Activation by anti-CD3 of tumor-draining lymph node cells for specific adoptive immunotherapy.

Lymph nodes draining progressive tumors contain tumor-sensitized but not functional preeffector T lymphocytes. These cells can acquire antitumor reactivity after stimulation with tumor cells and interleukin-2 (IL-2). We demonstrated here that, in the absence of tumor cells, preeffector cells could be stimulated and expanded by sequential culture with anti-CD3 monoclonal antibody and IL-2. The adoptive transfer of such activated cells mediated immunologically specific reductions of established pulmonary metastases. The therapeutic effects could be enhanced by the administration of IL-2. This activation represents a secondary immune response because effector cells could be generated only from tumor-draining but not from normal or adjuvant-stimulated lymph nodes. Furthermore, treatment of advanced metastases with these cells resulted in prolongation of survival and cure of the disease. Thus, anti-CD3 may serve as a universal reagent for activating tumor-sensitized T lymphocytes for cancer therapy.     

Effector phenotype and immunologic specificity of T-cell-mediated adoptive therapy for a murine tumor that lacks intrinsic immunogenicity

The MCA 102 sarcoma has been defined by a variety of immunologic studies as a tumor lacking intrinsic immunogenicity. Nevertheless, we have recently demonstrated the feasibility of generating therapeutically effective lymphocytes for adoptive immunotherapy of this tumor. Procedures to achieve this required in vivo priming of syngeneic mice to elicit preeffector cells followed by in vitro sensitization (IVS) with tumor cells in the presence of IL-2. By selective depletion of T cell subsets in vivo, we identified the involvement of both CD4+ (L3T4+) and CD8+ (Lyt-2+) T cells in mediating tumor regression. The CD4+ cells exerted their helper function via the secretion of IL-2 because antitumor effects abrogated by depletion of CD4+ cells could be reconstituted by exogenous IL-2. In order to elicit preeffector cells with reactivity against the MCA 102 tumor, we found that in vivo sensitization could be accomplished with either the MCA 102 or MCA 106 tumor but not with the MCA 101 or MCA 105 tumor. Analysis of specificity of tumor stimulation during IVS of MCA 102 tumor-primed preeffector cells demonstrated cross-reactivity between not only the MCA 102 and MCA 106 tumors but also the MCA 105 tumor whereas the MCA 101 tumor was ineffective. In adoptive immunotherapy, transfer of IVS cells generated from MCA 102 tumor-primed and stimulated lymph node cells was able to mediate reductions of pulmonary metastases established from the MCA 102, MCA 105, and MCA 106 tumors but not from the MCA 101 tumor. We conclude that regression of the MCA 102 tumor is probably mediated through T cell recognition of a set of common tumor-associated Ag shared by several other syngeneic tumors. Immunologically, the tumor-associated Ag are characteristically different from classical tumor-specific transplantation Ag (TSTA) because immunity to TSTA on the MCA 105 or MCA 106 tumor does not cross-react with the MCA 102 tumor. Thus, this study demonstrates that Ag other than TSTA on chemically induced tumors can serve as target molecules for T cell-mediated adoptive immunotherapy.     

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.     

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.     

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.     

Intratumoral interleukin-2/agonist CD40 antibody drives CD4<Superscript>+</Superscript>-independent resolution of treated-tumors and CD4<Superscript>+</Superscript>-dependent systemic and memory responses

Targeting interleukin-2 (IL-2) and/or agonist anti-CD40 antibody (Ab) into tumors represents an effective vaccination strategy that avoids systemic toxicity and resolves treated-site tumors. Here, we examined IL-2 and/or anti-CD40 Ab-driven local versus systemic T cell function and the installation of T cell memory. Single tumor studies showed that IL-2 induced a potent CD4⁺ and CD8⁺ T cell response that was limited to the draining lymph node and treated-site tumor, and lymph node tumor-specific CD8⁺ T cells did not upregulate CD44. A two-tumor model showed that while IL-2-treated-site tumors resolved, distal tumors continued to grow, implying limited systemic immunity. In contrast, anti-CD40 Ab treatment with or without IL-2 expanded the systemic T cell response to non-draining lymph nodes, and distal tumors resolved. Tumor-specific T cells in lymph nodes of anti-CD40 Ab ± IL-2-treated mice upregulated CD44, demonstrating activation and transition to effector/memory migratory cells. While CD40-activated CD4⁺ T cells were not required for eradicating treated-site tumors, they, plus CD8⁺ T cells, were crucial for removing distal tumors. Rechallenge/depletion experiments showed that the effector/memory phase required the presence of previously CD40/IL-2-activated CD4⁺ and CD8⁺ T cells to prevent recurrence. These novel findings show that different T cell effector mechanisms can operate for the eradication of local treated-site tumors versus untreated distal tumors and that signaling through CD40 generates a whole of body, effector/memory CD4⁺ and CD8⁺ T cell response that is amplified and prolonged via IL-2. Thus, successful immunotherapy needs to generate collaborating CD4⁺ and CD8⁺ T cells for a complete long-term protective cure.     

