Integrase-derived peptides together with CD24-targeted lentiviral particles inhibit the growth of CD24 expressing cancer cells.

The integration of viral DNA into the host genome is mediated by viral integrase, resulting in the accumulation of double-strand breaks. Integrase-derived peptides (INS and INR) increase the number of integration events, leading to escalated genomic instability that induces apoptosis. CD24 is a surface protein expressed mostly in cancer cells and is very rarely found in normal cells. Here, we propose a novel targeted cancer therapeutic platform based on the lentiviral integrase, stimulated by integrase-derived peptides, that are specifically delivered to cancerous cells via CD24 antigen-antibody targeting. INS and INR were synthesized and humanized and anti-CD24 antibodies were fused to the lentivirus envelope. The activity, permeability, stability, solubility, and toxicity of these components were analyzed. Cell death was measured by fluorescent microscopy and enzymatic assays and potency were tested in vitro and in vivo. Lentivirus particles, containing non-functional DNA led to massive cell death (40-70%). Raltegravir, an antiretroviral drug, inhibited the induction of apoptosis. In vivo, single and repeated administrations of INS/INR were well tolerated without any adverse effects. Tumor development in nude mice was significantly inhibited (by 50%) as compared to the vehicle arm. In summary, a novel and generic therapeutic platform for selective cancer cell eradication with excellent efficacy and safety are presented.

Integrase-Defective Lentiviral Vector Is an Efficient Vaccine Platform for Cancer Immunotherapy.

Integrase-defective lentiviral vectors (IDLVs) have been used as a safe and efficient delivery system in several immunization protocols in murine and non-human primate preclinical models as well as in recent clinical trials. In this work, we validated in preclinical murine models our vaccine platform based on IDLVs as delivery system for cancer immunotherapy. To evaluate the anti-tumor activity of our vaccine strategy we generated IDLV delivering ovalbumin (OVA) as a non-self-model antigen and TRP2 as a self-tumor associated antigen (TAA) of melanoma. Results demonstrated the ability of IDLVs to eradicate and/or controlling tumor growth after a single immunization in preventive and therapeutic approaches, using lymphoma and melanoma expressing OVA. Importantly, LV-TRP2 but not IDLV-TRP2 was able to break tolerance efficiently and prevent tumor growth of B16F10 melanoma cells. In order to improve the IDLV efficacy, the human homologue of murine TRP2 was used, showing the ability to break tolerance and control the tumor growth. These results validate the use of IDLV for cancer therapy.

Secretion of a mammalian chondroitinase ABC aids glial integration at PNS/CNS boundaries.

Schwann cell grafts support axonal growth following spinal cord injury, but a boundary forms between the implanted cells and host astrocytes. Axons are reluctant to exit the graft tissue in large part due to the surrounding inhibitory environment containing chondroitin sulphate proteoglycans (CSPGs). We use a lentiviral chondroitinase ABC, capable of being secreted from mammalian cells (mChABC), to examine the repercussions of CSPG digestion upon Schwann cell behaviour in vitro. We show that mChABC transduced Schwann cells robustly secrete substantial quantities of the enzyme causing large-scale CSPG digestion, facilitating the migration and adhesion of Schwann cells on inhibitory aggrecan and astrocytic substrates. Importantly, we show that secretion of the engineered enzyme can aid the intermingling of cells at the Schwann cell-astrocyte boundary, enabling growth of neurites over the putative graft/host interface. These data were echoed in vivo. This study demonstrates the profound effect of the enzyme on cellular motility, growth and migration. This provides a cellular mechanism for mChABC induced functional and behavioural recovery shown in in vivo studies. Importantly, we provide in vitro evidence that mChABC gene therapy is equally or more effective at producing these effects as a one-time application of commercially available ChABC.

Persistence of Integrase-Deficient Lentiviral Vectors Correlates with the Induction of STING-Independent CD8+ T Cell Responses.

