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* Department of Immunology, Mayo Clinic, Rochester, MN 55905; and
Department of Pathology, Ohio State University Medical Center, Columbus, OH 43210
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Introduction |
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Recent years have witnessed the rapid identification and characterization of new B7 family molecules and their receptors, whereas their role in the positive and negative control of immune responses is just being elucidated (2). B7-H3 was initially identified as a costimulatory molecule with limited amino acid sequence homology to other B7 family molecules for human T cell responses through a distinct receptor from CD28, CTLA-4, ICOS, and PD-1 (3). Although B7-H3 mRNA is widely detected in lymphoid and nonlymphoid organs in human and other species, B7-H3 protein is not constitutively expressed on these tissues (3, 4). Expression of cell surface B7-H3 could be induced on monocytes and dendritic cells by IFN-
(3). In addition to the originally identified two-domain structure (2IgB7-H3) containing an IgV and an IgC domain for both mouse and human B7-H3, a four-domain structure containing IgV-IgC-IgV-IgC (4IgB7-H3) has also been found in the genomic DNA of humans and monkey, but not in mouse and hamster (4, 5). A recent study using a mAb indicates that the mainly expressed form of B7-H3 on human dendritic cells may be 4IgB7-H3 (6).
Immunological function of B7-H3 is still controversial. Our original study demonstrates that immobilized and cell-associated human B7-H3 costimulates T cell proliferation, selectively induces IFN-, and promotes the growth and differentiation of CD8+ CTL against allogeneic Ags, indicating that B7-H3 preferentially costimulate the cellular immune response (3). Injection of mouse B7-H3 plasmid directly into established mouse EL4 lymphoma induced partial regression of tumors and enhanced T cell responses (7). Two recent studies, however, suggest a role of B7-H3 in the inhibition of T cell responses. Ling et al. (5) showed that immobilized 4B7-H3Ig inhibited T cell proliferation in vitro. Lung infiltration of inflammatory cells increased after challenge with a model Ag in B7-H3-deficient mice. The authors concluded that B7-H3 inhibited Th 1 T cell responses. In this study, however, inductions of experimental autoimmune encephalitis and CTL to lymphocytic choriomeningitis virus, which are largely dependent on Th 1 cytokines, were not affected (8). In this study, we report the role of B7-H3 in the stimulation of T cell-mediated tumor immunity in vivo.
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Materials and Methods |
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DBA/2, CByD2F1/J (DBA/2 x BALB/c), C57BL/6, and BALB/c nu/nu mice were purchased from the National Cancer Institute (Frederick, MD). Transgenic mice expressing TCR specific for tumor Ag P1A in the context of H-2Ld (P1CTL) were described previously (9). P815 mastocytomas were maintained in a complete medium of RPMI 1640 (Invitrogen Life Technologies, Rockville, MD) containing 10% FBS (HyClone, Logan, UT), 25 mM HEPES, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin G, and 100 µg/ml streptomycin sulfate.
Cloning of mouse B7-H3 cDNA and transfection
Full-length B7-H3 cDNA was amplified by RT-PCR from Con A-activated C57BL/6 splenocytes and cloned into pcDNA3 plasmid (BD Clontech, Palo Alto, CA) as previously described (3, 4). The resulting plasmid mB7-H3/pcDNA3 and wild-type (wt)3 pcDNA3 vector were transfected into P815 cells using the Fugene 6 transfection system (Boehringer Mannheim, Mannheim, Germany) according to the manufacturer’s instruction. Cell lines stably expressing mouse B7-H3 were selected by G418 and were screened by flow cytometry analysis with a mouse B7-H3-specific mAb.
Fusion proteins and mAb
The fusion protein containing the extracellular domain of mouse B7-H3 and mouse IgG2a Fc was prepared and cloned into pMIgV vector (10). The resulting pmB7-H3Ig plasmid was transfected into Chinese hamster ovary (CHO) cells and cultured in CHO medium (Invitrogen Life Technologies) containing 1% FBS. B7-H3Ig fusion protein was purified from the supernatants by a protein G-Sepharose column (Amersham Pharmacia Biotech, Uppsala, Sweden). A hybridoma (clone 9C11)-secreting IgG mAb against mouse B7-H3 was generated by immunizing an Armenian hamster with purified B7-H3Ig. The specificity of 9C11 was determined by negative staining of CHO cell transfectants expressing mouse B7-1, B7-2, B7-H1, B7-DC, and B7-H4. Hybridomas that produced mAb against CD4 (clone GK1.5) or CD8 (clone 53-6.72) were purchased from American Type Culture Collection. All mAb were purified from culture supernatants of the hybridomas by either protein A- or protein G-Sepharose columns as described previously (11). The mAb to mouse CD3; FITC-conjugated mAb to B220, CD4, or I-Ad; PE-conjugated mAb to mouse IgG2a; and CyChrome-conjugated mAb to CD8 were purchased from BD Pharmingen (San Diego, CA). Anti-asialo GM1 antiserum was purchased from Wako (Osaka, Japan). H-2Ld Dimer XI was purchased from BD Pharmingen and P1A35–43 peptide was incorporated into the dimer according to the manufacturer’s instructions.
