An unappreciated role for neutrophil-DC hybrids in immunity to invasive fungal infections.

Neutrophils are classically defined as terminally differentiated, short-lived cells; however, neutrophils can be long-lived with phenotypic plasticity. During inflammation, a subset of neutrophils transdifferentiate into a population called neutrophil-DC hybrids (PMN-DCs) having properties of both neutrophils and dendritic cells. While these cells ubiquitously appear during inflammation, the role of PMN-DCs in disease remains poorly understood. We observed the differentiation of PMN-DCs in pre-clinical murine models of fungal infection: blastomycosis, aspergillosis and candidiasis. Using reporter strains of fungal viability, we found that PMN-DCs associate with fungal cells and kill them more efficiently than undifferentiated canonical neutrophils. During pulmonary blastomycosis, PMN-DCs comprised less than 1% of leukocytes yet contributed up to 15% of the fungal killing. PMN-DCs displayed higher expression of pattern recognition receptors, greater phagocytosis, and heightened production of reactive oxygen species compared to canonical neutrophils. PMN-DCs also displayed prominent NETosis. To further study PMN-DC function, we exploited a granulocyte/macrophage progenitor (GMP) cell line, generated PMN-DCs to over 90% purity, and used them for adoptive transfer and antigen presentation studies. Adoptively transferred PMN-DCs from the GMP line enhanced protection against systemic infection in vivo. PMN-DCs pulsed with antigen activated fungal calnexin-specific transgenic T cells in vitro and in vivo, promoting the production of interferon-γ and interleukin-17 in these CD4+ T cells. Through direct fungal killing and induction of adaptive immunity, PMN-DCs are potent effectors of antifungal immunity and thereby represent innovative cell therapeutic targets in treating life-threatening fungal infections.

Th2/1 Hybrid Cells Occurring in Murine and Human Strongyloidiasis Share Effector Functions of Th1 Cells.

Infections by the soil-transmitted threadworm Strongyloides stercoralis affect 30-100 million people worldwide, predominantly in tropic and sub-tropic regions. Here we assessed the T helper cell phenotypes in threadworm-infected patients and experimental murine infections with focus on CD4+ T cells co-expressing markers of Th2 and Th1 differentiation. We show that mice infected with the close relative S. ratti generate strong Th2 responses characterized by the expansion of CD4+ GATA-3+ cells expressing IL-4/-5/-13 in blood, spleen, gut-draining lymph nodes, lung and gut tissue. In addition to conventional Th2 cells, significantly increased frequencies of GATA-3+T-bet+ Th2/1-hybrid cells were detected in all organs and co-expressed Th2- and Th1-cytokines at intermediate levels. Assessing the phenotype of blood-derived CD4+ T cells from South Indian patients infected with S. stercoralis and local uninfected control donors we found that GATA-3 expressing Th2 cells were significantly increased in the patient cohort, coinciding with elevated eosinophil and IgE/IgG4 levels. A fraction of IL-4+CD4+ T cells simultaneously expressed IFN-γ hence displaying a Th2/1 hybrid phenotype. In accordance with murine Th2/1 cells, human Th2/1 cells expressed intermediate levels of Th2 cytokines. Contrasting their murine counterparts, human Th2/1 hybrids were marked by high levels of IFN-γ and rather low GATA-3 expression. Assessing the effector function of murine Th2/1 cells in vitro we found that Th2/1 cells were qualified for driving the classical activation of macrophages. Furthermore, Th2/1 cells shared innate, cytokine-driven effector functions with Th1 cells. Hence, the key findings of our study are that T helper cells with combined characteristics of Th2 and Th1 cells are integral to immune responses of helminth-infected mice, but also occur in helminth-infected humans and we suggest that Th2/1 cells are poised for the instruction of balanced immune responses during nematode infections.

Dendritic-tumor cell hybrids induce tumor-specific immune responses more effectively than the simple mixture of dendritic and tumor cells.

