The role of Ly49E receptor expression on murine intraepithelial lymphocytes in intestinal cancer development and progression.

Ly49E is a member of the Ly49 family of NK receptors and is distinct from other members of this family on the basis of its structural properties, expression pattern and ligand recognition. Importantly, Ly49E receptor expression is high on small intestinal and colonic intraepithelial lymphocytes (IELs). Intestinal IELs are regulators of the mucosal immune system and contribute to front-line defense at the mucosal barrier, including anti-tumor immune response. Whereas most Ly49 receptors have MHC class-I ligands, we showed that Ly49E is instead triggered by urokinase plasminogen activator (uPA). uPA has been extensively implicated in tumor development, where increased uPA expression correlates with poor prognosis. As such, we investigated the role of Ly49E receptor expression on intestinal IELs in the anti-tumor immune response. For this purpose, we compared Ly49E wild-type mice to Ly49E knockout mice in two established tumor models: ApcMin/+-mediated and azoxymethane-induced intestinal cancer. Our results indicate that Ly49E expression on IELs does not influence the development or progression of intestinal cancer.

Notch3 activation is sufficient but not required for inducing human T-lineage specification.

Although the role for the individual Notch receptors in early hematopoiesis have been thoroughly investigated in mouse, studies in human have been mostly limited to the use of pan-Notch inhibitors. However, such studies in human are important to predict potential side effects of specific Notch receptor blocking reagents because these are currently being considered as therapeutic tools to treat various Notch-dependent diseases. In this study, we studied the individual roles of Notch1 and Notch3 in early human hematopoietic lineage decisions, particularly during T-lineage specification. Although this process in mice is solely dependent on Notch1 activation, we recently reported Notch3 expression in human uncommitted thymocytes, raising the possibility that Notch3 mediates human T-lineage specification. Although expression of a constitutive activated form of Notch3 (ICN3) results in the induction of T-lineage specification in human CD34(+) hematopoietic progenitor cells, similar to ICN1 overexpression, loss-of-function studies using blocking Abs reveal that only Notch1, but not Notch3, is critical in this process. Blocking of Notch1 activation in OP9-DLL4 cocultures resulted in a complete block in T-lineage specification and induced monocytic and plasmacytoid dendritic cell differentiation instead. In fetal thymus organ cultures, impeded Notch1 activation resulted in B and dendritic cell development. In contrast, Notch3 blocking Abs only marginally affected T-lineage specification and hematopoietic differentiation with a slight increase in monocyte development. No induction of B or dendritic cell development was observed. Thus, our results unambiguously reveal a nonredundant role for Notch1 in human T-lineage specification, despite the expression of other Notch receptors.

Differential Ly49e expression pathways in resting versus TCR-activated intraepithelial γδ T cells.

The Ly49 NK receptor family in mice is composed of several members that recognize MHC class I (MHC-I) or MHC-I-related molecules. We and others have shown before that Ly49E is a unique member, with a different expression pattern on NK cells and being triggered by the non-MHC-I-related protein urokinase plasminogen activator. Among the entire Ly49 receptor family, Ly49E is the only Ly49 member expressed by epidermal-localized γδ T cells and their fetal thymic TCRγδ precursors, and it is the most abundantly expressed member on intestinal intraepithelial γδ T cell lymphocytes. In this study, we provide mechanistic insights into the regulation of Ly49e expression in γδ T cells. First, we demonstrate that TCR-mediated activation of intraepithelial γδ T cells significantly increases Ly49E expression. This results from de novo Ly49E expression and is highly selective, because no other Ly49 family members are induced. TCR-mediated Ly49E induction is a conserved feature of skin- and gut-residing intraepithelial-localized γδ T cell subsets, whereas it is not observed in spleen γδ T cells. By investigating Ly49e promoter activities and lymphotoxin (LT) αß dependency in resting versus TCR-activated intraepithelial γδ T cells, we reveal two separate regulatory pathways for Ly49E expression, as follows: a LTαß-dependent pathway leading to basal Ly49E expression in resting cells that is induced by Pro2-mediated Ly49e transcription, and a LTαß-independent pathway leading to elevated, Pro3-driven Ly49E expression in TCR-stimulated cells.

