Immature Bone Marrow Research Articles

In vitro derivation of marginal zone B cells from CD23- precursors (82.8)

Abstract B cell development has been portrayed as proceeding in a linear fashion; specifically, newly formed B cells migrate from the bone marrow to the spleen where they develop into transitional B cells (T1 and T2), which give rise to marginal zone (MZ) and follicular mature (FM) B cells. Previously, we hypothesized that MZ B cells can develop from a T1 B cell without passing through a T2 developmental stage. We have developed an in vitro culture system that allows immature splenic and bone marrow cells, when cultured in the presence of B cell activating factor (BAFF) and Notch ligand, delta-like-1 (DL1), to develop into mature FM and MZ B cells. Cells cultured with only Notch or BAFF signaling did not assume a MZ phenotype. To further investigate the ability of immature B cells to develop into MZ B cells, we separated immature cells based on CD23 expression and cultured them in the presence of Notch and BAFF. CD23- populations isolated from total splenocytes or B220+ cells yielded the most MZ-like B cells in culture. When CD23+ cells were cultured, most cells retained expression of CD23 and failed to develop into MZ B cells. However, a small percentage of CD23+ cells decreased CD23 expression, yielding a population of cells with a MZ phenotype. These data suggest that the majority of MZ-like B cells in culture were derived from CD23- cells and support the hypothesis that many, if not all, MZ cells in vivo are derived from a CD23- T1-like intermediate.

Heterogeneity of COX-2 macrophages in acute and chronic mycobacterial inflammation (134.30)

Abstract Persistent up-regulation of cyclooxygenase-2 (COX-2) and over-production of PGE2 by activated macrophages (MØ) compromise host immunity in cancer immunotherapy, autoimmune diseases, and chronic mycobacterial inflammation such as TB. Previous studies showed that a population of COX-2+ MØ is induced in the spleen of mice given i.p. HK-Mycobacterium bovis BCG. These MØ are derived from radiosensitive immature bone marrow cells that migrate and localize in the spleen. Mature spleen MØ express active COX-2 (subcellularly associated with the nuclear envelope (NE)), and release increased PGE2 from day 5 to day 21 after BCG administration. To determine if local sites of inflammation caused by BCG accumulate similar COX-2+ MØ, C57Bl/6 mice were given i.p. or i.n. BCG (1 and 0.5 mg doses, respectively). We found that 1 - 2 days after i.p. administration, peritoneal and splenic MØ transiently exhibited COX-2 which was dissociated from the NE, inactive, and released no PGE2. However, unlike splenic MØ, neither active nor inactive COX-2 was found in peritoneal MØ over days 3 - 28. After i.n. administration, alveolar MØ persistently expressed inactive COX-2 over days 1 - 28. No alveolar MØ with active COX-2+ were detected. Normal splenic, peritoneal, and alveolar MØ treated in vitro with BCG expressed active COX-2 and increased PGE2 release within 24 h, indicating that in vitro MØ activation does not always mimic in vivo responses. Our results indicate that in vivo BCG preferentially induces active COX-2 in the splenic MØ. In addition, without suppression by PGE2, MØ with inactive COX-2 may be "hyper-active" and regulate tissue-dependent acute and chronic inflammation. (Supported by NIH HL71711, DOD DAMD 17-03-1-0004, BCCRP 06BS-04-9615)

Distinct and Overlapping Patterns of Cytokine Regulation of Thymic and Bone Marrow-Derived NK Cell Development

Although bone marrow (BM) represents the main site for postnatal NK cell development, recently a distinct thymic-dependent NK cell pathway was identified. These studies were designed to investigate the role of cytokines in regulation of thymic NK cells and to compare with established regulatory pathways of BM-dependent NK cell compartment. The common cytokine receptor gamma-chain (Il2rg) essential for IL-15-induced signaling, and FMS-like tyrosine kinase 3 (FLT3) receptor ligand (Flt3l) were previously identified as important regulatory pathways of the BM NK cell compartment based on lack of function studies in mice, however their complementary action remains unknown. By investigating mice double-deficient in Il2rg and Flt3l (Flt3l(-/-) Il2rg(-/-)), we demonstrate that FLT3L is important for IL2Rg-independent maintenance of both immature BM as well as peripheral NK cells. In contrast to IL-7, which is dispensable for BM but important for thymic NK cells, IL-15 has a direct and important role in both thymic and BM NK cell compartments. Although thymic NK cells were not affected in Flt3l(-/-) mice, Flt3l(-/-)Il2rg(-/-) mice lacked detectable thymic NK cells, suggesting that FLT3L is also important for IL-2Rg-independent maintenance of thymic NK cells. Thus, IL-2Rg cytokines and FLT3L play complementary roles and are indispensable for homeostasis of both BM and thymic dependent NK cell development, suggesting that the cytokine pathways crucial for these two distinct NK cell pathways are largely overlapping.

