ST6GAL1 sialyltransferase promotes acinar to ductal metaplasia and pancreatic cancer progression.

The role of aberrant glycosylation in pancreatic ductal adenocarcinoma (PDAC) remains an under-investigated area of research. In this study, we determined that ST6 ß-galactoside α2,6 sialyltransferase 1 (ST6GAL1), which adds α2,6-linked sialic acids to N-glycosylated proteins, was upregulated in patients with early-stage PDAC and was further increased in advanced disease. A tumor-promoting function for ST6GAL1 was elucidated using tumor xenograft experiments with human PDAC cells. Additionally, we developed a genetically engineered mouse (GEM) model with transgenic expression of ST6GAL1 in the pancreas and found that mice with dual expression of ST6GAL1 and oncogenic KRASG12D had greatly accelerated PDAC progression compared with mice expressing KRASG12D alone. As ST6GAL1 imparts progenitor-like characteristics, we interrogated ST6GAL1's role in acinar to ductal metaplasia (ADM), a process that fosters neoplasia by reprogramming acinar cells into ductal, progenitor-like cells. We verified ST6GAL1 promotes ADM using multiple models including the 266-6 cell line, GEM-derived organoids and tissues, and an in vivo model of inflammation-induced ADM. EGFR is a key driver of ADM and is known to be activated by ST6GAL1-mediated sialylation. Importantly, EGFR activation was dramatically increased in acinar cells and organoids from mice with transgenic ST6GAL1 expression. These collective results highlight a glycosylation-dependent mechanism involved in early stages of pancreatic neoplasia.

Probing the hydrolytic degradation of UF4 in humid air.

This manuscript describes the chemical transformations that occur during hydrolysis of uranium tetrafluoride (UF4) due to its storage in humid air (85% and 50% relative humidity) at ambient temperatures. This hydrolysis was previously reported to proceed slowly or not at all (depending on the percent relative humidity); however, previous reports relied primarily on X-ray diffraction methods to probe uranium speciation. In our report, we employ a battery of physiochemical probing techniques to explore potential hydrolysis, including Raman spectroscopy, powder X-ray diffraction, 19F nuclear magnetic resonance spectroscopy, scanning electron microscopy, and focused ion beam microscopy with energy-dispersive X-ray spectroscopy. Of these, only Raman spectroscopy proved to be particularly useful at observing chemical changes to UF4. It was found that anhydrous UF4 slightly oxidizes over the course of thirteen days to Schoepite-like uranium complexes and possibly UO3. In contrast, UF4 exposed to 50% relative humidity slightly decomposes into UO2F2, Schoepite-like uranium complexes, and possibly a high order uranium oxide that eluded chemical assignment (UxOy). Despite the rich chemical speciation observed in our Raman spectroscopy measurements, X-ray diffraction and 19F NMR measurements on the same material showed no changes. Microscopy measurements suggest that the observed reactions between UF4 and water occur primarily on the surface of UF4 particulates via a method that is visually similar to surface corrosion of metals. Therefore, we postulate that NMR spectroscopy and X-ray diffraction, which are well-suited for bulk analysis, are less suited than Raman spectroscopy to observe the surface-based reactions that occur to UF4 when exposed to humid air. Considering the importance of UF4 in the production of nuclear fuel and weapons, the results presented herein are widely applicable to numerous nuclear science fields where uranium detection and speciation in humid environments is of value, including nuclear nonproliferation and nuclear forensics.

The structures of 1:1 and 1:2 adducts of phosphane-tricarbo-nitrile with 1,4-di-aza-bicyclo[2.2.2]octa-ne.

