Genetically modified bone marrow-derived dendritic cells expressing tumor-associated viral or "self" antigens induce antitumor immunity in vivo

The clinical application of synthetic tumor peptide-based vaccines is currently limited to patients with specified major histocompatibility complex (MHC) class I alleles. Such logistic limitations may be overcome using tumor gene-based approaches. Here we describe the effective generation of dendritic cells (DC) expressing tumor peptide-MHC complexes as a result of particle-mediated transfer of genes encoding tumor-associated antigens (TAA). Bone marrow-derived DC were transfected with plasmid DNA encoding the tumor-associated viral antigen E7 derived from human papilloma virus (HPV) 16. When applied as a vaccine, these genetically modified DC induced antigen-specific CD8+ cytotoxic T lymphocytes (CTL) in vivo and promoted the rejection of a subsequent, normally lethal challenge with an HPV 16-transformed tumor cell line. Of greatest interest, immunization of mice with syngeneic DC genetically modified to enhance their presentation of a constitutive "self" epitope derived from the tumor-suppressor gene product p53 caused a significant reduction in the in vivo growth of a chemically induced p53-positive sarcoma. These results suggest that cancer vaccines consisting of DC genetically modified to express TAA of viral or "self" origin effectively induce antitumor immunity in vivo.     

Virus-mediated delivery of antigenic epitopes into dendritic cells as a means to induce CTL

Dendritic cells (DC) are potent inducers of CD8+ T cells and can stimulate protective antitumor immunity when pulsed with an antigenic peptide or protein. We used a replication-deficient adenovirus containing a Kb-restricted antigenic peptide of chicken OVA to study CTL induction in vitro and in vivo after adenovirus-mediated gene transfer into DC. The efficiency of adenovirus-infected DC in eliciting a specific CTL response was compared with immunizations with a recombinant vaccinia virus and DC pulsed with peptide or protein. An immortalized DC line derived from a C57BL/6 mouse and freshly isolated splenic DC from C57BL/6 mice were used in CTL induction. Virus-infected DC elicited the strongest Ag-specific CTL response in vitro and in vivo and induced protective antitumor immunity to a challenge with EG.7 tumors (EL-4 cell line expressing OVA). Direct immunization of mice with recombinant adenovirus resulted in the induction of high titers of neutralizing Abs, which precluded a boost of a CTL response after repeated inoculations. However, repeated injections of virus-infected DC induced only low titers of neutralizing Abs. Furthermore, the presence of neutralizing Abs specific for the virus did not affect the usefulness of infected DC as repeated applications of virus-infected DC boosted the CTL response even in mice previously infected with the recombinant vector. The use of DC infected with a recombinant virus has advantages over other forms of immunization and could provide an alternative approach for designing vaccination therapies.     

Bone marrow-generated dendritic cells pulsed with tumor extracts or tumor RNA induce antitumor immunity against central nervous system tumors

Recent studies have shown that the brain is not a barrier to successful active immunotherapy that uses gene-modified autologous tumor cell vaccines. In this study, we compared the efficacy of two types of vaccines for the treatment of tumors within the central nervous system (CNS): dendritic cell (DC)-based vaccines pulsed with either tumor extract or tumor RNA, and cytokine gene-modified tumor vaccines. Using the B16/F10 murine melanoma (B16) as a model for CNS tumor, we show that vaccination with bone marrow-generated DCs, pulsed with either B16 cell extract or B16 total RNA, can induce specific cytotoxic T lymphocytes against B16 tumor cells. Both types of DC vaccines were able to protect animals from tumors located in the CNS. DC-based vaccines also led to prolonged survival in mice with tumors placed before the initiation of vaccine therapy. The DC-based vaccines were at least as effective, if not more so, as vaccines containing B16 tumor cells in which the granulocytic macrophage colony-stimulating factor gene had been modified. These data support the use of DC-based vaccines for the treatment of patients with CNS tumors.     

