Graphical AbstractHighlights d S1PR-independent intrinsic S1P signaling activates PPARg to skew the Treg/Th17 balance d S1P-PPARg activation inversely correlates with lipolysis and regulates Tcm phenotype d Inhibiting SphK1/S1P/PPARg signaling improves T cellmediated tumor control Correspondence ogretmen@musc.edu (B.O.), mehrotr@musc.edu (S.M.) In BriefChakraborty et al. define the role for SphK1/S1P signaling via engaging lipid transcription factor PPARg to attenuate lipolysis and spare respiratory capacity in T cells. Genetic ablation or pharmacological inhibition of SphK1 expression limits intrinsic S1P levels and improves T cell-mediated anti-tumor immunotherapeutic control. SUMMARYSphingosine 1-phosphate (S1P), a bioactive lysophospholipid generated by sphingosine kinase 1 (SphK1), regulates lymphocyte egress into circulation via S1P receptor 1 (S1PR1) signaling, and it controls the differentiation of regulatory T cells (Tregs) and T helper-17 cells. However, the mechanisms by which receptor-independent SphK1-mediated intracellular S1P levels modulate T cell functionality remains unknown. We show here that SphK1-deficient T cells maintain central memory phenotype and exhibit higher mitochondrial respiration and reduced differentiation to Tregs. Mechanistically, we discovered a direct correlation between SphK1-generated S1P and lipid transcription factor PPARg (peroxisome proliferator-activated receptor gamma) activity, which in turn regulates lipolysis in T cells. Genetic and pharmacologic inhibition of SphK1 improved metabolic fitness and anti-tumor activity of T cells against murine melanoma. Further, inhibition of SphK1 and PD1 together led to improved control of melanoma. Overall, these data highlight the clinical potential of limiting SphK1/S1P signaling for enhancing anti-tumor-adoptive T cell therapy.(G) Cells obtained using strategy in (F) with different cytokines post day 3 (IL-2 at 100 IU/mL, and IL-15 at 100 ng/mL, respectively) were analyzed for determining the flow cytometry-based cell surface expression of CD62L and intracellular expression of SphK1. (H) Day 3 TCR-activated WT T cells were transfected with either WT vector or empty vector for SphK1 enzyme. Cells were subsequently cultured for another 3 days, either in presence of IL-2 (100 IU/mL), or IL-15 (100 ng/mL). After 3 days, expression of CD62L and SphK1 was evaluated in CD8 + T cells by flow cytometry. (I) Day 3 TCR-activated WT T cells were further cultured for 3 days, either in presence of IL-2 (100 IU/mL) or in presence of TGF-b (5 ng/mL) and IL-2 (100 IU/mL). Cells were then used to determine the intracellular expression of SphK1. Data were analyzed using FlowJo software. The numerical values within the fluorescence-activated cell sorting (FACS) overlay plots indicate MFI, and the adjacent bar diagrams represent cumulative data from at least three repeat experiments. Error bars represent mean ± SD; *p < 0.05. See also Figure S1.

Purpose: Adoptive T-cell therapy (ACT) of cancer, which involves the infusion of ex vivo-engineered tumor epitope reactive autologous T cells into the tumor-bearing host, is a potential treatment modality for cancer. However, the durable antitumor response following ACT is hampered either by loss of effector function or survival of the antitumor T cells. Therefore, strategies to improve the persistence and sustain the effector function of the antitumor T cells are of immense importance. Given the role of metabolism in determining the therapeutic efficacy of T cells, we hypothesize that inhibition of PIM kinases, a family of serine/threonine kinase that promote cell-cycle transition, cell growth, and regulate mTORC1 activity, can improve the potency of T cells in controlling tumor.Experimental Design: The role of PIM kinases in T cells was studied either by genetic ablation (PIM1 À/À PIM2 À/À PIM3 À/À ) or its pharmacologic inhibition (pan-PIM kinase inhibitor, PimKi). Murine melanoma B16 was established subcutane-ously and treated by transferring tumor epitope gp100-reactive T cells along with treatment regimen that involved inhibiting PIM kinases, anti-PD1 or both.Results: With inhibition of PIM kinases, T cells had significant reduction in their uptake of glucose, and upregulated expression of memory-associated genes that inversely correlate with glycolysis. In addition, the expression of CD38, which negatively regulates the metabolic fitness of the T cells, was also reduced in PimKi-treated cells. Importantly, the efficacy of antitumor T-cell therapy was markedly improved by inhibiting PIM kinases in tumor-bearing mice receiving ACT, and further enhanced by adding anti-PD1 antibody to this combination.Conclusions: This study highlights the potential therapeutic significance of combinatorial strategies where ACT and inhibition of signaling kinase with checkpoint blockade could improve tumor control.

