Specific antitumor activity of tumor-infiltrating lymphocytes expanded first in a culture with both anti-CD3 monoclonal antibody and activated B cells and then in a culture with interleukin-2

In order to expand tumor-infiltrating lymphocytes (TIL) efficiently and in order to use them for immunotherapy, we utilized lipopolysaccharide-activated B cells (LPS blasts) as costimulatory-signal-providing cells in an in vitro culture system. TIL, prepared from subcutaneously inoculated B16 melanoma, failed to expand when cultured with anti-CD3 monoclonal antibody (mAb) alone followed by a low dose of interleukin(IL)-2. In contrast, such TIL did expand efficiently in culture with both anti-CD3 mAb and LPS blasts followed by culture with IL-2. These findings suggest that the presence of LPS blasts in the initial culture was essential for the cell expansion. The expansion of TIL was partially blocked by the addition of CTLA4 Ig, which is an inhibitor of costimulatory molecules such as CD80 and CD86, and was almost blocked by the addition of anti-(Fc receptor γII)mAb. These findings thus indicate that such molecules, in conjunction with the receptor on the LPS blasts, participate in the efficient expansion of TIL. The B16-derived TIL, which expanded in our culture system, were predominantly CD8+T cells and showed a higher level of cytolytic activity against B16 melanoma than either lymphokine-activated killer cells or TIL cultured with a high dose of IL-2. In addition, the in vitro expanded B16-derived TIL produced interferon γ, but not IL-4, in response to B16 melanoma. What is more important, the adoptive transfer of such TIL had a significant antitumor effect against pulmonary metastasis in B16 melanoma, even without the concurrent administration of IL-2. Collectively, our results thus indicate the therapeutic efficacy of the protocol presented here for antitumor immunotherapy with TIL.     

Specific antitumor activity of tumor-infiltrating lymphocytes expanded first in a culture with both anti-CD3 monoclonal antibody and activated B cells and then in a culture with interleukin-2

In order to expand tumor-infiltrating lymphocytes (TIL) efficiently and in order to use them for immunotherapy, we utilized lipopolysaccharide-activated B cells (LPS blasts) as costimulatory-signal-providing cells in an in vitro culture system. TIL, prepared from subcutaneously inoculated B16 melanoma, failed to expand when cultured with anti-CD3 monoclonal antibody (mAb) alone followed by a low dose of interleukin(IL)-2. In contrast, such TIL did expand efficiently in culture with both anti-CD3 mAb and LPS blasts followed by culture with IL-2. These findings suggest that the presence of LPS blasts in the initial culture was essential for the cell expansion. The expansion of TIL was partially blocked by the addition of CTLA4 Ig, which is an inhibitor of costimulatory molecules such as CD80 and CD86, and was almost blocked by the addition of anti-(Fc receptor II)mAb. These findings thus indicate that such molecules, in conjunction with the receptor on the LPS blasts, participate in the efficient expansion of TIL. The B16-derived TIL, which expanded in our culture system, were predominantly CD8+T cells and showed a higher level of cytolytic activity against B16 melanoma than either lymphokine-activated killer cells or TIL cultured with a high dose of IL-2. In addition, the in vitro expanded B16-derived TIL produced interferon , but not IL-4, in response to B16 melanoma. What is more important, the adoptive transfer of such TIL had a significant antitumor effect against pulmonary metastasis in B16 melanoma, even without the concurrent administration of IL-2. Collectively, our results thus indicate the therapeutic efficacy of the protocol presented here for antitumor immunotherapy with TIL.This work was supported in part by a grant from the Ministry of Education, Science and Culture     

Critical role of CD11a (LFA-1) in therapeutic efficacy of systemically transferred antitumor effector T cells

The systemic adoptive transfer of activated T cells, derived from tumor-draining lymph nodes (LNs), mediates the regression of established tumors. In this study, the requirement of cell adhesion molecules, CD11a/CD18 (LFA-1), CD54 (ICAM-1), CD49d/CD29 (VLA-4), and CD106 (VCAM-1), for T cell infiltration into tumors and antitumor function was investigated. Administration of anti-CD11a mAb completely abrogated the efficacy of adoptive immunotherapy for both intracranial and pulmonary metastatic MCA 205 fibrosarcomas. In contrast, adoptive immunotherapy was effective in animals treated with anti-CD49d mAb, anti-CD106 mAb, anti-CD54 mAb, or in CD54 knockout recipients. Trafficking of transferred cells to the intracranial tumor was not affected by any of the mAb. However, the tumor-specific secretion of IFN-gamma by activated LN T cells was suppressed by anti-CD11a mAb or anti-CD54 mAb. To account for the different effects of CD11a and CD54 blockade in vivo, an additional CD11a/CD18 ligand, CD102 (ICAM-2), was demonstrated on tumor-associated macrophages but not on tumor cells. These results show that CD11a mediates a critical function in interactions between effector T cells, tumor cells, and host accessory cells in situ leading to tumor regression.     

