Importance of B7-1-expressing host antigen-presenting cells for the eradication of B7-2 transfected P815 tumor cells

We have previously shown that B7-2 (CD86)-transfected P815 tumor cells elicit tumor-eradicating immunity that leads to the regression of the B7-2+ P815 tumor after transient growth in normal DBA/2 mice. Here, we show that both the B7-2 and B7-1 (CD80) molecules contribute to the eradication of B7-2+ P815 tumors as treatment of the mice with both anti-B7-2 and anti-B7-1 mAb was required to prevent B7-2+ P815 tumor regression. The cells that expressed the B7-1 molecule following inoculation of B7-2+ P815 tumor cells into normal mice were not the tumor cells but rather host APCs including MAC-1+ cells present in the draining lymph nodes. Moreover, B7-1-expressing host APCs were found to be important for the rejection of B7-2+ P815 tumors as anti-B7-2 mAb alone, which was ineffective in preventing B7-2+ P815 tumor rejection by normal wild-type mice, was effective in preventing B7-2+ P815 tumor rejection by mice in which the B7-1 gene was disrupted. Finally, consistent with the importance of B7-1-expressing host APCs for the generation of tumor-eradicating immunity against B7-2+ P815 tumor cells, CD4+ T cells (not only CD8+ T cells) were found to participate in tumor-eradicating immunity against B7-2+ P815 tumor cells. Thus, in addition to eliciting tumor-eradicating immunity directly, B7-2+ P815 tumor cells elicit tumor-eradicating immunity indirectly through B7-1-expressing host APCs that present tumor-associated Ags to CD4+ T cells.     

Ganciclovir chemoablation of herpes thymidine kinase suicide gene-modified tumors produces tumor necrosis and induces systemic immune responses

The goal of this work was to identify potential host immune responses to thymidine kinase (TK) suicide gene-modified tumors undergoing chemoablation induced by the prodrug ganciclovir (GCV). The aims were to measure the efficacy and specificity of immunity induced against unmodified tumor, to identify qualitative or quantitative changes in the host response to TK+ tumors undergoing chemoablation that may contribute to the induction of antitumor immunity, and to compare critically the induction of immunity by chemoablation of TK-modified tumors with that of other methods of immunization in this tumor model and in response to other well-defined model antigens. Animals treated with TK+ tumors and GCV developed specific resistance to rechallenge with unmodified tumor. GCV induced significant tumor necrosis, which was associated with a pronounced host cell infiltrate composed of polymorphonuclear cells, both CD4+ and CD8+ T lymphocytes, and increased intratumoral IL-12. Cyclophosphamide-treated mice exhibited no such host response despite the induction of tumor necrosis. CTL responses to defined antigens in TK+ cells were greater in animals treated with prodrug than were those in animals not treated with prodrug but harboring live TK+ cells. Similar degrees of immunity were produced by immunization with irradiated cells.     

Eliciting T cell immunity against poorly immunogenic tumors by immunization with dendritic cell-tumor fusion vaccines

Dendritic cells (DCs) are the most effective APCs and are being studied as natural adjuvants or Ag delivery vehicles to elicit T cell-mediated antitumor immunity. This study examined whether inoculation of DCs fused with poorly immunogenic tumor cells elicited tumor-reactive T cells for adoptive immunotherapy. DCs derived from bone marrow of C57BL/6 (B6) mice were fused with syngeneic B16 melanoma or RMA-S lymphoma cells by polyethylene glycol. The B16/DC and RMA-S/DC fusion hybrids expressed MHC class I, class II Ags, costimulatory molecules, as well as DC-specific and tumor-derived surface markers. The tumor/DC hybrids were capable of processing and presenting tumor-derived Ags, and immunization of B6 mice with irradiated B16/DC or RMA-S/DC vaccine elicited tumor-specific CTL activities. Vaccination of B6 mice with irradiated B16/DC fusion preparations induced partial host protective immunity against B16 tumor challenge. Reduced tumor incidence and prolonged survival time were observed. Adoptive transfer of T cells derived from B16/DC vaccine-primed lymph nodes into B16 tumor-bearing mice greatly reduced the number of established pulmonary metastases with or without in vivo administration of IL-2. Moreover, adoptive transfer of RMA-S/DC vaccine-primed, cultured lymph node T cells eradicated disseminated FBL-3 tumor. The results demonstrate that tumor/DC fusion products are effective cellular vaccines for eliciting T cell-mediated antitumor immunity.     