Protective roles of brain-migrated immature bone marrow cells against NO-dependent neurotoxicity in a brain injury model

Protective roles of brain-migrated immature bone marrow cells against NO-dependent neurotoxicity in a brain injury model

Inhibitory effects of rat bone marrow-derived dendritic cells on naïve and alloantigen-specific CD4+ T cells: a comparison between dendritic cells generated with GM-CSF plus IL-4 and dendritic cells generated with GM-CSF plus IL-10

BackgroundUnlike mouse immature bone marrow (BM)-derived dendritic cells (DC), rat immature BMDC have not been thoroughly characterised in vitro for the mechanisms underlying their suppressive effect. To better characterise these mechanisms we therefore analysed the phenotypes and immune inhibitory properties of rat BMDC generated with GM-CSF plus IL-4 (= IL-4 DC) and with GM-CSF plus IL-10 (= IL-10 DC).ResultsBoth IL-4 DC and IL-10 DC exhibited lower surface expression of MHC class II and costimulatory molecules than mature splenic DC. They had a strong inhibitory effect on responsive T cells in vitro and despite their weak function as antigen-presenting cells they induced anergic T cells. However, only anergic T cells induced by IL-4 DC had a suppressive effect on responsive T cells. Induction of suppressive/tolerogenic T cells by IL-4 DC required direct contact between antigen-specific T cells and IL-4 DC. In addition, IL-4 DC and IL-10 DC prolonged allograft survival in an antigen-specific manner.ConclusionA unique phenotype of immature BMDC was isolated from the cultures. The mechanisms underlying the suppressive effect may be caused by their inability to deliver adequate costimulatory signals for T-cell activation. In addition, IL-4 DC but not IL-10 DC induce anergic T cells with suppressive function. This indicates that IL-4 DC and IL-10 DC may differ in the quality of their costimulation although no differences in the surface expression of costimulatory molecules were found.

Genetic correlates of autoreactivity and autoreactive potential in human Ig heavy chains

BackgroundImmature bone marrow B cells are known to have longer CDR3 than mature peripheral B cells, and this genetic characteristic has been shown to correlate with autoreactivity in these early cells. B-cell Central tolerance eliminates these cells, but it is known that autoreactive B cells nevertheless appear commonly in healthy human blood. We examined over 7,300 Ig genes from Genbank, including those annotated by their discoverers as associated with autoreactivity, to determine the genetic correlates of autoreactivity in mature B cells.ResultsWe find differential biases in gene segment usage and higher mutation frequency in autoreactivity-associated Ig genes, but the CDR3 lengths do not differ between autoreactive and non-autoreactive Ig genes. The most striking genetic signature of autoreactivity is an increase in the proportion of N-nucleotides relative to germline-encoded nucleotides in CDR3 from autoreactive genes.ConclusionWe hypothesize that peripheral autoreactivity results primarily from somatic mutation, and that the genetic correlates of autoreactivity in mature B-cells are not the same as those for autoreactivity in immature B cells. What is seen in mature autoreactive B cells are the correlates of autoreactive potential, not of autoreactivity per se. The autoreactive potential is higher for V(D)J rearrangements encoded to a large extent by N-nucleotides rather than by the gene segments that, we posit, have been selected in germline evolution for their suppression of autoreactive potential.

