫹/⫹ mice. Error bars indicate the SD. The dotted line indicates the limit of detection.

2321

FIGURE 1. CTL response following peptide immunization in 4-1BBL⫹/⫹ and 4-1BBL⫺/⫺ mice. 4-1BBL⫹/⫹ and 4-1BBL⫺/⫺ mice were immunized s.c. at the base of the tail with 10 ␮g of lipidated NP396 – 404 peptide. Seven or eight days after immunization, CTL generation in the spleen was measured by a secondary bulk CTL assay using NP396 – 404 peptide-coated target cells. Each point indicates the average ⫾ SEM of three independent experiments involving six mice per group.

GP33– 41 for 5 h in vitro with brefeldin A (Golgiplug, PharMingen). They were then stained with allophycocyanin-conjugated monoclonal anti-CD8 (clone 53-6.7, PharMingen) and stained for intracellular IFN-␥ using the Cytofix/Cytoperm staining kit (PharMingen) according to the manufacturer’s recommended protocol. FITC-conjugated monoclonal rat anti-mouse IFN-␥ (clone XMG1.2) and its control isotype Ab (rat IgG1) from PharMingen were used for intracellular IFN-␥ staining.

Quantitation of peptide-specific IFN-␥-secreting CD8 T cells by ELISPOT assay Peptide-specific CD8 T cell responses were measured by IFN-␥ ELISPOT assay as described previously (42, 43). Spleen cells were stimulated with 0.1 ␮g/ml of purified NP396 – 404 peptide. Unimmunized spleen cells had a frequency of ⱕ2 IFN-␥-producing cells/106 spleen cells with or without stimulation.

CTL assay Splenocytes that were not treated with NH4Cl were cultured in vitro in 24-well plates at 4 ⫻ 106 effector cells/well. The cells were stimulated in the presence of LCMV-infected splenocytes from congenitally infected C57BL/6 mice at 1 ⫻ 106 cells/well. After 5 days, the cells were harvested, and CTL activity was measured in a 51Cr release assay as described previously (40). The target cells used in the CTL assay were either uncoated or coated with NP396 – 404 peptide (0.1 ␮g/ml).

2322

4-1BB IS REQUIRED FOR PROTECTIVE IMMUNITY AFTER PEPTIDE VACCINATION

Results

POT were 1/6522 in 4-1BBL⫺/⫺ mice and 1/877 in 4-1BBL⫹/⫹ mice. IFN-␥ ELISPOT analysis of spleen cells indicated that 4-1BBL⫺/⫺ mice generated 3- to 10-fold fewer NP396 – 404-specific cells compared with 4-1BBL⫹/⫹ mice (Table I). Wild-type mice had 2– 6 ⫻ 104 NP396 – 404 cells compared with 0.2–2 ⫻ 104 cells in 4-1BBL⫺/⫺ mice. To determine whether signaling through 4-1BB could rescue the defective CD8 T cell response and increase the number of Ag-specific cells generated in these mice, 4-1BBL⫺/⫺ mice were immunized with NP396 – 404 peptide and treated with an agonistic anti-4-1BB Ab. Table I shows that anti4-1BB Ab treatment of 4-1BBL⫺/⫺ mice increased the number of NP396 – 404-specific cells 2- to 30-fold, so that these mice now generated 3–7 ⫻ 104 NP396 – 404-specific cells. These numbers are comparable to the response generated by 4-1BBL⫹/⫹ mice.

