Dormant and self-renewing hematopoietic stem cells and their niches.

In the mouse, over the last 20 years, a set of cell-surface markers and activities have been identified, enabling the isolation of bone marrow (BM) populations highly enriched in hematopoietic stem cells (HSCs). These HSCs have the ability to generate multiple lineages and are capable of long-term self-renewal activity such that they are able to reconstitute and maintain a functional hematopoietic system after transplantation into lethally irradiated recipients. Using single-cell reconstitution assays, various marker combinations can be used to achieve a functional HSC purity of almost 50%. Here we have used the differential expression of six of these markers (Sca1, c-Kit, CD135, CD48, CD150, and CD34) on lineage-depleted BM to refine cell hierarchies within the HSC population. At the top of the hierarchy, we propose a dormant HSC population (Lin(-)Sca1(+)c-Kit(+) CD48(-)CD150(+)CD34(-)) that gives rise to an active self-renewing CD34(+) HSC population. HSC dormancy, as well as the balance between self-renewal and differentiation activity, is at least, in part, controlled by the stem cell niches individual HSCs are attached to. Here we review the current knowledge about HSC niches and propose that dormant HSCs are located in niches at the endosteum, whereas activated HSCs are in close contact to sinusoids of the BM microvasculature.

Hard-wired heterogeneity in blood stem cells revealed using a dynamic regulatory network model.

MOTIVATION: Combinatorial interactions of transcription factors with cis-regulatory elements control the dynamic progression through successive cellular states and thus underpin all metazoan development. The construction of network models of cis-regulatory elements, therefore, has the potential to generate fundamental insights into cellular fate and differentiation. Haematopoiesis has long served as a model system to study mammalian differentiation, yet modelling based on experimentally informed cis-regulatory interactions has so far been restricted to pairs of interacting factors. Here, we have generated a Boolean network model based on detailed cis-regulatory functional data connecting 11 haematopoietic stem/progenitor cell (HSPC) regulator genes. RESULTS: Despite its apparent simplicity, the model exhibits surprisingly complex behaviour that we charted using strongly connected components and shortest-path analysis in its Boolean state space. This analysis of our model predicts that HSPCs display heterogeneous expression patterns and possess many intermediate states that can act as 'stepping stones' for the HSPC to achieve a final differentiated state. Importantly, an external perturbation or 'trigger' is required to exit the stem cell state, with distinct triggers characterizing maturation into the various different lineages. By focusing on intermediate states occurring during erythrocyte differentiation, from our model we predicted a novel negative regulation of Fli1 by Gata1, which we confirmed experimentally thus validating our model. In conclusion, we demonstrate that an advanced mammalian regulatory network model based on experimentally validated cis-regulatory interactions has allowed us to make novel, experimentally testable hypotheses about transcriptional mechanisms that control differentiation of mammalian stem cells. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Lassa virus entry in antigen presenting cells and evaluation of a novel nanoparticle vaccine platform against arena viruses

