Antibody treatment of human tumor xenografts elicits active anti-tumor immunity in nude mice

Athymic nude mice bearing subcutaneous tumor xenografts of the human anti-colorectal cancer cell line SW480 were used as a preclinical model to explore anti-tumor immunotherapies. Intratumor or systemic treatment of the mice with murine anti-SW480 serum, recombinant anti-SW480 polyclonal antibodies, or the anti-colorectal cancer monoclonal antibody CO17-1A, caused retardation or regression of SW480 tumor xenografts. Interestingly, when mice that had regressed their tumors were re-challenged with SW480 cells, these mice regressed the new tumors without further antibody treatment. Adoptive transfer of spleen cells from mice that had regressed their tumors conferred anti-tumor immunity to na?ve nude mice. Pilot experiments suggest that the transferred anti-tumor immunity is mediated by T cells of both gammadelta and alphabeta lineages. These results demonstrate that passive anti-tumor immunotherapy can elicit active immunity and support a role for extra-thymic gammadelta and alphabeta T cells in tumor rejection. Implications for potential immunotherapies include injection of tumor nodules in cancer patients with anti-tumor antibodies to induce anti-tumor T cell immunity.     

Dendritic cells modified to express fractalkine/CX3CL1 in the treatment of preexisting tumors

Fractalkine (CX3CL1) is a unique membrane-bound CX3C chemokine that serves as a potent chemoattractant for lymphocytes. The hypothesis of this study is that dendritic cells (DC) genetically modified ex vivo to overexpress fractalkine would enhance the T cell-mediated cellular immune response with a consequent induction of anti-tumor immunity to suppress tumor growth. To prove this hypothesis, established tumors of different mouse cancer cells (B16-F10 melanoma, H-2b, and Colon-26 colon adenocarcinoma, H-2d) were treated with intratumoral injection of bone marrow-derived DC that had been modified in vitro with an RGD fiber-mutant adenovirus vector expressing mouse fractalkine (Ad-FKN). In both tumor models tested, treatment of tumor-bearing mice with Ad-FKN-transduced DC gave rise to a significant suppression of tumor growth along with survival advantages in the treated mice. Immunohistochemical analysis of tumors treated with direct injection of Ad-FKN-transduced DC demonstrated that the treatment prompted CD8+ T cells and CD4+ T cells to accumulate in the tumor milieu, leading to activation of immune-relevant processes. Consistent with the finding, the intratumoral administration of Ad-FKN-transduced DC evoked tumor-specific cytotoxic T lymphocytes, which ensued from in vivo priming of Th1 immune responses in the treated host. In addition, the anti-tumor effect provided by intratumoral injection of Ad-FKN-transduced DC was completely abrogated in CD4+ T cell-deficient mice as well as in CD8+ T cell-deficient mice. These results support the concept that genetic modification of DC with a recombinant fractalkine adenovirus vector may be a useful strategy for cancer immunotherapy protocols.     

Use of a skin test assay to determine tumor-specific CD8+ T cell reactivity

We have observed delayed-type hypersensitivity (DTH) reactions in immunized mice challenged subcutaneously with class I-binding peptides related to rejection antigens recognized by cytotoxic T lymphocytes on mutagenized (tum^?) variants of mastocytoma P815. As observed by skin test in virally infected mice challenged with viral peptides, the intrafootpad injection of tum^? peptides resulted in a dose-dependent DTH that peaked at approximately 24 h. The response was mediated by CD8⁺ cells and could be induced by previous vaccination of mice with live tumor cells, intrasplenic deposition of the eliciting peptide, or adoptive transfer with peptide-pulsed syngeneic dendritic cells. These sensitization procedures resulted in an immunologically specific footpad reaction detectable for up to 2–6 months after priming. The evaluation by DTH in cancer patients of long-lived CD8⁺ anti-tumor T cell responses following local challenge with tumor-specific peptides may be of great interest in human immunotherapy trials involving immunization against identified tumor antigens.     

