Comparative analysis of murine dendritic cells derived from spleen and bone marrow.

In order to improve upon preclinical tumor vaccine strategies that employ dendritic cells (DC), we now have compared short-term cultures of spleen- and GM-CSF/IL-4-stimulated bone marrow (BM) to determine if differences exist in phenotype and function of murine DC derived from primary and secondary hematolymphoid organs. Although cultures of BM contained a lower percentage of DC compared to spleen, their capacity to stimulate a primary allogeneic mixed leukocyte reaction (MLR) and to uptake fluorescent dextran was substantially greater. In addition, the overall yields of DC per animal was at least twofold greater from BM compared to spleen. Cultures of BM harvested at day 3, 6, or 9 stimulated comparable levels of primary allo-MLR on a per-cell basis. However, there was a consistent loss (at least twofold) of all cells occurring beyond day 6 as compared with cell yields from earlier time points. Importantly, we also improved on methods to rapidly obtain highly enriched DC (> 90%) from BM, which has obviated the reported prior need for complex antibody and complement treatments to remove contaminating mature T and B lymphocytes, Ia-bearing cells, and granulocytes before DC generation. In contrast, although similar purity of DC with similar phenotype and function could be obtained from the spleen, substantial loss in yield occurred, suggesting a further difference in DC between the two tissue sources. The overall yield of DC derived from spleen and BM cultures could be substantially increased by in vivo pretreatment of the donor animals with recombinant Flt3-L. Collectively, these studies demonstrate that notable differences exist in DC preparations derived from spleen vs. BM and that BM provides the preferred source of DC that can be rapidly enriched to high purity for use in further vaccine development.

Genetic modification of human peripheral blood lymphocytes with a transdominant negative form of Rev: safety and toxicity.

A transdominant mutant form of the rev gene, M10, confers resistance to infection by the human immunodeficiency virus (HIV) in vitro and is currently under investigation as a potential intervention in acquired immunodeficiency syndrome (AIDS). In this report, we examine three issues relevant to the safety of autologous transfer of human T cells genetically modified with Rev M10. First, the potential for malignant transformation was assessed in vitro using interleukin-2 (IL-2) dependence and fibroblast transformation assays, and tumorigenicity was evaluated in severe combined immunodeficient (SCID) mice. Possible toxicity was evaluated by pathologic analysis following adoptive transfer of genetically modified human T cells into SCID mice. Second, methods were developed that permit T cell activation required for gene transfer but do not allow replication of endogenous HIV. Third, T cell function was evaluated in peripheral blood lymphocytes (PBL) of HIV-seropositive donors transduced with Rev M10 and compared to a negative control mutant, delta Rev M10. By all criteria, no oncogenicity or toxicity was observed. Human T cells transduced with these vectors did not grow in the absence of IL-2 in vitro, and no tumors were observed following transplantation of genetically modified human cells into recipient SCID mice. Histopathological analysis of heart, lung, liver, spleen, and kidney of animals 1-21 weeks following adoptive transfer of gene-modified human T cells revealed no significant abnormalities. Additionally, no differences were observed in the pattern of cytokine secretion in enriched human PBL expressing Rev M10 compared to delta Rev M10. (ABSTRACT TRUNCATED AT 250 WORDS)