An effective immunization and cancer treatment with activated dendritic cells transduced with full-length wild-type p53.

P53-based immunization is an attractive approach to cancer immunotherapy due to the accumulation of p53 protein in tumor, but not in normal cells. However, it was not known whether immune response against self-protein (p53) could be generated in vivo. Mouse dendritic cells (DCs) were transduced with adenoviral construct containing murine full-length wild-type p53 (Ad-p53). Repeated immunizations with these cells protected 60% of mice against challenge with MethA sarcoma cells bearing point mutations in p53 gene. Activation of DCs via ligation of CD40 significantly improved the results of immunization: all mice were protected against MethA sarcoma. The treatment of MethA tumor-bearing mice with activated Ad-p53-transduced DCs showed complete tumor rejection in four out of six mice. The specificity of antitumor immune response was confirmed by CTL assay. The analysis of phenotype and function of DCs demonstrated that the effect of CD40 ligation on these cells was enhanced by their infection with Ad-p53. The level of neutralizing anti-adenovirus antibody was moderately elevated in these mice. No signs of autoimmune reaction were evident during detailed pathological evaluation of treated mice. These data demonstrate that activated Ad-p53-infected DCs are able to break tolerance to this protein and can be used in immunotherapy of cancer.

Combination of gamma-irradiation and dendritic cell administration induces a potent antitumor response in tumor-bearing mice: approach to treatment of advanced stage cancer.

We investigated a new approach to the treatment of advanced stage cancer, a combination of apoptosis-inducing therapy and dendritic cell (DC) administration. MethA sarcoma and C3 tumor containing defined tumor-specific antigens in form of peptides' epitopes were selected as experimental mouse models. Sites of established subcutaneous tumors were gamma-irradiated with 10 Gy 3-5 times with 4-5 day interval. DCs generated from bone marrow of syngeneic mice were injected i.v. and s.c. after each irradiation. A large number of cell tracker-labeled DCs accumulated at the site of the irradiated tumor after s.c. injections. This effect was not observed in non-irradiated tumors. Almost all of these DCs bound GFP-labeled MethA cells in tumor tissue. Interferon-gamma production by splenocytes in response to the tumor-specific MHC class I matched peptides was determined by ELISPOT assays. The combination of gamma-irradiation and DC administration, but not each of the treatments alone resulted in a significant increase in T cell response to the specific, but not to the control peptides. An increased proportion of tumor peptide-specific CD8(+)-cells was found only in that group of mice using staining with tetramers. DCs with defective presentation of antigen via MHC class I or MHC class II pathways were unable to induce peptide-specific T cell response. The combination of gamma-irradiation and DC administration but not each of the treatments alone resulted in a dramatic antitumor effect. A substantial proportion of mice completely rejected their tumors (80% in case of MethA sarcoma and 40% in case of C3 tumor). In the rest of the mice, tumor growth was notably suppressed or completely blocked. These data suggest that the combination of apoptosis-inducing therapy and DC administration may be an attractive approach to the treatment of advanced cancer.

Notch-1 regulates NF-kappaB activity in hemopoietic progenitor cells.

We investigated the interaction between two elements critical for differentiation of hemopoietic cells, the Notch-1 receptor and the transcription factor NF-kappaB. These factors were studied in hemopoietic progenitor cells (HPC) using Notch-1 antisense transgenic (Notch-AS-Tg) mice. DNA binding of NF-kappaB as well as its ability to activate transcription was strongly decreased in HPC from Notch-AS-Tg mice. NF-kappaB-driven transcriptional activity was completely restored after transduction of the cells with retroviral constructs containing activated Notch-1 gene. HPC from Notch-AS-Tg mice have decreased levels of several members of the NF-kappaB family, p65, p50, RelB, and c-Rel and this is due to down-regulation of the gene expression. To investigate functional consequences of decreased NF-kappaB activity in transgenic mice, we studied LPS-induced proliferation of B cells and GM-CSF-dependent differentiation of dendritic cells from HPC. These two processes are known to be closely dependent on NF-kappaB. B cells from Notch-AS-Tg mice had almost 3-fold lower response to LPS than B cells isolated from control mice. Differentiation of dendritic cells was significantly affected in Notch-AS-Tg mice. However, it was restored by transduction of activated Notch-1 into HPC. Taken together, these data indicate that in HPC NF-kappaB activity is regulated by Notch-1 via transcriptional control of NF-kappaB.

