PHENOTYPE AND IMMUNOMODULATORY PROPERTIES OF PαS MSCs
3.1 Chapter Rationale and Aims
3.3.1 Chapter Summary
PαS MSCs were successfully isolated and cultured according to previously published protocols (Houlihan et al., 2012). These cells had characteristic spindle-‐shaped morphology, were able to form CFU-‐F and undergo tri-‐lineage differentiation into bone, fat and cartilage. PαS MSCs were also able to be expanded in standard culture conditions and expressed characteristic MSC markers, thereby meeting the ISCT criteria for MSCs (Dominici et al., 2006). The immunosuppressive phenotype of PαS MSCs have also been described for the first time, with different wild type mouse strains utilising distinct mechanisms to suppress T cell proliferation. Balb/c-‐derived PαS MSCs secreted NO to inhibit CD4 lymphocytes that were stimulated with anti-‐CD3e antibody and CD19 B cells. C57BL/6-‐derived PαS MSCs failed to suppress in the same assay, and required the development of a more complex in vitro system involving the use of TCR transgenic OT-‐1 mice. Preliminary results show reduction in CD8 T cell numbers after co-‐culture with C57BL/6-‐PαS MSCs in the splenocyte reaction, although more work is required to elucidate the mechanism behind this.
3.3.2 Phenotype of PαS MSCs
Protocols for the prospective isolation of murine MSCs represent a major advance in the field. PαS MSC yields achieved from C57BL/6 and Balb/c mice were similar in number to the ones reported by Morikawa et al. (2009). Approximately 5000 to 8000 PαS MSCs were isolated per mouse, and the flow cytometry plots looked similar to the original study. All
cells were fibroblastic in morphology, and cell surface staining revealed <1% positivity for haematopoietic or leukocytic markers in PαS cultures after one passage. This compares favourably with older protocols in which leukocytic cells persisted in culture for several weeks, even after serial passaging (Meirelles Lda and Nardi, 2003, Phinney et al., 1999). Population doubling times for PαS cells at early passage was approximately 55 hours, which is similar to the 50.6 hours reported by Morikawa and colleagues. Interestingly, we only observed a CFU-‐F forming efficiency of 1 in every 66 PαS MSCs, which is lower than the 1 in 50 reported by Morikawa et al. (2009). As CFU-‐F was performed on freshly-‐sorted PαS MSCs, cell death due to the sorting procedure may have negatively affected the CFU-‐F efficiency. Pinho et al. (2013) report similar CFU-‐F forming efficiency in PDGFRα+CD51+ MSCs, and
suggest that the “harsh” sorting procedure adversely affects their cell viability. I did observe some dead, floating cells in PαS MSC cultures 24 hours after cell sorting, which gives further evidence for this theory. As such, PDGFRα and Sca-‐1 enriches for cells with MSC-‐like phenotype from mouse BM, but not every PαS MSC can perform all the functions expected of MSCs. However, the CFU-‐F efficiency of PαS MSCs is significantly higher than those reported in plastic-‐adherent studies, which range from 1 in every 9000 BM cells (Meirelles Lda and Nardi, 2003) to 1 in 3.3x106 BM cells (Phinney et al., 1999).
The tri-‐lineage differentiation of PαS MSCs was demonstrated using well-‐established in vitro techniques. PαS MSCs readily underwent osteogenic and chondrogenic differentiation, although adipogenic differentiation was less impressive. Clonal PαS MSC differentiation studies by Morikawa et al. revealed all clones (6/6) could differentiate towards bone and
cartilage, but less than half (2/6) could differentiate towards fat (Morikawa et al., 2009). Poor adipogenic differentiation (relative to osteogenic differentiation) was also observed in previous studies of mouse (Cunha et al., 2013), sheep (Heidari et al., 2013), and rat (Peng et al., 2008) MSC populations. The reasons behind this disparity are unclear and could be due to heterogeneity in the PDGFRα+Sca-‐1+ population or the fact that BM MSCs are naturally primed towards bone and cartilage (skeletal tissue) over fat (connective tissue). Additionally, culture on a stiff surface has also been shown to enhance osteogenic differentiation in MSC populations (Engler et al., 2006).
3.2.3 Immunosuppressive phenotype of PαS MSCs
In vitro T cell suppression assays demonstrated that Balb/c-‐derived PαS MSCs suppressed
CD4 T cell proliferation in a dose-‐dependent manner. Blocking studies revealed that local release of NO by PαS MSCs was responsible for immunosuppression. Additional experiments using PαS MSCs isolated from transgenic iNOS-‐/-‐ mice provided further proof for this finding. A comparative study by Ren et al. (2009) identified that mouse MSCs exclusively use NO secretion to immunosuppress while human MSCs secrete IDO. Our findings back up this hypothesis, as we did not see any effect when using an IDO inhibitor. It is interesting to note that inhibition of NO secretion caused a complete reversal in the immunosuppressive phenotype of PαS MSCs. Past studies have noted that neutralisation of one factor secreted by mouse or human MSCs does not result in a complete reversal of suppression, suggesting that there are other factors in play (Ben-‐Ami et al., 2011). This degree of redundancy could be due to the heterogeneous stromal populations used in past studies, with each having a
different mechanism of immunosuppression. The purified population of PαS MSCs used in this study displayed a ‘unified’ response to the compounds tested and demonstrates nicely how prospective isolation could help reduce diversity in the MSC field.
