��-tocotrienol protects mouse and human hematopoietic progenitors from ��-irradiation through extracellular signal-regulated kinase/mammalian target of rapamycin signaling

Background

Exposure to γ-radiation causes rapid hematopoietic cell apoptosis and bone marrow suppression. However, there are no approved radiation countermeasures for the acute radiation syndrome. In this study, we demonstrated that natural δ-tocotrienol, one of the isomers of vitamin E, significantly enhanced survival in total body lethally irradiated mice. We explored the effects and mechanisms of δ-tocotrienol on hematopoietic progenitor cell survival after γ-irradiation in both in vivo and in vitro experiments.

Design and Methods

CD2F1 mice and human hematopoietic progenitor CD34⁺ cells were treated with δ-tocotrienol or vehicle control 24 h before or 6 h after γ-irradiation. Effects of δ-tocotrienol on hematopoietic progenitor cell survival and regeneration were evaluated by clonogenicity studies, flow cytometry, and bone marrow histochemical staining. δ-tocotrienol and γ-irradiation-induced signal regulatory activities were assessed by immunofluorescence staining, immunoblotting and short-interfering RNA assay.

Results

δ-tocotrienol displayed significant radioprotective effects. A single injection of δ-tocotrienol protected 100% of CD2F1 mice from total body irradiation-induced death as measured by 30-day post-irradiation survival. δ-tocotrienol increased cell survival, and regeneration of hematopoietic microfoci and lineage⁻/Sca-1⁺/ckit⁺ stem and progenitor cells in irradiated mouse bone marrow, and protected human CD34⁺ cells from radiation-induced damage. δ-tocotrienol activated extracellular signal-related kinase 1/2 phosphorylation and significantly inhibited formation of DNA-damage marker γ-H2AX foci. In addition, δ-tocotrienol up-regulated mammalian target of rapamycin and phosphorylation of its downstream effector 4EBP-1. These alterations were associated with activation of mRNA translation regulator eIF4E and ribosomal protein S6, which is responsible for cell survival and growth. Inhibition of extracellular signal-related kinase 1/2 expression by short interfering RNA abrogated δ-tocotrienol-induced mammalian target of rapamycin phosphorylation and clonogenicity, and increased γ-H2AX foci formation in irradiated CD34⁺ cells.

Conclusions

Our data indicate that δ-tocotrienol protects mouse bone marrow and human CD34⁺ cells from radiation-induced damage through extracellular signal-related kinase activation-associated mammalian target of rapamycin survival pathways.     

Oxygen tension plays a critical role in the hematopoietic microenvironment in vitro

Background

In the bone marrow mesenchymal stromal cells and osteoblasts form functional niches for hematopoietic stem and progenitor cells. This microenvironment can be partially mimicked using in vitro co-culture systems. In this study, we examined the oxygen tension in three distinct compartments in a co-culture system of purified CD34⁺ cells and mesenchymal stromal cells with regard to different spatial localizations.

Design and Methods

Hypoxic cells in the co-culture were visualized by pimonidazole staining. Hematopoietic cell distribution, and functional and phenotypic characteristics were analyzed by flow cytometry. The secretion of vascular endothelial growth factor and stromal-derived factor-1 by mesenchymal stromal cells in low oxygen co-cultures was determined by an enzyme-linked immunosorbent assay. The effect of co-culture medium on the hematopoietic cell migration potential was tested in a transwell assay.

Results

In co-cultures under atmospheric oxygen tension, regions of low oxygen tension could be detected beneath the feeder layer in which a reservoir of phenotypically more primitive hematopoietic cells is located in vitro. In low oxygen co-culture, the adhesion of hematopoietic cells to the feeder layer was decreased, whereas hematopoietic cell transmigration beneath mesenchymal stromal cells was favored. Increased vascular endothelial growth factor-A secretion by mesenchymal stromal cells under low oxygen conditions, which increased the permeability of the monolayer, was responsible for this effect. Furthermore, vascular endothelial growth factor-A expression in low oxygen mesenchymal stromal cells was induced via hypoxia-inducible factor signaling. However, stromal cell-derived factor-1 secretion by mesenchymal stromal cells was down-regulated under low oxygen conditions in a hypoxia-inducible factor-independent manner.

