Adoptive immunotherapy of breast cancer with lymph node cells primed by cryoablation of the primary tumor

Cryoablation of cancer leaves tumor-associated antigens intact in an inflammatory microenvironment that can stimulate a regional anti-tumor immune response. We examined whether cryoablated tumor draining lymph nodes (CTDLN) as adoptive immunotherapy may be an effective immunotherapeutic approach in the adjuvant treatment of breast cancer. BALB/c mice with MT-901 mammary adenocarcinoma tumors underwent cryoablation, resection or no treatment and tumor draining lymph nodes were harvested. Cryoablation resulted in only a mild increase in the absolute number of T-cells but a significant increase in the fraction of tumor-specific T-cells as evidenced on IFN-gamma release assay. FACS analysis demonstrated no significant relative shift in the proportion of CD4(+) or CD8(+) cells. The adoptive transfer of CTDLN resulted in a significant reduction of pulmonary metastases as compared to TDLN from either tumor-bearing mice or mice who underwent surgical excision. Cryoablation prior to surgical resection of breast cancer can be used as a method to generate effector T-cells for adjuvant adoptive cellular immunotherapy.     

Adoptive immunotherapy of advanced tumors with CD62 L-selectin(low) tumor-sensitized T lymphocytes following ex vivo hyperexpansion

Tumor-draining lymph nodes (TDLN) contain sensitized T cells with the phenotype CD62 L-selectin(low) (CD62L(low)) that can be activated ex vivo with anti-CD3 mAb and IL-2 to acquire potent dose-dependent effector function manifested upon adoptive transfer to secondary tumor-bearing hosts. In this study advanced tumor models were used as a stringent comparison of efficacy for the CD62L(low) subset, comprising 5-7% of the TDLN cells, vs the total population of TDLN cells following culture in high dose IL-2 (100 U/ml). During the 9-day activation period the total number of CD8+ T cells increased 1500-fold, with equivalent proliferation in the CD62L(low) vs the total TDLN cell cultures. Adoptive transfer of activated CD62L(low) cells eliminated 14-day pulmonary metastases and cured 10-day s.c. tumors, whereas transfer of maximally tolerated numbers of total TDLN cells was not therapeutic. Despite their propagation in a high concentration of IL-2, the hyperexpanded CD62L(low) subset of TDLN cells functioned in vivo without exogenous IL-2, and CD8+ T cells demonstrated relative helper independence. Moreover, the anti-tumor response was specific for the sensitizing tumor, and long term memory was established. The facile enrichment of tumor-reactive TDLN T cells, based on the CD62L(low) phenotype, circumvents the need for prior knowledge of the relevant tumor Ags. Coupling the isolation of pre-effector T cells with rapid ex vivo expansion to >3 logs could overcome some of the shortcomings of active immunotherapy or in vivo cytokine treatment, where selective robust expansion of effector cells has been difficult to achieve.     

Short-term activation induces multifunctional dendritic cells that generate potent antitumor T-cell responses in vivo

Dendritic cell (DC) vaccines have emerged as a promising strategy to induce antitumoral cytotoxic T cells for the immunotherapy of cancer. The maturation state of DC is of critical importance for the success of vaccination, but the most effective mode of maturation is still a matter of debate. Whereas immature DC carry the risk of inducing tolerance, extensive stimulation of DC may lead to DC unresponsiveness and exhaustion. In this study, we investigated how short-term versus long-term DC activation with a Toll-like receptor 9 agonist influences DC phenotype and function. Murine DC were generated in the presence of the hematopoietic factor Flt3L (FL-DC) to obtain both myeloid and plasmacytoid DC subsets. Short activation of FL-DC for as little as 4 h induced fully functional DC that rapidly secreted IL-12p70 and IFN-α, expressed high levels of costimulatory and MHC molecules and efficiently presented antigen to CD4 and CD8 T cells. Furthermore, short-term activated FL-DC overcame immune suppression by regulatory T cells and acquired high migratory potential toward the chemokine CCL21 necessary for DC recruitment to lymph nodes. In addition, vaccination with short-term activated DC induced a strong cytotoxic T-cell response in vivo and led to the eradication of tumors. Thus, short-term activation of DC generates fully functional DC for tumor immunotherapy. These results may guide the design of new protocols for DC generation in order to develop more efficient DC-based tumor vaccines.     

