The exception that reinforces the rule: crosspriming by cytosolic peptides that escape degradation.

The nature of crosspriming immunogens for CD8(+) T cell responses is highly controversial. By using a panel of T cell receptor-like antibodies specific for viral peptides bound to mouse D(b) major histocompatibility complex class I molecules, we show that an exceptional peptide (PA(224-233)) expressed as a viral minigene product formed a sizeable cytosolic pool continuously presented for hours after protein synthesis was inhibited. PA(224-233) pool formation required active cytosolic heat-shock protein 90 but not ER g96 and uniquely enabled crosspriming by this peptide. These findings demonstrate that exceptional class I binding oligopeptides that escape proteolytic degradation are potent crosspriming agents. Thus, the feeble immunogenicity of natural proteasome products in crosspriming can be attributed to their evanescence in donor cells and not an absolute inability of cytosolic oligopeptides to be transferred to and presented by professional antigen-presenting cells.

Human placental eXpanded (PLX) mesenchymal-like adherent stromal cells confer neuroprotection to nerve growth factor (NGF)-differentiated PC12 cells exposed to ischemia by secretion of IL-6 and VEGF.

Mesenchymal stem cells are potent candidates in stroke therapy due to their ability to secrete protective anti-inflammatory cytokines and growth factors. We investigated the neuroprotective effects of human placental mesenchymal-like adherent stromal cells (PLX) using an established ischemic model of nerve growth factor (NGF)-differentiated pheochromocytoma PC12 cells exposed to oxygen and glucose deprivation (OGD) followed by reperfusion. Under optimal conditions, 2 × 105 PLX cells, added in a trans-well system, conferred 30-60% neuroprotection to PC12 cells subjected to ischemic insult. PC12 cell death, measured by LDH release, was reduced by PLX cells or by conditioned medium derived from PLX cells exposed to ischemia, suggesting the active release of factorial components. Since neuroprotection is a prominent function of the cytokine IL-6 and the angiogenic factor VEGF165, we measured their secretion using selective ELISA of the cells under ischemic or normoxic conditions. IL-6 and VEGF165 secretion by co-culture of PC12 and PLX cells was significantly higher under ischemic compared to normoxic conditions. Exogenous supplementation of 10 ng/ml each of IL-6 and VEGF165 to insulted PC12 cells conferred neuroprotection, reminiscent of the neuroprotective effect of PLX cells or their conditioned medium. Growth factors as well as co-culture conditioned medium effects were reduced by 70% and 20% upon pretreatment with 240 ng/ml Semaxanib (anti VEGF165) and/or 400 ng/ml neutralizing anti IL-6 antibody, respectively. Therefore, PLX-induced neuroprotection in ischemic PC12 cells may be partially explained by IL-6 and VEGF165 secretion. These findings may also account for the therapeutic effects seen in clinical trials after treatment with these cells.

Innate immune and chemically triggered oxidative stress modifies translational fidelity.

Translational fidelity, essential for protein and cell function, requires accurate transfer RNA (tRNA) aminoacylation. Purified aminoacyl-tRNA synthetases exhibit a fidelity of one error per 10,000 to 100,000 couplings. The accuracy of tRNA aminoacylation in vivo is uncertain, however, and might be considerably lower. Here we show that in mammalian cells, approximately 1% of methionine (Met) residues used in protein synthesis are aminoacylated to non-methionyl-tRNAs. Remarkably, Met-misacylation increases up to tenfold upon exposing cells to live or non-infectious viruses, toll-like receptor ligands or chemically induced oxidative stress. Met is misacylated to specific non-methionyl-tRNA families, and these Met-misacylated tRNAs are used in translation. Met-misacylation is blocked by an inhibitor of cellular oxidases, implicating reactive oxygen species (ROS) as the misacylation trigger. Among six amino acids tested, tRNA misacylation occurs exclusively with Met. As Met residues are known to protect proteins against ROS-mediated damage, we propose that Met-misacylation functions adaptively to increase Met incorporation into proteins to protect cells against oxidative stress. In demonstrating an unexpected conditional aspect of decoding mRNA, our findings illustrate the importance of considering alternative iterations of the genetic code.

RNA binding targets aminoacyl-tRNA synthetases to translating ribosomes.

Here, we examine tRNA-aminoacyl synthetase (ARS) localization in protein synthesis. Proteomics reveals that ten of the twenty cytosolic ARSs associate with ribosomes in sucrose gradients: phenylalanyl-RS (FRS), and the 9 ARSs that form the multi-ARS complex (MSC). Using the ribopuromycylation method (RPM) for localizing intracellular translation, we show that FRS and the MSC, and to a lesser extent other ARSs, localize to translating ribosomes, most strikingly when translation is restricted to poxvirus or alphavirus factories in infected cells. Immunoproximity fluorescence indicates close proximity between MSC and the ribosome. Stress induced-translational shutdown recruits the MSC to stress-granules, a depot for mRNA and translation components. MSC binding to mRNA provides a facile explanation for its delivery to translating ribosomes and stress granules. These findings, along with the abundance of the MSC (9 × 10(6) copies per cell, roughly equimolar with ribosomes), is consistent with the idea that MSC specificity, recently reported to vary with cellular stress (Netzer, N., Goodenbour, J. M., David, A., Dittmar, K. A., Jones, R. B., Schneider, J. R., Boone, D., Eves, E. M., Rosner, M. R., Gibbs, J. S., Embry, A., Dolan, B., Das, S., Hickman, H. D., Berglund, P., Bennink, J. R., Yewdell, J. W., and Pan, T. (2009) Nature 462, 522-526) can be modulated at the level of individual mRNAs to modify decoding of specific gene products.