Delivery of genetic material to tissues in vivo is an important technique used in research settings and is the foundation upon which clinical gene therapy is built. The lung is a prime target for gene delivery due to a host of genetic, acquired, and infectious diseases that manifest themselves there, resulting in many pathologies. However, the in vivo delivery of genetic material to the lung remains a practical problem clinically and is considered the major obstacle needed to be overcome for gene therapy. Currently there are four main strategies for in vivo gene delivery to the lung: viral vectors, liposomes, nanoparticles, and electroporation. Viral delivery uses several different genetically modified viruses that enter the cell and express desired genes that have been inserted to the viral genome. Liposomes use combinations of charged and neutral lipids that can encapsulate genetic cargo and enter cells through endogenous mechanisms, thereby delivering their cargoes. Nanoparticles are defined by their size (typically less than 100 nm) and are made up of many different classes of building blocks, including biological and synthetic polymers, cell penetrant and other peptides, and dendrimers, that also enter cells through endogenous mechanisms. Electroporation uses mild to moderate electrical pulses to create pores in the cell membrane through which delivered genetic material can enter a cell. An emerging fifth category, exosomes and extracellular vesicles, may have advantages of both viral and non-viral approaches. These extracellular vesicles bud from cellular membranes containing receptors and ligands that may aid cell targeting and which can be loaded with genetic material for efficient transfer. Each of these vectors can be used for different gene delivery applications based on mechanisms of action, side-effects, and other factors, and their use in the lung and possible clinical considerations is the primary focus of this review.

While earlier studies have suggested that cells positive for hematopoietic markers can be found in dental tissues, it has yet to be confirmed. To conclusively demonstrate this, we utilized a unique transgenic model in which all hematopoietic cells are green fluorescent protein+ (GFP+). Pulp, periodontal ligament (PDL) and alveolar bone (AvB) cell culture analysis demonstrated numerous GFP+ cells, which were also CD45+ (indicating hematopoietic origin) and co-expressed markers of cellular populations in pulp (dentin matrix protein-1, dentin sialophosphoprotein, alpha smooth muscle actin [ASMA], osteocalcin), in PDL (periostin, ASMA, vimentin, osteocalcin) and in AvB (Runx-2, bone sialoprotein, alkaline phosphatase, osteocalcin). Transplantation of clonal population derived from a single GFP+ hematopoietic stem cell (HSC), into lethally irradiated recipient mice, demonstrated numerous GFP+ cells within dental tissues of recipient mice, which also stained for markers of cell populations in pulp, PDL and AvB (used above), indicating that transplanted HSCs can differentiate into cells in dental tissues. These hematopoietic-derived cells deposited collagen and can differentiate in osteogenic media, indicating that they are functional. Thus, our studies demonstrate, for the first time, that cells in pulp, PDL and AvB can have a hematopoietic origin, thereby opening new avenues of therapy for dental diseases and injuries.

Currently, there is no cure for osteogenesis imperfecta (OI)—a debilitating pediatric skeletal dysplasia. Herein we show that hematopoietic stem cell (HSC) therapy holds promise in treating OI. Using single-cell HSC transplantation in lethally irradiated oim/oim mice, we demonstrate significant improvements in bone morphometric, mechanics, and turnover parameters. Importantly, we highlight that HSCs cause these improvements due to their unique property of differentiating into osteoblasts/osteocytes, depositing normal collagen—an attribute thus far assigned only to mesenchymal stem/stromal cells. To confirm HSC plasticity, lineage tracing was done by transplanting oim/oim with HSCs from two specific transgenic mice—VavR, in which all hematopoietic cells are GFP+ and pOBCol2.3GFP, where GFP is expressed only in osteoblasts/osteocytes. In both models, transplanted oim/oim mice demonstrated GFP+ HSC-derived osteoblasts/osteocytes in bones. These studies unequivocally establish that HSCs differentiate into osteoblasts/osteocytes, and HSC transplantation can provide a new translational approach for OI.