Mechanism of immune dysfunction in cancer mediated by immature Gr-1+ myeloid cells.

The mechanism of tumor-associated T cell dysfunction remains an unresolved problem of tumor immunology. Development of T cell defects in tumor-bearing hosts are often associated with increased production of immature myeloid cells. In tumor-bearing mice, these immature myeloid cells are represented by a population of Gr-1(+) cells. In this study we investigated an effect of these cells on T cell function. Gr-1(+) cells were isolated from MethA sarcoma or C3 tumor-bearing mice using cell sorting. These Gr-1(+) cells expressed myeloid cell marker CD11b and MHC class I molecules, but they lacked expression of MHC class II molecules. Tumor-induced Gr-1(+) cells did not affect T cell responses to Con A and to a peptide presented by MHC class II. In sharp contrast, Gr-1(+) cells completely blocked T cell response to a peptide presented by MHC class I in vitro and in vivo. Block of the specific MHC class I molecules on the surface of Gr-1(+) cells completely abrogated the observed effects of these cells. Thus, immature myeloid cells specifically inhibited CD8-mediated Ag-specific T cell response, but not CD4-mediated T cell response. Differentiation of Gr-1(+) cells in the presence of growth factors and all-trans retinoic acid completely eliminated inhibitory potential of these cells. This may suggest a new approach to cancer treatment.

Human squamous cell carcinomas of the head and neck chemoattract immune suppressive CD34(+) progenitor cells.

CD34(+) progenitor cells have previously been shown to be mobilized in patients with squamous cell carcinoma of the head and neck (HNSCC). The present study showed that these CD34(+) cells inhibit the capacity of intratumoral lymphoid cells to become activated in response to stimulation through the TCR/CD3 complex. The mechanisms that could lead to the accumulation of CD34(+) cells within the tumor tissue were assessed. This was accomplished through in vitro studies that determined if HNSCC produce soluble factors that chemoattract CD34(+) cells. The migration of cord blood CD34(+) cells, which were used as a readily available source of progenitor cells, was stimulated by products derived from HNSCC explants and primary HNSCC cultures. This stimulated migration was due to chemotaxis because it was dependent on an increasing gradient of HNSCC-derived products. CD34(+) cells that were isolated from the peripheral blood of HNSCC patients were similarly chemoattracted to the HNSCC-derived products. The majority of the chemotactic activity produced by HNSCC could be attributed to vascular endothelial cell growth factor (VEGF). These studies indicate that HNSCC can chemoattract immune inhibitory CD34(+) progenitor cells through their production of VEGF.

Vascular endothelial growth factor effects on nuclear factor-kappaB activation in hematopoietic progenitor cells.

Vascular endothelial growth factor (VEGF) inhibits of the activation of transcription factor nuclear factor-kappaB (NF-kappaB) in hematopoietic progenitor cells (HPCs), and this is associated with alterations in the development of multiple lineages of hematopoietic cells and defective immune induction in tumor-bearing animals. Antibodies to VEGF have been shown to abrogate this effect. The mechanism by which VEGF antagonizes the induction of NF-kappaB was investigated in this study. Using supershift electrophoretic mobility shift analysis, we found that although tumor necrosis factor alpha (TNF-alpha) induced the nuclear translocation and DNA binding of p65-containing complexes, VEGF alone induced nuclear translocation and DNA binding of the complexes containing RelB. These results were confirmed by immunofluorescence confocal microscopy. VEGF effectively blocked TNF-alpha-induced NF-kappaB activation in HPCs from RelB-/- mice, however, similar to the effect observed in HPCs obtained from RelB+/- and RelB+/+ mice. This suggests that RelB is not required for VEGF to inhibit NF-kappaB activation. However, although TNF-alpha induced rapid activation of IkappaB kinase (IKK) as expected, this activity was substantially reduced in the presence of VEGF. This decreased IKK activation correlated with the inhibition of IkappaB alpha phosphorylation and degradation of IkappaB alpha and IkappaB epsilon in HPCs. VEGF alone, however, did not have any effect on phosphorylation of IkappaB alpha or degradation of IkappaB alpha and other inhibitory molecules IkappaB beta, IkappaB epsilon, or Bcl-3. SU5416, a potent inhibitor of the VEGF receptor I (VEGFR1) and VEGFR2 receptor tyrosine kinases, did not abolish the inhibitory effect of VEGF, indicating that the VEGF effect is mediated by a mechanism unrelated to VEGFR1 or VEGFR2 tyrosine kinase activity. Thus, VEGF appears to inhibit TNF-alpha-induced NF-kappaB activation by VEGFR kinase-independent inhibition of IKK. Therapeutic strategies aimed at overcoming VEGF-mediated defects in immune induction in tumor-bearing hosts will need to target this kinase-independent pathway.