FGD2, a CDC42-specific Exchange Factor Expressed by Antigen-presenting Cells, Localizes to Early Endosomes and Active Membrane Ruffles

Members of the Fgd (faciogenital dysplasia) gene family encode a group of critical guanine nucleotide exchange factors (GEFs), which, by specifically activating Cdc42, control cytoskeleton-dependent membrane rearrangements. In its first characterization, we find that FGD2 is expressed in antigen-presenting cells, including B lymphocytes, macrophages, and dendritic cells. In the B lymphocyte lineage, FGD2 levels change with developmental stage. In both mature splenic B cells and immature bone marrow B cells, FGD2 expression is suppressed upon activation through the B cell antigen receptor. FGD2 has a complex intracellular localization, with concentrations found in membrane ruffles and early endosomes. Although endosomal localization of FGD2 is dependent on a conserved FYVE domain, its C-terminal pleckstrin homology domain mediates recruitment to membrane ruffles. FGD2 overexpression promotes the activation of Cdc42 and leads to elevated JNK1 activity in a Cdc42- but not Rac1-dependent fashion. These findings are consistent with a role of FGD2 in leukocyte signaling and vesicle trafficking in cells specialized to present antigen in the immune system.

Quantitative Dynamics of Flow Cytometric Aberrancies during Treatment with Erythropoietin/G-CSF Are Predictive for Responses in LOW/INT-I Risk Myelodysplastic Syndromes.

Flow cytometry (FCM) in myelodysplastic syndromes (MDS) is recognized as a useful tool in identifying aberrancies in expression of differentiation antigens on immature and mature bone marrow cells of erythroid and myelomonocytic lineages. FCM in MDS correlates significantly to morphology according to WHO and the WHO-based Prognostic Scoring System (WPSS). Moreover, certain aberrancies in pure RA+/−RS patients, i.e. CD7 on myeloid blasts, seemed to predict transfusion dependency and disease progression independent of classical prognostic parameters such as WHO, cytogenetics and IPSS (Van de Loosdrecht et al., Blood 2008, 111). Flow cytometric aberrancies in bone marrow of 29 patients with low or int-I risk MDS were analyzed at diagnosis and after approximately 1 year of treatment with a standardized regimen of Epo (NeoRecormon®) and G-CSF (median: 12 months (range: 4–24) in order to evaluate the value of FCM in disease monitoring. Patients were classified as RA+/−RS (n=11), RCMD+/−RS (n=15), 1 hypoplastic MDS, 1 MDS-U and 1 MDS/MPD. Progression was defined as an increase in WHO subgroup to at least RAEB-1 within 18 months after diagnosis of MDS. Before treatment, median number of aberrancies in the myelomonocytic lineage, as assessed by FCM, was 4 in RA+/−RS (range 1–8) and 5 in RCMD+/−RS (range 3–10); other MDS subgroups were too small. In age-matched normal bone marrow samples the median number of aberrancies was 1 (range 0–3, n=18). Aberrant expression of CD5, CD7 or CD56 was observed on myeloid blasts at diagnosis in 13 out of 18 patients that were transfusion-dependent or suffered from progressive disease during follow-up (4 RA+/−RS, 7 RCMD+/−RS, 1 MDS-U and 1MDS/MPD). Interestingly, expression of these markers was only detected in 1 out of 11 patients that were not or low transfusion dependent. This particular RCMD-RS patient had short response duration. The number of blast aberrancies correlated significantly to transfusion dependency and disease progression (r=0.459, p=0.012). Nine patients showed hematological improvement upon Epo/G-CSF treatment. In most of these patients (4/6 RA+/−RS and 2/3 RCMD+/−RS) the number of flow cytometric aberrancies decreased (median 4 at diagnosis to 2.5 at follow-up in all responders). This decrease was caused by a diminished number of monocytic aberrancies (p=0.028); a stable blast aberrancy was seen in only one patient. In 15 patients with stable disease, no significant changes in the number of flow cytometric aberrancies could be detected (median 5 and 5, at diagnosis and follow-up, respectively); whereas in 4 of the 5 patients with progressive disease during Epo/G-CSF (2/2 RA+/−RS, 1/2 RCMD+/−RS and 1 MDS/MPD) the number of aberrancies increased (median 7 and 9, at diagnosis and follow-up, respectively). This increase was mainly caused by changes in the number of aberrancies in blast and granulocyte subpopulations. The response to therapy seems to depend on the number of blast aberrancies (r=0.351, p=0.062). From these data we hypothesize that FCM can be used as an objective sensitive disease monitoring parameter in low/int-I risk MDS. Furthermore, in a subset of these patients treatment with Epo/G-CSF might have altered disease progression reflected by normalization or stabilization of aberrancies on myelomonocytic cells. This underscores observations that Epo/G-CSF improves overall survival and/or leukemia free survival. Prospective studies are being conducted to validate flow cytometric analysis in the diagnosis, prognostication and disease monitoring of low/int-I risk MDS during Epo/G-CSF and lenalidomide. To conclude, FCM can contribute to the management of low/int-I risk MDS patients as changes in the number of flow cytometric aberrancies reflect response to treatment.