Conclusions

We demonstrate for the first time that differences in oxygen tension cause selective modification of hematopoietic cell and mesenchymal stromal cell interactions in a co-culture system, thus confirming that oxygen tension plays a critical role in the interaction between hematopoietic cells and the niche environment.     

Histone deacetylase inhibition modulates cell fate decisions during myeloid differentiation

Background

The clinical use of chromatin-modulating drugs, such as histone deacetylase inhibitors, for the treatment of bone marrow failure and hematopoietic malignancies has increased dramatically over the last few years. Nonetheless, little is currently known concerning their effects on myelopoiesis.

Design and Methods

We utilized an ex vivo differentiation system in which umbilical cord blood-derived CD34⁺ cells were treated with trichostatin A, sodium butyrate and valproic acid to evaluate the effect of histone deacetylase inhibitor treatment on myeloid lineage development, colony-forming potential, proliferation, and terminal neutrophil differentiation.

Results

Trichostatin A treatment modestly reduced progenitor proliferation, while sodium butyrate and valproic acid resulted in concentration-dependent effects on proliferation and apoptosis. Addition of valproic acid uniquely stimulated CD34⁺ proliferation. Sodium butyrate treatment inhibited terminal neutrophil differentiation both quantitatively and qualitatively. Addition of 100 μM valproic acid resulted in increased numbers of mature neutrophils with a block in differentiation at increasing concentrations. Sodium butyrate and valproic acid treatment resulted in increased acetylation of histones 3 and 4 while trichostatin A, sodium butyrate and valproic acid had differential effects on the acetylation of non-histone proteins.

Conclusions

Individual histone deacetylase inihibitors had specific effects on cell fate decisions during myeloid development. These data provide novel insights into the effects of histone deacetylase inhibitors on the regulation of normal hematopoiesis, which is of importance when considering utilizing these compounds for the treatment of myeloid malignancies and bone marrow failure syndromes.     

Rac1 and Rac2 GTPases are necessary for early erythropoietic expansion in the bone marrow but not in the spleen

Background

The small Rho GTPases Rac1 and Rac2 have both overlapping and distinct roles in actin organization, cell survival, and proliferation in various hematopoietic cell lineages. The role of these Rac GTPases in erythropoiesis has not yet been fully elucidated.

Design and Methods

Cre-recombinase-induced deletion of Rac1 genomic sequence was accomplished on a Rac2-null genetic background, in mouse hematopoietic cells in vivo. The erythroid progenitors and precursors in the bone marrow and spleen of these genetically engineered animals were evaluated by colony assays and flow cytometry. Apoptosis and proliferation of the different stages of erythroid progenitors and precursors were evaluated by flow cytometry.

Results

Erythropoiesis in Rac1^−/−;Rac2^−/− mice is characterized by abnormal burst-forming unit-erythroid colony morphology and decreased numbers of megakaryocyte-erythrocyte progenitors, erythroid colony-forming units, and erythroblasts in the bone marrow. In contrast, splenic erythropoiesis is increased. Combined Rac1 and Rac2 deficiency compromises proliferation of the megakaryocyte-erythrocyte progenitor population in the bone marrow, while it allows increased survival and proliferation of megakaryocyte-erythrocyte progenitors in the spleen.

Conclusions

These data suggest that Rac1 and Rac2 GTPases are essential for normal bone marrow erythropoiesis but that they are dispensable for erythropoiesis in the spleen, implying different signaling pathways for homeostatic and stress erythropoiesis.     

Low-Dose Rapamycin (Sirolimus) Effects in Autosomal Dominant Polycystic Kidney Disease: An Open-Label Randomized Controlled Pilot Study

Background and objectives

The two largest studies of mammalian target of rapamycin inhibitor treatment of autosomal dominant polycystic kidney disease (ADPKD) demonstrated no clear benefit on the primary endpoint of total kidney volume (TKV) or on eGFR. The present study evaluated two levels of rapamycin on the 12-month change in ¹²⁵I-iothalamate GFR (iGFR) as the primary endpoint and TKV secondarily.

Design, setting, participants, & measurements

In a 12-month open-label pilot study, 30 adult patients with ADPKD were randomly assigned to low-dose (LD) rapamycin (rapamycin trough blood level, 2–5 ng/ml) (LD group, n=10), standard-dose (STD) rapamycin trough level (>5–8 ng/ml) (STD group, n=10), or standard care (SC group, n=10). They were evaluated with iGFR and noncontrast computed tomography.

