Mesothelial Cells Exhibit Characteristics of Perivascular Cells in an In Vitro Angiogenesis Assay.

Mesothelial cells have been shown to have remarkable plasticity towards mesenchymal cell types during development and in disease situations. Here, we have characterized the potential of mesothelial cells to undergo changes toward perivascular cells using an in vitro angiogenesis assay. We demonstrate that GFP-labeled mesothelial cells (GFP-MCs) aligned closely and specifically with endothelial networks formed when human dermal microvascular endothelial cells (HDMECs) were cultured in the presence of VEGF-A165 on normal human dermal fibroblasts (NHDFs) for a 7-day period. The co-culture with GFP-MCs had a positive effect on branch point formation indicating that the cells supported endothelial tube formation. We interrogated the molecular response of the GFP-MCs to the angiogenic co-culture by qRT-PCR and found that the pericyte marker Ng2 was upregulated when the cells were co-cultured with HDMECs on NHDFs, indicating a change towards a perivascular phenotype. When GFP-MCs were cultured on the NHDF feeder layer, they upregulated the epithelial-mesenchymal transition marker Zeb1 and lost their circularity while increasing their size, indicating a change to a more migratory cell type. We analyzed the pericyte-like behavior of the GFP-MCs in a 3D cardiac microtissue (spheroid) with cardiomyocytes, cardiac fibroblasts and cardiac endothelial cells where the mesothelial cells showed alignment with the endothelial cells. These results indicate that mesothelial cells have the potential to adopt a perivascular phenotype and associate with endothelial cells to potentially support angiogenesis.

Enhancing Efficacy of TCR-engineered CD4 + T Cells Via Coexpression of CD8α.

Adoptive cell therapy with T cells expressing affinity-enhanced T-cell receptors (TCRs) is a promising treatment for solid tumors. Efforts are ongoing to further engineer these T cells to increase the depth and durability of clinical responses and broaden efficacy toward additional indications. In the present study, we investigated one such approach: T cells were transduced with a lentiviral vector to coexpress an affinity-enhanced HLA class I-restricted TCR directed against MAGE-A4 alongside a CD8α coreceptor. We hypothesized that this approach would enhance CD4 + T-cell helper and effector functions, possibly leading to a more potent antitumor response. Activation of transduced CD4 + T cells was measured by detecting CD40 ligand expression on the surface and cytokine and chemokine secretion from CD4 + T cells and dendritic cells cultured with melanoma-associated antigen A4 + tumor cells. In addition, T-cell cytotoxic activity against 3-dimensional tumor spheroids was measured. Our data demonstrated that CD4 + T cells coexpressing the TCR and CD8α coreceptor displayed enhanced responses, including CD40 ligand expression, interferon-gamma secretion, and cytotoxic activity, along with improved dendritic cell activation. Therefore, our study supports the addition of the CD8α coreceptor to HLA class I-restricted TCR-engineered T cells to enhance CD4 + T-cell functions, which may potentially improve the depth and durability of antitumor responses in patients.

Characterisation of Cultured Mesothelial Cells Derived from the Murine Adult Omentum.

The human omentum has been long regarded as a healing patch, used by surgeons for its ability to immunomodulate, repair and vascularise injured tissues. A major component of the omentum are mesothelial cells, which display some of the characteristics of mesenchymal stem/stromal cells. For instance, lineage tracing studies have shown that mesothelial cells give rise to adipocytes and vascular smooth muscle cells, and human and rat mesothelial cells have been shown to differentiate into osteoblast- and adipocyte-like cells in vitro, indicating that they have considerable plasticity. However, so far, long-term cultures of mesothelial cells have not been successfully established due to early senescence. Here, we demonstrate that mesothelial cells isolated from the mouse omentum could be cultured for more than 30 passages. While epithelial markers were downregulated over passages in the mesothelial cells, their mesenchymal profile remained unchanged. Early passage mesothelial cells displayed clonogenicitiy, expressed several stem cell markers, and up to passage 5 and 13, respectively, could differentiate along the adipogenic and osteogenic lineages, demonstrating stem/progenitor characteristics and differentiation potential.