T cell proliferation and cytokine assay
T cell costimulation assays were performed as described previously (12). Briefly, nylon wool-purified T cells from C57BL/6 mice were added at 2.5 x 105 cells/well in triplicate into 96-well flat-bottom microplates that were precoated with anti-CD3 mAb overnight at 4°C and subsequently coated with B7-H3Ig or control Ig for 4 h at 37°C. The cells were incubated at 37°C for 72 h and [3H]TdR was added at 1 µCi/well during the last 6 h. The incorporation of [3H]TdR was counted using the MicroBeta Trilux liquid scintillation counter (Wallac, Turku, Finland). To detect cytokines, culture supernatants were collected at 24–72 h and the concentration of cytokines was determined by sandwich ELISA (BD Pharmingen) according to the manufacturer’s instructions.
P1A-specific CTL generation and cytotoxicity assay
Naive or regressor (spontaneous regression after E3 tumor inoculation) mice were challenged with 1 x 106 wt P815 cells. Three weeks later, spleens were removed and cultured with irradiated (260 Gy) wt P815 cells at the indicated responder:stimulator ratio for 5 days. The cultured cells were collected on day 5 and CTL activity against wt P815 was measured in a standard 4-h 51Cr release assay. To generate P1A-specific CTL, CD8+ T cells were negatively selected from a pool of spleen and lymph node cells from P1CTL transgenic mice by incubation with mixed FITC-conjugated mAbs against mouse B220, CD4, and I-Ad, subsequently incubated with anti-FITC-coated magnetic beads, and selected with MACS LD separation columns (Miltenyi Biotec, Auburn, CA). Purified CD8+ T cells (>99% CD3+ T cell) were stimulated with irradiated P815/mock or E3 lines or with 0.1 µg/ml P1A peptide in the presence of irradiated syngeneic splenocytes for 4 days. CTL activity was measured in a standard 4-h 51Cr release assay.
Animal studies
The methods to determine the growth and measurement of tumors in mice were described previously (13). For depletion of CD4, CD8, and asialo GM1 cells in vivo, 500 µg anti-CD4, anti-CD8, or 50 µl of anti-asialoGM1 Ab was injected i.p. on days 0, 7, and 14, respectively, after E3 tumor injection. In a P1CTL reconstitution model, a pool of spleen and lymph node cells from P1CTL transgenic mice were injected i.v. into CByD2F1/J mice at 1 x 106/mouse. The mice were then challenged s.c. with tumor cells on the same day. To determine cell division, the MACS LD-purified CD8+ T cells from P1CTL mice were labeled with CFSE and adoptively transferred into CByD2F1/J mice that were inoculated with P815/mock or E3 cells 6 days before. Tumor-draining lymph nodes (TDLN) and spleens were removed on days 2, 3, and 5 and double-stained with P1A/H-2Ld dimer and CyChrome-conjugated anti-CD8 mAb. Cell divisions were measured by using flow cytometry analysis to calculate the dilution of CFSE intensity as described previously (14, 15).
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Results |
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production (Fig. 1B) in the presence of suboptimal doses of mAb against mouse CD3. This costimulatory effect of B7-H3Ig is dose dependent. Isotype-matched control IgG2a stimulates neither proliferation nor IFN- production of T cells. We conclude that mouse B7-H3 has similar costimulatory functions compared with its human homologue.
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We next determined the cellular mechanisms of B7-H3-mediated tumor immunity. DBA/2 mice were first inoculated with E3 cells and injected with various Abs to deplete CD4, CD8, or NK cells. Injection of Abs against CD4 or asialo GM1 had a small but not significant effect on the tumorigenicity of E3 cells. However, injection of mAb against CD8 completely eliminated the growth resistance and all mice died of tumors within 30 days (Fig. 3A). Our results thus support a central role for CD8+ T cells in B7-H3-mediated tumor immunity. Furthermore, the mice, which experienced regression of E3 tumor, were also resistant to challenge by lethal doses of wt P815 cells (Fig. 3B) and developed high levels of CTL activity against wt P815 (Fig. 3C), indicating the development of memory T cells. Naive mice after challenged with the same dose of P815 cells developed progressively growing tumors without the induction of specific CTL (Fig. 3, B and C).
