Tumor Vaccine Draining Lymph Nodes Research Articles

Anti-Gr-1 Antibody Provides Short-Term Depletion of MDSC in Lymphodepleted Mice with Active-Specific Melanoma Therapy.

Lymphodepletion, reconstitution and active-specific tumor cell vaccination (LRAST) enhances the induction of tumor-specific T cells in a murine melanoma model. Myeloid-derived suppressor cells (MDSC) may counteract the induction of tumor-reactive T cells and their therapeutic efficacy. Thus, the aim of the study was to evaluate a possible benefit of MDSC depletion using anti-Gr-1 antibodies (Ab) in combination with LRAST. Female C57BL/6 mice with 3 days established subcutaneous (s.c.) D5 melanoma were lymphodepleted with cyclophosphamide and reconstituted with naive splenocytes. Vaccination was performed with irradiated syngeneic mGM-CSF-secreting D5G6 melanoma cells. MDSC depletion was performed using anti-Gr-1 Ab (clone RB6-8C5). Induction of tumor-specific T cells derived from tumor vaccine draining lymph nodes (TVDLN) was evaluated by the amount of tumor-specific interferon (IFN)-γ release. LRAST combined with anti-Gr-1 mAb administration enhanced the induction of tumor-specific T cells in TVDLN capable of releasing IFN-γ in a tumor-specific manner. Additional anti-Gr-1 mAb administration in LRAST-treated mice delayed growth of D5 melanomas by two weeks. Furthermore, we elucidate the impact of anti-Gr-1-depleting antibodies on the memory T cell compartment. Our data indicate that standard of care treatment regimens against cancer can be improved by implementing agents, e.g., depleting antibodies, which target and eliminate MDSC.

In vitro-activated tumor-bearing host T cells and the effectiveness of tumor vaccine immunotherapy

BACKGROUND AND OBJECTIVEVaccination during periods of lymphopenia may facilitate immune responses to weak self-antigens and enhance antitumor immunity. The objective of this study was to determine the effectiveness of tumor vaccine immunotherapy combined with immune reconstruction using tumor-bearing host immune cells in lymphopenia, and to investigate the role of tumor-bearing host T cells activated in vitro during immunotherapy.DESIGN AND SETTINGAnimal study conducted in the First Affiliated Hospital of Xi’an Jiaotong University from January 2009 to January 2010.PATIENTS AND METHODSLymphopenia was induced by cyclophosphamide. A reconstituted immune system with different syngeneic lymphocytes was employed, including lymphocytes from naïve rats (unsensitized group), tumor-bearing rats (tumor-bearing group), and tumor-bearing rats activated in vitro (activated group). All rats were immunized with granulocyte-macrophage colony-stimulating factor (GM-CSF)-modified NuTu-19 ovarian cancer (GM-CSF/NuTu-19) cells. Tumor vaccine-draining lymph nodes (TVDLNs) were harvested, and then stimulated to induce effector T cells (TE). TE were then adoptively transferred to rats bearing a 3-day pre-established abdominal tumor (NuTu-19), and the survival rate was calculated.RESULTSCompared with the unsensitized group, the levels of interleukin-2 (IL-2) were significantly lower in the tumor-bearing group, whereas that of IL-4 were significantly higher (P<.05). The number of CD4+ T cells secreting interferon-γ and the specific cytotoxicity of CD8+ cytotoxic T lymphocytes were significantly lower (P<.05). The survival was significantly higher in the activated group compared with the other groups.CONCLUSIONSLymphocytes from tumor-bearing rats activated in vitro can effectively reverse the immunosuppressive effects of tumor-bearing hosts.

Therapeutic T cells induce tumor-directed chemotaxis of innate immune cells through tumor-specific secretion of chemokines and stimulation of B16BL6 melanoma to secrete chemokines.

