1021 - Transcriptional correlation analysis of the CD180/MD-1 complex and toll-like receptors and signaling lymphocyte activation family receptors in chronic lymphocytic leukemia.

Introduction: CD180 is a toll-like receptor (TLR) which is highly co-expressed with the satellite molecule MD-1 on surface of normal B cells and heterogeneously on the surface of CLL cells[1]. The natural ligand for CD180 is presently unknown although evidence suggests that CD180/MD-1 signals in concert with other surface receptors including signaling lymphocyte activation receptor 1 (SLAMF1) in CLL[2] and TLR9 in healthy B cells[3]. Little is known however about the interactions between CD180/MD-1 and TLRs and other members of the SLAMF family in CLL. We therefore set out to identify novel signaling partners for CD180/MD-1 by analyzing the co-expression CD180 and MD-1 relative to TLRs and SLAMF receptor genes at the transcriptional level.

Methods: We accessed dataset GSE126595[4] from the gene expression omnibus which contained normalized gene expression for 726 CLL patients (untreated = 337; relapsed = 389). We screened for the transcriptional expression of TLRs and SLAMF receptors. We considered the receptor to be expressed by CLL cells if the mRNA expression was higher than the CD3G gene which is exclusively expressed by T cells. The CD19 gene, which is known to be highly expressed by CLL cells, was used as a positive control. Pearson’s correlation was applied to measure relationships between CD180/MD-1 and TLR/SLAMF gene expression.

Results: CD180 and MD-1 mRNA were found to be highly expressed compared to CD19 (Figure 1a). Among the classical TLR genes, TLR7 was the most highly expressed followed by TLR9, TLR6 and TLR10. TLR2, TLR4 and the TLR4-associated satellite molecule MD-2 were all expressed at very low levels (Figure 1a). TLR3 and TLR8 were not expressed by CLL cells (Figure 1a).

SLAMF2 was the most highly expressed by CLL cells followed by SLAMF3, SLAMF6 and SLAMF5. SLAMF1, SLAMF3, SLAMF5 and SLAMF6 genes were highly expressed by CLL cells (Figure 1b). SLAMF1 was also expressed albeit at lower levels (Figure 1b). The remaining SLAMF receptor genes were found to be negative or marginally positive and were therefore excluded from the correlation analysis.
CD180/MD-1 mRNA was found to be highly correlated with TLR1 and TLR6 mRNA (Table 1) which code for surface TLRs. Given that CD180/MD-1 is expressed on the cell surface, it is possible that the complex may dimerise with TLR1 or TLR6 the cell surface to detect pathogen-associated ligands. TLR1 and TLR6 are therefore rational novel targets for studying receptors which topologically associate with CD180/MD-1 for ligand detection.
As expected, there was a negative correlation between CD180/MD-1 and TLR4 given that human B cells do not express this receptor, but a positive correlation with MD-2 (Table 1), the satellite molecule which is co-expressed with TLR4[5]. The reasons for this are presently unclear however, it is hypothesised that MD-1 and MD-2 are evolved from a common ancestral molecule which appeared during the early development of vertebrates[6]. This could therefore mean that the same transcriptional regulation pathways exist for both MD-1/CD180 and MD-2 resulting strong corelations of the two genes. Studies should be conducted to determine if there is aberrant expression of MD-2 protein by CLL cells which may be of biological relevance.

CD180/MD-1 were also both found to be highly correlated with TLR7, TLR9, and TLR10 which code for TLRs expressed in the endosomes (Table 1). This indicates potential role for CD180/MD-1 in modulating immune responses to intracellular pathogens in CLL.
SLAMF2, which serves as a ligand for CD2 and SLAMF4[7], was highly correlated with CD180/MD-1 indicating a potential role for the complex in modulating the interactions between CLL cells and various other haematopoietic cell types. There was also a high correlation between CD180/MD-1 and SLAMF6 and SLAMF5.

Conclusions: Co-expression analysis of CD180/MD-1 at the transcriptional level has revealed potential novel signaling partners for this TLR complex, providing rationale for further phenotypic and functional studies in CLL. Since CD180 can rewire IgM-mediated signaling to a pro-apoptotic pathway in CLL[8], it will be important to determine if CD180/MD-1 has synergistic or antagonistic effects on the signaling partners proposed here. The high correlation between CD180/MD-1 and TLR10 and SLAMF6 mRNA suggest these receptors as rational starting points for such studies.