Known expression and interactions

CD248 is a relatively new molecule that has been identified as having a role in promoting the growth and invasiveness and metastasis of tumours (Valdez et al., 2012, Maia et al., 2011), as well as having been identified on a number of stromal cell markers, making this molecule an interesting target in cancer and in other inflammatory diseases. The expression of CD248 has been shown to be mainly on fibroblasts and pericytes, and in the lymph node capsule, as previously mentioned (MacFadyen et al., 2005, Virgintino et al., 2007, Christian et al., 2008, Simonavicius et al., 2008, Tomkowicz et al., 2010, Simonavicius et al., 2012).

Studies using the CD248 knockout mouse to investigate the role of this molecule in the progression of cancer have found that these mice exhibit reduced cancer growth following implantation of cancerous cells, compared to wild type animals (Nanda et al., 2006). Investigation of the expression of CD248 within tumours have found that CD248 is expressed on pericytes that surround the tumour vasculature, and so may it may potentially play a role in supporting tumour angiogenesis and growth (Bagley et al., 2009, Tomkowicz et al., 2007, MacFadyen et al., 2005, MacFadyen et al., 2007). An article published by Nanda et al. (Nanda et al., 2006) has shown that the progression of tumour development in different tumour models in both immunocompromised and immunocompetent mice was compromised in the absence of CD248. In this study, CD248-/- _{nude mice not only had an increased}

survival compared to the WT counterparts, but also increased incidence of metastases. This study found expression of CD248 on the tumour vasculature in the WT mice, and hypothesised that it is involved in angiogenesis, as the authors also observed abnormal tumour development, with the

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CD248-/- _{mice developing much longer, flatter and less rounded tumours which had a decreased}

invasiveness compared to the WT mice. Importantly, the authors were unable to find any evidence for a defect in s.c. vascularisation in a wound healing model. This differential response in the normal angiogenesis and in the development of the tumour vasculature indicates that CD248 plays an interestingly varied role in these different disease processes. However, the authors were unable to explain how CD248 is able to produced this nuanced response (Nanda et al., 2006). Interestingly, the lack of CD248 appears to result in a significant increase in the migratory velocity of cells, this was observed both in CD248-/- _{MEF, but also when the human osteosarcoma cell line is transfected with}

CD248, the migratory velocity of the CD248-expressing cells in significantly increased (Lax et al., 2010). This is relevant when compared to the decreased invasiveness observed by Nanda et al. (Nanda et al., 2006).

Expression of CD248 has also been studied in human disease states, and it has been found on fibroblasts in the synovium from rheumatoid arthritis patients and in chronic kidney disease. Other diseases that are associated with an increased expression of CD248 include cirrhotic end stage liver disease, where CD248 mRNA has been identified on the hepatic stellate cells (HSC) (Wilhelm et al., 2016). In a variety of human tumours, including small intestine, renal cell carcinoma, melanoma and brain metastasis, CD248 expression has been found to be highly upregulated when compared to the very low resting expression (MacFadyen et al., 2005).

CD248 is expressed in the developing embryo in a number of different developing tissues at different stages of development, as early as embryonic day 8.5 (Opavsky et al., 2001). Weak expression can be observed in the heart at E10 (Huang et al., 2011) and in the developing vessels surrounding the brain (Rupp et al., 2006). Expression on mesenchymal cells and in the developing lymphoid organs can be observed from embryonic day 15 onwards, with the expression becoming restricted specifically to lymphoid structures as development progresses (MacFadyen et al., 2007, Lax et al., 2007).

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Mesenchymal cells expressing CD248 are able to differentiate into various lineages, including adipose tissue and bone (Naylor et al., 2012, Bechar, 2012). The importance of the CD248 expression by these cell types is evidenced by work done by this laboratory, which has shown that the CD248 knockout mice develop stronger bones than their wild type counterparts at the expense of the development of adipose tissues, resulting in stronger bones and less fat in the CD248 knockout mice (Naylor et al., 2012, Bechar, 2012). However, the role of CD248 in maintaining the balance of these three distinct lineages requires further investigation in order to fully understand the differentiation of these cell types. In vitro cultured cells are also understood to express CD248, notably mouse embryonic fibroblasts and other mesenchymal stem cells as well as pericytic cells (Christian et al., 2008, Bagley et al., 2009). Expression of CD248 cannot be observed on human umbilical vein endothelial cells, even upon stimulation with a variety of different factors (Rettig et al., 1992, MacFadyen et al., 2005, Carson- Walter et al., 2009). The stromal cell scaffold of lymphoid organs has been shown to be derived from mesenchymal stem cells (Castagnaro et al., 2013). Interestingly, during lymph node embryogenesis, CD248 is highly expressed in the spleen, thymus and lymph node. This expression becomes progressively restricted to the capsule (Lax et al., 2007). After birth in resting conditions, the capsule is the only CD248+ _{structure in the organ, excluding the pericytes that surround bloods vessels that}

express CD248 in the secondary lymphoid organs (MacFadyen et al., 2005, Lax et al., 2007, Teicher, 2007, Armulik et al., 2011). This would indicate that CD248 plays some role in controlling the development of the lymphatic tissue, although the mechanism by which this is able to proceed is not understood.

During the immune response, the expression of CD248, which is restricted to the lymph node capsule in homeostatic conditions, has been shown to be significantly upregulated throughout the spleen in response to salmonella infection (Lax et al., 2007). Further investigation into this mechanism, exploiting the CD248-/- mice has demonstrated that expansion of popliteal lymph nodes (pLN) in response to paw pad immunisation with NP-CGG is compromised, resulting in a significant reduction

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in the size of these LN at the peak of the immune response. Interestingly, the authors were unable to identify any defects in the immune response, most notably with normal antibody production in the CD248-/- mice (Lax et al., 2010). The role of CD248 in other inflammatory processes was investigated in a model of liver inflammation following carbon tetrachloride (CCI4) injury (Wilhelm et al., 2016). The

authors found no difference in the inflammatory response to this stimulus as there was no significant difference in the expression of hepatic CD45. The CD248-/- _{mice also demonstrated a significant}

reduction in the levels of fibrosis compared to WT mice, a key regulator of which is understood to be the cytokine TGF-β, which was shown to be reduced in the WT mice compared to the CD248-/-_.

However, there is no difference in a number of other pro-fibrotic factors, including matrix metalloproteinase-2 (MMP-2), MMP-9 and tissue inhibitor of matrix metalloproteinase-1. This reduction in TGF-β appears to result in a significant reduction in the accumulation of collagen in the portal area of the liver, as well as a reduction in the mRNA expression of procollagen α-1, which combine to result in a significant reduction in liver fibrosis in CD248-/- _{mice. However, the resolution}

of fibrosis following injury shows no significant difference between the WT and CD248-/- _{mice. This}

data is particularly interesting as it appears to show that CD248 plays a very subtle role in the progression of this disease, as inflammation is unaffected in the CD248-/-, whereas there is a reduction in the level of fibrosis, which was thought to be a product of the inflammation. This interestingly nuanced role of CD248 was also reported on in previous work published by this group (Lax et al., 2010), and also in the context of cancer progression as discussed before in the report by Nanda et al. (Nanda et al., 2006).