Established B16 tumors are rejected following treatment with GM-CSF-secreting tumor cell immunotherapy in combination with anti-4-1BB mAb

Immunization with irradiated tumor cells engineered to secrete granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates potent, specific and long lasting anti-tumor immunity in clinical and preclinical settings. Efforts to further increase immunotherapy efficacy with immune-modulatory agents are under evaluation. Based on the immune-modulatory properties of 4-1BB (CD137), it has been postulated that agonistic 4-1BB antibodies may add additional anti-tumor efficacy to GM-CSF-secreting tumor cell immunotherapy. The combination of GM-CSF-secreting tumor cell immunotherapy and anti-4-1BB monoclonal antibody (mAb) treatment resulted in rejection of established tumors in the B16 melanoma model. These anti-tumor effects correlated with persistent tumor-specific CD8(+) T cell responses. In addition, early tumor infiltration of functional CD8(+) T cells and a greater expansion of antigen-specific memory T cells were found in mice treated with the combination therapy. In summary, an agonistic anti-4-1BB mAb combined with GM-CSF-secreting tumor cell immunotherapy may provide a novel and potent treatment strategy for patients with cancer.     

Immunotherapy of the rat 13762SC mammary adenocarcinoma by vaccinia virus augmentation of tumor immunity

We studied whether vaccinia virus (VV) functioned as an immunogenic carrier in augmenting anti-tumor immunity in rats bearing a syngeneic metastatic tumor. The primary tumor was induced by injecting 10⁶ 13762SC mammary adenocarcinoma cells subcutaneously into the right hind footpad of Fischer 344 rats. A concomitant anti-tumor response is induced by the tumor as demonstrated by the inhibited growth of a second tumor challenge given in the contralateral footpad 3–15 days later. Attempts were made to increase the concomitant immunity by injecting tumor-bearing animals intramuscularly with irradiated, VV-infected or uninfected 13762SC cells without adjuvant. Provided the immunotherapy was done within 5 days of the tumor challenge, administration of 10⁶–10⁷ irradiated, VV-infected 13762SC cells resulted in significantly slower tumor growth, or led to complete tumor regression, compared to control animals given no treatment. In contrast, tumor growth in animals given only VV or given irradiated, uninfected 13762SC cells, alone or mixed with VV, was the same as that in control animals. Kinetics of early primary tumor growth were predictive of a longer-term anti-tumor effect. Rechallenge of 13762SC tumor-cured animals with either the homologous or with a heterologous syngeneic mammary adenocarcinoma showed the animals to be specifically 13762SC tumor-resistant, since only rats challenged with the heterologous mammary adenocarcinoma developed progressive tumors. We interpret these results to mean that early immunotherapy with irradiated, VV-infected 13762SC cells enhances an on-going anti-tumor immune response sufficiently to cause rejection of the primary tumor and any metastases that have occurred. We also believe that later immunotherapy with irradiated, VV-infected cells has no effect due to tumor-induced immunosuppression becoming paramount.To whom correspondence should be addressed.     

Augmentation of anti-tumor activity by immunization with Mycobacterium tuberculosis (Tbc) and tuberculin-coupled tumor cells

The anti-tumor effect of immunization with heat-killed Mycobacterium tuberculosis (Tbc) and Tuberculin (PPD)-coupled syngeneic tumor cells was examined in vivo. Three tumor cell lines were employed. Immunization of Tbc-primed BALB/c mice with PPD-coupled syngeneic Meth-A tumor cells displayed a potent anti-tumor effect on viable Meth-A cells inoculated subcutaneously. Neither PPD-coupled LLC (Lewis Lung Carcinoma) cells nor sonicated PPD-coupled Meth-A cells were capable of immunizing these mice. PPD-coupled syngeneic whole tumor cells were indispensable for induction of this tumor-specific resistance. Immunization of Tbc-primed C3H/He mice with PPD-coupled syngeneic MH134 tumor cells did not elicit anti-tumor activity against MH134, but additional pretreatment of mice with cyclophosphamide brought on an anti-tumor effect. Antimetastatic reactivity was investigated in C57BL/6 mice bearing LLC, with a reduction in metastases noted. This antimetastatic effect was observed even when the mice were immunized with PPD-coupled LLC cells three days after removal of the initial tumor. Immunization with Tbc and PPD-coupled Meth-A cells together with intraperitoneal administration of murine or rat interleukin 2 (IL 2) further augmented anti-Meth-A resistance. Murine IL 2 further inhibited tumor growth during the early stage, while rat IL 2 showed an anti-tumor effect throughout the course of tumor growth.     

