Wayne Aldrich

Phenotypic and functional differences between human dendritic cells derived in vitro from hematopoietic progenitors and from monocytes/macrophages.

We compared dendritic cells (DC) derived from CD34+ hematopoietic progenitor cells with tumor necrosis factor α and granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) to DC derived from monocytes/macrophages with interleukin‐4 (IL‐4) and GM‐CSF. Monocyte/macrophage‐derived DC demonstrated higher levels of CD1a, lower levels of CD14, greater stimulatory activity in mixed lymphocyte reactions, and greater capacity to present soluble protein antigen than CD34+ cell‐derived DC. Lymphocytes stimulated with antigen‐pulsed, monocyte/macrophage‐derived DC produced more IL‐10 than those stimulated with antigen‐pulsed, CD34+‐derived DC. Whereas CD1a+ DC could be derived from CD34+ cells in serum‐free‐ and human‐sera‐containing cultures, the derivation of CD1a+ DC from monocytes/macrophages required the presence of fetal calf serum. The spectrum of cytokine mRNA expression, the presentation of peptide antigen, and the sensitivity to human immunodeficiency virus‐1 infection of CD34+‐ and monocyte/ macrophage‐derived DC were comparable. Although cells derived by both methods are potent antigen‐presenting cells, there are differences between DC derived in vitro from hematopoietic progenitors and from monocytes/macrophages that may influence their in vivo activity. J. Leukoc. Biol. 61: 600–608; 1997.

Bisphosphonates Inhibit the Growth of Mesothelioma Cells In vitro and In vivo

Purpose: Bisphosphonates (such as risedronate and zoledronate) are widely used inhibitors of bone resorption. Despite their in vitro antiproliferative effects in various cancer cells, bisphosphonates have not exhibited significant antitumor efficacy in animal models of visceral cancer, which may be due to their poor bioavailability. The diagnostic use of radioactive bisphosphonates has revealed the accumulation of bisphosphonates in mesothelioma, which prompted us to test the antitumor efficacy of bisphosphonates in this disease. Experimental Design and Results: Treatment with either risedronate or zoledronate (2 × 10−4 to 2 × 10−6 mol/L) inhibited the growth of AB12 and AC29 mouse mesothelioma cells and induced the accumulation of unprenylated Rap1A in these cells. Both these in vitro effects were reversed by geranygeraniol, an end product of the mevalonate pathway that these bisphosphonates inhibit. Both bisphosphonates also induced the phosphorylation of the p38 mitogen-activated protein kinase in AB12 and AC29 cells. The inhibition of p38 augmented bisphosphonate-induced growth inhibition in these cells. Bisphosphonate-induced p38 phosphorylation was not reversible by geranylgeraniol. Risedronate (15 mg/kg) and zoledronate (0.5 mg/kg) inhibited the growth of s.c. tumors and increased the median survival of mice with i.p. mesothelioma tumors in vivo. Discussion: In conclusion, risedronate and zoledronate inhibit the mevalonate pathway and induce p38 activation in mesothelioma cells in vitro. The effects on the mevalonate pathway dominate because the net result is growth inhibition. Both bisphosphonates also inhibit mesothelioma tumor growth in vivo and prolong the survival of mesothelioma-bearing mice. These results support further study of bisphosphonates in the management of mesothelioma.

Phase I study of a plasmid DNA vaccine encoding MART-1 in patients with resected melanoma at risk for relapse

Immunization with plasmid DNA represents an attractive method for increasing cellular immune responses against cancer antigens. The safety and immunologic response of a plasmid encoding the MART-1 melanocyte differentiation antigen was evaluated in 12 patients with resected melanoma at risk for relapse. As a control, patients were also administered a plasmid encoding hepatitis B surface antigen (HBsAg). After establishing immunologic activity of the vaccines in mice, groups of three to six HLA-A2-positive patients were enrolled into one of three cohorts in which they received intramuscular injections of the MART-1 plasmid into the right deltoid and the HBsAg plasmid into the left deltoid at doses of 0.1, 0.3, or 1.0 mg on days 1, 43, 85, and 127. Injections were well tolerated. Toxicity was limited to grade 1 pain and injection site tenderness. Systemic toxicity was not observed. Although baseline MART-1-specific lymphoproliferative and ELISPOT responses were evident, no patient manifested increases after injection of the MART-1 plasmid. Furthermore, changes in MART-1-specific precursors were not evident after immunization as assessed by an in vitro stimulation assay. No patients manifested a lymphoproliferative response to HBsAg antigen, and significant antibody responses to HBsAg were also not observed. Although injections were safe, the authors could not show significant immunologic responses to plasmid encoding MART-1 or HBsAg using the dose, schedule, and route of administration applied. This study underscores species differences in the ability to respond to plasmid immunogens.

