PD-1 alters T-cell metabolic reprogramming by inhibiting glycolysis and promoting lipolysis and fatty acid oxidation.

During activation, T cells undergo metabolic reprogramming, which imprints distinct functional fates. We determined that on PD-1 ligation, activated T cells are unable to engage in glycolysis or amino acid metabolism but have an increased rate of fatty acid ß-oxidation (FAO). PD-1 promotes FAO of endogenous lipids by increasing expression of CPT1A, and inducing lipolysis as indicated by elevation of the lipase ATGL, the lipolysis marker glycerol and release of fatty acids. Conversely, CTLA-4 inhibits glycolysis without augmenting FAO, suggesting that CTLA-4 sustains the metabolic profile of non-activated cells. Because T cells utilize glycolysis during differentiation to effectors, our findings reveal a metabolic mechanism responsible for PD-1-mediated blockade of T-effector cell differentiation. The enhancement of FAO provides a mechanistic explanation for the longevity of T cells receiving PD-1 signals in patients with chronic infections and cancer, and for their capacity to be reinvigorated by PD-1 blockade.

The cyclin dependent kinase inhibitor (R)-roscovitine mediates selective suppression of alloreactive human T cells but preserves pathogen-specific and leukemia-specific effectors.

Graft versus host disease (GvHD), mediated by donor T cells, remains the primary cause of non-relapse mortality after allogeneic hematopoietic stem cell transplantation and novel therapeutic approaches are required. Cdk2 is a critical node of signal integration and programming of T cell responses towards immunity versus anergy but is dispensable for hematopoiesis and thymocyte development. We examined the effects of pharmacologic Cdk2 inhibition on alloreactive human T cells. Inhibition of Cdk2 blocked expansion of alloreactive T cells upon culture with HLA-mismatched dendritic cells and prevented generation of IFN-γ-producing alloantigen-specific effectors. In contrast, Cdk2 inhibition preserved effectors specific for Wilms' tumor 1 (WT1) leukemia antigen and for CMV as determined by WT1-specific and CMV-specific pentamers. Cdk2 inhibition preserved Treg cells, which have the ability to prevent GvHD while maintaining GvL. Thus, Cdk inhibitors may improve allogeneic HSCT by reducing alloreactivity and GvHD without loss of pathogen-specific and leukemia-specific immunity.

CD31+ cells represent highly angiogenic and vasculogenic cells in bone marrow: novel role of nonendothelial CD31+ cells in neovascularization and their therapeutic effects on ischemic vascular disease.

RATIONALE: Bone marrow (BM) cells play an important role in physiological and therapeutic neovascularization. However, it remains unclear whether any specific uncultured BM cell populations have higher angiogenic and vasculogenic activities. Moreover, there has been controversy regarding the vasculogenic ability of BM cells. OBJECTIVE: Preliminary flow cytometric analysis showed that CD31, traditionally a marker for endothelial cells, is expressed in certain nonendothelial BM mononuclear cells in both human and mouse. Based on the conserved CD31 expression in the axis of hematopoietic stem/progenitor cells (HSC/HPCs) to endothelial cells, we further sought to determine the comprehensive vasculogenic and angiogenic characteristics of human and mouse BM-derived CD31(+) cells. METHODS AND RESULTS: Flow cytometric analysis demonstrated that all CD31(+) cells derived from BM were CD45(+) and expressed markers for both HSC/HPCs and endothelial cells. Comprehensive gene expression analyses revealed that BM-CD31(+) cells expressed higher levels of angiogenic genes than CD31(-) cells. Endothelial progenitor cells, as well as HSC/HPCs, were almost exclusively confined to the CD31(+) cell fraction, and culture of CD31(+) cells under defined conditions gave rise to endothelial cells. Finally, injection of CD31(+) cells into ischemic hindlimb repaired ischemia, increased expression of angiogenic and chemoattractive factors, and, in part, directly contributed to vasculogenesis, as demonstrated by both 3D confocal microscopy and flow cytometry. CONCLUSIONS: These data indicate that BM-CD31(+) cells represent highly angiogenic and vasculogenic cells and can be a novel and highly promising source of cells for cell therapy to treat ischemic cardiovascular diseases.

In vivo gene transfer to the CNS using recombinant SV40-derived vectors.

