A Long-Lasting Antitumor Vaccine Into Converting a B Cell Lymphoma

4-1BBL Cooperates with B7-1 and B7-2 in Converting a B Cell Lymphoma Cell Line into a Long-Lasting Antitumor Vaccine1

Barbara-ann Guinn,* Mark A. DeBenedette,* Tania H. Watts,* and Neil L. Berinstein2*†‡§¶

A20 is a B cell lymphoma that constitutively expresses the costimulatory molecule B7-2 yet grows readily as a tumor in syngeneic BALB/c mice. We have compared the tumorigenicity of A20 variants expressing either B7-1 (A20/B7-1) or B7-2 (A20/B7-2) with an A20 variant expressing B7-1 and B7-2 with 4-1BBL (A20/4-1BBL), a costimulatory member of the TNF family. Mice injected with tumors expressing the vector backbone (A20/CMV) or B7-1 developed tumors within 25 days of s.c. injection. In contrast, mice injected with A20/4-1BBL were tumor free for the 150-day follow-up period, while 25% of mice injected with A20/B7-2 developed tumors. Tumorigenicity experiments using nude mice indicated the requirement for T cells for variant rejection. Almost all mice that resisted the initial tumor challenge were resistant to further challenge with the parental tumor. Splenocytes from these mice showed high CTL lytic activity against the parental tumor, A20, as well as the syngeneic BALB/c lymphoma K46J, but showed background levels of lytic activity against the congenic SCID thymoma line ST-D2 or the allogeneic EL4 thymoma. In vitro blocking experiments with anti-B7-1 plus anti-B7-2 and/or soluble 4-1BB receptor showed B7-1, B7-2, and 4-1BBL all contributed to the CTL activity. Thus, the data show that neither B7-1 or B7-2 alone can confer full immunogenicity to the A20 lymphoma but that the addition of 4-1BBL results in a tumor that is highly immunogenic and can confer long-lasting protection against challenge with parental tumor in vivo. The Journal of Immunology, 1999, 162: 5003–5010.

ostimulatory molecules on APCs have been shown to be CD4ϩ and CD8ϩ T cells (20–22). Engagement of the 4-1BB re- the necessary second signal required to activate naive T ceptor has been shown to relay strong costimulatory signals within C cells. The first signal comes from the binding of pro- activated T cells, which leads to their enhanced proliferation and cessed Ag presented on MHC molecules by the TCR/CD3 com- cytokine secretion (23, 24). Such signaling prevents activation- plex on T cells. The absence of the second signal has been shown induced cell death (25) following TCR cross-linking in the absence to lead to T cell anergy (1, 2) and is thought to be one mechanism of other accessory signals. 4-1BBL (20) is a high-affinity ligand for by which tumors evade the immune system (3, 4). The B7 family 4-1BB, expressed on the surface of activated APCs (20, 21, 26, of costimulatory molecules consists of two members, B7-1 (CD80) 27). It is a type II membrane protein that shows homology to and B7-2 (CD86) (5–8), which have been shown to be equally able members of the TNF receptor family (20, 28, 29). T cells purified to activate T cells. These molecules are generally expressed on from CD28Ϫ/Ϫ mice have been shown to secrete cytokines and APCs, including macrophages, dendritic cells, and activated B proliferate in response to lymphomas expressing 4-1BBL, a re- cells (5, 9, 10) and have more recently been shown to be expressed sponse that can be inhibited by the soluble 4-1BB receptor fusion on T cells (11, 12). Most APCs require activation for the expres- protein (27). In the absence of a CD28 signal, the 4-1BBL:4-1BB sion of B7-1 and B7-2, and B7-2 is induced more rapidly than interaction has been shown to have a role in the production of a B7-1 after various stimuli (13, 14). The receptors for the B7 mol- Th2 response in mixed lymphocyte reactions (MLR)3 (30). ecules are CD28 and CTLA4. Positive signals that lead to T cell A number of investigators have transfected B7-1 or B7-2 into proliferation and cytokine secretion