In the structures of 1:1 and 1:2 adducts of phosphanetricarbo-nitrile (C3N3P) with 1,4-di-aza-bicyclo-[2.2.2]octane (C6H12N2), the 1:1 adduct crystallizes in the ortho-rhom-bic space group, Pbcm, with four formula units in the unit cell (Z' = 0.5). The P(CN)3 unit lies on a crystallographic mirror plane while the C6H12N2 unit lies on a crystallographic twofold axis passing through one of the C-C bonds. The P(CN)3 moiety has close to C 3v symmetry and is stabilized by forming adducts with two symmetry-related C6H12N2 units. The phospho-rus atom is in a five-coordinate environment. As a result of the symmetry, the two trans angles are equal so τ5 = 0.00 and thus the geometrical description could be considered to be square pyramidal. However, the electronic geometry is distorted octa-hedral with the lone pair on the phospho-rous occupying the sixth position. As would be expected from VSEPR considerations, the repulsion of the lone-pair electrons with the equatorial bonding electrons means that the trans angles for the latter are considerably reduced from 180° to 162.01 (4)°, so the best description of the overall geometry for phospho-rus is distorted square pyramidal. The 1:2 adduct crystallizes in the monoclinic space group, P21/m with two formula units in the asymmetric unit (i.e. Z' = 1/2). The P(CN)3 moiety lies on a mirror plane and one of the two C6H12N2 (dabco) mol-ecules also lies on a mirror plane. The symmetry of the P(CN)3 unit is close to C 3v. There are three P⋯N inter-actions and consequently the mol-ecular geometry of the phospho-rus atom is distorted octa-hedral. This must mean that the lone pair of electrons on the phospho-rus atom is not sterically active. For the 1:1 adduct, there are weak associations between the phospho-rus atom and one of the terminal nitro-gen atoms from the C≡ N moiety, forming chains in the a-axis direction. In addition there are weak C-H⋯N inter-actions between a terminal nitro-gen atoms from the C≡N moiety and the C6H12N2 mol-ecules, which form sheets perpendicular to the a axis.

Methylation of dual-specificity phosphatase 4 controls cell differentiation.

Mitogen-activated protein kinases (MAPKs) are inactivated by dual-specificity phosphatases (DUSPs), the activities of which are tightly regulated during cell differentiation. Using knockdown screening and single-cell transcriptional analysis, we demonstrate that DUSP4 is the phosphatase that specifically inactivates p38 kinase to promote megakaryocyte (Mk) differentiation. Mechanistically, PRMT1-mediated methylation of DUSP4 triggers its ubiquitinylation by an E3 ligase HUWE1. Interestingly, the mechanistic axis of the DUSP4 degradation and p38 activation is also associated with a transcriptional signature of immune activation in Mk cells. In the context of thrombocytopenia observed in myelodysplastic syndrome (MDS), we demonstrate that high levels of p38 MAPK and PRMT1 are associated with low platelet counts and adverse prognosis, while pharmacological inhibition of p38 MAPK or PRMT1 stimulates megakaryopoiesis. These findings provide mechanistic insights into the role of the PRMT1-DUSP4-p38 axis on Mk differentiation and present a strategy for treatment of thrombocytopenia associated with MDS.

Characterizing the solid hydrolysis product, UF4(H2O)2.5, generated from neat water reactions with UF4 at room temperature.

Uranium tetrafluoride (UF4) is an important intermediate in the production of UF6 and uranium metal. Room temperature hydrolysis of UF4 was investigated using a combination of Fluorine-19 nuclear magnetic resonance spectroscopy (19F NMR), Raman and infrared spectroscopy, powder X-ray diffraction, and microscopy measurements. UF4(H2O)2.5 was identified as the primary solid hydrolysis product when anhydrous UF4 was stirred in deionized water. Static NMR and 19F magic angle spinning NMR measurements revealed that a small amount of uranyl fluoride can also form when anhydrous UF4 is left in water, although this species comprises less than 5% of the total sample with the remaining parts being UF4(H2O)2.5. Since UF4 is generally considered to be stable under ambient conditions, these findings mark the first time that a room temperature reaction between UF4 and water has been detected and analyzed without interference from additional chemical reagents. The Raman characterization of UF4(H2O)2.5 presented herein is the first on record. Since UF4 is one of the most used intermediates during chemical conversion of uranium ore to uranium metal for nuclear fuel and weapons, the results presented herein are applicable to numerous nuclear science fields where solid state detection of uranium is of value, including nuclear nonproliferation, nuclear forensics, and environmental remediation.

Mapping oscillating magnetic fields around rechargeable batteries.

Power storage devices such as batteries are a crucial part of modern technology. The development and use of batteries has accelerated in the past decades, yet there are only a few techniques that allow gathering vital information from battery cells in a nonivasive fashion. A widely used technique to investigate batteries is electrical impedance spectroscopy (EIS), which provides information on how the impedance of a cell changes as a function of the frequency of applied alternating currents. Building on recent developments of inside-out MRI (ioMRI), a technique is presented here which produces spatially-resolved maps of the oscillating magnetic fields originating from the alternating electrical currents distributed within a cell. The technique works by using an MRI pulse sequence synchronized with a gated alternating current applied to the cell terminals. The approach is benchmarked with a current-carrying wire coil, and demonstrated with commercial and prototype lithium-ion cells. Marked changes in the fields are observed for different cell types.