Inhibitory receptors specific for MHC class I educate murine NK cells but not CD8αα intestinal intraepithelial T lymphocytes.

The engagement of inhibitory receptors specific for major histocompatibility complex class I (MHC-I) molecules educates natural killer (NK) cells, meaning the improvement of the response of activation receptors to subsequent stimulation. It is not known whether inhibitory MHC-I receptors educate only NK cells or whether they improve the responsiveness of all cell types, which express them. To address this issue, we analyzed the expression of inhibitory MHC-I receptors on intestinal intraepithelial lymphocytes (iIELs) and show that T-cell receptor (TCR)-αß CD8αα iIELs express multiple inhibitory receptors specific for MHC-I molecules, including CD94/NKG2A, Ly49A, and Ly49G2. However, the presence of MHC-I ligand for these receptors did not improve the response of iIELs to activation via the TCR. The absence of iIEL education by MHC-I receptors was not related to a lack of inhibitory function of these receptors in iIELs and a failure of these receptors to couple to the TCR. Thus, unlike NK cells, iIELs do not undergo an MHC-I-guided education process. These data suggest that education is an NK cell-specific function of inhibitory MHC-I receptors.

Jagged2 acts as a Delta-like Notch ligand during early hematopoietic cell fate decisions.

Notch signaling critically mediates various hematopoietic lineage decisions and is induced in mammals by Notch ligands that are classified into 2 families, Delta-like (Delta-like-1, -3 and -4) and Jagged (Jagged1 and Jagged2), based on structural homology with both Drosophila ligands Delta and Serrate, respectively. Because the functional differences between mammalian Notch ligands were still unclear, we have investigated their influence on early human hematopoiesis and show that Jagged2 affects hematopoietic lineage decisions very similarly as Delta-like-1 and -4, but very different from Jagged1. OP9 coculture experiments revealed that Jagged2, like Delta-like ligands, induces T-lineage differentiation and inhibits B-cell and myeloid development. However, dose-dependent Notch activation studies, gene expression analysis, and promoter activation assays indicated that Jagged2 is a weaker Notch1-activator compared with the Delta-like ligands, revealing a Notch1 specific signal strength hierarchy for mammalian Notch ligands. Strikingly, Lunatic-Fringe- mediated glycosylation of Notch1 potentiated Notch signaling through Delta-like ligands and also Jagged2, in contrast to Jagged1. Thus, our results reveal a unique role for Jagged1 in preventing the induction of T-lineage differentiation in hematopoietic stem cells and show an unexpected functional similarity between Jagged2 and the Delta-like ligands.

Presence of CXCR4-using HIV-1 in patients with recently diagnosed infection: correlates and evidence for transmission.

BACKGROUND: The prevalence and correlates of CXCR4-use in recently diagnosed patients and the impact of X4/DM transmission remain largely unknown. METHOD: Genotypic coreceptor use determination on the baseline sample of 539 recently diagnosed individuals. Correlation of coreceptor use with clinical, viral and epidemiological data and with information on transmission events as obtained through phylogenetic analysis of protease and reverse transcriptase sequences. Results. CXCR4-use was predicted in 12 to 19% of the patients, depending on the interpretative cutoff used. CXCR4-use was correlated with lower CD4(+) T cell counts and subtype 01_AE infection. No association with viral load was observed. Seven (11%) of 63 transmission clusters and 4 (31%) of 13 donor-source pairs resulted from X4/DM transmission. CONCLUSION: The results confirmed the relation between CXCR4-use at diagnosis and low baseline CD4+ T cell counts. Significantly more CXCR4-use was predicted in 01_AE infections, which may impose constraints on the use of CCR5 antagonists in certain regions of the world. Observations from the transmission cluster analysis contradict the hypothesis that R5 viruses are selected at transmission, and support the idea that R5 or X4/DM infections result from a stochastic process.

RHAMM/HMMR (CD168) is not an ideal target antigen for immunotherapy of acute myeloid leukemia.