To examine the role of 4-1BB costimulation in the development of T cell responses after peptide immunization, 4-1BBL⫹/⫹ and 4-1BBL⫺/⫺ mice were immunized s.c. with the lipidated LCMV MHC class I-restricted peptide NP396 – 404 covalently linked to an OVA Th epitope. Seven or eight days following immunization, spleen cells were harvested and cultured in vitro for 5 days with LCMV. The presence of peptide-specific CTL was determined in a cytolytic T cell assay using 51Cr-labeled NP396 – 404-coated target cells. As shown in Fig. 1, CTL responses in 4-1BBL⫺/⫺ mice were 10-fold lower than those in 4-1BBL⫹/⫹ mice (10-fold more effector cells from 4-1BBL⫺/⫺ mice were required to achieve the same percentage of specific lysis in 4-1BBL⫹/⫹ mice). The number of CD8 T cells specific to NP396 – 404 in 4-1BBL⫹/⫹ and 4-1BBL⫺/⫺ mice was quantitated by intracellular IFN-␥ staining. The percentage of NP396 – 404-specific CD8 T cells in 4-1BBL⫹/⫹ peptide-immunized mice was 1%, whereas it was only 0.2% in peptide-immunized 4-1BBL⫺/⫺ mice (Fig. 2A). The percentage observed was not due to any background staining, because ⬍0.01% of CD8 T cells were Ag specific in the absence of peptide stimulation in both 4-1BBL⫹/⫹ and 4-1BBL⫺/⫺ mice. 4-1BBL⫺/⫺ mice generated 3-fold fewer epitope-specific cells compared with 4-1BBL⫹/⫹ mice as measured by intracellular IFN-␥ staining. 4-1BBL⫺/⫺ mice had 1.7 ⫻ 104 NP396 – 404-specific cells in the spleen compared with 5.6 ⫻ 104 cells in 4-1BBL⫹/⫹ mice (Fig. 2B). The frequencies of IFN-␥-secreting NP396 – 404-specific cells in the draining inguinal lymph nodes as measured by IFN-␥ ELIS-

To examine whether 4-1BB costimulation affects T cell memory following peptide immunization, 4-1BBL⫹/⫹ and 4-1BBL⫺/⫺ mice were analyzed 2 mo postimmunization. The percentage and total numbers of Ag-specific memory CD8 T cells were measured by intracellular IFN-␥ staining. Some 0.3% of CD8 T cells were NP396 – 404 specific in 4-1BBL⫹/⫹ mice. In 4-1BBL⫺/⫺ mice, the percentage of NP396 – 404-specific cells was ⬍0.01% or below the level of detection (Fig. 3A). These numbers corresponded to 1.4 ⫻ 104 NP396 – 404 specific cells in 4-1BBL⫹/⫹ mice and ⬍7 ⫻ 103 in 4-1BBL⫺/⫺ mice (Fig. 3B). A more stringent test of T cell memory is protection after live viral challenge. The clone 13 variant of LCMV causes chronic infection in unimmunized mice. In contrast, LCMV-immune mice are able to generate secondary recall responses and control the infection quickly. To examine whether peptide vaccination could confer long term protective immunity and whether 4-1BB costimulation plays a role in this immunity, 4-1BBL⫹/⫹ and 4-1BBL⫺/⫺ mice were challenged with clone 13 70 days post-NP396 – 404 immunization. Unimmunized 4-1BBL⫹/⫹ and 4-1BBL⫺/⫺ mice were also infected with clone 13 as control groups. Eight days following clone 13 challenge, levels of virus in the serum were measured by plaque assay. Although immunized 4-1BBL⫹/⫹ mice had low viral titers and were clearly resolving the infection, four of six immunized 4-1BBL⫺/⫺ mice had elevated viral titers comparable to those found in unimmunized mice (Fig. 4). Among the

Table I. Number of NP396-404-specific cells/spleena

Expt.

1 2 3

⫹/⫹ Rat IgG

3.7 ⫻ 104b 2.0 ⫾ 0.5 ⫻ 104 5.7 ⫾ 1.9 ⫻ 104

4-1BBL⫺/⫺ Rat IgG

4-1BBL⫺/⫺ Anti-41BB Ab

0.5 ⫾ 0.01 ⫻ 104 0.2 ⫾ 0.01 ⫻ 104 1.8 ⫾ 0.8 ⫻ 104

3.1 ⫾ 1.0 ⫻ 104 6.7 ⫾ 5.3 ⫻ 104 2.8 ⫾ 1.0 ⫻ 104

a The number of NP396-404-specific cells in the spleen of ⫹/⫹ and 4-1BBL⫺/⫺ mice 7– 8 days after NP396-404 peptide immunization was determined using the IFN-␥ ELSIPOT assay. Mice were treated with 200 ␮g of either rat IgG or an agonistic anti-4-1BB Ab on days 0, 2, 4, and 6 after immunization. The numbers indicate the average of two mice per experiment ⫾ the range. b Data from one mouse is shown.

Impaired CD8 T cell responses in 4-1BBL⫺/⫺ mice following immunization with NP396 – 404 peptide

FIGURE 3. Analysis of peptide-specific memory T cells. Memory T cell responses in 4-1BBL⫺/⫺ mice were measured by intracellular IFN-␥ staining 58 days after peptide immunization. A, Representative flow cytometric analysis. Some 0.3% of CD8 T cells are NP396 – 404 specific in 4-1BBL⫹/⫹ mice, whereas the percentage was ⬍0.01% or below the level of detection in 4-1BBL⫺/⫺ mice. B, Absolute number of NP396 – 404-specific memory CD8 T cells was quantitated. Some 1.4 ⫻ 104 cells were Ag specific in 4-1BBL⫹/⫹ mice, and 4-1BBL⫺/⫺ mice had lower than detectable numbers of Ag-specific cells.

