Dendritic cell-based combined immunotherapy with autologous tumor-pulsed dendritic cell vaccine and activated T cells for cancer patients: rationale, current progress, and perspectives

Effective adoptive cancer immunotherapy depends on an ability to generate tumor-antigen-presenting cells and tumor-reactive effector lymphocytes and to deliver these effector cells to the tumor. Dendritic cells (DCs) are the most potent antigen-presenting cells, capable of sensitizing T cells to new and recall antigens. Many studies have shown that tumors express unique proteins that can be loaded on DCs to trigger an immune response. The current experimental and clinical statuses of adoptive transfer of tumor antigen-pulsed DCs and vaccine-primed activated T cells are summarized herein. Clinical trials of antigen-pulsed DCs have been conducted in patients with various types of cancer, including non-Hodgkin lymphoma, multiple myeloma, prostate cancer, renal cell carcinoma, malignant melanoma, colorectal cancer, and non-small cell lung cancer. These studies have shown that antigen-loaded DC vaccination is safe and promising for the treatment of cancer. In addition, tumor vaccine-primed T cells have been shown to induce antitumor activity in vivo. Several clinical studies are being conducted on the use of vaccine-primed T cells such as tumor-drainage lymph node. It is reasonable to consider using both tumor antigen-pulsed DCs and vaccine-primed lymphocytes as adjuvants. We are now investigating the use of autologous whole tumor antigen-pulsed DCs and the DC vaccine-primed activated lymphocytes in patients with multiple metastasis of solid tumors.     

Immunotherapy with dendritic cells pulsed with tumor-derived gp96 against murine lung cancer is effective through immune response of CD8+ cytotoxic T lymphocytes and natural killer cells

Background and purpose Immunization with heat shock proteins, gp96, elicits specific protective immunity against parent tumors. However, it is marginally effective as a therapeutic tool against established tumors. In the present study, we evaluated the efficacy and mechanism of immunotherapy with bone marrow-derived dendritic cells (DCs) pulsed with tumor-derived gp96 against murine lung cancer. Methods Mice were transplanted subcutaneously with ovalbumin (OVA)-transfected Lewis Lung Cancer (LLC-OVA) cells and immunized with gp96 derived from LLC-OVA, DCs, or DCs pulsed with gp96 derived from LLC-OVA. Results The antitumor effect was significantly enhanced in the mice immunized with DCs pulsed with gp96 derived from LLC-OVA, compared to mice immunized with gp96 or DCs (P < 0.05). The antitumor effect was significantly dependent on natural killer (NK) cells and CD8⁺ cells and partially dependent on CD4⁺ cells. Analysis by laser confocal microscopy demonstrated that gp96 was shown on the cell surface at 15 min, and after 30 min internalized in the endosomes and not in the endoplasmic reticulum or lysosomes. OVA-specific⁺ CD8⁺ cells were more readily recruited into the draining lymph nodes and higher CD8⁺ cytotoxic T cell activity against LLC-OVA was observed in splenocytes from mice immunized with DCs pulsed with gp96 derived from LLC-OVA. Re-challenge of the surviving mice with LLC-OVA tumors after the initial tumor inoculation showed dramatic retardation in tumor growth. Conclusion In conclusion, immunotherapy of DCs pulsed with tumor-derived gp96 against murine lung cancer is effective through immune response of CD8⁺ cytotoxic T lymphocytes and NK cells.     

Uptake and presentation of malignant glioma tumor cell lysates by monocyte-derived dendritic cells

Malignant glioma of the CNS is a tumor with a very bad prognosis. Development of adjuvant immunotherapy is hampered by interindividual and intratumoral antigenic heterogeneity of gliomas. To evaluate feasibility of tumor vaccination with (autologous) tumor cells, we have studied uptake of tumor cell lysates by dendritic cells (DCs), and the T-cell stimulatory capacity of the loaded DCs. DCs are professional antigen-presenting cells, which have already been used as natural adjuvants to initiate immune responses in human cancer. An efficacious uptake of tumor cell proteins, followed by processing and presentation of tumor-associated antigens by the DCs, is indeed one of the prerequisites for a potent and specific stimulation of T lymphocytes. Human monocytes were differentiated in vitro to immature DCs, and these were loaded with FITC-labeled tumor cell proteins. Uptake of the tumor cell proteins and presentation of antigens in the context of both MHC class I and II could be demonstrated using FACS analysis and confocal microscopy. After further maturation, the loaded DCs had the capacity to induce specific T-cell cytotoxic activity against tumor cells. We conclude that DCs loaded with crude tumor lysate are efficacious antigen-presenting cells able to initiate a T-cell response against malignant glioma tumor cells.     