are mediated through CD28, tumor cells and assessed the affect on tumor immunogenicity. while CTLA4 appears to transduce an “off” signal limiting the Most of these studies have shown that elevating B7-1 levels con- period of T cell activation and proliferation (15–18). fers an antitumor response in a number of tumor cell lines (4, Another more recently described costimulatory receptor, 4-1BB 31–34). While most studies have indicated that B7-1 was more (19, 20), has been shown to have a pattern of expression that fol- potent than B7-2 at conferring antitumor immunity (34–36), some lows the primary activation of T cells and is restricted to activated studies have shown that B7-2 was more effective in other model systems (37). The variation in antitumor response conferred by the B7 molecules appears to lie in the immunogenicity of the tumor *Department of Immunology, †Institute of Medical Sciences, ‡Department of Medi- cell line used (32, 37). In the present report, we analyze the efficacy cine, and §Department of Medical Biophysics, University of Toronto, Toronto, On- tario, Canada; and ¶Toronto Sunnybrook Regional Cancer Centre, Toronto, Ontario, of the 4-1BBL molecule in eliciting an antitumor immune response Canada against A20 B cell lymphomas. Recent investigations have indi- Received for publication October 28, 1998. Accepted for publication January cated the potential for the 4-1BB:4-1BBL interaction to confer 21, 1999. antitumor immunity (38, 39), and we now show that transfected The costs of publication of this article were defrayed in part by the payment of page 4-1BBL in combination with B7-1 and B7-2 is more effective than charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. cells transfected with either the B7-1 and B7-2 molecules alone in conferring antitumor immunogenicity in a model system. We also 1 This work was funded by Connaught Canadian Universities Research Program (to N.L.B) and Medical Research Council of Canada (to T.H.W.). B.G. is funded by demonstrate that A20 cells that express high levels of 4-1BBL Leukemia Research Fund of Canada and Faculty of Medicine Edward Christie Stevens Postdoctoral Fellowship Awards. 2 Address correspondence and reprint requests to Dr. Neil Berinstein, Toronto-Sun- 3 Abbreviations used in this paper: MLR, mixed lymphocyte reaction; AP, alkaline nybrook Regional Cancer Centre, 2075 Bayview Avenue, Toronto, Ontario, Canada phosphatase; A20/B7-1 and A20/B7-2, A20 transfected with B7-1 and B7-2, M4N 3 M5. E-mail address: [email protected] respectively.

Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00 5004 4-1BBL ROLE IN CONVERTING B CELL LINE INTO ANTITUMOR VACCINE together with B7-1 and B7-2 are able to mediate full, long-lasting Gillian Wu (Ontario Cancer Institute, University of Toronto, Toronto, tumor immunogenicity preventing tumor development and produc- Canada). ing an in vivo resistance to further tumor challenge, which is mediated through CTL activity. Thus, a combinatorial approach in- Abs and reagents volving several costimulatory molecules may offer a more effective strategy for antitumor vaccine regimens. The hybridomas used in these studies were obtained from ATCC and used as described previously (30). T cell phenotype checks before use in MLR were performed using anti-TCR, anti-CD4, anti-CD8 (PharMingen, San Materials and Methods Diego, CA), and anti-CD3 (Cedarlane Laboratories, Hornby, Ontario, Can- Cell lines, transfection, and cloning ada) Abs in addition to those described above. FITC-conjugated anti- The A20 (40) BALB/c B cell lymphoma line originally derived from a mouse IgG (Sigma) and streptavidin-FITC (Molecular Probes, Eugene, spontaneous reticulum cell neoplasm type B was obtained from the Amer- OR) were used as secondary Abs, the latter in conjunction with D-biotin ican Type Culture Collection (ATCC, Manassas, VA). All A20 cells and (Molecular Probes)-conjugated (44) primary Abs. Isotype controls of rat variants were maintained as described previously (41). The full-length IgG2a and rat IgG2b were obtained from Cedarlane, and murine IgG2a was 4-1BBL cDNA was isolated by RT-PCR from the K46J cell line and sub- obtained from PharMingen and used at equivalent concentrations to test cloned into the pRc/CMV vector (Invitrogen, San Diego, CA) as described Abs in each experiment. elsewhere (42). Cells were electroporated with 100 ␮g of plasmid, cloned, NIH3T3 cells secreting the fusion protein of the extracellular domain of and subcloned as described previously (41). The A20/4-1BBL clone 1 4-1BB linked to alkaline phosphatase (AP) were a kind gift from Dr. By- (Cl.1) was derived from an independent clone to the A20/4-1BBL clone 2 oung Kwon (Indiana University, Indianapolis, IN) (26). 4-1BB-AP was (Cl.2). puriﬁed as described previously (27), and AP was used as an irrelevant The X63 IL-2- and IL-4-secreting cells lines were a kind gift from Dr. binding control. Fritz Melchers (Basel Institute for Immunology, Basel, Switzerland) (43). The CTLL-2 IL-2-dependent line was obtained from ATCC, and the CT.4S IL-4-dependent line was a kind gift from Dr. William Paul (National In- Cell counts and phenotyping stitute of Allergy and Infectious Disease, Bethesda, MD). The BALB/c B cell lymphoma line K46J (40) and the C57BL/6 lymphoma line EL4 were Cell counts were performed using 0.04% trypan blue/PBS. FACS pheno- obtained from ATCC. The SCID thymoma line ST-D2 was derived from a typing of cell surface markers and the costimulatory molecules was per- mouse with a congenic BALB/c background and was obtained from Dr. formed as described previously (41).

FIGURE 1. FACS phenotyping of the A20 cell line variants. Shown on the x-axis are relative ﬂuorescence levels (FL1-H) following Ab and FITC- labeled second-step staining; shown on the y-axis are cell counts; 106 cells were analyzed in each test. Isotype control staining of the cells appears as shaded histograms, and test staining for cell surface markers appears as outlined histograms. Staining is 1 ␮gofAbper106 cells. Similar phenotypic analyses were performed before every experimental assay. The Journal of Immunology 5005

Animal studies Female BALB/c and C57BL/6 mice age 8–10 wk were purchased from Charles River Laboratories (St. Constant, Quebec, Canada). All mice were maintained in accordance with University of Toronto guidelines and following the approval of independently reviewed protocols by the Animal Facility Committee at the University of Toronto. Mice were sacrificed when the tumors reached 2 cm in diameter. Allogeneic MLR assays MLR assays were performed as described previously (30). Irradiated A20 tumor variants were plated at 2 ϫ 105 cells per well along with 2 ϫ 105 C57BL/6 T cells in 96-well plates (Nalge Nunc International, Rochester, NY). Cells were incubated for 3 days before measurement of IL-2 and 5 days before the measurement of IL-4 using bioassays. Bioassays to measure the levels of biologically active IL-2 or IL-4 MLR supernatants were harvested and levels of biologically active IL-2 and IL-4 were measured in bioassays as described elsewhere (41). Tumor implantation and growth Tumor variants were washed free of serum with RPMI 1640 and 106 cells implanted s.c. into the right flank of naive BALB/c mice. Surviving mice, in which no tumor formed, were challenged by s.c. injection in the left flank. Mice were checked for tumor formation at least three times weekly throughout the experimental period, with daily checks at times of peak tumor incidence. CTL assays Mice that survived injection with tumor variants and challenge with the parental tumor line were sacrificed and their spleens removed. CTL assays were performed as described previously (30). For Ab blocking experiments, cells were incubated in the presence of 20 ␮g/ml Ab during the effector phase.

Results Generation and phenotypic analysis of A20 variants expressing 4-1BBL FIGURE 2. Allogeneic MLR to assess IL-2 and IL-4 secretion induced A20 variants derived from independent clones expressing elevated by tumor cell variants. Naive C57BL/6 T cells (105) were incubated with levels of 4-1BBL were generated as described in Materials and irradiated cell line variants (105). Bioassays were used to measure IL-2 and Methods. Two A20 variants expressing high levels of 4-1BBL IL-4 production. MLR supernatants were serially diluted in culture media were selected for further study and compared with the A20 paren- as indicated on the x-axis to ensure that optimal measurement of cytokine tal line, A20 transfected with vector alone (A20/CMV), A20 trans- output was made. To measure biologically active IL-2 secretion, [methyl- 3 fected with B7-1 (A20/B7-1), and A20 transfected with B7-2 H]thymidine incorporation by proliferating IL-2-dependent CTLL-2 cells (A20/B7-2) (Fig. 1). Weekly FACS phenotyping indicated that the was measured (A). IL-4 production was similarly measured using the IL- 4-dependent CT.4S cells (B). Only the A20/B7-1 cells elicited a MLR expression of the costimulatory molecules and phenotypic markers response from the T cells with high IL-2 secretion and a moderate IL-4 were stable. Cell cycle analyses and 4-1BBL expression were si- response. Data are representative of at least two independent experiments; multaneously investigated by propidium iodide staining and FACS error bars indicate the SD from triplicate samples used in the experiment and indicated no cell cycle variation of 4-1BBL expression in the shown, in which three ratios of responder:stimulator cells were assessed at parental A20 cells or the A20/4-1BBL clones. Furthermore, the three time points. cell lines showed no variation in growth rates as determined by daily cell counts of plated cells (data not shown). The A20 variants MLRs indicate that B7-1 induces an IL-2 and IL-4 response by showed similar levels of IgG, MHC I, and MHC II expression. As allogeneic CD4ϩ T cells, while all other variants do not expected, the parental A20 cell line expressed B7-2 but undetect- able levels of B7-1 and 4-1BBL. A20 transfected with the vector We performed MLR to determine the response of allogeneic T control showed a slight elevation of B7-1 and 4-1BBL, and this is cells to the A20 cell line variants. Using bioassays we found that attributed to clonal variation during the selection process. The the responding T cells produced biologically active IL-2 and IL-4 A20/B7-1 variant showed elevated B7-1 and levels of B7-2 similar in response to highly elevated expression of B7-1 (A20/B7-1) (Fig. to those of the parental line. Surprisingly, the A20/B7-2 line also 2, A and B). However, no detectable IL-2 or IL-4 was produced by showed enhanced 4-1BBL expression, and the A20/4-1BBL clones the responding T cells to the parental or vector controls or to the showed enhanced B7-1 and B7-2 expression. However, the A20/ A20/B7-2 or A20/4-1BBL clones. 4-1BBL line showed the highest level of 4-1BBL of all the variants. We attribute the acquisition of 4-1BBL on the B7-2 trans- Tumor variants expressing B7-2 and 4-1BBL are immunogenic fectant and B7-1 on the 4-1BBL transfectant to the cloning/ and protect against further systemic challenge with unmodiﬁed selection process. Nevertheless, the panel of transfectants parental A20 cells described in Fig. 1 provides a useful set of variants for assessing Injections of 106 A20, A20/CMV vector control, or A20/B7-1 cells the impact of 4-1BBL on the tumorigenicity of the A20 lymphoma. in the right ﬂank of the BALB/c mice resulted in similar rates of 5006 4-1BBL ROLE IN CONVERTING B CELL LINE INTO ANTITUMOR VACCINE