Tumor infiltration by adoptively transferred T cells is independent of immunologic specificity but requires down-regulation of L-selectin expression.

Adoptive immunotherapy with anti-CD3/IL-2 activated tumor-draining lymph node (LN) T cells is capable of eradicating tumor established at various histological sites. Tumor-specific effector lymphocytes have recently been identified to be LN T cells with down-regulated L-selectin (L-selectin-). Using fluorochrome labeling, the present study determined the early trafficking pattern of systemically transferred cells. In mice with 10-day established pulmonary 3-methylcholanthrene (MCA) 205 metastases, accumulation of cells in tumors was evident as early as 2 h after i.v. cell transfer, and, by 24 h, >50-fold higher numbers of cells were seen in metastases than in normal tissues. Similarly, transferred cells selectively infiltrated s.c. tumors, albeit at a lower rate. Analysis of the transferred cells isolated from recipient mice revealed that tumor-infiltrating cells were mostly L-selectin- (>95%). By contrast, only 24% and 58% L-selectin- cells were found in the LN and spleen, respectively. The ability of L-selectin- cells to accumulate at tumor sites was confirmed by the transfer of purified cell populations. Despite this selective tumor infiltration, the trafficking pattern did not reflect antigenetic specificity, and tumor regression occurred only after the transfer of tumor-specific effector cells. These results, thus, suggest that there are two distinct mechanisms operative in successful adoptive immunotherapy. Early infiltration of tumors by transferred cells is dictated by the physiological properties of cells and is independent on their immunologic specificity. Tumor regression, however, requires immunologically specific interactions at the site of tumor.     

The antitumor effect of tumor-draining lymph node cells activated by both anti-CD3 monoclonal antibody and activated B cells as costimulatory-signal-providing cells

To establish an efficient cell-culture system for adoptive immunotherapy, we attempted to use lipopolysacharide (LPS)-activated B cells (LPS blasts) as costimulatory-signal-providing cells in the in vitro induction of antitumor effector cells. Both normal and tumor-draining lymph node cells were efficiently activated by both anti-CD3 monoclonal antibody (mAb) and LPS blasts, and subsequently expanded by a low dose of interleukin-2 (IL-2; anti-CD3 mAb and LPS blasts/IL-2). The expanded cells were predominantly CD8⁺ T cells and showed a low level of tumor-specific cytotoxic T lymphocyte (CTL) activity. The adoptive transfer of B16-melanoma-draining lymph node cells expanded by anti-CD3 mAb and LPS blasts/IL-2 showed significant antitumor effect against the established metastases of B16 in combination with intraperitoneal injections of IL-2. This treatment cured all B16-bearing mice. In addition, these mice also showed tumorspecific protective immunity against B16 at the rechallenge. Considering that activated B cells express several kinds of costimulatory molecules, these findings thus indicate an efficacy of costimulation that is derived from activated B cells for the in vitro induction of tumor-specific CTL, in co-operation with anti-CD3 mAb. The culture system presented here may thus be therapeutically useful, providing potent effectors for adoptive immunotherapy against various types of cancer.     