Dendritic cells efficiently acquire and present antigen derived from lung cancer cells and induce antigen-specific T-cell responses

Active immunotherapy of cancer requires the availability of a source of tumor antigens. To date, no such antigen associated with lung cancer has been identified. We have therefore investigated the ability of dendritic cells (DC) to capture whole irradiated human lung tumor cells and to present a defined surrogate antigen derived from the ingested tumor cells. We also describe an in vitro system using a modified human adenocarcinoma cell line (A549-M1) that expresses the well-characterized, immunogenic influenza M1 matrix protein as a surrogate tumor antigen. Peripheral blood monocyte-derived DC, when co-cultured with sub-lethally irradiated A549 cells or primary lung tumor cells derived from surgical resection of non-small cell carcinoma (NSCLC), efficiently ingested the tumor cells as determined by flow cytometry analysis and confocal microscopic examination. More importantly, DC loaded with irradiated A549-M1 cells efficiently processed and presented tumor cell-derived M1 antigen to T cells and elicited antigen-specific immune responses that included IFNgamma release from an M1-specific T-cell line, expansion of M1 peptide-specific Vbeta17+ and CD8+ peripheral T cells and generation of M1-specific cytotoxic T lymphocytes (CTL). We also compared DC loaded with irradiated tumor cells to those loaded with tumor cell lysate or killed tumor cells and found that irradiated lung tumor cells as a source of tumor antigen for DC loading is superior to tumor cell lysate or killed tumor cells in efficient induction of antigen-specific T-cell responses. Our results demonstrate the feasibility of using lung tumor cell-loaded DC to induce immune responses against lung cancer-associated antigens and support ongoing efforts to develop a DC-based lung cancer vaccine.     

Reduced L-selectin (CD62LLow) 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 (CD62LLow) or high levels of CD62L (CD62LHigh). Effector T cells generated from CD62LLow 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 CD62LLow or CD62LHigh TVDLN were polarized towards a dominant type 1 or type 2 cytokine profile, respectively. For CD62LLow 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.     

Adoptive Transfer of siRNA Cblb-Silenced CD8(+) T Lymphocytes Augments Tumor Vaccine Efficacy in a B16 Melanoma Model

The ubiquitin ligase Cbl-b is an established regulator of T cell immune response thresholds. We recently showed that adoptive cell transfer (ACT) of cblb(-/-) CD8(+) T cells enhances dendritic cell (DC) immunization-mediated anti-tumor effects in immune-competent recipients. However, translation of cblb targeting to clinically applicable concepts requires that inhibition of cblb activity be transient and reversible. Here we provide experimental evidence that inhibition of cblb using chemically synthesized siRNA has such potential. Silencing cblb expression by ex vivo siRNA transfection of polyclonal CD8(+) T cells prior to ACT increased T cell tumor infiltration, significantly delayed tumor outgrowth, and increased survival rates of tumor-bearing mice. As shown by ex vivo recall assays, cblb silencing resulted in significant augmentation of intratumoral T cell cytokine response. ACT of cblb-silenced polyclonal CD8(+) T cells combined with DC-based tumor vaccines predominantly mediated anti-tumor immune responses, whereas no signs of autoimmunity could be detected. Importantly, CBLB silencing in human CD8(+) T cells mirrored the effects observed for cblb-silenced and cblb-deficient murine T cells. Our data validate the concept of enhanced anti-tumor immunity by repetitive ACT of ex vivo cblb siRNA-silenced hyper-reactive CD8(+) T cells as add-on adjuvant therapy to augment the efficacy of existing cancer immunotherapy regimens in clinical practice.     

