The chimeric antigen receptor NK-92 (CAR NK-92) cell targeting the prostate-specific membrane antigen (PSMA) has shown antitumour effects in castration-resistant prostate cancer (CRPC). However, the expression changes of programmed death ligand 1 (PD-L1) and its mechanisms on CAR NK-92 and CRPC cells and the effect of the anti-PD-L1 monoclonal antibody (mAb) on PD-L1 expressed on CAR NK-92 cells remain unknown.

Human dendritic cells and CD8+ T cells were acquired from blood samples of healthy donors and cocultured with C4-2 cells. Changes in PD-L1 expression were detected by flow cytometry. Differential gene expressions were investigated by RNA sequence analysis, while the regulation of PD-L1 molecular signaling was explored using western blotting. In vitro cytotoxicity was evaluated using the Cell Counting Kit-8 assay and the bioluminescent intensity (BLI) of green fluorescent protein-labelled C4-2 cells. CRPC growth in vivo was monitored using callipers and BLI in male NOD/SCID mice subcutaneously injected with C4-2 cells and treated intravenously with anti-PD-L1/PD-1 mAb, CAR NK-92 or cocultured CD8+ T cells.

Significantly upregulated expression of PD-L1k was observed in cocultured C4-2 and CAR NK-92 cells. In addition, upregulation of PD-L1 expression was dependent on interferon-γ in C4-2 cells, while it was dependent on direct cell-to-cell interaction via the NK group 2 member D/ phosphatidylinositol 3-kinase/AKT pathway in CAR NK-92 cells. The anti-PD-L1 mAb directly acted on PD-L1 expressed on CAR NK-92 cells and augmented the cytotoxicity of CAR NK-92 cells against C4-2 and CRPC cells from one patient in vitro. Anti-PD-L1 mAb significantly enhanced the antitumour effect of CAR NK-92 cells against CRPC cells in vivo when compared to treatment with CAR NK-92 cells or combined with anti-PD-1 mAb in the absence or presence of cocultured CD8+ T cells.

Combined treatment with CAR NK-92 and anti-PD-L1 mAb improved the antitumour efficacy against CRPC, which is of extraordinary translational value in the clinical treatment of CRPC.

To determine whether an autologous dendritic cell (DC) vaccine could induce antitumor immune responses in patients after resection of colorectal cancer metastases and whether these responses could be enhanced by activating DCs with CD40L.

Twenty-six patients who had undergone resection of colorectal metastases were treated with intranodal injections of an autologous tumor lysate- and control protein [keyhole limpet hemocyanin (KLH)]-pulsed DC vaccine. Patients were randomized to receive DCs that had been either activated or not activated with CD40L. All patients were followed for a minimum of 5.5 years.

Immunization induced an autologous tumor-specific T-cell proliferative or IFNγ enzyme-linked immunospot response in 15 of 24 assessable patients (63%) and a tumor-specific DTH response in 61%. Patients with evidence of a vaccine-induced, tumor-specific T-cell proliferative or IFNγ response 1 week after vaccination had a markedly better recurrence-free survival (RFS) at 5 years (63% versus 18%, P = 0.037) than nonresponders. In contrast, no association was observed between induction of KLH-specific immune responses and RFS. CD40L maturation induced CD86 and CD83 expression on DCs but had no effect on immune responses or RFS.

Adjuvant treatment of patients after resection of colorectal metastases with an autologous tumor lysate-pulsed, DC vaccine-induced, tumor-specific immune responses in a high proportion of patients. There was an association between induction of tumor-specific immune responses and RFS. Activation of this DC vaccine with CD40L did not lead to increased immune responses.

Dendritic cells (DCs) transfected with mRNA encoding tumor-associated antigens (TAAs) can induce tumor-specific T-cell responses. To potentiate this, we transfected mature DCs (mDCs) with mRNA encoding TAA targeted to the proteasome. DCs were generated from bone marrow cells by culture with 20 ng/ml GM-CSF and maturation with 1 microg/ml LPS. These mDCs were then electroporated with 10 microg of mRNA. Antigen presentation after electroporation with in vitro transcribed mRNA was compared with mRNA from a construct of the TAA preceded by ubiquitin. Proteasomal targeting of mRNA encoding cotranslationally ubiquitinated antigen was found to enhance intracellular degradation of target protein, and result in more efficient priming and expansion of TAA-specific CD8(+) T-cells. We therefore suggest that RNA-transfected DC vaccine efficacy could be improved by the use of mRNA targeted to the proteasome.