BackgroundThe mechanisms by which tumor-specific T cells induce regression of established metastases are not fully characterized. In using the poorly immunogenic B16BL6-D5 (D5) melanoma model we reported that T cell-mediated tumor regression can occur independently of perforin, IFN-γ or the combination of both. Characterization of regressing pulmonary metastases identified macrophages as a major component of the cells infiltrating the tumor after adoptive transfer of effector T cells. This led us to hypothesize that macrophages played a central role in tumor regression following T-cell transfer. Here, we sought to determine the factors responsible for the infiltration of macrophages at the tumor site.MethodsThese studies used the poorly immunogenic D5 melanoma model. Tumor-specific effector T cells, generated from tumor vaccine-draining lymph nodes (TVDLN), were used for adoptive immunotherapy and in vitro analysis of chemokine expression. Cellular infiltrates into pulmonary metastases were determined by immunohistochemistry. Chemokine expression by the D5 melanoma following co-culture with T cells, IFN-γ or TNF-α was determined by RT-PCR and ELISA. Functional activity of chemokines was confirmed using a macrophage migration assay. T cell activation of macrophages to release nitric oxide (NO) was determined using GRIES reagent.ResultsWe observed that tumor-specific T cells with a type 1 cytokine profile also expressed message for and secreted RANTES, MIP-1α and MIP-1β following stimulation with specific tumor. Unexpectedly, D5 melanoma cells cultured with IFN-γ or TNF-α, two type 1 cytokines expressed by therapeutic T cells, secreted Keratinocyte Chemoattractant (KC), MCP-1, IP-10 and RANTES and expressed mRNA for MIG. The chemokines released by T cells and cytokine-stimulated tumor cells were functional and induced migration of the DJ2PM macrophage cell line. Additionally, tumor-specific stimulation of wt or perforin-deficient (PKO) effector T cells induced macrophages to secrete nitric oxide (NO), providing an additional effector mechanism for T cell-mediated tumor regression.ConclusionThese data suggest two possible sources for chemokine secretion that stimulates macrophage recruitment to the site of tumor metastases. Both appear to be initiated by T cell recognition of specific antigen, but one is dependent on the tumor cells to produce the chemokines that recruit macrophages.

Tumor-specific T cells signal tumor destruction via the lymphotoxin β receptor

BackgroundPreviously, we reported that adoptively transferred perforin k/o (PKO), and IFN-γ k/o (GKO), or perforin/IFN-γ double k/o (PKO/GKO) effector T cells mediated regression of B16BL6-D5 (D5) pulmonary metastases and showed that TNF receptor signaling played a critical role in mediating tumor regression. In this report we investigated the role of lymphotoxin-α (LT-α) as a potential effector molecules of tumor-specific effector T cells.MethodsEffector T cells were generated from tumor vaccine-draining lymph node (TVDLN) of wt, GKO, LT-α deficient (LKO), or PKO/GKO mice and tested for their ability to mediate regression of D5 pulmonary metastases in the presence or absence of LT-βR-Fc fusion protein or anti-IFN-γ antibody. Chemokine production by D5 tumor cells was determined by ELISA, RT-PCR and Chemotaxis assays.ResultsStimulated effector T cells from wt, GKO, or PKO/GKO mice expressed ligands for LT-β receptor (LT-βR). D5 tumor cells were found to constitutively express the LT-βR. Administration of LT-βR-Fc fusion protein completely abrogated the therapeutic efficacy of GKO or PKO/GKO but not wt effector T cells (p < 0.05). Consistent with this observation, therapeutic efficacy of effector T cells deficient in LT-α, was greatly reduced when IFN-γ production was neutralized. While recombinant LT-α1β2 did not induce apoptosis of D5 tumor cells in vitro, it induced secretion of chemokines by D5 that promoted migration of macrophages.ConclusionThe contribution of LT-α expression by effector T cells to anti-tumor activity in vivo was not discernable when wt effector T cells were studied. However, the contribution of LT-β R signaling was identified for GKO or PKO/GKO effector T cells. Since LT-α does not directly induce killing of D5 tumor cells in vitro, but does stimulate D5 tumor cells to secrete chemokines, these data suggest a model where LT-α expression by tumor-specific effector T cells interacts via cross-linking of the LT-βR on tumor cells to induce secretion of chemokines that are chemotactic for macrophages. While the contribution of macrophages to tumor elimination in our system requires additional study, this model provides a possible explanation for the infiltration of inate effector cells that is seen coincident with tumor regression.

Induction of Tumor Immunity Early after Syngeneic Bone Marrow Transplantation (BMT) by Transient Transfection of Mouse Neuroblastoma with Plasmid DNA Vectors Encoding a Panel of Immune Stimulatory Molecules.