Arenaviruses are a large and diverse family of viruses that merit significant attention as causative agents of severe hemorrhagic fevers in humans. Lassa virus (LASV) in Africa and the South American hemorrhagic fever viruses Junin (JUNV), Machupo (MACV), and Guanarito (GTOV) have emerged as important human pathogens and represent serious public health problems in their respective endemic areas. A hallmark of fatal arenaviruses hemorrhagic fevers is a marked immunosuppression of the infected patients. Antigen presenting cells (APCs) such as macrophages and in particular dendritic cells (DCs) are early and preferred targets of arenaviruses infection. Instead of being recognized and presented as foreign antigens by DCs, arenaviruses subvert the normal mechanisms of pathogen recognition, invade DCs and establish a productive infection. Viral replication perturbs the DCs' ability to present antigens and to activate T and B cells, contributing to the marked virus-induced immunosuppression observed in fatal disease. Considering their crucial role in the development of an anti-viral immune response, the mechanisms by which arenaviruses, and in particular LASV, invade DCs are of particular interest. The C-type lectin DC-specific Intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) was recently identified as a potential entry receptor for LASV. The first project of my thesis focused therefore on the investigation of the role of DC-SIGN in LASV entry into primary human DCs. My data revealed that DC-SIGN serves as an attachment factor for LASV on human DCs and can facilitate capture of free virus and subsequent cell entry. However, in contrast to other emerging viruses, of the phlebovirus family, I found that DC-SIGN does likely not function as an authentic entry receptor for LASV. Moreover, I was able to show that LASV enters DCs via an unusually slow pathway that depends on actin, but is independent of clathrin and dynamin. Considering the lack of effective treatments and the limited public health infrastructure in endemic regions, the development of protective vaccines against arenaviruses is an urgent need. To address this issue, the second project of my thesis aimed at the development of a novel recombinant arenavirus vaccine based on a nanoparticle (NPs) platform and its evaluation in a small animal model. During the first phase of the project I designed, produced, and characterized suitable vaccine antigens. In the second phase of the project, I generated antigen-conjugated NPs, developed vaccine formulations, and tested the NPs for their ability to elicit anti-viral T cell responses as well as anti-viral antibodies. I demonstrated that the NPs platform is able to activate both cellular and humoral branches of the adaptive anti-viral immunity, providing proof-of-principle. In sum, my first project will allow, in a long term perspective, a better understanding of the viral pathogenesis and contribute to the development of novel antiviral strategies. The second project will expectidly offer a new treatment option against arenaviruses.

Relevance of biallelic versus monoallelic TNFRSF13B mutations in distinguishing disease-causing from risk-increasing TNFRSF13B variants in antibody deficiency syndromes.

TNFRSF13B encodes transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI), a B cell- specific tumor necrosis factor (TNF) receptor superfamily member. Both biallelic and monoallelic TNFRSF13B mutations were identified in patients with common variable immunodeficiency disorders. The genetic complexity and variable clinical presentation of TACI deficiency prompted us to evaluate the genetic, immunologic, and clinical condition in 50 individuals with TNFRSF13B alterations, following screening of 564 unrelated patients with hypogammaglobulinemia. We identified 13 new sequence variants. The most frequent TNFRSF13B variants (C104R and A181E; n=39; 6.9%) were also present in a heterozygous state in 2% of 675 controls. All patients with biallelic mutations had hypogammaglobulinemia and nearly all showed impaired binding to a proliferation-inducing ligand (APRIL). However, the majority (n=41; 82%) of the pa-tients carried monoallelic changes in TNFRSF13B. Presence of a heterozygous mutation was associated with antibody deficiency (P< .001, relative risk 3.6). Heterozygosity for the most common mutation, C104R, was associated with disease (P< .001, relative risk 4.2). Furthermore, heterozygosity for C104R was associated with low numbers of IgD(-)CD27(+) B cells (P= .019), benign lymphoproliferation (P< .001), and autoimmune complications (P= .001). These associations indicate that C104R heterozygosity increases the risk for common variable immunodeficiency disorders and influences clinical presentation.

Intestinal bacteria condition dendritic cells to promote IgA production.

Immunoglobulin (Ig) A represents the predominant antibody isotype produced at the intestinal mucosa, where it plays an important role in limiting the penetration of commensal intestinal bacteria and opportunistic pathogens. We show in mice that Peyer's Patch-derived dendritic cells (PP-DC) exhibit a specialized phenotype allowing the promotion of IgA production by B2 cells. This phenotype included increased expression of the retinaldehyde dehydrogenase 1 (RALDH1), inducible nitric oxide synthase (iNOS), B cell activating factor of the tumor necrosis family (BAFF), a proliferation-inducing ligand (APRIL), and receptors for the neuropeptide vasoactive intestinal peptide (VIP). The ability of PP-DC to promote anti-CD40 dependent IgA was partially dependent on retinoic acid (RA) and transforming growth factor (TGF)-beta, whilst BAFF and APRIL signaling were not required. Signals delivered by BAFF and APRIL were crucial for CD40 independent IgA production, although the contribution of B2 cells to this pathway was minimal. The unique ability of PP-DC to instruct naïve B cells to differentiate into IgA producing plasma cells was mainly imparted by the presence of intestinal commensal bacteria, and could be mimicked by the addition of LPS to the culture. These data indicate that exposure to pathogen-associated molecular patterns present on intestinal commensal bacteria condition DC to express a unique molecular footprint that in turn allows them to promote IgA production.