Downregulation of endogenous STAT3 augments tumoricidal activity of interleukin 15 activated dendritic cell against lymphoma and leukemia via TRAIL

Effector functions in tumor resistance by dendritic cells (DCs) are less well characterized. In this study, we describe that the murine DCs upon stimulation with recombinant IL-15 in vitro or in vivo, expresses TNF superfamily member TRAIL which mediates cytotoxicity and growth inhibition against a murine lymphoma called Dalton lymphoma (DL) via apoptosis. Presence of tumor lysate or intact tumor cells significantly reduces the DC mediated tumoricidal effect, possibly via masking and down-regulating TRAIL in DCs. The antitumor effect of DC derived TRAIL was further augmented by deactivation of STAT3 in tumor cells by cucurbitacin I, which makes it more susceptible to DC derived TRAIL Treatment of tumor cells with cucurbitacin I upregulates TRAIL receptor expression in addition to activation of caspases. Compared to naïve DCs, DCs from tumor bearing mice are significantly impaired in TRAIL expression and consequent antitumor functions against DL which was partially restored by activation with IL-15 or LPS. Priming with recombinant IL-15 prolongs the survival of tumor bearing mice treated with cucurbitacin I. Naïve peripheral blood DCs derived from chronic myeloid leukemia (CML) patients have significant impairment in expression of TRAIL and consequent tumoricidal properties against TRAIL sensitive lymphoma cell lines and primary tumor cells compared to normal control.     

Cytotoxic dendritic cells generated from cancer patients

Known for years as professional APCs, dendritic cells (DCs) are also endowed with tumoricidal activity. This dual role of DC as killers and messengers may have important implications for tumor immunotherapy. However, the tumoricidal activity of DCs has mainly been investigated in animal models. Cancer cells inhibit antitumor immune responses using numerous mechanisms, including the induction of immunosuppressive/ tolerogenic DCs that have lost their ability to present Ags in an immunogenic manner. In this study, we evaluated the possibility of generating tumor killer DCs from patients with advanced-stage cancers. We demonstrate that human monocyte-derived DCs are endowed with significant cytotoxic activity against tumor cells following activation with LPS. The mechanism of DC-mediated tumor cell killing primarily involves peroxynitrites. This observed cytotoxic activity is restricted to immature DCs. Additionally, after killing, these cytotoxic DCs are able to activate tumor Ag-specific T cells. These observations may open important new perspectives for the use of autologous cytotoxic DCs in cancer immunotherapy strategies.     

Comparative analysis of DC fused with allogeneic hepatocellular carcinoma cell line HepG2 and autologous tumor cells as potential cancer vaccines against hepatocellular carcinoma

Fusions of patient-derived dendritic cells (DCs) and autologous tumor cells induce T-cell responses against autologous tumors in animal models and human clinical trials. These fusion cells require patient-derived tumor cells, which are not, however, always available. Here we fused autologous DCs from patients with hepatocellular carcinoma (HCC) to an allogeneic HCC cell line (HepG2). These fusion cells co-expressed tumor-associated antigens (TAAs) and DC-derived costimulatory and MHC molecules. Both CD4⁺ and CD8⁺ T cells were activated by the fusion cells. Cytotoxic T lymphocytes (CTLs) induced by the fusion cells were able to kill autologous HCC by HLA-A2- and/or HLA-A24-restricted mechanisms. CTL activity against shared TAAs indicates that the presence of alloantigens does not prevent the development of CTLs with activity against autologous HCC cells. These fusion cells may have applications in anti-tumor immunotherapy through cross-priming against shared tumor antigens and may provide a platform for adoptive immunotherapy.     