ing the highest level of 4-1BBL on the cells surface) developed tumors in the 150-day follow-up period in any of the experiments. Mice injected with A20/B7-2 cells that expressed elevated levels of 4-1BBL (but not to the extent of the A20/4-1BBL variant) showed a tumor development frequency of 25% compared with 100% in mice injected with the A20 parental line, 81.25% in mice injected with the A20/CMV control, and 93.75% in mice injected with A20/B7-1. In addition, tumors that formed in mice injected with A20/B7-2 developed at a later time point (Fig. 3A). FACS analysis of three of four of these tumors indicated that 4-1BBL expression had been completely down-regulated compared with the original A20/B7-2 clone injected into these mice and in two of three of the tumors B7-2 had also been fully down-regulated (Fig. 4). Mice surviving the primary injection with the A20 variants were challenged on the opposite flank with the parental A20 line. All but two mice resisted this systemic challenge, one originally injected with A20/B7-2 (1 of 16) and one originally injected with A20/4- 1BBL (1 of 32). To control for the tumorigenicity of these A20 parental cells used for the challenge, naive BALB/c mice were also injected with the same cells. The naive mice all formed tumors on the left flank within 25 days of injection (Fig. 3B). To ensure that the A20/B7-2 and A20/4-1BBL clones were all potentially tumorigenic in the absence of an intact immune system, nude mice were injected with 106 cells of A20/B7-1, A20/B7-2, or A20/4-1BBL clones 1 or 2. All of these mice formed tumors with no variation in tumor development rates (Fig. 3C). Mice injected with tumor variants expressing B7-2 or 4-1BBL showed ex vivo CTL activity against the parental tumor A20 and K46J following secondary restimulation in vitro Mice injected with the A20 cell line variants were analyzed at days 9, 15, and 21 postinjection for CTL activity against the A20 parental clone following in vitro stimulation with irradiated A20. No CTL activity against A20 was observed except in one of three mice injected with A20/B7-2 and one of three mice injected with A20/ 4-1BBL at days 15 and 21 (data not shown). Mice that had resisted tumor challenge were sacrificed at greater than day 150 and analyzed for CTL activity against A20 and a panel of cell line controls following ex vivo stimulation of the splenocytes using irradiated parental A20 cells. CTL activity against A20 and another BALB/c lymphoma, K46J, was observed in splenocytes from mice injected with A20/B7-2 and both clones of A20/4-1BBL (Fig. 5, A and B). A small amount of background CTL activity was seen against the SCID thymoma ST-D2 and the C57BL/6 lymphoma EL4 cell lines by splenocytes from mice in these groups. FIGURE 3. Tumorigenicity of A20 cell variants in syngeneic mice. Mice CTL activity was mediated by B7-1, B7-2, and 4-1BBL 6 were injected with 10 tumor cell variants and sacrificed when tumors reached interactions a diameter of 2 cm. Data shown are conglomerated from two independent experiments. Eight mice were used in each group. A, Survival of the mice To test the importance of the different costimulatory ligands in (y-axis) over a 150-day follow-up period (x-axis) indicated that there was no CTL activation, soluble 4-1BB receptor (4-1BB-AP) or anti- significant difference in the tumorigenicity of the A20 parental, A20/CMV B7-1 or anti-B7-2 was added during the in vitro secondary re- vector control, or the A20/B7-1 cells. All mice injected with the A20/4-1BBL activation as described in Materials and Methods. Splenocytes clones did not develop tumors at any time during the study. B, Mice that did from mice immunized with A20/B7-2 showed diminished CTL not form tumors when injected with the A20/B7-2 or A20/4-1BBL clones activity following blocking with anti-B7-1 and anti-B7-2 but were challenged with A20 parental cells on the opposing flanks, and naive not anti-B7-2 alone. However, CTL activity was further mice were similarly injected to ensure the tumorigenicity