Nuclear magnetic resonance spectroscopy of rechargeable pouch cell batteries: beating the skin depth by excitation and detection via the casing.

Rechargeable batteries are notoriously difficult to examine nondestructively, and the obscurity of many failure modes provides a strong motivation for developing efficient and detailed diagnostic techniques that can provide information during realistic operating conditions. In-situ NMR spectroscopy has become a powerful technique for the study of electrochemical processes, but has mostly been limited to laboratory cells. One significant challenge to applying this method to commercial cells has been that the radiofrequency, required for NMR excitation and detection, cannot easily penetrate the battery casing due to the skin depth. This complication has limited such studies to special research cell designs or to 'inside-out' measurement approaches. This article demonstrates that it is possible to use the battery cell as a resonator in a tuned circuit, thereby allowing signals to be excited inside the cell, and for them to subsequently be detected via the resonant circuit. Employing this approach, 7Li NMR signals from the electrolyte, as well as from intercalated and plated metallic lithium in a multilayer (rolled) commercial pouch cell battery were obtained. Therefore, it is anticipated that critical nondestructive device characterization can be performed with this technique in realistic and even commercial cell designs.

Defining the epigenetic status of blood cells using a cyanine-based fluorescent probe for PRMT1.

Dynamic regulation of histone modification enzymes such as PRMT1 (protein arginine methyltransferase 1) determines the ordered epigenetic transitions in hematopoiesis. Sorting cells according to the expression levels of histone modification enzymes may further define subpopulations in hematopoietic lineages with unique differentiation potentials that are presently defined by surface markers. We discovered a vital near infrared dye, E84, that fluoresces brightly following binding to PRMT1 and excitation with a red laser. The staining intensity as measured by flow cytometry is correlated with the PRMT1 expression level. Importantly, E84 staining has no apparent negative effect on the proliferation of the labeled cells. Given that long-term hematopoietic stem cells (LT-HSCs) produce low levels of PRMT1, we used E84 to sort LT-HSCs from mouse bone marrow. We found that SLAM (the signalling lymphocyte activation molecule family) marker-positive LT-HSCs were enriched in the E84low cell fraction. We then performed bone marrow transplantations with E84high or E84low Lin-Sca1+Kit+ (LSK) cells and showed that whole blood cell lineages were successfully reconstituted 16 weeks after transplanting 200 E84low LSK cells. Thus, E84 is a useful new tool to probe the role of PRMT1 in hematopoiesis and leukemogenesis. Developing E84 and other small molecules to label histone modification enzymes provides a convenient approach without modifying gene loci to study the interaction between hematopoietic stem/progenitor cell epigenetic status and differentiation state.

Optorelaxers: Achieving real-time control of NMR relaxation.

We present an approach to increase the detection sensitivity of NMR by shortening the spin-lattice relaxation time using transient paramagnetic species created by light irradiation of "optorelaxer" molecules. In the ultimate implementation of this concept, not yet realized here, these transient species are absent during the detection period, thereby avoiding the loss of spectral resolution caused by inhomogeneous broadening from paramagnetic species. Real-time control of NMR relaxation by visible light is demonstrated with Fe(II)(ptz)6(BF4)2, (ptz = 1-propyltetrazole), abbreviated FePTZ. Illumination of FePTZ at 30 K results in a decrease of the 1H NMR spin-lattice relaxation time T1 due to formation of a high spin photoexcited state. The 1H NMR of polystyrene containing a low concentration of FePTZ molecules shows a similar reduction in T1, establishing that FePTZ can act as an optorelaxer for the protons of a matrix. Numerical modeling of the spin-diffusion processes from the protons in a FePTZ core to those in a shell of polystyrene accounts for the observed T1 effects under both dark and light conditions. Additionally, 1H MAS (magic-angle spinning) NMR results for pure FePTZ provide information on the isotropic and anisotropic portions of the electron-nuclear hyperfine interactions.

Investigations of Uranyl Fluoride Sesquihydrate (UO2F2·1.57H2O): Combining 19F Solid-State MAS NMR Spectroscopy and GIPAW Chemical Shift Calculations.