Dendritic cells transduced with full-length wild-type p53 generate antitumor cytotoxic T lymphocytes from peripheral blood of cancer patients.

Accumulation of wild-type or mutant p53 protein occurs in approximately 50% of human malignancies. This overexpression may generate antigenic epitopes recognized by CTLs. Because normal cells have undetectable levels of p53, these CTLs are likely to be tumor specific. Here, for the first time, we test the hypothesis that full-length wild-type p53 protein can be used for generation of an immune response against tumor cells with p53 overexpression. T cells obtained from nine HLA-A2-positive cancer patients and three HLA-A2-positive healthy individuals were stimulated twice with dendritic cells (DCs) transduced with an adenovirus wild-type p53 (Ad-p53) construct. Significant cytotoxicity was detected against HLA-A2-positive tumor cells with accumulation of mutant or wild-type p53 but not against HLA-A2-positive tumor cells with normal (undetectable) levels of p53 or against HLA-A2-negative tumor cells. This response was specific and mediated by CD8+ CTLs. These CTLs recognized HLA-A2-positive tumor cells expressing normal levels of p53 protein after their transduction with Ad-p53 but not with control adenovirus. Stimulation of T cells with Ad-p53-transduced DCs resulted in generation of CTLs specific for p53-derived peptide. These data demonstrate that DCs transduced with the wild-type p53 gene were able to induce a specific antitumor immune response. This offers a new promising approach to immunotherapy of cancer.

Increased production of immature myeloid cells in cancer patients: a mechanism of immunosuppression in cancer.

Defective dendritic cell (DC) function caused by abnormal differentiation of these cells is an important mechanism of tumor escape from immune system control. Previously, we have demonstrated that the number and function of DC were dramatically reduced in cancer patients. This effect was closely associated with accumulation of immature cells (ImC) in peripheral blood. In this study, we investigated the nature and functional role of those ImC. Using flow cytometry, electron microscopy, colony formation assays, and cell differentiation in the presence of different cell growth factors, we have determined that the population of ImC is composed of a small percentage (<2%) of hemopoietic progenitor cells, with all other cells being represented by MHC class I-positive myeloid cells. About one-third of ImC were immature macrophages and DC, and the remaining cells were immature myeloid cells at earlier stages of differentiation. These cells were differentiated into mature DC in the presence of 1 microM all-trans-retinoic acid. Removal of ImC from DC fractions completely restored the ability of the DC to stimulate allogeneic T cells. In two different experimental systems ImC inhibited Ag-specific T cell responses. Thus, immature myeloid cells generated in large numbers in cancer patients are able to directly inhibit Ag-specific T cell responses. This may represent a new mechanism of immune suppression in cancer and may suggest a new approach to cancer treatment.

Clinical significance of defective dendritic cell differentiation in cancer.