Lentiviruses are among the most promising viral vectors for in vivo gene delivery. To overcome the risk of insertional mutagenesis, integrase-deficient lentiviral vectors (IDLVs) have been developed. We show here that strong and persistent specific cytotoxic T cell (CTL) responses are induced by IDLVs, which persist several months after a single injection. These responses were associated with the induction of mild and transient maturation of dendritic cells (DCs) and with the production of low levels of inflammatory cytokines and chemokines. They were independent of the IFN-I, TLR/MyD88, interferon regulatory factor (IRF), retinoic acid induced gene I (RIG-I), and stimulator of interferon genes (STING) pathways but require NF-κB signaling in CD11c+ DCs. Despite the lack of integration of IDLVs, the transgene persists for 3 months in the spleen and liver of IDLV-injected mice. These results demonstrate that the capacity of IDLVs to trigger persistent adaptive responses is mediated by a weak and transient innate response, along with the persistence of the vector in tissues.

A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells.

Lentiviral vectors are an ideal choice for delivering gene-editing components to cells due to their capacity for stably transducing a broad range of cells and mediating high levels of gene expression. However, their ability to integrate into the host cell genome enhances the risk of insertional mutagenicity and thus raises safety concerns and limits their usage in clinical settings. Further, the persistent expression of gene-editing components delivered by these integration-competent lentiviral vectors (ICLVs) increases the probability of promiscuous gene targeting. As an alternative, a new generation of integrase-deficient lentiviral vectors (IDLVs) has been developed that addresses many of these concerns. Here the production protocol of a new and improved IDLV platform for CRISPR-mediated gene editing and list the steps involved in the purification and concentration of such vectors is described and their transduction and gene-editing efficiency using HEK-293T cells was demonstrated. This protocol is easily scalable and can be used to generate high titer IDLVs that are capable of transducing cells in vitro and in vivo. Moreover, this protocol can be easily adapted for the production of ICLVs.

Multiple cellular proteins interact with LEDGF/p75 through a conserved unstructured consensus motif.

Lens epithelium-derived growth factor (LEDGF/p75) is an epigenetic reader and attractive therapeutic target involved in HIV integration and the development of mixed lineage leukaemia (MLL1) fusion-driven leukaemia. Besides HIV integrase and the MLL1-menin complex, LEDGF/p75 interacts with various cellular proteins via its integrase binding domain (IBD). Here we present structural characterization of IBD interactions with transcriptional repressor JPO2 and domesticated transposase PogZ, and show that the PogZ interaction is nearly identical to the interaction of LEDGF/p75 with MLL1. The interaction with the IBD is maintained by an intrinsically disordered IBD-binding motif (IBM) common to all known cellular partners of LEDGF/p75. In addition, based on IBM conservation, we identify and validate IWS1 as a novel LEDGF/p75 interaction partner. Our results also reveal how HIV integrase efficiently displaces cellular binding partners from LEDGF/p75. Finally, the similar binding modes of LEDGF/p75 interaction partners represent a new challenge for the development of selective interaction inhibitors.

Unbiased detection of off-target cleavage by CRISPR-Cas9 and TALENs using integrase-defective lentiviral vectors.

The utility of CRISPR-Cas9 and TALENs for genome editing may be compromised by their off-target activity. We show that integrase-defective lentiviral vectors (IDLVs) can detect such off-target cleavage with a frequency as low as 1%. In the case of Cas9, we find frequent off-target sites with a one-base bulge or up to 13 mismatches between the single guide RNA (sgRNA) and its genomic target, which refines sgRNA design.

Optimization of mucosal responses after intramuscular immunization with integrase defective lentiviral vector.

Many infectious agents infiltrate the host at the mucosal surfaces and then spread systemically. This implies that an ideal vaccine should induce protective immune responses both at systemic and mucosal sites to counteract invasive mucosal pathogens. We evaluated the in vivo systemic and mucosal antigen-specific immune response induced in mice by intramuscular administration of an integrase defective lentiviral vector (IDLV) carrying the ovalbumin (OVA) transgene as a model antigen (IDLV-OVA), either alone or in combination with sublingual adjuvanted OVA protein. Mice immunized intramuscularly with OVA and adjuvant were compared with IDLV-OVA immunization. Mice sublingually immunized only with OVA and adjuvant were used as a positive control of mucosal responses. A single intramuscular dose of IDLV-OVA induced functional antigen-specific CD8+ T cell responses in spleen, draining and distal lymph nodes and, importantly, in the lamina propria of the large intestine. These results were similar to those obtained in a prime-boost regimen including one IDLV immunization and two mucosal boosts with adjuvanted OVA or vice versa. Remarkably, only in groups vaccinated with IDLV-OVA, either alone or in prime-boost regimens, the mucosal CD8+ T cell response persisted up to several months from immunization. Importantly, following IDLV-OVA immunization, the mucosal boost with protein greatly increased the plasma IgG response and induced mucosal antigen-specific IgA in saliva and vaginal washes. Overall, intramuscular administration of IDLV followed by protein boosts using the sublingual route induced strong, persistent and complementary systemic and mucosal immune responses, and represents an appealing prime-boost strategy for immunization including IDLV as a delivery system.