Combined treatment of an intratumoral injection of dendritic cells and systemic chemotherapy (Paclitaxel) for murine fibrosarcoma

A novel combined treatment of conventional chemotherapy with an intratumoral injection of syngeneic dendritic cells (DCs) has emerged as a potent cancer treatment strategy. In this study, we evaluated the synergistic effect of an intraperitoneal (i.p.) injection of a chemotherapeutic drug, paclitaxel, and an intratumoral (i.t.) injection of syngeneic bone marrow-derived DCs for the treatment of pre-existing fibrosarcoma. Subcutaneous tumors were established using MCA102 fibrosarcoma cells in syngeneic C57BL/6 mice. The results demonstrated that the combined treatment of paclitaxel chemotherapy and the injection of DCs led to complete tumor regression, in contrast to only partial eradication of the tumors with chemotherapy or DCs alone. Furthermore, the tumor-free mice were able to resist a repeat challenge with the same type of tumor. These findings suggest that a combination therapy of systemic chemotherapy along with the intratumoral administration of DCs is a potent treatment strategy for fibrosarcoma.     

CD4+ helper T cells are required for resistance to a highly metastatic murine tumor

A role of CD4+ T helper cells in induction of tumor transplant rejection leading to complete regression of a highly metastatic DBA/2 mouse lymphoma was analyzed. Using an anti-CD4 monoclonal antibody (GK1.5) which eliminates T helper cells in vivo and in vitro, we found that CD4+ cells are required for tumor resistance in syngeneic DBA/2 mice or allogeneic but major histocompatibility complex-identical B10.D2 mice. In contrast, in allogeneic C57BL/6 mice tumor rejection was independent of CD4+ cells. An analogous requirement for immune CD4+ cells for in vitro induction of CD8+ tumor-specific cytotoxic T cells was found in these respective strains. The requirement for immune CD4+ cells in vitro could be replaced by recombinant interleukin 2. These results demonstrate a role of CD4+ regulatory T cells and T-T cell cooperation in the induction of anti-tumor immunity and tumor rejection, and point to possible therapeutic interventions in the afferent phase of anti-tumor immune responses.     

The augmentation of tumor-specific immunity by virus help. II. Enhanced induction of cytotoxic T lymphocyte and antibody responses to tumor antigens by vaccinia virus-reactive helper T cells

The present study investigates the role of vaccinia virus-reactive helper T cells in causing enhanced induction of syngeneic tumor immunity. Vaccinia virus-reactive helper T cell activity capable of inducing the augmented generation of cytotoxic T lymphocyte (CTL) or antibody responses was generated in C3H/HeN mice by inoculating i.p. live virus. Immunization of these mice with vaccinia virus-infected syngeneic X5563 plasmacytoma or MH134 hapatoma cells led to augmented induction of immune resistance against the challenge with corresponding viable tumor cells when compared with the incidence of resistance observed in control mice not primed to vaccinia virus. In vitro cytotoxicity tests utilizing spleen cells and serum from mice which resulted in the augmented tumor resistance by virus help have revealed that spleen cells from C3H/HeN mice immune to the X5563 plasmacytoma exhibited appreciable anti-X5563 CTL activity, whereas serum from these mice failed to display any antibody response. In contrast, MH134-immune mice exhibited potent anti-MH134 antibody, but not CTL responses. Such an anti-tumor CTL or antibody response augmented by vaccinia virus-reactive helper T cells was found to be tumor specific. These results are discussed in the context of (a) the functional diversity of tumor antigens, and (b) mechanisms of virus help that are involved in various forms of augmented induction of syngeneic tumor immunity.     