5-Androstene-3b,17b-Diol Administration of Mice Exposed to Total Body Irradiation Results in Rapid Reconstitution of Immature Bone Marrow Progenitor Cells and Synergizes with Thrombopoietin but Not with Granulocyte-Colony Stimulating Factor.

5-AED (5-androstene-3β,17β-diol) is a naturally occurring adrenal cortical steroid, which displays radioprotective effects in both rodents and non-human primates, resulting in accelerated multilineage hematopoiesis and enhanced survival after total body irradiation (TBI), including a 1-log accelerated CD34+ cell reconstitution in bone marrow of non-human primates. Pegylated granulocyte-colony stimulating factor (Peg-G-CSF) is known to stimulate lineage-specific recovery of neutrophils, whereas the effects of thrombopoietin (TPO) are broader and include protection of short-term spleen repopulating immature cells as well as platelet recovery. To gain insight into the mechanism of 5-AED on immature hematopoietic cells, the effects of 5-AED on multilineage hematopoiesis and recovery of specific repopulating stem and progenitor cell subsets after TBI was evaluated in combination with and relative to either Peg-G-CSF or TPO. For direct measurements of the radioprotective effect of 5-AED, BALB/c mice were exposed to a midlethal dose of 6 Gy TBI. Two hours after TBI, mice were injected IM with 40 mg/kg 5-AED or the carrier, with or without 0.225 mg TPO or 10 mg Peg-G-CSF IP. Radioprotective effects of 5-AED on immature repopulating cell subsets were assessed by exposing BALB/c donor mice to 3 fractions of 2 Gy TBI, separated by 24 hours, and treatment with 40 mg/kg/d 5-AED or the carrier IM, or 0.7 mg TPO IP after each fraction or a single injection of 10 mg Peg-G-CSF IP after the first fraction. Twenty four hours after the last fraction, bone marrow of donor mice was examined for immature cell content per femur using the marrow repopulating ability (MRA day 13) assay and the CFU-S day 12 after transplantation in 8 Gy irradiated mice. After 6 Gy TBI, BALB/c mice treated with 5-AED displayed an accelerated multilineage recovery with increased white blood cells (p<0.001), blood platelets (p<0.0001) and red blood cells (p<0.03), as well as increased bone marrow cellularity (p<0.0001) and elevated numbers of bone marrow colony forming cells (p<0.00001) at 14 days post-TBI in comparison to placebo-treated animals. Increasing the 5-AED dose up to 200 mg/kg did not augment this effect. Combined treatment with 5-AED and Peg-G-CSF or TPO treatment did not result in an additive effect in this setting. However, after the fractionated 3x2 Gy, a 5- and 7- fold increase in CFU-S relative to radiation controls was observed in the 5-AED and TPO groups, respectively, and a synergistic 20-fold increase in CFU-S day 12 was observed when 5-AED and TPO were used simultaneously. Consistent with earlier observations, Peg-GCSF alone did not affect CFU-S day 12 and appeared to dampen the effect of 5-AED. MRA, expressed as GM-CFU per femur at 13 days after transplantation, was found to be increased 5- to 6-fold with 1002 colonies (range 0-5785) for 5-AED versus 174 (5-360) for radiation controls. This is in contrast to TPO, which promotes CFU-S reconstitution at the expense of the more immature MRA (Neelis et al. 1998: Blood 92, 1586). Thus, 5-AED as a single agent stimulates multilineage hematopoiesis and increases bone marrow cellularity following TBI. This effect is mediated by increased survival and/or reconstitution of immature repopulating cells in a pattern distinct from that of TPO. Consistently, 5-AED strongly synergizes with TPO at the level of immature cells from which reconstitution originates, thus revealing a novel mechanism of bone marrow protection in cytoreductive therapy.