Results

Change in iGFR at 12 months was significantly higher in the LD group (7.7±12.5 ml/min per 1.73 m²; n=9) than in the SC group (−11.2±9.1 ml/min per 1.73 m²; n=9) (LD versus SC: P<0.01). Change in iGFR at 12 months in the STD group (1.6±12.1 ml/min per 1.73 m²; n=8) was not significantly greater than that in the SC group (P=0.07), but it was in the combined treatment groups (LD+STD versus SC: P<0.01). Neither eGFR calculated by the CKD-Epidemiology Collaboration equation nor TKV (secondary endpoint) changed significantly from baseline to 12 months in any of the groups. On the basis of results of the mixed model, during the study, patients in the LD group had significantly lower trough blood levels of rapamycin (mean range±SD, 2.40±0.64 to 2.90±1.20 ng/ml) compared with those in the STD group (3.93±2.27 to 5.77±1.06 ng/ml) (P<0.01).

Conclusion

Patients with ADPKD receiving LD rapamycin demonstrated a significant increase in iGFR compared with those receiving standard care, without a significant effect on TKV after 12 months.     

Stability of human rapamycin-expanded CD4+CD25+ T regulatory cells

Background

The clinical use of ex vivo-expanded T-regulatory cells for the treatment of T-cell-mediated diseases has gained increasing momentum. However, the recent demonstration that FOXP3⁺ T-regulatory cells may contain interleukin-17–producing cells and that they can convert into effector cells once transferred in vivo raises significant doubts about their safety. We previously showed that rapamycin permits the ex vivo expansion of FOXP3⁺ T-regulatory cells while impairing the proliferation of non-T-regulatory cells. Here we investigated the Th17-cell content and the in vivo stability of rapamycin-expanded T-regulatory cells as pertinent aspects of cell-based therapy.

Design and Methods

T-regulatory-enriched cells were isolated from healthy volunteers and were expanded ex vivo with rapamycin with a pre-clinical applicable protocol. T-regulatory cells cultured with and without rapamycin were compared for their regulatory activity, content of pro-inflammatory cells and stability.

Results

We found that CD4⁺CCR6⁺CD161⁺ T cells (i.e., precursor/committed Th17 cells) contaminate the T-regulatory cells cultured ex vivo in the absence of rapamycin. In addition, Th17 cells do not expand when rapamycin-treated T-regulatory cells are exposed to a “Th17-favorable” environment. Rapamycin-expanded T-regulatory cells maintain their in vitro regulatory phenotype even after in vivo transfer into immunodeficient NOD-SCID mice despite being exposed to the irradiation-induced pro-inflammatory environment. Importantly, no additional rapamycin treatment, either in vitro or in vivo, is required to keep their phenotype fixed.

Conclusions

These data demonstrate that rapamycin secures ex vivo-expanded human T-regulatory cells and provide additional justification for their clinical use in future cell therapy-based trials.     

Prolonged ex vivo culture of human bone marrow mesenchymal stem cells influences their supportive activity toward NOD/SCID-repopulating cells and committed progenitor cells of B lymphoid and myeloid lineages

Background

Bone marrow mesenchymal stem cells support proliferation and differentiation of hematopoietic progenitor cells in vitro. Since these cells constitute a rare subset of bone marrow cells, mesenchymal stem cell preparations for clinical purposes require a preparative step of ex vivo multiplication. The aim of our study was to analyze the influence of culture duration on mesenchymal stem cell supportive activity.

Design and Methods

Mesenchymal stem cells were expanded for up to ten passages. These cells and CD34+ cells were seeded in cytokine-free co-cultures after which the phenotype, clonogenic capacity and in vivo repopulating activity of harvested hematopoietic cells were assessed.

Results

Early passage mesenchymal stem cells supported hematopoietic progenitor cell expansion and differentiation toward both B lymphoid and myeloid lineages. Late passage mesenchymal stem cells did not support hematopoietic progenitor cell and myeloid cell outgrowth but maintained B-cell supportive ability. In vitro maintenance of NOD/SCID mouse repopulating cells cultured for 1 week in contact with mesenchymal stem cells was effective until the fourth passage of the mesenchymal cells and declined thereafter. The levels of engraftment of CD34⁺ cells in NOD/SCID mice was higher when these cells were co-injected with early passage mesenchymal stem cells; however mesenchymal cells expanded beyond nine passages were ineffective in promoting CD34⁺ cell engraftment. Non-contact cultures indicated that mesenchymal stem cell supportive activity involved diffusible factors. Among these, interleukins 6 and 8 contributed to the supportive activity of early passage mesenchymal stem cells but not to those of late passage cells. The phenotype, as well as fat, bone and cartilage differentiation capacity, of mesenchymal stem cells did not change during their culture.