Direct visualization of the dynamics of antigen presentation in human cells infected with cytomegalovirus revealed by antibodies mimicking TCR specificity.

There are no direct means to study class I MHC presentation in human normal or diseased cells. Using CMV-infected human cells and applying novel mAb that mimic T-cell receptor specificity directed toward the immunogenic epitope of the viral pp65 protein presented on HLA-A2 molecules, we directly imaged the dynamics of Ag presentation in infected cells. We demonstrate that following infection large intracellular pools of HLA-A2/pp65 complexes are localized to the Golgi. These HLA-A2/pp65 pools account for the majority of total HLA-A2 molecules in infected cells. Interestingly, these large pools are sequestered inside infected cells and only a small portion of them are exported to the cell surface. Virus-induced class I MHC down-regulation did not affect the intracellular pool of HLA-A2/pp65 complexes. Our data also suggest that proteasome function influences the release of class I complexes to the membrane. We present herein a new and direct molecular tool to study the dynamics of viral Ag presentation that may further elucidate the balance between immune response versus viral escape.

Tr1 cell-dependent active tolerance blunts the pathogenic effects of determinant spreading.

Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated autoimmune disease of the CNS. The current study shows that even in an acute episode of disease the autoimmune response spreads from one determinant on myelin basic protein (MBP) to the other linked determinant and that this spread plays a functional role in the pathogenesis of disease. The soluble form of each determinant could be used to induce Ag-specific T cell tolerance and reverse an ongoing disease. We show that the rapid effect of soluble peptide therapy is due to repolarization of autoimmune T cells undergoing activation. We suggest that at least two different types of regulatory T cells participate in the induction of active tolerance. The first, yet to be fully characterized, functions in an IL-4-dependent manner. The second produces high levels of IL-10 and low levels of IL-4 (Tr1). We bring about completing evidence showing that these Tr1 cells play a pivotal role in the regulation of T cell tolerance during determinant spread and that soluble peptide therapy with the determinant to which the autoimmune response spreads amplifies a de novo regulatory mechanism aimed to reduce the pathological consequences of determinant spreading.

Long-Term Efficacy of GMP Grade Xeno-Free hESC-Derived RPE Cells Following Transplantation.

PURPOSE: Retinal pigment epithelium (RPE) dysfunction underlies the retinal degenerative process in age-related macular degeneration (AMD), and thus RPE cell replacement provides an optimal treatment target. We characterized longitudinally the efficacy of RPE cells derived under xeno-free conditions from clinical and xeno-free grade human embryonic stem cells (OpRegen) following transplantation into the subretinal space of Royal College of Surgeons (RCS) rats. METHODS: Postnatal (P) day 20 to 25 RCS rats (n = 242) received a single subretinal injection of 25,000 (low)-, 100,000 (mid)-, or 200,000 (high)-dose xeno-free RPE cells. BSS+ (balanced salt solution) (vehicle) and unoperated eyes served as controls. Optomotor tracking (OKT) behavior was used to quantify functional efficacy. Histology and immunohistochemistry were used to evaluate photoreceptor rescue and transplanted cell survival at 60, 100, 150, and 200 days of age. RESULTS: OKT was rescued in a dose-dependent manner. Outer nuclear layer (ONL) was significantly thicker in cell-treated eyes than controls up to P150. Transplanted RPE cells were identified in both the subretinal space and integrated into the host RPE monolayer in animals of all age groups, and often contained internalized photoreceptor outer segments. No pathology was observed. CONCLUSIONS: OpRegen RPE cells survived, rescued visual function, preserved rod and cone photoreceptors long-term in the RCS rat. Thus, these data support the use of OpRegen RPE cells for the treatment of human RPE cell disorders including AMD. TRANSLATIONAL RELEVANCE: Our novel xeno-free RPE cells minimize concerns of animal derived contaminants while providing a promising prospective therapy to the diseased retina.

Mitigation of Lethal Radiation Syndrome in Mice by Intramuscular Injection of 3D Cultured Adherent Human Placental Stromal Cells.