BACKGROUND AIMS: Dendritic cell (DC)-tumor cell hybrids have been used clinically in cancer immunotherapy, but their advantage over the simple mixture of tumor cells and DCs is still a matter of controversy. In this study, we compared DC-tumor cell hybrids with the non-fused mixture of DC and tumor cells directly in their ability to induce a specific immune response. METHODS: Hybrids were obtained by electrofusion of tumor cells and monocyte-derived DCs. Cell phenotype was evaluated by flow cytometry and antigen-presenting ability by co-culture with syngeneic T cells followed by tetramer analysis and interferon (IFN)-γ ELISPOT. RESULTS: Less than half the cells in the mixture expressed DC co-stimulatory molecules. Furthermore, DCs in the mixture had significantly lower expression of MHC class I molecules than DCs in the fusion. Conversely, nearly all CD11c(+)Her2/neu(+) hybrids expressed CD80, CD86, CD83, HLA-DR and MHC class I from both tumor cells and DCs. Using tumor cells constitutively expressing a cytomegalovirus (CMV) antigen, we show that expansion of CMV-specific cytotoxic T lymphocytes (CTLs) restricted by DCs' MHC class I molecules was higher when DC-tumor hybrids were the stimulators. Furthermore, only hybrids stimulated CTLs to produce IFN-γ in response to CMV-positive target cells. CONCLUSIONS: These data show the superiority of DC-tumor cell hybrids over their simple mixture as T-cell stimulators. Hybrids expressed more co-stimulatory and MHC molecules, induced higher antigen-specific T-cell expansion and were the only cells able to induce IFN-γ-producing antigen-specific T cells. Thus, these data offer further support for cancer immunotherapeutic approaches using DC-tumor cell hybrids.

Chimeric cells of maternal origin do not appear to be pathogenic in the juvenile idiopathic inflammatory myopathies or muscular dystrophy.

INTRODUCTION: Microchimeric cells have been studied for over a decade, with conflicting reports on their presence and role in autoimmune and other inflammatory diseases. To determine whether microchimeric cells were pathogenic or mediating tissue repair in inflammatory myopathies, we phenotyped and quantified microchimeric cells in juvenile idiopathic inflammatory myopathies (JIIM), muscular dystrophy (MD), and noninflammatory control muscle tissues. METHOD: Fluorescence immunophenotyping for infiltrating cells with sequential fluorescence in situ hybridization was performed on muscle biopsies from ten patients with JIIM, nine with MD and ten controls. RESULTS: Microchimeric cells were significantly increased in MD muscle (0.079 ± 0.024 microchimeric cells/mm(2) tissue) compared to controls (0.019 ± 0.007 cells/mm(2) tissue, p = 0.01), but not elevated in JIIM muscle (0.043 ± 0.015 cells/mm(2)). Significantly more CD4+ and CD8+ microchimeric cells were in the muscle of patients with MD compared with controls (mean 0.053 ± 0.020/mm(2) versus 0 ± 0/mm(2) p = 0.003 and 0.043 ± 0.023/mm(2) versus 0 ± 0/mm(2) p = 0.025, respectively). No differences in microchimeric cells between JIIM, MD, and noninflammatory controls were found for CD3+, Class II+, CD25+, CD45RA+, and CD123+ phenotypes, and no microchimeric cells were detected in CD20, CD83, or CD45RO populations. The locations of microchimeric cells were similar in all three conditions, with MD muscle having more microchimeric cells in perimysial regions than controls, and JIIM having fewer microchimeric muscle nuclei than MD. Microchimeric inflammatory cells were found, in most cases, at significantly lower proportions than autologous cells of the same phenotype. CONCLUSIONS: Microchimeric cells are not specific to autoimmune disease, and may not be important in muscle inflammation or tissue repair in JIIM.

Cell fusion between dendritic cells and whole tumor cells.

We have developed cell fusion vaccines generated with dendritic cells (DCs) and whole tumor cells to induce antigen-specific antitumor immunity. This approach allows DCs to be exposed to the entire repertoire of tumor-associated antigens (TAAs) originally expressed by the tumor cell, to process them endogenously, and to present antigenic epitopes thought the MHC class I and class II pathways to activate both CD8+ and CD4+ T cells, respectively. The therapeutic efficacy of DC/tumor fusion cell vaccines requires the improved immunogenicity of both cells. Here, we describe the strategy to generate DC/tumor fusion cells.

Novel dendritic cell-based vaccination in late stage melanoma.