Use of Ceramic Scaffolds as Inductors of Extramedullar Hematopoiesis

The extracellular matrix in skeletal tissue besides being responsible for its structure and function also provides key regulatory signals for cell proliferation and differentiation by interacting directly with cell receptors, controlling the diffusion of soluble growth factors. Immature bone marrow cells can migrate through capillaries to systemic circulation as well as return to bone marrow. This phenomenon is the biological basis for bone marrow transplant. The mechanisms that control the migration and the return of these immature bone marrow cells are still to be described in a generally accepted model. Thirty days-old Wistar rats were employed as recipients for the implantation of ceramic blocks or demineralized femur diaphysis. The material was implanted subcutaneously at the dorsum. The rats were euthanized with CO2 at 15, 40 and 68 days after implantation. The whole area of implantation was harvested from skin deep up to the muscle. Liver, spleen, lungs, femurs and the vertebral spine, ribs and sternum were also harvested. The harvested material was fixed in Millonig formalin and decalcified with EDTA, if applicable. The material was then progressively dehydrated in alcohol, immersed in xylol, impregnated and embedded in paraffin. The paraffin blocks were then sliced in 5µm sections with a microtome, stained in Haematoxilin – Eosin, Lennert's Giemsa and Gomori's reticulin for histological analysis under brightfield optical microscopy. All implants produced an intense fibroblastic reaction, angiogenesis and subsequent modulation into a loose stroma. However, hematopoesis was not observed. Implantation of endogenous haematopoetic cells in an inductive stroma, which might propitiate hematopoesis is a model scarcely studied. Experiments such as the one described here might be good models for understanding the sequence of events leading to formation of the stroma and implantation and differentiation of haematopoetic precursors.

Hematopoietic Stem Cell Engraftment and Seeding Permits Multi-Lymphoid Chimerism in Vascularized Bone Marrow Transplants

Vascularized bone marrow transplantation (VBMT) across a MHC barrier under a 7-day alphabeta-TCR mAb and CsA protocol facilitated multiple hematolymphoid chimerism via trafficking of the immature (CD90) bone marrow cells (BMC) between donor and recipient compartments. Early engraftment of donor BMC [BN(RT1(n))] into the recipient BM compartment [LEW(RT1(l))] was achieved at 1 week posttransplant and this was associated with active hematopoiesis within allografted bone and correlated with high chimerism in the hematolymphoid organs. Two-way trafficking between donor and recipient BM compartments was confirmed by the presence of recipient MHC class I cells (RT1(l)) within the allografted bone up to 3 weeks posttransplant. At 10 weeks posttransplant, decline of BMC viability in allografted bone corresponded with bone fibrosis and lack of hematopoiesis. In contrast, active hematopoiesis was present in the recipient bone as evidenced by the presence of donor-specific immature (CD90/RT1(n)) cells, which correlated with chimerism maintenance. Clonogenic activity of donor-origin cells (RT1(n)) engrafted into the host BM compartment was confirmed by colony-forming units (CFU) assay. These results confirm that hematolymphoid chimerism is developed early post-VBMT by T-cell lineage and despite allografted bone fibrosis chimerism maintenance is supported by B-cell linage and active hematopoiesis of donor-origin cells in the host BM compartment.

Thrombopoietin (TPO) regulates HIF-1α levels through generation of mitochondrial reactive oxygen species

Hypoxia inducible factor (HIF)-1 is a master transcriptional regulator mediating the cellular adaptation to hypoxia. In addition, HIF-1 is also vital for the development of hematopoietic stem cells (HSCs). In a previous study we found that thrombopoietin (TPO), an important and non-redundant cytokine for HSC maintenance and expansion, induces HIF-1alpha expression in HSCs by enhancing the stability of HIF-1alpha under normoxic conditions. However, the molecular mechanisms of these effects are not yet fully understood. In this study, we explored the mechanisms and found that TPO-induced mitochondrial reactive oxygen species (ROS) played a crucial role in stabilization of HIF-1. Both ROS scavengers and inhibitors of mitochondrial electron transport completely blocked HIF-1alpha induction by TPO in UT-7/TPO cells and in primary immature mouse bone marrow cells. We also found that TPO-induced HIF-1alpha induction was tightly coupled with glucose metabolism. Inhibition of glucose transporter or glycolytic enzyme blocked HIF-1alpha elevation of TPO. These results indicate that TPO induces HIF-1alpha expression in a manner very similar to that of hypoxia.