Protective immunity in NP396 – 404 peptide-immunized mice

The Journal of Immunology

FIGURE 4. Peptide-vaccinated 4-1BBL⫺/⫺ mice are impaired in their ability to resolve LCMV challenge. Seventy days after peptide immunization, 4-1BBL⫹/⫹ and 4-1BBL⫺/⫺ mice were injected with 2 ⫻ 106 PFU of LCMV clone 13. Eight days after LCMV challenge, levels of virus in the serum were determined by plaque assay (left panel). Immunized 4-1BBL⫹/⫹ mice had lower titers of virus than unimmunized mice at this time point, indicating that they had an enhanced response to the infection. In contrast, four of the six 4-1BBL⫺/⫺ mice still had viral titers similar to those found in unimmunized mice. Unimmunized 4-1BBL⫹/⫹ and 4-1BBL⫺/⫺ mice were unable to resolve the infection 8 days after clone 13 LCMV infection (right panel). Each dot represents one mouse. The dashed line indicates the limit of detection.

unimmunized mice, 4-1BBL⫹/⫹ and 4-1BBL⫺/⫺ mice had comparable viral titers, showing that the initial viral burden was similar in both groups of mice, and that the impaired viral clearance observed in immunized 4-1BBL⫺/⫺ mice was due to a weaker memory CD8 T cell response. By 23 days after challenge, all immunized 4-1BBL⫹/⫹ mice had cleared the infection. In contrast, only two of four immunized 4-1BBL⫺/⫺ mice cleared LCMV by this time point, and these mice still had 8.3 ⫻ 103 to 1.4 ⫻ 105 PFU/ml in the serum (data not shown). Ag-specific CD8 T cells in PBL were visualized directly using tetramers of Db complexed to the NP396 – 404 peptide (Fig. 5A). Eight days after clone 13 challenge, there was a strong skewing toward the NP396 – 404 epitope in 4-1BBL⫹/⫹ mice (70% of CD8 T cells were NP396 – 404 specific) that was 6-fold greater than that found in 4-1BBL⫹/⫹ unimmunized mice (13% of CD8 T cells were NP396 – 404 specific). In contrast, the skewing toward the NP396 – 404 epitope was less pronounced in 4-1BBL⫺/⫺ mice,

FIGURE 5. Analysis of Ag-specific cells in 4-1BBL⫹/⫹ and 4-1BBL⫺/⫺ mice after LCMV challenge. PBL from peptide-immunized 4-1BBL⫹/⫹ and 4-1BBL⫺/⫺ mice 8 days after LCMV challenge were examined for the percentage of Ag-specific cells. Ag-specific cells were visualized directly with the Db NP396 – 404 tetramer. A, Representative flow cytometric analysis. A strong skewing of NP396 – 404-specific cells was observed in immunized 4-1BBL⫹/⫹ mice (70% of CD8 T cells were Ag specific), whereas only 39% of CD8 T cells were Ag specific in immunized 4-1BBL⫺/⫺ mice. Note that the skewing of the NP396 – 404-specific cells was also reflected in the percentage of GP33– 41 (unimmunized epitope)specific cells (B). Some 3% of CD8 T cells in 4-1BBL⫹/⫹ mice were GP33– 41 specific, whereas 10% were GP33– 41 specific in 4-1BBL⫺/⫺ mice. Numbers indicate the percentage of CD8 T cells that are tetramer positive.