Dendritic cells (DCs) are professional antigen-presenting cells (APCs) that play an important role in stimulating an immune response of both CD4(+) T helper cells and CD8(+) cytotoxic T lymphocytes (CTLs). As such, DCs have been studied extensively in cancer immunotherapy for their capability to induce a specific anti-tumor response when loaded with tumor antigens. However, when the most relevant antigens of a tumor remain to be identified, alternative approaches are required. Formation of a dentritoma, a fused DC and tumor cells hybrid, is one strategy. Although initial studies of these hybrid cells are promising, several limitations interfere with its clinical and commercial application. Here we present early experience in clinical trials and an alternative approach to manufacturing this DC/tumor cell hybrid for use in the treatment of late stage and metastatic melanoma.

Induction of antigen-specific cytotoxic T lymphocytes by fusion cells generated from allogeneic plasmacytoid dendritic and tumor cells.

Previous work has demonstrated that fusion cells generated from autologous monocyte-derived dendritic cells (MoDCs) and whole tumor cells induce efficient antigen-specific cytotoxic T lymphocytes. A major limitation to the use of this strategy is the availability of adequate amounts of autologous tumor cells. Moreover, MoDCs from cancer patients are often defective in their antigen-processing and presentation machinery. In this study, two types of allogeneic cells, a leukemia plasmacytoid dendritic cell (pDC) line (PMDC05) and pancreatic cancer cell lines (PANC-1 or MIA PaCa-2), were fused instead of autologous MoDCs and tumor cells. We created four types of pDC/tumor fusion cells by alternating fusion partners and treating with lipopolysaccharide (LPS): i) PMDC05 fused with PANC-1 (pDC/PANC-1), ii) PMDC05 fused with MIA PaCa-2 (pDC/MIA PaCa-2), iii) LPS-stimulated pDC/PANC-1 (LPS-pDC/PANC-1) and iv) LPS-stimulated pDC/MIA PaCa-2 (LPS-pDC/MIA PaCa-2) and examined their antitumor immune responses. The LPS-pDC/tumor cell fusions were the most active, as demonstrated by their: i) upregulated expression of HLA-DR and CD86 on a per-fusion-cell basis, ii) increased production of IL-12p70, iii) generation of a higher percentage of IFN-γ-producing CD4⁺ and CD8⁺ T cells and iv) augmented induction of MUC1-specific CD8⁺ T cells that lyse target tumor cells. This study provides the first evidence for an in vitro induction of antigen-specific cytotoxic T lymphocytes by LPS-stimulated fusion cells generated from leukemia plasmacytoid DCs and tumor cells and suggests that this strategy has potential applicability to the field of adoptive immunotherapy.

Purified dendritic cell-tumor fusion hybrids supplemented with non-adherent dendritic cells fraction are superior activators of antitumor immunity.

BACKGROUND: Strong evidence supports the DC-tumor fusion hybrid vaccination strategy, but the best fusion product components to use remains controversial. Fusion products contain DC-tumor fusion hybrids, unfused DCs and unfused tumor cells. Various fractions have been used in previous studies, including purified hybrids, the adherent cell fraction or the whole fusion mixture. The extent to which the hybrids themselves or other components are responsible for antitumor immunity or which components should be used to maximize the antitumor immunity remains unknown. METHODS: Patient-derived breast tumor cells and DCs were electro-fused and purified. The antitumor immune responses induced by the purified hybrids and the other components were compared. RESULTS: Except for DC-tumor hybrids, the non-adherent cell fraction containing mainly unfused DCs also contributed a lot in antitumor immunity. Purified hybrids supplemented with the non-adherent cell population elicited the most powerful antitumor immune response. After irradiation and electro-fusion, tumor cells underwent necrosis, and the unfused DCs phagocytosed the necrotic tumor cells or tumor debris, which resulted in significant DC maturation. This may be the immunogenicity mechanism of the non-adherent unfused DCs fraction. CONCLUSIONS: The non-adherent cell fraction (containing mainly unfused DCs) from total DC/tumor fusion products had enhanced immunogenicity that resulted from apoptotic/necrotic tumor cell phagocytosis and increased DC maturation. Purified fusion hybrids supplemented with the non-adherent cell population enhanced the antitumor immune responses, avoiding unnecessary use of the tumor cell fraction, which has many drawbacks. Purified hybrids supplemented with the non-adherent cell fraction may represent a better approach to the DC-tumor fusion hybrid vaccination strategy.

