Roy, Abhishek

Abstract Introduction The ability of the adaptive immune system to have memory of previous pathogen encounters is the basis for vaccination as one of the most successful medical interventions. With increasing age, the ability to develop protective immunity after vaccination declines. Differentiation into T memory cells is at least in part determined during the early cell divisions after activation when cells undergo assymmetric cell division (ACD). Fate decisions depend on the uneven distribution of signaling molecules and transcription factors with accumulation of mTORC1 activity biasing for an effector and TCF1 for a memory state. We explored whether ACD of naïve CD4 T cells is impaired in older adults. Methods We used human primary cells from young and older adults. Human naive CD4 T cells were isolated and cultured in RPMI, 10% FBS and antibiotics on plates coated with anti-CD3 Ab, anti-CD28 Ab and huICAM1 for 3 days. We used transfection by electroporation to modify gene expression. Read out systems included confocal microscopy, flow cytometry and lysosome function assays. Results were compared by t-test with values of p < 0.05 considered significant. Results Naive CD4 T cell responses from older adults is characterized by increased activation of the AKT-mTORC1 pathway and reduced ACD of mTORC1 activity. Increased RICTOR expression resulting in increased mTORC2 activity in naive CD4 T cells from older individuals accounted for the activation of the AKT-mTORC1 pathway. AKT inhibition or silencing of RICTOR restores ACD of mTORC1 activity as well as of TCF1. Conversely, ACD was also improved through inducing WNT signaling by GSK3β inhibition irrespective of RICTOR expression. Conclusion Our data show that increased RICTOR expression in naïve T cells from older adults at least in part accounts for defective memory cell generation. Silencing of RICTOR or downstream AKT inhibition can enforce both mTORC1 ACD and TCF1 ACD suggesting that mTORC2 is a promising target to improve vaccine responses. Funding Source n/a Topic Categories Lymphocyte Differentiation and Peripheral Maintenance (LYM)

Abstract Extracellular vesicles (EVs) are key mediators of cell–cell communication and are involved in transferring specific biomolecular cargo to recipient cells to regulate their physiological functions. A major challenge in the understanding of EV function in vivo is the difficulty ascertaining the origin of the EV particles. The recent development of the “Snorkel-tag,” which includes EV-membrane-targeted CD81 fused to a series of extra-vesicular protein tags, can be used to mark EVs originating from a specific source for subsequent isolation and characterization. We developed an in vivo mouse model, termed “CAGS-Snorkel,” which expresses the Snorkel-tag under the control of the Cre-lox system, and crossed this mouse with either Prx1-Cre (mesenchymal progenitors) or Ocn-Cre (osteoblasts/osteocytes) and isolated Snorkel-tagged EVs from the mouse bone marrow plasma using a magnetic bead affinity column. miRNA-sequencing was performed on the isolated EVs, and although similar profiles were observed, a few key miRNAs involved in bone metabolism (miR-106b-5p, miRs-19b-3p, and miRs-219a-5p) were enriched in the Ocn-derived relative to the Prx1-derived EV subpopulations. To characterize the effects of these small EVs on a bone cell target, cultured mouse bone marrow stromal cells were treated with Prx1 or Ocn EVs, and mRNA-sequencing was performed. Pathways involved in ossification, bone development, and extracellular matrix interactions were regulated by both EV subpopulations, whereas a few pathways including advanced glycation end-products signaling were uniquely regulated in the Ocn EV subpopulation, underlying important biological effects of specific EV subpopulations within the bone marrow microenvironment. These data demonstrate that EV isolation in vivo using the CAGS-Snorkel mouse model is a useful tool in characterizing the cargo and understanding the biology of tissue-specific EVs. Moreover, while bone mesenchymal cell populations share a common EV secretory profile, we uncover key differences based on the stage of osteoblastic differentiation that may have important biological consequences.