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The hematopoietic stem cell (HSC) is probably the best characterized somatic stem cell and is still the only one regularly used in clinical practice. Nevertheless, expansion of HSCs in vitro has been surprisingly unsuccessful, limiting their full therapeutic potential. During homeostasis, the vast majority of HSCs are found in the bone marrow (BM) localized to specific microenvironments called stem cell "niches." Over the last few years our knowledge of cellular niche components and the signaling molecules that coordinate the crosstalk between HSCs and niche cells has dramatically increased. Here we review the two main niche types found in the BM: the endosteal and the vascular niches, and provide an overview of the different signaling and cell adhesion molecules that form the HSC-niche synapse. Signals from BM niches not only control HSC dormancy, but also regulate the balance between self-renewal and differentiation. In the future, successful expansion of HSCs for therapeutic use will require three-dimensional reconstruction of a stem cell-niche unit.

T-cell and NK-cell infiltration into solid tumors: a key limiting factor for efficacious cancer immunotherapy.

Cancer immunotherapy has great promise, but is limited by diverse mechanisms used by tumors to prevent sustained antitumor immune responses. Tumors disrupt antigen presentation, T/NK-cell activation, and T/NK-cell homing through soluble and cell-surface mediators, the vasculature, and immunosuppressive cells such as myeloid-derived suppressor cells and regulatory T cells. However, many molecular mechanisms preventing the efficacy of antitumor immunity have been identified and can be disrupted by combination immunotherapy. Here, we examine immunosuppressive mechanisms exploited by tumors and provide insights into the therapies under development to overcome them, focusing on lymphocyte traffic.

Toll-Interacting Protein Deficiency Leads to Increased Susceptibility to Acute and Chronic Colitis.

BACKGROUND: Sensing of bacterial products via Toll-like receptors is critical to maintain gut immune homeostasis. The Toll-Interacting Protein (Tollip) inhibits downstream signaling through the IL-1 receptor, TLR-2 and TLR-4. Here,we aimed to address the role of Tollip in acute and chronic inflammatory responses in the gut. MATERIAL AND METHODS: WT or Tollip-deficient mice were exposed to dextran sulfate sodium (DSS) 1.5% in the drinking water during 7 days. To generate bone-marrow chimeras, WT or Tollip deficient mice were 900-rads irradiated, transplanted with WT or Tollip deficient bone-marrow cells and challenged with DSS 2-3 months after transplantation. IL-10 deficient mice were bred with Tollip deficient mice and colitis was compared at various time points. RESULTS: Upon DSS exposure, Tollip-deficient mice had increased body weight loss and increased pro-inflammatory cytokine expression compared to WT controls. Challenge of bone-marrow chimeras showed that colitis susceptibility was also increased when Tollip deficiency was restricted to non-hematopoietic cells. DSS-exposure lead to a disorganized distribution of zona-occludens-1, a tight junction marker and increased number of apoptotic, cleaved caspase 3 positive, epithelial cells in Tollip-deficient compared to WT mice. Chronic colitis was also affected by Tollip deficiency as Tollip/IL-10 deficient mice had more severe histological stigmata of colitis and higher IL-17 expression than IL-10 deficient controls. CONCLUSION: Tollip in non-hematopoietic cells is critical for adequate response to a chemical-induced stress in the gut and to hamper chronic bacteria-driven colitis. Modulation of epithelial cell integrity via Tollip likely contributes to the observed defects.

Extrafollicular plasmablast B cells play a key role in carrying retroviral infection to peripheral organs.

B cells can either differentiate in germinal centers or in extrafollicular compartments of secondary lymphoid organs. Here we show the migration properties of B cells after differentiation in murine peripheral lymph node infected with mouse mammary tumor virus. Naive B cells become activated, infected, and carry integrated retroviral DNA sequences. After production of a retroviral superantigen, the infected B cells receive cognate T cell help and differentiate along the two main differentiation pathways analogous to classical Ag responses. The extrafollicular differentiation peaks on day 6 of mouse mammary tumor virus infection, and the follicular one becomes detectable after day 10. B cells participating in this immune response carry a retroviral DNA marker that can be detected by using semiquantitative PCR. We determined the migration patterns of B cells having taken part in the T cell-B cell interaction from the draining lymph node to different tissues. Waves of immigration and retention of infected cells in secondary lymphoid organs, mammary gland, salivary gland, skin, lung, and liver were observed correlating with the two peaks of B cell differentiation in the draining lymph node. Other organs revealed immigration of infected cells at later time points. The migration properties were correlated with a strong up-regulation of alpha(4)beta(1) integrin expression. These results show the migration properties of B cells during an immune response and demonstrate that a large proportion of extrafolliculary differentiating plasmablasts can escape local cell death and carry the retroviral infection to peripheral organs.