In vitro generated anti-tumor T lymphocytes exhibit distinct subsets mimicking in vivo antigen-experienced cells

The T-lymphocyte pool can be subdivided into naïve (Tn), effector memory (Tem), and central memory (Tcm) T cells. In this study, we characterized in vitro short-term cultured anti-tumor human T lymphocytes generated by lentiviral transduction with an anti-tumor antigen TCR vector. Within 2 weeks of in vitro culture, the cultured T cells showed a Tcm-like phenotype illustrated by a high percentage of CD62L and CD45RO cells. When the cells were sorted into populations that were CD45RO+/CD62L-(Tem), CD45RO+/CD62L+(Tcm), or CD45RO^low/CD62L+(Tn) and co-cultured with antigen-matched tumor lines, the magnitude of cytokine release from these populations for IFNγ (Tn < Tcm < Tem) and IL-2 (Tn > Tcm > Tem) mimicked the types of immune cell responses observed in vivo. In comparing cell-mediated effector function, Tn were found to be deficient (relative to Tcm and Tem) in the ability to form conjugates with tumor cells and subsequent lytic activity. Moreover, analysis of the gene expression profiles of the in vitro cultured and sorted T-cell populations also demonstrated patterns consistent with their in vivo counterparts. When Tcm and Tem were tested for the ability to survive in vivo, Tcm displayed significantly increased engraftment and persistence in NOD/SCID/γc^?/? mice. In general, a large percentage of in vitro generated anti-tumor T lymphocytes mimic a Tcm-like phenotype (based on phenotype, effector function, and increased persistence in vivo), which suggests that these Tcm-like cultured T cells may be optimal for adoptive immunotherapy.     

Naive, effector, and memory CD8 T cells in protection against pulmonary influenza virus infection: homing properties rather than initial frequencies are crucial.

The goal of adoptive immunotherapy is to target a high number of persisting effector cells to the site of a virus infection or tumor. In this study, we compared the protective value of hemagglutinin peptide-specific CD8 T cells generated from the clone-4 TCR-transgenic mice, defined by different stages of their differentiation, against lethal pulmonary influenza infection. We show that the adoptive transfer of high numbers of Ag-specific unprimed, naive CD8 T cells failed to clear the pulmonary virus titer and to promote host survival. The same numbers of in vitro generated primary Ag-specific Tc1 effector cells, producing high amounts of IFN-gamma, or resting Tc1 memory cells, generated from these effectors, were protective. Highly activated CD62Llow Tc1 effectors accumulated in the lung with rapid kinetics and most efficiently reduced the pulmonary viral titer early during infection. The resting CD62Lhigh naive and memory populations first increased in cell numbers in the draining lymph nodes. Subsequently, memory cells accumulated more rapidly and to a greater extent in the lung lavage as compared with naive cells. Thus, effector cells are most effective against a localized virus infection, which correlates with their ability to rapidly distribute at the infected tissue site. The finding that similar numbers of naive Ag-specific CD8 T cells are not protective supports the view that qualitative differences between the two resting populations, the naive and the memory population, may play a major role in their protective value against disease.     

Low molecular weight hyaluronan preconditioning of tumor-pulsed dendritic cells increases their migratory ability and induces immunity against murine colorectal carcinoma