BACKGROUND: Criteria for good candidate antigens for immunotherapy of acute myeloid leukemia are high expression on leukemic stem cells in the majority of patients with acute myeloid leukemia and low or no expression in vital tissues. It was shown in vaccination trials that Receptor for Hyaluronic Acid Mediated Motility (RHAMM/HMMR) generates cellular immune responses in patients with acute myeloid leukemia and that these responses correlate with clinical benefit. It is not clear however whether this response actually targets the leukemic stem cell, especially since it was reported that RHAMM is expressed maximally during the G2/M phase of the cell cycle. In addition, tumor specificity of RHAMM expression remains relatively unexplored. DESIGN AND METHODS: Blood, leukapheresis and bone marrow samples were collected from both acute myeloid leukemia patients and healthy controls. RHAMM expression was assessed at protein and mRNA levels on various sorted populations, either fresh or after manipulation. RESULTS: High levels of RHAMM were expressed by CD34(+)CD38(+) and CD34(-) acute myeloid leukemia blasts. However, only baseline expression of RHAMM was measured in CD34(+)CD38(-) leukemic stem cells, and was not different from that in CD34(+)CD38(-) hematopoietic stem cells from healthy controls. RHAMM was significantly up-regulated in CD34(+) cells from healthy donors during in vitro expansion and during in vivo engraftment. Finally, we demonstrated an explicit increase in the expression level of RHAMM after in vitro activation of T cells. CONCLUSIONS: RHAMM does not fulfill the criteria of an ideal target antigen for immunotherapy of acute myeloid leukemia. RHAMM expression in leukemic stem cells does not differ significantly from the expression in hematopoietic stem cells from healthy controls. RHAMM expression in proliferating CD34+ cells of healthy donors and activated T cells further compromises RHAMM-specific T-cell-mediated immunotherapy.

Continuous CD27 triggering in vivo strongly reduces NK cell numbers.

NK cells are important mediators of the early defense. In mice, immature and mature NK (mNK) cells constitutively express the TNF receptor family member CD27; however, mNK cells eventually lose CD27 expression and become resting NK cells. Interaction of CD27 with its ligand, CD70, enhances proliferation and effector functions of NK cells. We used mice that constitutively express CD70 on B cells (CD70-Tg) to study the in vivo effects of continuous triggering of CD27 on NK cells. Continuous CD70-CD27 interaction resulted in strongly down-modulated CD27 expression on NK cells and gradually reduced absolute NK cell numbers. This reduction was most prominent in the mNK cell subpopulation and was at least partially due to increased apoptosis. Residual NK cells showed lower expression of activating Ly49 receptors and normal (liver) or decreased (spleen) IFN-gamma production. Nevertheless, NK cells from CD70-Tg mice displayed higher YAC-1 killing capacities. CD70-Tg NK cells exhibited up-regulated expression of NKG2D, which is in accordance with the increased YAC-1 lysis, as this is mainly NKG2D-dependent. Taken together, this study is the first to demonstrate that continuous CD70 triggering of CD27 on NK cells in vivo results in a severe reduction of NK cells. On a single cell basis, however, residual NK cells display enhanced cytotoxicity.

CD27-deficient mice show normal NK-cell differentiation but impaired function upon stimulation.

Natural killer (NK) cells are part of the first line defense against tumors, parasites and virus-infected cells. Therefore, factors that control NK-cell numbers and their function are important. CD27 is constitutively expressed on NK cells and its expression correlates with sequential phases in NK-cell development, discriminating phenotypically and functionally different subsets within the NK-cell population. Although CD27 has been described to have an important regulatory role in effector and memory T and B lymphocytes, its role in NK-cell biology remains to be addressed. In this study, we used CD27(-/-) mice to investigate the role of CD27 in NK-cell development and function, both during the resting state and upon stimulation. The results show that NK-cell numbers are not impaired in CD27(-/-) mice. Moreover, CD27(-/-) NK cells reach full phenotypic maturity, evidenced by normal expression of CD49b, CD43 and CD11b. Expression of activating receptors is unaltered, whereas expression of several inhibitory receptors is increased. Cytotoxicity and interferon-γ production by NK cells from CD27(-/-) mice in the resting state are normal. However, upon in vivo anti-CD40- or poly-I:C-mediated activation, or in vitro interleukin-15 priming plus anti-NKp46 stimulation, the absence of CD27 results in decreased cytolytic activity and cytokine production by spleen and liver NK cells. In conclusion, this study demonstrates that CD27 is dispensable for the development of functional NK cells. However, upon stimulation of NK cells, CD27 displays an important role in their activation and functionality.