Development and selection of Valpha l4i NKT cells.

Valpha14 invariant natural killer T (Valpha14i NKT) cells are a unique lineage of mouse T cells that share properties with both NK cells and memory T cells. Valpha14i NKT cells recognize CDld-associated glycolipids via a semi-invariant T cell receptor (TCR) composed of an invariant Valpha14-Jalpha 18 chain paired preferentially with a restricted set of TCRbeta chains. During development in the thymus, rare CD4+ CD8+ (DP) cortical thymocytes that successfully rearrange the semi-invariant TCR are directed to the Valpha14i NKT cell lineage via interactions with CD d-associated endogenous glycolipids expressed by other DP thymocytes. As they mature, Valphal4i NKT lineage cells upregulate activation markers such as CD44 and subsequently express NK-related molecules such as NKI.1 and members of the Ly-49 inhibitory receptor family. The developmental program of Valpha l4i NKT cells is critically regulated by a number of signaling cues that have little or no effect on conventional T cell development, such as the Fyn/SAP/SLAM pathway, NFkappaB and T-bet transcription factors, and the cytokine IL-15. The unique developmental requirements of Valphal4i NKT cells may represent a paradigm for other unconventional T cell subsets that are positively selected by agonist ligands expressed on hematopoietic cells.

Innate immune responses to "Aspergillus fumigatus"

AbstractAspergillus fumigatus is a ubiquitous mould that can cause invasive aspergillosis, a potentially lethal infection in onco-hematological patients. With an incidence rate ranging from 5 to 15%, invasive aspergillosis (IA) is one of the most frequent infections in patients undergoing intensive myeloablative chemotherapy for acute leukaemia or allogenic hematopoietic stem cell transplantation (HSCT). Toll-like receptors (TLRs) are transmembrane proteins located in immune cells, such as macrophages sand dendritic cells, that detect molecular motifs from invading pathogens to initiate immune response mechanisms. Studies suggested a role for TLR2 and TLR4 in the detection of A. fumigatus. However, few data are available on the role of TLR1 and TLR6, both known as TLR2 co-receptors, in innate immune responses to this pathogen.In this study, we used an immunogenic mutant strain of A. fumigatus, together with a wild-type strain, to analyse the role of TLRs and their signalling pathways in the innate immune response to this mould. We show for the first time that this response involves both TLR1 and TLR6 in mouse and TLR1, but not TLR6, in human. We show that, despite the high sequence homology between TLR1 and TLR6, the specificity in the sensing of A. fumigatus relies on the human TLR1 and TLR6 ectodomains. Furthermore, we show that two human single nucleotide polymorphisms (SNPs) (G1805T [S6021] and G239C [R80T]) affect the response to this pathogen. Our work also confirms the role of TLR2 and TLR4 in the detection of A. fumigatus, together with their co-receptors CD 14 and MD2, in both mouse and human, and highlights the nature of the intracellular signaling pathway used by these receptors to mediate the immune response against this pathogen.This study provides a comprehensive analysis of the role of TLRs and their signalling pathways in the innate immune recognition of A. fumigatus and may have important consequences for diagnosis, management and treatment of IA in high risk patients.RésuméAspergillus fumigatus est un champignon saprophyte ubiquitaire qui peut causer l'aspergillose invasive (AI), une infection potentiellement mortelle chez les patients onco-hématologiques. Avec un taux d'incidence de 5 à 15%, l'AI est l'une des infections les plus fréquentes chez les patients subissant une chimiothérapie intensive pour une leucémie aiguë ou une allogreffe de cellules souches hématopoïétiques. Les récepteurs Toll-like (Toll-like receptors, TLRs) sont des protéines transmembranaires placés stratégiquement à la surface de certaines cellules immunitaires, comme les macrophages et les cellules dendritiques. Ces protéines sont capables de détecter des motifs moléculaires à la surface des pathogènes et de déclencher la réponse immunitaire innée. Des études ont suggéré l'implication de TLR2 et TLR4 dans la détection dΆ. fumigatus. Cependant, peu de données sont disponibles sur le rôle de TLR1 et TLR6, qui sont les co-récepteurs de TLR2, dans ce mécanisme de défense immunitaire.Dans cette étude, nous avons utilisé une souche particulièrement immunogénique d'A. fumigatus, ainsi qu'une souche sauvage, pour analyser l'implication des récepteurs TLRs dans la réponse immunitaire à ce champignon filamenteux. Nous montrons pour la première fois que cette détection implique TLR1 et TLR6 chez la souris, et TLR1, mais pas TLR6, chez l'homme. Nous montrons également que la spécificité de détection chez l'homme est due à des séquences spécifiques du domaine extra- membranaire de TLR1 et TLR6, et que des polymorphismes mono-nucléotidiques du récepteur (G1805T [S602I] and G239C [R80T]) influencent la réponse à ce pathogène. Nous confirmons également l'implication de TLR2 et TLR4, avec leurs co-récepteurs CD14 et MD2, dans la détection d'A. fumigatus, chez l'homme et la souris, et mettons en évidence les voies de signalisation cellulaires impliquées dans la réponse immunitaire à ce pathogène.Ces nouvelles connaissances sur le rôle des TLRs et de leurs voies de signalisation cellulaire dans la détection immunitaire innée d'A. fumigatus pourraient influencer le diagnostic, la prévention et le traitement de l'AI chez les patients à haut risque de développer cette infection.