We have recently shown that systemic administration of low molecular weight hyaluronan (LMW HA) significantly reduces colorectal carcinoma (CRC) growth in vivo. The elicited response is partially mediated by activated dendritic cells (DC). To potentiate the ability of DC loaded with whole tumor lysate (DC/TL) to induce immunity against CRC in mice, we aimed to study the effects of preconditioning DC with LMW HA for therapeutic vaccination. LMW HA improved maturation of ex vivo generated DC, increased IL-12, decreased IL-10 production, and enhanced a MLR activity in vitro. Although TNF-α showed a similar capacity to mature DC, preconditioning of DC/TL with LMW HA increased their ability to migrate in vitro toward CCL19 and CCL-21 in a CD44- and a TLR4-independent manner; this effect was superior to Poly(I:C), LPS, or TNF-α and partially associated with an increase in the expression of CCR7. Importantly, LMW HA dramatically enhanced the in vivo DC recruitment to tumor-regional lymph nodes. When these LMW HA-treated CRC tumor lysate-pulsed DC (DC/TL/LMW HA) were administered to tumor-bearing mice, a potent antitumor response was observed when compared to DC pulsed with tumor lysate alone and matured with TNF-α. Then, we showed that splenocytes isolated from animals treated with DC/TL/LMW HA presented a higher proliferative capacity, increased IFN-γ production, and secreted lower levels of the immunosuppressive IL-10. Besides, increased specific CTL response was observed in DC/TL/LMW HA-treated animals and induced long-term protection against tumor recurrence. Our data show that LMW HA is superior to other agents at inducing DC migration; therefore, LMW HA could be considered a new adjuvant candidate in the preparation of DC-based anticancer vaccines with potent immunostimulatory properties.     

Reinforcement of cancer immunotherapy by adoptive transfer of cblb-deficient CD8+ T cells combined with a DC vaccine

The success of cancer immunotherapy is limited by potent endogenous immune-evasion mechanisms, which are at least in part mediated by transforming growth factor-β (TGF-β). The E3 ubiquitin ligase Cbl-b is a key regulator of T cell activation and is established to regulate TGF-β sensitivity. cblb-deficient animals reject tumors via CD8(+) T cells, which make Cbl-b an ideal target for improvement of adoptive T-cell transfer (ATC) therapy. In this study, we show that cblb-deficient CD8(+) T cells are hyper-responsive to T-cell receptor (TCR)/CD28-stimulation and are in part protected against the negative cues induced by TGF-β in vitro. Notably, adoptive transfer of polyclonal, non-TCR transgenic cblb-deficient CD8(+) T cells is not sufficient to reject B16-ova or EG7 tumors in vivo. Thus, cblb-deficient ATC requires proper in vivo re-activation by a dendritic cell (DC) vaccine. In strict contrast to ATC monotherapy, this approach delayed tumor outgrowth and significantly increased survival rates, which is paralleled by increased CD8(+) T-cells infiltration to the tumor site and enrichment of ova-specific and interferon-γ (IFN-γ)-secreting CD8(+) T cell in the draining lymph node (LN). Moreover, CD8(+) T cells from cblb-deficient mice vaccinated with the DC vaccine show increased cytolytic activity in vivo. In summary, our data using cblb-deficient polyclonal, non-TCR-transgenic adoptively transferred CD8(+) T cells into immuno-competent non-lymphodepleted recipients suggest that targeting Cbl-b might serve as a novel 'adjuvant approach', suitable to augment the effectiveness of established anti-cancer immunotherapies.     

GMP production of anti-tumor cytotoxic T-cell lines for adoptive T-cell therapy in patients with solid neoplasia

BACKGROUND: The adoptive transfer of ex vivo-induced tumor-specific T-cell lines provides a promising approach for cancer immunotherapy. We have demonstrated previously the feasibility of inducing in vitro long-term anti-tumor cytotoxic T-cell (CTL) lines directed against different types of solid tumors derived from both autologous and allogeneic PBMC. We have now investigated the possibility of producing large amounts of autologous anti-tumor CTL, in compliance with good manufacturing practices, for in vivo use. METHODS: Four patients with advanced solid tumors (two sarcoma, one renal cell cancer and one ovarian cancer), who had received several lines of anticancer therapy, were enrolled. For anti-tumor CTL induction, patient-derived CD8-enriched PBMC were stimulated with DC pulsed with apoptotic autologous tumor cells (TC) as the source of tumor Ag. CTL were then restimulated in the presence of TC and expanded in an Ag-independent way. RESULTS: Large amounts of anti-tumor CTL (range 14-20 x 10(9)), which displayed high levels of cytotoxic activity against autologous TC, were obtained in all patients by means of two-three rounds of tumor-specific stimulation and two rounds of Ag-independent expansion, even when a very low number of viable TC was available. More than 90% of effector cells were CD3(+) CD8(+) T cells, while CD4(+) T lymphocytes and/or NK cells were less than 10%. DISCUSSION: Our results demonstrate the feasibility of obtaining large quantities of anti-tumor specific CTL suitable for adoptive immunotherapy approaches.     

