A study of signal transduction events involved in anti CD40 therapy lymphoma.

Abstract

CD40 is essential in enabling antigen-presenting cells to process and present antigen effectively to T cells. Previous data shows that distribution of antibody raised against CD40 in mice with syngeneic lymphoma resulted in a rapid cytotoxic T-cell response independent of T-helper cells. This response eradicated the established lymphoma and provided protection against secondary tumor re-challenge without further antibody treatment. Although precise mechanism of in vivo action of anti CD40 antibodies is not clear, indirect evidence suggests that dendritic cells (DCs) play a major role in establishment of the response.

The intracellular signalling events that lead to CD40 ligation-induction activation of IL6 gene transcription in a murine DC line, FSDC, were determined. IL6 RT-PCR and promoter assays established the responsiveness of FSDCs to anti CD40 antibody ligation. Further promoter assays showed that the transcription factors NFκB and AP1 are downstream transcriptional mediators of CD40 induced IL6 gene expression. Anti CD40 treatment of FSDCs stimulated increased expression of specific NFκB (p50:p65) and AP1 (c-Jun, JunB, JunD and c-Fos) DNA:protein complexes. Over-expression of an IκBα dominant negative repressor or a dominant negative JunD resulted in a strong inhibition of CD40 inducible IL6 promoter activity supporting a role of both transcription factors.

Upstream signal transduction events were studied by transfection of wild type and mutant human CD40 expression constructs into FSDCs followed by stimulation with an anti human CD40 antibody. These experiments revealed that anti-CD40 stimulation of NFκB and IL6 gene transcription requires specific amino acid residues in the cytoplasmic region of CD40 involved in the recruitment of TRAF2. Induction of IL6 mRNA by anti CD40 treatment was found to be a transient event and was followed by a diminution of IL6 transcripts to levels below those found in unstimulated cells. This loss of IL6 expression was associated with reduced p50:p65 NFκB binding and elevated binding of CBF1 to a site overlapping the NFκB site. Over-expression of CBF1 resulted in a profound inhibition of basal and anti CD40 induced IL6 promoter activities indicating that prolonged induction of CBF1 may contribute to the transient nature of IL6 response.

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