Autologous hematopoietic stem cell transplantation has resulted in improved event-free survival for patients with neuroblastoma, but the majority of these patients still relapse. We have shown that transient transfection (nucleofection) of mouse neuroblastoma (Neuro-2a) cells with plasmid DNA vectors encoding immune stimulatory molecules generates cell-based vaccines capable of inducing potent anti-tumor T cell immunity. We are exploring tumor vaccine administration early after hematopoietic stem cell transplantation because this may be an ideal setting to induce effective tumor immunity due to altered immune regulation and altered T cell homeostasis. The goal of this study was to determine the cellular components required to induce tumor-protective immunity in mice early after lethal total body irradiation (TBI) and syngeneic BMT. A/J mice were given 1100 cGy TBI, and one day later, transplanted with 107 syngeneic bone marrow cells with or without purified T cells or total splenocytes (as a source of T cells). An aggressive variant of Neuro-2a (AGN2a) was nucleofected with plasmid vectors encoding CD54 (ICAM-1), CD80, CD86 and CD137L (4-1BBL). The nucleofected cells were irradiated and then given to transplant recipients as a cell-based vaccine on days 7 and 14 post-transplant. To assess development of tumor immunity, groups of transplanted/vaccinated mice were challenged with different doses of tumor cells one week after the last vaccine. Vaccination of mice given BMT without added T cells failed to induce any protective anti-tumor immunity, and all of these mice died after challenge with 105 or 106 viable tumor cells. In contrast, 78.5% of vaccinated mice given 5x106 T cells at the time of BMT survived tumor challenge. Tumor protective immunity in the transplant recipients was dependent upon both CD4+ and CD8+ T cells, and tumor-specific CD8+ T cells in the tumor vaccine draining lymph nodes and spleens of vaccinated mice could be detected in IFN-g ELISPOT assays. From these results, we concluded that the adoptive transfer of T cells was absolutely required for induction of protective immunity by the tumor vaccine. We examined whether in vivo CD25 depletion of the transplant recipients, or depletion of CD25+ regulatory T cells from the adoptively-transferred T cells, would further increase vaccine-induced tumor immunity. This was based on previous results in non-transplanted mice, where in vivo-depletion of CD25+ cells increased effectiveness of cell-based neuroblastoma vaccines. Depletion of CD25+ cells in recipient mice or in the adoptively transferred T cells did not increase anti-tumor immunity as determined from tumor challenge experiments, suggesting that the lethal TBI itself had eliminated the majority of CD25+ regulatory T cells. Collectively, these results demonstrate that anti-neuroblastoma immunity can be induced early after BMT using a novel cell-based cancer vaccine; however, sufficient numbers of T cells must be included in the graft to achieve protective anti-tumor immunity.

Reduced L-selectin (CD62L Low) expression identifies tumor-specific type 1 T cells from lymph nodes draining an autologous tumor cell vaccine

Reduced expression of CD62L can identify tumor-specific T cells in lymph nodes draining murine tumors. Here, we examined whether this strategy could isolate tumor-specific T cells from vaccinated patients. Tumor vaccine-draining lymph node (TVDLN) T cells of seven patients were separated into populations with reduced (CD62L Low) or high levels of CD62L (CD62L High). Effector T cells generated from CD62L Low cells maintained or enriched the autologous tumor-specific type 1 cytokine response compared to unseparated TVDLN T cells in four of four patients showing tumor-specific cytokine secretion. Interestingly, effector T cells generated from CD62L Low or CD62L High TVDLN were polarized towards a dominant type 1 or type 2 cytokine profile, respectively. For CD62L Low T cells the type 1 cytokine profile appeared determined prior to culture. Since a tumor-specific type 1 cytokine profile appears critical for mediating anti-tumor activity in vivo, this approach might be used to isolate T cells for adoptive immunotherapy.

Regression of bone metastases following adoptive transfer of anti-CD3-activated and IL-2-expanded tumor vaccine draining lymph node cells

As many as 80% of patients with breast, prostate, or lung cancer develop bone metastases during the course of their illness. However, thus far, no attempts have been made to explore the potential value of adoptive immunotherapy with antigen-specific T lymphocytes specifically for the treatment of skeletal metastases. Here, we demonstrate tumor regression in a preclinical model of bone metastases from the murine B16BL6 melanoma following adoptive transfer of effector T lymphocytes obtained from tumor vaccine draining lymph nodes. The antitumor effect required transfer of high number of effector cells, which was dependent on CD8+ cells as demonstrated by in vivo depletion of different T cell subsets, and was magnified if effector cells were administered to the arterial supply of the bone/bone marrow. Using flow cytometric analysis, CFSE-labelled Thy1.1+ donor T cells were isolated from the bone marrow of tumor-bearing mice at 24 h and 6 days following adoptive transfer. At the latter time point cell division of the transferred effector cells was detectable. Currently, no curative treatment is known for skeletal metastases in clinical practice. Considering the promising early findings in the present study, further studies exploring the therapeutic potential of adoptive immunotherapy for metastatic disease to the skeleton are warranted.