Conclusions

Extended culture of mesenchymal stem cells alters the ability of these cells to support hematopoietic progenitor cells without causing concomitant changes in their phenotype or differentiation capacity.     

Heparan sulfate mimetics can efficiently mobilize long-term hematopoietic stem cells

Background

Although mobilization of hematopoietic stem cells and hematopoietic progenitor cells can be achieved with a combination of granulocyte colony-stimulating factor and plerixafor (AMD3100), improving approaches for hematopoietic progenitor cell mobilization is clinically important.

Design and Methods

Heparan sulfate proteoglycans are ubiquitous macromolecules associated with the extracellular matrix that regulates biology of hematopoietic stem cells. We studied the effects of a new family of synthetic oligosaccharides mimicking heparan sulfate on hematopoietic stem cell mobilization. These oligosaccharides were administered intravenously alone or in combination with granulocyte colony-stimulating factor and/or AMD3100 in mice. Mobilized hematopoietic cells were counted and phenotyped at different times and the ability of mobilized hematopoietic stem cells to reconstitute long-term hematopoiesis was determined by competitive transplantation into syngenic lethally irradiated mice followed by secondary transplantation.

Results

Mimetics of heparan sulfate induced rapid mobilization of B-lymphocytes, T-lymphocytes, hematopoietic stem cells and hematopoietic progenitor cells. They increased the mobilization of hematopoietic stem cells and hematopoietic progenitor cells more than 3-fold when added to the granulocyte colony-stimulating factor/AMD3100 association. Hematopoietic stem cells mobilized by mimetics of heparan sulfate or by the granulocyte colony-stimulating factor/AMD3100/mimetics association were as effective as hematopoietic stem cells mobilized by the granulocyte colony-stimulating factor/AMD3100 association for primary and secondary hematopoietic reconstitution of lethally irradiated mice.

Conclusions

This new family of mobilizing agents could alone or in combination with granulocyte colony-stimulating factor and/or AMD3100 mobilize a high number of hematopoietic stem cells that were able to maintain long-term hematopoiesis. These results strengthen the role of heparan sulfates in the retention of hematopoietic stem cells in bone marrow and support the use of small glyco-drugs based on heparan sulfate in combination with granulocyte colony-stimulating factor and AMD3100 to improve high stem cell mobilization, particularly in a prospect of use in human therapeutics.     

MicroRNA126 contributes to granulocyte colony-stimulating factor-induced hematopoietic progenitor cell mobilization by reducing the expression of vascular cell adhesion molecule 1

Background

Mobilization of hematopoietic stem/progenitor cells from the bone marrow to the peripheral blood by granulocyte colony-stimulating factor is the primary means to acquire stem cell grafts for hematopoietic cell transplantation. Since hematopoietic stem/progenitor cells represent a minority of all blood cells mobilized by granulocyte colony-stimulating factor, the underlying mechanisms need to be understood in order to develop selective drugs.

Design and Methods

We analyzed phenotypic, biochemical and genetic changes in bone marrow cell populations from granulocyte colony-stimulating factor-mobilized and control mice, and linked such changes to effective mobilization of hematopoietic stem/progenitor cells.

Results

We show that granulocyte colony-stimulating factor indirectly reduces expression of surface vascular cell adhesion molecule 1 on bone marrow hematopoietic stem/progenitor cells, stromal cells and endothelial cells by promoting the accumulation of microRNA-126 (miR126)-containing microvescicles in the bone marrow extracellular compartment. We found that hematopoietic stem/progenitor cells, stromal cells and endothelial cells readily incorporate these miR126-loaded microvescicles, and that miR126 represses vascular cell adhesion molecule 1 expression on bone marrow hematopoietic stem/progenitor cells, stromal cells and endothelial cells. In line with this, miR126-null mice displayed a reduced mobilization response to granulocyte colony-stimulating factor.