Defective dendritic cell (DC) function has been described previously in cancer patients and tumor-bearing mice. It can be an important factor in the escape of tumors from immune system control. However, the mechanism and clinical significance of this phenomenon remain unclear. Here, 93 patients with breast, head and neck, and lung cancer were investigated. The function of peripheral blood and tumor draining lymph node DCs was equally impaired in cancer patients, consistent with a systemic rather than a local effect of tumor on DCs. The number of DCs was dramatically reduced in the peripheral blood of cancer patients. This decrease was associated with the accumulation of cells lacking markers of mature hematopoietic cells. The presence of these immature cells was closely associated with the stage and duration of the disease. Surgical removal of tumor resulted in partial reversal of the observed effects. The presence of immature cells in the peripheral blood of cancer patients was closely associated with an increased plasma level of vascular endothelial growth factor but not interleukin 6, granulocyte macrophage colony-stimulating factor, macrophage colony-stimulating factor, interleukin 10, or transforming growth factor-beta and was decreased in lung cancer patients receiving therapy with antivascular endothelial growth factor antibodies. These data indicate that defective DC function in cancer patients is the result of decreased numbers of competent DCs and the accumulation of immature cells. This effect may have significant clinical implications.

Antibodies to vascular endothelial growth factor enhance the efficacy of cancer immunotherapy by improving endogenous dendritic cell function.

Inadequate function of dendritic cells (DCs) in tumor-bearing hosts is one mechanism of tumor escape from immune system control and may compromise the efficacy of cancer immunotherapy. Vascular endothelial growth factor (VEGF), produced by most tumors, not only plays an important role in tumor angiogenesis but also can inhibit the maturation of DCs from hematopoietic progenitors. Here, we investigate a novel combination of antiangiogenic and immunotherapy based on this dual role of VEGF. Two s.c. mouse tumor models were used: D459 cells, expressing mutant human p53; and MethA sarcoma with point mutations in the endogenous murine p53 gene. Therapy with anti-mouse VEGF antibody (10 microg i.p. twice a week over 4 weeks) was initiated when tumors became palpable. Treatment of established tumors with anti-VEGF antibody alone did not affect the rate of tumor growth. However, anti-VEGF antibody significantly improved the number and function of lymph node and spleen DCs in these tumor-bearing animals. To investigate the possible effects of this antibody on the immunotherapy of established tumors, tumor-bearing mice were immunized with DCs pulsed with the corresponding mutation-specific p53 peptides, together with injections of anti-VEGF antibody. Therapy with peptide-pulsed DCs alone resulted in considerable slowing of tumor growth but only during the period of treatment, and tumor growth resumed after the end of the therapy. Combined treatment with peptide-pulsed DCs and anti-VEGF antibody resulted in a prolonged and much more pronounced antitumor effect. This effect was associated with the induction of significant anti-p53 CTL responses only in this group of mice. These data suggest that inhibition of VEGF may be a valuable adjuvant in the immunotherapy of cancer.

Effect of vascular endothelial growth factor and FLT3 ligand on dendritic cell generation in vivo.

The cytokine FLT3 ligand (FL) enhances dendritic cell (DC) generation and has therefore been proposed as a means to boost antitumor immunity. Vascular endothelial growth factor (VEGF) is produced by a large percentage of tumors and is required for development of tumor neovasculature. We previously showed that VEGF decreases DC production and function in vivo. In this study, we tested the hypothesis that VEGF regulates FL effects on DC generation. In seven experiments, four groups of mice were treated with PBS, VEGF alone (100 ng/h), FL alone (10 microgram/day), or with the combination of FL and VEGF. VEGF and PBS were administered continuously for 14 days via s.c. pumps. FL was given s.c. daily for 9 days, beginning on day 4. Tissues were collected and the number, phenotype, and function of lymph node, splenic, and thymic DCs were analyzed on day 14. As expected, treatment with FL resulted in a marked increase in the number of lymph node and spleen DCs and a smaller increase in thymic DC. Pretreatment of mice with VEGF inhibited these FL effects in lymph nodes and thymus by about 50%, whereas spleen DC numbers were undiminished by VEGF. VEGF treatment in vivo also inhibited the ability of FL to increase the number of hemopoietic precursor cells and the level of maturity exhibited by DC derived from these hemopoietic precursor cells in vitro. VEGF inhibited FL-inducible activation of transcription factor NF-kappaB. These data suggest that VEGF interferes with the ability of FL to promote dendritic cell differentiation from bone marrow progenitor cells in mice and therefore may decrease the therapeutic efficacy of FL in settings where increased numbers of DCs might provide clinical benefits.