Lymphocytes generated by in vivo priming and in vitro sensitization demonstrate therapeutic efficacy against a murine tumor that lacks apparent immunogenicity

The adoptive transfer of sensitized lymphocytes is an effective means to mediate the regression of established tumors. However, successful therapy can only be demonstrated in animal models where tumors are intrinsically immunogenic, capable of eliciting systemic immunity. To explore the potential of this therapeutic approach to tumors of less immunogenicity, we have selected and used a murine tumor, MCA 102, for the current study because all attempts to immunize syngeneic mice failed. We report here that inoculation of mice with a mixture of tumor cells and a bacterial adjuvant, Corynebacterium parvum led to the production of sensitized, but not fully functional, lymphocytes in the draining lymph nodes (LN). These cells, termed pre-effector cells, could nevertheless further differentiate to acquire full immunologic function by an established in vitro sensitization culture method. In adoptive immunotherapy experiments, transfer of as few as 1.5 X 10(7) in vitro sensitized cells not only reduced established pulmonary MCA 102 metastases but also prolonged survival and cured tumors in a majority of the treated animals. In order to elicit pre-effector cells in vivo, inoculation with both tumor cells and C. parvum was essential. Although a broad range of numbers of MCA 102 tumor cells appeared to be effective, generation of pre-effector cells was dependent on the dose of C. parvum. We have found that a C. parvum dose of 25 micrograms was optimal, whereas higher doses of the adjuvant had suppressive effects. Analysis of the kinetics of their appearance revealed that the generation of pre-effector cells was transient. They were detectable 7 days after in vivo priming followed by a rapid decline. Furthermore, pre-effector cells were detected only in the regional draining LN. No reactivity was demonstrable in the spleen, mesenteric LN, PBL, or bone marrow. Taken together, these results expand the scope of immunotherapy by demonstrating the feasibility of manipulating a limited and obscure immune response to the MCA 102 tumor for therapeutic efficacy.     

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     

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.     

In vitro sensitization and expansion with viable tumor cells and interleukin 2 in the generation of specific therapeutic effector cells

We have investigated the efficacy and immunologic characteristics of immune effector cells generated from cultures containing large numbers of viable tumor cells and interleukin 2 (IL 2) in the adoptive immunotherapy of experimentally induced pulmonary metastases from the newly developed, weakly immunogenic MCA 105 sarcoma in mice. The current culture conditions allowed increases of either normal or MCA 105 immune spleen cells up to 94-fold in 15 days. The in vitro expanded normal and MCA 105 immune cells displayed nonspecific in vitro cytotoxicity against several syngeneic tumor targets. However, therapeutically effective cells could only be obtained from cultures initiated with MCA 105 immune spleen cells. Immunotherapy with expanded immune effector cells could lead to the reduction of established 3 day pulmonary metastases, prolongation of survival, and cure of tumor in the majority of animals. The generation and proliferation of therapeutic effector cells in vitro depended on the presence in cultures of specific tumor stimulator cells as well as the presence of IL 2. Although immunotherapy with either fresh noncultured or secondarily in vitro-sensitized (IVS) MCA 105 immune spleen cells was immunologically specific, the efficacy of the adoptive cellular therapy with cultured but not fresh immune cells could be improved by the administration to tumor-bearing hosts of exogenous IL 2. In addition to numerical expansion, the IVS immune cells, on a per cell basis, afforded an eightfold to 10-fold increase in therapeutic efficacy when compared with fresh noncultured MCA 105 immune cells. Our results indicate that the current culture procedure induced in vitro antigenic stimulation and expansion of tumor-specific immune effector cells that was otherwise not possible by conventional mixed lymphocyte-tumor cultures.     

Cellular basis of immunologic interactions in adoptive T cell therapy of established metastases from a syngeneic murine sarcoma