BACKGROUND: Gene transfer to the CNS has been approached using various vectors. OBJECTIVE: We illustrate how SV40-derived vectors may be useful to deliver long-term gene expression to the brain, locally or diffusely. RESULTS/CONCLUSION: SV40-derived vectors transduce neurons and microglial cells. The potential utility of both localized and widespread gene delivery in treating neuroAIDS and other CNS diseases characterized by excessive oxidative stress is demonstrated. Finally, direct injection of rSV40 vectors into rat femoral bone marrow (BM) led to transgene expression in CNS neurons and microglia, mostly in the dentate gyrus and in the periventricular subependymal zone, suggesting that BM-derived cells may be progenitors of some CNS cells in adult animals, and that gene delivery to BM may allow transgene expression in newly formed neurons.

Delivering genes to the organ-localized immune system: long-term results of direct intramarrow transduction.

We studied the distribution of transgene-expressing cells after direct gene transfer into the bone marrow (BM). Rats received direct injection into the femoral BM of SV(Nef-FLAG), a Tag-deleted recombinant SV40 carrying a marker gene (FLAG epitope). Controls received an unrelated rSV40 or saline. Blood cells (5%) and femoral marrow cells (25%) expressed FLAG throughout. FLAG expression was assessed in different organs at 1, 4 and 16 months. FLAG+ macrophages were seen throughout the body, and were prominent in the spleen. FLAG+ cells were common in pulmonary alveoli. The former included alveolar macrophages and type II pneumocytes. These cells were not detected at 1 month, occasional at 4 months and common at 16 months after intramarrow injection. Rare liver cells were positive for both FLAG and ferritin, indicating that some hepatocytes also expressed this BM-delivered transgene. Control animals were negative. Thus: (a) fixed tissue phagocytes may be accessible to gene delivery by intramarrow transduction of their progenitors; (b) transduced BM-resident cells or their derivatives may migrate to other organs (lungs) and may differentiate into epithelial cells; and (c) intramarrow injection of rSV40s does not detectably transduce parenchymal cells of other organs.

Long-term gene expression in dividing and nondividing cells using SV40-derived vectors.

Among the goals of gene therapy is long-term expression of delivered transgenes. Recombinant Tagdeleted SV40 vectors (rSV40s) are especially well suited for this purpose. rSV40s deliver transgene expression that endures for extended periods of time in tissue culture and in vivo, in both dividing and nondividing cells. These vectors are particularly effective in transducing some cell types that have been almost unapproachable using other gene delivery systems, such as quiescent hematopoietic progenitor cells and their differentiated derivatives. Other cellular targets include neurons, brain microglia, hepatocytes, dendritic cells, vascular endothelium, and others. Because rSV40s do not elicit neutralizing antibodies they are useful for in vivo gene delivery in settings where more than one administration may be desirable. The key characteristics of these vectors include their high production titers and therefore suitability for large cell pools, effectiveness in delivering intracellular proteins, and untranslated RNAs, maintenance of transgene expression at constant levels for extended times, suitability for constitutive or conditional promoters and for combinatorial gene delivery and ability to integrate into genomes of both dividing and nondividing cells.

Rat bone marrow progenitor cells transduced in situ by rSV40 vectors differentiate into multiple central nervous system cell lineages.

Using bone marrow-directed gene transfer, we tested whether bone marrow-derived cells may function as progenitors of central nervous system (CNS) cells in adult animals. SV40-derived gene delivery vectors were injected directly into femoral bone marrow, and we examined transgene expression in blood and brain for 0-16 months thereafter by immunostaining for FLAG epitope marker. An average of 5% of peripheral blood cells and 25% of femoral marrow cells were FLAG(+) throughout the study. CNS FLAG-expressing cells were mainly detected in the dentate gyrus (DG) and periventricular subependymal zone (PSZ). Although absent before 1 month and rare at 4 months, DG and PSZ FLAG(+) cells were abundant 16 months after bone marrow injection. Approximately 5% of DG cells expressed FLAG, including neurons (48.6%) and microglia (49.7%), and occasional astrocytes (1.6%), as determined by double immunostaining for FLAG and lineage markers. These data suggest that one or more populations of cells resident within adult bone marrow can migrate to the brain and differentiate into CNS-specific cells.