Dendritic cells transduced with wild-type p53 gene elicit potent anti-tumour immune responses.

In this study we have tested the concept of using wild-type p53 gene for immunotherapy of cancer. Dendritic cells (DC) were transduced with a human wild-type p53 containing recombinant adenovirus (Ad-p53). About a half of DC transduced with this virus expressed p53 protein by FACS analysis 48 h after infection. Mice immunized twice with Ad-p53 DC developed substantial cytotoxic T lymphocyte (CTL) responses against tumour cells expressing wild-type and different mutant human and murine p53 genes. Very low CTL responses were observed against target cells infected with control adenovirus (Ad-c). Immunization with Ad-p53 provided complete tumour protection in 85% of mice challenged with tumour cells expressing human mutant p53 and in 72.7% of mice challenged with tumour cells with murine mutant p53. Treatment with Ad-p53-transduced DC significantly slowed the growth of established tumours. Thus, DC transduced with wild-type p53 may be a promising new tool for the immunotherapy of cancer.

Chemoattraction of femoral CD34+ progenitor cells by tumor-derived vascular endothelial cell growth factor.

Patients and animals with GM-CSF-producing tumors have an increased number of mobilized CD34+ progenitor cells within their peripheral blood and tumor tissue. These CD34+ cells are inhibitory to the activity of intratumoral T-cells. The present study used the murine Lewis lung carcinoma (LLC) model to assess mechanisms that could lead to the accumulation of CD34+ cells within the tumor tissue. In vitro analyses showed that LLC tumor explants released chemoattractants for normal femoral CD34+ cells. The LLC tumor cells contributed to the production of this activity since CD34+ cell chemoattractants were also released by cultured LLC cells. Antibody neutralization studies showed that most, although not all, of the chemotactic activity that was produced by LLC cells could be attributed to VEGF. In vivo studies with fluorescent-tagged CD34+ cells showed their accumulation within the tumor tissue, but not within the lungs, spleen or bone marrow, suggesting a selective accumulation within the tumor. Whether or not VEGF could chemoattract CD34+ cells in vivo was measured with a VEGF-containing Matrigel plug assay. Infusion of fluorescent-tagged CD34+ cells into mice after the plugs became vascularized revealed the accumulation of fluorescent-tagged cells within the plugs. However, these CD34+ cells failed to accumulate within the VEGF-containing Matrigel plugs when they were infused together with neutralizing anti-VEGF antibody. Through a combination of in vitro and in vivo analyses, the LLC cells were shown to be capable of chemoattracting CD34+ cells, with most of the tumor-derived chemotactic activity being due to tumor release of VEGF.

Defective function of Langerhans cells in tumor-bearing animals is the result of defective maturation from hemopoietic progenitors.

Langerhans cells (LC), the APCs in the skin, serve as a model for investigation of dendritic cell (DC) function in tissues. DC play a crucial role in the generation of antitumor immune responses. In this study, we investigated the effect of the presence of tumor in vivo on the ability of LC to take up Ag, migrate to draining lymph nodes, and stimulate primary T cell responses. In two animal models, these functions were substantially inhibited. This effect was not restricted to LC located in the skin near a tumor but was also seen at sites distant from the tumor. The duration of tumor exposure, and not its ultimate size, were found to be important, suggesting that tumors could be inhibiting the maturation of LC rather than directly inhibiting their function. Model experiments with radiation chimeras supported this hypothesis. To investigate the potential role of vascular endothelial growth factor (VEGF) in these effects we used anti-VEGF-neutralizing Ab to treat animals bearing tumors. Treatment with the Ab at a dose of 10 microg i.p. per mouse, twice a week for 4 wk, significantly improved the number and function of LC as measured by their ability to migrate to lymph nodes and stimulate primary T cell responses, even at doses that do not affect the growth of these established poorly immunogenic tumors. Thus, inhibition of VEGF signaling may improve DC function in tumor-bearing hosts and possibly serve to improve the efficacy of cancer immunotherapy.