Dendritic cell immunotherapy combined with gemcitabine chemotherapy enhances survival in a murine model of pancreatic carcinoma

Pancreatic cancer is an extremely aggressive malignancy with a dismal prognosis. Cancer patients and tumor-bearing mice have multiple immunoregulatory subsets including regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSC) that may limit the effectiveness of anti-tumor immunotherapies for pancreatic cancer. It is possible that modulating these subsets will enhance anti-tumor immunity. The goal of this study was to explore depletion of immunoregulatory cells to enhance dendritic cell (DC)-based cancer immunotherapy in a murine model of pancreatic cancer. Flow cytometry results showed an increase in both Tregs and MDSC in untreated pancreatic cancer–bearing mice compared with control. Elimination of Tregs alone or in combination with DC-based vaccination had no effect on pancreatic tumor growth or survival. Gemcitabine (Gem) is a chemotherapeutic drug routinely used for the treatment for pancreatic cancer patients. Treatment with Gem led to a significant decrease in MDSC percentages in the spleens of tumor-bearing mice, but did not enhance overall survival. However, combination therapy with DC vaccination followed by Gem treatment led to a significant delay in tumor growth and improved survival in pancreatic cancer–bearing mice. Increased MDSC were measured in the peripheral blood of patients with pancreatic cancer. Treatment with Gem also led to a decrease of this population in pancreatic cancer patients, suggesting that combination therapy with DC-based cancer vaccination and Gem may lead to improved treatments for patients with pancreatic cancer.     

Linkage of CD40L to a self-tumor antigen enhances the antitumor immune responses of dendritic cell-based treatment

CD40-CD40 ligand (CD40L) interaction is an important costimulatory signal in the interaction between T cells and antigen-presenting cells (APC). In the present study, we determined whether the linkage of CD40L to the tumor-specific idiotype (Id) derived from a murine B-cell lymphoma, 38C13, could enhance its immunogenicity when presented by dendritic cells (DC). We showed that bone marrow-derived DC pulsed with Id-CD40L upregulated the expression of CD40, CD80, CD86, and major histocompatibility complex (MHC) class II molecules with the increased production of interleukin-12 (IL-12). Mice immunized with DC loaded with Id-CD40L showed high levels of anti-Id antibody response of both IgG2a and IgG1 isotypes. In addition, nylon wool-enriched T cells from these immunized mice showed a tumor-specific T-cell proliferative response upon stimulation with Id protein. Mice immunized with DC pulsed with Id alone failed to show any of these immune responses. Immunization with DC pulsed with Id-CD40L showed increased resistance to the challenge by 38C13 tumor, and tumor growth was significantly retarded. Together, these results show that linkage of CD40L to a self-tumor antigen enhances the anti-tumor immune response in DC-based treatment.     

Therapeutic immune response induced by electrofusion of dendritic and tumor cells

To elicit a therapeutic antitumor immune response, dendritic cells (DCs) have been employed as a cellular adjuvant. Among various DC-based approaches, fusion of DCs and tumor cells potentially confers not only DC functionality, but also a continuous source of unaltered tumor antigens. We have recently demonstrated successful generation of fusion hybrids by a large-scale electrofusion technique. The immunogenicity and therapeutic potential of fusion hybrids were further analyzed in a model system of a murine melanoma cell line expressing beta-galactosidase (beta-gal) as a surrogate tumor antigen. A single vaccination with fusion hybrids plus IL-12 induced a therapeutic immune response against 3-day established pulmonary metastases. This immunotherapy was beta-gal specific and involved both CD4 and CD8 T cells. In vitro, fusion hybrids stimulated specific IFN-gamma secretion from both CD4 and CD8 immune T cells. They also nonspecifically induced IL-10 secretion from CD4 but not CD8 T cells. Compared to other DC loadings, our results demonstrate the superior immunogenicity of fusion. The current technique of electrofusion is adequately developed for clinical use in cancer immunotherapy.     