Exposure to high lethal dose of ionizing radiation results in acute radiation syndrome with deleterious systemic effects to different organs. A primary target is the highly sensitive bone marrow and the hematopoietic system. In the current study C3H/HeN mice were total body irradiated by 7.7 Gy. Twenty four hrs and 5 days after irradiation 2×10(6) cells from different preparations of human derived 3D expanded adherent placental stromal cells (PLX) were injected intramuscularly. Treatment with batches consisting of pure maternal cell preparations (PLX-Mat) increased the survival of the irradiated mice from ∼27% to 68% (P<0.001), while cell preparations with a mixture of maternal and fetal derived cells (PLX-RAD) increased the survival to ∼98% (P<0.0001). The dose modifying factor of this treatment for both 50% and 37% survival (DMF50 and DMF37) was∼1.23. Initiation of the more effective treatment with PLX-RAD injection could be delayed for up to 48 hrs after irradiation with similar effect. A delayed treatment by 72 hrs had lower, but still significantly effect (p<0.05). A faster recovery of the BM and improved reconstitution of all blood cell lineages in the PLX-RAD treated mice during the follow-up explains the increased survival of the cells treated irradiated mice. The number of CD45+/SCA1+ hematopoietic progenitor cells within the fast recovering population of nucleated BM cells in the irradiated mice was also elevated in the PLX-RAD treated mice. Our study suggests that IM treatment with PLX-RAD cells may serve as a highly effective "off the shelf" therapy to treat BM failure following total body exposure to high doses of radiation. The results suggest that similar treatments may be beneficial also for clinical conditions associated with severe BM aplasia and pancytopenia.

Targeted overexpression of IL-18 binding protein at the central nervous system overrides flexibility in functional polarization of antigen-specific Th2 cells.

The current study shows that functional polarization of Ag-specific CD4(+) Th2 cells entering the CNS during the accelerating phase of experimental autoimmune encephalomyelitis is flexible and dependent on the cytokine milieu there. Thus, targeted cell/gene therapy by Ag-specific T cells overexpressing IL-18 binding protein overrides this flexibility and induces infectious spread of T cell tolerance. Using a congenic system, we demonstrated that at this time, Ag-specific Th2 cells accumulate at the CNS but then arrest of IL-4 production. A manipulation of targeted cell/gene delivery was then used to detect whether this function is dependent on the cytokine milieu there. Targeted overexpression of IL-18 binding protein, a natural inhibitor of IL-18, restored the ability of these Ag-specific Th2 cells to produce IL-4 and subsequently induce protective spread of Th2 polarization. These findings not only suggest a novel way of therapy, but also explain why shifting the balance of Ag-specific T cells toward Th2 suppresses ongoing experimental autoimmune encephalomyelitis, whereas a direct transfer of these cells is ineffective.

Plasmid DNA encoding IFN-gamma-inducible protein 10 redirects antigen-specific T cell polarization and suppresses experimental autoimmune encephalomyelitis.

IFN-gamma-inducible protein 10 (IP-10) is a CXC chemokine that stimulates the directional migration of activated T cells, particularly Th1 cells. We demonstrate in this work that during activation this chemokine drives naive CD4(+) T cells into Th1 polarization. Administration of plasmid DNA encoding self IP-10 was found capable of breaking down immunological tolerance to IP-10, resulting in the generation of self-specific immunity to the gene product of the vaccine. Despite the CpG motif that drives T cells into Th1, the vaccine redirected the polarization of myelin basic protein-specific T cells into Th2 and conferred the vaccinated recipients a high state of resistance against experimental autoimmune encephalomyelitis, a T cell-mediated autoimmune disease of the CNS. The vaccine also suppressed full-blown ongoing disease in a mouse model of multiple sclerosis. Self-specific Ab to IP-10 developed in protected animals could inhibit leukocyte migration, alter the in vitro Th1/Th2 balance of autoimmune T cells, and adoptively transfer disease suppression. This demonstrates not only the pivotal role of a chemokine in T cell polarization and function but also its potential implications for plasmid DNA gene therapy.

Targeting the function of IFN-gamma-inducible protein 10 suppresses ongoing adjuvant arthritis.

IFN-gamma-inducible protein 10 (IP-10) is a CXC chemokine that is thought to manifest a proinflammatory role because it stimulates the directional migration of activated T cells, particularly Th1 cells. It is an open question whether this chemokine is also directly involved in T cell polarization. We show here that during the course of adjuvant-induced arthritis the immune system mounts a notable Ab titer against self-IP-10. Upon the administration of naked DNA encoding IP-10, this titer rapidly accelerates to provide protective immunity. Self-specific Ab to IP-10 developed in protected animals, as well as neutralizing Ab to IP-10 that we have generated in rabbits, could inhibit leukocyte migration, alter the in vivo and in vitro Th1/Th2 balance toward low IFN-gamma, low TNF-alpha, high IL-4-producing T cells, and adoptively transfer disease suppression. This not only demonstrates the pivotal role of this chemokine in T cell polarization during experimentally induced arthritis but also suggests a practical way to interfere in the regulation of disease to provide protective immunity. From the basic science perspective, this study challenges the paradigm of in vivo redundancy. After all, we did not neutralize the activity of other chemokines that bind CXCR3 (i.e., macrophage-induced gene and IFN-inducible T cell alpha chemoattractant) and yet significantly blocked not only adjuvant-induced arthritis but also the in vivo competence to mount delayed-type hypersensitivity.