Vascular endothelial growth factor affects dendritic cell maturation through the inhibition of nuclear factor-kappa B activation in hemopoietic progenitor cells.

Vascular endothelial growth factor (VEGF), produced by almost all tumor cells, affects the ability of hemopoietic progenitor cells (HPC) to differentiate into functional dendritic cells (DC) during the early stages of their maturation. In this study we demonstrate specific binding of VEGF to HPC. This binding was efficiently competed by placenta growth factor (PIGF), a ligand reportedly specific for the Flt-1 receptor. The number of binding sites for VEGF decreased during DC maturation in vitro associated with decreased levels of mRNA for Flt-1. VEGF significantly inhibited nuclear factor-kappa B (NF-kappa B)-dependent activation of reporter gene transcription during the first 24 h in culture. The presence of VEGF significantly decreased the specific DNA binding of NF-kappa B as early as 30 min after induction with TNF-alpha. This was followed on days 7 to 10 by decreases in the mRNA for RelB and c-Rel, two subunits of NF-kappa B. Blockade of NF-kappa B activity in HPC at early stages of differentiation with an adenovirus expressing a dominant I kappa B inhibitor of NF-kappa B reproduced the pattern of effects observed with VEGF. Thus, NF-kappa B plays an important role in maturation of HPCs to DC, and VEGF activation of the Flt-1 receptor is able to block the activation of NF-kappa B in this system. Blockade of NF-kappa B activation in HPCs by tumor-derived factors may therefore be a mechanism by which tumor cells can directly down-modulate the ability of the immune system to generate effective antitumor immune responses.

Decreased antigen presentation by dendritic cells in patients with breast cancer.

We evaluated T-cell responses to mitogens and to defined antigens in breast cancer patients. Significant defects in responses to tetanus toxoid and influenza virus were observed in patients with advanced-stage breast cancer. To define whether these defects were associated with a defect in antigen presentation [dendritic cells (DCs)] or effector function (T cells), these cells were studied separately. Purified DCs from 32 patients with breast cancer demonstrated a significantly decreased ability to stimulate control allogeneic T cells, but stimulation of patient T cells with either control allogeneic DCs or immobilized anti-CD3 antibody resulted in normal T-cell responses, even in patients with stage IV tumors. These data suggest that reduced DC function could be one of the major causes of the observed defect in cellular immunity in patients with advanced breast cancer. We then tested whether stem cells from these patients could give rise to functional DCs after in vitro growth with granulocyte/macrophage colony-stimulating factor and interleukin 4. Normal levels of control allogeneic and tetanus toxoid-dependent T-cell proliferation were observed when DCs obtained from precursors were used as stimulators. Those cells also induced substantially higher levels of influenza virus-specific CTL responses than mature DCs from the peripheral blood of these patients, although responses did not quite reach control values. Thus, defective T-cell function in patients with advanced breast cancer can be overcome by stimulation with DCs generated from precursors, suggesting that these cells may better serve as autologous antigen carriers for cancer immunotherapy than mature peripheral blood DCs.

IL-12 and mutant P53 peptide-pulsed dendritic cells for the specific immunotherapy of cancer.

Mice bearing palpable tumors expressing a mutant p53 gene were treated with intravenous injection of mutant p53-specific peptide-pulsed dendritic cells (DCs). Control groups were treated with control peptide-pulsed DCs. All mice received three injections of 10(5) DCs on days 0, 5, and 10 after the start of the therapy. Half of the animals in each group were also treated with intraperitoneal injections of interleukin (IL)-12 (300 ng per mouse every other day, from day 0 to day 20). Mice treated with control peptide-pulsed DCs showed fast tumor progression. This growth was not substantially affected by treatment with IL-12 alone. Tumor growth in mice treated with mutant p53 peptide-pulsed DCs was significantly slowed during therapy, but resumed after cessation of therapy. In contrast, tumor growth in mice receiving both specific peptide-pulsed DCs and IL-12 remained very slow even 4 weeks after termination of the immunotherapy. To investigate the immunological basis for these effects, mutant p53 specific cytotoxic T lymphocyte (CTL) response was tested in mice 2 weeks after the last injection of DCs. Significant levels of CTLs were registered only in mice treated with IL-12 in addition to specific immunization. Thus, IL-12 dramatically improved the effect of the mutant p53-specific immunotherapy of palpable, preexisting tumors, supporting the use of IL-12 as an immunoadjuvant in clinical trials of tumor-specific peptide-pulsed DC.