High-resolution 19F magic-angle spinning (MAS) NMR spectra were obtained for the uranium-bearing solid uranyl fluoride sesquihydrate (UO2F2·1.57H2O). While there are seven distinct crystallographic fluorine sites, the 19F NMR spectrum reveals six peaks at -33.3, 9.1, 25.7, 33.0, 39.0, and 48.2 ppm, with the peak at 33.0 ppm twice the intensity of all the others and therefore corresponding to two sites. To assign the peaks in the experimental spectra to crystallographic sites, 19F chemical shifts were calculated using the gauge including projector augmented waves (GIPAW) plane-wave pseudopotential approach for a DFT-optimized crystal structure. The peak assignments from DFT are consistent with two-dimensional double-quantum 19F MAS NMR experiments.

Automated detection of broad NMR spectra: 19F NMR of paramagnetic UF4 and 195Pt NMR of supported Pt catalysts.

The automated detection of broad NMR spectra via the controlled stepwise motion of the NMR probe along the field axis of the superconducting solenoid is demonstrated for the detection of 19F NMR of paramagnetic UF4 and 195Pt NMR of supported metal catalysts. The sensitivity advantages of performing these measurements at 2.3 T are discussed with reference to applying this methodology to room temperature in situ electrochemical 195Pt NMR.

Utility of PEGylated dithiolane ligands for direct synthesis of water-soluble Au, Ag, Pt, Pd, Cu and AuPt nanoparticles.

Water soluble metallic nanoparticles are being developed for a variety of roles ranging from catalysis to drug delivery and as potential contrast agents. We demonstrate direct synthesis of high-quality water-soluble Au, Ag, Pt, Pd, Cu and alloyed AuPt nanoparticles as well as ligand-exchange of FePt, cubic Pt and Au/Pt core/shell nanoparticles using bidentate dithiolane PEG as a universal ligand. Simple chemistry can greatly expand the applications of metal nanoparticles.

Enhanced Hematopoietic Stem Cell Self-Renewal-Promoting Ability of Clonal Primary Mesenchymal Stromal/Stem cells Versus Their Osteogenic Progeny.

Long-term self-renewing hematopoietic stem cell (LT-HSC) homeostasis within the bone marrow (BM) of adult mammals is regulated by complex interactions between LT-HSC and a number of niche-associated cell types including mesenchymal stromal/stem cells (MSC), osteoblasts (OB), macrophage, and neuronal cells in close proximity with the vasculature. Here, we cloned and functionally characterized a murine BM MSC subpopulation that was uniformly Nestin+ Lepr + Sca-1+ CD146+ and could be stably propagated with high colony-forming unit fibroblast re-cloning efficiency. MSC synergized with SCF and IL-11 to support a 20-fold expansion in true LT-HSC after 10-days of in vitro coculture. Optimal stimulation of LT-HSC expansion was minimally dependent on Notch signaling but was significantly enhanced by global inhibition of Wnt signaling. The self-renewal-promoting activity of MSC was progressively lost when MSC clones were differentiated into mature OB. This suggests that the stage of osteoblast development may significantly impact the ability of osteolineage cells to support LT-HSC homeostasis in vivo. Stem Cells 2017;35:473-484.

The Tumor-Associated Glycosyltransferase ST6Gal-I Regulates Stem Cell Transcription Factors and Confers a Cancer Stem Cell Phenotype.

The glycosyltransferase ST6Gal-I, which adds α2-6-linked sialic acids to substrate glycoproteins, has been implicated in carcinogenesis; however, the nature of its pathogenic role remains poorly understood. Here we show that ST6Gal-I is upregulated in ovarian and pancreatic carcinomas, enriched in metastatic tumors, and associated with reduced patient survival. Notably, ST6Gal-I upregulation in cancer cells conferred hallmark cancer stem-like cell (CSC) characteristics. Modulating ST6Gal-I expression in pancreatic and ovarian cancer cells directly altered CSC spheroid growth, and clonal variants with high ST6Gal-I activity preferentially survived in CSC culture. Primary ovarian cancer cells from patient ascites or solid tumors sorted for α2-6 sialylation grew as spheroids, while cells lacking α2-6 sialylation remained as single cells and lost viability. ST6Gal-I also promoted resistance to gemcitabine and enabled the formation of stably resistant colonies. Gemcitabine treatment of patient-derived xenograft tumors enriched for ST6Gal-I-expressing cells relative to pair-matched untreated tumors. ST6Gal-I also augmented tumor-initiating potential. In limiting dilution assays, subcutaneous tumor formation was inhibited by ST6Gal-I knockdown, whereas in a chemically induced tumor initiation model, mice with conditional ST6Gal-I overexpression exhibited enhanced tumorigenesis. Finally, we found that ST6Gal-I induced expression of the key tumor-promoting transcription factors, Sox9 and Slug. Collectively, this work highlighted a previously unrecognized role for a specific glycosyltransferase in driving a CSC state. Cancer Res; 76(13); 3978-88. ©2016 AACR.