To investigate the in-vitro activation of cytotoxic T lymphocytes (CTLs) by fusion of mouse hepatocellular carcinoma (HCC) cells and lymphotactin gene-modified dendritic cells (DCs).

Lymphotactin gene modified DCs (DCLptn) were prepared by lymphotactin recombinant adenovirus transduction of mature DCs which differentiated from mouse bone marrow cells by stimulation with granulocyte/macrophage colony-stimulating factor (GM-CSF), interleukin-4 (IL-4) and tumor necrosis factor alpha (TNF-alpha). DCLptn and H22 fusion was prepared using 50% PEG. Lymphotactin gene and protein expression levels were measured by RT-PCR and ELISA, respectively. Lymphotactin chemotactic responses were examined by in-vitro chemotaxis assay. In-vitro activation of CTLs by DCLptn/H22 fusion was measured by detecting CD25 expression and cytokine production after autologous T cell stimulation. Cytotoxic function of activated T lymphocytes stimulated with DCLptn/H22 cells was determined by LDH cytotoxicity assay.

Lymphotactin gene could be efficiently transduced to DCs by adenovirus vector and showed an effective biological activity. After fusion, the hybrid DCLptn/H22 cells acquired the phenotypes of both DCLptn and H22 cells. In T cell proliferation assay, flow cytometry showed a very high CD25 expression, and cytokine release assay showed a significantly higher concentration of IFN-gamma and IL-2 in DCLptn/H22 group than in DCLptn, DCLptn+H22, DC/H22 or H22 groups. Cytotoxicity assay revealed that T cells derived from DCLptn/H22 group had much higher anti-tumor activity than those derived from DCLptn, H22, DCLptn+H22, DC/H22 groups.

Lymphotactin gene-modified dendritoma induces T-cell proliferation and strong CTL reaction against allogenic HCC cells. Immunization-engineered fusion hybrid vaccine is an attractive strategy in prevention and treatment of HCC metastases.

Dendritic cells (DCs) play a crucial role in the induction of antigen-specific T-cell responses, and therefore their use for the active immunotherapy of malignancies has been studied with considerable interest. More than a decade has passed since the publication of the first clinical data of DC-based vaccines, and through this and subsequent studies, a number of important developmental insights have been gleaned. These include the ideal source and type of DCs, the discovery of novel antigens and methods of loading DCs, the role of DC maturation, and the most efficient route of immunization. The generation of immune responses against tumor antigens after DC immunization has been demonstrated, and favorable clinical responses have been reported in some patients; however, it is difficult to pool the results as a whole, and thus the body of data remains inconclusive, in part because of varying DC preparation and vaccination protocols, the use of different forms of antigens, and, most importantly, a lack of rigorous criteria for defining clinical responses. As such, the standardization of clinical and immunologic criteria utilized, as well as DC preparations employed, will allow for the comparison of results across multiple clinical studies and is required in order for future trials to measure the true value and role of this treatment modality. In addition, issues regarding the optimal dose and clinical setting for the application of DC vaccines remain to be resolved, and recent clinical studies have been designed to begin to address these questions.

Tumor-associated peptides isolated by acid elution are frequently used for therapeutic immunization against various tumors both in mice and in humans. In acute myeloid leukemia (AML), the frequent accessibility of a large tumor burden allows for extraction of peptides from leukemia cells by using either citrate-phosphate (CP) or trifluoroacetic acid (TFA) buffer. To develop an optimal immunotherapeutic protocol for AML patients, we evaluated both in mice and in humans, the immunogenicity of peptides eluted from leukemia cells with the two acids (TFA or CP). Although ex vivo studies in mice showed that both prophylactic immunizations with mature dendritic cells (DC) loaded with TFA-peptides (DC/TFA), or CP-peptides (DC/CP), were able to stimulate specific antileukemia immune responses, only vaccination with DC/TFA was able to prevent leukemia outgrowth. Moreover, in humans, only DC/TFA generated significant antileukemia CD4(+) and cytotoxic CD8(+) T cell responses in vitro. In summary, these data demonstrate that the choice of the acid elution procedure to isolate immunogenic peptides strongly influences the efficacy of the antileukemia immune responses. These finding raise essential considerations for the development of immunotherapeutic protocols for cancer patients. In our model, our results argue for the use of the TFA elution method to extract immunogenic AML-associated peptides.