2323 where 39% of CD8 T cells were NP396 – 404 specific. This corresponded to a 4-fold increase compared with unimmunized 4-1BBL⫺/⫺ mice (10% of CD8 T cell were Ag specific). Similar results were observed when Ag-specific cells were quantitated by intracellular IFN-␥ staining (Fig. 6). In immunized 4-1BBL⫹/⫹ mice, 47% of CD8 T cells were Ag specific, a 7-fold increase compared with that in unimmunized 4-1BBL⫹/⫹ mice. In contrast, 26% of CD8 T cells in 4-1BBL⫺/⫺ mice were Ag specific, a 4-fold increase compared with that in unimmunized 4-1BBL⫺/⫺ mice. To examine how immunization with NP396 – 404 peptide would affect responses to other epitopes of LCMV, PBL from LCMV clone 13-infected mice were stained with the GP33– 41 tetramer (Fig. 5B). Eight days following clone 13 infection, about 10% of CD8 T cells were GP33– 41 specific in unimmunized mice. Interestingly, the percentage of CD8 T cells that were GP33– 41 specific in 4-1BBL⫹/⫹ mice after NP396 – 404 peptide immunization was reduced to 3% and reflected the strong skewing toward the NP396 – 404 epitope. In 4-1BBL⫺/⫺ mice, the percentage of GP33– 41-specific cells was 10% (comparable to that in unimmunized mice). The higher percentage of GP33– 41-specific cells in NP396 – 404 peptide-immunized 4-1BBL⫺/⫺ mice reflected the weaker skewing toward the NP396 – 404 epitope.

Discussion In this study we examined the role of 4-1BB costimulation in CD8 T cell responses following immunization with the LCMV peptide NP396 – 404. We showed that in the absence of 4-1BB costimulation, peptide-specific primary and memory CD8 T cell responses were reduced. Furthermore, agonistic anti-4-1BB Ab rescued peptide-specific CD8 T cell responses to numbers comparable to those in 4-1BBL⫹/⫹ mice, indicating that 4-1BB signals are important for the generation of CD8 T cell responses and that 4-1BBL⫺/⫺ mice do not have any inherent defect in their ability to signal through 4-1BB. In addition, 4-1BBL⫺/⫺ mice had low numbers of memory CD8 T cells and an impaired ability to generate secondary responses to LCMV challenge. 4-1BBL⫺/⫺ mice had higher viral titers in serum than 4-1BBL⫹/⫹ mice, because they generated lower numbers of Ag-specific cells. 4-1BB costimulation is therefore important for the elimination of LMCV infection and the generation of Ag-specific CD8 T cells after LCMV challenge in NP396 – 404 peptide-immunized mice. Peptide immunization induced weaker CD8 T cell responses than infection with LCMV. One percent of Ag-specific CD8 T

2324

4-1BB IS REQUIRED FOR PROTECTIVE IMMUNITY AFTER PEPTIDE VACCINATION

FIGURE 6. Intracellular IFN-␥ staining of virus-specific cells after LCMV challenge. The percentage of Ag-specific cells 8 days after clone 13 challenge was measured by intracellular IFN-␥ staining of PBL. Note that immunized 4-1BBL⫺/⫺ mice generated lower percentages of NP396 – 404specific cells than 4-1BBL⫹/⫹ mice. Unimmunized 4-1BBL⫹/⫹ and 4-1BBL⫺/⫺ mice had similar percentages of Ag-specific cells.

cells were generated following peptide immunization compared with 20% after LCMV infection (42, 44). The percentages correspond to about 5 ⫻ 104 cells in the spleen after immunization, whereas the number was about 1.2 ⫻ 107 cells 8 days after LCMV infection. The reasons why the lipidated NP396 – 404 peptide is a weaker stimulator of CD8 T cell responses compared with LCMV infection may be that there is a fixed amount of the peptide, the immunogen does not replicate, and the Ag is not systemically distributed. In contrast, much more Ag is present after systemic viral infections, where high amounts of Ag are produced, so that the total number of cells expressing the epitope and the amount of the epitope expressed by each of these cells are much greater than those after peptide immunization. In our previous studies when T cell responses were measured after acute LCMV infection, we observed that expansion of CD8 T cells was 2- to 3-fold lower in 4-1BBL⫺/⫺ mice than in 4-1BBL⫹/⫹ mice, whereas CTL generation in 4-1BBL⫺/⫺ mice was comparable to that in 4-1BBL⫹/⫹ mice (37). In this study responses to a nonreplicating Ag were 3- to 10-fold weaker in 4-1BBL⫺/⫺ mice. These results show that 4-1BB costimulation clearly plays a more important role in CD8 T cell activation in cases of limiting Ag, such as peptide immunization, than in cases where there is a lot of Ag, such as after systemic viral infection. A reason for this may be that LCMV infection provides a sufficiently strong stimulus for TCR so that some CD8 T cell responses do not require 4-1BB costimulation. Previous studies have shown that when no costimulation was provided, many more TCRs needed to be engaged for effective T cell activation, whereas fewer TCRs needed to be engaged when CD28 costimulation was provided (45). Therefore, during peptide immunization, where lower amounts of Ag are presented, the optimal number of TCRs required for T cell activation may not be available. Consequently, costimulatory molecules such as 4-1BB may be required for optimal T cell responses. Previous studies using agonistic anti-4-1BB Abs have shown that 4-1BB costimulation is important for promoting tumor-specific CTL generation, proliferation of CD8 T cells, induction of T cell activation markers, and enhancing CD8 T cell survival (30, 31, 34).