Phase I Clinical Trial of Autologous Ascites-derived Exosomes Combined With GM-CSF for Colorectal Cancer

Exosomes are small membrane vesicles that are secreted by a multitude of cell types. The exosomes derived from dendritic cells (Dex), tumor cells (Tex), and malignant effusions demonstrate immunomodulatory functions, and are even under clinical trial for cancer treatments. In this study we report the phase I clinical trial of the ascites-derived exosomes (Aex) in combination with the granulocyte–macrophage colony-stimulating factor (GM-CSF) in the immunotherapy of colorectal cancer (CRC). The Aex isolated by sucrose/D2O density gradient ultracentrifugation are 60–90-nm vesicles that contain the diverse immunomodulatory markers of exosomes and tumor-associated carcinoembryonic antigen (CEA). Totally 40 patients (HLA-A0201+CEA+) with advanced CRC were enrolled in the study, and randomly assigned to treatments with Aex alone or Aex plus GM-CSF. Patients in both groups received a total of four subcutaneous immunizations at weekly intervals. We found that both therapies were safe and well tolerated, and that Aex plus GM-CSF but not Aex alone can induce beneficial tumor-specific antitumor cytotoxic T lymphocyte (CTL) response. Therefore, our study suggests that the immunotherapy of CRC with Aex in combination with GM-CSF is feasible and safe, and thus can serve as an alternative choice in the immunotherapy of advanced CRC.

SWAP‐70 regulates mast cell FcϵRI‐mediated signaling and anaphylaxis

Mast cells, perhaps best known by their ability to trigger allergic reactions after stimulation through the FcepsilonRI, express the unusual phosphatidylinositol 3-kinase (PI3K)-dependent, Rac-binding protein SWAP-70. Here, we show that the IgE-mediated passive cutaneous and the systemic anaphylactic responses are strongly reduced in SWAP-70(-/-) mice. Cultured SWAP-70(-/-) immature bone marrow mast cells (BMMC) are also impaired in FcepsilonRI-mediated degranulation, which can be restored by expression of exogenous wild-type SWAP-70, but less so if a phosphatidylinositol trisphosphate (PIP(3)) binding mutant is expressed. SWAP-70 itself supports inositol-3-phosphate and PIP(3) production, the latter indicating a potential feedback from SWAP-70 towards PI3K. FcepsilonRI-stimulated transcription and release of cytokines is controlled by SWAP-70. Key FcepsilonRI signal transduction events like activation of LAT by phosphorylation, activation of Akt/PKB and of p38 MAP kinase are reduced in SWAP-70(-/-) BMMC, but ERK is strongly hyperactivated. Some requirements for SWAP-70 were apparent only under limited-strength signaling conditions. We suggest that SWAP-70 defines a new element of efficient mast cell activation upon FcepsilonRI signaling, important for the control of mast cell-dependent anaphylaxis.

Transitional B Cells Lose Their Ability to Receptor Edit but Retain Their Potential for Positive and Negative Selection

Ligation of B cell receptors on immature bone marrow B cells, either by an endogenous Ag or by an anti-B cell receptor Ab induces secondary V(D)J gene rearrangements, termed receptor editing. Whether the same signal induces receptor editing in transitional B cells is not clear. In this study, we examined the responses of immature and transitional B cells from V(H)12Vkappa1A Ig transgenic mice to stimulation with an anti-Igbeta Ab. Our results demonstrated that immature B cells stimulated with a low concentration of anti-Igbeta Ab, mimicking Ag stimulation, underwent receptor editing both in vivo and in vitro, as evidenced by the detection of dsDNA breaks at Jkappa recombination signal sequences, whereas transitional B cells did not. The lack of dsDNA breaks in transitional B cells contrasts with their increased expression of RAG1 and RAG2, suggesting a novel mechanism that may prevent rearrangements. Furthermore, treatment of transitional B cells with high concentrations of anti-Igbeta Abs induced apoptosis, whereas low concentrations induced differentiation. Our results support the idea that transitional B cells lose the capacity to edit, but are sensitive to positive and negative selection.

NKG2D-Mediated Marrow Injury in Paroxysmal Nocturnal Hemoglobinuria and Its Related Disorders.