Retrovirally transduced bone marrow-derived dendritic cells require CD4+ T cell help to elicit protective and therapeutic antitumor immunity

It has been extensively documented that murine dendritic cells loaded with tumor-associated Ag (TAA)-derived peptides or protein can prime Ag-specific CD8+ cytotoxic T cells in vivo and can elicit Ag-specific immunity. Optimal presentation of TAA might be achieved by retroviral transduction of DCs allowing long term and stable expression of the TAA-peptides as well as the presentation of multiple epitopes in the context of MHC class I and/or class II molecules. Here we show that retroviral transduction of bone marrow-derived dendritic cells (DCs) with chicken OVA cDNA or the reporter gene green fluorescent protein retained their potent stimulatory capacity and that the transduced DCs could process and present the endogenously expressed OVA protein. The DCs transduced with cDNA encoding native OVA protein presented OVA-derived peptides in the context of MHC class I as well as MHC class II and induced a strong Ag-specific CTL response. DCs expressing a cytosolic form of OVA presented OVA peptides only in the context of MHC class I and failed to induce an OVA-specific CTL response in vivo when they had been cultured in the absence of exogenous protein. Immunization with retrovirally transduced DCs resulted in an Ag-specific immunity and rejection of a tumor cell challenge and a significant survival advantage in tumor-bearing mice. These results obtained in this rapidly lethal tumor model suggest that DCs transduced with TAA may be useful for tumor immunotherapy and underscore the importance of the simultaneous delivery of T cell help in the development of Ag-specific cytotoxic T-cells.     

Myeloma bone marrow plasma cells: evidence for their capacity as antigen-presenting cells

Myeloma plasma cells constitute 10% to 90% of the total bone marrow cell count in patients with multiple myeloma (MM). These cells express a variety of cell surface markers, such as HLA-ABC and HLA-DR, and surface antigens that are necessary for professional antigen-presenting cells, including adhesion and costimulatory molecules. In this study, we examined the expression of major histocompatability complex (MHC) and costimulatory molecules on CD38(bright,++) plasma cells in bone marrow aspirates from eight MM patients. Small percentages of plasma cells expressed weak but detectable levels of HLA-DR, HLA-DQ, CD40, CD80, and CD86, which could be upregulated by interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha. CD38++ plasma cell and CD38(dim,+) cells were sorted from freshly isolated bone marrow mononuclear cells and tested for their capacity to act as antigen-presenting cells. Indeed, both CD38++ plasma cells and CD38+ cells were able to stimulate allogeneic T cells and present the soluble antigens purified protein derivative and tetanus toxoid to autologous T cells. Recognition of the antigens led to T-cell proliferation and secretion of IFN-gamma and was MHC class-I and -II restricted. Antigen processing and presentation by CD38++ and CD38+ cells were abolished by treatment of the cells with chloroquine. Hence, our study provides for the first time evidence that myeloma plasma cells may act as antigen-presenting cells. Further studies are warranted to examine in detail the molecules required for inducing T-cell stimulation.     

Comparison of the abilities of MHC-compatible bone marrow cells and lymph node cells to induce tolerance of skin allografts in rats

A comparison has been made of the abilities of bone marrow cells and lymph node cells to induce tolerance of skin when inoculated into neonatal rats known to differ only with regard to non-MHC incompatibilities, including putative skin-specific (Skn) antigens. Each recipient received 50 x 10(6) cells, and tolerance was confirmed by the permanent acceptance of donor-strain neonatal heart tissue transplanted to the pinna of the ear. In 5 of the 8 MHC-compatible strain combinations tested, BMC were significantly more effective than LNC in inducing tolerance of skin, whereas in one situation LNC proved more efficient. Although the results are in accord with the occurrence of Skn antigens in rats, it appears that at least some of the antigens involved must also be expressed by BMC or LNC, but not equally by both of these tissues. The results also provide evidence that in rats, as in mice, the MHC can play a major role in determining the response to, and/or the immunogenicity of, Skn antigens.     

