Assessing the impact of immunosuppressive drugs on regulatory T cell therapy

Immunosuppressive drugs have facilitated the progression of solid organ transplantation from experimental therapy to routine practice, however transplant recipients are still susceptible to chronic rejection and co-morbidities. The emergence of regulatory T cells (Treg) as a key regulator of the immune system, together with an abundance of evidence from experimental transplant models, has led to clinical trials asking whether Treg can improve transplant outcomes. However, given that Treg cellular therapy will only be acceptable if introduced into current immunosuppressive regimens, a critical question is how Treg will respond in the presence of concomitant immunosuppression. Whilst in vitro data are available, very few credible experiments have been done asking whether individual immunosuppressive drugs have a positive, a neutral or a detrimental impact on the Treg function in vivo. Thus the aims of this thesis were firstly to generate sufficient numbers of adaptive Treg for extensive experimental use and secondly to evaluate their ability to control transplant rejection in vivo in the presence of biologically valid doses of individual, clinically relevant immunosuppressive drugs. Importantly, the model chosen was the heterotopic heart transplant model in lymphoreplete mice to avoid possible artefacts that can occur in cell reconstituted lympho-depleted mice. The model also has the added advantage that by dealing with an intact immune system, it perhaps represents a small step closer to the clinical situation.
Generating sufficient numbers of stable Treg was necessary for planned in vivo experiments. Incubating CD4+ T cells with anti-CD44 antibody, prior to driving them with bone-marrow derived dendritic cells, enriched for a stable population of Treg and importantly yielded sufficient numbers of cells for in vivo experiments. It is frequently stated that alloantigen-driven Treg are more efficacious than activated autologous nTreg, however there was no difference in rejection kinetics in either a skin or heart allograft model when comparing alloantigen-driven Treg with nTreg. As generating alloantigen-driven Treg is less efficient than nTreg, pursuing the former as a potential therapy might therefore be unnecessary. This could have a considerable impact on the logistics and the practicality of clinical Treg cellular therapeutics.
The timing of Treg administration is an important consideration to maximise efficacy. Pre-transplant administration led to the longest graft survival times, suggesting that this is the most effective time for cell delivery.
Preclinical models provide a useful tool to ask how immunosuppressive drugs will affect adoptively transferred Treg. The data presented in this thesis suggest that combining Treg with Rapamycin, Mycophenolate Mofetil (MMF) or Tacrolimus did not completely prevent Treg function. However, Methylprednisolone (MP) did prevent Treg function, suggesting it cannot be used with adoptively transferred Treg. Overall, these results provide important data for the design of immunosuppressive regimens for future clinical trials assessing the efficacy of Treg in transplant recipients.

Advisor: Bushell, A