Recognition of major histoincompatibilities after transplantation with marrow from HLA closely matched donors.

BACKGROUND: To determine the extent to which major histoincompatibilities are recognized after bone marrow transplantation, we characterized the specificity of the cytotoxic T lymphocytes isolated during graft-versus-host disease. We studied three patients transplanted with marrow from donors who were histoincompatible for different types of HLA antigens. METHODS: Patient 1 was mismatched for one "ABDR-antigen" (HLA-A2 versus A3). Two patients were mismatched for antigens that would usually not be taken into account by standard selection procedures: patient 2 was mismatched for an "HLA-A subtype" (A*0213 versus A*0201), whereas patient 3 was mismatched for HLA-C (HLA-C*0501 versus HLA-C*0701). All three HLA class I mismatches were detected by a pretransplant cytotoxic precursor test. RESULTS: Analysis of the specificity of the cytotoxic T lymphocyte clones isolated after transplantation showed that the incompatibilities detected by the pretransplant cytotoxic precursor assay were the targets recognized during graft-versus-host disease. CONCLUSIONS: Independent of whether the incompatibility consisted of a "full" mismatch, a "subtype" mismatch, or an HLA-C mismatch, all clones recognized the incompatible HLA molecule. In addition, some of these clones had undergone antigen selection and were clearly of higher specificity than the ones established before transplantation, indicating that they had been participating directly in the antihost immune response.

Early intensification and autologous stem cell transplantation in patients with systemic AL amyloidosis: a single-centre experience.

Primary systemic amyloidosis (AL amyloidosis) continues to have a very poor prognosis. Most therapeutic strategies remain unsatisfactory. Conventional chemotherapy is known to offer at best only moderate efficacy. Several studies have yielded higher complete response rates after high-dose chemotherapy and autologous stem cell transplantation (ASCT) in addition to improving outcomes in a subgroup of patients. However, the superiority of an intensive approach in AL amyloidosis has not been confirmed in a randomised trial. The precise role of ASCT remains unclear. We report our experience in 16 patients diagnosed with AL amyloidosis and treated in a multidisciplinary approach with high-dose melphalan and ASCT. Median age was 59 (39-71) years. The kidneys were predominantly affected in 75% of cases; two or more organs were affected in 38%. Median time from diagnosis to transplantation was 2 (1-4) months. Three patients (19%) developed acute renal failure and required transient dialysis. Transplant-related mortality was 6% after 100 days. Haematological complete response (CR) was obtained in nine (56%) and organ response in six (38%) patients. Nine out of 12 patients (75%) with kidney involvement exhibited a sustained clinical benefit at 12 months. Half of all the patients (n = 8) were alive after a median follow-up of 33 months, including two in continuous CR. This suggests that high-dose chemotherapy and ASCT are still valid treatment options in AL amyloidosis and that a significant number of patients with renal involvement might benefit from this approach.

A novel approach to characterize clonality and differentiation of human melanoma-specific T cell responses: spontaneous priming and efficient boosting by vaccination.