Conclusions

Our results implicate miR126 in the regulation of hematopoietic stem/progenitor cell trafficking between the bone marrow and peripheral sites, clarify the role of vascular cell adhesion molecule 1 in granulocyte colony-stimulating factor-mediated mobilization, and have important implications for improved approaches to selective mobilization of hematopoietic stem/progenitor cells.     

Tyrosine kinase inhibitor therapy can cure chronic myeloid leukemia without hitting leukemic stem cells

Background

Tyrosine kinase inhibitors, such as imatinib, are not considered curative for chronic myeloid leukemia – regardless of the significant reduction of disease burden during treatment – since they do not affect the leukemic stem cells. However, the stochastic nature of hematopoiesis and recent clinical observations suggest that this view must be revisited.

Design and Methods

We studied the natural history of a large cohort of virtual patients with chronic myeloid leukemia under tyrosine kinase inhibitor therapy using a computational model of hematopoiesis and chronic myeloid leukemia that takes into account stochastic dynamics within the hematopoietic stem and early progenitor cell pool.

Results

We found that in the overwhelming majority of patients the leukemic stem cell population undergoes extinction before disease diagnosis. Hence leukemic progenitors, susceptible to tyrosine kinase inhibitor attack, are the natural target for chronic myeloid leukemia treatment. Response dynamics predicted by the model closely match data from clinical trials. We further predicted that early diagnosis together with administration of tyrosine kinase inhibitor opens the path to eradication of chronic myeloid leukemia, leading to the wash out of the aberrant progenitor cells, ameliorating the patient’s condition while lowering the risk of blast transformation and drug resistance.

Conclusions

Tyrosine kinase inhibitor therapy can cure chronic myeloid leukemia, although it may have to be prolonged. The depth of response increases with time in the vast majority of patients. These results illustrate the importance of stochastic effects on the dynamics of acquired hematopoietic stem cell disorders and have direct relevance for other hematopoietic stem cell-derived diseases.     

Hematopoietic stem and progenitor cells are differentially mobilized depending on the duration of Flt3-ligand administration

Background

Flt3-ligand is a cytokine that induces relatively slow mobilization of hematopoietic cells in animals and humans in vivo. This provides a time-frame to study hematopoietic stem and progenitor cell migration kinetics in detail.

Design and Methods

Mice were injected with Flt3-ligand (10 μg/day, intraperitoneally) for 3, 5, 7 and 10 days. Mobilization of hematopoietic stem and progenitor cells was studied using colony-forming-unit granulocyte/monocyte and cobblestone-area-forming-cell assays. The radioprotective capacity of mobilized peripheral blood mononuclear cells was studied by transplantation of 1.5×10⁶ Flt3-ligand-mobilized peripheral blood mononuclear cells into lethally irradiated (9.5 Gy) recipients.

Results

Hematopoietic progenitor cell mobilization was detected from day 3 onwards and prolonged administration of Flt3-ligand produced a steady increase in mobilized progenitor cells. Compared to Flt3-ligand administration for 5 days, the administration of Flt3-ligand for 10 days led to a 5.5-fold increase in cobblestone-area-forming cells at week 4 and a 5.0-fold increase at week 5. Furthermore, transplantation of peripheral blood mononuclear cells mobilized by 5 days of Flt3-ligand administration did not radioprotect lethally irradiated recipients, whereas peripheral blood mononuclear cells mobilized by 10 days of Flt3-Ligand administration did provide 100% radioprotection of the recipients with significant multilineage donor chimerism. Compared to the administration of Flt3-ligand or interleukin-8 alone, co-administration of interleukin-8 and Flt3-ligand led to synergistic enhancement of hematopoietic stem and progenitor cell mobilization on days 3 and 5.

Conclusions

These results indicate that hematopoietic stem and progenitor cells show different mobilization kinetics in response to Flt3-ligand, resulting in preferential mobilization of hematopoietic progenitor cells at day 5, followed by hematopoietic stem cell mobilization at day 10.     

Cryohydrocytosis: increased activity of cation carriers in red cells from a patient with a band 3 mutation

Background

Cryohydrocytosis is an inherited dominant hemolytic anemia characterized by mutations in a transmembrane segment of the anion exchanger (band 3 protein). Transfection experiments performed in Xenopus oocytes suggested that these mutations may convert the anion exchanger into a non-selective cation channel. The present study was performed to characterize so far unexplored ion transport pathways that may render erythrocytes of a single cryohydrocytosis patient cation-leaky.