NO is a potent signalling molecule with a short half-‐life that is involved in many physiological processes ranging from vasodilation to immune regulation (Bogdan, 2001). NO production by macrophages has been shown to suppress T cell proliferation via the inhibition of STAT5 phosphorylation, resulting in cell cycle arrest (Mazzoni et al., 2002, Bingisser et al., 1998). Interestingly, Sato et al. (2007) identified the same mechanism at play with murine MSCs, highlighting the importance of the NO-‐STAT5 axis. Future work could include western blotting for phosphorylated STAT5 in CD4 T cells to identify whether the same mechanism occurs with PαS MSC co-‐culture.
Recent studies have identified MSCs as key players in the HSC niche, as they are the precursors of multiple niche components and can secrete factors that are crucial for HSC maintenance (Frenette et al., 2013). However, the physiological role for MSC-‐mediated immunosuppression and NO release in the BM niche is unclear. Some authors have suggested that MSCs may function to protect HSCs from immune mediated damage by creating an ‘immunoprivileged zone’ around these cells (Hsu and Fuchs, 2012). However, an elegant imaging study by Fujisaki et al. (2011) demonstrates that Tregs co-‐localise with HSCs
in vivo to create an immunoprivileged site that enabled allogeneic-‐HSCs to avoid rejection
remains to be seen whether they do play an immunosuppressive role in the BM niche. Data from the Matsuzaki group suggest that mismatched naïve PαS MSCs can trigger the onset of GvHD in a mouse model of BM transplantation (Ogawa et al., 2012). The selective depletion of PαS MSCs from BM grafts reduced fibrosis across all organs. From these findings, it can be speculated that naïve PαS MSCs directly isolated from their niche are not suppressive and that immunosuppression is a property acquired through in vitro culture. This hypothesis is further backed up by the various human phase I/II trials using in vitro cultured MSCs in the treatment of GvHD that report favourable outcomes (Le Blanc et al., 2008, Le Blanc et al., 2004b). Future studies using freshly-‐isolated PαS cells in immunosuppression assays would be required to test this hypothesis.
3.2.4 Strain-‐specific differences in Immunosuppression
PαS MSCs isolated from C57BL/6 mice failed to suppress T cell proliferation in our standard assay, leading us to hypothesise that there might be strain-‐specific differences in the immunosuppressive mechanism of MSCs isolated from Balb/c and C57BL/6 mice. Hashemi et al. (2013) compared the immunosuppressive properties of conditioned media (CM) isolated from adipose-‐derived MSCs of Balb/c and C57BL/6 mice. They report that Balb/c MSC CM is more suppressive than C57BL/6-‐derived CM, partly due to higher levels of IDO, TGF-‐β and NO in Balb/C MSC supernatants. Although a comparative study of immunosuppression from BM-‐derived MSCs has not yet been performed, one can assume with the differences observed in CFU-‐F and differentiation from different mouse strains that immunosuppression may vary as well.
We hypothesised that C57BL/6-‐derived PαS MSCs were exerting an indirect effect on T cell proliferation by inhibiting antigen presentation or polarising macrophages towards a regulatory phenotype. To test this hypothesis, we moved away from a ‘purified’ system where no antigen presentation was required to a more complex splenocyte culture containing several different immune cell subsets. After unsuccessful attempts to stimulate splenocyte cultures with ConA or Dynabeads®, the OT-‐1 transgenic mouse was chosen as our model system. CD8 T cells from OT-‐1 mice have a TCR specific for the ovalbumin protein (Wright et al., 2005). Exogenous addition of OVA peptide into OT-‐1 splenocyte cultures causes antigen-‐specific proliferation of CD8 lymphocytes (Clarke et al., 2000). OVA peptide needs to be processed and presented in a MHC class I context to CD8 lymphocytes to cause activation, thereby making this system more physiologically relevant than the use of TCR-‐ binding antibodies or beads. It is also a good in vitro representation of the OVA-‐Bil mouse model, where ectopic expression of OVA on the biliary epithelium of the liver results in an OT-‐1 mediated immune reaction and inflammation (Buxbaum et al., 2006).
Our preliminary findings show that C57BL/6-‐derived PαS MSCs can suppress CD8 lymphocyte proliferation in a dose-‐dependent manner. We also saw reductions in the IFNγ production of CD8 cells after MSC co-‐culture, a finding that has been shown before by Hof-‐Nahor and colleagues for human MSCs (Hof-‐Nahor et al., 2012). Interestingly, although there were large drops in total numbers of CD8 T cells after PαS MSC co-‐culture, we only observed minor differences in the proliferation status of CD8 cells that remained viable. This suggests that MSCs could induce CD8 lymphocyte apoptosis and that any lymphocytes which escaped
MSC-‐mediated immunosuppression were still proliferating in response to OVA antigen. The induction of CD8 T cell apoptosis has been reported previously for human MSC populations due to IDO-‐mediated depletion of tryptophan from the local microenvironment (Plumas et al., 2005). Further experiments are needed to see whether a similar mechanism is in play for PαS MSCs. Further repeats are also needed to increase the sample size and to understand the significance of these findings. Small molecule inhibitors of known immunosuppressive pathways can be added to the splenocyte reaction to try and identify a mechanism of action. Additionally, individual immune cell subsets (e.g. monocytes/macrophages, B cells, DCs) can be selectively removed from the splenocyte mixture to study indirect effects on T cell proliferation.