In December 2005, the UNAIDS and WHO reported that the global epidemic known as acquired immunodeficiency syndrome (AIDS) has claimed the lives of more than 25 million adults and children over the past 26 years. These figures included an estimated 3.1 million AIDS-related deaths in 2005. Despite enormous efforts to control the spread of human immunodeficiency virus (HIV) new infection rates are on the rise. An estimated 40.3 million people are now living with HIV, including 4.9 million new infections this past year. Nearly half of new HIV infections are in young people between the ages of 15 and 24. While drug therapies have helped sustain the lives of infected individuals in wealthy regions, they are relatively unavailable to the poorest global regions. This includes sub-Saharan Africa which has approximately 25.8 million infected individuals, more than triple the number of infections of any other region in the world. It is widely believed that the greatest hope for controlling this devastating pandemic is a vaccine. In this review, we will discuss the current state of DNA-based vaccines and how they compare to other vaccination methods currently under investigation. We will also discuss innovative ideas for enhancing DNA vaccine efficacy and the progress being made toward developing an effective vaccine.

Immunotherapy for malignant brain tumors by active immunization or adoptive transfer of tumor antigen-specific T lymphocytes has the potential to make up for some of the limitations of current clinical therapy. In this study, the authors tested whether active immunotherapy is curative in mice bearing advanced, rapidly progressive intracranial tumors.

Tumor vaccines were created through electrofusion of dendritic cells (DCs) and irradiated tumor cells to form multinucleated heterokaryons that retained the potent antigen processing and costimulatory function of DCs as well as the entire complement of tumor antigens. Murine hosts bearing intracranial GL261 glioma or MCA 205 fibrosarcoma were treated with a combination of local cranial radiotherapy, intrasplenic vaccination with DC/tumor fusion cells, and anti-OX40R (CD134) monoclonal antibody (mAb) 7 days after tumor inoculation. Whereas control mice had a median survival of approximately 20 days, the treated mice underwent complete tumor regression that was immunologically specific. Seven days after vaccination treated mice demonstrated robust infiltration of CD4+ and CD8+ T cells, which was exclusively confined to the tumor without apparent neurological toxicity. Cured mice survived longer than 120 days with no evidence of tumor recurrence and resisted intracranial tumor challenge.

These data indicate a strategy to achieve an antitumor response against tumors in the central nervous system that is highly focused from both immunological and anatomical perspectives.

APC are used extensively to induce and expand Ag-specific T cells as well as to test their specificity and function. In the treatment of malignant and infectious diseases, APC are used to stimulate and expand Ag-specific T cells for adoptive transfer, or used directly in vivo to present Ag. The choice of APC to use depends on the particular application and on practical considerations, which include ease of production, availability, reproducibility and (for clinical use) established safety. The diversity of APC in use partly reflects the fact that no single technique of Ag presentation is ideal. For the clinician and laboratory worker alike the field can seem illogical and confusing. In this review we outline the functional requirements of APC for the induction of T cells, classify the APC in common use and describe their laboratory and clinical applications.

We demonstrated previously that tumor lysate-pulsed dendritic cells (TP-DC) could mediate a specific and long-lasting antitumor immune response against a weakly immunogenic breast tumor during early lymphoid reconstitution. The purpose of this study was to examine the potential therapeutic efficacy of bone marrow transplants from TP-DC-vaccinated donors. In 2 aggressive metastatic models, bone marrow transplantation with donor bone marrow cells from TP-DC-immunized mice mediated a tumor-specific immune response in the recipient, and this caused regressions of preexisting tumor metastases. After vaccination with TP-DC, donors harbored increased numbers of both activated CD4+ and CD8+ T-cell populations in the bone marrow. Adoptive transfer of T cells purified from the bone marrow of TP-DC-vaccinated mice led to a reduction in preestablished lung metastases, whereas depletion of T cells from bone marrow abolished this effect. By using T cells derived from the bone marrow of TP-DC-vaccinated major histocompatibility complex class I and class II knockout mice, the effector cells required for the observed antitumor effect were determined to be major histocompatibility complex class I-restricted CD8+ T cells. Additionally, the tumor burden in TP-DC-immunized transplant recipients could be reduced further by repetitive TP-DC immunizations after bone marrow transplantation. Collectively, these results demonstrate an important therapeutic role of bone marrow from TP-DC-immunized donors and raise the potential for this approach in patients with advanced cancer.