Successful therapeutic vaccination with integrase defective lentiviral vector expressing nononcogenic human papillomavirus E7 protein.

Persistent infection with high risk genotypes of human papillomavirus (HPV) is the cause of cervical cancer, one of most common cancer among woman worldwide, and represents an important risk factor associated with other anogenital and oropharyngeal cancers in men and women. Here, we designed a therapeutic vaccine based on integrase defective lentiviral vector (IDLV) to deliver a mutated nononcogenic form of HPV16 E7 protein, considered as a tumor specific antigen for immunotherapy of HPV-associated cervical cancer, fused to calreticulin (CRT), a protein able to enhance major histocompatibility complex class I antigen presentation (IDLV-CRT/E7). Vaccination with IDLV-CRT/E7 induced a potent and persistent E7-specific T cell response up to 1 year after a single immunization. Importantly, a single immunization with IDLV-CRT/E7 was able to prevent growth of E7-expressing TC-1 tumor cells and to eradicate established tumors in mice. The strong therapeutic effect induced by the IDLV-based vaccine in this preclinical model suggests that this strategy may be further exploited as a safe and attractive anticancer immunotherapeutic vaccine in humans.

Gene delivery to overcome astrocyte inhibition of axonal growth: an in vitro model of the glial scar.

After injury to the central nervous system, a glial scar develops that physically and biochemically inhibits axon growth. In the scar, activated astrocytes secrete inhibitory extracellular matrix, of which chondroitin sulfate proteoglycans (CSPGs) are considered the major inhibitory component. An inhibitory interface of CSPGs forms around the lesion and prevents axons from traversing the injury, and decreasing CSPGs can enhance axon growth. In this report, we established an in vitro interface model of activated astrocytes and subsequently investigated gene delivery as a means to reduce CSPG levels and enhance axon growth. In the model, a continuous interface of CSPG producing astrocytes was created with neurons seeded opposite the astrocytes, and neurite crossing, stopping, and turning were evaluated as they approached the interface. We investigated the efficacy of lentiviral delivery to degrade or prevent the synthesis of CSPGs, thereby removing CSPG inhibition of neurite growth. Lentiviral delivery of RNAi targeting two key CSPG synthesis enzymes, chondroitin polymerizing factor and chondroitin synthase-1, decreased CSPGs, and reduced inhibition by the interface. Degradation of CSPGs by lentiviral delivery of chondroitinase also resulted in less inhibition and more neurites crossing the interface. These results indicate that the interface model provides a tool to investigate interventions that reduce inhibition by CSPGs, and that gene delivery can be effective in promoting neurite growth across an interface of CSPG producing astrocytes.

Phylogenetic analysis of small ruminant lentiviruses detected in Slovenia.

Small ruminant lentiviruses (SRLV), which belong to the Retroviridae family, infect goats and sheep worldwide. The aim of this study was to characterize the SRLV strains circulating in Slovenia, by phylogenetic analysis of two genomic regions, 1.8 kb gag-pol fragment and 1.2kb pol fragment. The results of our study revealed that Slovenian SRLV strains are highly heterogeneous, with ovine strains belonging to genotype A and caprine strains to genotypes A and B. The closest relatives of sheep virus sequences from two flocks that clustered together (SLO 35, 36) were found to be in subtype A5. A cluster composed of four sheep virus sequences (SLO 31) was clearly divergent from all other subtypes in group A and could not be assigned to any of them. The virus sequences from one goat flock belonged solely to subtype B1, whereas virus sequences from more than one genotype were found to circulate within the other two goat flocks, belonging to subtype B1 (SLO 1 and SLO 37) and to genotype A (SLO 2 and 78-88 g). Two goat virus sequences (SLO 2) were found to belong to genotype A and could not be assigned to existing subtypes. One goat virus sequence (37-88 g) from flock 37 was clearly different from other sequences of this flock and was more closely related to genotype A sequences. We propose two new subtypes within genotype A, subtype A14 (SLO 2) and A15 (SLO 31).