The adoptive transfer of specifically sensitized T lymphocytes can effectively mediate the regression of established local and metastatic tumors. Previous experiments using the weakly immunogenic MCA 105 sarcoma indicated that cellular interactions between transferred L3T4+ helper and Lyt-2+ cytotoxic immune T cells were necessary for mediating tumor regression. In this study, the kinetics of T-T cell interactions were analyzed by in vivo depletion of T cell subsets with mAb. The anti-tumor efficacy of transferred immune cells was abrogated by in vivo administration of either L3T4 or Lyt-2 mAb on the day of cellular therapy. However, if mAb were given 3 days after the transfer of immune cells, depletion of Lyt-2+ but not L3T4+ cells abrogated anti-tumor efficacy. T cell depletion on day 6 after transfer of immune cells had no adverse effect on tumor regression, indicating the period required for T cell reactivity in vivo. Furthermore, depletion of Ia+ cells by in vivo mAb treatment abrogated the anti-tumor efficacy of immune cells. It is thus hypothesized that there are two distinct but sequential phases of in vivo T cell interactions leading to the regression of established tumors after adoptive immunotherapy. An initial "helper/inducer" phase apparently requires the interaction of L3T4+ immune cells and the tumor Ag involving Ia+ cells. The inducement of L3T4+ cell activation is to provide helper function via the secretion of IL-2. The second phase designated as an "effector phase" involves differentiation of immune Lyt-2+ cells under the influence of IL-2 secreted during the helper/inducer phase for generation of mature Lyt-2+ effector cells. To further support the hypothesis of a two-phase process we have examined the phenotype and kinetics of tumor regression mediated by effector cells generated by secondary in vitro sensitization (IVS). Although the IVS cells were generated from fresh MCA 105 immune spleen cells, their anti-tumor efficacy was mediated solely by Lyt-2+ lymphocytes. Kinetic studies revealed that the in vivo requirement of IVS Lyt-2+ effector cells to mediate tumor regression was less than 3 days, and the anti-tumor reactivity of these cells was not affected by in vivo depletion of Ia+ cells. Thus, the IVS reaction is likely representative of the in vivo counterpart of the helper/inducer phase leading to the generation of mature Lyt-2+ immune effector cells. Tumor regression after transfer of Lyt-2+ cells generated in IVS therefore required a relatively shorter period of time than that required after the transfer of fresh noncultured MCA 105 immune spleen cells.     

Cross-presentation of tumor antigens to effector T cells is sufficient to mediate effective immunotherapy of established intracranial tumors

The systemic adoptive transfer of tumor-sensitized T cells, activated ex vivo, can eliminate established intracranial tumors. Regression of MHC class II negative MCA 205 fibrosarcomas occurs optimally following adoptive transfer of both CD4 and CD8 tumor-sensitized T cells, indicating an important function for tumor-infiltrating APC. Here, we demonstrate that during an effector response, indirect presentation of tumor Ags to transferred T cells is sufficient to mediate intracranial tumor regression. BALB/c --> CB6F1 (H-2bxd) bone marrow chimeras were challenged with the MCA 205 fibrosarcoma (H-2b). The tumor grew progressively in the H-2b-tolerant chimeras and stimulated an immune response in tumor-draining lymph nodes. Tumor-sensitized lymph node T cells were activated ex vivo with anti-CD3 and IL-2, then adoptively transferred to sublethally irradiated BALB/c or C57BL/6 recipients bearing established intracranial MCA 205 tumors. The transferred T cells eradicated MCA 205 tumors in BALB/c recipients and demonstrated tumor specificity, but had no therapeutic efficacy in the C57BL/6 recipients. These data establish that tumor-associated host cell constituents provide sufficient Ag presentation to drive effector T cell function in the complete absence of direct tumor recognition. This effector mechanism has an evident capacity to remain operative in circumstances of immune escape, where the tumor does not express the relevant MHC molecules, and may have importance even at times when direct CTL recognition also remains operative.     

Adoptive immunotherapy of a newly induced sarcoma: immunologic characteristics of effector cells

A newly induced syngeneic transplantable sarcoma, MCA 105, was used for studies of the biologic characteristics of fresh noncultured and secondarily in vitro sensitized (IVS) cells with antitumor reactivity. Fresh spleen cells harvested from mice immunized to the MCA 105 tumor by a mixture of viable tumor cells and Corynebacterium parvum exhibited no detectable cytotoxic activity to MCA 105 tumor targets in a 4-hr chromium-release assay, and adoptive transfer of these cells mediated the specific regression of established MCA 105 tumors. Phenotypic analysis of fresh, noncultured immune cells revealed that the therapeutically effective cells expressed both the Lyt-1 and the Lyt-2 T cell differentiation antigens. The therapeutic efficacy of fresh noncultured immune cells was not augmented by the concomitant administration of exogeneous interleukin 2 (IL 2). Secondary IVS of fresh immune cells with irradiated MCA 105 tumor stimulator cells resulted in the generation of tumor-specific cytotoxic effector cells. The generation of cytotoxic effector cells required Lyt-1+, 2+ cytotoxic precursor cells. Effective adoptive immunotherapy with these IVS immune cells, unlike fresh noncultured immune cells, depended on the concomitant administration of IL 2. Furthermore, the generation of therapeutically effective cells did not require the specific stimulation by MCA 105 tumor cells, because cultures of MCA 105 immune spleen cells with FBL-3 lymphoma cells in vitro also contained in vivo functional immune effector cells. These cells, however, possessed no detectable MCA 105 cytotoxic activity in vitro. Although this observation suggests that a noncytotoxic cell population is sufficient to initiate tumor regression in vivo, it does not exclude the possibility that cytolytic cells are generated in vivo after adoptive transfer of these cells. As a whole, our results indicate that secondary IVS functional immune effector cells are characteristically distinct from freshly harvested immune cells.     