The histone H2A deubiquitinase Usp16 regulates hematopoiesis and hematopoietic stem cell function.

Epigenetic mechanisms play important regulatory roles in hematopoiesis and hematopoietic stem cell (HSC) function. Subunits of polycomb repressive complex 1 (PRC1), the major histone H2A ubiquitin ligase, are critical for both normal and pathological hematopoiesis; however, it is unclear which of the several counteracting H2A deubiquitinases functions along with PRC1 to control H2A ubiquitination (ubH2A) level and regulates hematopoiesis in vivo. Here we investigated the function of Usp16 in mouse hematopoiesis. Conditional deletion of Usp16 in bone marrow resulted in a significant increase of global ubH2A level and lethality. Usp16 deletion did not change HSC number but was associated with a dramatic reduction of mature and progenitor cell populations, revealing a role in governing HSC lineage commitment. ChIP- and RNA-sequencing studies in HSC and progenitor cells revealed that Usp16 bound to many important hematopoietic regulators and that Usp16 deletion altered the expression of genes in transcription/chromosome organization, immune response, hematopoietic/lymphoid organ development, and myeloid/leukocyte differentiation. The altered gene expression was partly rescued by knockdown of PRC1 subunits, suggesting that Usp16 and PRC1 counterbalance each other to regulate cellular ubH2A level and gene expression in the hematopoietic system. We further discovered that knocking down Cdkn1a (p21cip1), a Usp16 target and regulated gene, rescued the altered cell cycle profile and differentiation defect of Usp16-deleted HSCs. Collectively, these studies identified Usp16 as one of the histone H2A deubiquitinases, which coordinates with the H2A ubiquitin ligase PRC1 to regulate hematopoiesis, and revealed cell cycle regulation by Usp16 as key for HSC differentiation.

Surveying the serologic proteome in a tissue-specific kras(G12D) knockin mouse model of pancreatic cancer.

We have applied a serologic proteomic workflow involving three complementary MS approaches to a tissue-specific Kras(G12D) -knockin mouse model of pancreatic cancer that consistently forms precancerous lesions by 4 months of age. The three proteomics applications were highly complementary and allowed us to survey the entire range of low to high molecular weight serologic proteins. Combined, we identified 121 (49↓, 72↑) unique and statistically relevant serologic biomarkers with 88% previously reported to be associated with cancer and 38% specifically correlated with pancreatic cancer. Four markers, lysozyme C2, cytokeratin 19, Serpina1A and Pcf11, were further verified by Western blotting. When applying systems analysis, the top-associated gene ontology functions were tied to wound healing, RXR signaling, growth, differentiation and innate immune activation through the JAK/STAT pathway. Upon further investigation of the apparent immune response using a multiplex cytokine screen, we found that IFN-γ, VEGF and GM-CSF were significantly increased in serum from the Kras(G12D) animals compared to littermate controls. By combining three complementary MS applications, we were able to survey the native intact peptidome and the global proteome in parallel, unveiling pathways that may be biologically relevant to promotion of pancreatic cancer progression and serologic markers of noninvasive early-stage neoplasia.

Validation of a robust proteomic analysis carried out on formalin-fixed paraffin-embedded tissues of the pancreas obtained from mouse and human.