Protein-binding site at the immunoglobulin mu membrane polyadenylylation signal: possible role in transcription termination.

mRNAs specifying immunoglobulin mu and delta heavy chains are encoded by a single large, complex transcription unit (mu + delta gene). The transcriptional activity of delta gene segments in terminally differentiated, IgM-secreting B lymphocytes is 10-20 times lower than in earlier B-lineage cells expressing delta mRNA. We find that transcription of the mu + delta gene in IgM-secreting murine myeloma cells terminates within a region of 500-1000 nucleotides immediately following the mu membrane (mu m) polyadenylylation site. Transcription decreases only minimally through this region in murine cell lines representative of earlier stages in B-cell development. A DNA fragment containing the mu m polyadenylylation signal gives protein-DNA complexes with different mobilities in gel retardation assays with nuclear extracts from myeloma cells than with nuclear extracts from earlier B-lineage cells. However, using a recently developed "footprinting" procedure in which protein-DNA complexes resolved in gel retardation assays are subjected to nucleolytic cleavage while still in the polyacrylamide gel, we find that the DNA sequences protected by factors from the two cell types are indistinguishable. The factor-binding site on the DNA is located 5' of the mu m polyadenylylation signal AATAAA and includes the 15-nucleotide-long A + T-rich palindrome CTGTAAACAAATGTC. This type of palindromic binding site exhibits orientation-dependent activity consistent with the reported properties of polymerase II termination signals. This binding site is followed by two sets of directly repeated DNA sequences with different helical conformation as revealed by their reactivity with the chemical nuclease 1,10-phenanthroline-copper. The close proximity of these features to the signals for mu m mRNA processing may reflect a linkage of the processes of developmentally regulated mu m polyadenylylation and transcription termination.

Muscle recovery after repair of short and long peripheral nerve gaps using fibrin conduits.

Peripheral nerve injuries with loss of nervous tissue are a significant clinical problem and are currently treated using autologous nerve transplants. To avoid the need for donor nerve, which results in additional morbidity such as loss of sensation and scarring, alternative bridging methods have been sought. Recently we showed that an artificial nerve conduit moulded from fibrin glue is biocompatible to nerve regeneration. In this present study, we have used the fibrin conduit or a nerve graft to bridge either a 10 mm or 20 mm sciatic nerve gap and analyzed the muscle recovery in adult rats after 16 weeks. The gastrocnemius muscle weights of the operated side were similar for both gap sizes when treated with nerve graft. In contrast, muscle weight was 48.32 ± 4.96% of the contra-lateral side for the 10 mm gap repaired with fibrin conduit but only 25.20 ± 2.50% for the 20 mm gap repaired with fibrin conduit. The morphology of the muscles in the nerve graft groups showed an intact, ordered structure, with the muscle fibers grouped in fascicles whereas the 20 mm nerve gap fibrin group had a more chaotic appearance. The mean area and diameter of fast type fibers in the 20 mm gap repaired with fibrin conduits were significantly (P<0.01) worse than those of the corresponding 10 mm gap group. In contrast, both gap sizes treated with nerve graft showed similar fiber size. Furthermore, the 10 mm gaps repaired with either nerve graft or fibrin conduit showed similar muscle fiber size. These results indicate that the fibrin conduit can effectively treat short nerve gaps but further modification such as the inclusion of regenerative cells may be required to attain the outcomes of nerve graft for long gaps.

Immuno-monitoring of CD8+ T cells in whole blood versus PBMC samples.