Further characterization of cytotoxic T cells generated by short-term culture of human peripheral blood lymphocytes with interleukin-2 and anti-CD3 mAb

 In this study we have specifically investigated the participation of T cells in the cytotoxic activity of peripheral blood lymphocytes (PBL) activated by interleukin-2 (IL-2, 50 U/ml) alone or in combination with an anti-CD3 mAb (BMA030, 10 ng/ml, IgG2a). Purified CD3⁺ T cells, incubated in the presence of the anti-CD3 mAb for 4 days, mediated a cytotoxic activity against HL60 and U937 tumor cell lines. Several findings suggested the involvement of a redirected-cytotoxicity phenomenon, since the lytic process was restricted to target cell lines bearing the high-affinity Fcγ receptor (FcγRI) and T lymphocytes stimulated by IL-2 alone did not lyse these cell lines. Furthermore, anti-CD3 mAb F(ab′)₂, anti-CD3 IgG1 (UCHT1), phytohemagglutinin or staphylococcal enterotoxin A did not induce a similar cytotoxic activity in T lymphocytes. The cytotoxic process occurred in the presence of a very low level of anti-CD3 antibodies (in the nanomolar range). The cytotoxic activity of T cells stimulated by IL-2 or by IL-2 + BMA030, against OVCAR-3 cells (MOv18⁺ ovarian tumor cell line), was also compared in the presence of a bispecific antibody (OC/TR, anti-CD3 × MOv18). The stimulation by IL-2 + BMA030 induced approximately a twofold higher cytotoxic activity than IL-2-activated T cells. This could be related to the state of activation of effector cells stimulated by IL-2 + BMA030, since the phenotypic analysis showed an increased proportion of T cells expressing several activation/differentiation markers (CD25, HLA-DR, CD45R0, adhesion molecules). These findings could be applied to the design of therapeutic protocols using anti-CD3 ×antitumoral bispecific antibodies. Received: 6 December 1995 / Accepted: 4 June 1996     

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.     

Distinct immunologic specificity of tumor regression mediated by effector cells isolated from immunized and tumor-bearing mice

Sensitized T lymphocytes can mediate potent antitumor effects when transferred to tumor-bearing animals. Employing the MCA 105 and MCA 106 sarcomas, we were able to generate antitumor effector cells by immunization of syngeneic mice with tumor cells admixed with Corynebacterium parvum. These immune splenocytes could be further sensitized and expanded in culture by the in vitro sensitization (IVS) method utilizing tumor stimulator cells and IL-2. Adoptive immunotherapy of pulmonary metastases mediated by noncultured splenocytes from immunized mice or immune IVS cells showed exquisite specificity between the two sarcomas. These results demonstrate the presence of tumor-specific antigens on MCA 105 and MCA 106 tumor cells which can serve as target molecules for immunotherapy. Recently, we have generated therapeutic T lymphocytes from mice bearing progressively growing tumors by the IVS method. However, IVS cells from tumor-bearing mice showed cross-reactivity between the MCA 105 and 106 sarcomas in adoptive immunotherapy experiments. Since these IVS cells did not affect other control tumors, the limited cross-reactivity suggests the presence of common tumor-associated antigens on MCA 105 and MCA 106 tumor cells which can also serve as the target for tumor rejection. Therefore, immune responses to progressive tumor growth and to immunization are distinct with respect to antigen recognition by T lymphocytes.     

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.