Therapeutic immune response induced by intratumoral expression of the fusogenic membrane protein of vesicular stomatitis virus and cytokines encoded by adenoviral vectors

We assessed whether intratumoral expression of the fusogenic membrane protein of vesicular stomatitis virus (VSV-G), encoded by a replication-defective adenovirus vector (Ad.VSV-G), alone or in combination with local coexpression of cytokines induces tumor-specific immune responses in a syngeneic murine colon cancer model. We confirmed in vitro by dye colocalization that transduction of murine cells with Ad.VSV-G induces cell-cell fusion. In a bilateral syngeneic subcutaneous colon cancer model in C57BL/6 and BALB/c mice, we demonstrated that intratumoral injection of Ad.VSV-G leads to a significant growth reduction of the directly vector-treated tumor, but also of the contralateral not directly vector-treated tumor. When compared to monotherapy, the anti-neoplastic efficacy was significantly enhanced when intratumoral Ad.VSV-G administration was combined with adenovirus vectors encoding IL-2, IL-12, IL-18, IL-21, or GM-CSF. The anti-tumor effects of the first three cytokines in combination with VSV-G expression were somewhat greater than those of the latter two. However, the differences did not reach statistical significance. The combination therapy resulted also in a significantly enhanced survival when compared to monotherapy. In addition, we demonstrated that intratumoral expression of VSV-G in combination with the tested cytokines induced a strong tumor-specific cytotoxic T lymphocyte (CTL) response and infiltration of tumors with macrophages. The effects of the combination therapy were clearly greater than those of the monotherapy. Our experimental data indicate that intratumoral expression of VSV-G, particularly in combination with cytokines, is a promising novel tool for the development of in situ tumor vaccination approaches.     

Chitosan hydrogel containing GMCSF and a cancer drug exerts synergistic anti-tumor effects via the induction of CD8<Superscript>+</Superscript> T cell-mediated anti-tumor immunity

Cancer treatments consisting of a combination of chemotherapy and immunotherapy have been vigorously exploited to further improve the efficacy of cancer therapies. In this study, we utilized a chitosan hydrogel (CH) system loaded with GMCSF and a cancer drug as a chemo-immunotherapeutic agent in an effort to assess the effects on tumor growth in mice using TC-1 cervical tumor cells, which express the tumor-specific antigen, HPV-16 E7. The growth of TC-1 tumors was significantly reduced in mice treated with a CH harboring a cancer drug (doxorubicin (DOX), cisplatin (CDDP), or cyclophosphamide (CTX)) and GMCSF (CH-a cancer drug + GMCSF), as compared to other groups that were treated with CH containing only a cancer drug(CH-a cancer drug) or GMCSF(CH-GMCSF). Among the cancer drugs, CTX exerted the most potent anti-tumor effects. Interestingly, the intra-tumoral injection of CH-a cancer drug + GMCSF induced a significant E7-specific CD8⁺ T cell immune response as compared to CH-GMCSF or CH-a cancer drug. This enhancement of tumor antigen-specific CD8⁺ T cell immunity was associated principally with the anti-tumor effects induced by CH-CTX + GMCSF, as demonstrated by antibody depletion. Collectively, the aforementioned results indicate that co-treatment of tumors with a combination of GMCSF and a cancer drug incorporated into a CH system results in synergistic anti-tumor effects, which occur via the induction of a tumor antigen-specific CD8⁺ T cell-mediated anti-tumor immunity. This study demonstrates the use of a biodegradable hydrogel system for the co-delivery of an immunoadjuvant and an anti-cancer drug for successful chemo-immunotherapy. Soo Hong Seo and Hee Dong Han contributed equally to this paper.     