IL-12 administered in vivo to young and aged mice. Discrepancy between the effects on tumor growth in vivo and cytotoxic T lymphocyte generation ex vivo: dependence on IFN-gamma

Because IL-12 restores allogeneic cytotoxic T lymphocyte (CTL) activity by T cells of aged mice in vitro, we initially assessed whether IL-12 could overcome age-related deficits when given to aged mice in vivo. Growth of P815(H-2(d)) was enhanced in aged compared with young BALB/c (H-2(d)) mice and tumor growth was curtailed by IL-12 in both age groups. Unexpectedly, secondary CTL stimulated ex vivo with P815 were reduced in IL-12-treated mice compared with controls. Primary CTL generated ex vivo across MHC differences in IL-12 treated BALB/c and C57BL/6 young mice were reduced by 90-99%, were dose- and time-dependent, and were associated with reduced allo-stimulated NK-like activity and [3H]thymidine incorporation. IFN-gamma was elevated in sera and in supernatants from allo-stimulated cultures from IL-12-treated mice, while IL-4 was reduced in such supernatants, suggesting that, despite reduced CTL, IL-12 was associated with increased Th1- and reduced Th2-type cytokine production. IL-12 also induced splenomegaly, primarily due to increased numbers of cells lacking markers of mature T, B and NK cells, or macrophages, or polymorphonuclear leukocyte morphology. IFN-gamma mutant mice exhibited reduced splenic enlargement in response to IL-12, suggesting that the splenomegaly was due, in part, to IFN-gamma production. However, reduced CTL generation was not due entirely to dilution of CTL precursor cells because spleen cellularity and size increased 3-fold while CTL activity decreased 10- to 100-fold, and CTL generation normalized to CD8(+) T effector cells was still significantly reduced in IL-12-treated mice. Interestingly, purified CD4(+) and CD8(+) T cells from IL-12-treated normal mice exhibited greater proliferative and cytolytic activities respectively compared with controls. Thus, effector T cells in IL-12-treated mice were not impaired, but exhibited augmented responsiveness, suggesting that IL-12 induced complex interactions among spleen cell populations and that these effects, in part, are mediated by IFN-gamma.     

Delayed progression of murine AIDS in C57BL/6 mice pre-immunized with a highly antigenic 10-mer peptide encoded by the murine AIDS defective virus gag p12 gene

C57BL/6 (B6) mice were immunized with a highly antigenic 10-mer peptide (P12-10), which is encoded by the murine AIDS (MAIDS) defective virus gag p12 gene, emulsified in incomplete Freund's adjuvant (ICFA). One week later, the mice were inoculated with the MAIDS virus to see if the immunization affects progression of MAIDS. It was demonstrated that the immunization significantly delayed progression of MAIDS, although it failed to induce appreciable cytotoxic T lymphocyte (CTL) responses against the P12-10 antigen. In contrast, immunization of B6 mice with the P12-10 coupled with liposome induced substantial CTL responses but failed to protect the mice against MAIDS development. This segregation between CTL activity and in vivo protection efficacy might be worth considering when we exploit vaccines for augmenting cellular immunity mediated by CD8+ T cells.     