Allogeneic tumor lysate can serve as both antigen source and protein supplementation for dendritic cell culture

BACKGROUND: Recent preclinical and clinical evidence suggests the use of allogeneic tumor as a source of antigen for DC-based immunotherapy against cancer. We hypothesized that addition of allogeneic tumor lysate to monocyte-derived DC culture could serve a dual purpose: (1) antigen source and (2) protein supplementation of DC culture media. Protein supplementation whether of known origin (human serum/plasma, fetal bovine serum, human serum albumin) or undeclared origin ("serum-free" media) is a source of variability and bias. We addressed the question whether protein supplementation can be omitted in the presence of allogeneic tumor lysate. MATERIALS AND METHODS: Human DC cultured in the presence of lysate from medullary thyroid carcinoma (MTC) cell line SHER-I (TuLy-DC) and DC pulsed with the same lysate but cultured in the presence of FBS (FBS-DC) were assessed for morphology, phenotype, maturation and functional properties. RESULTS: In comparison of FBS-DC/TuLy-DC no significant differences in morphology, phenotype and maturation could be detected. Both culture conditions produced CD1a(high), CD14(low) DC with high expression of costimulatory molecules and CD83 upon stimulation. TuLy-DC gave significantly better yields and produced more IL12p70. DC showed high (allo)stimulatory capacity toward T-cells. TuLy-DC induced more intracellular IFNgamma in CD8+T-cells of vaccinated MTC patients. Both types of DC induced killing of SHER-I after short in vitro restimulation. Tumor lysate from SHER-I can substitute for further protein supplementation in DC culture. Allogeneic tumor lysates should be taken into consideration as both source of antigen and protein supplementation in monocyte-derived DC culture.     

Secondary lymphoid tissue chemokine mediates T cell-dependent antitumor responses in vivo

Secondary lymphoid tissue chemokine (SLC, also referred to as Exodus 2 or 6Ckine) is a recently identified high endothelial-derived CC chemokine. The ability of SLC to chemoattract both Th1 lymphocytes and dendritic cells formed the rationale to evaluate this chemokine in cancer immunotherapy. Intratumoral injection of recombinant SLC evidenced potent antitumor responses and led to complete tumor eradication in 40% of treated mice. SLC-mediated antitumor responses were lymphocyte dependent as evidenced by the fact that this therapy did not alter tumor growth in SCID mice. Studies performed in CD4 and CD8 knockout mice also revealed a requirement for both CD4 and CD8 lymphocyte subsets for SLC-mediated tumor regression. In immunocompetent mice, intratumoral SLC injection led to a significant increase in CD4 and CD8 T lymphocytes and dendritic cells, infiltrating both the tumor and the draining lymph nodes. These cell infiltrates were accompanied by the enhanced elaboration of Th1 cytokines and chemokines monokine induced by IFN-gamma and IFN-gamma-inducible protein 10 but a concomitant decrease in immunosuppressive cytokines at the tumor site. In response to irradiated autologous tumor, splenic and lymph node-derived cells from SLC-treated tumor-bearing mice secreted significantly more IFN-gamma, GM-CSF, and IL-12 and reduced levels of IL-10 than did diluent-treated tumor-bearing mice. After stimulation with irradiated autologous tumor, lymph node-derived lymphocytes from SLC-treated tumor-bearing mice demonstrated enhanced cytolytic capacity, suggesting the generation of systemic immune responses. These findings provide a strong rationale for further evaluation of SLC in tumor immunity and its use in cancer immunotherapy.     