Alternatively, the reduced CD8 T cell expansion observed in the absence of 4-1BBL-dependent signals may not be due to a direct interaction of 4-1BB on the CD8 T cell with its ligand, but could be due to a lack of CD4 T cell help. The immunizing NP396 – 404 peptide contains an OVA Th epitope for generating CD4 T cell responses. Because peptide immunization provides a limiting, nonreplicating stimulus, CD8 T cell responses may be CD4 Th dependent. 4-1BB costimulation may be required for inducing CD4 T cell responses to provide help for developing CD8 T cell responses. Although previous work has shown that 4-1BB costimulation is not required for anti-viral CD4 T cell expansion (37), it cannot be ruled out that 4-1BB could be important for the induction of peptide-specific CD4 Th responses that facilitate the generation of CD8 T cell responses. Our results confirm previous studies showing that the lipidated NP396 – 404 peptide is an effective vaccine and extend these studies by showing that peptide vaccination can induce protective immunity against a viral challenge 2 mo after immunization. Previous work has shown that peptide immunization can induce protective immunity against viral infection ⬃2 wk after peptide immunization (10, 11). We show in this study that memory T cells could be detected 2 mo or more after immunization, and immunized mice were able to mount strong epitope-specific secondary responses and quickly control a viral challenge. Previous studies have shown that in chronically infected mice, cells responsive to NP396 – 404 are deleted at later time points after infection (⬍1% of CD8 T cells were NP396 – 404 specific by 45 days postinfection (46). In contrast, peptide-immunized mice did not delete the NP396 – 404 epitope at later time points following clone 13 challenge (50 and 30% of CD8 T cells were NP396 – 404 specific on days 23 and 45 postchallenge, respectively; data not shown). The skewing toward the NP396 – 404 epitope of NP396 – 404 peptideimmunized mice was so strong that 70% of CD8 T cells in NP396 – 404-immunized mice were NP396 – 404 specific 8 days after clone 13 infection. Interestingly, this strong skewing toward the NP396 – 404 epitope was reflected in the low percentages of GP33– 41-specific CD8 T cells. These results suggest that the number of NP396 – 404-specific cells generated was so high in NP396 – 404 peptide-immunized mice after clone 13 challenge that the viral infection was quickly eliminated and the deletion of the NP396 – 404-responsive cells was prevented. In addition, these results show that most of the CD8 T cell response after viral challenge is specific to the immunizing peptide. The strong recall response induced by peptide vaccination is Ag specific and not due to nonspecific bystander effects. Together, these results demonstrate that immunization with a single epitope confers protective immunity and illustrates the importance of costimulation for the effectiveness of vaccines. This study shows that the costimulatory molecule, 4-1BB, is important for establishing a strong memory CD8 T cell response following peptide immunization to induce protective immunity against a viral challenge. Our study therefore points to 4-1BB costimulation as an important therapeutic target for the development of vaccine strategies to boost immune responses against pathogenic agents.

Acknowledgments We thank Rose Hendrix, Carol Tucker-Burden, Shannon Cowan, and Kaja Madhavi Krishna for excellent technical assistance.

References 1. Feltkamp, M. C., H. L. Smits, M. P. Vierboom, R. P. Minnaar, B. M. de Jongh, J. W. Drijfhout, J. ter Schegget, C. J. Melief, and W. M. Kast. 1993. Vaccination with cytotoxic T lymphocyte epitope-containing peptide protects against a tumor induced by human papillomavirus type 16-transformed cells. Eur. J. Immunol. 23:2242.