Anti-tumor T cell response and protective immunity in mice that received sublethal irradiation and immune reconstitution.

To test whether homeostasis-driven T cell proliferation in reconstituted lymphodepleted hosts would improve the therapeutic efficacy of tumor vaccines, normal mice and reconstituted lymphopenic mice (RLM; C57BL/6 mice rendered lymphopenic with sublethal total-body irradiation and reconstituted with naive splenocytes) were used in the vaccination and challenge experiments with weakly immunogenic F10 melanoma cells. Only limited protection was observed in vaccinated normal mice (16.7%), whereas significantly greater protection was induced in vaccinated RLM (63.2%). Protective immunity in RLM depended on CD8 T cells. Following vaccination, a significant increase in the percentage of CD44(hi)CD62L(lo) T cells was detected in the tumor vaccine-draining lymph node (TVDLN) of vaccinated RLM compared to that of vaccinated normal mice. After in vitro stimulation, effector T cells generated from TVDLN of vaccinated RLM produced more IFN-gamma than T cells from vaccinated normal mice, and contained more melanoma-specific T cells, as assessed by ELISA and intracellular cytokine staining. This study suggests that vaccination of reconstituted lymphopenic hosts could elicit superior anti-tumor immunity compared to normal hosts, highlighting the potential clinical benefit of performing tumor vaccination during immune reconstitution.

Evaluation of a Preclinical Model of Bone Metastasis for the Study of Adoptive Immunotherapy

The development of new therapeutic strategies for the treatment of bone metastases strongly depends on the availability of valid animal models. In this paper, we evaluate a preclinical model of bone metastases using a technique of tumor cell injection into the left heart ventricle of mice to study the efficacy of adoptive immunotherapy. Using flow cytometric analysis and histopathological and radiological examination, we investigated whether this experimental model of bony metastases using two murine cell lines of melanoma and breast cancer would be suitable for the study of adoptive immunotherapy for these diseases. We further report that anti-CD3-activated and IL-2-expanded tumor vaccine draining lymph node cells cause regression of tumor metastases, including bone metastases, following adoptive transfer to mice bearing 3-day metastases from the D5 melanoma cell line. These promising early results lead us to conclude the following: (1) this model of experimental bone metastases is suitable for the study of immunotherapy, and (2) further studies are warranted to extend these promising early findings of the therapeutic effects of adoptive immunotherapy in this animal model.

CD28, TNF Receptor, and IL-12 Are Critical for CD4-Independent Cross-Priming of Therapeutic Antitumor CD8+ T Cells

Previously, we have shown that priming of therapeutic CD8(+) T cells in tumor vaccine-draining lymph nodes of mice vaccinated with GM-CSF secreting B16BL6 melanoma cells occurs independent of CD4 T cell help. In this study, we examined the contribution of the major costimulatory molecules, CD40 ligand (CD40L), CD80, and CD86, in the priming of CD8(+) T cells. Priming of therapeutic CD8(+) T cells by a GM-CSF-transduced tumor vaccine did not require CD40 and CD40L interactions, as therapeutic T cells could be generated from mice injected with anti-CD40L Ab and from CD40L knockout mice. However, costimulation via either CD80 or CD86 was required, as therapeutic T cells could be generated from mice injected with either anti-CD80 or anti-CD86 Ab alone, but administration of both Abs completely inhibited the priming of therapeutic T cells. Blocking experiments also identified that priming of therapeutic T cells in MHC class II-deficient mice required TNFR and IL-12 signaling, but signaling through CD40, lymphotoxin-betaR, or receptor activator of NF-kappaB was not essential. Thus, cross-priming of therapeutic CD8(+) T cells by a tumor vaccine transduced with GM-CSF requires TNFR, IL-12, and CD28 signaling.