Interstitial brachytherapy procedures for brain tumors

This report characterizes the immunological host response to a syngeneic murine mammary carcinoma along with variants genetically modified to express B7-1 or secrete GM-CSF and interleukin-12 (IL-12). MT-901 is a subline of a mammary adenocarcinoma that was chemically induced in the Balb/c host. It was found to be weakly immunogenic by immunization/ challenge experiments, and it induced tumor-specific T-cell responses in lymph nodes (LN) draining progressive subcutaneous tumors. Tumor clones expressing B7-1 or secreting GM-CSF exhibited reduced tumorigenicity without completely abrogating tumor growth, whereas IL-12 elaboration lead to complete tumor growth inhibition. In vivo subcutaneous inoculation of a transgenic cell clone secreting GM-CSF (240 ng/10(6) cells/24 hours) resulted in significantly enhanced T-cell reactivity of tumor-draining lymph node (TDLN) cells as compared to wild-type TDLN cells. This finding was obtained from observations assessed by several different methods, including: 1) in vitro cytotoxicity, 2) in vitro interferon-gamma release, and 3) adoptive transfer in mice with established tumor. Moreover, the transfer of activated LN cells derived from mice inoculated with GM-CSF-secreting tumor cells resulted in the prolonged survival of animals with macroscopic metastatic disease, which was not evident utilizing LN cells from mice inoculated with wild-type tumor. By contrast, clones that expressed B7-1 or IL-12 (4 ng/10(6) cells/24 hours) did not elicit enhanced tumor-reactive TDLN cells compared with wild-type tumor when assessed in the adoptive transfer model. The autocrine secretion of GM-CSF by transduced tumor cells was found to serve as an effective immune adjuvant in the host response to this weakly immunogenic tumor.     

The shared tumor-specific antigen encoded by mouse gene P1A is a target not only for cytolytic T lymphocytes but also for tumor rejection

A number of human tumor antigens have been characterized recently using cytolytic T lymphocytes (CTL) as screening tools. Some of them are encoded by MAGE-type genes, which are silent in normal tissues except in male germ cells, but are activated in a variety of tumors. These tumor-specific shared antigens appear to be promising targets for cancer immunotherapy. However, the choice of these antigens as targets has been questioned because of the lack of direct evidence that in vivo responses against such antigens can lead to tumor rejection. The antigen encoded by the mouse gene P1A represents the only available animal model system for MAGE-type tumor antigens. We show here that mice immunized by injection of L1210 leukemia cells expressing P1A and B7-1 (L1210.P1A.B7-1) are efficiently protected against a challenge with a lethal dose of mastocytoma P815 tumor cells, which express P1A. Mice immunized with L1210 cells expressing B7-1 but not P1A were not protected. Furthermore, we observed that P1A-transgenic mice, which are tolerant to P1A, were not protected after immunization with L1210.P1A.B7-1. These results demonstrate that the immune response to P1A is the major component of the tumor rejection response observed in normal mice, and support the use of tumor-specific shared antigens as targets for the immunotherapy of human cancer.     

Antimetastatic vaccination against Lewis lung carcinoma with autologous tumor cells modified to express murine interleukin 12

Interleukin 12 (IL-12) is a disulfide-linked heterodimer molecule produced predominantly by professional antigen presenting cells. It promotes the induction of sundry biological effects with significant relevance to antitumor immunity, such as enhancing a T(H)1 helper response, an in vivo antiangiogenic effect, induction of adhesion molecules that assist in lymphocyte homing to sites of tumor growth, and a direct stimulatory effect on both T-cells and NK cells. We tested the efficacy of an antimetastatic vaccine composed of autologous murine D122 cells transfected with both subunits of IL-12 cDNA to express biologically-active IL-12 molecule. Expression of IL-12 by D122 cells significantly reduced their tumorigenicity and metastatic potential in immunocompetent syngeneic hosts. Furthermore, vaccination of mice with 2 x 10(6) irradiated IL-12-transfected D122 cells engendered a protective CTL response which rejected a subsequent challenge with parental D122 cells and eradicated lung micrometastasis in animals whose primary tumors have been surgically removed. The antitumor effects of IL-12 were mediated primarily by its ability to induce gammaIFN expression in vivo. CD8+ T-cells as well as NK cells were crucial in the execution of the antitumor effects of IL-12. These results suggest that autologous tumor cells expressing IL-12 by gene transfer are a potent antitumor vaccine able to induce a systemic immune response against poorly immunogenic and spontaneously metastatic tumors.     