Superior anti-tumor protection and therapeutic efficacy of vaccination with dendritic cell/tumor cell fusion hybrids for murine Lewis lung carcinoma.

BACKGROUND: The development of protocols for the ex vivo generation of dendritic cells (DCs) has led to intensive research into their potential use in immunotherapy in the treatment of cancer. In this study, we examined the efficacy of dendritic cell-tumor cell fusion hybrid vaccines in eliciting an immune response against Lewis lung carcinoma (LLC) cells, as compared to other types of tumor vaccines. In addition, we also tested whether the efficacy of the vaccines was affected by the route of administration. Four different tumor vaccines were compared: (1) HC (hybrid cell), consisting of DC/LLC hybrids; (2) DC+LLC (DCs pulsed with apoptotic LLCs); (3) DC without antigen loading/pulsing; (4) LLC (apoptotic/irradiated tumor cells). We also compared four different routes of administration for each vaccine: (1) Preimmunization; (2) Vaccination therapy; (3) Adoptive immunotherapy; (4) Vaccination therapy combined with adoptive immunotherapy. Anti-tumor immunity was assessed in vivo and the CTL (cytotoxic T lymphocyte) response as well as the expression of key cytokines, IFN-γ and IL-10 were further evaluated using in vitro assays. RESULTS: Our data demonstrate that vaccination with HC hybrids provides more effective anti-tumor protective immunity and significantly greater therapeutic immunity than vaccination with DC+LLC, DC or LLC. Most remarkably, vaccination therapy with HC hybrids was more successful than combination (vaccination + adoptive) therapy for the induction of anti-tumor responses. Splenocytes harvested from mice immunized with HC hybrids demonstrated the greatest cytotoxic T lymphocyte (CTL) activity and their production of IFN-γ was high, while their production of IL-10 was very low. CONCLUSIONS: Our results suggest that vaccination therapy with DC-tumor cell fusion hybrids provides more effective protection against lung cancer.

Specific mtDNA mutations in mouse carcinoma cells suppress their tumor formation via activation of the host innate immune system.

In mammalian species, mitochondrial DNA (mtDNA) with pathogenic mutations that induce mitochondrial respiration defects has been proposed to be involved in tumor phenotypes via induction of enhanced glycolysis under normoxic conditions (the Warburg effects). However, because both nuclear DNA and mtDNA control mitochondrial respiratory function, it is difficult to exclude the possible contribution of nuclear DNA mutations to mitochondrial respiration defects and the resultant expression of tumor phenotypes. Therefore, it is important to generate transmitochondrial cybrids sharing the same nuclear DNA background but carrying mtDNA with and without the mutations by using intercellular mtDNA transfer technology. Our previous studies isolated transmitochondrial cybrids and showed that specific mtDNA mutations enhanced tumor progression as a consequence of overproduction of reactive oxygen species (ROS). This study assessed whether mtDNA mutations inducing ROS overproduction always enhance tumor progression. We introduced mtDNA from senescence-accelerated mice P1 (SAMP1) into C57BL/6J (B6) mice-derived Lewis lung carcinoma P29 cells, and isolated new transmitochondrial cybrids (P29mtSAMP1 cybrids) that overproduced ROS. The inoculation of the cybrids into B6 mice unexpectedly showed that mtDNA from SAMP1 mice conversely induced tumor suppression. Moreover, the tumor suppression of P29mtSAMP1 cybrids in B6 mice occurred as a consequence of innate immune responses of the host B6 mice. Enzyme pretreatment experiments of P29mtSAMP1 cybrids revealed that some peptides encoded by mtDNA and expressed on the cell surface of P29mtSAMP1 cybrids induce increased IL-6 production from innate immune cells (dendritic cells) of B6 mice, and mediate augmented inflammatory responses around the tumor-inoculated environment. These observations indicate presence of a novel role of mtDNA in tumor phenotype, and provide new insights into the fields of mitochondrial tumor biology and tumor immunology.

Antigen presenting cell/ tumor cell fusion vaccines for cancer immunotherapy.