Haematopoietic capacity of colony-forming cells mobilized in hepatic inflammatory reactions as compared to that of normal bone marrow cells

Chronic inflammatory periovular granulomatous reactions elicited in liver by schistosomal infection are a site of active myelopoiesis. We quantified the colony-forming cells (CFCs) in granulomas and found that the whole liver contains a number of CFCs roughly equivalent to 50% of a femur. Clonogenic analysis showed the presence of committed as well as pluripotent and totipotent CFCs. Long-term Dexter-type cultures showed that the granuloma-derived totipotent CFCs do not have self-renewal capacity. Hence, they did not correspond functionally to haematopoietic stem cells, despite the fact that the stroma established by adherent cells harvested from granulomas had the capacity to sustain long-term proliferation of bone-marrow-derived haematopoietic stem cells. We conclude that myelopoietic cytokines produced by inflammatory reactions in schistosomiasis elicit mobilization of bone marrow CFCs into the circulation, which can settle in hepatic granulomas. This environment may induce their proliferation and differentiation, but not their self-renewal, sustaining temporary production of myeloid cell lineages which nevertheless depends upon cell renewal from the bone marrow pool of haematopoietic precursors.     

Tumor defense by murine cytotoxic T cells specific for peptide bound to nonclassical MHC class I

Cytotoxic T Cells (CTLs) can exhibit considerable antitumor activity. Thus far, the characterized tumor peptide antigens recognized by CTLs are all presented by classical MHC class Ia molecules [human lymphocyte antigen A (HLA-A), HLA-B, and HLA-C in humans and H-2K, H-2D, and H-2L in mice]. Here we show that CTLs recognized peptides presented by nonclassical MHC class Ib molecule Qa-1b expressed by tumor cells. These CTLs conferred in vivo protection by delaying the growth of Qa-1b-expressing B78H1 melanoma cells pulsed with Qa-1b-binding peptides Cw4L or B35L and injected s.c. in C57BL/6 mice. A hierarchy of the peptides was found with regard to their ability to trigger CTLs; Cw4L stimulated a strong CTL response. The closely related and cross-reactive peptide B35L induced a weaker CTL response but was still efficient in sensitizing the target cells. Finally, Qa-1b-expressing melanoma cells without exogenous peptides were not immunogenic but possibly expressed endogenous cross-reactive antigenic peptides. The data are compatible with earlier findings that CTL activation requires relatively strong peptide antigens, whereas subsequent effector functions are also mediated by weak peptide analogues. In conclusion, CTLs mediated tumor immunity through the recognition of peptides presented by nonclassical MHC class Ib molecules. The identification of similar CTLs in humans may facilitate the vaccination of cancer patients because MHC class Ib/peptide complexes are much less polymorphic than MHC class Ia/peptide complexes.     

Peptide-antibody conjugates for tumour therapy: a MHC-class-II-restricted tetanus toxin peptide coupled to an anti-Ig light chain antibody can induce cytotoxic lysis of a human B-cell lymphoma by specific CD4 T cells

Anti-idiotype antibody therapy of B-cell lymphomas, despite numerous promising experimental and clinical studies, has so far met with limited success. Tailor-made monoclonal anti-idiotype antibodies have been injected into a large series of lymphoma patients, with a few impressive complete tumour remissions but a large majority of negative responses. The results presented here suggest that, by coupling to antilymphoma idiotype antibodies a few molecules of the tetanus toxin universal epitope peptide P2 (830-843), one could markedly increase the efficiency of this therapy. We show that after 2-hr incubation with conjugates consisting of the tetanus toxin peptide P2 coupled by an S-S bridge to monoclonal antibodies directed to the lambda light chain of human immunoglobulin, human B-lymphoma cells can be specifically lysed by a CD4 T-lymphocyte clone specific for the P2 peptide. Antibody without peptide did not induce B-cell killing by the CD4 T-lymphocyte clone. The free cysteine-peptide was also able to induce lysis of the B-lymphoma target by the T-lymphocyte clone, but at a molar concentration 500 to 1000 times higher than that of the coupled peptide. Proliferation assays confirmed that the antibody-peptide conjugate was antigenically active at a much lower concentration than the free peptide. They also showed that antibody-peptide conjugates required an intact processing function of the B cell for peptide presentation, which could be selectively inhibited by leupeptin and chloroquine.(ABSTRACT TRUNCATED AT 250 WORDS)     

