IMMUNE REGULATION AND TOLERANCE II

T. Searcy, R. Krishnan
Renal Transplantation Immunology Laboratory, Royal Adelaide Hospital, Adelaide/SA/AUSTRALIA

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Aims: Mesenchymal stem cells (MSC) induce T cell hyporesponsiveness and inhibit dendritic cell maturation. Thus MSC may have a role in the treatment of transplant rejection. However, clinicaltransplantation involving MSC therapy will need to be performed under the cover of immunosuppressive agents (ISA). Therefore, the aim of this study was to characterise the complimentary interactionsbetween MSC and specific ISA. Methods: Human MSC were co-cultured with: (i) Monocyte-derived DC during differentiation with IL-4, GM-CSF and TNF-alpha and (ii) T cells stimulated with the mitogenPHA. To assess the influence of ISA, MSC were pretreated with ISA prior to their use in assays or directly added to cocultures. Flow cytometry was used to characterize the changes in dendritic cellphenotypic markers. Results: Flow cytometry showed that unmodified MSC inhibited the generation of mature CD83+ DC by 50% when co-cultured with monocytes at a ratio of 1:10 compared to monocytes thatwere differentiated in the absence of MSC. MSC pre-treated with 10nM Sirolimus (Siro-MSC) or 50µM Mycophenolate (MPA-MSC) further inhibited the numbers of CD83+ DC by 40% and 16%compared to unmodified MSC, respectively. In contrast, MSC pre-treated with 100nM Cyclosporin A (CsA-MSC) increased the number of CD83+ DC by 36%. Interestingly, in the cocultures MSC maintained highexpression of CD14 on the monocyte precursors when differentiated to DC with IL-4, GMCSF and TNF-alpha but did not modify the expression of DC-SIGN on these cells. In proliferation assays MSCinhibited PHA-activated T cell proliferation by 30%, when co-cultured at a ratio of 1:100. Siro-MSC further inhibited T cell proliferation by 39% compared to unmodified MSC. However, MPA-MSC andCsA-MSC displayed no enhanced ability to inhibit T cell proliferation. To determine whether MSC synergise with ISA to inhibit T cell proliferation, MSC and ISA were applied alone or in combination toPHA stimulated T cells. MSC added alone at a ratio of 1:100 to T cells demonstrated a 7% (±2% SD) inhibition and 1nM Siro added alone demonstrated 16% (±3% SD) inhibition of PHAmediated T cell proliferation. However, in combination (1nM Siro and 1% MSC) demonstrated 70% (±1% SD) inhibition of T cell proliferation. In addition, while 10nM CsA alone inhibitedPHA-mediated T cell proliferation by 10% (±1% SD), in combination with 1% MSC demonstrated a 41% (±9% SD) inhibitition. Synergy was not observed when MSC were used in combination withMPA. Conclusion: In conclusion, the data show that MSC have potent immunomodulatory effects, which can be further enhanced by pretreatment with ISA. In particular, the inhibition of the calcineurinand mTOR pathways at subtherapeutic concentrations of ISA appears to show profound immunomodulation when combined with MSC and hence may be optimal combinations to use in clinical transplantation tofacilitate allograft tolerance.

Disclosure: All authors have declared no conflicts of interest.