For cancer immunotherapy the loading of dendritic cells (DCs) with whole tumor cell lysate preparations represents a simple and promising approach for presentation of tumor-associated antigens (TAAs), avoiding the disadvantages of HLA-matching and definition of TAAs. The aim of this study was to investigate whether lysate-pulsed DCs efficiently cross-prime CD8+ T cells and induce a strong T(H)1 cell response, as compared to DCs pulsed with specific peptides (FLU M1 and Melan-A/Mart-1). As a model system breast carcinoma cell lysate from either MCF-7 or MDA-MB-231 cell lines (both HLA-A*0201+) expressing the TAA MUC1 were selected. Both cell lines expressed MUC1, the epithelial mucin, which is a large molecular weight O-glycosylated protein expressed in the majority of breast, ovarian, and other epithelial malignancies and is under evaluation as a target antigen in cancer immunotherapy. We developed a simple lysate preparation method to solubilize all cell proteins without degradation. For loading of monocyte-derived dendritic cells, 100 microgmL(-1) of breast carcinoma cell lysate was used, accompanied by an adjuvant consisting of tumor necrosis factor-alpha (TNF-alpha) and prostaglandin-E2. T cells were co-cultivated with lysate or peptide pulsed DCs and were restimulated weekly. Before cultivation, and after the 3rd stimulation, tetramer frequencies for the MUC1 epitopes M1.2 and F7 as well as for the FLU M1 and Melan-A/Mart-1 epitopes were determined. After stimulation with lysate, higher frequencies for M1.2-specific T cells were observed compared with the F7 epitope. Furthermore, we found expansion factors for M1.2-specific T cells that had been stimulated with MCF-7 lysate-pulsed DCs of up to 43-fold. The analysis of typical T(H)1/T(H)2 cytokines (IFN-gamma, TNF-alpha, IL-12p70, IL-2, IL-4, IL-5, and IL-10) revealed a strong T(H)1 response. These results provide evidence for a strong T(H)1 polarization and cross-priming of MUC1-specific CD8+ T cells and demonstrate the feasibility of using lysate-pulsed dendritic cells in breast cancer immunotherapy.

Although there are several ways to load tumor antigens to DCs, in vitro preparation of tumor antigens and manipulation of DCs are usually required. Therefore, to develop a simple antitumor immunization method, we examined if direct injection of DCs into tumor apoptosed by ionizing IR could induce efficient antitumor immunity. Ionizing IR with 15 Gy induced apoptosis in tumor maximally after 6 hr. Injection of DCs i.t. into IR tumor induced strong cytotoxicity of splenocytes against tumor cells compared to i.t. injection of DCs or ionizing IR of tumor, both of which induced weak cytotoxicity. In an animal study, i.t. injection of DCs into IR tumor induced therapeutic antitumor immunity against a tumor established at a distant site. Moreover, when TNF-alpha or LPS was added as a danger/maturation signal to DC suspension before i.t. injection, antitumor immunity was significantly potentiated compared to a group treated with i.t. injection of DCs into IR tumor. Our results suggest that injection of DCs into tumor apoptosed by ionizing IR might be a simple and efficient method of immunization against tumor.

Dendritic cells (DC) based vaccinations have been widely used for the induction of anti-tumoral immunity in clinical studies. Antigen loading of DC with whole tumor cell preparations is an attractive method whenever tumor cell material is available. In order to determine parameters for the loading procedure, we performed dose finding and timing experiments. We found that apoptotic and necrotic melanoma cells up to a ratio of one-to-one, equivalent to 1mg/ml protein per 1 x 10(6) DC, can be added to monocyte derived DC without effecting DC recovery extensively. Using the isolated protein content of tumor cells (lysate) as a parameter, up to 5 mg/ml protein per 1 x 10(6) DC can be added. To achieve significant protein uptake at least 1 mg/ml of protein have to be added for more than 24 h as tested with FITC-labelled ovalbumin. Maturation inducing cytokines can be added simultaneously with the tumor cell preparations to immature DC without affecting the uptake. Furthermore, we tested the feasibility of cryopreservation of loaded and matured DC to facilitate the generation of ready to use aliquots. DC were cryopreserved in a mix of human serum albumin, DMSO and 5% glucose. After thawing, surface expression of molecules indicating the mature status (CD83, costimulatory and MHC molecules), was found to be unaltered. Furthermore, cryopreserved DC kept the capability to stimulate allogenic T-cell proliferation in mixed leukocyte reactions at full level. Loaded and matured DC pulsed with influenza matrix peptide (IMP) retained the capacity to induce the generation of IMP-specific cytotoxic T-lymphocytes after cryopreservation as measured by ELISPOT and tetramer staining. The expression of the chemokine receptor CXCR-4 and CCR-7 remained unaltered during cryopreservation and the migratory responsiveness towards MIP-3beta was unaltered as measured in a migration assay. Thus we conclude that the large scale loading and maturation of DC with whole tumor cell preparations can be performed in a single session. These data will facilitate the clinical application of DC loaded with whole tumor cell preparations.