The study of natural T cell responses against pathogens or tumors, as well as the assessment of new immunotherapy strategies aimed at boosting these responses, requires increasingly precise ex vivo analysis of blood samples. For practical reasons, studies are often performed using purified PBMC samples, usually cryopreserved. Here, we report on FACS analyses of peripheral blood T cells, performed by direct antibody staining of non-purified total blood. For comparison, fresh PBMC, purified by Ficoll, were analysed. Our results show that the latter method can induce a bias in subpopulation distribution, in particular of CD8+ T cells, and sometimes lead to inaccurate measurement of antigen specific CD8+ T cell responses. Direct analysis of total blood can be applied to longitudinal immuno-monitoring of T cell-based therapy. While the need to purify and cryopreserve PBMC for subsequent studies is obvious, the use of whole blood has the advantage of providing unbiased results and only small amounts of blood are used.

Proliferation capacity and cytotoxic activity are mediated by functionally and phenotypically distinct virus-specific CD8 T cells defined by interleukin-7R{alpha} (CD127) and perforin expression.

Cytotoxicity and proliferation capacity are key functions of antiviral CD8 T cells. In the present study, we investigated a series of markers to define these functions in virus-specific CD8 T cells. We provide evidence that there is a lack of coexpression of perforin and CD127 in human CD8 T cells. CD127 expression on virus-specific CD8 T cells correlated positively with proliferation capacity and negatively with perforin expression and cytotoxicity. Influenza virus-, cytomegalovirus-, and Epstein-Barr virus/human immunodeficiency virus type 1-specific CD8 T cells were predominantly composed of CD127(+) perforin(-)/CD127(-) perforin(+), and CD127(-)/perforin(-) CD8 T cells, respectively. CD127(-)/perforin(-) and CD127(-)/perforin(+) cells expressed significantly more PD-1 and CD57, respectively. Consistently, intracellular cytokine (gamma interferon, tumor necrosis factor alpha, and interleukin-2 [IL-2]) responses combined to perforin detection confirmed that virus-specific CD8 T cells were mostly composed of either perforin(+)/IL-2(-) or perforin(-)/IL-2(+) cells. In addition, perforin expression and IL-2 secretion were negatively correlated in virus-specific CD8 T cells (P &lt; 0.01). As previously shown for perforin, changes in antigen exposure modulated also CD127 expression. Based on the above results, proliferating (CD127(+)/IL-2-secreting) and cytotoxic (perforin(+)) CD8 T cells were contained within phenotypically distinct T-cell populations at different stages of activation or differentiation and showed different levels of exhaustion and senescence. Furthermore, the composition of proliferating and cytotoxic CD8 T cells for a given antiviral CD8 T-cell population appeared to be influenced by antigen exposure. These results advance our understanding of the relationship between cytotoxicity, proliferation capacity, the levels of senescence and exhaustion, and antigen exposure of antiviral memory CD8 T cells.

Characterization of the murine high Km glucose transporter GLUT2 gene and its transcriptional regulation by glucose in a differentiated insulin-secreting cell line.

In pancreatic beta-cells, the high Km glucose transporter GLUT2 catalyzes the first step in glucose-induced insulin secretion by glucose uptake. Expression of the transporter has been reported to be modulated by glucose either at the protein or mRNA levels. In this study we used the differentiated insulinoma cell line INS-1 which expresses high levels of GLUT2 and show that the expression of GLUT2 is regulated by glucose at the transcriptional level. By run-on transcription assays we showed that glucose induced GLUT2 gene transcription 3-4-fold in INS-1 cells which was paralleled by a 1.7-2.3-fold increase in cytoplasmic GLUT2 mRNA levels. To determine whether glucose regulatory sequences were present in the promoter region of GLUT2, we cloned and characterized a 1.4-kilobase region of mouse genomic DNA located 5' of the translation initiation site. By RNase protection assays and primer extension, we determined that multiple transcription initiation sites were present at positions -55, -64, and -115 from the first coding ATG and which were identified in liver, intestine, kidney, and beta-cells mRNAs. Plasmids were constructed with the mouse promoter region linked to the reporter gene chloramphenicol acetyltransferase (CAT), and transiently and stably transfected in the INS-1 cells. Glucose induced a concentration-dependent increase in CAT activity which reached a maximum of 3.6-fold at 20 mM glucose. Similar CAT constructs made of the human GLUT2 promoter region and the CAT gene displayed the same glucose-dependent increase in transcriptional activity when transfected into INS-1 cells. Comparison of the mouse and human promoter regions revealed sequence identity restricted to a few stretches of sequences which suggests that the glucose responsive element(s) may be conserved in these common sequences.