Isolation of immune cells from primary tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors. Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4(+) T helper cells via adoptive transfer, promotes CD8(+) T cells to maintain pro-inflammatory effector functions. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.     

Application of immunotherapy based on dendritic cells stimulated by tumor cell-derived exosomes in a syngeneic breast tumor mouse model

We here evaluated the therapeutic effect of tumor cell-derived exosomes (TEXs)-stimulated dendritic cells (DCs) in a syngeneic orthotopic breast tumor model. The DC line DC2.4 and breast cancer cell line E0771 originally isolated from C57BL/6 mice were used. E0771 cells stably expressing the exosomal CD63-RFP or luciferase (Luc) and DC2.4 cells stably expressing GFP were produced using lentivirus. TEXs were purified from conditioned medium of E0771/CD63-RFP cells. Breast tumor model was established by injecting E0771/Luc cells into mammary gland fat pad of mice. TEXs contained immune modulatory molecules such as HSP70, HSP90, MHC I, MHC II, TGF-β, and PD-L1. TEXs were easily taken by DC2.4 cells, resulting in a significant increase in the in vitro proliferation and migration abilities of DC2.4 cells, accompanied by the upregulation of CD40. TEX-DC-treated group exhibited a decreased tumor growth compared with control group. CD8⁺ cells were more abundant in the tumors and lymph nodes of TEX-DC-treated group than in those of control group, whereas many CD4⁺ or FOXP3+ cells were localized in those of control group. Our results suggest a potential application of TEX-DC-based cancer immunotherapy.     

The role of dendritic cells in the initiation of immune responses to contact sensitizers. II. Studies in nude mice

Twenty-four hours after skin painting nude mice with picryl chloride, there was an increase in the number of dendritic cells (DC) isolated from the draining lymph nodes. This increased inflow or retention of DC in lymph nodes following skin painting is therefore unlikely to depend on interaction of DC with T cells. The DC obtained initiated primary proliferative responses in vitro in lymph node cells from congenic euthymic mice. Contact sensitivity developed in congenic mice when they received footpad injections of 60,000 DC from the lymph nodes of nude mice skin sensitized 1 day previously with picryl chloride or oxazolone. The initiation of delayed hypersensitivity was therefore independent of T-cell contamination within the donor DC.     

Anti-tumor activity and trafficking of self, tumor-specific T cells against tumors located in the brain

It is commonly believed that T cells have difficulty reaching tumors located in the brain due to the presumed "immune privilege" of the central nervous system (CNS). Therefore, we studied the biodistribution and anti-tumor activity of adoptively transferred T cells specific for an endogenous tumor-associated antigen (TAA), gp100, expressed by tumors implanted in the brain. Mice with pre-established intracranial (i.c.) tumors underwent total body irradiation (TBI) to induce transient lymphopenia, followed by the adoptive transfer of gp100(25-33)-specific CD8+ T cells (Pmel-1). Pmel-1 cells were transduced to express the bioluminescent imaging (BLI) gene luciferase. Following adoptive transfer, recipient mice were vaccinated with hgp100(25-33) peptide-pulsed dendritic cells (hgp100(25-33)/DC) and systemic interleukin 2 (IL-2). This treatment regimen resulted in significant reduction in tumor size and extended survival. Imaging of T cell trafficking demonstrated early accumulation of transduced T cells in lymph nodes draining the hgp100(25-33)/DC vaccination sites, the spleen and the cervical lymph nodes draining the CNS tumor. Subsequently, transduced T cells accumulated in the bone marrow and brain tumor. BLI could also detect significant differences in the expansion of gp100-specific CD8+ T cells in the treatment group compared with mice that did not receive either DC vaccination or IL-2. These differences in BLI correlated with the differences seen both in survival and tumor infiltrating lymphocytes (TIL). These studies demonstrate that peripheral tolerance to endogenous TAA can be overcome to treat tumors in the brain and suggest a novel trafficking paradigm for the homing of tumor-specific T cells that target CNS tumors.