Abstract
Dendritic cells (DC) play a key role in the pathogenesis of Graft versus Host Disease (GvHD), a complication of haematopoietic stem cell transplantation and offer an attractive target for therapy. Regulatory T cells (Treg) have a potent immunoregulatory effect on the maturation and the antigen-presenting cell (APC) function of DC and adoptive transfer of Treg is highly efficacious in the induction of tolerance in an experimental model of GvHD and has entered Phase I clinical trials. Several mechanisms of suppression have been proposed, including Treg acting directly on DCs, attenuating their antigen-presenting and co-stimulatory functions by arresting their maturation. However, the molecular basis underpinning these effects in DCs remains ill-defined. We investigated the effect of Treg treatment on DCs by conducting gene expression profiling and confirmed the functional consequences using downstream assays.
Immature, mature and Treg-treated DCs were generated from immuno-magnetic isolated monocytes (im-DC, mat-DC and Treg-DC, respectively) and moDC populations were generated using a well-established 6 day culture with GM-CSF and IL-4, followed by 24 hour LPS maturation. Treg were added on day 3 of culture at a 3:1 ratio. All cell populations were harvested on day 7 and sorted via FSC/SSC/CD3neg gating to remove Treg present in the co-culture and control for any changes in gene expression caused by shear stress. Gene expression profiling was carried out using the Illumina HumanHT12 microarray platform. Data was processed using R/Bioconductor workflows and the functional significance of differentially expressed genes was evaluated using Ingenuity Pathway Analysis software.
Mat-DC and Treg-DC expression profiles were compared relative to the im-DC for data analysis. Upon LPS treatment, the levels of 1834 unique genes were differentially regulated in mat-DC by at least twofold (862 genes upregulated/972 downregulated) compared to the im-DC counterparts. In the Treg-DC, 1326 unique genes were differentially modulated (633 genes upregulated/693 genes downregulated). Microarray analysis of the CD markers identified a higher expression of the previously identified surface markers CD80, CD83 and CD86 in the mat-DC compared to the Treg-treated counterpart (validated by flow cytometry), confirming the semi-mature phenotype.
Novel findings from the dataset include the reduction of the endocytotic-related genes, CD206 and CD209, in the Treg-DCs compared to the im-DC and this reduction manifested functionally in an impaired antigen uptake, as assessed by FITC-Dextran. Additionally, the surface marker, CD38, was downregulated in the Treg-DC compared to the mat-DC, confirmed by flow cytometry. CD38 has been shown to be NFκB-dependent and a marker of maturation in monocyte-derived DCs, further supporting the semi-mature phenotype. Furthermore, CD38 is functionally involved in CD83 expression and IL-12 induction. We assessed IL-12 cytokine secretion by Treg-treated DCs and showed a significantly reduced level of induction compared to mat-DC (p=0.0079).
Pathway analysis revealed NFκB-related genes to be downregulated in the Treg-DC compared to the mat-DC. These differentially expressed genes included the TLR-adaptor protein, MYD88, the NFκB subunit, RELB and an inhibitor of NFκB, NFκB1A. This finding, coupled to the importance of NFκB signalling pathway in DC function, prompted us to investigate it at the functional level by measuring levels of phosphorylation of serine 536 of the RelA subunit as a marker of activity in response to LPS stimulation. DC cultured in the absence of Tregs (mat-DC) showed significantly higher levels of Ser536 phosphorylation when compared to those unstimulated cells (im-DC) (p= 0.0018). Concordant with the gene expression data, Treg-treated DCs (Treg-DC), showed a significantly attenuated NFκB activation when compared to their LPS-stimulated DCs counterparts (p = 0.0191), however, signalling was not completely abolished compared to those unstimulated DCs (p= 0.0003).
In conclusion, gene expression profiles of Treg-treated DCs are significantly different to their mat-DC and im-DC counterparts. Here, we present the novel finding that Tregs modulate DC function, in part, by attenuation of the NFkB signalling pathway, arresting the DCs at a semi-mature phenotype, as evidenced by expression arrays and functional assays.
Disclosures
No relevant conflicts of interest to declare.
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