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Using the P1CTL adoptive transfer system, we examined the role of B7-H3 on the expansion of tumor-specific CTL. F1 mice were first inoculated with E3 cells. Seven days after inoculation, naive spleen cells from P1CTL transgenic mice were labeled with CFSE and transferred into tumor-bearing F1 mice. Two, 3, and 5 days after T cell transfer, cells from TDLN and spleens were prepared and stained by the P1A dimer, which specifically stains P1A-specific T cells, and gated on CFSE for analysis. Although there was no significant CTL division observed on day 2, a dramatic expansion of P1A-specific CTL were found in both the TDLN and the spleen of E3-bearing mice on day 3. Significant T cell division was also found in P815/mock TDLN, albeit to a lesser degree than those from E3-bearing mice. On day 5, the majority of P1A-specific CTL had undergone at least one cycle of division in E3-bearing mice while some CTL remained unactivated in P815/mock-bearing mice (Fig. 4A). Our results thus support that expression of B7-H3 on tumor cells accelerates division of tumor-specific CTL in lymphoid organs, which may be responsible for enhanced tumor immunity.
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Discussion |
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We reported previously that the receptor for B7-H3, which could be distinguished from CD28, CTLA-4, and PD-1, is not expressed on resting T cells (3), supporting the hypothesis that B7-H3 is involved in the expansion rather than initial priming of T cell responses. Although our results here support an important role of B7-H3 costimulation in the expansion of T cells in vivo, the effect of B7-H3 in the stimulation of naive or early primed CTL could not be excluded. The observation that B7-H3+ P815 cells induced rapid proliferation and cell division on naive P1TCR CTL in vitro and in vivo (Fig. 4) supports this possibility. Clonal expansion of P1A-specific CTL occurs first in the TDLN and the expression of B7-H3 accelerates this process. Taken together with the observation of the rapid appearance of dividing cells in the spleen, these findings indicate that B7-H3 costimulation rapidly induces not only local expansion of tumor-specific CTL in the TDLN but also systemic responses in the spleen.
High-level expression of costimulatory molecules on tumor cells could enhance direct and bystander costimulation of T cells (16). It is unknown at this time whether these mechanisms, individually or collectively, operate in our model. Direct stimulation/expansion, however, is normally not a major mechanism for tumor Ag-mediated activation of T cells because solid tumors often do not express high levels of such molecules. To test whether the expression of B7-H3 by tumor cells enhances direct stimulation of T cells, we purified P1A-specifiic CTL CD8+ T cells to move professional APC by MACS bead-guided negative selection (>99% purity) and stimulated them with irradiated P815/mock or E3 cells. Stimulation with E3 cells induced an increased proliferation of T cells, in contrast to P815/mock, in a wide range of responder:stimulator ratios from 1:5 to 1:40. Consistent with this finding, incubation of purified P1CTL with E3 cells induced elevated division of T cells on day 3 and stimulated a stronger CTL response in comparison to the P815/mock cells (Fig. 4C). Our results thus indicate that expression of B7-H3 on tumor cells could directly stimulate T cells in the absence of professional APC. In a recent report, intratumoral injection of a mouse B7-H3 plasmid induced regression of established EL4 tumor in a portion of tumor-bearing mice by CD8+ T cells and NK cells (7). In our system, we did not find the role of NK/NKT cells for the regression of B7-H3+ tumors. One possibility is that plasmid injection led to expression of B7-H3 on APC to enhance an interaction with NK cells because it is unclear whether or not B7-H3 was expressed exclusively by tumor cells or other APC after plasmid injection.
Inoculation of B7-H3+ tumors led to regression on 
50% of mice, whereas others eventually developed large tumors and died (Fig. 2). The escaping mechanism underlying this observation is yet to be elucidated. Progressively growing B7-H3+ tumors do not lose the expression of B7-H3 and MHC molecules based on flow cytometry analysis (L. Luo and L. Chen, unpublished data), thus excluding clonal selection of tumor variants. One possible interpretation is elevated regulatory T cell activity in the mice escaping from tumor rejection. We found that depletion of CD25+ T cells by mAb in naive mice before challenge of B7-H3+ tumor led to accelerated rejection (L. Luo and L. Chen, unpublished data), suggesting involvement of regulatory T cells in the regulation oftumor immunity in our system. However, frequency of Ag-specific T cells may play a key role because adoptive transfer of P1 TCR T cells, which increase frequency of tumor Ag-specific T cells, could lead to regression of 100% tumors in syngeneic mice (Fig. 3). P815 tumors grow very rapidly after inoculation into the mice and it is thus likely that expansion of tumor Ag-specific T cells by B7-H3 expression is suboptimal and does not outpace the growth of tumor cells in the mice.