The sialomucin CD164 (MGC-24v) is an adhesive glycoprotein expressed by human hematopoietic progenitors and bone marrow stromal cells that serves as a potent negative regulator of hematopoiesis

Mucin-like molecules represent an emerging family of cell surface glycoproteins expressed by cells of the hematopoietic system. We report the isolation of a cDNA clone that encodes a novel transmembrane isoform of the mucin-like glycoprotein MGC-24, expressed by both hematopoietic progenitor cells and elements of the bone marrow (BM) stroma. This molecule was clustered as CD164 at the recent workshop on human leukocyte differentiation antigens. CD164 was identified using a retroviral expression cloning strategy and two novel monoclonal antibody (MoAb) reagents, 103B2/9E10 and 105.A5. Both antibodies detected CD164/MGC-24v protein expression by BM stroma and subpopulations of the CD34(+) cells, which include the majority of clonogenic myeloid (colony-forming unit-granulocyte-macrophage [CFU-GM]) and erythroid (blast-forming unit-erythroid [BFU-E]) progenitors and the hierarchically more primitive precursors (pre-CFU). Biochemical and functional characterization of CD164 showed that this protein represents a homodimeric molecule of approximately 160 kD. Functional studies demonstrate a role for CD164 in the adhesion of hematopoietic progenitor cells to BM stromal cells in vitro. Moreover, antibody ligation of CD164 on primitive hematopoietic progenitor cells characterized by the cell surface phenotype CD34(BRIGHT)CD38(-) results in the decreased recruitment of these cells into cell cycle, suggesting that CD164 represents a potent signaling molecule with the capacity to suppress hematopoietic cell proliferation.     

Fluorescent in situ hybridization on flow-sorted cells as a tool for evaluating minimal residual disease or chimerism after allogenic bone marrow transplantation

The objective is to determine the blood lead levels of the children (7-8 years of age) living in some of Madrid's satellite towns, and to establish links between these levels and different socioeconomic items, life styles and environmental factors, among other variables. A cross-sectional study using a sample based on 205 schoolchildren of Second Grade Primary Education, was under taken during 1995. Venous blood samples were analysed through atomic absorption spectrophotometry. Parents answered a questionnaire with items used in this study. The average blood lead level found was 3.9 mud/dl (CI 95% 3.7-4.1), with higher levels among the children of the Northern zone of Madrid. Between the associated factors to blood lead level was found to be the habit of nail biting, mother's studies and some variables associated with habitual dwelling. We reached the conclusion that the blood lead levels of Madrid's suburban children are low.     

MHC class Ib-restricted cells contribute to antilisterial immunity: evidence for Qa-1b as a key restricting element for Listeria-specific CTLs