Clathrin facilitates the morphogenesis of retrovirus particles.

The morphogenesis of retroviral particles is driven by Gag and GagPol proteins that provide the major structural component and enzymatic activities required for particle assembly and maturation. In addition, a number of cellular proteins are found in retrovirus particles; some of these are important for viral replication, but many lack a known functional role. One such protein is clathrin, which is assumed to be passively incorporated into virions due to its abundance at the plasma membrane. We found that clathrin is not only exceptionally abundant in highly purified HIV-1 particles but is recruited with high specificity. In particular, the HIV-1 Pol protein was absolutely required for clathrin incorporation and point mutations in reverse transcriptase or integrase domains of Pol could abolish incorporation. Clathrin was also specifically incorporated into other retrovirus particles, including members of the lentivirus (simian immunodeficiency virus, SIVmac), gammaretrovirus (murine leukemia virus, MLV) and betaretrovirus (Mason-Pfizer monkey virus, M-PMV) genera. However, unlike HIV-1, these other retroviruses recruited clathrin primarily using peptide motifs in their respective Gag proteins that mimicked motifs found in cellular clathrin adaptors. Perturbation of clathrin incorporation into these retroviruses, via mutagenesis of viral proteins, siRNA based clathrin depletion or adaptor protein (AP180) induced clathrin sequestration, had a range of effects on the accuracy of particle morphogenesis. These effects varied according to which retrovirus was examined, and included Gag and/or Pol protein destabilization, inhibition of particle assembly and reduction in virion infectivity. For each retrovirus examined, clathrin incorporation appeared to be important for optimal replication. These data indicate that a number of retroviruses employ clathrin to facilitate the accurate morphogenesis of infectious particles. We propose a model in which clathrin contributes to the spatial organization of Gag and Pol proteins, and thereby regulates proteolytic processing of virion components during particle assembly.

Hepatocyte-targeted expression by integrase-defective lentiviral vectors induces antigen-specific tolerance in mice with low genotoxic risk.

UNLABELLED: Lentiviral vectors are attractive tools for liver-directed gene therapy because of their capacity for stable gene expression and the lack of preexisting immunity in most human subjects. However, the use of integrating vectors may raise some concerns about the potential risk of insertional mutagenesis. Here we investigated liver gene transfer by integrase-defective lentiviral vectors (IDLVs) containing an inactivating mutation in the integrase (D64V). Hepatocyte-targeted expression using IDLVs resulted in the sustained and robust induction of immune tolerance to both intracellular and secreted proteins, despite the reduced transgene expression levels in comparison with their integrase-competent vector counterparts. IDLV-mediated and hepatocyte-targeted coagulation factor IX (FIX) expression prevented the induction of neutralizing antibodies to FIX even after antigen rechallenge in hemophilia B mice and accounted for relatively prolonged therapeutic FIX expression levels. Upon the delivery of intracellular model antigens, hepatocyte-targeted IDLVs induced transgene-specific regulatory T cells that contributed to the observed immune tolerance. Deep sequencing of IDLV-transduced livers showed only rare genomic integrations that had no preference for gene coding regions and occurred mostly by a mechanism inconsistent with residual integrase activity. CONCLUSION: IDLVs provide an attractive platform for the tolerogenic expression of intracellular or secreted proteins in the liver with a substantially reduced risk of insertional mutagenesis.

Mesenchymal stem cells modified to express lentivirus TNF-α Tumstatin(45-132) inhibit the growth of prostate cancer.

Mesenchymal stem cells (MSCs) are a potential novel delivery system for cell-based gene therapies. Although tumour necrosis factor (TNF)-α has been shown to have antitumour activity, its use in therapy is limited by its systemic toxicity. For the present study, we designed lentivirus-mediated signal peptide TNF-α-Tumstatin(45-132) -expressing mesenchymal stem cells (SPTT-MSCs) as a novel anti-cancer approach. We evaluated the effects of this approach on human prostate cancer cells (PC3 and LNCaP) by co-culturing them with either SPTT-MSCs or supernatants from their culture medium in vitro. The antitumour effects and possible mechanisms of action of SPTT-MSCs were then determined in PC3 cells in vivo. The results showed that efficient TNF-α-Tumstatin(45-132) -expressing MSCs had been established, and demonstrated that SPTT-MSCs inhibited the proliferation of and induced apoptosis in prostate cancer cells and xenograft tumours. As would be expected, given the properties of the individual proteins, the TNF-α-Tumstatin(45-132) fusion exerted potent cytotoxic effects on human prostate cancer cells and tumours via the death receptor-dependent apoptotic pathway and via antiangiogenic effects. Our findings suggest that SPTT-MSCs have significant activity against prostate cancer cells, and that they may represent a promising new therapy for prostate cancer.