Comparison of phenotypic and genotypic tropism determination in triple-class-experienced HIV patients eligible for maraviroc treatment.

BACKGROUND: Determination of HIV-1 tropism is a pre-requisite to the use of CCR5 antagonists. This study evaluated the potential of population genotypic tropism tests (GTTs) in clinical practice, and the correlation with phenotypic tropism tests (PTTs) in patients accessing routine HIV care. METHODS: Forty-nine consecutive plasma samples for which an original Trofile(TM) assay was performed were obtained from triple-class-experienced patients in need of a therapy change. Viral tropism was defined as the consensus of three or more tropism calls obtained from the combination of two independent population PTT assays (Trofile Biosciences, San Francisco, CA, USA, and Virco, Beerse, Belgium), population GTTs and GTTs based on ultra-deep sequencing. If no consensus was reached, a clonal PTT was performed in order to finalize the tropism call. This two-step approach allowed the definition of a reference tropism call. RESULTS: According to the reference tropism result, 35/49 samples were CCR5 tropic (R5) (patients eligible for maraviroc treatment) and 14/49 were assigned as non-R5 tropic. The non-R5 samples [patients not eligible for maraviroc treatment according to the FDA/European Medicines Agency (EMEA) label] group included both the CXCR4 (X4) samples and the dual and mixed CCR5/CXCR4 (R5/X4) samples. Compared with Trofile(TM) population PTTs, population GTTs showed a higher sensitivity (97%) and a higher negative predictive value (91%), but almost equal specificity and an equal positive predictive value. CONCLUSIONS: In line with recent reports from clinical trial data, our data support the use of population genotypic tropism testing as a tool for tropism determination before the start of maraviroc.

Notch signaling is required for proliferation but not for differentiation at a well-defined beta-selection checkpoint during human T-cell development.

Notch signaling is absolutely required for beta-selection during mouse T-cell development, both for differentiation and proliferation. In this report, we investigated whether Notch has an equally important role during human T-cell development. We show that human CD34(+) thymocytes can differentiate into CD4(+)CD8beta(+) double positive (DP) thymocytes in the absence of Notch signaling. While these DP cells phenotypically resemble human beta-selected cells, they lack a T-cell receptor (TCR)-beta chain. Therefore, we characterized the beta-selection checkpoint in human T-cell development, using CD28 as a differential marker at the immature single positive CD4(+)CD3(-)CD8alpha(-) stage. Through intracellular TCR-beta staining and gene expression analysis, we show that CD4(+)CD3(-)CD8alpha(-)CD28(+) thymocytes have passed the beta-selection checkpoint, in contrast to CD4(+)CD3(-)CD8alpha(-)CD28(-) cells. These CD4(+)CD3(-)CD8alpha(-)CD28(+) thymocytes can efficiently differentiate into CD3(+)TCRalphabeta(+) human T cells in the absence of Notch signaling. Importantly, preselection CD4(+)CD3(-)CD8alpha(-)CD28(-) thymocytes can also differentiate into CD3(+)TCRalphabeta(+) human T cells without Notch activation when provided with a rearranged TCR-beta chain. Proliferation of human thymocytes, however, is clearly Notch-dependent. Thus, we have characterized the beta-selection checkpoint during human T-cell development and show that human thymocytes require Notch signaling for proliferation but not for differentiation at this stage of development.

An early decrease in Notch activation is required for human TCR-alphabeta lineage differentiation at the expense of TCR-gammadelta T cells.

