Abstract
In carcinomas, the nature of CD40 ligand shapes the outcome of CD40 ligation. To date, the consequences of membrane-bound CD40L (mCD40L) on its immune-stimulatory function are unknown. Here, we examined the impact of mCD40L versus soluble CD40L (sCD40L) on T24 bladder carcinoma gene expression profiling. Of 410 differentially expressed genes, 286 were upregulated and 124 downregulated by mCD40L versus sCD40L. Gene ontology enrichment analysis revealed immune-stimulatory function as the most significant enriched biological process affected by upregulated transcripts, while those downregulated were critical for cell growth and division. Furthermore, immature dendritic cells (iDC) responded to mCD40L with enhanced maturation and activation over sCD40L evidenced by higher expression levels of CD83, CD86, HLA-DR and CD54, increased secretion of IL12 and IL10 and higher tumour-antigen (TA) uptake capacity. Furthermore, autologus CD3+ T cells responded to TA-loaded mCD40L-activated DC with increased proliferation and cytotoxic response (CD107a and IFN-γ-producing CD3+ CD8+ T cells) to the tumour-loaded autologous PBMCs compared to sCD40L. Thus, these data indicate that mCD40L enhances the immunostimulatory capacity over sCD40L. Furthermore, the ability of mCD40L to also directly induce cell death in CD40-expressing carcinomas, subsequently releasing tumour-specific antigens into the tumour microenvironment highlights the potential for mCD40L as a multi-faceted anti-cancer immunotherapeutic.
Highlights
In carcinomas, the nature of CD40 ligand shapes the outcome of CD40 ligation
The membrane-bound and the soluble forms of CD40L differentially influence the outcome of CD40-CD40L interaction in carcinomas, with membrane-bound CD40L directly inducing apoptosis[11,12,13], whilst a blockade of the protein synthesis machinery is a prerequisite for cell death induction by soluble CD40L14
We have previously generated a mutant form of CD40L that is resistant to proteolytic cleavage, with retained expression at the cell membrane and we have shown that this membrane-bound CD40L (mCD40L) can induce apoptosis in CD40-expressing carcinomas, indicating its potential as an anti-cancer therapy
Summary
Transcriptional profiling to examine the differential effects of membrane-bound CD40L (mCD40L) and soluble CD40L (sCD40L) on CD40-expressing carcinoma cells with a focus on immune function. In line with our previous findings, indicating that mCD40L induces cell death in CD40 positive carcinomas by influencing the balance between apoptotic and survival signals, through posttranscriptional stabilization of TNFR-associated factor 3 (TRAF3) and destabilization of TRAF6, with no evidence of mCD40L inducing transcriptional activity in regards to TRAF3 and TRAF612, no changes in the transcriptional levels of TRAF2, TRAF3, TRAF5 and TRAF6 have been detected, TRAF1 was upregulated in RAdnCD40L compared to sCD40L (AdnL/sL; FC 30.5: p = 2.8 × 1013) and confirmed as well by RT-PCR analysis (Fig. 4D). DC co-cultured with CFPAC-1 cells expressing mCD40L exhibited higher expression levels maturation and activation (Fig. 5A) with higher detection levels of IL-12 and IL-10 in samples collected at 24 and 48 hours points (Fig. 5B) compared to sCD40L.
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