T cell polarization in tumor vaccine draining lymph nodes (TVDLN) correlates with the tumor immunogenicity

T cell polarization in tumor vaccine draining lymph nodes (TVDLN) correlates with the tumor immunogenicity

Spontaneous mammary carcinomas fail to induce an immune response in syngeneic FVBN202 neu transgenic mice

FVBN202 mice, which are transgenic for the rat neu gene, spontaneously develop mammary carcinomas between 6 and 7 months of age. We investigated whether these spontaneous tumors (spontaneous breast carcinoma cells, SBCC) could elicit an immune response in naive 6- to 8-week-old FVBN202 transgenic and FVBN nontransgenic mice. After s.c. injection of SBCC, the recently activated T cells, which were identified by their reduced expression of CD62L (L-selectin), were isolated from the draining lymph nodes, expanded with anti-CD3 and IL-2, and their cytokine response to tumor cells in vitro was analyzed. Tumor-vaccine draining lymph node lymphocytes (TVDLN) from transgenic mice failed to make IFN-gamma in response to the tumor cells. However, TVDLN from the nontransgenic mice exhibited a tumor-specific IFN-gamma response against the SBCC. This indicates that the SBCC are immunogenic. The lack of response in transgenic mice could not be attributed to cytokine immune deviation or T-cell signaling defects. Although transgenic mice were tolerant to their own tumors, their immune competence was established by their ability to respond in an allogeneic mixed lymphocyte reaction, to reject an allogeneic breast carcinoma cell line, and to produce a tumor-specific IFN-gamma response against a syngeneic cancer cell line. This transgenic mouse model provides the opportunity to investigate the immune response against a primary tumor cell culture rather than cell lines or clones and should prove useful for developing immunotherapies that overcome tolerance to self-tumor antigens.

Tumor Regression After Adoptive Transfer of Effector T Cells Is Independent of Perforin or Fas Ligand (APO-1L/CD95L)

The adoptive transfer of tumor-specific effector T cells can result in complete regression and cure mice with systemic melanoma, but the mechanisms responsible for regression are not well characterized. Perforin- and Fas ligand (APO-1/CD95 ligand)-mediated cytotoxicity have been proposed as mechanisms for T cell-mediated tumor destruction. To determine the role of perforin and Fas ligand (FasL) in T cell-mediated tumor regression in a murine melanoma model, B16BL6-D5 (D5), we generated D5-specific effector T cells from tumor vaccine-draining lymph nodes of wild type (wt), perforin knock out (PKO), or FasL mutant (gld) mice and treated established D5 metastases in mice with the same genotype. Effector T cells from wt, PKO and gld mice induced complete regression of pulmonary metastases and significantly prolonged survival of the treated animals regardless of their genotype. Complete tumor regression induced by PKO effector T cells was also observed in a sarcoma model (MCA-310). Furthermore, adoptive transfer of PKO and wt effector T cells provided long-term immunity to D5. Therapeutic T cells from wt, PKO, or gld mice exhibit a tumor-specific type 1 cytokine profile; they secrete IFN-gamma, but not IL-4. In these models, T cell-mediated tumor regression and long-term antitumor immunity are perforin and FasL independent.

Gene-Modified Tumor Vaccine with Therapeutic Potential Shifts Tumor-Specific T Cell Response from a Type 2 to a Type 1 Cytokine Profile

Vaccination with a poorly immunogenic/nonimmunogenic tumor fails to protect the host from a subsequent challenge with the same tumor. The mechanisms underlying the failure of these tumors to sensitize therapeutic T cells are not clearly understood, but the inability of host T cells to recognize tumor has been implicated. In this study, vaccination with the poorly immunogenic B16BL6-D5 (D5 H-2b) tumor fails to generate therapeutic T cells from the tumor vaccine-draining lymph nodes (TVDLN) in our adoptive immunotherapy model. However, if vaccination is performed with an allogeneic MHC class I gene (H-2 Kd)-modified tumor, the T cells obtained from the TVDLN are therapeutic after activation with anti-CD3 and IL-2. Lymph nodes (LN) draining both D5 and D5-Kd tumor vaccines contained increased numbers of cells with reduced expression of L-selectin (L-selectin(low/-)) compared with naive LN. This implies that vaccination led to sensitization of T cells even in LN draining the unmodified D5 tumor. L-selectin(low/-) cells from D5-Kd, but not D5, TVDLN were therapeutic in our animal model. No antitumor activity was seen in the high level L-selectin T cells. L-selectin(low/-) T cells exhibited tumor-specific cytokine release that was type 2 (IL-4, IL-10) following vaccination with native D5 and type 1 (IFN-gamma) following vaccination with gene-modified D5-Kd. Our data suggest that the failure of unmodified D5 to generate therapeutic T cells is not due to an inability to recognize tumor Ags, but, rather, to the induction of an immune response that is ineffective in mediating tumor regression, i.e., immune deviation.