In vivo gene transfer into rat bone marrow progenitor cells using rSV40 viral vectors.

Hematopoietic stem cell (HSC) gene transfer has been attempted almost entirely ex vivo and has been limited by cytokine-induced loss of self-renewal capacity and transplantation-related defects in homing and engraftment. Here, we attempted to circumvent such limitations by injecting vectors directly into the bone marrow (BM) to transduce HSCs in their native environment. Simian virus 40 (SV40)-derived gene delivery vectors were used because they transduce resting CD34+ cells very efficiently. Rats received SV-(Nef-FLAG), carrying FLAG marker epitope--or a control recombinant SV40 (rSV40)--directly into both femoral marrow cavities. Intracellular transgene expression by peripheral blood (PB) or BM cells was detected by cytofluorimetry. An average of 5.3% PB leukocytes expressed FLAG for the entire study--56 weeks. Transgene expression was sustained in multiple cell lineages, including granulocytes (average, 3.3% of leukocytes, 20.4% of granulocytes), CD3+ T lymphocytes (average, 0.53% of leukocytes, 1% of total T cells), and CD45R+ B lymphocytes, indicating gene transfer to long-lived progenitor cells with multilineage capacity. An average of 15% of femoral marrow cells expressed FLAG up to 16.5 months after transduction. Thus, direct intramarrow administration of rSV40s yields efficient gene transfer to rat BM progenitor cells and may be worthy of further investigation.

What they are, how they work and why they do what they do? The story of SV40-derived gene therapy vectors and what they have to offer.

The natural function of viruses is to deliver their genetic material to cells. Among the most effective of viruses in doing that is Simian Virus-40 (SV40). The properties that make SV40 a successful virus make it an attractive candidate for use as a gene delivery vehicle: high titer replication, infectivity for almost all nucleated cell types whether the cells are dividing or resting, potential for integration into cellular DNA, a peculiar pathway for entering cells that bypasses the cells' antigen processing apparatus, very high stability, and the apparent ability to activate expression of its own capsid genes in trans. Exploiting these and other characteristics of wild type (wt) SV40, increasing numbers of laboratories are studying recombinant (r) SV40-derived vectors. Among the uses to which these vectors have been applied are: delivering therapy to inhibit HIV, hepatitis C virus (HCV) and other viruses; correction of inherited hepatic and other protein deficiencies; immunizing against lentiviral and other antigens; treatment of inherited and acquired diseases of the central nervous system; protecting the lung and other organs from free radical-induced injury; and many others. The effectiveness of these vectors is a reflection of the adaptive evolution that produced their parent virus, wt SV40. This article explores how and why these vectors work, their strengths and their limitations, and provides a functional model for their exploitation for experimental and clinical applications.

Homing defect of cultured human hematopoietic cells in the NOD/SCID mouse is mediated by Fas/CD95.

OBJECTIVE: To determine the bone marrow homing efficiency (20 hours) of cultured compared to noncultured umbilical cord blood (UCB)-derived human hematopoietic cells in the nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse, and to explain the difference in homing between these populations. METHODS: Human UCB CD34+ cells were cultured for up to 5 days, reselected, and used for transplantation, phenotype analysis, and functional studies, including adhesion and trans-endothelial migration assays. Seeding of CD34+ cells was measured after labeling of cells with 111-Indium, while homing of colony-forming cells (CFC) and SCID-repopulating (SRC) cells was determined using functional assays. RESULTS: Short-term culture was associated with a decrease in the 20-hour homing of CD34+ cells, CFC, and SRC to the BM. Although cultured compared to noncultured cells showed increased expression and function (adhesion/migration) of several cell adhesion molecules described to play a role in homing and engraftment, culture also induced expression of Fas/CD95 and rendered cells more susceptible to apoptosis. Finally, we demonstrate that the level of Fas/CD95 on cultured cells was inversely related to the ability of CFC to home to the BM, and that the homing of cultured CFC could be restored by incubating cells prior to transplantation with Fas/CD95-blocking mAb ZB4. CONCLUSION: These data implicate Fas/CD95 in the homing defect of cultured human hematopoietic cells in the NOD/SCID transplant model and suggest that prevention of apoptosis may be an important strategy to improve engraftment of ex vivo-manipulated HSC in a clinical setting.