Fusions of antigen presenting cells and tumor cells have been investigated in animal models and phase I/II clinical trials as candidate cancer vaccines. In animal studies there have been numerous reports of induction of protective immunity against a wide range of tumor types. Results of clinical trials have been less dramatic, but tumor-specific immune responses have been reported in many patients, with clinical responses to the vaccination in a subset. In this commentary article, I review the current status of antigen presenting cell/tumor cell fusion vaccines for cancer immunotherapy.

Utilização da vacina Escherichia coli J5 na imunização de vacas leiteiras contra mastites causadas por E. coli / Use of an Escherichia coli J5 vaccine on immunization of dairy cows against mastitis caused by E. coli

Avaliou-se a utilização da vacina Escherichia coli J5, na imunização de vacas leiteiras, para prevenção e controle da mastite causada por E. coli através da análise da prevalência das infecções intramamárias (IMM) no pós-parto, ocorrência e intensidade dos casos clínicos de mastite nos primeiros 100 dias de lactação, influência na contagem de células somáticas (CCS) e produção de leite. O grupo experimental foi composto de 187 animais, divididos em 2 grupos, vacas vacinadas e não vacinadas. As imunizações ocorreram 60 dias antes do parto, 30 dias antes do parto e na primeira semana pós-parto. No dia da secagem e sete dias após o parto foram coletadas amostras para diagnóstico microbiológico dos patógenos causadores de mastite. A ocorrência de casos clínicos foi verificada pelo teste da caneca durante as ordenhas sendo registrados os dados relacionados à intensidade. Amostras foram coletadas mensalmente, a partir do décimo dia de lactação, para avaliação da CCS. A produção de leite foi registrada mensalmente nos primeiros 100 dias de lactação. Verificou-se no grupo vacinado, redução na prevalência de E. coli no pós-parto, na ocorrência de casos clínicos por E.coli nos primeiros 100 dias de lactação bem como na intensidade destes casos clínicos. Não foram observadas diferenças estatísticas significativas na CCS, entretanto vacas vacinadas apresentaram maior produção de leite, comparadas às vacas não vacinadas. A vacinação com E. coli J5 foi eficaz em reduzir a prevalência de infecções intramamárias (IMM) ao parto, ocorrência e intensidade dos casos clínicos e aumento na produção de leite nos primeiros 100 dias de lactação.

We evaluated the use of Escherichia coli J5 vaccine immunization in dairy cows for the prevention and control of mastitis caused by E. coli. Were analyzed the prevalence of postpartum intramammary infections (IMM), occurrence and severity of clinical cases of mastitis in the first 100 days of lactation, influence on somatic cell count (SCC) and milk production. The experimental group consisted of 187 animals that were divided into two groups, vaccinated and unvaccinated cows. Immunizations occurred 60 days before calving, 30 days before calving and the first week postpartum. On the day of drying and seven days after birth, samples were collected for microbiological diagnosis of mastitis pathogens. The occurrence of clinical cases was verified by testing during the milking and data was recorded related to the intensity and duration. Samples were collected monthly starting at day ten of lactation to evaluate the CCS. Milk production was recorded monthly for the first 100 days of lactation. It was found in the vaccinated group reduction in the prevalence of E. coli in the postpartum period, the occurrence of clinical cases of E. coli in the first 100 days of lactation as well the intensity of these clinical cases. There were no statistically significant differences in CCS, however vaccinated cows had higher milk production, compared to unvaccinated cows. Vaccination with E. coli J5 was effective in reducing the prevalence of intramammary infections (IMM) at calving, occurrence and intensity of clinical cases and an increase in milk production in the first 100 days of lactation.

Long-lived fusions of human haematological tumour cells and B-lymphoblastoid cells induce tumour antigen-specific cytotoxic T-cell responses in vitro.

Tumour-specific cytotoxic T-cells (CTL) are important anti-cancer immune effectors. Most tumour cells, however, do not stimulate effective anti-tumour immune responses, in vivo or in vitro. To enhance tumour cell immunogenicity, we fused human tumour cells from haematological malignancies with the B-lymphoblastoid cell line (LCL), HMy2, to generate a panel of long-lived, self replicating LCL/tumour hybrid cell lines. The LCL/tumour hybrid cell lines expressed HLA class I and class II molecules, CD80 and CD86, and a range of known tumour associated antigens (TAAs). In vitro stimulation of PBLs from healthy, HLA-A2+ individuals by hybrid cell lines induced tumour antigen-specific CTLs to TAAs, including survivin, MAGE-A1, NY-ESO-1 and WT-1. Individual hybrid cell lines simultaneously induced CTL to multiple TAAs. In vitro stimulation of PBL from 2 patients with acute myeloid leukaemia by autologous LCL/tumour hybrid cell lines induced CTL capable of killing the patient's own tumour cells. Our data show, for the first time, that hybrid cell lines formed by fusion of HMy2 cells and haematological tumour cells induce tumour- and tumour antigen-specific cytotoxic T-cell responses in vitro. Hybrid cell lines such as those described may represent novel reagents for use in the immunotherapy of haematological malignancies.

