Optimizing Donor-derived, patient alloantigen-specific type-1 regulatory cells for the Suppression of graft vs. host disease.

Transplantation of allogeneic hematopoietic stem cells can be a life-saving treatment for many patients with hematological malignancies, however, can result in complications such as organ rejection or graft vs host disease (GvHD). Dr. Roncarolo and her team have developed an in vitro system (T-allo10) to generate donor-derived, patient alloantigen-specific type 1 regulatory cells (Tr1), which can suppress GvHD. This system leverages a mixed lymphocyte reaction between donor’s CD4+ T cells and patient-derived tolerogenic dendritic cells called DC-10, producing Tr1 cells that suppress primary alloantigen responses. Studies have shown that T cells undergo metabolic changes during differentiation and proliferation, however, the metabolic pathways required to support human Tr1 cell differentiation and survival remain unknown. Previous RNA-seq data suggested upregulation of genes involved in fatty acid oxidation (FAO), a metabolic process that contributes to oxidative phosphorylation (OXPHOS). We hypothesized that T-allo10 induction is accompanied by a metabolic switch, specifically FAO, which would be marked by higher OXPHOS capacity. The Mito Stress Test on the Seahorse XF analyzer allows researchers to quantitate the maximum capacity of OXPHOS of cells. In this study we investigated the oxygen consumption rate (OCR, indicative of OXPHOS) and extracellular acidification rate (ECAR, indicative of glycolysis) of donor-derived T-allo10 cultures. Determining the metabolic processes necessary for induction of CD4+T cells into Tr1 cells could be pivotal to determining appropriate culture conditions for maximal Tr1 yield. 

To address these concerns, I isolated monocytes from peripheral blood mononuclear cells, and induced tolerogenic differentiation into DC-10 by culturing the monocytes for 7 days with recombinant human (rh) IL-4, rhIL-10, and granulocyte macrophage colony-stimulating factor (GMCSF). A control mature DC (matDC) line was generated with the rhIL-4 and GMCSF only. On day 7 the DC-10 and matDC were co-cultured with PBMC-isolated CD4+T cells from a separate donor for T-allo10 and T-allo mixed lymphocyte reactions (MLR), respectively. On day 10 of the MLR the cells were harvested, phenotype was analyzed for Tr1 cell abundance (marked by CD4+CD3+CD45RA-CD49b+LAG3+). T-allo10 and T-allo cells were subsequently prepared for the Mito Stress Test on the Seahorse XF analyzer. 

The Seahorse assay showed that T-allo10 cells exhibited slightly higher OCR and substantially higher ECAR than the T-allo cells, suggesting that glycolysis is utilized more by T-allo10 than T-allo cells. In the future we will perform the same Mito Stress Test with a FAO substrate to determine the intrinsic rates and capacity of fluorescence-activated cell sorting (FACS)-sorted Tr1 cells to oxidize exogenous fatty acids. If the Tr1 cells utilize FAO as an energy source, changes can be made to T-allo10 cultures, such as the addition of long chain fatty acids to improve yield of Tr1 cells in the T-allo10 culture. 

During my time at Stanford University, I have completed training and certification for use on BDFACSAria, BD Fortessa, and Becton Coulter Cytoflex flow cytometry/FACS analysis