of the A20 cells at blocked in the presence of a combination of anti-B7-1, anti- that time. C, BALB/c nu/nu mice were injected with the tumor variants A20/ B7-1, A20/B7-2, or the A20/4-1BBL clones to determine whether these lines B7-2, and 4-1BB-AP (Fig. 6A). Splenocytes from mice immu- were tumorigenic in the absence of a mature T cell population. The two A20/ nized with A20/4-1BBL showed reduced CTL activity in the 4-1BBL clones were used in every study. presence of the combination of anti-B7-1, anti-B7-2 and 4-1BB-AP but not when any of the other Ab and 4-1BB-AP combinations were used (Fig. 6B). Cells only, Ab, and AP con- tumor formation and were highly tumorigenic despite the expres- trols were also used as negative controls for blocking reagents, sion of some level of B7-2 on the cell surface (Fig. 3A). None of and ST-D2 was used as a control for background lysis. The data the mice injected with either of the A20/4-1BBL clones (express- show that in splenocytes from mice immunized with A20/B7-2 The Journal of Immunology 5007

FIGURE 4. Analysis of escaping tumors in mice inoculated with the A20/B7-2 variants. Tumors that developed in mice injected with A20/B7-2 were excised and stained for the expression of B7-1, B7-2, 4-1BBL, IgG, MHC I, and MHC II. The tumor phenotype was compared with the parental A20/B7-2 cells and the A20 parental line. These tumor cells were also cultured to demonstrate viability and their capacity to survive the original culture conditions.

or A20/4-1BBL, combinations of B7-1, B7-2, and 4-1BBL sig- al. (38) showed that administration of anti-4-1BB mAbs into naling were all contributing factors in the induction of CTL DBA/2 mice following the injection of the P815 mastocytoma activity against the parental tumor. or Ag104A sarcoma cell line prevented the development of tumors in most mice. This tumor destruction was shown to be long lasting and specific against the parental tumor. The treat- Discussion ment was shown to lead to the proliferation of both CD4ϩ and In this study, we have shown that neither B7-2 on the A20 parental CD8ϩ cells ex vivo, and the role of both populations of T cells cells nor B7-1 on A20/B7-1 variants were able to mediate long- in the antitumor response was confirmed by anti-CD4 and anti- term antitumor immunity. However, expression of transfected CD8 depletion studies in vivo. Melero et al. (39) showed in 4-1BBL in conjunction with elevated B7-1 and B7-2 levels on the subsequent studies that P815 tumor cells expressing transfected A20/4-1BBL variants resulted in long-term immunity. None of the 4-1BBL did not form tumors in the majority of mice and that mice injected with 106 A20 variants expressing transfected these cells conferred long-term immunity against later chal- 4-1BBL developed tumors in a 150-day follow-up period (n ϭ 32), lenge with the wild-type tumor. CD4ϩ and CD8ϩ depletion while all mice injected with the A20 parental or vector control studies indicated that CD8ϩ but not CD4ϩ cells were respon- cells formed tumors within 25 days (n ϭ 32). In contrast, 25% of sible for the immunogenicity observed in vivo. We have ex- mice injected with A20/B7-2 succumbed to tumor development, tended the above studies by analyzing the effects of 4-1BBL in albeit at a delayed time point (n ϭ 4) compared with the controls. conferring antitumor immunity to the A20 tumor cell line in the Mice that resisted tumor formation following injection with A20/ presence of the B7-1 or B7-2 molecules. We have shown that 4-1BBL or A20/B7-2 demonstrated long-term immunity against the long-term immunity conferred to the mice by the variants later systemic challenge with the A20 parental cell line. This im- was dependent on an intact immune system. Tumor destruction munity lasted at least 150 days, at which time the mice were sac- appeared to be mediated by T cells as shown by experiments rificed for