Allogeneic dendritic cell vaccination against metastatic renal cell carcinoma with or without cyclophosphamide

In this phase I/II study, we evaluated the feasibility, safety and efficacy of allogeneic dendritic cells (DCs) with or without cyclophosphamide in the treatment of patients with metastatic renal cell carcinoma (RCC). Immunomagnetic beads were used to isolate CD14⁺ monocytes from healthy donor leukapheresis products, and CD83⁺ antigen-pulsed monocyte-derived DCs (moDCs) loaded with tumor lysate and keyhole limpet hemocyanin (KLH) were generated. Twelve patients were treated with allogeneic moDCs alone, while ten patients also received cyclophosphamide on days 4 and 3 prior to vaccination. Of the 22 patients enrolled, 20 received full treatment consisting of at least three vaccinations at monthly intervals. Two mixed responses with substantial tumor regression were observed. In 3 patients, disease stabilization occurred, in 13 patients disease progressed and 4 patients were lost to follow-up. Overall, immune responses against KLH and tumor lysate were weak or absent; however, the strongest increases in antigen-independent and KLH-specific responses were observed in the 2 patients with mixed responses. In addition, 1 of them showed a substantial increase in oncofetal antigen (OFA)-specific IFN- production. Importantly, the 2 mixed responders and 1 patient with stable disease belonged to the cyclophosphamide group. Median overall survival in the cyclophosphamide group was 23.2 and 20.3 months in the group that received allogeneic moDCs alone. Allogeneic immunotherapy with moDCs is feasible and well tolerated. However, the immunogenicity of allogeneic moDCs is clearly less pronounced than that of autologous moDC immunotherapy. Cyclophosphamide may have the capacity to augment DC-induced antitumor immunity.     

Heteroconjugate antibody-directed killing of autologous human renal carcinoma cells by in vitro-activated lymphocytes

Tumor cell lysis can be enhanced significantly in vitro when heteroconjugate (HC) antibodies (anti-CD3 x anti-tumor mAb) are used to specifically direct lymphocyte effector cells to the tumor cell target. In order to effectively utilize HC antibodies in an immunotherapy protocol, methods must be identified for the optimum expansion, activation, and retargeting of lymphocyte-effector populations from cancer patients. In this study, we have compared the proliferative responses of different normal and renal cell carcinoma (RCC) patient lymphocyte preparations (PBL, tumor-infiltrating lymphocytes) stimulated in vitro for periods up to 12 days with a variety of growth factor combinations (anti-CD3, rIL-2, rIL-4). These activated lymphocyte preparations were then tested in vitro for their ability to kill RCC tumor cells and tumor cell lines in the presence of HC preparations (anti-CD3 mAb covalently linked to mAb reactive to different RCC tumor-associated Ag). RCC patient PBL cultured with anti-CD3 plus rIL-2 for 12 days resulted in a 3- to 160-fold expansion of effector cells. These cells, as well as tumor infiltrating lymphocytes, when retargeted with appropriate HC antibodies were capable of mediating high levels of killing of autologous tumor cells. No constitutive autologous anti-tumor cell response was detected in the absence of added HC antibodies. Of the five anti-RCC mAb tested (A6H, K29, K20, UR07, and URO 3), HC containing URO 3 x anti-CD3 and K20 x anti-CD3 elicited the highest level of tumor cell lysis by the activated lymphocyte effector cells. Together these results demonstrate that HC antibodies may be a useful imunotherapeutic reagent for directing the killing of RCC tumor cells by autologous lymphocytes.     

Patient-derived renal cell carcinoma cells fused with allogeneic dendritic cells elicit anti-tumor activity: in vitro results and clinical responses

Renal cell carcinoma (RCC) has been shown to be susceptible to immunotherapeutic treatment strategies. In the present study, patient-derived tumor cells were fused with allogeneic dendritic cells (DC) to elicit anti-tumor activity against RCC. DC from HLA-A2+ healthy donors were fused with primary RCC cells from ten patients. Phenotype of fusion cells were characterized by flow cytometer and confocal microscopy. In vitro, T cell proliferation, IFN-γ secretion and cytotocic T lymphocytes (CTL) activity elicited by allogeneic DC/RCC fusion cells were assessed. Clinically, ten patients were vaccinated with allogeneic DC/RCC fusion vaccine. The adverse effects and toxicity were observed. The clinical response was evaluated by CT scans. After fusion, the created hybrids expressed both tumor associated antigen and DC-derived molecules and could stimulate the proliferation and IFN-γ secretion of T cells as well as elicit strong CTL activity against RCC cells in vitro. In vivo, no serious adverse effects, toxicity, or signs of autoimmune disease were observed after vaccination therapy. Percentage of T lymphocyte subsets in peripheral blood of patients was increased significantly. One of ten patients exhibited a partial response with regression of lung metastases. Six patients showed stable disease with stabilization of previously progressive disease (follow up 1.5 years). The PR and SD responses, exhibited by 7/10 patients who received the allogeneic DC/RCC fusion vaccine treatment, suggest that this approach is safe and can elicit immunological responses in a significant portion of patients with RCC. J. Zhou and D. Weng contributed equally.     

Eradication of metastatic renal cell carcinoma after adenovirus-encoded TNF-related apoptosis-inducing ligand (TRAIL)/CpG immunotherapy

Despite evidence that antitumor immunity can be protective against renal cell carcinoma (RCC), few patients respond objectively to immunotherapy and the disease is fatal once metastases develop. We asked to what extent combinatorial immunotherapy with Adenovirus-encoded murine TNF-related apoptosis-inducing ligand (Ad5mTRAIL) plus CpG oligonucleotide, given at the primary tumor site, would prove efficacious against metastatic murine RCC. To quantitate primary renal and metastatic tumor growth in mice, we developed a luciferase-expressing Renca cell line, and monitored tumor burdens via bioluminescent imaging. Orthotopic tumor challenge gave rise to aggressive primary tumors and lung metastases that were detectable by day 7. Intra-renal administration of Ad5mTRAIL+CpG on day 7 led to an influx of effector phenotype CD4 and CD8 T cells into the kidney by day 12 and regression of established primary renal tumors. Intra-renal immunotherapy also led to systemic immune responses characterized by splenomegaly, elevated serum IgG levels, increased CD4 and CD8 T cell infiltration into the lungs, and elimination of metastatic lung tumors. Tumor regression was primarily dependent upon CD8 T cells and resulted in prolonged survival of treated mice. Thus, local administration of Ad5mTRAIL+CpG at the primary tumor site can initiate CD8-dependent systemic immunity that is sufficient to cause regression of metastatic lung tumors. A similar approach may prove beneficial for patients with metastatic RCC.     

BCG preparations,cultured homogeneously dispersed or as a surface pellicle,elicit different immunopotentiating effects but have similar antitumor activity in a murine fibrosarcoma

Summary Four preparations of Bacillus Calmette-Guérin (BCG), cultured either homogeneously dispersed or as a surface pellicle, were compared with reference to their immunomodulating capacities and antitumor effects. The BCG preparations included two vaccines that originated from the same seed strain, but one had subsequently been produced in each way. The immunological assays included in vivo stimulation of lymph nodes in a mouse model, in vitro stimulation of murine spleen lymphocytes, and in vivo stimulation of macrophages in a Listeria monocytogenes clearance model in the mouse. The antitumor effect was determined in a non-immunogenic, non-metastasizing murine fibrosarcoma. The results indicated that vaccines produced as a homogeneous culture in general induced a higher lymphocyte stimulation both in vivo and in vitro. In the Listeria clearance model a markedly enhanced clearance was established with three of the four preparations, the phenomenon being related to the number of culturable particles administered. This difference was not attributed to the production method, but to other factors, including the actual composition of the vaccine.The results found in the immunological assays were not connected to the observed antitumor activities, as for each preparation a combination of route, dose, and time interval for tumor regression was found. Prophylactic administration of BCG had no effect at all; enhancement was observed after intratumoral administration of the two preparations prepared as surface pellicles.It is concluded that a protocol for the quality control of bacterial vaccines used for cancer immunotherapy should include both immunological assays and a range of different animal tumor models.Fellow of the Koningin Wilhelmina Fonds of the National Cancer League of the Netherlands The Abbreviations used are: BCG, Bacillus Calmette-Guérine; c.p., culturable particles; Con A, Concanavalin A; MEM, Minimum Essential Medium; FCS, fetal calf serum; cpm, counts per minute; IT, intratumoral     

Renal Cell Carcinoma-Infiltrating CD3low Vγ9Vδ1 T Cells Represent Potentially Novel Anti-Tumor Immune Players