Despite major progress in T lymphocyte analysis in melanoma patients, TCR repertoire selection and kinetics in response to tumor Ags remain largely unexplored. In this study, using a novel ex vivo molecular-based approach at the single-cell level, we identified a single, naturally primed T cell clone that dominated the human CD8(+) T cell response to the Melan-A/MART-1 Ag. The dominant clone expressed a high-avidity TCR to cognate tumor Ag, efficiently killed tumor cells, and prevailed in the differentiated effector-memory T lymphocyte compartment. TCR sequencing also revealed that this particular clone arose at least 1 year before vaccination, displayed long-term persistence, and efficient homing to metastases. Remarkably, during concomitant vaccination over 3.5 years, the frequency of the pre-existing clone progressively increased, reaching up to 2.5% of the circulating CD8 pool while its effector functions were enhanced. In parallel, the disease stabilized, but subsequently progressed with loss of Melan-A expression by melanoma cells. Collectively, combined ex vivo analysis of T cell differentiation and clonality revealed for the first time a strong expansion of a tumor Ag-specific human T cell clone, comparable to protective virus-specific T cells. The observed successful boosting by peptide vaccination support further development of immunotherapy by including strategies to overcome immune escape.

TWEAK, via its receptor Fn14, is a novel regulator of mesenchymal progenitor cells and skeletal muscle regeneration.

Inflammation participates in tissue repair through multiple mechanisms including directly regulating the cell fate of resident progenitor cells critical for successful regeneration. Upon surveying target cell types of the TNF ligand TWEAK, we observed that TWEAK binds to all progenitor cells of the mesenchymal lineage and induces NF-kappaB activation and the expression of pro-survival, pro-proliferative and homing receptor genes in the mesenchymal stem cells, suggesting that this pro-inflammatory cytokine may play an important role in controlling progenitor cell biology. We explored this potential using both the established C2C12 cell line and primary mouse muscle myoblasts, and demonstrated that TWEAK promoted their proliferation and inhibited their terminal differentiation. By generating mice deficient in the TWEAK receptor Fn14, we further showed that Fn14-deficient primary myoblasts displayed significantly reduced proliferative capacity and altered myotube formation. Following cardiotoxin injection, a known trigger for satellite cell-driven skeletal muscle regeneration, Fn14-deficient mice exhibited reduced inflammatory response and delayed muscle fiber regeneration compared with wild-type mice. These results indicate that the TWEAK/Fn14 pathway is a novel regulator of skeletal muscle precursor cells and illustrate an important mechanism by which inflammatory cytokines influence tissue regeneration and repair. Coupled with our recent demonstration that TWEAK potentiates liver progenitor cell proliferation, the expression of Fn14 on all mesenchymal lineage progenitor cells supports a broad involvement of this pathway in other tissue injury and disease settings.

In situ RHAMM protein expression in acute myeloid leukemia blasts suggests poor overall survival.

Treatment options for patients with high-risk acute myeloid leukemia (AML) include high-dose chemotherapy regimens in combination with allogeneic hematopoietic stem cell transplantation, which takes advantage of the donor T-cell-mediated graft-versus-leukemia effect. Together with beneficial responses observed in assays targeted at leukemia-associated antigens (LAA), this encouraged research on cancer vaccines and adoptive cellular therapies in AML. The receptor for hyaluronic acid-mediated motility (RHAMM, CD168) was identified as one of the most promising LAA in AML. Thus far, little is known about in situ expression in leukemic bone marrow blasts or the prognostic role of RHAMM and its interaction partners in AML. We immunohistochemically analyzed the expression and prognostic significance of RHAMM on trephine bone marrow biopsies from 71 AML cases that had been evaluated for cytogenetics and presence of FLT3-internal tandem duplications and NPM1 mutations. Fifty-five patients (77%) were treated with curative intent, while 16 (23%) received the most appropriate supportive care. Twenty of 71 (28%) AML cases were considered RHAMM+. Receiver operating characteristic curves showed significant discriminatory power considering overall survival (OS) in AML patients treated curatively for RHAMM (p = 0.015). Multivariable analysis revealed that expression of RHAMM in >5% of leukemic blasts identifies a subgroup of curatively treated cases with adverse OS independent of failures to achieve complete remission. RHAMM not only represents a promising LAA with specific T-cell responses in AML but, if assessed in situ on blasts, also a probable prognostic factor.

Notch signaling in the immune system.

The Notch signaling pathway regulates many aspects of embryonic development, as well as differentiation processes and tissue homeostasis in multiple adult organ systems. Disregulation of Notch signaling is associated with several human disorders, including cancer. In the last decade, it became evident that Notch signaling plays important roles within the hematopoietic and immune systems. Notch plays an essential role in the development of embryonic hematopoietic stem cells and influences multiple lineage decisions of developing lymphoid and myeloid cells. Moreover, recent evidence suggests that Notch is an important modulator of T cell-mediated immune responses. In this review, we discuss Notch signaling in hematopoiesis, lymphocyte development, and function as well as in T cell acute lymphoblastic leukemia.