Identification of a critical T(Q/D/E)x5ADx2(I/L) motif from primate lentivirus Vif proteins that regulate APOBEC3G and APOBEC3F neutralizing activity.

Primate lentiviruses are unique in that they produce several accessory proteins to help in the establishment of productive viral infection. The major function of these proteins is to clear host resistance factors that inhibit viral replication. Vif is one of these proteins. It functions as an adaptor that binds to the cytidine deaminases APOBEC3G (A3G) and APOBEC3F (A3F) and bridges them to a cullin 5 (Cul5) and elongin (Elo) B/C E3 ubiquitin ligase complex for proteasomal degradation. So far, 11 discontinuous domains in Vif have been identified that regulate this degradation process. Here we report another domain, T(Q/D/E)x(5)ADx(2)(I/L), which is located at residues 96 to 107 in the human immunodeficiency virus type 1 (HIV-1) Vif protein. This domain is conserved not only in all HIV-1 subtypes but also in other primate lentiviruses, including HIV-2 and simian immunodeficiency virus (SIV), which infects rhesus macaques (SIVmac) and African green monkeys (SIVagm). Mutations of the critical residues in this motif seriously disrupted Vif's neutralizing activity toward both A3G and A3F. This motif regulates Vif interaction not only with A3G and A3F but also with Cul5. When this motif was inactivated in the HIV-1 genome, Vif failed to exclude A3G and A3F from virions, resulting in abortive HIV replication in nonpermissive human T cells. Thus, T(Q/D/E)x(5)ADx(2)(I/L) is a critical functional motif that directly supports the adaptor function of Vif and is an attractive target for inhibition of Vif function.

Placenta-specific gene activation and inactivation using integrase-defective lentiviral vectors with the Cre/LoxP system.

Transgenic and knockout studies have advanced our understanding of the genetic control of embryonic development over the past decades. However, interpretation of the phenotype of mutant mice is potentially complicated, since the commonly used knockout approach modifies both the fetal and placental genome. To circumvent this problem, we previously developed a placenta-specific gene manipulation system by lentiviral vector transduction of embryos at the blastocyst stage. In the present study, by combination with the Cre/LoxP system, we successfully demonstrate placenta-specific gene activation and inactivation in EGFP reporter mice and Ets2 floxed mice, respectively. Transient expression using integrase-defective lentiviral (IDLV) vectors diminished the toxic effect of Cre expression and solved the dilemma of mosaic recombination with lower concentrations and toxic effects with higher concentrations of Cre recombinase. We also show that placenta-specific Ets2 disruption causes embryonic lethality and reconfirmed the critical role of Ets2 during placentation. This technology facilitates both gain and loss of gene function analyses in placental development during pregnancy. Since IDLV vectors can efficiently transduce a variety of cell types similarly to wild-type vectors, our IDLV-Cre strategy is potentially useful for a wide range of applications.

In vitro proliferation of cells derived from adult human beta-cells revealed by cell-lineage tracing.

