In vivo engineering of mobilized stem cell grafts with the immunomodulatory drug FTY720 for allogeneic transplantation

The immunological attributes of stem cell grafts play an important role in the outcome of allogeneic stem cell transplants. Currently, ex vivo manipulation techniques such as bulk T cell depletion or positive selection of CD34(+) cells are utilized to improve the immunological attributes of grafts and minimize the potential for graft-versus-host disease (GvHD). Here, we demonstrate a novel graft engineering technique, which utilizes the immunomodulatory drug FTY720 for in vivo depletion of naïve T (TN ) cells from donor G-CSF-mobilized grafts without ex vivo manipulation. We show that treatment of donor mice with FTY720 during mobilization depletes grafts of TN cells and prevents lethal GvHD following transplantation in a major mismatch setting. Importantly, both stem cells and NK cells are retained in the FTY720-treated grafts. FTY720 treatment does not negatively affect the engraftment potential of stem cells as demonstrated in our congenic transplants or the functionality of NK cells. In addition, potentially useful memory T cells may be retained in the graft. These findings suggest that FTY720 may be used to optimize the immunological attributes of G-CSF-mobilized grafts by removing potentially deleterious TN cells which can contribute to GvHD, and by retaining useful cells which can promote immunity in the recipient

Role of Active Site Rigidity in Activity: MD Simulation and Fluorescence Study on a Lipase Mutant

Relationship between stability and activity of enzymes is maintained by underlying conformational flexibility. In thermophilic enzymes, a decrease in flexibility causes low enzyme activity while in less stable proteins such as mesophiles and psychrophiles, an increase in flexibility is associated with enhanced enzyme activity. Recently, we identified a mutant of a lipase whose stability and activity were enhanced simultaneously. In this work, we probed the conformational dynamics of the mutant and the wild type lipase, particularly flexibility of their active site using molecular dynamic simulations and time-resolved fluorescence techniques. In contrast to the earlier observations, our data show that active site of the mutant is more rigid than wild type enzyme. Further investigation suggests that this lipase needs minimal reorganization/flexibility of active site residues during its catalytic cycle. Molecular dynamic simulations suggest that catalytically competent active site geometry of the mutant is relatively more preserved than wild type lipase, which might have led to its higher enzyme activity. Our study implies that widely accepted positive correlation between conformation flexibility and enzyme activity need not be stringent and draws attention to the possibility that high enzyme activity can still be accomplished in a rigid active site and stable protein structures. This finding has a significant implication towards better understanding of involvement of dynamic motions in enzyme catalysis and enzyme engineering through mutations in active site

High Log-Scale Expansion of Functional Human Natural Killer Cells from Umbilical Cord Blood CD34-Positive Cells for Adoptive Cancer Immunotherapy

Immunotherapy based on natural killer (NK) cell infusions is a potential adjuvant treatment for many cancers. Such therapeutic application in humans requires large numbers of functional NK cells that have been selected and expanded using clinical grade protocols. We established an extremely efficient cytokine-based culture system for ex vivo expansion of NK cells from hematopoietic stem and progenitor cells from umbilical cord blood (UCB). Systematic refinement of this two-step system using a novel clinical grade medium resulted in a therapeutically applicable cell culture protocol. CD56+CD3− NK cell products could be routinely generated from freshly selected CD34+ UCB cells with a mean expansion of >15,000 fold and a nearly 100% purity. Moreover, our protocol has the capacity to produce more than 3-log NK cell expansion from frozen CD34+ UCB cells. These ex vivo-generated cell products contain NK cell subsets differentially expressing NKG2A and killer immunoglobulin-like receptors. Furthermore, UCB-derived CD56+ NK cells generated by our protocol uniformly express high levels of activating NKG2D and natural cytotoxicity receptors. Functional analysis showed that these ex vivo-generated NK cells efficiently target myeloid leukemia and melanoma tumor cell lines, and mediate cytolysis of primary leukemia cells at low NK-target ratios. Our culture system exemplifies a major breakthrough in producing pure NK cell products from limited numbers of CD34+ cells for cancer immunotherapy

Elucidating the Mechanisms of Influenza Virus Recognition by Ncr1

Natural killer (NK) cells are innate cytotoxic lymphocytes that specialize in the defense against viral infection and oncogenic transformation. Their action is tightly regulated by signals derived from inhibitory and activating receptors; the later include proteins such as the Natural Cytotoxicity Receptors (NCRs: NKp46, NKp44 and NKp30). Among the NCRs, NKp46 is the only receptor that has a mouse orthologue named Ncr1. NKp46/Ncr1 is also a unique marker expressed on NK and on Lymphoid tissue inducer (LTI) cells and it was implicated in the control of various viral infections, cancer and diabetes. We have previously shown that human NKp46 recognizes viral hemagglutinin (HA) in a sialic acid-dependent manner and that the O-glycosylation is essential for the NKp46 binding to viral HA. Here we studied the molecular interactions between Ncr1 and influenza viruses. We show that Ncr1 recognizes influenza virus in a sialic acid dependent manner and that N-glycosylation is important for this binding. Surprisingly we demonstrate that none of the predicted N-glycosilated residues of Ncr1 are essential for its binding to influenza virus and we thus conclude that other, yet unidentified N-glycosilated residues are responsible for its recognition. We have demonstrated that N glycosylation play little role in the recognition of mouse tumor cell lines and also showed the in-vivo importance of Ncr1 in the control of influenza virus infection by infecting C57BL/6 and BALB/c mice knockout for Ncr1 with influenza