Recombinant IL-7 enhances the potency of GM-CSF-secreting tumor cell immunotherapy

IL-7 is known for its role in lymphopoiesis and T-cell homeostasis. In addition, its capacity to augment the immune response to weak or low affinity antigens makes it an ideal candidate to evaluate in combination with a GM-CSF-secreting tumor cell immunotherapy, which has been shown to elicit broad humoral and cellular immune responses. The studies reported here show that IL-7, when combined with a GM-CSF-secreting tumor cell immunotherapy, significantly prolonged the survival of tumor-bearing mice. The enhanced anti-tumor protection correlated with an increased number of activated dendritic cells (DC) and T cells in lymphoid tissues, such as the draining lymph nodes (DLN) and spleen. Moreover, an increased number of activated effector T cells were found in the tumor microenvironment, correlating with a more potent systemic tumor-specific T-cell response than each monotherapy alone. Taken together, these studies demonstrate that IL-7 augments the anti-tumor response of a GM-CSF-secreting tumor cell immunotherapy in preclinical models.     

Dendritic cell vaccine immunotherapy of cancer targeting MUC1 mucin

The use of dendritic cells (DCs) for cancer vaccination is effective in suppressing cancer progression. This is because the DCs play a crucial role in priming tumor-specific immunity efficiently as antigen-presenting cells. In this study, we analyzed the ability of DCs to elicit tumor-specific immunity and clinical effects of DC vaccine immunotherapy targeting MUC1 tumor antigens. DCs from 14 patients with advanced or metastatic breast or lung cancer (9 positive for MUC1 and 5 negative for MUC1) were loaded with MUC1 antigens or tumor lysate and used for therapeutic vaccination. After vaccination, all the MUC1-positive patients acquired antigen-specific immunity whereas only 1 case with MUC1-negative cancer showed the specific immunity. Clinically, marked effects such as reduction in tumor sizes or tumor marker levels or disappearance of malignant pleural effusion were observed in 7 of the 9 MUC1-positive cases. However, MUC1-negative patients did not respond to DC vaccines, with the exception of 1 case with MAGE3-positive lung cancer. Survival of MUC1-positive patients was significantly prolonged in comparison with MUC1-negative patients (mean survival: 16.75 versus 3.80 months, p=0.0101). These data suggest that MUC1 is sufficiently immunogenic to elicit strong anti-tumor immunity as a tumor antigen and that DC vaccines targeting MUC1 are useful for immunotherapy of cancer.     

Bacteriophages support anti-tumor response initiated by DC-based vaccine against murine transplantable colon carcinoma

Bacteriophages in eukaryotic hosts may behave as particulate antigens able to activate the innate immune system and generate adaptive immunity. Dendritic cells (DCs) play a key role in the initiation of the immune response, mainly by priming T cell-mediated immunity. For this reason, they are increasingly applied as an adjuvant for effective anti-tumor therapies in animal models as well as in a few clinical trials. The presented study focused on the application of mouse DCs which were activated with T4 bacteriophages (T4 phages, T4) and further loaded with tumor antigens (TAg) in inducing an anti-tumor response. The activation of bone marrow-derived DCs with T4 phages and TAg resulted in augmentation of their differentiation marker expression accompanied by an enhanced ability to prime T cells for IFN-gamma production. These activated DCs (BM-DC/T4+TAg) were used in experimental immunotherapy of C57BL/6 mice bearing advanced MC38 colon carcinoma tumors. As a result of their triple application, a significant tumor growth delay, up to 19 days, was observed compared with the controls - treated with BM-DCs activated only with T4 phages, TAg, or lipopolysaccharide solution ["solvent"], where the tumor growth delay did not exceed 7 days. The percentage of tumor growth inhibition estimated 10 days after the third cell injection ranged from 32% (for animals treated with BM-DC/TAg cells) to 76% (for animals treated with BM-DC/T4+TAg cells) over the tumor-bearing untreated control mice. The obtained data indicate that in vitro interactions between T4 phages and BM-DCs followed by TAg activation caused augmentation of the anti-tumor effect when DCs were used as a vaccine for tumor-bearing mice treatment. Therefore, pretreatment of DCs with the phages may be considered as a beneficial element of a novel strategy in anti-tumor immunotherapy.     