OBJECTIVE: Expansion of insulin-producing beta-cells from adult human islets could alleviate donor shortage for cell-replacement therapy of diabetes. A major obstacle to development of effective expansion protocols is the rapid loss of beta-cell markers in the cultured cells. Here, we report a genetic cell-lineage tracing approach for following the fate of cultured beta-cells. RESEARCH DESIGN AND METHODS: Cells dissociated from isolated human islets were infected with two lentiviruses, one expressing Cre recombinase under control of the insulin promoter and the other, a reporter cassette with the structure cytomegalovirus promoter-loxP-DsRed2-loxP-eGFP. RESULTS: Beta-cells were efficiently and specifically labeled by the dual virus system. Label(+), insulin(-) cells derived from beta-cells were shown to proliferate for a maximum of 16 population doublings, with an approximate doubling time of 7 days. Isolated labeled cells could be expanded in the absence of other pancreas cell types if provided with medium conditioned by pancreatic non-beta-cells. Analysis of mouse islet cells by the same method revealed a much lower proliferation of labeled cells under similar culture conditions. CONCLUSIONS: Our findings provide direct evidence for survival and dedifferentiation of cultured adult human beta-cells and demonstrate that the dedifferentiated cells significantly proliferate in vitro. The findings confirm the difference between mouse and human beta-cell proliferation under our culture conditions. These findings demonstrate the feasibility of cell-specific labeling of cultured primary human cells using a genetic recombination approach that was previously restricted to transgenic animals.

Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery.

Achieving the full potential of zinc-finger nucleases (ZFNs) for genome engineering in human cells requires their efficient delivery to the relevant cell types. Here we exploited the infectivity of integrase-defective lentiviral vectors (IDLV) to express ZFNs and provide the template DNA for gene correction in different cell types. IDLV-mediated delivery supported high rates (13-39%) of editing at the IL-2 receptor common gamma-chain gene (IL2RG) across different cell types. IDLVs also mediated site-specific gene addition by a process that required ZFN cleavage and homologous template DNA, thus establishing a platform that can target the insertion of transgenes into a predetermined genomic site. Using IDLV delivery and ZFNs targeting distinct loci, we observed high levels of gene addition (up to 50%) in a panel of human cell lines, as well as human embryonic stem cells (5%), allowing rapid, selection-free isolation of clonogenic cells with the desired genetic modification.

LEDGF/p75 interacts with divergent lentiviral integrases and modulates their enzymatic activity in vitro.

Transcriptional co-activator LEDGF/p75 is the major cellular interactor of HIV-1 integrase (IN), critical to efficient viral replication. In this work, a series of INs from the Betaretrovirus, Gammaretrovirus, Deltaretrovirus, Spumavirus and Lentivirus retroviral genera were tested for interaction with the host factor. None of the non-lentiviral INs possessed detectable affinity for LEDGF in either pull-down or yeast two-hybrid assays. In contrast, all lentiviral INs examined, including those from bovine immunodeficiency virus (BIV), maedi-visna virus (MVV) and equine infectious anemia virus (EIAV) readily interacted with LEDGF. Mutation of Asp-366 to Asn in LEDGF ablated the interaction, suggesting a common mechanism of the host factor recognition by the INs. LEDGF potently stimulated strand transfer activity of divergent lentiviral INs in vitro. Unprecedentedly, in the presence of the host factor, EIAV IN almost exclusively catalyzed concerted integration, whereas HIV-1 IN promoted predominantly half-site integration, and BIV IN was equally active in both types of strand transfer. Concerted BIV and EIAV integration resulted in 5 bp duplications of the target DNA sequences. These results confirm that the interaction with LEDGF is conserved within and limited to Lentivirus and strongly argue that the host factor is intimately involved in the catalysis of lentiviral DNA integration.

Expression changes in tolerant murine cardiac allografts after gene therapy with a lentiviral vector expressing alpha1,3 galactosyltransferase.

Comparison of intragraft gene expression changes in tolerant cardiac allograft models may provide the basis for identifying pathways involved in graft survival. Our laboratory has previously demonstrated that tolerance to the gal alpha1,3 gal epitope, the major target of rejection of wild-type pig hearts in human cardiac transplantation, can be achieved after transplantation with bone marrow transduced with a lentiviral vector expressing alpha1,3 galactosyltransferase. We now present intracardiac gene expression changes associated with long-term tolerance in this model. Biotin-labeled cRNA was hybridized to Affymetrix GeneChip 430 2.0 Mouse Genome Arrays. Data were subjected to functional annotation analysis to identify genes of known function in which expression was increased or decreased by at least 2-fold (t-test, P < .05) in tolerant gal+/+ wild-type hearts as compared to transplanted syngeneic controls. Tolerant hearts demonstrated increased expression of genes associated with the stress response, modulation of immune function and cell survival (HSPa9a, CD56, and Akt1s1), and decreased expression of several immunoregulatory genes (CD209, CD26, and PDE4b). These data suggest that tolerance may be associated with activation of immunomodulatory and survival pathways.