Immune mechanism is considered to exert in the pathogenesis of marrow failure in paroxysmal nocturnal hemoglobinuria (PNH), idiopathic aplastic anemia (AA) and myelodysplastic syndromes (MDS); however, the molecular events are unknown. We have currently reported the appearance of NKG2D ligands such as cytomegalovirus glycoprotein UL16 binding proteins (ULBPs) and MHC class I-related chains A and B (MICA/B) on granulocytes and CD34+ marrow cells of some patients with PNH and its related diseases (Hanaoka N, et al. Blood . 2006; 107:1184–1191). ULBP and MICA/B are stress-inducible membrane proteins that appear in infection and transformation. The ligands share NKG2D receptor on lymphocytes such as NK, CD8+ T, and γδ T-cells and promote activation of the lymphocytes. Cells expressing the ligands are then deadly injured by NKG2D+ lymphocytes (Groh, PNAS 1996; Cosman, Immunity 2001). Indeed, cells expressing NKG2D ligands were killed in vitro by autologous NKG2D+ lymphocytes of our patients (Hanaoka N, et al. Blood . 2005; 106:304a; Blood . 2006; 108:295a). In further analysis, ligands were detected on granulocytes in 47 (53%) of 88 patients: 11 (58%) of 19 PNH, 28 (60%) of 47 AA, and 8 (36%) of 22 refractory anemia. Ligands were also detected on immature bone marrow cells in all 11 patients (3 PNH, 5 AA, and 3 refractory anemia) who permitted analysis of their marrow cells. In the patients, it is conceivable that blood cells were exposed to a certain stress to induce NKG2D ligands, leading to NKG2D-mediated marrow injury. We also observed a close association of the ligand expression with pancytopenia and favorable response to immunosuppressive therapy by prospective analysis of 5 patients (3 AA-PNH syndrome and 2 AA) for more than one year up to 5 years. Thus, we here propose that NKG2D-mediated immunity, which drives both NK and T-cells, is critically implicated in the pathogenesis of bone marrow failure of PNH and its related disorders.

Correction of the Skin Defect in Murine Recessive Dystrophic Epidermolysis Bullosa by Bone Marrow Derived SLAM Family Receptor Enriched Cells.

Dystrophic epidermolysis bullosa (EB) is a disorder of incurable skin fragility, blistering and perturbations in anchoring fibrils caused by mutations in the type VII collagen gene (COL7A1). Since bone marrow (BM) includes cells with capacity to differentiate into diverse cell types and can seed various organs, including skin, we hypothesized that if type VII collagen producing cells are present in BM, BM infusions could be a beneficial therapy for EB. To test this hypothesis this in vivo, we assessed multiple candidate populations for cellular therapy of murine recessive dystrophic EB (RDEB, col7a1 −/−). As the col7a1 −/− newborn mice develop blisters and do not survive after two weeks of life, we can readily identify the mutant pups and the lethality provided a robust readout for the cellular interventions. The following cell populations were tested: epidermal stem cells and transit amplifying cells (from epidermis); total BM; BM derived multipotent adult progenitor cells and mesenchymal stem cells; fetal liver cells; and lineage positive and lineage negative BM fractions. None of these cell populations, however, provided any correction in col7a1 −/− recipients (N=223), even when the infusion regimen was varied by cell dose, age of donor, in utero and postnatal age at the time of infusion, and intravenous versus intrahepatic infusion. In a search for alternatives, we used CD150+ and CD48− BM cells, since signaling molecule SLAM family receptor positive populations have been shown to have significant pluripotentiality. We used several doses and infused them early postnatally into unconditioned col7a1 −/− mutant animals. One of these conditions, namely CD150+/CD48− BM population from C57Bl/6 GFP transgenic mice at a dose of 8 million cells per animal administered on day 3 or 4 after birth intravenously, resulted in survival of 3 out of 13 (23%) animals in two independent experiments. One of them was harvested at postnatal day 55; two of them were electively harvested at postnatal day 70. The animals were confirmed to be mutants by genotyping using PCR. The weights of these three animals were lower than that of their normal litter mates. Donor engraftment was up to 3% in peripheral blood and up to 12% in BM. Strikingly, the skin blisters characteristic of EB healed. Tissue analyses showed that the adoptive transfer of donor SLAM CD150+ selected BM resulted in skin engraftment of GFP+ donor cells and in the production of VII collagen mRNA in the skin, as assessed by RT- PCR. BM cells explanted from the 3 surviving recipients showed collagen VII producing cells. Ultrastructurally, we were able to identify anchoring fibrils, structures in the skin that are comprised exclusively of type VII collagen protein. This is consistent with the hypothesis that adoptive transfer of enriched wild-type BM cells resulted in, at least partial, functional correction of RDEB. Experiments are ongoing to identify the type VII collagen producing cells in CD150+ and CD48− BM population, and, in parallel, to identify immature BM cells that could be induced into becoming a skin stem cell. Collectively, these data demonstrate proof-of-principle of BM transfer for correction of the basement membrane zone defect in col7a1 −/− animals that may offer a valuable approach for treatment of human RDEB.