Design and Methods

Cold-induced changes in cell volume were monitored using ektacytometry and density gradient centrifugation. Kinetics, temperature and inhibitor-dependence of the cation and water movements in the cryohydrocytosis patient’s erythrocytes were studied using radioactive tracers and flame photometry. Response of the membrane potential of the patient’s erythrocyte membrane to the presence of ionophores and blockers of anion and cation channels was assessed.

Results

In the cold, the cryohydrocytosis patient’s erythrocytes swelled in KCl-containing, but not in NaCl-containing or KNO₃-containing media indicating that volume changes were mediated by an anion-coupled cation transporter. In NaCl-containing medium the net HOE-642-sensitive Na⁺/K⁺ exchange prevailed, whereas in KCl-containing medium swelling was mediated by a chloride-dependent K⁺ uptake. Unidirectional K⁺ influx measurements showed that the patient’s cells have abnormally high activities of the cation-proton exchanger and the K⁺,Cl⁻ co-transporter, which can account for the observed net movements of cations. Finally, neither chloride nor cation conductance in the patient’s erythrocytes differed from that of healthy donors.

Conclusions

These results suggest that cross-talk between the mutated band 3 and other transporters might increase the cation permeability in cryohydrocytosis.     

Cathepsin X is secreted by human osteoblasts, digests CXCL-12 and impairs adhesion of hematopoietic stem and progenitor cells to osteoblasts

Background

Hematopoietic stem cells are retained within discrete bone marrow niches through the effects of cell adhesion molecules and chemokine gradients. However, a small proportion of hematopoietic stem cells can also be found trafficking in the peripheral blood. During induced stem cell mobilization a proteolytic microenvironment is generated, but whether proteases are also involved in physiological trafficking of hematopoietic stem cells is not known. In the present study we examined the expression, secretion and function of the cysteine protease cathepsin X by cells of the human bone marrow.

Design and Methods

Human osteoblasts, bone marrow stromal cells and hematopoietic stem and progenitor cells were analyzed for the secretion of cathepsin X by western blotting, active site labeling, immunofluorescence staining and activity assays. A possible involvement of cathepsin X in cell adhesion and CXCL-12-mediated cell migration was studied in functional assays. Matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) analysis revealed the digestion mechanism of CXCL-12 by cathepsin X.

Results

Osteoblasts and stromal cells secrete cathepsin X, whereas hematopoietic stem and progenitor cells do not. Using a cathepsin X-selective substrate, we detected the catalytic activity of cathepsin X in cell culture supernatants of osteoblasts. Activated cathepsin X is able to reduce cellular adhesive interactions between CD34⁺ hematopoietic stem and progenitor cells and adherent osteoblasts. The chemokine CXCL-12, a highly potent chemoattractant for hematopoietic stem cells secreted by osteoblasts, is readily digested by cathepsin X.

Conclusions

The exo-peptidase cathepsin X has been identified as a new member of the group of CXCL-12-degrading enzymes secreted by non-hematopoietic bone marrow cells. Functional data indicate that cathepsin X can influence hematopoietic stem and progenitor cell trafficking in the bone marrow.     

CD8+ T-cell immunity against cancer-testis antigens develops following allogeneic stem cell transplantation and reveals a potential mechanism for the graft-versus-leukemia effect

Background

Allogeneic stem cell transplantation is associated with a powerful ‘graft-versus-leukemia’ effect that is generally considered to result from an alloreactive T-cell immune response. However, disease remission can also be observed after syngeneic transplantation and we investigated whether a T-cell immune response to cancer-testis antigens can be detected in patients in the post-transplant period.

Design and Methods

The T-cell immune response against cancer-testis antigens was studied in a cohort of 41 patients who underwent allogeneic stem cell transplantation for the management of acute myeloid leukemia or multiple myeloma. The cytokine secretion assay was combined with magnetic selection to allow detection of an interferon-γ-secreting T-cell response to a panel of cancer-testis antigen peptides.

Results

A cancer-testis antigen-specific CD8⁺ T-cell immune response was observed in the peripheral blood of five patients with an average magnitude of 0.045% of the CD8⁺ T-cell repertoire. Four of these patients had undergone reduced intensity conditioning transplantation with alemtuzumab for the treatment of acute myeloid leukemia and three remain in long-term remission. T-cell immunity was focused against peptides derived from MAGE proteins and was markedly increased within the bone marrow.

