End-stage renal disease does not impair the large-scale generation of potent alloantigen-specific regulatory T cells for immunotherapy


N.H.R. Litjens, K. Boer, J.M. Zuijderwijk, M. Klepper, A.M.A. Peeters, W. Verschoor, R. Kraaijeveld, C.C. Baan, M.G.H. Betjes

Thursday 10 march 2016

9:40 - 9:50h at Zaal 1 & 2

Categories: Parallelsessie (basaal)

Parallel session: Parallelsessie VII - Basaal immunologie

Background:
Alloantigen-specific natural occurring regulatory T cells (nTregs) have the potential to offer a more targeted approach of immunosuppression and are the cell type of interest for inducing tolerance in kidney transplantation. We have recently developed an expansion protocol for highly enriched nTregs thereby obtaining sufficient numbers of potent nTregs for immunotherapy. Patients suffering from end-stage renal disease (ESRD) have a dysfunctional T cell immune system but it is not known how this affects nTreg function and expansion potential. In this study, frequencies, phenotype, expansion capacity, stability and function of nTregs from ESRD patients were compared to those from healthy individuals (HI).

Material and Methods:
Flow cytometry-based isolated nTregs of both ESRD patients (either not or on renal replacement therapy, RRT) and HI were expanded using (donor-derived) allogeneic mature monocyte-derived dendritic cells (moDC) and extensively characterized by analysis of the demethylation status of the TSDR of the FOXP3 gene and the expression of typical nTreg markers, i.e. FOXP3, HELIOS and CTLA4. In addition, the suppressive capacity of allogeneic mature moDC-expanded nTregs was tested in a mixed lymphocyte reaction (MLR).

Results:
Compared to age- and gender-matched HI, similar frequencies of nTregs were present within the circulation of ESRD patients either not or on RRT, i.e. median percentages nTregs within CD4+ T cells amounted to 5.6, 5.2 and 5.9, respectively. The isolated nTregs of ESRD patients could be equally well or even better expanded using allogeneic mature moDC, i.e. median fold expansions after 10-11 days amounted to 12.6 and 30.2 (ESRD patients not or on RRT, respectively) versus 8.7 (HI). Extensive phenotypical characterization did not reveal significant differences. The demethylation status of the TSDR was maintained or even further promoted as was the expression of characteristic nTregs markers. In addition, even at low ratios of Tregs: Teffectors (i.e. 1:320), the median percentage of inhibition of donor-alloantigen-induced proliferation by allogeneic mature moDC-expanded nTregs of ESRD patients either not or on RRT was similar to HI.

Conclusion:
Circulating nTregs of ESRD patients either not or on RRT can be highly enriched and alloantigen-specific expanded to numbers needed for clinical applications. Phenotype, stability and functionality of these alloreactive nTregs is similar to results obtained from HI.