Redirecting T Cells to Ewing's Sarcoma Family of Tumors by a Chimeric NKG2D Receptor Expressed by Lentiviral Transduction or mRNA Transfection

We explored the possibility to target Ewing's sarcoma family of tumors (ESFT) by redirecting T cells. To this aim, we considered NKG2D-ligands (NKG2D-Ls) as possible target antigens. Detailed analysis of the expression of MICA, MICB, ULBP-1, -2, and -3 in fourteen ESFT cell lines revealed consistent expression of at least one NKG2D-L. Thus, for redirecting T cells, we fused a CD3ζ/CD28-derived signaling domain to the ectodomain of NKG2D, however, opposite transmembrane orientation of this signaling domain and NKG2D required inverse orientation fusion of either of them. We hypothesized that the particularly located C-terminus of the NKG2D ectodomain should allow reengineering of the membrane anchoring from a native N-terminal to an artificial C-terminal linkage. Indeed, the resulting chimeric NKG2D receptor (chNKG2D) was functional and efficiently mediated ESFT cell death triggered by activated T cells. Notably, ESFT cells with even low NKG2D-L expression were killed by CD8pos and also CD4pos cells. Both, mRNA transfection and lentiviral transduction resulted in high level surface expression of chNKG2D. However, upon target-cell recognition receptor surface levels were maintained by tranfected RNA only during the first couple of hours after transfection. Later, target-cell contact resulted in strong and irreversible receptor down-modulation, whereas lentivirally mediated expression of chNKG2D remained constant under these conditions. Together, our study defines NKG2D-Ls as targets for a CAR-mediated T cell based immunotherapy of ESFT. A comparison of two different methods of gene transfer reveals strong differences in the susceptibility to ligand-induced receptor down-modulation with possible implications for the applicability of RNA transfection

Chemotherapy-resistant osteosarcoma is highly susceptible to IL-15-activated allogeneic and autologous NK cells

High-grade osteosarcoma occurs predominantly in adolescents and young adults and has an overall survival rate of about 60%, despite chemotherapy and surgery. Therefore, novel treatment modalities are needed to prevent or treat recurrent disease. Natural killer (NK) cells are lymphocytes with cytotoxic activity toward virus-infected or malignant cells. We explored the feasibility of autologous and allogeneic NK cell–mediated therapies for chemotherapy-resistant and chemotherapy-sensitive high-grade osteosarcoma. The expression by osteosarcoma cells of ligands for activating NK cell receptors was studied in vitro and in vivo, and their contribution to NK cell–mediated cytolysis was studied by specific antibody blockade. Chromium release cytotoxicity assays revealed chemotherapy-sensitive and chemotherapy-resistant osteosarcoma cell lines and osteosarcoma primary cultures to be sensitive to NK cell–mediated cytolysis. Cytolytic activity was strongly enhanced by IL-15 activation and was dependent on DNAM-1 and NKG2D pathways. Autologous and allogeneic activated NK cells lysed osteosarcoma primary cultures equally well. Osteosarcoma patient–derived NK cells were functionally and phenotypically unimpaired. In conclusion, osteosarcoma cells, including chemoresistant variants, are highly susceptible to lysis by IL-15-induced NK cells from both allogeneic and autologous origin. Our data support the exploitation of NK cells or NK cell–activating agents in patients with high-grade osteosarcoma

Excision of HIV-1 Proviral DNA by Recombinant Cell Permeable Tre-Recombinase

Over the previous years, comprehensive studies on antiretroviral drugs resulted in the successful introduction of highly active antiretroviral therapy (HAART) into clinical practice for treatment of HIV/AIDS. However, there is still need for new therapeutic approaches, since HAART cannot eradicate HIV-1 from the infected organism and, unfortunately, can be associated with long-term toxicity and the development of drug resistance. In contrast, novel gene therapy strategies may have the potential to reverse the infection by eradicating HIV-1. For example, expression of long terminal repeat (LTR)-specific recombinase (Tre-recombinase) has been shown to result in chromosomal excision of proviral DNA and, in consequence, in the eradication of HIV-1 from infected cell cultures. However, the delivery of Tre-recombinase currently depends on the genetic manipulation of target cells, a process that is complicating such therapeutic approaches and, thus, might be undesirable in a clinical setting. In this report we demonstrate that E.coli expressed Tre-recombinases, tagged either with the protein transduction domain (PTD) from the HIV-1 Tat trans-activator or the translocation motif (TLM) of the Hepatitis B virus PreS2 protein, were able to translocate efficiently into cells and showed significant recombination activity on HIV-1 LTR sequences. Tre activity was observed using episomal and stable integrated reporter constructs in transfected HeLa cells. Furthermore, the TLM-tagged enzyme was able to excise the full-length proviral DNA from chromosomal integration sites of HIV-1-infected HeLa and CEM-SS cells. The presented data confirm Tre-recombinase activity on integrated HIV-1 and provide the basis for the non-genetic transient application of engineered recombinases, which may be a valuable component of future HIV eradication strategies