A number of reports have recently emerged with focus on extraction of proteins from formalin-fixed paraffin-embedded (FFPE) tissues for MS analysis; however, reproducibility and robustness as compared to flash frozen controls is generally overlooked. The goal of this study was to identify and validate a practical and highly robust approach for the proteomics analysis of FFPE tissues. FFPE and matched frozen pancreatic tissues obtained from mice (n = 8) were analyzed using 1D-nanoLC-MS(MS)(2) following work up with commercially available kits. The chosen approach for FFPE tissues was found to be highly comparable to that of frozen. In addition, the total number of unique peptides identified between the two groups was highly similar, with 958 identified for FFPE and 1070 identified for frozen, with protein identifications that corresponded by approximately 80%. This approach was then applied to archived human FFPE pancreatic cancer specimens (n = 11) as compared to uninvolved tissues (n = 8), where 47 potential pancreatic ductal adenocarcinoma markers were identified as significantly increased, of which 28 were previously reported. Further, these proteins share strongly overlapping pathway associations to pancreatic cancer that include estrogen receptor α. Together, these data support the validation of an approach for the proteomic analysis of FFPE tissues that is straightforward and highly robust, which can also be effectively applied toward translational studies of disease.

GM-CSFRα: the sex-chromosome link to t(8;21)(+) AML?

In this issue of Blood,Matsuura and colleagues provide evidence that loss of GMCSF signaling promotes leukemic progression in association with one of the most frequently observed cytogenetic abnormalities in AML, the t(8;21)(q22;q22) that generates the RUNX1-ETO fusion protein.

Distinct classes of c-Kit-activating mutations differ in their ability to promote RUNX1-ETO-associated acute myeloid leukemia.

The t(8;21) RUNX1-ETO translocation is one of the most frequent cytogenetic abnormalities in acute myeloid leukemia (AML). In RUNX1-ETO(+) patient samples, differing classes of activating c-KIT receptor tyrosine kinase mutations have been observed. The most common (12%-48%) involves mutations, such as D816V, which occur in the tyrosine kinase domain, whereas another involves mutations within exon 8 in a region mediating receptor dimerization (2%-13% of cases). To test whether distinct subtypes of activating c-KIT mutations differ in their leukemogenic potential in association with RUNX1-ETO, we used a retroviral transduction/transplantation model to coexpress RUNX1-ETO with either c-Kit(D814V) or c-Kit(T417IΔ418-419) in murine hematopoietic stem/progenitor cells used to reconstitute lethally irradiated mice. Analysis of reconstituted animals showed that RUNX1-ETO;c-Kit(D814V) coexpression resulted in 3 nonoverlapping phenotypes. In 45% of animals, a transplantable AML of relatively short latency and frequent granulocytic sarcoma was noted. Other mice exhibited a rapidly fatal myeloproliferative phenotype (35%) or a lethal, short-latency pre-B-cell leukemia (20%). In contrast, RUNX1-ETO;c-Kit(T417IΔ418-419) coexpression promoted exclusively AML in a fraction (51%) of reconstituted mice. These observations indicate that c-Kit(D814V) promotes a more varied and aggressive leukemic phenotype than c-Kit(T417IΔ418-419), which may be the result of differing potencies of the activating c-Kit alleles.

Limited transplantation of antigen-expressing hematopoietic stem cells induces long-lasting cytotoxic T cell responses.

Harnessing the ability of cytotoxic T lymphocytes (CTLs) to recognize and eradicate tumor or pathogen-infected cells is a critical goal of modern immune-based therapies. Although multiple immunization strategies efficiently induce high levels of antigen-specific CTLs, the initial increase is typically followed by a rapid contraction phase resulting in a sharp decline in the frequency of functional CTLs. We describe a novel approach to immunotherapy based on a transplantation of low numbers of antigen-expressing hematopoietic stem cells (HSCs) following nonmyeloablative or partially myeloablative conditioning. Continuous antigen presentation by a limited number of differentiated transgenic hematopoietic cells results in an induction and prolonged maintenance of fully functional effector T cell responses in a mouse model. Recipient animals display high levels of antigen-specific CTLs four months following transplantation in contrast to dendritic cell-immunized animals in which the response typically declines at 4-6 weeks post-immunization. Majority of HSC-induced antigen-specific CD8+ T cells display central memory phenotype, efficiently kill target cells in vivo, and protect recipients against tumor growth in a preventive setting. Furthermore, we confirm previously published observation that high level engraftment of antigen-expressing HSCs following myeloablative conditioning results in tolerance and an absence of specific cytotoxic activity in vivo. In conclusion, the data presented here supports potential application of immunization by limited transplantation of antigen-expressing HSCs for the prevention and treatment of cancer and therapeutic immunization of chronic infectious diseases such as HIV-1/AIDS.