Regulatory T cells (Tregs) have shown great promise as a means of cellular therapy in a multitude of allo- and auto-immune diseases—due in part to their immunosuppressive potency. Nevertheless, the clinical efficacy of human Tregs in patients has been limited by their poor in vivo homeostasis. To avert apoptosis, Tregs require stable antigenic (CD3ζ/T-cell-receptor-mediated), co-stimulatory (CD28-driven), and cytokine (IL-2-dependent) signaling. Notably, this sequence of signals supports an activated Treg phenotype that includes a high expression of granzymes, particularly granzyme B (GrB). Previously, we have shown that aside from the functional effects of GrB in lysing target cells to modulate allo-immunity, GrB can leak out of the intracellular lysosomal granules of host Tregs, initiating pro-apoptotic pathways. Here, we assessed the role of inhibiting mechanistic target of rapamycin complex 1 (mTORC1), a recently favored drug target in the transplant field, in regulating human Treg apoptosis via GrB. Using ex vivo models of human Treg culture and a humanized mouse model of human skin allotransplantation, we found that by inhibiting mTORC1 using rapamycin, intracytoplasmic expression and functionality of GrB diminished in host Tregs; lowering human Treg apoptosis by in part decreasing the phosphorylation of S6K and c-Jun. These findings support the already clinically validated effects of mTORC1 inhibition in patients, most notably their stabilization of Treg bioactivity and in vivo homeostasis.

BackgroundKidney transplant recipients are currently treated with nonspecific immunosuppressants that cause severe systemic side effects. Current immunosuppressants were developed based on their effect on T-cell activation rather than the underlying mechanisms driving alloimmune responses. Thus, understanding the role of the intragraft microenvironment will help us identify more directed therapies with lower side effects.MethodsTo understand the role of the alloimmune response and the intragraft microenvironment in cellular rejection progression, we conducted a Single nucleus RNA sequencing (snRNA-seq) on one human non-rejecting kidney allograft sample, one borderline sample, and T-cell mediated rejection (TCMR) sample (Banff IIa). We studied the differential gene expression and enriched pathways in different conditions, in addition to ligand-receptor (L-R) interactions.ResultsPathway analysis of T-cells in borderline sample showed enrichment for allograft rejection pathway, suggesting that the borderline sample reflects an early rejection. Hence, this allows for studying the early stages of cellular rejection. Moreover, we showed that focal adhesion (FA), IFNg pathways, and endomucin (EMCN) were significantly upregulated in endothelial cell clusters (ECs) of borderline compared to ECs TCMR. Furthermore, we found that pericytes in TCMR seem to favor endothelial permeability compared to borderline. Similarly, T-cells interaction with ECs in borderline differs from TCMR by involving DAMPS-TLRs interactions.ConclusionOur data revealed novel roles of T-cells, ECs, and pericytes in cellular rejection progression, providing new clues on the pathophysiology of allograft rejection.

Chordomas account for approximately 1–4% of all malignant bone tumors and 20% of primary tumors of the spinal column. It is a rare disease, with an incidence estimated to be approximately 1 per 1,000,000 people. The underlying causative mechanism of chordoma is unknown, which makes it challenging to treat. Chordomas have been linked to the T-box transcription factor T (TBXT) gene located on chromosome 6. The TBXT gene encodes a protein transcription factor TBXT, or brachyury homolog. Currently, there is no approved targeted therapy for chordoma. Here, we performed a small molecule screening to identify small chemical molecules and therapeutic targets for treating chordoma. We screened 3730 unique compounds and selected 50 potential hits. The top three hits were Ribociclib, Ingenol-3-angelate, and Duvelisib. Among the top 10 hits, we found a novel class of small molecules, including proteasomal inhibitors, as promising molecules that reduce the proliferation of human chordoma cells. Furthermore, we discovered that proteasomal subunits PSMB5 and PSMB8 are increased in human chordoma cell lines U-CH1 and U-CH2, confirming that the proteasome may serve as a molecular target whose specific inhibition may lead to better therapeutic strategies for chordoma.