The Journal of Immunology 2. Mayordomo, J. I., T. Zorina, W. J. Storkus, L. Zitvogel, C. Celluzzi, L. D. Falo, C. J. Melief, S. T. Ildstad, W. M. Kast, A. B. Deleo, et al. 1995. Bone marrowderived dendritic cells pulsed with synthetic tumour peptides elicit protective and therapeutic antitumour immunity. Nat. Med. 1:1297. 3. Mandelboim, O., E. Vadai, M. Fridkin, A. Katz-Hillel, M. Feldman, G. Berke, and L. Eisenbach. 1995. Regression of established murine carcinoma metastases following vaccination with tumour-associated antigen peptides. Nat. Med. 1:1179. 4. Schulz, M., R. M. Zinkernagel, and H. Hengartner. 1991. Peptide-induced antiviral protection by cytotoxic T cells. Proc. Natl. Acad. Sci. USA 88:991. 5. Kast, W. M., L. Roux, J. Curren, H. J. Blom, A. C. Voordouw, R. H. Meloen, D. Kolakofsky, and C. J. Melief. 1991. Protection against lethal Sendai virus infection by in vivo priming of virus-specific cytotoxic T lymphocytes with a free synthetic peptide. Proc. Natl Acad. Sci. USA 88:2283. 6. Aichele, P., H. Hengartner, R. M. Zinkernagel, and M. Schulz. 1990. Antiviral cytotoxic T cell response induced by in vivo priming with a free synthetic peptide. J. Exp. Med. 171:1815. 7. Ciupitu, A. M., M. Petersson, C. L. O’Donnell, K. Williams, S. Jindal, R. Kiessling, and R. M. Welsh. 1998. Immunization with a lymphocytic choriomeningitis virus peptide mixed with heat shock protein 70 results in protective antiviral immunity and specific cytotoxic T lymphocytes. J. Exp. Med. 187:685. 8. Schild, H., M. Norda, K. Deres, K. Falk, O. Rotzschke, K. H. Wiesmuller, G. Jung, and H. G. Rammensee. 1991. Fine specificity of cytotoxic T lymphocytes primed in vivo either with virus or synthetic lipopeptide vaccine or primed in vitro with peptide. J. Exp. Med. 174:1665. 9. Vitiello, A., G. Ishioka, H. M. Grey, R. Rose, P. Farness, R. LaFond, L. Yuan, F. V. Chisari, J. Furze, R. Bartholomeuz, et al. 1995. Development of a lipopeptide-based therapeutic vaccine to treat chronic HBV infection. I. Induction of a primary cytotoxic T lymphocyte response in humans. J. Clin. Invest. 95:341. 10. van der Most, R. G., A. Sette, C. Oseroff, J. Alexander, K. Murali-Krishna, L. L. Lau, S. Southwood, J. Sidney, R. W. Chesnut, M. Matloubian, et al. 1996. Analysis of cytotoxic T cell responses to dominant and subdominant epitopes during acute and chronic lymphocytic choriomeningitis virus infection. J. Immunol. 157:5543. 11. van der Most, R. G., K. Murali-Krishna, J. L. Whitton, C. Oseroff, J. Alexander, S. Southwood, J. Sidney, R. W. Chesnut, A. Sette, and R. Ahmed. 1998. Identification of Db- and Kb-restricted subdominant cytotoxic T-cell responses in lymphocytic choriomeningitis virus-infected mice. Virology 240:158. 12. Whitmire, J. K., R. A. Flavell, I. S. Grewal, C. P. Larsen, T. C. Pearson, and R. Ahmed. 