A double recombinant adenovirus expressing the costimulatory molecule B7-1 (murine) and human IL-2 induces complete tumor regression in a murine breast adenocarcinoma model

Tumors that express tumor-specific antigens can maintain growth in an immunocompetent organism. Current hypotheses tend toward T cell anergy as a key component for the inhibition of immunoreactivity against such tumors. Anergy is thought to occur from hyperactive stimulation of the TCR in the absence of costimulation (costimulation leads to proliferation via IL-2 production). Subcutaneous injection of transgenic polyoma middle T transformed breast adenocarcinoma tumor cells (PyMT) in the hind flank of FVB/n mice results in the formation of tumor nodules at this site. We determined the MHC class I and class II, B7-1, and B7-2 expression in the tumor cells by flow cytometry and showed positive staining for only MHC class I. We show that a single E1-deleted adenovirus constructed to express both the costimulatory molecule B7-1 (murine) and human IL-2 genes (Ad5E1 mB7-1/human IL-2) elicits a very potent antitumor response when administered intratumorally. Ad5E1 mB7-1/human IL-2 induced rapid and complete regression (100%) of all tumors compared with Ad5 E1 mB7-1 (38%), Ad CAIL-2 (42%), and Ad5E1 dl70-3 (control vector) (0%). All mice that exhibited complete tumor regression were fully protected in tumor cell challenge experiments. The systemic immunity generated by intratumoral administration of the Ad vectors was associated with a strong anti-PyMT CTL response. These observations indicate that augmenting the immunogenicity of the tumor with coincident expression of B7-1 in combination with IL-2 may prove beneficial in direct tumor immunotherapy.     

Interleukin 12 and B7-1 costimulatory molecule expressed by an adenovirus vector act synergistically to facilitate tumor regression

Stimulation of antitumor immune mechanisms is the primary goal of cancer immunotherapy, and accumulating evidence suggests that effective alteration of the host-tumor relationship involves immunomodulating cytokines and also the presence of costimulatory molecules. To examine the antitumor effect of direct in vivo gene transfer of murine interleukin 12 (IL-12) and B7-1 into tumors, we developed an adenovirus (Ad) vector, AdIL12-B7-1, that encodes the two IL-12 subunits in early region 1 (E1) and the B7-1 gene in E3 under control of the murine cytomegalovirus promoter. This vector expressed high levels of IL-12 and B7-1 in infected murine and human cell lines and in primary murine tumor cells. In mice bearing tumors derived from a transgenic mouse mammary adenocarcinoma, a single intratumoral injection with a low dose (2.5 x 10(7) pfu/mouse) of AdIL12-B7-1 mediated complete regression in 70% of treated animals. By contrast, administration of a similar dose of recombinant virus encoding IL-12 or B7-1 alone resulted in only a delay in tumor growth. Interestingly, coinjection of two different viruses expressing either IL-12 or B7-1 induced complete tumor regression in only 30% of animals treated at this dose. Significantly, cured animals remained tumor free after rechallenge with fresh tumor cells, suggesting that protective immunity had been induced by treatment with AdIL12-B7-1. These results support the use of Ad vectors as a highly efficient delivery system for synergistically acting molecules and show that the combination of IL-12 and B7-1 within a single Ad vector might be a promising approach for in vivo cancer therapy.     