Due to the highly immunogenic nature of renal cell carcinoma (RCC), the tumor microenvironment (TME) is enriched with various innate and adaptive immune subsets. In particular, gamma-delta (γδ) T cells can act as potent attractive mediators of adoptive cell transfer immunotherapy because of their unique properties such as non-reliance on major histocompatibility complex expression, their ability to infiltrate human tumors and recognize tumor antigens, relative insensitivity to immune checkpoint molecules, and broad tumor cytotoxicity. Therefore, it is now critical to better characterize human γδ T-cell subsets and their mechanisms in RCCs, especially the stage of differentiation. In this study, we aimed to identify γδ T cells that might have adaptive responses against RCC progression. We characterized γδ T cells in peripheral blood and tumor-infiltrating lymphocytes (TILs) in freshly resected tumor specimens from 20 RCC patients. Furthermore, we performed a gene set enrichment analysis on RNA-sequencing data from The Cancer Genome Atlas (TCGA) derived from normal kidneys and RCC tumors to ascertain the association between γδ T-cell infiltration and anti-cancer immune activity. Notably, RCC-infiltrating CD3^low Vγ9Vδ1 T cells with a terminally differentiated effector memory phenotype with up-regulated activation/exhaustion molecules were newly detected as predominant TILs, and the cytotoxic activity of these cells against RCC was confirmed in vitro. In an additional analysis of the TCGA RCC dataset, γδ T-cell enrichment scores correlated strongly with those for CTLs, Th1 cells, “exhausted” T cells, and M1 macrophages, suggesting active involvement of γδ T cells in anti-tumor rather than pro-tumor activity, and Vδ1 cells were more abundant than Vδ2 or Vδ3 cells in RCC tumor samples. Thus, we posit that Vγ9Vδ1 T cells may represent an excellent candidate for adoptive immunotherapy in RCC patients with a high risk of relapse after surgery.     

Aminobisphosphonate-pretreated dendritic cells trigger successful Vγ9Vδ2 T cell amplification for immunotherapy in advanced cancer patients

Hepatocellular carcinoma (HCC) and colorectal carcinoma with hepatic metastases (mCRC) are cancers with poor prognosis and limited therapeutic options. New approaches are needed and adoptive immunotherapy with Vγ9Vδ2 T lymphocytes represents an attractive strategy. Indeed, Vγ9Vδ2 T cells were shown to exhibit efficient lytic activity against various human tumor cell lines, and in vitro Vγ9Vδ2 T expansion protocol based on single phosphoantigen stimulation could be easily performed for healthy donors. However, a low proliferative response of Vγ9Vδ2 T cells was observed in about half of the cancer patients, leading to an important limitation in the development of Vγ9Vδ2 T cell-based immunotherapy. Here, for the first time in the context of cancer patients, Vγ9Vδ2 T cell expansions were performed by co-culturing peripheral blood mononuclear cell (PBMCs) with autologous dendritic cells (DCs) pretreated with aminobisphosphonate zoledronate. For patients not responding to the conventional culture protocol, co-culture of PBMC with zoledronate-pretreated DCs induced strong cell expansion and allowed reaching a minimal rate of purity of 70% of Vγ9Vδ2 T cells. The potent immunostimulatory activity of zoledronate-treated DCs was associated with higher amount of isopentenyl pyrophosphate (IPP) in the culture and was correlated with better ability to activate Vγ9Vδ2 T cells as measured by IFN-γ production. Moreover, we demonstrated that the cytotoxic level of Vγ9Vδ2 T cells against freshly autologous tumor cells isolated from patients could be significantly increased by pretreating the tumor cells with zoledronate. Thus, this method of generating Vγ9Vδ2 T cells leads eligible for Vγ9Vδ2 T cell adoptive immunotherapy the HCC and mCRC patients.     