Because dendritic cells (DC) are central to the induction of antigen-specific T cell responses, their use for the active immunotherapy of malignancies has been of considerable interest. Since clinical trials with DC-based vaccines have been initiated, a number of important developmental issues have become apparent. These include the ideal source and type of DC, the form of antigen and method of loading DC, whether to induce maturation, the route and timing of immunization, and the optimal clinical scenario. Clinical responses such as stability of disease and tumor regressions have been reported in some patients, particularly with melanoma, myeloma, and prostate cancer.

Dendritic cells (DCs) are important for the induction of primary T-cell responses and may serve as "biologic adjuvants" in therapeutic protocols. However, given the "plasticity" of this antigen-presenting cell, it remains unclear which DC type (source, subtype, and stage of differentiation) should be applied clinically. To provide additional insight in this selection process, we have, for the first time, analyzed the in vitro differentiation of CD34(+) precursor-derived and monocyte-derived DCs for ultrastructure, phenotype, and function. The ultrastructural intracytoplasmic differentiation of DCs correlated with increasing T-cell stimulatory activity of these cells. "Early-stage"-DCs proliferate, exhibit high levels of soluble antigen uptake, and moderate T-cell stimulatory capacity, and are characterized by centrally located nuclei and numerous enlarged mitochondria. "Intermediate-stage"-DCs are enlarged cells with enhanced T-cell stimulatory activity and pronounced cytoplasmic protein synthesis machinery. "Late-stage" (LS)-DCs exhibit a mature secretory cell phenotype and low proliferative index. They express high levels of the HLA-DR, CD40L, B7-1, and B7-2 molecules and CD83, a specific marker of mature DCs, and appear maximally stimulatory to T cells. Ultrastructurally, LS-DCs feature an accentric nucleus, an enlarged cytoplasm, containing numerous secretory storage vesicles, along with a fully developed Golgi complex. LS-DCs exhibited numerous multivesicular and multilaminar structures containing major histocompatibility complex class II molecules, consistent with the MIIC (peptide-loading) compartment. In extended studies, cultured CD14(+) monocyte-derived DCs displayed a similar, but accelerated, temporal differentiation staging pattern.

Dendritic cells (DCs) are the most important antigen-presenting cells. Many recent studies have compared the function of immature DCs (iDCs) and mature DCs (mDCs), but there have been few reports of the molecular changes that occur in DCs during maturation. Here, we report on differential gene expression in iDCs generated from peripheral blood monocytes compared with mDCs. Gene expression was evaluated using the differential display method after activation of iDCs with a low concentration of lipopolysaccharide (LPS) to induce maturation. Proteasome subunit alpha type 3 (PSMA3), transcription factor EC (TFEC) isoform and BTK region clone 2f10-rpi were transiently upregulated. Tryptophanyl-tRNA synthetase and CD63 antigen were upregulated for at least 24 h. Neuronal apoptosis inhibitory protein (NAIP) and transforming growth factor-beta-induced 68 kDa protein were downregulated. This is the first report of NAIP expression in human DCs. By comparing the expression of NAIP with that of other members of the inhibitor of apoptosis protein (IAP) family and the Bcl-2 family, only NAIP was found to be strongly expressed in iDCs before stimulation by LPS. PSMA3 was also induced in the DCs stimulated with immune complex. These findings might contribute to our understanding of DC maturation and the effectiveness of DC-based vaccines.

Reports of novel developments in tumor vaccines that have appeared in the year ending May 1, 2002 are reviewed here. Antigenic moieties were revealed for tumors previously considered nonimmunogenic. The use of peptides spanning mutations detected exclusively in tumor tissue avoids the common concern for autoimmune responses. Carbohydrate biology is revealing novel antigenic moieties. The search for helper epitopes from tumor antigens has come into full swing. Humoral immunity is regaining terrain, particularly through the development of antiidiotypic antibodies. Major steps forward have been made in optimizing modes and routes of antigen delivery and in the use of immune adjuvants. In the clinic, phase I/II trials support the notion that tumor vaccines are safe. Because these trials are conducted in patients in whom tumor remission is not a realistic endpoint, patient responses were established by immune monitoring strategies to detect subtle changes in antitumor reactivity. Both clinical and laboratory data stress the vast potential of tumor vaccines for the treatment of cancer.