Conclusions

Functional cancer-testis antigen-specific CD8⁺ T-cell immune responses develop in the early period following reduced intensity allogeneic stem cell transplantation and are preferentially localized to bone marrow. These immune responses are likely to contribute to the cellular basis of the graft-versus-leukemia effect.     

Enhanced L-type calcium currents in cardiomyocytes from transgenic rats overexpressing SERCA2a

BACKGROUND:

Previous research reported that transgenic rats overexpressing the sarco(endo)plasmic reticulum Ca²⁺-ATPase SERCA2a exhibit improved contractile function of the myocardium. Furthermore, impaired Ca²⁺ uptake and reduced relaxation rates in rats with diabetic cardiomyopathy were partially rescued by transgenic expression of SERCA2a in the heart.

OBJECTIVE:

To explore whether enhanced Ca²⁺ cycling in the cardiomyocytes of SERCA2a transgenic rats is associated with changes in L-type Ca²⁺ (I_Ca-L) currents.

METHODS:

The patch-clamp technique was used to measure whole-cell currents in cardiomyocytes from transgenic rats overexpressing SERCA2a and from wild-type (nontransgenic) animals.

RESULTS:

The amplitudes of I_Ca-L currents at depolarizing pulses ranging from −45 mV to 0 mV (350 ms duration, 1 Hz) were significantly higher in cardiomyocytes of SERCA2a transgenic rats than in nontransgenic rats (1985±48 pA [n=32] versus 1612±55 pA [n=28], respectively). The inactivation kinetics of I_Ca-L showed subtle differences with increased tau fast and tau slow decay constants in cardiomyocytes of SERCA2a transgenic animals. Beta-adrenergic stimulation with 50 nM isoproterenol reduced tau fast and tau slow decay constants in cardiomyocytes of transgenic rats to values that were not significantly different from those in normal cardiomyocytes. Furthermore, isoproterenol enhanced I_Ca-L currents 3.2-fold and 2.3-fold in cardiomyocytes with and without the SERCA2a transgene, respectively, and this effect was abolished by buffering intracellular Ca²⁺ with BAPTA.

CONCLUSIONS:

These findings indicate that enhanced Ca²⁺ cycling in the hearts of SERCA2a transgenic rats, both under normal conditions and during beta-adrenergic stimulation, involves changes in I_Ca-L currents. Modified I_Ca-L kinetics may contribute, to some extent, to the improved contractile function of the myocardium of transgenic rats.     

Identification of 4 novel HLA-B*40:01 restricted minor histocompatibility antigens and their potential as targets for graft-versus-leukemia reactivity

Background

Patients with hematologic malignancies can be successfully treated with donor lymphocyte infusion after HLA-matched allogeneic hematopoietic stem cell transplantation. The effect of donor lymphocyte infusion is mediated by donor T cells recognizing minor histocompatibility antigens. T cells recognizing hematopoietic restricted minor histocompatibility antigens may induce selective graft-versus-leukemia reactivity, whereas broadly-expressed antigens may be targeted in graft-versus-host disease.

Design and Methods

We analyzed in detail CD8⁺ T-cell immunity in a patient with relapsed chronic myelogenous leukemia who responded to donor lymphocyte infusion with minimal graft-versus-host disease of the skin. CD8⁺ T-cell clones specific for 4 HLA-B*40:01 restricted minor histocompatibility antigens were isolated which were identified by screening a plasmid cDNA library and whole genome association scanning. Detailed T-cell reactivity and monitoring experiments were performed to estimate the clinical and therapeutic relevance of the novel antigens.

Results

Three antigens were demonstrated to be expressed on primary leukemic cells of various origins as well as subtypes of non-malignant hematopoietic cells, whereas one antigen was selectively recognized on malignant hematopoietic cells with antigen presenting cell phenotype. Skin derived fibroblasts were only recognized after pre-treatment with IFN-γ by two T-cell clones.

Conclusions

Our data show evidence for different roles of the HLA-B*40:01 restricted minor histocompatibility antigens in the onset and execution of the anti-tumor response. All antigens may have contributed to a graft-versus-leukemia effect, and one minor histocompatibility antigen (LB-SWAP70-1Q) has specific therapeutic value based on its in vivo immunodominance and strong presentation on leukemic cells of various origins, but absence of expression on cytokine-treated fibroblasts.