Cord blood-derived Vδ2+ and Vδ2- T cells acquire differential cell state compositions upon in vitro expansion.

Human cord blood-derived γδ T cells (CBγδ) display a highly diverse TCRγδ repertoire and have a unique subtype composition different from fetal or adult peripheral blood counterparts. We expanded CBγδ in vitro using an irradiated Epstein-Barr virus-transformed feeder cell-based modified rapid expansion protocol (REP). Single-cell RNA sequencing tracked progressive differentiation of naïve CBγδ into cells expressing neoantigen-reactive tumor-infiltrating lymphocyte as well as tissue-resident memory precursor-like and antigen-presenting cell-like gene signatures. TCRγδ clonal tracing revealed a bias toward cytotoxic effector differentiation in a much larger proportion of Vδ2- clones compared to Vδ2+ clones, resulting in the former being more cytotoxic at the population level. These clonotype-specific differentiation dynamics were not restricted to REP and were recapitulated upon secondary nonviral antigen stimulations. Thus, our data showed intrinsic cellular differences between major subtypes of human γδ T cells already in operation at early postnatal stage and highlighted key areas of consideration in optimizing cell manufacturing processes.

A role for Rac3 GTPase in the regulation of autophagy.

The process of autophagy is situated at the intersection of multiple cell signaling pathways, including cell metabolism, growth, and death, and hence is subject to multiple forms of regulation. We previously reported that inhibition of isoprenylcysteine carboxylmethyltransferase (Icmt), which catalyzes the final step in the post-translational prenylation of so-called CAAX proteins, results in the induction of autophagy which enhances cell death in some cancer cells. In this study, using siRNA-mediated knockdown of a group of small GTPases that are predicted Icmt substrates, we identify Rac3 GTPase as a negative regulator of the process of autophagy. Knockdown of Rac3, but not the closely related isoforms Rac1 and Rac2, results in induction of autophagy. Ectopic expression of Rac3, significantly rescues cells from autophagy and cell death induced by Icmt inhibition, strengthening the notion of an isoform-specific autophagy regulatory function of Rac3. This role of Rac3 was observed in multiple cell lines with varying Rac subtype expression profiles, suggesting its broad involvement in the process. The identification of this less-studied Rac member as a novel regulator provides new insight into autophagy and opens opportunities in identifying additional regulatory inputs of the process.