Antitumor response independent of functional B or T lymphocytes induced by the local and sustained release of interleukin-2 by the tumor cells

Transfection of tumor cells with a vector containing the entire coding sequence of human interleukin-2 (hIL-2) was previously shown to convert the tumorigenic murine fibrosarcoma line CMS-5 into a non-tumorigenic line. The failure of the IL-2-secreting tumor to grow in conventional (immunocompetent) mice was attributed to the activation of CD8+ T cells that exhibited tumor specificity and memory. In order to determine whether or not the IL-2 produced by the tumor may be activating tumor cytotoxic effector cells other than B or T cells we have repeated this study using immunodeficient SCID and SCID-beige mice as syngeneic tumor recipients. In contrast to the rapid growth of the wild-type tumor, the hIL-2-transfected cells (N2A/IL2/CMS5) did not grow, or grew more slowly and regressed, in the mice that lack functional B and T cells. The inhibition of tumor growth associated with the local release of IL-2 was reversed in mice treated with antiasialo-GM1 antibodies specific for natural killer (NK) lineage cells. In contrast to the studies with conventional mice, the IL-2-dependent effector cells in the immunodeficient mice exhibited no evidence of memory. In vitro analysis of spleen cells from tumor-bearing mice revealed the presence of effector cells able to lyse YAC-1 target cells as well as the wild-type CMS-5 and the IL-2-transfected variant tumor lines but unable to lyse P815 cells. The pattern of selective target cell killing and the kinetics of killing were indistinguishable from those observed using tumor necrosis factor alpha (TNF alpha) the mediator associated with natural cytotoxicity cell killing of tumor cells. Histopathology of the IL-2-secreting tumors in SCID mice reveals the presence of infiltrating lymphoid cells and macrophages that were not observed in the CMS-5 tumors. Consistent with the notion that the tumor killing in the SCID mice was mediated by TNF alpha, mice bearing IL-2-secreting tumors had elevated levels of serum TNF alpha and little or no effector cell activity, or TNF alpha was found in tumor-bearing mice treated with anti-asialo-GM1 antibody. The results indicate that the cytokine-induced tumor regression observed in the IL-2-transfected tumors is a more complex phenomenon than previously recognized and one that is mediated by effector cells of the NK cell and/or monocyte/macrophage lineages, in addition to CD8+ T cells.     

IL-10 inhibits alloreactive cytotoxic T lymphocyte generation in vivo

In this report, we present evidence that the CTL response directed against MHC Class I allo-determinants can be inhibited as a result of IL-10 expression in vivo. The presence of localized IL-10 secretion at the site of allogeneic tumor cell challenge resulted in marked inhibition of the CTL response and allowed growth of the tumor in the allogeneic host. Using purified CD4+ T cells from mice immunized in the presence or absence of IL-10, we have shown that the loss of alloreactivity as a consequence of IL-10 expression results from the inhibition of CD4+ T cell function. The expression of either IL-2 or IFN-gamma with IL-10 locally at the time of allogeneic cell challenge completely restored CTL alloreactivity, suggesting that the action of IL-10 could be bypassed by providing helper T lymphocyte-derived cytokines of the Th1 type at the site of immunization. Inhibition of alloreactivity by IL-10 was observed using either purified macrophages or dendritic cells as APC in an in vitro assay. Thus, the expression of IL-10 following antigenic challenge (such as that observed in Th2-like immune responses) may profoundly limit the ability for generating functional CTL in vivo.     

Passive but not active CD8+ T cell-based immunotherapy interferes with liver tumor progression in a transgenic mouse model

To evaluate tumor immunotherapies, we used transgenic mice that harbor a progressive liver tumor associated with the expression of the SV40 large tumor T oncoprotein (SV40-T). To induce "self" tumor Ag-specific CD8+ T cells, mice were injected with an immunodominant SV40-T CTL epitope mixed with a heterologous helper peptide. Despite repeated injections, this vaccine failed to raise a tumor-specific CD8+ T cell response that was efficient enough to counteract tumors. Although coimmunization with SV40-T CTL epitope and heterologous helper peptide efficiently recruited the respective Th cells, only low-avidity SV40-T-specific CD8+ T cells were activated. Furthermore, major alterations in SV40-T-specific B and Th cell responses were characterized. In contrast, transfers of higher-avidity CTLs specific for the same SV40-T epitope were effective in counteracting tumors. These results suggest that passive therapies targeted to self tumor Ag may be more suitable than active immunization in the treatment of spontaneous tumors.     