Induction of CTL responses by simultaneous administration of liposomal peptide vaccine with anti-CD40 and anti-CTLA-4 mAb

Activation of APC via CD40-CD40 ligand pathway induces up-regulation of costimulatory molecules such as B7 and production of IL-12. Interaction between B7 on APC and CD28 on naive T cells is necessary for priming the T cells. On the other hand, interaction between B7 on APC and CTLA-4 on activated T cells transduces a negative regulatory signal to the activated T cells. In the present study, we attempted to generate tumor-specific CTL by s.c. administration of antigenic peptides encapsulated in multilamellar liposomes (liposomal peptide vaccine) with anti-CD40 mAb and/or anti-CTLA-4 mAb. Liposomal OVA257-264 and anti-CD40 mAb or anti-CTLA-4 mAb were administrated to C57BL/6 mice and the splenocytes were cocultured with OVA257-264 for 4 days. The splenic CD8+ T cells showed a significant cytotoxicity against EL4 cells transfected with cDNA of OVA. In addition, administration of both anti-CD40 and anti-CTLA-4 mAb enhanced the CTL responses. Considerable CTL responses were induced in MHC class II deficient mice by the same procedure. This finding indicated that CTL responses could be generated even in the absence of Th cells. When BALB/c mice were immunized with pRL1a peptide that are tumor-associated Ag of RLmale symbol1 leukemia cells using the same procedure, significant CTL responses were induced and prolonged survival of the BALB/c mice was observed following RLmale symbol1 inoculation. These results demonstrate that anti-CD40 mAb and anti-CTLA-4 mAb function as immunomodulators and may be applicable to specific cancer immunotherapy with antitumor peptide vaccine.     

Activation of dendritic cells that cross-present tumor-derived antigen licenses CD8+ CTL to cause tumor eradication

The fate of naive CD8(+) T cells is determined by the environment in which they encounter MHC class I presented peptide Ags. The manner in which tumor Ags are presented is a longstanding matter of debate. Ag presentation might be mediated by tumor cells in tumor draining lymph nodes or via cross-presentation by professional APC. Either pathway is insufficient to elicit protective antitumor immunity. We now demonstrate using a syngeneic mouse tumor model, expressing an Ag derived from the early region 1A of human adenovirus type 5, that the inadequate nature of the antitumor CTL response is not due to direct Ag presentation by the tumor cells, but results from presentation of tumor-derived Ag by nonactivated CD11c(+) APC. Although this event results in division of naive CTL in tumor draining lymph nodes, it does not establish a productive immune response. Treatment of tumor-bearing mice with dendritic cell-stimulating agonistic anti-CD40 mAb resulted in systemic efflux of CTL with robust effector function capable to eradicate established tumors. For efficacy of anti-CD40 treatment, CD40 ligation of host APC is required because adoptive transfer of CD40-proficient tumor-specific TCR transgenic CTL into CD40-deficient tumor-bearing mice did not lead to productive antitumor immunity after CD40 triggering in vivo. CpG and detoxified LPS (MPL) acted similarly as agonistic anti-CD40 mAb with respect to CD8(+) CTL efflux and tumor eradication. Together these results indicate that dendritic cells, depending on their activation state, orchestrate the outcome of CTL-mediated immunity against tumors, leading either to an ineffective immune response or potent antitumor immunity.     

Generation propagation, and targeting of human CD4+ helper/killer T cells induced by anti-CD3 monoclonal antibody plus recombinant IL-2. An efficient strategy for adoptive tumor immunotherapy.