Neutrophil apoptosis mediated by nicotinic acid receptors (GPR109A)

G protein-coupled receptor (GPR)109A (HM74A) is a G(i) protein-coupled receptor, which is activated by nicotinic acid (NA), a lipid-lowering drug. Here, we demonstrate that mature human neutrophils, but not eosinophils, express functional GPR109A receptors. The induction of the GPR109A gene appears to occur late in the terminal differentiation process of neutrophils, since a mixed population of immature bone marrow neutrophils did not demonstrate evidence for its expression. NA accelerated apoptosis in cultured neutrophils in a concentration-dependent manner, as assessed by phosphatidylserine redistribution, caspase-3 activation, and DNA fragmentation assays. The pro-apoptotic effect of NA was abolished by pertussis toxin, which was used to block G(i) proteins, suggesting a receptor-mediated mechanism. Activation of GPR109A by NA resulted in decreased levels of cyclic adenosine monophosphate (cAMP), most likely due to G(i)-mediated inhibition of adenylyl cyclase activity. NA-induced apoptosis was reversed by the addition of cell-permeable cAMP, pointing to the possibility that reduced cAMP levels promote apoptosis in neutrophils. Distal mechanism involved in this process may include the post-translational modification of members of the Bcl-2 family, such as dephosphorylation of pro-apoptotic Bad and antiapoptotic Mcl-1 proteins. Taken together, following maturation in the bone marrow, neutrophils express functional GPR109A receptors, which might be involved in the regulation of neutrophil numbers. Moreover, this study identified a new cellular target of NA and future drugs activating GPR109A receptors, the mature neutrophil.

Class Switch Recombination and Somatic Hypermutation in Early Mouse B Cells Are Mediated by B Cell and Toll-like Receptors

Activation-induced cytidine deaminase (AID) is required for immunoglobulin (Ig) gene class switch recombination (CSR), somatic hypermutation (SHM), and somatic hyperconversion. In general, high AID expression is found in mature B cells that are responding to antigens. However, AID expression and SHM have also been detected in developing B cells from transgenic mice that have a limited Ig repertoire. Here we demonstrate that AID expression, ongoing CSR, and active SHM occur in developing B cells from wild-type mice. Further, our results suggest that somatic variants arising from developing B cells in the bone marrow further diversify in the spleen of unimmunized mice. AID expression in developing B cells is T cell independent but involves engagement of B cell receptors and Toll-like receptors. Early AID expression can increase the preimmune repertoire of developing B cells, may provide an innate population of IgG- and IgA-expressing cells, and could be involved in receptor editing of self-reactive immature B cells.

Categorical selection of the antibody repertoire in splenic B cells

In the bone marrow, the passage of developing B cells through critical checkpoints of differentiation is associated with a reduction of specific categories of CDR3 of the Ig heavy chain (CDR-H3), particularly those with excessive hydrophobic or charged amino acids and those with a length of eight or fewer residues. To gain insight into the role of CDR-H3 content in the development of B cells in the spleen, we compared the sequences of V(H)7183DJCmu transcripts from sorted transitional T1, marginal zone, and follicular B cell subsets to those expressed by immature IgM(+)IgD(-) and mature IgM(lo)IgD(hi) B cells in the bone marrow. Although differences in V(H) utilization were noted, the T1 CDR-H3 repertoire showed extensive similarity to that of immature bone marrow B cells, and the follicular CDR-H3 repertoire most resembled that of mature bone marrow B cells. Unlike the splenic follicular and bone marrow mature B cell CDR-H3 repertoires, the marginal zone B cell CDR-H3 repertoire retained both short and highly charged amino acid motifs, including those with two arginines. Our findings suggest that antigen binding sites containing specific categories of CDR-H3 sequence content may inhibit, permit, or even facilitate passage of the host B cell through critical checkpoints in peripheral as well as central development.