CpG DNA can induce strong Th1 humoral and cell-mediated immune responses against hepatitis B surface antigen in young mice

Successful neonatal immunization of humans has proven difficult. We have evaluated CpG-containing oligonucleotides as an adjuvant for immunization of young mice (1-14 days old) against hepatitis B virus surface antigen. The protein-alum-CpG formulation, like the DNA vaccine, produced seroconversion of the majority of mice immunized at 3 or 7 days of age, compared with 0-10% with the protein-alum or protein-CpG formulations. All animals, from neonates to adults, immunized with the protein-alum vaccine exhibited strong T helper (Th)2-like responses [predominantly IgG1, weak or absent cytotoxic T lymphocytes (CTL)]. Th2-type responses also were induced in young mice with protein-CpG (in 1-, 3-, and 7-day-old mice) and protein-alum-CpG (in 1- and 3-day-old mice) but immunization carried out at older ages gave mixed Th1/Th2 (Th0) responses. DNA vaccines gave Th0-like responses when administered at 1 and 7 days of age and Th1-like (predominantly IgG2a and CTL) responses with 14-day-old or adult mice. Surprisingly, the protein-alum-CpG formulation was better than the DNA vaccine for percentage of seroconversion, speed of appearance, and peak titer of the antibody response, as well as prevalence and strength of CTL. These findings may have important implications for immunization of human infants.     

B7-CD28 costimulation unveils the hierarchy of tumor epitopes recognized by major histocompatibility complex class I-restricted CD8+ cytolytic T lymphocytes

Immunization of mice with tumors genetically engineered to express the B7 costimulatory molecules amplifies the antitumor immune response mediated by CD8+ cytolytic T lymphocytes (CTL). In this report, we examined the effect of B7-CD28 costimulation on the hierarchy of tumor epitopes. Using a combination of affinity chromatography/reversed-phase high performance liquid chromatography and CTL cloning, we show that major histocompatibility complex (MHC) class I molecules from EL4 lymphoma cells can present at least six distinct CTL epitopes presented by MHC class I molecules. Nevertheless, mice immunized with wild-type B7-negative EL4 cells develop CTL only to one immunodominant epitope. In contrast, immunization with B7-transduced EL4 cells led to not only the amplification of the CTL response to this immunodominant epitope, but also to the recognition of five otherwise silent subdominant epitopes. The adoptive transfer of a CTL clone against such a subdominant epitope cured mice bearing EL4 lymphoma growing as an ascites tumor. The fact that CTL response can be spread to normally silent epitopes as a result of B7-CD28 costimulation suggests a novel approach to manipulate the hierarchy of CTL epitopes and offers an opportunity to explore novel targets for T cell-mediated cancer therapy.     

Construction and characterization of a triple-recombinant vaccinia virus encoding B7-1, interleukin 12, and a model tumor antigen