We developed a culture system for the rapid generation of CD4+ T cells that have both helper and killer functions. CD4+ T cells isolated from human PBL did not proliferate or develop significant cytotoxicity when treated with rIL-2 because of the lack of p75 IL-2R expression. However, culture of isolated CD4+ T cells with immobilized anti-CD3 mAb plus rIL-2 resulted in a marked proliferation (500-fold increase in 14 days) of CD4+ T cells. The proliferating CD4+ T cells produced IL-2 (92 U/ml) and showed strong cytotoxicity against OKT3 hybridoma cells and Daudi, K562, and U937 tumor cells in an anti-CD3 mAb-dependent manner. The CD4+ T cells contained significant amounts of cytolytic granule-related proteins such as serine esterase and perforin. Activated CD4+ helper/killer cells can be generated from both healthy donors and tumor patients and can be propagated in vitro for 14 to 35 days by biweekly restimulation with immobilized anti-CD3 mAb plus rIL-2. This culture yielded about 20,000-fold increase in cell number after a 21-day culture. Bispecific antibody containing anti-CD3 and anti-glioma Fab components enhanced the cytotoxicity of activated CD4+ helper/killer cells against IMR32 glioma cells. Moreover, the activated CD4+ helper/killer cells showed both helper and antitumor activity in vivo and prevented growth of anti-CD3 hybridoma cells in nude mice whether or not IL-2 was administered. These results indicate that anti-CD3 mAb plus IL-2-activated CD4+ helper/killer cells may provide an effective strategy for adoptive tumor immunotherapy of cancer.     

Specific adoptive immunotherapy mediated by tumor-draining lymph node cells sequentially activated with anti-CD3 and IL-2.

Lymph nodes (LN) draining progressively growing tumors contain tumor-sensitized but not fully functional preeffector lymphocytes. These cells could acquire therapeutic efficacy and be expanded upon sequential culture with anti-CD3 mAb for 2 days followed by incubation in IL-2 for 3 days. Using the weakly immunogenic MCA 106 and MCA 205 murine sarcomas, we have further defined conditions of this anti-CD3/IL-2 activation with which preeffector cells differentiated into immune effector cells. In vitro activation and expansion of effector cells required sequential but independent stimulation with anti-CD3 and IL-2 because the simultaneous presence of both anti-CD3 and IL-2 at either stage did not enhance the efficacy of activation. Generation of effector cells by this two-stage activation was critically dependent on the optimal concentrations of anti-CD3 (1.0 microgram/ml) and IL-2 (2-10 U/ml). However, these conditions were not optimal for inducing the greatest cellular proliferation. In adoptive immunotherapy experiments, although the transfer of anti-CD3/IL-2-activated cells alone could mediate the regression of established metastases, the concomitant administration of IL-2 enhanced the in vivo activity of these cells. More importantly, tumor regression mediated by the anti-CD3/IL-2-activated cells was found to be immunologically specific. The specificity was determined by the tumor that stimulated the preeffector cell response. In spite of their in vivo antitumor effects, the anti-CD3/IL-2-activated tumor-draining LN cells did not exhibit detectable in vitro cytotoxicity against the tumor target in the 4-h 51Cr-release assay. In mice bearing progressive tumor, draining LN contained most preeffector cells. Some preeffector cells were also detected in the spleen whereas mesenteric LN did not demonstrate any reactivity. In kinetics studies, sensitization of preeffector cells in the draining LN occurred between 4 to 6 days after tumor inoculation. As the tumor progressed, the presence of preeffector cells declined gradually suggesting a tumor-induced suppression. These results define the conditions whereby tumor-draining LN cells could be stimulated, in the absence of tumor Ag, to develop into specific therapeutic effector cells. Our findings also raise the possibility of using similar approaches for isolating immune effector cells from cancer patients for adoptive immunotherapy.     

Regression of extensive pulmonary metastases in mice by adoptive transfer of antigen-specific CD8(+) CTL reactive against tumor cells expressing a naturally occurring rejection epitope