Quantification, self-renewal, and genetic tracing of FL1+ tumor-initiating cells in a large cohort of human gliomas.

Evidence has emerged that the initiation and growth of gliomas is sustained by a subpopulation of cancer-initiating cells (CICs). Because of the difficulty of using markers to tag CICs in gliomas, we have previously exploited more robust phenotypic characteristics, including a specific morphology and intrincic autofluorescence, to identify and isolate a subpopulation of glioma CICs, called FL1(+). The objective of this study was to further validate our method in a large cohort of human glioma and a mouse model of glioma. Seventy-four human gliomas of all grades and the GFAP-V(12)HA-ras B8 mouse model were analyzed for in vitro self-renewal capacity and their content of FL1(+). Nonneoplastic brain tissue and embryonic mouse brain were used as control. Genetic traceability along passages was assessed with microsatellite analysis. We found that FL1(+) cells from low-grade gliomas and from control nonneoplasic brain tissue show a lower level of autofluorescence and undergo a restricted number of cell divisions before dying in culture. In contrast, we found that FL1(+) cells derived from many but not all high-grade gliomas acquire high levels of autofluorescence and can be propagated in long-term cultures. Moreover, FL1(+) cells show a remarkable traceability over time in vitro and in vivo. Our results show that FL1(+) cells can be found in all specimens of a large cohort of human gliomas of different grades and in a model of genetically induced mouse glioma as well as nonneoplastic brain. However, their self-renewal capacity is variable and seems to be dependent on the tumor grade.

Identification of an expanded population of activated CD4(+) CD25(+) T cells expressing CD45RO and IL-7R in kidney transplant recipients

Recent studies have shown that CD4+ CD25+ T cells belong to two functionally different T lymphocytes, i.e. regulatory T cells (Treg) or activated T cells (Tact), which can be distinguished based on the expression of CD45RO and IL-7R: Treg (FoxP3+) are CD45RO+ IL-7R- , whereas Tact (FoxP3- ) are CD45RO+ IL- 7R+. In order to determine if a CD4+ CD25+ CD45RO+ IL-7R+ activated T cell population might be identified in kidney transplant recipients, we studied 27 healthy subjects (HS) and 23 kidney recipients, of whom 17 had stable graft function under standard immunosuppression (IS), 5 had biopsy-proven chronic humoral rejection (CHR), and one was a stable "tolerant" patient who had discontinued IS for more than 2 years. Phenotypical analysis by flow cytometry and functional assays by MLR were performed. Overall, the Tact population was found to be significantly increased in 87% of the transplant recipients (mean: 18.8±10.1% of CD4+ CD25+ T cells) compared to HS (mean: 4.5±2.0%; P<0.0001). In the 5 patients with CHR, this Tact population was highly expanded (31.3±9.3%; P<0.0001), whereas it was comparable to HS in the "tolerant" recipient (4.7%). Intermediate levels (16.0±6.9%; P<0.0001) were found in the 17 stable recipients. In CHR, the proliferative capacity of the Tact population was found to be 5-fold higher when stimulated by irradiated donor PBMC as compared to a stimulation by irradiated 3rd party PBMC. After kidney transplantation, an expanded circulating CD4+ CD25+ T cell population characterized by the expression of CD45RO and IL-7R was found in most recipients, particularly in those with CHR. In a patient with long-term operational tolerance, this Tact population was similar to HS. Measuring circulating Tact may become a useful monitoring tool after transplantation.

Long-term health-related quality of life and walking capacity of lung recipients with and without bronchiolitis obliterans syndrome.