HER-2/neu tolerant and non-tolerant mice for fine assessment of antimetastatic potency of dendritic cell-tumor cell hybrid vaccines.

Main obstacles to cancer vaccine efficacy are pre-existing antigenic load and immunoescape mechanisms, including tolerance against self tumor-associated antigens. Here we explored the role of tolerance in an antimetastatic vaccine approach based on dendritic cell-tumor cell (DC-TC) hybrids, thanks to the comparison between BALB-neuT mice, transgenic for and tolerant to rat HER-2/neu, with their non-tolerant strain of origin BALB/c. Allogeneic DC-TC hybrid vaccine displayed a high antimetastatic activity in non-tolerant mice, but was far less effective in tolerant mice, even with intensified vaccine schedule. Tolerant BALB-neuT mice revealed a reduced ability to mount polarized Th1 responses. A further attempt to increase the antimetastatic activity by using LPS-matured DC hybrids failed. Allogeneic LPS-matured DC-TC hybrids induced high IFN-γ levels, but concomitantly also the highest production of IL-4 and IL-10 suggesting activation of mechanisms sustaining regulatory cells able to blunt vaccine efficacy. Our data in tolerant versus non-tolerant hosts suggest that clinical translation of effective DC-based strategies could benefit from more extensive investigations in tolerant transgenic models.

Immune response to cancer and its regulation in regional lymph nodes.

Regional draining lymph nodes (LNs) play a pivotal role in initiating immune responses. However, the presence of metastases may compromise their normal immunological function. Preclinical studies indicate that despite metastases, early tumor-draining LNs are still a rich source of sensitized T cells. Recently, we found that dendritic (DC)-tumor fusion hybrids were capable of stimulating therapeutic T-cell generation in the LN. However, this response is regulated by a tumor-specific suppression mechanism(s). Reversal of these dysfunctions would help the success of immunotherapy.

Immunopathogenic behaviors of canine transmissible venereal tumor in dogs following an immunotherapy using dendritic/tumor cell hybrid.

Canine transmissible venereal tumor (CTVT) is a naturally occurring tumor that can be transmitted between dogs via live tumor cell inoculation. It is also a spontaneous self-regression tumor and its behavior is closely related to host immune responses. Since CTVT had been widely used for tumor models in canine cancers, whether this self-regression may overtake the immunity elicited from an exogenous tumor vaccine remains unclear and certainly worthwhile to be investigated. In this study, we used DCs/tumor hybrids as a tumor vaccine to evaluate the CTVT model. We prepared mature allogeneic dendritic cells from bone marrow and then assessed their phenotype (CD80, CD83, CD86, CD1a, CD11c, CD40 and MHC II), antigen uptake and presenting abilities. Fused dendritic cell/CTVT hybrids were then used as a vaccine, administered three times at two-week intervals via subcutaneous injection near the bilateral auxiliary and inguinal lymph nodes. In comparison with unvaccinated dogs (spontaneous regressed group), within a period of 2.5 months, the vaccinations substantially inhibited tumor progression (p<0.05) and accelerated the rate of regression by a mechanism involving amplification of the host tumor-specific adaptive immune responses and NK cytotoxicity (p<0.001). Pathologic examination revealed early massive lymphocyte infiltration resulting in final tumor necrosis. In addition, there are not any detectable effects on routine physical, body temperature or blood chemistry examinations. In conclusion, our data furnishes a reference value showing that CTVT is a model of potential use for the study of immunity elicited by vaccines against tumors, and also enable early-phase evaluation of the dendritic cell/tumor vaccine in terms of raising host immunity.

Dendritic cell/tumor hybrids enhances therapeutic efficacy against colorectal cancer liver metastasis in SCID mice.