Dendritic cells charged with apoptotic tumor cells induce long-lived protective CD4+ and CD8+ T cell immunity against B16 melanoma

Dendritic cells (DCs) are potent APCs and attractive vectors for cancer immunotherapy. Using the B16 melanoma, a poorly immunogenic experimental tumor that expresses low levels of MHC class I products, we investigated whether DCs loaded ex vivo with apoptotic tumor cells could elicit combined CD4(+) and CD8(+) T cell dependent, long term immunity following injection into mice. The bone marrow-derived DCs underwent maturation during overnight coculture with apoptotic melanoma cells. Following injection, DCs migrated to the draining lymph nodes comparably to control DCs at a level corresponding to approximately 0.5% of the injected inoculum. Mice vaccinated with tumor-loaded DCs were protected against an intracutaneous challenge with B16, with 80% of the mice remaining tumor-free 12 wk after challenge. CD4(+) and CD8(+) T cells were efficiently primed in vaccinated animals, as evidenced by IFN-gamma secretion after in vitro stimulation with DCs loaded with apoptotic B16 or DCs pulsed with the naturally expressed melanoma Ag, tyrosinase-related protein 2. In addition, B16 melanoma cells were recognized by immune CD8(+) T cells in vitro, and cytolytic activity against tyrosinase-related protein 2(180-188)-pulsed target cells was observed in vivo. When either CD4(+) or CD8(+) T cells were depleted at the time of challenge, the protection was completely abrogated. Mice receiving a tumor challenge 10 wk after vaccination were also protected, consistent with the induction of tumor-specific memory. Therefore, DCs loaded with cells undergoing apoptotic death can prime melanoma-specific helper and CTLs and provide long term protection against a poorly immunogenic tumor in mice.     

The duration of signaling through CD40 directs biological ability of dendritic cells to induce antitumor immunity

Although it has been demonstrated that the functions of dendritic cells (DCs), including Ag capture, Ag presentation, and migratory activity, change dynamically with their maturation, the most appropriate conditioning of DCs for anticancer immunotherapy is still unclear. The help signal is one of the most potent stimuli for DC maturation and is provided by the interaction of CD40 expressed on DCs with CD40 ligand on CD4(+) T cells. To elucidate the appropriate conditioning of DCs for anticancer immunotherapy, we examined the biological activity of DCs stimulated with immobilized anti-CD40 Ab. DCs stimulated for 3 h (3h-DCs) still showed an immature phenotype, but exhibited augmented migration toward secondary lymphoid tissues. Subcutaneous injection of 3h-DCs facilitated priming of T cells, which could mediate potent antitumor therapeutic efficacy, in draining lymph nodes and successfully induced protective immunity. In contrast, 24h-DCs showed a mature phenotype with good Ag presentation ability to induce cell killing by adoptively transferred CD8(+) T cells when injected at tumor sites; however, they showed no migratory activity and were unable to induce protective immunity when injected s.c. This is the first report that functionally distinct DCs, either for the priming phase or for the effector phase, could be obtained by conditioning with CD40 stimulation and that the duration of stimulation determines the biological outcome. The usage of DCs conditioned for the priming phase might provide significant advantages in anticancer immunotherapy.     

Natural killer (NK) and lymphokine-activated killer (LAK) cell functions from healthy dogs and 29 dogs with a variety of spontaneous neoplasms

To investigate natural killer (NK) and lymphokine-activated killer (LAK) cell functions from 10 healthy dogs and 29 dogs with a variety of spontaneous neoplasms, large granular lymphocytes (LGLs) from blood samples were separated by a 58.5% Percoll density gradient. LGLs were stimulated with a low dose of recombinant human interleukin 2 (rhIL-2) for 7 days. Cytotoxicity of effector cells against the susceptible CTAC cell line was measured before and after stimulation. Compared with those before stimulation, the percentage of LGLs after stimulation with rhIL-2 was found to be significantly increased (P<0.01) in both dogs with tumors and controls. However, the increase was significantly higher in control animals, indicating a defect in proliferation ability of NK cells in canine tumor patients. After stimulation with rhIL-2, lymphokine-activated killer (LAK) cell activity in dogs with tumors was significantly lower (P<0.01) when compared with controls. Reduced cytotoxicity of rhIL-2–activated NK cells in dogs with tumors seems to be attributable to the presence of a diminished proliferative capacity of NK cells and a decreased ability of LAK cells to lyse target cells. Further knowledge of the precise function of IL-2–activated NK cells in dogs with tumors may help to optimize new and therapeutically beneficial treatment strategies in canine and human cancer patients. Our findings suggest that the dog could also serve as a relevant large animal model for cancer immunotherapy with IL-2.     