In this study, we developed a mouse model of adoptive immunotherapy reflecting immune recognition of syngeneic tumor cells naturally expressing an endogenous rejection Ag. Specifically, in a pulmonary metastases model, we examined the potency and maintenance of an antitumor CD8(+) CTL response in vivo, as well as its effectiveness against an "extensive" tumor burden. The approach taken was to first generate tumor-specific CTL from mice challenged with the CMS4 sarcoma coadministered with anti-CTLA4 mAb, which has been shown to facilitate the induction of Ag-specific T cell responses in vivo. An H-2L(d)-restricted nonamer peptide, derived from an endogenous murine leukemia provirus was identified as a CMS4-reactive CTL epitope based upon the following: CTL cross-recognition of another syngeneic tumor cell line (CT26 colon carcinoma) previously characterized to express that gene product; sensitization of Ag-negative lymphoblasts or P815 targets with the peptide; and by cold target inhibition assays. In vivo, the adoptive transfer of CMS4-reactive CTL (> or =1 x 10(6)) resulted in nearly the complete regression of 3-day established lung metastases. Furthermore, mice that rejected CMS4 following a single adoptive transfer of CTL displayed antitumor activity to a rechallenge 45 days later, not only in the lung, but also at a s.c. distal site. Lastly, the adoptive transfer of CTL to mice harboring extensive pulmonary metastases (> 150 nodules) led to a substantial reduction in tumor burden. Overall, these data suggest that the adoptive transfer of tumor-specific CTL may have therapeutic potential for malignancies that proliferate in or metastasize to the lung.     

Potent tumor-specific protection ignited by adoptively transferred CD4+ T cells

Administration of anti-CD25 mAb before an aggressive murine breast tumor inoculation provoked effective antitumor immunity. Compared with CD4(+) T cells purified from anti-CD25 mAb-pretreated mice that did not reject tumor, CD4(+) T cells purified from anti-CD25 mAb-pretreated mice that rejected tumor stimulated by dendritic cells (DCs) produced more IFN-gamma and IL-2, and less IL-17 in vitro, and ignited protective antitumor immunity in vivo in an adoptive transfer model. Tumor Ag-loaded DCs activated naive CD8(+) T cells in the presence of these CD4(+) T cells in vitro. Tumor Ag and adoptively transferred CD4(+) T cells were both required for inducing a long-term tumor-specific IFN-gamma-producing cellular response and potent protective antitumor activity. Although adoptively transferred CD4(+) T cells ignited effective tumor-specific antitumor immunity in wild-type mice, they failed to do so in endogenous NK cell-depleted, Gr-1(+) cell-depleted, CD40(-/-), CD11c(+) DC-depleted, B cell(-/-), CD8(+) T cell-depleted, or IFN-gamma(-/-) mice. Collectively, the data suggest that adoptively transferred CD4(+) T cells orchestrate both endogenous innate and adaptive immunity to generate effective tumor-specific long-term protective antitumor immunity. The data also demonstrate the pivotal role of endogenous DCs in the tumor-specific protection ignited by adoptively transferred CD4(+) T cells. Thus, these findings highlight the importance of adoptively transferred CD4(+) T cells, as well as host immune components, in generating effective tumor-specific long-term antitumor activity.     

Generation of tumor-specific cytotoxic T lymphocytesin vivo by combined treatment with inactivated tumor cells and recombinant interleukin-2

In order to search for a new therapy that would maximize the effect of interleukin-2 (IL-2) in evoking antitumor immunity in vivo, the therapeutic effect of a combination of mitomycin-C(MMC)-treated tumor cells and recombinant IL-2 was examined for its induction of antitumor activity against established melanoma metastasis. In C57BL/6 mice intravenously (i. v.) injected with B16 melanoma cells on day 0, the combined treatment with an intraperitoneal (i. p.) injection of MMC-treated melanoma cells on day 6 and 2500 U rIL-2 (twice daily) on days 7 and 8 markedly reduced the number of pulmonary metastases. This antitumor activity was more effective than that in untreated controls and mice that were injected with MMC-treated melanoma cells alone or rIL-2 alone. When the i. p. injection of MMC-treated tumor cells was replaced by other syngeneic tumor cells, antitumor activity against metastatic melanoma was not induced. The antitumor activity induced by this treatment increased in parallel with an increase in the dose of rIL-2 injected. In contrast, an i. p. injection of soluble tumor-specific antigens alone could induce only a marginal level of antitumor activity, and this activity was not augmented by subsequent i. p. injections of rIL-2. In vivo treatment with anti-CD8 monoclonal antibody (mAb), but not with anti-CD4 mAb or anti-asialo-GM1 antibody, abrogated the antitumor activity induced by this combined therapy. This suggests that the antitumor effect was dependent on CD8⁺ T cells. Lung-infiltrating lymphocytes from mice that had been i. v. injected with melanoma cells 11 days before and were treated with this combined therapy, showed melanoma-specific cytolytic activity. This combined therapy also showed significant antitumor activity against subcutaneously inoculated melanoma cells. These results demonstrate that the combined therapy of an i. p. injection of MMC-treated tumor cells and subsequent and consecutive i. p. administration of rIL-2 increases antitumor activity against established metastatic melanoma by generating tumor-specific CD8⁺ CTL in vivo.     