Notch 1-deficient common lymphoid precursors adopt a B cell fate in the thymus.

We have recently reported that Notch 1, a member of the Notch multigene family, is essential for the development of murine T cells. Using a mouse model in which Notch 1 is inactivated in bone marrow (BM) precursors we have shown that B cells instead of T cells are found in the thymus of BM chimeras. However, it is not clear whether these B cells develop by default from a common lymphoid precursor due to the absence of Notch 1 signaling, or whether they arise as a result of perturbed migration of BM-derived B cells and/or altered homeostasis of normal resident thymic B cells. In this report we show that Notch 1-deficient thymic B cells resemble BM B cells in phenotype and turnover kinetics and are located predominantly in the medulla and corticomedullary junction. Peripheral blood lymphocyte analysis shows no evidence of recirculating Notch1(-/)- BM B cells. Furthermore, lack of T cell development is not due to a failure of Notch1(-/)- precursors to home to the thymus, as even after intrathymic reconstitution with BM cells, B cells instead of T cells develop from Notch 1-deficient precursors. Taken together, these results provide evidence for de novo ectopic B cell development in the thymus, and support the hypothesis that in the absence of Notch 1 common lymphoid precursors adopt the default cell fate and develop into B cells instead.

Phosphorylation of CARMA1 by HPK1 is critical for NF-kappaB activation in T cells.

Activation of the NF-kappaB pathway in T cells is required for induction of an adaptive immune response. Hematopoietic progenitor kinase (HPK1) is an important proximal mediator of T-cell receptor (TCR)-induced NF-kappaB activation. Knock-down of HPK1 abrogates TCR-induced IKKbeta and NF-kappaB activation, whereas active HPK1 leads to increased IKKbeta activity in T cells. Yet, the precise molecular mechanism of this process remains elusive. Here, we show that HPK1-mediated NF-kappaB activation is dependent on the adaptor protein CARMA1. HPK1 interacts with CARMA1 in a TCR stimulation-dependent manner and phosphorylates the linker region of CARMA1. Interestingly, the putative HPK1 phosphorylation sites in CARMA1 are different from known PKC consensus sites. Mutations of residues S549, S551, and S552 in CARMA1 abrogated phosphorylation of a CARMA1-linker construct by HPK1 in vitro. In addition, CARMA1 S551A or S5549A/S551A point mutants failed to restore HPK1-mediated and TCR-mediated NF-kappaB activation and IL-2 expression in CARMA1-deficient T cells. Thus, we identify HPK1 as a kinase specific for CARMA1 and suggest HPK1-mediated phosphorylation of CARMA1 as an additional regulatory mechanism tuning the NF-kappaB response upon TCR stimulation.

Mobilization of hemopoietic stem cells with high-dose methotrexate plus granulocyte-colony-stimulating factor in patients with primary central nervous system lymphoma.

BACKGROUND: High-dose therapy with autologous stem cell support after standard dose induction is a promising approach for therapy of primary central nervous system lymphoma (PCNSL). High-dose methotrexate (HD-MTX) is a standard drug for induction of PCNSL; however, data about the capacity of HD-MTX plus granulocyte-colony-stimulating factor (G-CSF) to mobilize hemopoietic progenitors are lacking. STUDY DESIGN AND METHODS: This investigation describes the data from stem cell mobilization and apheresis procedures after one or two cycles of HD-MTX for induction of PCNSL within the East German Study Group for Haematology and Oncology 053 trial. Eligible patients proceeded to high-dose busulfan/thiotepa after induction therapy and mobilization. RESULTS: Data were available from nine patients with a median age of 58 years. The maximal CD34+ cell count per microL of blood after the first course of HD-MTX was 13.89 (median). Determination was repeated in six patients after the second course with a significantly higher median CD34+ cell count of 33.69 per microL. Five patients required two apheresis procedures and in four patients a single procedure was sufficient. The total yield of CD34+ cells per kg of body weight harvested by one or two leukapheresis procedures was 6.60 x 10(6) (median; range, 2.68 x 10(6)-15.80 x 10(6)). The yield of CD34+ cells exceeded the commonly accepted lower threshold of 3 x 10(6) cells per kg of body weight in eight of nine cases. Even in the ninth, hemopoietic recovery after stem cell reinfusion was rapid and safe. CONCLUSION: HD-MTX plus G-CSF is a powerful combination for stem cell mobilization in patients with PCNSL and permits safe conduction of time-condensed and dose-intense protocols with high-dose therapy followed by stem cell reinfusion after HD-MTX induction.