Recently, human dendritic cells (DCs) pulsed with mRNA encoding a broad range of tumor antigens have proven to be potent activators of a primary anti-tumor-specific T-cell response in vitro. The aim of this study was to improve the mRNA pulsing of murine DC. Compared to a standard lipofection protocol and passive pulsing, electroporation was, in our hands, the most efficient method. The optimal conditions to electroporate murine bone marrow-derived DCs with mRNA were determined using enhanced green fluorescent protein and a truncated form of the nerve growth factor receptor. We could obtain high transfection efficiencies around 70-80% with a mean fluorescence intensity of 100-200. A maximal expression level was reached 3 hours after electroporation. A clear dose-response effect was seen depending on the amount of mRNA used. Importantly, the electroporation process did not affect the viability nor the allostimulatory capacity or phenotype of the DC. To study the capacity of mRNA-electroporated DCs to present antigen in the context of MHC classes I and II, we made use of chimeric constructs of ovalbumin. The dose-dependent response effect and the duration of presentation were also determined. Together, these results demonstrate that mRNA electroporation is a useful method to generate genetically modified murine DC, which can be used for preclinical studies testing immunotherapeutic approaches.

The immunotherapy of cancer is predicated on the belief that it is possible to generate a clinically meaningful antitumor response that provides patient benefit, such as improvement in the time to progression or survival. Indeed, immunotherapeutics with dendritic cells (DC) as antigen-presenting delivery vehicles for cell-based vaccines have already improved patient outcome against a wide range of tumor types (1-9). This approach stimulates the patient's own antitumor immunity through the induction or enhancement of T-cell immunity. It is generally believed that the activity of cytotoxic T lymphocytes (CTL), the cells directly responsible for killing the tumor cells in vivo, are directed by DC. Therefore, the goal of many current designs for DC-based vaccines is to induce strong tumor-specific CTL responses in patients with cancer. In practice, most studies for DC-based cancer vaccine development have focused on the development of methods that can effectively deliver exogenous tumor antigens to DC for cross-priming of CD8+ T cells through the endogenous MHC class I processing and presentation pathway (10). To date, many methods have been developed or evaluated for the delivery of defined and undefined tumor antigens to DC. This review provides a brief summary on these methods, the techniques used in these methods, as well as the advantages and disadvantages of each method.

Murine dendritic cells (DCs) are widely used for experimental vaccinations in mouse models. A high-yield method for freezing and thawing batches of these cells, if compatible with retention of cell immunophenotype, would reduce the time required for repeated preparations from DC precursors in bone marrow (BM), as well as variability among lots. Following depletion of specific lineages, murine bone marrow cells from C57BL/6 inbred-strain mice were grown in medium containing 10% fetal calf serum (FCS) and granulocyte/macrophage colony-stimulating factor (GM-CSF); after 6 days, large numbers of immature DCs were obtained. The immature cells were frozen in complete medium with GM-CSF and 10% DMSO, at a cell density of 5x10(6) DCs/ml. After thawing, 80% of DCs survived; they were induced to mature by addition of lipopolysaccharide (LPS). In comparison with fresh DCs, the thawed DCs had similar morphology, purity, and expression of class I (H-2D(b) and H-2K(b)) and class II major histocompatibility complex (MHC) proteins, as well as CD11b, CD11c, CD40, CD80, and CD86 molecules. Freeze-thawing did not affect trafficking to T cell areas of spleen, nor reduce the capacity to stimulate an alloresponse. Frozen-thawed cells were also proficient at uptake, processing, and presentation of native or denatured ovalbumin (OVA) protein to a peptide-specific T cell hybridoma, and were able to induce T cell responses in vivo after being loaded with denatured OVA protein. The ability to freeze and thaw DCs, and to obtain high yields without altering their essential properties, will facilitate future immunotherapy experiments in laboratory mouse models.

Until recently, immunotherapies have been of limited success, particularly against cancer. However, recent insights into the cells, molecules and signalling pathways that regulate immune responsiveness are providing new approaches for immunotherapy. In this article, I review some of the most promising molecular and cellular targets for immunotherapy and discuss approaches that use these targets to amplify immune responses and potentially break antigen-specific tolerance. These strategies provide a blueprint for the development of successful immunotherapy over the next decade.