BACKGROUND: Construction of recombinant viruses that can serve as vaccines for the treatment of experimental murine tumors has recently been achieved. The cooperative effects of immune system modulators, including cytokines such as interleukin 12 (IL-12) and costimulatory molecules such as B7-1, may be necessary for activation of cytotoxic T lymphocytes. Thus, we have explored the feasibility and the efficacy of inclusion of these immunomodulatory molecules in recombinant virus vaccines in an experimental antitumor model in mice that uses Escherichia coli beta-galactosidase as a target antigen. METHODS: We developed a "cassette" system in which three loci of the vaccinia virus genome were used for homologous recombination. A variety of recombinant vaccinia viruses were constructed, including one virus, vB7/beta/IL-12, that contains the following five transgenes: murine B7-1, murine IL-12 subunit p35, murine IL-12 subunit p40, E. coli lacZ (encodes beta-galactosidase, the model antigen), and E. coli gpt (xanthine-guanine phosphoribosyltransferase, a selection gene). The effects of the recombinant viruses on lung metastases and survival were tested in animals that had been given an intravenous injection of beta-galactosidase-expressing murine colon carcinoma cells 3 days before they received the recombinant virus by intravenous inoculation. RESULTS: Expression of functional B7-1 and IL-12 by virally infected cells was demonstrated in vitro. Lung tumor nodules (i.e., metastases) were reduced in mice by more than 95% after treatment with the virus vB7/beta/IL-12; a further reduction in lung tumor nodules was observed when exogenous IL-12 was also given. Greatest survival of tumor-bearing mice was observed in those treated with viruses encoding beta-galactosidase and B7-1 plus exogenous IL-12. CONCLUSION: This study shows the feasibility of constructing vaccinia viruses that express tumor antigens and multiple immune cofactors to create unique immunologic microenvironments that can modulate immune responses to cancer.     

Tumor-infiltrating lymphocytes exhibiting high ex vivo cytolytic activity fail to prevent murine melanoma tumor growth in vivo

The identification of tumor-associated Ags recognized by CD8+ CTL and prevention of tumor outgrowth by adoptive transfer of these CTL demonstrates that CD8+ T cells play a major role in antitumor immunity. We have generated B16.F10 melanoma cells that express the glycoprotein epitope amino acid 33-41 (GP33) of the lymphocytic choriomeningitis virus (LCMV) to examine antitumor CD8+ T cell response in C57BL/6 mice immune to LCMV and in mice transgenic for the LCMV GP33-specific P14 TCR (P14 TCR mice). We find that B16.F10GP33 tumor cells grew in syngeneic C57BL/6 mice without inducing T cell tolerance. LCMV infection or adoptive transfer of LCMV-specific effector T cells delayed but did not prevent growth of preestablished tumors in these mice. However, B16.F10GP33 tumor cells were rejected in mice immune to LCMV and in mice treated with LCMV-specific effector T cells on the same day as the tumor. Surprisingly, B16.F10GP33 tumor cells grew in P14 TCR transgenic mice despite an abundance of tumor-associated Ag-specific CD8+ T cells. In these mice, freshly isolated tumor-infiltrating lymphocytes exhibited an activated phenotype and displayed high GP33-specific cytolytic activity when assessed ex vivo. Thus, B16.F10GP33 melanoma cells are able to initiate, but not to sustain, a GP33-specific CTL response sufficient to clear the tumor enduringly.     

Involvement of NK1+ CD4- CD8- alphabeta T cells and endogenous IL-4 in non-MHC-restricted rejection of embryonal carcinoma in genetically resistant mice

Non-MHC-restricted rejection mechanisms against the murine MHC-negative F9 embryonal carcinoma cells were analyzed. Strains of C57BL/6 (B6) background were resistant to the tumors irrespective of H-2 haplotypes, while others, including BALB/c background, were susceptible. This resistance was suggested to be mediated primarily by the host thymus-dependent alphabeta T cells, since both athymic B6 nude and normal B6 mice depleted with alphabeta T cells showed susceptible phenotype. The difference of the nature of alphabeta T cells infiltrating in H-2-identical B6- and BALB.B-derived tumors was then comparatively analyzed. It was revealed that unique T cells with NK1+ CD4- CD8- (double negative (DN)) alphabeta TCR+ phenotype were accumulated significantly in B6, but few in BALB.B mice. The population freshly isolated from the F9 tumor tissues preferentially expressed potent IL-4 mRNA, and was suggested to be mostly responsible for the endogenous IL-4 production. Indeed, the injection of either anti-NK1.1 or anti-IL-4-neutralizing Ab into the normal B6 rendered them significantly susceptible to the tumor cells. These results strongly suggested that NK1+ DN alphabeta T cells were responsible primarily for the rejection mechanisms against F9 tumors. Histologically, F9 tumors in B6 mice were characterized by abundant macrophage infiltration and massive tumor necrosis, neither of which was observed in those in BALB.B nor B6 mice preinjected with anti-IL-4 Ab, indicating that both histologic features in the resistant strain were dependent on the endogenous IL-4. Present results provide one of the first instances in which a recently emerging minor T cell subpopulation, thymus-dependent NK1+ DN alphabeta T cells, plays an essential role in anti-tumor responses in vivo.     