Regulatory T-cell depletion synergizes with gp96-mediated cellular responses and antitumor activity

Despite its potent immunostimulatory properties, vaccination with autologous tumor-derived gp96 has relatively modest antitumor effect in a range of clinical trials. Based on our previous study showing a gp96-mediated immune balance between CTL and Tregs, here we investigated possible synergy between gp96 vaccine and systemic Treg depletion on induction of antitumor T-cell immunity and the mechanisms accounting for synergistic efficacy. In gp96-peptide complex immunized BALB/c mice, anti-CD25 mAb treatment significantly increased IFN-γ-producing CD8(+) and CD4(+) T cells by about 1-2-fold in spleen and 40-50% in lymph node. A significantly higher number of peptide-specific CTL were observed under anti-CD25 mAb treatment compared with no treatment. Moreover, Treg depletion synergistically improved the anticancer activity of tumor-derived gp96 vaccine in the poorly immunogenic and highly tumorigenic B16 melanoma model in C57BL/6?J mice. While gp96 immunization alone led to the modest enhancement of CTL activities in spleen, the combination with Treg depletion dramatically increased tumor-specific CTL responses. In addition, the combination resulted in a significant increase of CD8(+) T-cell infiltration in tumor, which correlated with an enhanced inhibition of tumor growth. Our results provide evidence that targeting Tregs may provide a more efficient strategy to potentiate gp96-mediated T-cell responses and enhance the antitumor efficiency of gp96-based therapeutic vaccine.     

Potent in vivo anti-tumor activity of isolated CD62L(low) lymph node cells sensitized in vivo with tumor lysate-pulsed DC-based vaccines

BACKGROUND: DC are potent APC that can activate both CD4 and CD8 T cells in vitro and in vivo. Although the efficacy of DC-based cancer vaccines is currently being evaluated in clinical trials, the systemic immune suppression in cancer patients negatively impacts the clinical benefit of this therapeutic approach. Therefore, in this study we tested the feasibility and anti-tumor effect of adoptive immunotherapy using in vitro-activated CD62L(low) lymph node cells that were isolated from DC-vaccinated draining lymph nodes (VDLN). METHODS: DC were prepared from BM cells and loaded with tumor lysate for inoculating into naive mice. Subsequently, the VDLN were removed and CD62L(low) cells in the VDLN population isolated, expanded in vitro by 5-day culture with IL-2 and immobilized anti-CD3 stimulation, then injected into mice with established pulmonary tumors. Eighteen days after treatment, mice were killed in order to enumerate pulmonary tumor nodes. RESULTS: DC phagocytosed the tumor lysate efficiently and induced detectable T-cell responses and significant cell expansion in the draining lymph nodes. After induction of maturation by LPS treatment, DC expressed higher levels of CD40, CD86 and MHC class II molecules. When CD62L(low) VDLN cells that had been isolated and expanded in vitro were transferred into tumor-bearing mice, as few as 3 x 10(6) cells were able to cure metastatic pulmonary tumors in vivo. DISCUSSION: DC-based VDLN T cells are an important source of anti-tumor effector for adoptive immunotherapy. This study provides a novel and an effective protocol using T-cell adoptive immunotherapy for application in cancer patients; therefore, clinical trials based on this protocol may be warranted.     

Phenotypic and functional analysis of murine CD3+,CD4-,CD8- TCR-gamma delta-expressing peripheral T cells