Concomitant presence of anti-tumor effector cells and suppressor cells in the spleen of tumor-bearing mice : the nature of suppressor cells

In order to investigate the immunological responsiveness of tumor-bearing hosts to tumor cells, splenic suppressor cells from Ehrlich tumor-bearing mice that inhibited anti-tumor effector cell activity were characterized. In vitro cell-mediated cytoxicity and cytostasis assays were performed to test for the existence of anti-tumor immunity. suppressive activity assayed by cell mixture experiments became apparent with decline of anti-tumor immunity and progressive tumor growth. The cells mediating the suppression were found to be nylon wool column adherent T cells and inhibited T cell dependent cytotoxicity rather than non-T cell dependent cytostasis. In vivo cell transfer experiments demonstrated that intravenous injection of suppressor cells to a host already inoculated with tumor cells mixed with antitumor effector cells resulted in significant enhancement of tumor growth. This inhibition of in vivo neutralization assay be suppressor cells was found in not only allogeneic but also syngeneic tumor system. Splenectomy at the time of tumor resection endowed the host with stronger resistance against subsequent reinoculated tumor than sham-splenectomy did, reflected by prolonged survival times. These results suggest that splenectomy combined with surgical removal of the tumor is a useful treatment of clinical malignancies.     

Combination tumor immunotherapy with radiotherapy and Th1 cell therapy against murine lung carcinoma

Mice bearing established Lewis lung carcinoma (LLC) expressing model tumor antigen, ovalbumin (OVA) (LLC-OVA) marginally responded to local radiotherapy, but none of the mice was cured. In contrast, treatment of the tumor-bearing mice with intratumoral injection of tumor-specific T helper type 1 (Th1) cells and tumor antigen (OVA) after radiotherapy dramatically prolonged the survival days and induced complete cure of the mice at high frequency (80%). Radiation therapy combined with Th1 cells or OVA alone showed no significant therapeutic activity against LLC-OVA. Such a strong therapeutic activity was not induced by intratumoral injection of Th1 cells plus OVA. Compared with other treatment, radiation therapy combined with Th1 cells and OVA was superior to induce the generation of OVA/H-2^b tetramer⁺ tumor-specific cytotoxic T lymphocyte (CTL) with a strong cytotoxicity against LLC-OVA in draining lymph node (DLN). Moreover, the combined therapy is demonstrated to inhibit the growth of tumor mass, which grew at contralateral side. These results indicated that radiotherapy combined with Th1 cell/vaccine therapy induced a systemic antitumor immunity. These findings suggested that combination therapy with radiotherapy and Th1 cell/vaccine therapy may become a practical strategy for cancer treatment. Hiroshi Yokouchi and Kenji Chamoto are equally contributed.     

Therapeutic anti-tumor immunity triggered by injections of immunostimulating single-stranded RNA

Stabilized synthetic RNA oligonucleotides (ORN) and protected messenger RNA (mRNA) were recently discovered to possess an immunostimulatory capacity through their recognition by TLR 7 and 8. We wanted to find out whether this danger signal is capable of triggering anti-tumor immunity when injected locally into an established tumor. Using the mouse glioma tumor cell line SMA-560 in syngenic VM/Dk mice, we were able to show that intra-tumor injections of protamine-stabilized mRNA do indeed induce tumor regression and long-term anti-tumor immunity. Residual RNA-injected tumors show CD8 infiltration. Distant injections of protamine-protected mRNA and intra-tumor injection of naked mRNA also result in anti-tumor immunity. Although they are strong danger signals, RNA are labile molecules with a short half-life: they do not trigger side effects such as long-term, uncontrolled immunostimulation evidenced by splenomegaly in CpG DNA-treated mice. In conclusion, RNA molecules are potent and safe danger signals that are relevant for active immunotherapy strategies aimed at the eradication of solid tumors.     

Peritransplantation vaccination with chaperone-rich cell lysate induces antileukemia immunity.

We have reported that chaperone-rich cell lysate (CRCL) is an effective anticancer vaccine in immunocompetent mice. In this study, we explored the therapeutic applicability of CRCL in the context of syngeneic hematopoietic cell transplantation (HCT) to treat preexisting leukemia. Our results demonstrate that tumor growth is significantly delayed in mice receiving syngeneic HCT from 12B1 tumor CRCL-immunized donors compared with animals receiving HCT from nonimmunized donors. CRCL immunization after immune HCT further hindered tumor growth when compared with immune HCT without posttransplantation vaccination. The magnitude of the immune response was consistent with the antitumor effects observed in vivo. Rechallenge of surviving mice with 12B1 or A20 cells in opposite groins confirmed that mice had developed long-term tumor-specific immunity against 12B1 tumor cells. In addition, we documented that both T cells and natural killer cells contributed to the antitumor effect of CRCL vaccination, because depletion of either subset hampered tumor growth delay. Thus, our results indicate that CRCL is a promising vaccine capable of generating specific immune responses. This antitumor immunity can be effectively transferred to a host via HCT and further enhanced after HCT with additional tumor CRCL immunizations.     

Bryostatin 1/ionomycin (B/I) ex vivo stimulation preferentially activates L-selectinlow tumor-sensitized lymphocytes

We have shown that tumor vaccine-sensitized draining lymph node (vDLN) cells activated ex vivo with bryostatin and ionomycin (B/I) were capable of inducing antigen-specific regression of a murine mammary tumor, 4T07. vDLN cells not activated with B/I were ineffective. We hypothesized that B/I selectively activates tumor-sensitized (CD62Llow) lymphocytes, to account for the highly potent and tumor-specific activity. We hypothesized that CD8+ CD62Llow cells may be preferentially activated by B/I treatment, infiltrate the tumors and mediate tumor regression in mice. 4T07-IL2 tumor cells were injected into one hind footpad of BALB/c mice. Ten days later, vDLN were harvested and separated based on CD62L expression. After separation, cells were activated with B/I, expanded with IL2 (40 IU/ml) for 10 days, and adoptively transferred to 4T07 tumor bearing mice. Naive mice were also treated with different subsets of T cells and later were challenged with 4T07 tumor cells. To test in vitro responses to antigen, expanded lymphocytes were cultured either alone or with irradiated 4T07 tumor cells. Supernatants were harvested after 24 h and tested by ELISA for IFN-gamma. The importance of the host immune response was tested by AIT into 4T07-bearing nude athymic mice. Host mice were depleted in vivo of CD4 or CD8 T cells after vDLN AIT to ascertain the mediators of tumor regression. In order to track B/I activated vDLN cells, they were prestained with CFSE prior to adoptive transfer into tumor-bearing hosts. At various time points, tumors, spleens and lymph nodes of host mice were harvested, dual stained for activation marker expression and analyzed by flow cytometry. CD62Llow cells expanded 12-fold more than CD62Lhigh lymphocytes during the 10 day culture period. Supernatant from CD62Llow cells + 4T07 cultures contained 33-fold more IFN-gamma than supernatant from CD62Lhigh cells + 4T07 cultures (843.9 pg/ml +/- 135.8 vs 25.89 pg/ml +/- 0.01). Adoptive transfer of CD62Llow lymphocytes induced complete tumor regressions in all mice, while tumors regressed in only 17% of mice treated with CD62Lhigh lymphocytes. Naive mice that received B/I-activated CD62Llow cells were protected from future tumor challenges, while mice given CD62Lhigh cells did not exhibit the same resistance to tumor growth. Tumors in nude host mice regressed after AIT treatment. In vivo depletion of CD4 T cells after AIT did not inhibit tumor regression, but CD8 T cell depletion abrogated tumor regression. vDLN cells tracked preferentially to tumor draining lymph nodes and proliferated in vivo, persisting for at least 21 days, and were 95% CD44+ and 39% CD69+. Bryostatin 1 and ionomycin, by increasing PKC activity and intracellular calcium, respectively, mimic intracellular signals that result in T cell activation. CD62Llow cells are preferentially activated by B/I, leading to a highly effective anti-tumor T cell population.