Fas ligand-mediated lysis of self bystander targets by human papillomavirus-specific CD8+ cytotoxic T lymphocytes

Mouse cytotoxic T lymphocytes (CTL) reactive with a H-2Db-presented 9-mer peptide of the human papillomavirus type 16 protein E7(49-57) (RAHYNIVTF) were generated from the spleen cells of wild-type C57BL/6 (B6) or B6 perforin-deficient (B6.P0) mice. CD8(+) B6 CTL displayed peptide-specific perforin- and Fas-mediated lysis of E7-transfected mouse RMA lymphoma cells (RMA-E7), while CD8(+) CTL from B6.P0 mice lysed RMA-E7 cells via Fas ligand (FasL) exclusively. Rapid and efficient lysis of syngeneic bystander B6 blasts or RMA cells by either B6 or B6.P0 Ag-activated CTL was mediated by a FasL-Fas mechanism. Fas-resistant bystanders were not lysed, nor were allogeneic Fas-sensitive C3H/HeJ (H-2(k)) or BALB/c (H-2(d)) bystander blasts. Interestingly, however, phorbol myristate acetate-ionomycin preactivation of B6.P0 effectors enabled lysis of allogeneic H-2(k) and H-2(d) bystanders even in the absence of antigenic stimulation. Lysis of syngeneic bystander cells was always FasL-Fas dependent and required effector-bystander contact and, in particular, an interaction between CTL LFA-1 and bystander ICAM-1. Thus, in the context of major histocompatibility complex class I molecule-peptide ligation of the T-cell receptors of CD8(+) CTL, neighboring bystander cells that are syngeneic and Fas sensitive and express the adhesion molecule ICAM-1 are potential targets of CTL attack.     

Dissecting the immune response to moloney murine sarcoma/leukemia virus-induced tumors by means of a DNA vaccination approach

The intramuscular inoculation of Moloney murine sarcoma/leukemia (M-MSV/M-MuLV) retroviral complex gives rise to sarcomas that undergo spontaneous regression due to the induction of a strong immune reaction mediated primarily by cytotoxic T lymphocytes (CTL). We used a DNA-based vaccination approach to dissect the CTL response against the Gag and Env proteins of M-MSV/M-MuLV in C57BL/6 (B6) mice and to evaluate whether plasmid DNA-immunized mice would be protected against a subsequent challenge with syngeneic tumor cells expressing the viral antigens. Intramuscular DNA vaccination induced CTL against both Gag and Env proteins. A detailed analysis of epitopes recognized by CTL generated in mice inoculated with the whole virus and with the Gag-expressing plasmid confirmed the presence of an immunodominant peptide in the leader sequence of Gag protein (Gag85-93, CCLCLTVFL) that is identical to that described in B6 mice immunized with Friend MuLV-induced leukemia cells. Moreover, CTL generated by immunization with the Env-encoding plasmid recognized a subdominant Env peptide (Env189-196, SSWDFITV), originally described in the B6.CH-2(bm13) mutant strain. B6 mice immunized with the Gag-expressing plasmid were fully protected against a lethal tumor challenge with M-MuLV-transformed MBL-2 leukemia cells, while vaccination with the Env-expressing plasmid resulted in rejection of the tumor in 44% of the mice and in increased survival of an additional 17% of the animals. Taken together, these results indicate the existence of a hierarchy in the capacity of different structural viral proteins to induce a protective immune response against retrovirus-induced tumors.