Murine CD3+,CD4-,CD8- peripheral T cells, which express various forms of the TCR-gamma delta on their cell surface, have been characterized in terms of their cell-surface phenotype, proliferative and lytic potential, and lymphokine-producing capabilities. Three-color flow cytofluorometric analysis demonstrated that freshly isolated CD3+,CD4-, CD8- TCR-gamma delta lymph node cells were predominantly Thy-1+,CD5dull,IL-2R-,HSA-,B220-, and approximately 70% Ly-6C+ and 70% Pgp-1+. After CD3+,CD4-,CD8-splenocytes were expanded for 7 days in vitro with anti-CD3-epsilon mAb (145-2C11) and IL-2, the majority of the TCR-gamma delta cells expressed B220 and IL-2R, and 10 to 20% were CD8+. In comparison to CD8+ TCR-alpha beta T cells, the population of CD8+ TCR-gamma delta-bearing T cells exhibited reduced levels of CD8, and about 70% of the CD8+ TCR-gamma delta cells did not express Lyt-3 on the cell surface. Functional studies demonstrated that splenic TCR-gamma delta cells proliferated when stimulated with mAb directed against CD3-epsilon, Thy-1, and Ly-6C, but not when incubated with an anti-TCR V beta 8 mAb, consistent with the lack of TCR-alpha beta expression. In addition, activated CD3+,CD4-,CD8- peripheral murine TCR-gamma delta cells were capable of lysing syngeneic FcR-bearing targets in the presence of anti-CD3-epsilon mAb and the NK-sensitive cell line, YAC-1, in the absence of anti-CD3-epsilon mAb. Finally, activated CD3+, CD4-,CD8-,TCR-gamma delta+ splenocytes were also capable of producing IL-2, IL-3, IFN-gamma, and TNF when stimulated in vitro with anti-CD3-epsilon mAb.     

Antitumor Activity of T Cells Generated from Lymph Nodes Draining the SEA-expressing Murine B16 Melanoma and Secondarily Activated with Dendritic Cells

The successful use of tumor-draining lymph nodes (TDLN) as a source of effector cells for cancer immunotherapy depends largely on the immunogenicity of the tumor drained by the lymph nodes as well as the methods for secondary in vitro T cell activation and expansion. We transferred the bacterial superantigen staphylococcal enterotoxin A (SEA) gene into B16 murine melanoma tumor cells, and used them to induce TDLN (SEA TDLN) in syngeneic hosts. Wild-type (wt) TDLN induced by parental B16 tumor was used as a control. In vitro, SEA TDLN cells proliferated more vigorously, produced more IFNγ and demonstrated higher CTL activity than wt TDLN cells when activated with anti-CD3/anti-CD28/IL-2. In vivo, SEA TDLN cells mediated tumor eradication more effectively than similarly activated wt TDLN cells (p<0.01). Furthermore, use of dendritic cells (DC) plus tumor antigen in vitro in addition to anti-CD3/anti-CD28/IL-2 stimulation further amplified the immune function and therapeutic efficacy of SEA TDLN cells. DC-stimulated SEA TDLN cells eliminated nearly 90% of the pulmonary metastasis in mice bearing established B16 melanoma micrometastases. These results indicate that enforced expression of superantigen SEA in poorly immunogenic tumor cells can enhance their immunogenicity as a vaccine in vivo. The combined use of genetically modified tumor cells as vaccine to induce TDLN followed by secondary stimulation using antigen-presenting cells and tumor antigen in a sequential immunization/activation procedure may represent a unique method to generate more potent effector T cells for adoptive immunotherapy of cancer.     

Generation of human autologous melanoma-specific cytotoxic T cells from tumor-involved lymph nodes

Cytotoxic T lymphocytes (CTL) specific for autologous human melanoma have been successfully generated in vitro from tumor bearing lymph nodes without any stimulation by the autologous tumor. Tumor-involved lymph node cells (LNC) were cultured in serum free medium (AIM-V) containing 1,000 U/ml of recombinant interleukin-2. The best expansion and specific cytotoxicity of CTL were achieved in 4 to 6 weeks of culture. The predominant populations in cultured LNC-derived CTL were CD2+, CD3+, CD4-, CD8+, CD56-, and HLA-DR+ T cells. These data suggested that tumor-involved LNC may provide an alternative source for the generation of tumor-specific CTL in adoptive immunotherapy.     