Subclinical infection of BALB/c mice with the intracellular pathogen Listeria monocytogenes results in the development of MHC class Ia- and Ib-restricted CTLs. L. monocytogenes-infected TAP-/- bone marrow macrophage targets are not lysed by MHC class Ia- or Ib-restricted CTLs, showing a requirement for transport of peptides into the endoplasmic reticulum for development of the MHC class Ib-peptide target. L. monocytogenes-infected B6.Tla(a)-derived bone marrow macrophages (Kb Qa-1a) are not lysed by BALB/c (Kd Qa-1b)-derived antilisterial CTLs, confirming an earlier finding that the Ib-restricting element is T region encoded. We have further determined that Qa-1b is a restricting element for antilisterial CTLs using L. monocytogenes-infected Qa-1b-transformed mouse L cells as well as human-derived HeLa cells as target populations. These L. monocytogenes-infected Qa-1b-transformed cell lines are lysed by BALB/c (Qa-1b)- or C57BL/6 (Qa-1b)-derived antilisterial CTLs, but are not lysed by B6.AKM (Qa-1a)-derived antilisterial CTLs. Using L. monocytogenes-infected targets, we found that MHC class Ia- and Ib-restricted CTLs are evident within 4 days following infection, peak on day 5 following infection, and although Ib-restricted CTLs disappear by day 6 postinfection, la-restricted antilisterial CTL activity can still be detected. These results demonstrate that Qa-1b is a restricting element for antilisterial CTLs, and expression of the MHC class Ib-presented target at the cell surface is TAP dependent. In addition, these results show that following L. monocytogenes infection, MHC class Ib-restricted CTLs are evident in vivo.     

Efficacy and toxicity of 9-beta-D-arabinofuranosylguanine (araG) as an agent to purge malignant T cells from murine bone marrow: application to an in vivo T-leukemia model

9-beta-D-Arabinofuranosylguanine (araG), an analog of deoxyguanosine which is not degraded by purine nucleoside phosphorylase, has been previously shown in in vitro studies by our laboratory to be effective in purging malignant T cells from human bone marrow (1). We now describe studies in a murine model of T-cell acute lymphoblastic leukemia (ALL) in which we tested whether bone marrow, contaminated with malignant T cells and purged ex vivo with araG, could reconstitute both the lymphoid and myeloerythroid lineages in the absence of leukemic relapse. The model utilized 6C3HED tumor cells, derived from a Thy 1.2+ malignant murine T-cell line, which were shown to cause lethal leukemia in C3H/HeN mice. Intravenous injection of 10(6) 6C3HED cells resulted in 100% mortality within 18 days, with autopsy revealing tumor infiltration of multiple organs. 100% of non-leukemia bearing lethally irradiated C3H/HeN mice transplanted with syngeneic bone marrow, treated ex vivo with 100 microM of araG for 18 hours, survived > 365 days post-transplant with full lymphohematopoietic reconstitution. Evidence of araG's ability to purge bone marrow of malignant tumor cells without causing significant toxicity to normal marrow derived hematopoietic progenitor cells was documented in experiments in which 75% of lethally irradiated mice transplanted with syngeneic bone marrow, contaminated with 6C3HED tumor cells and treated ex vivo with 100 microM araG for 18 hours, survived for > 250 to > 400 days. Death in 25% of mice was secondary to infection which developed before marrow reconstitution occurred. Reconstitution of the lymphoid, myeloid, and erythroid lineages with donor cells in surviving mice was documented. The data presented indicate that araG may effectively purge bone marrow of malignant T cells without irreversible toxicity to hematopoietic stem cells. This purging regimen is recommended for consideration for clinical trials in patients with T-cell malignancies undergoing autologous bone marrow transplantation and may also be a viable option for T-cell depletion as a strategy to prevent graft-versus-host disease.     

Extensive apoptosis of bone marrow cells as evaluated by the in situ end-labelling (ISEL) technique may be the basis for ineffective haematopoiesis in patients with myelodysplastic syndromes