1999. CD40-CD40 ligand costimulation is required for generating antiviral CD4 T cell responses but is dispensable for CD8 T cell responses. J. Immunol. 163:3194. 13. Shahinian, A., K. Pfeffer, K. P. Lee, T. M. Kundig, K. Kishihara, A. Wakeham, K. Kawai, P. S. Ohashi, C. B. Thompson, and T. W. Mak. 1993. Differential T cell costimulatory requirements in CD28-deficient mice. Science 261:609. 14. Larsen, C. P., E. T. Elwood, D. Z. Alexander, S. C. Ritchie, R. Hendrix, C. Tucker-Burden, H. R. Cho, A. Aruffo, D. Hollenbaugh, P. S. Linsley, et al. 1996. Long-term acceptance of skin and cardiac allografts after blocking CD40 and CD28 pathways. Nature 381:434. 15. Grewal, I. S., H. G. Foellmer, K. D. Grewal, J. Xu, F. Hardardottir, J. L. Baron, C. A. Janeway, Jr., and R. A. Flavell. 1996. Requirement for CD40 ligand in costimulation induction, T cell activation, and experimental allergic encephalomyelitis. Science 273:1864. 16. Chen, L., S. Ashe, W. A. Brady, I. Hellstrom, K. E. Hellstrom, J. A. Ledbetter, P. McGowan, and P. S. Linsley. 1992. Costimulation of antitumor immunity by the B7 counterreceptor for the T lymphocyte molecules CD28 and CTLA-4. Cell 71:1093. 17. Townsend, S. E., and J. P. Allison. 1993. Tumor rejection after direct costimulation of CD8⫹ T cells by B7-transfected melanoma cells. Science 259:368. 18. Cao, X., W. Zhang, L. He, Z. Xie, S. Ma, Q. Tao, Y. Yu, H. Hamada, and J. Wang. 1998. Lymphotactin gene-modified bone marrow dendritic cells act as more potent adjuvants for peptide delivery to induce specific antitumor immunity. J. Immunol. 161:6238. 19. Gurunathan, S., K. R. Irvine, C. Y. Wu, J. I. Cohen, E. Thomas, C. Prussin, N. P. Restifo, and R. A. Seder. 1998. CD40 ligand/trimer DNA enhances both humoral and cellular immune responses and induces protective immunity to infectious and tumor challenge. J. Immunol. 161:4563. 20. Tsuji, T., K. Hamajima, N. Ishii, I. Aoki, J. Fukushima, K. Q. Xin, S. Kawamoto, S. Sasaki, K. Matsunaga, Y. Ishigatsubo, et al. 1997. Immunomodulatory effects of a plasmid expressing B7-2 on human immunodeficiency virus-1-specific cellmediated immunity induced by a plasmid encoding the viral antigen. Eur. J. Immunol. 27:782. 21. Pollok, K. E., Y. J. Kim, Z. Zhou, J. Hurtado, K. K. Kim, R. T. Pickard, and B. S. Kwon. 1993. Inducible T cell antigen 4-1BB: analysis of expression and function. J. Immunol. 150:771. 22. Kwon, B. S., and S. M. Weissman. 1989. cDNA sequences of two inducible T-cell genes. Proc. Natl Acad. Sci. USA 86:1963. 23. Goodwin, R. G., W. S. Din, T. Davis-Smith, D. M. Anderson, S. D. Gimpel, T. A. Sato, C. R. Maliszewski, C. I. Brannan, N. G. Copeland, N. A. Jenkins, et al. 1993. Molecular cloning of a ligand for the inducible T cell gene 4-1BB: a