CTL analyses. Our tumorigenicity studies have clearly using nude mice. Although we did not demonstrate any T helper demonstrated that 4-1BBL enhances the capacity of elevated B7-1 activity induced by the A20/B7-2 or A20/4-1BBL variants in and B7-2 in converting this tumorigenic cell line into a long-last- MLR assays, significant CTL activity against the parental line ing anticancer vaccine. was observed in T cells from mice that had rejected A20 chal- Only two other reports have investigated the role of the lenge following A20/B7-2 and A20/4-1BBL injections. The de- 4-1BB:4-1BBL interaction in tumor immunogenicity. Melero et velopment of CTL effectors was stimulated synergistically 5008 4-1BBL ROLE IN CONVERTING B CELL LINE INTO ANTITUMOR VACCINE

FIGURE 5. CTL generation in mice rejecting tumor challenge with the A20/B7-2 or A20/4-1BBL variants. Mice that survived injection with A20/ FIGURE 6. CTL activity was blocked in the effector phase using 20 B7-2 or A20/4-1BBL were sacriﬁced at 150 days postinjection and 58 days ␮g/ml anti-B7-1 and anti-B7-2, anti-B7-2 alone, 4-1BB-AP alone, or all post-systemic challenge with the parental tumor. Splenocytes were ex- three reagents together. Ab control and AP-only incubations were concur- panded in the presence of irradiated tumor cell lines. We assessed CTL rently performed. Ex vivo expanded splenocytes from mice injected with activity against the parental tumor A20 and the congenic BALB/c SCID A20/B7-2 (A) or A20/4-1BBL (B) were incubated with chromium-labeled thymoma cell line ST-D2 (A), the syngeneic tumor K46J and the allogeneic A20 parental cells, and lytic activity was determined as a percentage of C57BL/6 lymphoma cell line EL4 (B). Naive mice were used as controls. total lysis following adjustments for spontaneous lysis. Chromium-labeled Lytic activity was determined as a percentage of total lysis following ad- ST-D2 cells were used as a control for speciﬁc lysis of the target cell justments made for spontaneous lysis. Data are representative of two in- population. Data are representative of at least two mice assessed with all dependent experiments. Ab combinations and controls in two independent experiments.

through B7-1, B7-2, and 4-1BBL signaling pathways as dem- The level of cytokine expression induced by the A20/B7-1 cells onstrated by blocking experiments. appears to have been below the level necessary for an antitumor Tumors that formed in mice injected with A20/B7-2 all showed response in syngeneic mice, as these cells showed no delay in a down-regulation of B7-1, B7-2, and 4-1BBL. Ab blocking during tumor formation in mice compared with vector and parental cell the in vitro reactivation of CTLs further indicated that in mice line controls. However, in vitro stimulated splenocytes from mice injected with either A20/B7-2 or A20/4-1BBL, it was a synergy injected with either A20/B7-2 or A20/4-1BBL demonstrated a between B7-1, B7-2, and 4-1BBL signaling that played a role in strong CTL response against the parental tumor ex vivo. Many of the secondary reactivation of CTLs against the parental tumor ex the whole-cell cancer vaccine model systems have shown that the vivo. 4-1BBL is thought to enhance the effects of B7-1 and B7-2 antitumor effects conferred by tumors expressing B7-1, B7-2, or levels and has been shown to prolong T cell activation when CD28 4-1BBL were via a direct CTL response (4, 31, 36, 37, 39, 46). unresponsiveness may otherwise develop (45). Our data suggest a It was interesting to find that the restimulated CTLs from mice close association between B7-1, B7-2, and 4-1BBL expression and injected with A20/4-1BBL or A20/B7-2 were also able to lyse the a strong link between the expression of B7-2 and 4-1BBL and syngeneic K46J cell line (40). In fact, there was slightly more CTL antitumor immunity in this model system. activity against these unmodified K46J cells than the original A20 A20 cells transfected with 4-1BBL were only able to form tu- parental cell line. This may in part be due to a slightly increased mors in the absence of an intact immune system, as seen following immunogenicity of the K46J cells compared with A20, as indi- the injection of these tumor cells into BALB/c nu/nu mice. Other cated by their decreased tumorigenicity in BALB/c mice (our un- studies have shown that 4-1BBL may stimulate naive T cells to published data). In addition, the A20 and K46J cell lines were secrete IL-2 (23, 24, 30, 42, 46) and IFN-␥ (46) in MLR assays. developed in the same study from spontaneous B cell lymphomas However, in our MLR assays, only the A20/B7-1 variant elicited that had developed in older (Ͼ15 months of age) BALB/c mice. an IL-2 and IL-4 cytokine response from naive allogeneic T cells. Hence, it is possible that these tumor cells may share a common The Journal of Immunology 5009 tumor-associated Ag. Only background levels of CTL activity bution of the B7-2 costimulator in mice: abundant expression on dendritic cells were seen against the congenic BALB/c SCID thymoma line in situ and during maturation in vitro. J. Exp. Med. 180:1849. 11. Azuma, M., H. Yessel, J. H. Phillips, H. Spits, and L. L. Lanier. 1993. Func- ST-D2 or the allogeneic C57BL/6 lymphoma cell line EL4, sug- tional expression of B7/BB1 on activated T-lymphocytes. J. Exp. Med. 177: gesting that the observed lysis of A20 and K46J was specific and 845. 12. Prabhu Das, M. R., S. S. Zamvil, F. Borriello, H. L. Weiner, A. H. Sharpe, and not due to contaminating NK activity. No difference in the level of V. K. Kuchroo. 1995. Reciprocal expression of co-stimulatory molecules, CTL activity against the parental tumor was observed between B7-1 and B7-2, on murine T-cells following activation. Eur. J. Immunol. mice injected with A20/B7-2 or either of the A20/4-1BBL clones, 25:207. 13. Lenschow, D. J., G. H-T. Su, L. A. Zuckerman, N. Nabavi, C. L. Jellis, and very low CTL activity was observed against any of the tumor G. S. Gray, J. Miller, and J. A. Bluestone. 1993. Expression and functional sig- cell lines by splenocytes from naive mice. nificance of an additional ligand for CTLA-4. Proc. Natl. Acad. Sci. USA 90: Future studies in other tumor models should help determine 11054. 14. Hathcock, K. S., G. Laszlo, C. Pucillo, P. Linsley, and R. J. Rhodes. 1994. whether the 4-1BBL globally enhances antitumor immunity in Comparative analysis of B7-1 and B7-2 costimulatory ligands: expression and combination with the B7 molecules or whether this capacity function. J. Exp. Med. 180:631. varies with tumor type and the immunogenicity of the tumor 15. Gribben, J. G., G. J. Freeman, V. A. Boussiotis, P. Rennert, C. L. Jellis, E. Greenfield, M. Barber, V. A. Restivo, Jr., X. Ke, G. S. Gray, and L. M. Nadler. line. We have shown that in this B cell lymphoma model, 1995. CTLA-4 mediates antigen-specific apoptosis of human T cells. Proc. Natl. 4-1BBL has converted the tumorigenic A20 cell line into a po- Acad. Sci. USA 92:811. 16. Walunas, T. L., D. J. Lenschow, C. Y. Bakker, P. S. Linsley, G. J. Freeman, tent and long-lasting antitumor vaccine. We have demonstrated J. M. Green, C. B. Thompson, and J. A. Bluestone. 1994. CTLA-4 can function antitumor immunogenicity protective not only against systemic as a negative regulator of T cell activation. Immunity 1:501. wild-type tumor challenge with the parental cell line in vivo but 17. Tivol, E. A., F. Borriello, A. N. Schweitzer, W. P. Lynch, J. A. Bluestone, and A. H. Sharpe. 1995. Loss of CTLA-4 leads to massive lymphoproliferation and immunogenicity that may also lead to the development of CTLs fatal multiorgan tissue destruction, revealing a critical negative regulatory role of able to kill cells from a syngeneic tumor cell line in vitro. The CTLA-4-Ig. 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