Enhanced transduction of mouse bone marrow-derived dendritic cells by repetitive infection with self-complementary adeno-associated virus 6 combined with immunostimulatory ligands

The potential of adeno-associated virus (AAV)-based vectors in human gene therapy is being explored for several diseases. Although sustained transgene expression and low vector-associated cellular immunity are attractive features of recombinant (r) AAV, the wider application of rAAV vectors encapsidated in serotype 2 capsid is hampered by poor transduction efficiency in many target tissues. These include ex vivo-generated dendritic cells (DC), which have demonstrated promising immunotherapeutic activity. We report here that efficient transduction of mouse bone marrow-derived DC can be achieved with self-complementary (sc) rAAV encapsidated in serotype 6 capsid. Sequential exposure of DC precursor cultures to IL-4 and GM-CSF with sc rAAV6 encoding the human tumor antigen, carcinoembryonic antigen (CEA), for 7 days followed by activation with CpG oligodeoxynucleotides (ODN) and anti-mouse CD40 antibody resulted in highly efficient transduction of DC. DC surface markers as determined by flow cytometry analysis of sc rAAV6-transduced DC were comparable to nontransduced DC. Efficiency of vector transduction and transgene expression were confirmed by immunostaining and real-time PCR. Microarray analysis of RNA from CpG ODN and CD40 antibody stimulated sc AAV6-transduced DC revealed upregulation of transcription factors and cytokines involved in immune activation and downregulation of inhibitory factors, suggesting a possible role of transcriptional activation in the observed effect. The adoptive transfer into syngeneic mice of the ex vivo-transduced and activated DC resulted in the development of CEA-specific antibody and T-helper 1-associated immune responses. Immunized mice also developed antibody to AAV6 capsid protein, which did not crossreact with AAV2 capsid protein. These studies demonstrate the potential utility of sc rAAV serotype 6-based vectors in transduction of DC for genetic vaccination approaches.

Infusion of Unpulsed Dendritic Cells Derived from Granulocyte/Macrophage Colony-Stimulating Factor-Mobilized Peripheral Blood CD34+ Cells and Monocytes in Patients with Advanced Carcinoma

Dendritic cells are potent antigen-presenting cells that are reduced in number and function in cancer patients. The infusion of dendritic cells pulsed with tumor-associated antigens has demonstrated antitumor immunologic activity. The effects of dendritic cells derived from granulocyte/macrophage colony-stimulating factor (GM-CSF)-mobilized peripheral blood CD34(+) cell and monocyte precursors when administered without antigen pulsing was examined. Patients with metastatic pancreatic and colorectal cancer received GM-CSF for 5 days. Blood was collected by a 250-ml phlebotomy. Dendritic cells were derived from CD34(+) cells with culture in GM-CSF, tumor necrosis factor-alpha, and serum-free media or from monocytes with culture in GM-CSF, interleukin-4, and autologous serum. From 2.0 to 9.4 x 10(6) dendritic cells were generated from CD34(+) cells and from 71 x 10(6) to 210 x 10(6) dendritic cells were generated from monocytes. Dendritic cells generated from CD34(+) cells expressed more CD1a than dendritic cells generated from monocytes; the ability to stimulate mixed lymphocyte reactions in vitro was not significantly different. Six patients received a single intravenous infusion of up to 5 x 10(6) autologous CD34(+) cell derived, and 6 patients, up to 50 x 10(6) monocyte-derived dendritic cells. The infusion was well tolerated. Increases in skin test reactivity and peripheral blood proliferative responses to the recall antigen, candida, were observed after the infusion of dendritic cells of both derivations. Changes in skin test reactivity and peripheral blood proliferative responses to tumor-associated peptides, including Ras and Muc1, were not. Significant numbers of functionally competent dendritic cells can be generated from patients with advanced carcinoma after GM-CSF mobilization. The infusion of these dendritic cells has nonspecific immunomodulatory activity that may have clinical significance.

Cellular Immunotherapy of Advanced Human Immunodeficiency Virus Type 1 Infection Using Autologous Lymph Node Lymphocytes: Effects on Chemokine Production

A pilot study was undertaken in patients with human immunodeficiency virus type 1 (HIV-1) infection to examine the effects of infusing autologous lymph node lymphocytes that had been cultured ex vivo in conditions designed to maximize the specific secretion of HIV-1-suppressive factors, including beta chemokines. Ten patients with CD4 cell counts between 119 and 436/microliter on antiretroviral drugs received a single infusion of CD4 and CD8 lymph node lymphocytes. There were no serious acute or chronic adverse clinical effects. Increases in serum levels of macrophage inflammatory protein 1beta (MIP-1beta) and increases in the production of MIP-1beta by peripheral blood lymphocytes in response to HIV-1 env were observed. Increases in CD4 and CD8 cell counts and skin test reactivity to recall antigens and decreases in HIV-1 virus load were also observed. This cellular immunotherapy can modulate beta chemokine production in patients with advanced HIV-1 infection and may contribute immunorestorative and antiviral activities.