Production of biologically active equine interleukin 12 through expression of p35, p40 and single chain IL-12 in mammalian and baculovirus expression systems.

Interleukin-12 (IL-12) is a key cytokine in the development of cell-mediated immune responses. Bioactive IL-12 is a heterodimeric cytokine composed of disulphide linked p35 and p40 subunits. The aim of this study was to verify biologically activity of the products expressed from equine interleukin-12 (IL-12) p35 and p40 cDNAs and to establish whether equine IL-12 could be expressed as a p35/p40 fusion polypeptide, as has been reported for IL-12a of several mammalian species. We report production of equine IL-12 through expression of p35 and p40 subunits in mammalian and insect cells and of a p35:p40 fusion polypeptide in mammalian cells. Conditioned medium recovered from cultures transiently transfected with constructs encoding equine p35 and p40 subunits or single chain IL-12 enhanced IFN-gamma production in cells derived from equine lymph nodes. Preincubation of IFN-gamma inducing preparations with anti-p40 monoclonal antibody resulted in a significant decrease in IFN-gamma induction capacity. Medium recovered from p35 and p40-expressing baculovirus infected cultures enhanced target cell IFN-gamma production and proliferation. Experimental studies in mice and other animals have revealed a therapeutic benefit of IL-12 in cancer, inflammatory and infectious disease and an adjuvant effect in prophylactic regimes. Production of a bioactive species-specific IL-12 is a first step towards an investigation of its potential application in equine species.

Apparent bypass of negative selection in CD8+ tumours in CD2-myc transgenic mice.

A role for antigen stimulation in lymphoid neoplasia has been postulated and is supported by indirect evidence that suggests that the interaction of antigen with both T cells and B cells may constitute an epigenetic event that can contribute to tumour induction or tumour progression. Using myc-bearing transgenic mice that develop mainly clonal T-cell lymphomas we have investigated the possibility that endogenous antigen-mediated clonal deletion might be overridden in tumorigenesis. CD2-myc transgenic mice were backcrossed on to a CBA/Ca background to ensure Mtv-mediated deletion of V beta 11-expressing T cells in the resultant offspring. Lymphomas arising from these mice were subsequently screened for V beta 11 expression. There was a clear correlation between the age at which mice developed neoplasia and the tumour phenotype. Mice with CD4- CD8+ tumours succumbed to thymic lymphoma at a significantly younger age than mice developing CD4+ CD8+ tumours. A small number of tumours consisted of the 'forbidden' V beta 11 phenotype, showing that cells vulnerable to transformation could escape negative selection. The majority of the V beta 11-positive tumours were CD4- CD8+ and were only observed in mice showing clinical evidence of tumour development at a relatively young age. The phenotype of these cells and the age at which tumours arose suggests that T cells escaping tolerance may be susceptible to transformation.