CD40 ligation activates murine macrophages via an IFN-gamma-dependent mechanism resulting in tumor cell destruction in vitro

We have shown previously that agonistic anti-CD40 mAb induced T cell-independent antitumor effects in vivo. In this study, we investigated mechanisms of macrophage activation with anti-CD40 mAb treatment, assessed by the antitumor action of macrophages in vitro. Intraperitoneal injection of anti-CD40 mAb into C57BL/6 mice resulted in activation of peritoneal macrophages capable of suppressing B16 melanoma cell proliferation in vitro, an effect that was greatly enhanced by LPS and observed against several murine and human tumor cell lines. Anti-CD40 mAb also primed macrophages in vitro to mediate cytostatic effects in the presence of LPS. The tumoristatic effect of CD40 ligation-activated macrophages was associated with apoptosis and killing of tumor cells. Activation of macrophages by anti-CD40 mAb required endogenous IFN-gamma because priming of macrophages by anti-CD40 mAb was abrogated in the presence of anti-IFN-gamma mAb, as well as in IFN-gamma-knockout mice. Macrophages obtained either from C57BL/6 mice depleted of T and NK cells by Ab treatment, or from scid/beige mice, were still activated by anti-CD40 mAb to mediate cytostatic activity. These results argued against the role of NK and T cells as the sole source of exogenous IFN-gamma for macrophage activation and suggested that anti-CD40 mAb-activated macrophages could produce IFN-gamma. We confirmed this hypothesis by detecting intracytoplasmic IFN-gamma in macrophages activated with anti-CD40 mAb in vivo or in vitro. IFN-gamma production by macrophages was dependent on IL-12. Taken together, the results show that murine macrophages are activated directly by anti-CD40 mAb to secrete IFN-gamma and mediate tumor cell destruction.     

Successful adoptive immunotherapy of murine poorly immunogenic tumor with specific effector cells generated from gene-modified tumor-primed lymph node cells

We previously reported that cytokine gene transfer into weakly immunogenic tumor cells could enhance the generation of precursor cells of tumor-reactive T cells and subsequently augment antitumor efficacy of adoptive immunotherapy. We investigated whether such potent antitumor effector T cells could be generated from mice bearing poorly immunogenic tumors. In contrast to similarly modified weakly immunogenic tumors, MCA102 cells, which are chemically induced poorly immunogenic fibrosarcoma cells transfected with cDNA for IL-2, IL-4, IL-6, IFN-gamma, failed to augment the host immune reaction. Because priming of antitumor effector T cells in vivo requires two important signals provided by tumor-associated Ags and costimulatory molecules, these tumor cells were cotransfected with a B7-1 cDNA. Transfection of both IFN-gamma and B7-1 (MCA102/B7-1/IFN-gamma) resulted in regression of s.c. tumors, while tumor transfected with other combinations of cytokine and B7-1 showed progressive growth. Cotransfection of IFN-gamma and B7-1 into other poorly immunogenic tumor B16 and LLC cells also resulted in the regression of s.c. tumors. Cells derived from lymph nodes draining MCA102/B7-1/IFN-gamma tumors showed potent antitumor efficacy, eradicating established pulmonary metastases, but this effect was not seen with parental tumors. This mechanism of enhanced antitumor efficacy was further investigated, and T cells with down-regulated L-selectin expression, which constituted all the in vivo antitumor reactivity, were significantly increased in lymph nodes draining MCA102/B7-1/IFN-gamma tumors. These T cells developed into potent antitumor effector cells after in vitro activation with anti-CD3/IL-2. The strategy presented here may provide a basis for developing potent immunotherapy for human cancers.     

Anti-CD3 + IL-2-stimulated murine killer cells. In vitro generation and in vivo antitumor activity

The growth, phenotype, in vitro cytolytic characteristics, and in vivo antitumor activity of murine splenocytes stimulated with anti-murine CD3 mAb in combination with IL-2 as compared with IL-2 alone was investigated. When cultured for 12 days with anti-CD3 mAb + IL-2, murine splenocytes increased 100- to 4000-fold in number compared with only 6- to 20-fold for cultures stimulated with IL-2 alone. Anti-CD3 mAb + IL-2 activated cultures developed high lymphokine-activated killer activity against NK-resistant targets including the P815 mastocytoma cell line and fresh MCA 106 sarcoma. Peak cytotoxicity on a per cell basis developed by day 8 after anti-CD3 mAb + IL-2 activation. A large proportion of the total cytolytic activity of long term anti-CD3 mAb + IL-2-stimulated cultures was related to the presence of anti-CD3 in the assay, indicating enhancement of cytotoxicity by activated CD3+ T cells. Phenotypic analysis indicated that anti-CD3 mAb + IL-2-stimulated cultures contained heterogeneous populations of T cells with increased percentages of both CD4+ and CD8+ phenotypes compared with cultures stimulated with IL-2 alone. Anti-CD3 mAb + IL-2-stimulated cells were tested for their in vivo antitumor activity by using C57BL/6 mice bearing MCA 106 sarcoma pulmonary metastases. IL-2-activated murine killer cells were given in combination with in vivo IL-2 and indomethacin, the latter of which was shown to potentiate the antitumor effect of IL-2. When given on day 5 after tumor inoculation, cell doses as low as 5 x 10(6) anti-CD3 mAb + IL-2-stimulated cells per mouse significantly reduced the number of pulmonary metastases (p less than 0.005). Thus, activation with the combination of anti-CD3 mAb + IL-2 produces rapidly expanding cultures of cytolytic cells with demonstrated in vivo antitumor efficacy.     

Augmentation versus inhibition: effects of conjunctional OX-40 receptor monoclonal antibody and IL-2 treatment on adoptive immunotherapy of advanced tumor.

Therapeutic efficacy of adoptive immunotherapy of malignancies is proportional to the number of effector T cells transferred. Traditionally, exogenous IL-2 treatment has been used to promote the survival and function of transferred cells. Recently, we described the therapeutic effects of in vivo ligation of the costimulatory receptor, OX-40R, on activated T cells during early tumor growth. In this study, we examined the effects of IL-2 and OX-40R mAb on adoptive immunotherapy of advanced tumors. For treatment of 10-day 3-methylcholanthrene 205 pulmonary metastases, systemic transfer of 50 x 10(6) activated tumor-draining lymph node T cells resulted in >99% reduction of metastatic nodules. With either IL-2 or OX-40R mAb conjunctional treatment, only 20 x 10(6) cells were required. Advanced 10-day 3-methylcholanthrene 205 intracranial tumors could be cured by the transfer of 15 x 10(6) L-selectin(low) T cells derived from draining lymph nodes. In this situation, IL-2 administration inhibited therapeutic effects of the transferred cells. By contrast, 5 x 10(6) T cells were sufficient to cure all mice if OX-40R mAb was administrated. Studies on trafficking of systemically transferred T cells revealed that IL-2, but not OX-40R mAb, impeded tumor infiltration by T cells. Tumor regression required participation of both CD4 and CD8 T cells. Because only CD4 T cells expressed OX-40R at cell transfer, direct CD4 T cell activation is possible. Alternatively, OX-40R might be up-regulated on transferred T cells at the tumor site, rendering them reactive to the mAb. Our study suggests OX-40R mAb to be a reagent of choice to augment T cell adoptive immunotherapy in clinical trials.     

Ex vivo activation of tumor-draining lymph node T cells reverses defects in signal transduction molecules

 The adoptive transfer of tumor-draining lymph node (LN) T cells activated ex vivo with anti-CD3 and interleukin 2 (IL-2) mediates the regression of the poorly immunogenic murine melanoma D5. The efficacy of the activated LN cells is augmented when the sensitizing tumor is a genetically modified variant (designated D5G6) that secretes granulocyte/macrophage-colony-stimulating factor. In contrast to anti-CD3/IL-2-activated LN cells, adoptive transfer of freshly isolated tumor-draining LN T cells has no therapeutic activity. To determine whether the acquisition of antitumor function during ex vivo activation is associated with modifications in signal transduction capacity, the protein tyrosine kinases p56 ^lck and p59 ^fyn and proteins of the NF-κB family were analyzed in tumor-draining LN T cells. The levels of p56 ^lck and p59 ^fyn were lower in tumor-draining than in normal LN T cells and production of tyrosine-phosphorylated substrates was markedly depressed following anti-CD3 stimulation. After 5-day anti-CD3/IL-2 activation, levels of p56 ^lck and p59 ^fyn and protein tyrosine kinase activity increased. Interestingly, the levels of p56 ^lck , p59 ^fyn , and tyrosine kinase activity were higher in activated T cells derived from LN that drained D5G6 than they were in those from D5 tumors. In contrast, the cytoplasmic levels of c-Rel and Rel A were normal in freshly isolated tumor-draining LN, as was nuclear κB DNA-binding activity induced by anti-CD3 mAb or phorbol myristate acetate. Stimulation of activated LN cells with D5 tumor cells induced the nuclear translocation of NF-κB. These findings indicate that the recovery of proteins mediating signal transduction through the T cell receptor/CD3 complex in LN T cells activated ex vivo was associated with the acquisition of antitumor function. Received: 28 August 1997 / Accepted: 23 February 1998