BACKGROUND: Outcome after lung transplantation (LTx) is affected by the onset of bronchiolitis obliterans syndrome (BOS) and lung function decline. Reduced health-related quality of life (HRQL) and physical mobility have been shown in patients developing BOS, but the impact on the capacity to walk is unknown. We aimed to compare the long-term HRQL and 6-minute walk test (6MWT) between lung recipients affected or not by BOS Grade > or =2. METHODS: Fifty-eight patients were prospectively followed for 5.6 +/- 2.9 years after LTx. Assessments included the St George's Respiratory Questionnaire (SGRQ) and the 6MWT, which were performed yearly. Moreover, clinical complications were recorded to estimate the proportion of the follow-up time lived without clinical intercurrences after transplant. Analyses were performed using adjusted linear regression and repeated-measures analysis of variance. RESULTS: BOS was a significant predictor of lower SGRQ scores (p < 0.01) and reduced time free of clinical complications (p = 0.001), but not of 6MWT distance (p = 0.12). At 7 years post-transplant, results were: 69.0 +/- 21.8% vs 86.9 +/- 5.6%, p < 0.05 (SGRQ); 58.5 +/- 21.6% vs 88.7 +/- 11.4%, p < 0.01 (proportion of time lived without clinical complications); and 82.2 +/- 10.9% vs 91.9 +/- 14.2%, p = 0.27 (percent of predicted 6MWT), respectively, for patients with BOS and without BOS. CONCLUSIONS: Despite significantly less time lived without clinical complications and progressive decline of self-reported health status, the capacity to walk of patients affected by BOS remained relatively stable over time. These findings may indicate that the development of moderate to severe BOS does not prevent lung recipients from walking independently and pursuing an autonomous life.

NPY regulates catecholamine secretion from human adrenal chromaffin cells.

The aim of the present work was to find out whether NPY synthesized in human adrenal chromaffin cells controls in an autocrine/paracrine fashion the release of catecholamines by these cells. Accordingly, the constitutive and regulated release of both NPY and catecholamines was measured simultaneously in cultured human chromaffin cells. In addition, by using both RT-PCR and a combination of specific agonists and antagonists, we characterized the expression of NPY receptors on these cells as well as their pharmacology. Our results were as follows. 1) Human chromaffin cells constitutively secrete NPY. 2) Nicotine elicits a rapid increase in the release of both catecholamines and NPY; this release of NPY is more sustained than that of catecholamines. 3) RT-PCR shows expression of Y1, Y2, Y4, and Y5 receptor mRNA by chromaffin cells; these receptors are functional, as various receptor specific agonists elicit an increase in intracellular calcium. 4) Peptide YY, in contrast to NPY, is not able to stimulate the release of catecholamines. This finding was corroborated by the observation that no receptor-specific antagonists were able to reduce constitutive catecholamine release, whereas an NPY-immunoneutralizing antibody markedly attenuated the secretion. Taken together, these data suggest that NPY originating from the adrenal medulla locally enhances the secretion of catecholamines, presumably by acting via the putative y3 receptor.

A long, naturally presented immunodominant epitope from NY-ESO-1 tumor antigen: implications for cancer vaccine design.

The tumor antigen NY-ESO-1 is a promising cancer vaccine target. We describe here a novel HLA-B7-restricted NY-ESO-1 epitope, encompassing amino acids 60-72 (APRGPHGGAASGL), which is naturally presented by melanoma cells. The tumor epitope bound to HLA-B7 by bulging outward from the peptide-binding cleft. This bulged epitope was not an impediment to T-cell recognition, however, because four of six HLA-B7(+) melanoma patients vaccinated with NY-ESO-1 ISCOMATRIX vaccine generated a potent T-cell response to this determinant. Moreover, the response to this epitope was immunodominant in three of these patients and, unlike the T-cell responses to bulged HLA class I viral epitopes, the responding T cells possessed a remarkably broad TCR repertoire. Interestingly, HLA-B7(+) melanoma patients who did not receive the NY-ESO-1 ISCOMATRIX vaccine rarely generated a spontaneous T-cell response to this cryptic epitope, suggesting a lack of priming of such T cells in the natural anti-NY-ESO-1 response, which may be corrected by vaccination. Together, our results reveal several surprising aspects of antitumor immunity and have implications for cancer vaccine design.