Production of vascular endothelial growth factor by human tumors inhibits the functional maturation of dendritic cells.

Inadequate presentation of tumor antigens by host professional antigen-presenting cells (APCs), including dendritic cells (DCs), is one potential mechanism for the escape of tumors from the host immune system. Here, we show that human cancer cell lines release a soluble factor or factors that dramatically affect DC maturation from precursors without affecting the function of relatively mature DCs. One factor responsible for these effects was identified as vascular endothelial growth factor (VEGF). Thus, VEGF may play a broader role in the pathogenesis of cancer than was previously thought, and therapeutic blockade of VEGF action may improve prospects for immunotherapy as well as inhibit tumor neovasculature.

Mechanism for dendritic cell dysfunction in retroviral infection of mice.

Dendritic cells (DC) from mice infected with the murine retrovirus Rauscher leukaemia virus (RLV) are poor stimulators of allogeneic and syngeneic T cells and express lower, but still significant, levels of MHC class II. In this paper we further investigated the mechanism of the dysfunction of DC. DC from infected animals did not cause anergy of T cells during coculture for 3 or 6 days. They did not release a substantial amount of soluble factors which could suppress T cell responses. The low T cell responses on stimulation using RLV-infected DC could be overcome by the addition of control DC. Pretreatment of these control DC with monoclonal antibody against MHC class II molecules completely blocked their ability to restore stimulation of T cells in the presence of infected DC. However, antibody against MHC class I or mismatched MHC class II molecules did not prevent restoration of function. The reduced labeling of surface MHC class II molecules previously reported was shown to reflect a loss in total class II molecules within the cells; MHC class I levels were unaltered by exposure to the virus. In DC from RLV-infected mice biosynthesis of MHC class II was decreased by around 50% at the transcriptional level in comparison with beta-actin. Thus, the down-regulation of surface class II molecules observed in DC following RLV infection is a consequence of a specific block in its biosynthesis and the failure of DC to stimulate T cells may be a direct consequence of the reduced class II levels. Since reduced stimulation by DC is also seen in HIV-1 infection in humans we speculate that a similar mechanism might operate in retroviral infection in man.

Dendritic cells in antitumor immune responses. I. Defective antigen presentation in tumor-bearing hosts.

Induction of specific antitumor cytotoxic T cell responses was studied in BALB/c mice bearing tumors transfected with a mutant human p53 minigene. We observed that mice were resistant to the induction of peptide-specific CTL as early as 5 days after challenge with a minimal lethal dose of tumor cells, and the cell types responsible for this effect were further characterized. The contribution of CD4+ and CD8+ T cells in this response was studied after peptide-pulsed dendritic cell (DC) immunization. In vitro depletion of CD4+ cells during peptide restimulation reduced the level of specific lysis in control mice, and depletion of CD8+ T cells completely abrogated it. Substitution of CD8+ cells from immunized control mice during restimulation of cells from immunized tumor-bearing mice did not change the level of specific lysis. Substitution of the CD4+ from tumor-bearing mice by CD4+ cells from control mice improved CTL response, although this response did not reach control values. Peptide-pulsed dendritic cells isolated from tumor-bearing mice showed a significantly reduced ability to induce specific CTL in control animals and reduced ability to restimulate immune T cells from control mice in vitro. DC from tumor-bearing mice also had a reduced ability to stimulate control allogeneic T cells. Restimulation of T cells from immunized tumor-bearing mice with DC from control animals, but not from tumor-bearing mice, dramatically increased specific CTL responses to control levels. Macrophages at the same concentration were not able to improve CTL function. Thus, defective antigen presentation by DC appears to be a major determinant for CTL nonresponsiveness to peptide antigens in tumor-bearing mice, and addition of control DC can restore specific lysis. These data provide a basis for new approaches to peptide-based cancer immunotherapy.