Human Herpesvirus 8 (HHV8) Sequentially Shapes the NK Cell Repertoire during the Course of Asymptomatic Infection and Kaposi Sarcoma

The contribution of innate immunity to immunosurveillance of the oncogenic Human Herpes Virus 8 (HHV8) has not been studied in depth. We investigated NK cell phenotype and function in 70 HHV8-infected subjects, either asymptomatic carriers or having developed Kaposi's sarcoma (KS). Our results revealed substantial alterations of the NK cell receptor repertoire in healthy HHV8 carriers, with reduced expression of NKp30, NKp46 and CD161 receptors. In addition, down-modulation of the activating NKG2D receptor, associated with impaired NK-cell lytic capacity, was observed in patients with active KS. Resolution of KS after treatment was accompanied with restoration of NKG2D levels and NK cell activity. HHV8-latently infected endothelial cells overexpressed ligands of several NK cell receptors, including NKG2D ligands. The strong expression of NKG2D ligands by tumor cells was confirmed in situ by immunohistochemical staining of KS biopsies. However, no tumor-infiltrating NK cells were detected, suggesting a defect in NK cell homing or survival in the KS microenvironment. Among the known KS-derived immunoregulatory factors, we identified prostaglandin E2 (PGE2) as a critical element responsible for the down-modulation of NKG2D expression on resting NK cells. Moreover, PGE2 prevented up-regulation of the NKG2D and NKp30 receptors on IL-15-activated NK cells, and inhibited the IL-15-induced proliferation and survival of NK cells. Altogether, our observations are consistent with distinct immunoevasion mechanisms that allow HHV8 to escape NK cell responses stepwise, first at early stages of infection to facilitate the maintenance of viral latency, and later to promote tumor cell growth through suppression of NKG2D-mediated functions. Importantly, our results provide additional support to the use of PGE2 inhibitors as an attractive approach to treat aggressive KS, as they could restore activation and survival of tumoricidal NK cells

Formation of Trans-Activation Competent HIV-1 Rev:RRE Complexes Requires the Recruitment of Multiple Protein Activation Domains

The HIV-1 Rev trans-activator is a nucleocytoplasmic shuttle protein that is essential for virus replication. Rev directly binds to unspliced and incompletely spliced viral RNA via the cis-acting Rev Response Element (RRE) sequence. Subsequently, Rev oligomerizes cooperatively and interacts with the cellular nuclear export receptor CRM1. In addition to mediating nuclear RNA export, Rev also affects the stability, translation and packaging of Rev-bound viral transcripts. Although it is established that Rev function requires the multimeric assembly of Rev molecules on the RRE, relatively little is known about how many Rev monomers are sufficient to form a trans-activation competent Rev:RRE complex, or which specific activity of Rev is affected by its oligomerization. We here analyzed by functional studies how homooligomer formation of Rev affects the trans-activation capacity of this essential HIV-1 regulatory protein. In a gain-of-function approach, we fused various heterologous dimerization domains to an otherwise oligomerization-defective Rev mutant and were able to demonstrate that oligomerization of Rev is not required per se for the nuclear export of this viral trans-activator. In contrast, however, the formation of Rev oligomers on the RRE is a precondition to trans-activation by directly affecting the nuclear export of Rev-regulated mRNA. Moreover, experimental evidence is provided showing that at least two protein activation domains are required for the formation of trans-activation competent Rev:RRE complexes. The presented data further refine the model of Rev trans-activation by directly demonstrating that Rev oligomerization on the RRE, thereby recruiting at least two protein activation domains, is required for nuclear export of unspliced and incompletely spliced viral RNA

NK cell receptor NKG2D sets activation threshold for the NCR1 receptor early in NK cell development

The activation of natural killer (NK) cells depends on a change in the balance of signals from inhibitory and activating receptors. The activation threshold values of NK cells are thought to be set by engagement of inhibitory receptors during development. Here, we found that the activating receptor NKG2D specifically set the activation threshold for the activating receptor NCR1 through a process that required the adaptor DAP12. As a result, NKGD2-deficient (Klrk1-/-) mice controlled tumors and cytomegalovirus infection better than wild-type controls through the NCR1-induced production of the cytokine IFN-γ. Expression of NKG2D before the immature NK cell stage increased expression of the adaptor CD3ζ. Reduced expression of CD3ζ in Klrk1-/- mice was associated with enhanced signal transduction through NCR1, and CD3ζ deficiency resulted in hyper-responsiveness to stimulation via NCR1. Thus, an activating receptor developmentally set the activity of another activating receptor on NK cells and determined NK cell reactivity to cellular threats