Enhancing the immunotherapeutic potential of mycobacteria by transfection with tumour necrosis factor-alpha

Abstract

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.

Figure 1

The effect of wild-type and recombinant forms of M. smegmatis on ICAM-1 expression. Bladder tumour cells were stained following 24, 48 and 72 hr incubation with 5×10⁴ CFU/ml mycobacteria and ICAM-1 levels were compared with cells incubated with medium alone. Results show significantly greater up-regulation of ICAM-1 expression by the TNF-α-transfectant compared with wild-type and vector-transfectant. A typical experimental outcome is presented for each cell line from four similar repeat determinations and means±standard deviations are calculated from triplicate assessments.

Figure 2

The quenching of ICAM-1 expression by anti-huTNF-α antibody. MGH-U1 tumour cells were incubated for 48 hr with medium, TNF-α-transfected or mock-transfected M. smegmatis clones both with and without the addition of 5 μmg/ml anti-huTNF-α. The mean percentage ICAM⁺ cells±standard deviation are calculated from triplicate determinations.

Figure 3

A comparison of wild-type and recombinant mycobacterial antiproliferative potency measured by incorporation of [³H]thymidine by bladder tumour cell lines. Cells were incubated with medium alone or with 5×10⁴ CFU/ml wild-type, TNF-α-transfected or vector-transfected M. smegmatis over 72 hr. Results show TNF-α-transfected M. smegmatis clones are significantly more effective than wild-type and marginally more effective than vector-transfected strains at arresting tumour cell growth. A typical result is presented for each cell line showing the mean±standard deviation of six replicate wells. Each experiment was performed on four separate occasions with reproducible results.