Experimental tumors could be categorized into immunogenic and poorly immunogenic based on degree of immune responses they elicit in syngeneic hosts and it is, in general, difficult to elicit T cell responses against poorly immunogenic tumors (17). This is partially due to loss of Ag or Ag presentation/processing molecules as well as microenvironment by which a tumor creates during progression. P815 is an immunogenic tumor (17) and, similar to B7-H3, expression of immunomodulatory molecules including the B7 family costimulatory molecules and cytokines could enhance its immunogenicity (18). It remains to be tested whether B7-H3 could also enhance immunogenicity of poorly immunogenic tumors. In our preliminary experiments, we found that transfection to express B7-H3 in a poorly immunogenic C3 tumor (11) failed to enhance its immunogenicity (L. Luo and L. Chen, unpublished data). Since T cells in the mice with growing C3 tumor is in ignorant status (11), our data suggest that the expression of B7-H3 could not break T cell ignorance. In addition, we also found that repeated injection of irradiated B7-H3+ P815 cells did not induce regression of preinoculated P815 tumors (L. Luo and L. Chen, unpublished data). Taken together, our data suggest that B7-H3 costimulation should combine with other immune stimulatory modalities for cancer immunotherapy of established tumors.
A recent study by Suh et al. (8) using B7-H3-deficient mice indicates an enhanced lung inflammatory infiltration by macrophages and lymphocytes and increased IFN- secretion after exposure to OVA Ag. They concluded that B7-H3 is inhibitory for Th1-mediated responses. However, there was only a small difference in Th1-mediated responses including experimental autoimmune encephalitis and CTL responses to LCMV viral infection (8), which are believed to be mediated or dependent on Th1 T cell responses. Therefore, it is unclear whether or not B7-H3 preferentially regulates Th1-mediated responses. In our experiments, CD4+ T cells are not required for the induction of CD8+ CTL for tumor immunity since depletion of CD4+ T cells did not diminish the resistance of mice to B7-H3-transfected P815 tumor. One possibility is that B7-H3 may preferentially regulate CD4-independent induction of CD8+ CTL responses as shown in our tumor system. Our findings, however, do not exclude the possibility that absence of CD4+ T cells impairs the generation of memory CD8+ CTL, as indicated in several recent studies (19, 20, 21).
Although B7-H3 mRNA has been found in various tissues, surface expression of B7-H3 protein is not evident. B7-H3 is normally undetectable on resting hemopoietic cells and normal organs by immunohistochemistry analysis (L. Luo and L. Chen, unpublished data). However, the expression of B7-H3 protein could be up-regulated by various stimuli on dendritic cells, B cells, and T cells (3). It is thus likely that the expression of B7-H3 is tightly controlled by local assaults such as infection and inflammation. Despite this limited expression pattern, enhanced expression of B7-H3 on tumor cells, as shown in our experiments, has a strong stimulatory function for the expansion of T cell responses. These findings implicate that B7-H3 could be applied alone or incorporated with other immunostimulatory methods, ideally with those promoting T cell priming, to enhance immune responses against cancers.
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Footnotes |
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1 This study was supported by National Institutes of Health Grants CA98731, CA58033, and CA69091 and by the Mayo Foundation. 
2 Address correspondence and reprint requests to Dr. Lieping Chen, Johns Hopkins Medical Institutions, 600 North Wolfe Street, Jefferson Street Building 1-121, Baltimore, MD 21287. E-mail address: lchen42{at}jhmi.edu
3 Abbreviations used in this paper: wt, wild type; CHO, Chinese hamster Ovary; TDLN, tumor-draining lymph node.
Received for publication December 10, 2003. Accepted for publication August 10, 2004.
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production. Nat. Immunol. 2:269.[Medline]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) or control Ig (
) in the presence of a suboptimal dose of immobilized mAb to CD3. Data represent one experiment of five and [3H]TdR incorporation is presented as cpm ± SD. B, IFN-
, 
). Tumor sizes (mean diameter ± SD) were measured (B) every 4 days, starting on day 8, and survival time (C) was monitored. The data were obtained from three independent experiments. The mice with progressive (
