Enhancing the immunotherapeutic potential of mycobacteria by transfection with tumour necrosis factor-alpha.
In an attempt to enhance the anti-tumour properties of mycobacteria we have developed recombinant forms of Mycobacterium smegmatis which express and secrete biologically active human tumour necrosis factor-alpha (TNF-alpha). This was achieved by transfecting M. smegmatis using shuttle plasmids incorporating the cDNA sequence for the human TNF-alpha mature peptide. In vitro experiments on a panel of human bladder tumour cell lines (EJ18, MGH-U1, RT4, RT112) indicate that our genetically modified mycobacteria are more effective than wild-type at inducing or up-regulating the expression of intracellular adhesion molecule-1 and the secretion of an array of proinflammatory cytokines [interleukin-1 (IL-1), IL-6, IL-8, granulocyte-macrophage colony-stimulating factor]. We have also demonstrated increased adhesion molecule and cytokine expression in response to mycobacteria transfected with vector containing no gene insert. However, this was not as pronounced as that observed following tumour cell stimulation by the TNF-alpha-transfected strain. In contrast, in three out of four tumour cell lines all M. smegmatis strains were found to down-regulate the secretion of the anti-inflammatory cytokine transforming growth factor-beta1. Our studies have also confirmed that M. smegmatis is a powerful inhibitor of bladder tumour cell growth and revealed that its antiproliferative potency is enhanced by transfecting with human TNF-alpha and, to a lesser extent, with vector alone. All M. smegmatis strains were effective in the activation of peripheral blood leucocyte cultures. However, no differences were observed in the ability of the TNF-alpha-transfected, mock-transfected and wild-type mycobacteria to induce tumour cell killing activity. These results suggest that the immunomodulatory effects of M. smegmatis can be enhanced by transfection with vectors which allow the secretion of human TNF-alpha, thus increasing mycobacterial immunotherapeutic potential.
Haley
J L
JL
Department of Surgery, Lister Surgical Research Laboratories, University of Edinburgh, Royal Infirmary, Edinburgh, UK.
Young
D G
DG
Alexandroff
A
A
James
K
K
Jackson
A M
AM
D003001:Cloning, Molecular; D016207:Cytokines / Q000096:biosynthesis; D003601:Cytotoxicity Tests, Immunologic; D004926:Escherichia coli / Q000235:genetics; D005434:Flow Cytometry; D005822:Genetic Vectors; D006801:Humans; D016219:Immunotherapy, Adoptive; D020102:Mycobacterium smegmatis / Q000235:genetics*; D009369:Neoplasms / Q000276:immunology / Q000628:therapy; D011994:Recombinant Proteins / Q000235:genetics; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*
D016207:Cytokines; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha