CD317 profiling distinguishes bone marrow stromal cell subtypes with distinct functional impact upon T cell proliferation and polarisation

Abstract

Purpose: The development of enhanced therapeutic interventions using human mesenchymal stromal cells (MSCs) for regenerative medicine is a priority in osteoarthritis research. MSCs are a heterogeneous cell population with immunomodulatory and anti-inflammatory properties. We have identified a novel biomarker (CD317) that allows the isolation of MSCs subpopulations with tissue regenerative properties (Y201, CD317-) and non-differentiating MSCs with an enriched pro-inflammatory transcriptional profile (Y202, CD317+) that make up an average of 9% of the total MSCs population. We investigated the immunomodulatory function of these subpopulations to understand their role in disease processes and potential impact on regenerative therapies. Methods: MSC subpopulations (Y201 and Y202) were co-cultured with human T cells to examine the effects on proliferation and polarisation. Primary T cell populations labelled with VPD450 proliferation dye and activated with CD3/CD28 beads were cultured for 5 days with MSCs (either Y201 or Y202) or alone. T cells without activation were used as controls. After 5 days, CD4+ T cells were assessed for reduction in VPD450 intensity and cultures examined for T helper polarisation. CD4+ cells were evaluated for interferon gamma, interleukin 17a, interleukin 4 and nuclear protein FOXP3. These T helper subsets are representative of pro-inflammatory (Th1, Th17) and anti-inflammatory (Th2, regulatory T cells (Tregs)) cells respectively. Results: T cells cultured alone underwent increased proliferative cycles (mean 6.283±0.383) compared with those co-cultured with Y201 (1.95±0.17, P<0.001) and Y202 (3.76±0.55, P<0.005). Y202 MSCs were less effective at suppressing T cell proliferation compared to Y201 MSCs (P<0.05). A proliferative index was calculated to represent proportions of cells undergoing multiple divisions. This index reduced from T cell alone (7.60±1.00) when T cells are co-cultured with Y201 (1.47±0.11, P<0.001) or Y202 (3.16±0.40, P<0.005). When assessed for T helper polarisation, a highly significant proportion of CD4+ cells did not express any of the markers examined (80.29±2.33%) compared to co-cultures with Y201 (21.23±6.60%) or Y202 (7.54±4.60%) (P<0.001). T cells co-cultured with Y201 and Y202 MSCs differentiated predominantly towards anti-inflammatory Th2 cell types (55.32±8.95% and 59.72±6.02% respectively) compared with control T cells alone (9.70±0.51%, P<0.005). However, Y202 induced an increase in pro-inflammatory Th1 cells (14.43±2.03%) compared to Y201 (7.04±0.62%) and T cells cultured alone (5.23±1.96%) (P<0.05). Both Y202 and Y201 MSCs also increased pro-inflammatory Th17 polarisation (6.61±1.00%, 7.40±1.40% respectively) compared to T cells alone (1.78±0.24, P<0.05). Only Y202 MSCs prompted a significant increase in Treg production (11.63±2.55%) compared to T cells alone (3.01±0.68%) (P<0.05). These results demonstrate clear and distinct functional interactions between Y201 and Y202 MSCs and T cell populations. Conclusions: These data demonstrate that the transcriptional profile of CD317+ Y202 MSCs translates to functional outcomes through direct impact upon the cells of the immune system. Y202 MSCs encourage increased polarisation of T helper cell subsets with a more pro-inflammatory profile compared to Y201. Further investigation of the impact of CD317+ MSCs on the immune system is underway to assess the impact of these cells in osteoarthritis and autoimmunity. Stem cell based therapies do not account for the 9% of MSCs that are CD317+. The inclusion of a cell fraction of non-migratory, non-differentiating, proliferative and pro-inflammatory population of cells in cell therapies could contribute to poor tissue regenerative responses in clinical interventions. These results strengthen the case for negative selection of MSCs using biomarker CD317 for tissue regenerative applications and highlight the potential for an association between CD317+ MSCs and autoimmune disorders.