OBJECTIVE: Colorectal cancer (CRC) is one of the most common malignancies in the western world. More than 60% among patients will develop liver metastases. Although surgical resection is the first choice worldwide, at this point an effective approach for the treatment of patients with liver metastasis and cancer recurrence postoperation has not yet been found. The aim of this study is to investigate the role of the allogeneic dendritomas from fusion of DCs and metastatic colon cancer cells in the activation of anti-tumor immunity against colorectal cancer liver metastases. MATERIAL AND METHODS: Hybrids were generated by fused allogeneic human peripheral blood dendritic cells with metastatic colon cancer SW620 cells using 50% polyethylene glycol (PEG). Induction of immune responses was assessed by ex vivo ELISPOT assays. A murine model of CRC liver metastasis was used by intrasplenic injection. The validity of the vaccine was observed by Vaccination CRC liver metastasis murine model with DC/tumor hybrids. RESULTS: The hybrids highly express the major molecules of DCs and tumor cells. The number of hybrids pulsed CTL secreting IFN-gamma was significantly higher when compared to the DC controls (p < 0.01). In a therapeutic setting mice vaccinated with in vitro cultured hybrids produced strong cellular immune responses and significant inhibition of tumor growth, compared to sham vaccinated controls. CONCLUSIONS: Vaccination with hybrids can induces strong cellular responses and significant protection from challenge in SCID mouse metastatic CRC model.

Dendritic/pancreatic carcinoma fusions for clinical use: Comparative functional analysis of healthy- versus patient-derived fusions.

Fetal calf serum (FCS)-independent pancreatic cancer cells were established in plasma protein fraction (PPF)-supplemented medium that is an agent of good manufacturing practice (GMP) grade. Dendritic cells (DCs) were activated with the Toll-like receptor agonist, penicillin-inactivated Streptococcus pyogenes (OK-432) that is also a GMP grade agent. Therefore, sufficient amounts of FCS-independent fusions were successfully generated with decreased potential hazards of FCS. The FCS-independent fusions expressed tumor-associated antigens, HLA-DR, costimulatory molecules, IL-12, and IL-10. Stimulation of T cells with fusions from healthy donors resulted in proliferation of T cells with high expression levels of perforin/granzyme B and IFN-gamma and efficient induction of antigen-specific cytotoxic T lymphocytes (CTLs). Selection and expansion of T-cell clones were confirmed by TCR Vbeta analysis. However, fusions from patients with metastatic pancreatic cancer induced increased expression levels of TGF-beta1 in CD4+ CD25high T cells and low levels of CTLs with decreased IFN-gamma production.

Generation of highly pure fusions of colorectal carcinoma and antigen-presenting cells.

PURPOSE: The induction of potent T cell responses against tumors is the goal of tumor immunotherapy. One approach is the fusion of antigen-presenting cells (APCs) with tumor cells. Hybrid cells combine the antigenicity of tumors with the immunostimulatory capacity of APCs. However, contaminating unfused cells present in the fusion reaction may prevent the induction of antitumoral immune responses. Here, we present a simple and effective protocol to substantially elevate the purity of hybrid cells. METHODS: Colorectal tumor cell lines and CD40-activated B cells as APCs were fused using polyethylene glycol. Important parameters including cell numbers, concentrations, and handling and detection procedures were optimized. Combination of these optimized fusion conditions with both magnetic cell sorting and selective adherence delivered very pure preparations of APC/tumor cell hybrids. The T cell stimulatory capacity of these hybrids was tested using ELISpot. RESULTS: The optimization of the fusion resulted in maximal fusion efficiencies of 31.6% (n = 10, range 13.5-46.6%). Prelabeling of APCs with magnetic beads allowed for easy elimination of up to 94.3% of unfused tumor cells from the cell mixture by magnetic separation. Hybrid cell capacity to firmly adhere to plastic was then used to remove unfused B cells from the remaining cell mixture by simple washing. The obtained 85.0% pure hybrids cells readily induced antitumoral T cell responses. CONCLUSIONS: Our protocol delivers pure hybrid cell preparations with strong immunostimulatory potential. In subsequent experiments, the ability of hybrid cells to stimulate specific antitumoral T cell responses must be tested in vivo.