2325

24.

25.

26. 27.

28.

29.

30.

31. 32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44. 45. 46.

member of an emerging family of cytokines with homology to tumor necrosis factor. Eur. J. Immunol. 23:2631. Alderson, M. R., C. A. Smith, T. W. Tough, T. Davis-Smith, R. J. Armitage, B. Falk, E. Roux, E. Baker, G. R. Sutherland, and W. S. Din. 1994. Molecular and biological characterization of human 4-1BB and its ligand. Eur. J. Immunol. 24:2219. Pollok, K. E., Y. J. Kim, J. Hurtado, Z. Zhou, K. K. Kim, and B. S. Kwon. 1994. 4-1BB T-cell antigen binds to mature B cells and macrophages, and costimulates anti-␮-primed splenic B cells. Eur. J. Immunol. 24:367. DeBenedette, M. A., A. Shahinian, T. W. Mak, and T. H. Watts. 1997. Costimulation of CD28⫺ T lymphocytes by 4-1BB ligand. J. Immunol. 158:551. DeBenedette, M. A., N. R. Chu, K. E. Pollok, J. Hurtado, W. F. Wade, B. S. Kwon, and T. H. Watts. 1995. Role of 4-1BB ligand in costimulation of T lymphocyte growth and its upregulation on M12 B lymphomas by cAMP. J. Exp. Med. 181:985. Hurtado, J. E. C., S. H. Kim, K. E. Pollok, Z. H. Lee, and B. S. Kwon. 1995. Potential role of 4-1BB in T cell activation: comparison with costimulatory molecule CD28. J. Immunol. 155:3360. Saoulli, K., S. Y. Lee, J. L. Cannons, W. C. Yeh, A. Santana, M. D. Goldstein, N. Bangia, M. A. DeBenedette, T. W. Mak, Y. Choi, et al. 1998. CD28-independent, TRAF2-dependent costimulation of resting T cells by 4-1BB ligand. J. Exp. Med. 187:1849. Shuford, W. W., K. Klussman, D. D. Tritchler, D. T. Loo, J. Chalupny, A. W. Siadak, T. J. Brown, J. Emswiler, H. R. Cho, C. P. Larsen, et al. 1997. 4-1BB costimulatory signals preferentially induce CD8⫹ T cell proliferation and lead to the amplification in vivo of cytotoxic T cell responses. J. Exp. Med. 186:47. Takahashi, C., R. S. Mittler, and A. T. Vella. 1999. Cutting edge: 4-1BB is a bona fide CD8 T cell survival signal. J. Immunol. 162:5037. DeBenedette, M. A., T. Wen, M. F. Bachmann, P. S. Ohashi, B. H. Barber, K. L. Stocking, J. J. Peschon, and T. H. Watts. 1999. Analysis of 4-1BB ligand (4-1BBL)-deficient mice and of mice lacking both 4-1BBL and CD28 reveals a role for 4-1BBL in skin allograft rejection and in the cytotoxic T cell response to influenza virus. J. Immunol. 163:4833. Hurtado, J. C., Y. J. Kim, and B. S. Kwon. 1997. Signals through 4-1BB are costimulatory to previously activated splenic T cells and inhibit activation-induced cell death. J. Immunol. 158:2600. Melero, I., W. W. Shuford, S. A. Newby, A. Aruffo, J. A. Ledbetter, K. E. Hellstrom, R. S. Mittler, and L. Chen. 1997. Monoclonal antibodies against the 4-1BB T-cell activation molecule eradicate established tumors. Nat. Med. 3:682. Guinn, B. A., M. A. DeBenedette, T. H. Watts, and N. L. Berinstein. 1999. 4-1BBL cooperates with B7-1 and B7-2 in converting a B cell lymphoma cell line into a long-lasting antitumor vaccine. J. Immunol. 162:5003. Melero, I., N. Bach, K. E. Hellstrom, A. Aruffo, R. S. Mittler, and L. Chen. 1998. Amplification of tumor immunity by gene transfer of the co-stimulatory 4-1BB ligand: synergy with the CD28 co-stimulatory pathway. Eur. J. Immunol. 28: 1116. Tan, J. T., J. K. Whitmire, R. Ahmed, T. C. Pearson, and C. P. Larsen. 1999. 4-1BB ligand, a member of the TNF family, is important for the generation of antiviral CD8 T cell responses. J. Immunol. 163:4859. Ahmed, R., C. S. Hahn, T. Somasundaram, L. Villarete, M. Matloubian, and J. H. Strauss. 1991. Molecular basis of organ-specific selection of viral variants during chronic infection. J. Virol. 65:4242. Matloubian, M., S. R. Kolhekar, T. Somasundaram, and R. Ahmed. 1993. Molecular determinants of macrophage tropism and viral persistence: importance of single amino acid changes in the polymerase and glycoprotein of lymphocytic choriomeningitis virus. J. Virol. 67:7340. Ahmed, R., A. Salmi, L. D. Butler, J. M. Chiller, and M. B. Oldstone. 1984. Selection of genetic variants of lymphocytic choriomeningitis virus in spleens of persistently infected mice: role in suppression of cytotoxic T lymphocyte response and viral persistence. J. Exp. Med. 160:521. King, C. C., R. de Fries, S. R. Kolhekar, and R. Ahmed. 1990. In vivo selection of lymphocyte-tropic and macrophage-tropic variants of lymphocytic choriomeningitis virus during persistent infection. J. Virol. 64:5611. Murali-Krishna, K., J. D. Altman, M. Suresh, D. J. Sourdive, A. J. Zajac, J. D. Miller, J. Slansky, and R. Ahmed. 1998. Counting antigen-specific CD8 T cells: a reevaluation of bystander activation during viral infection. Immunity 8:177. Whitmire, J. K., M. S. Asano, K. Murali-Krishna, M. Suresh, and R. Ahmed. 1998. Long-term CD4 Th1 and Th2 memory following acute lymphocytic choriomeningitis virus infection. J. Virol. 72:8281. Butz, E. A., and M. J. Bevan. 1998. Massive expansion of antigen-specific CD8⫹ T cells during an acute virus infection. Immunity 8:167. Viola, A., and A. Lanzavecchia. 1996. T cell activation determined by T cell receptor number and tunable thresholds. Science 273:104. Zajac, A. J., J. N. Blattman, K. Murali-Krishna, D. J. Sourdive, M. Suresh, J. D. Altman, and R. Ahmed. 1998. Viral immune evasion due to persistence of activated T cells without effector function. J. Exp. Med. 188:2205.