By using data from the Bremer Adolescent Study, this report presents findings on the frequency, comorbidity, and psychosocial impairment of panic disorder and panic attacks among 1,035 adolescents. The adolescents were randomly selected from 36 schools in the province of Bremen, Germany. Panic disorder and other psychiatric disorders were coded based on DSM-IV criteria using the computerized-assisted personal interview of the Munich version of the Composite International Diagnostic Interview. Panic disorder occurred rather rare, with only 0.5% of all the adolescents met the DSM-IV criteria for this disorder sometimes in their live. Panic attack occurred more frequently, with 18% of the adolescents reported having had at least one panic attack. Slightly more girls than boys had panic attack and panic disorder. The occurrence of panic attack and panic disorder were the greatest among the 14-15 year olds. The experience of having a panic attack was associated with a number of problems, the most frequent being avoiding the situation for fear of having another attack. Four most common symptoms associated with a panic attack were that of palpitations, trembling/shaking, nausea or abdominal distress, and chills or hot flushes. Panic disorder comorbid highly with other psychiatric disorder covered in our study, especially with that of major depression. Among those with a panic disorder, about 40% of them were severely impaired during the worst episode of their illness. Only one out of five adolescents with panic disorder sought professional help for emotional and psychiatric problems. The implication of our findings for research and clinical practice are discussed.     

Nonmyeloablative stem cell transplantation and cell therapy as an alternative to conventional bone marrow transplantation with lethal cytoreduction for the treatment of malignant and nonmalignant hematologic diseases

Myeloablative conditioning associated with hazardous immediate and late complications is considered as a mandatory first step in preparation for allogeneic blood or marrow transplantation (allogeneic BMT) for the treatment of malignant hematologic disorders and genetic diseases. Immune-mediated graft-versus-leukemia (GVL) effects constitute the major benefit of allogeneic BMT. Therefore, we have introduced the use of relatively nonmyeloablative conditioning before allogeneic BMT aiming for establishing host-versus-graft tolerance for engraftment of donor immunohematopoietic cells for induction of GVL effects to displace residual malignant or genetically abnormal host cells. Our preliminary data in 26 patients with standard indications for allogeneic BMT, including acute leukemia (n = 10); chronic leukemia (n = 8), non-Hodgkin's lymphoma (n = 2), myelodysplastic syndrome (n = 1), multiple myeloma (n = 1), and genetic diseases (n = 4) suggest that nonmyeloablative conditioning including fludarabine, anti-T-lymphocyte globulin, and low-dose busulfan (8 mg/kg) is extremely well tolerated, with no severe procedure-related toxicity. Granulocyte colony-stimulating factor mobilized blood stem cell transplantation with standard dose of cyclosporin A as the sole anti-graft-versus-host disease (GVHD) prophylaxis resulted in stable partial (n = 9) or complete (n = 17) chimerism. In 9 patients absolute neutrophil count (ANC) did not decrease to below 0.1 x 10(9)/L whereas 2 patients never experienced ANC < 0.5 x 10(9)/L. ANC > or = 0.5 x 10(9)/L was accomplished within 10 to 32 (median, 15) days. Platelet counts did not decrease to below 20 x 10(9)/L in 4 patients requiring no platelet support at all; overall platelet counts > 20 x 10(9)/L were achieved within 0 to 35 (median 12) days. Fourteen patients experienced no GVHD at all; severe GVHD (grades 3 and 4) was the single major complication and the cause of death in 4 patients, occurring after early discontinuation of cyclosporine A. Relapse was reversed by allogeneic cell therapy in 2/3 cases, currently with no residual host DNA (male) by cytogenetic analysis and polymerase chain reaction. To date, with an observation period extending over 1 year (median 8 months), 22 of 26 patients (85%) treated by allogeneic nonmyeloablative stem cell transplantation are alive, and 21 (81%) are disease-free. The actuarial probability of disease-free survival at 14 months is 77.5% (95% confidence interval, 53% to 90%). Successful eradication of malignant and genetically abnormal host hematopoietic cells by allogeneic nonmyeloablative stem cell transplantation represents a potential new approach for safer treatment of a large variety of clinical syndromes with an indication for allogeneic BMT. Transient mixed chimerism which may protect the host from severe acute GVHD may be successfully reversed postallogeneic BMT with graded increments of donor lymphocyte infusions, thus resulting in eradication of malignant or genetically abnormal progenitor cells of host origin.