Selection of an antibody library identifies a pathway to induce immunity by targeting CD36 on steady-state CD8 alpha+ dendritic cells

Improvement of the strategy to target tumor Ags to dendritic cells (DCs) for immunotherapy requires the identification of the most appropriate ligand/receptor pairing. We screened a library of Ab fragments on mouse DCs to isolate new potential Abs capable of inducing protective immune responses. The screening identified a high-affinity Ab against CD36, a multi-ligand scavenger receptor primarily expressed by the CD8alpha+ subset of conventional DCs. The Ab variable regions were genetically linked to the model Ag OVA and tested in Ag presentation assays in vitro and in vivo. Anti-CD36-OVA was capable of delivering exogenous Ags to the MHC class I and MHC class II processing pathways. In vivo, immunization with anti-CD36-OVA induced robust activation of naive CD4+ and CD8+ Ag-specific T lymphocytes and the differentiation of primed CD8+ T cells into long-term effector CTLs. Vaccination with anti-CD36-OVA elicited humoral and cell-mediated protection from the growth of an Ag-specific tumor. Notably, the relative efficacy of targeting CD11c/CD8alpha+ via CD36 or DEC205 was qualitatively different. Anti-DEC205-OVA was more efficient than anti-CD36-OVA in inducing early events of naive CD8+ T cell activation. In contrast, long-term persistence of effector CTLs was stronger following immunization with anti-CD36-OVA and did not require the addition of exogenous maturation stimuli. The results identify CD36 as a novel potential target for immunotherapy and indicate that the outcome of the immune responses vary by targeting different receptors on CD8alpha+ DCs.     

Peritonitis-induced antitumor activity of peritoneal macrophages from uremic patients

The macrophages belong to the effector cells of both nonspecific and specific immune response. These cells generally express little cytotoxicity unless activated. The present work was intended to determine if peritoneal macrophages collected from patients on Continuous Ambulatory Peritoneal Dialysis (CAPD) during episodes of peritonitis were active against human tumor cell lines without further in vitro stimulation. We also compared macrophage antitumor potential with effectiveness of drugs used in cancer therapy (taxol and suramin). Conditioned medium (CM) of macrophages collected during inflammation-free periods did not exhibit cytostatic and cytotoxic activity against both tumor (A549 and HTB44) and non-transformed (BEAS-2B and CRL2190) cells. Exposure of tumor cells to CM of macrophages harvested during peritonitis resulted in significant suppression of proliferation, impairment of viability and induction of apoptosis, in contrast to non-transformed cells, which remained unaffected. The efficacy of CM of inflammatory macrophages as an antitumor agent appeared to be comparable to cytostatic and cytotoxic potency of taxol and suramin or, in the case of HTB44 cells, even higher. The results obtained suggest that activated human macrophages might represent a useful tool for cancer immunotherapy.     

Antitumor effect of intratumoral administration of dendritic cell combination with vincristine chemotherapy in a murine fibrosarcoma model

A new antitumor therapeutic strategy utilizing the combined effect of chemotherapy and DC (dendritic cell)-based immunotherapy was designed, and the effect of intratumoral injections of unpulsed, immature DCs was evaluated after in vivo pretreatment of vincristine on tumor growth in a murine fibrosarcoma tumor model. Vincristine exerted a much more potent apoptosis/necrosis-inducing effect on MCA-102 tumor cells than on DCs both in vitro and in vivo. Moreover, CD11c, CD40, CD80 and CD86 molecules on DCs were not downregulated after treatment with vincristine either in vitro or in vivo. The growth of tumor significantly regressed in the group which received the combined vincristine chemotherapy with intratumoral administration of DCs in contrast to the untreated group, the group treated with DCs alone, and the group treated with vincristine alone. In particular, an upregulated expression of CD40, CD80 and CD86 molecules on DCs was found in the combination treatment group. Furthermore, the number of CD4+ and CD8+ T cells and the staining intensity of their CD4 and CD8 surface molecules also increased after the combination treatment. Therefore, our results indicate the feasibility of this combination therapy with vincristine chemotherapy and DC-based immunotherapy as an efficient antitumor strategy for the treatment of fibrosarcoma.