Although well characterized in the mouse, the role of Notch signaling in the human T-cell receptor alphabeta (TCR-alphabeta) versus TCR-gammadelta lineage decision is still unclear. Although it is clear in the mouse that TCR-gammadelta development is less Notch dependent compared with TCR-alphabeta differentiation, retroviral overexpression studies in human have suggested an opposing role for Notch during human T-cell development. Using the OP9-coculture system, we demonstrate that changes in Notch activation are differentially required during human T-cell development. High Notch activation promotes the generation of T-lineage precursors and gammadelta T cells but inhibits differentiation toward the alphabeta lineage. Reducing the amount of Notch activation rescues alphabeta-lineage differentiation, also at the single-cell level. Gene expression analysis suggests that this is mediated by differential sensitivities of Notch target genes in response to changes in Notch activation. High Notch activity increases DTX1, NRARP, and RUNX3 expression, genes that are down-regulated during alphabeta-lineage differentiation. Furthermore, increased interleukin-7 levels cannot compensate for the Notch dependent TCR-gammadelta development. Our results reveal stage-dependent molecular changes in Notch signaling that are critical for normal human T-cell development and reveal fundamental molecular differences between mouse and human.

T-lymphoid differentiation potential measured in vitro is higher in CD34+CD38-/lo hematopoietic stem cells from umbilical cord blood than from bone marrow and is an intrinsic property of the cells.

BACKGROUND: Human bone marrow and umbilical cord blood are sources of allogeneic hematopoietic stem cells for transplantation, which is a life-saving treatment in a variety of diseases but is burdened by delayed T-cell reconstitution. Observational studies evaluating T-cell reconstitution in post-transplant recipients suggest that cord blood hematopoietic stem cells have a more effective capacity for T-cell reconstitution. This study focuses on the comparison of the capacity of cord blood and bone marrow hematopoietic stem cells to generate T cells in vitro. DESIGN AND METHODS: Hematopoietic stem cells were cultured in OP9-delta-like-1 and OP9-green fluorescent protein co-cultures to estimate T and myeloid generation capacity, respectively. Phenotypic markers of T-lineage or myeloid differentiation were measured by flow cytometry and used to analyze their kinetics as a function of culture time. Hematopoietic stem cells were labeled with carboxyfluorescein diacetate succinamidyl ester and analyzed after culture to track their phenotypic progression in consecutive generations. Mixed OP9-delta-like-1 co-cultures were done with either carboxyfluorescein diacetate succinamidyl ester-labeled bone marrow and unlabeled cord blood hematopoietic stem cells, or vice versa, to evaluate their mutual influence on T-lineage differentiation. The T-cell potential of hematopoietic stem cells was addressed quantitatively by limiting dilution analysis. RESULTS: Bulk cultures showed faster and more extensive T-cell differentiation by cord blood hematopoietic stem cells. Furthermore, the T-lymphoid differentiation capacity of cord blood and bone marrow hematopoietic stem cells can be discriminated very early based on the coordinated expression of CD34 and CD7. Mixing experiments with cord blood hematopoietic stem cells and bone marrow hematopoietic stem cells showed that these differences are cell intrinsic. Quantitative clonal analyses demonstrated that CD34(+)CD38(-/lo) hematopoietic stem cells from cord blood contained a two-fold higher T-lineage generation capacity than CD34(+)CD38(-/lo) bone marrow hematopoietic stem cells, whereas the myeloid differentiation was similar. CONCLUSIONS: Our data shows that cord blood hematopoietic stem cells have higher T-lymphoid differentiation potential than bone marrow hematopoietic stem cells and that this property is cell autonomous.

Generation of T cells from human embryonic stem cell-derived hematopoietic zones.

Human embryonic stem cells (hESC) are pluripotent stem cells. A major challenge in the field of hESC is the establishment of specific differentiation protocols that drives hESC down a particular lineage fate. So far, attempts to generate T cells from hESC in vitro were unsuccessful. In this study, we show that T cells can be generated in vitro from hESC-derived hematopoietic precursor cells present in hematopoietic zones (HZs). These zones are morphologically similar to blood islands during embryonic development, and are formed when hESC are cultured on OP9 stromal cells. Upon subsequent transfer of these HZs on OP9 cells expressing high levels of Delta-like 1 and in the presence of growth factors, cells expand and differentiate to T cells. Furthermore, we show that T cells derive exclusively from a CD34(high)CD43(low) population, further substantiating the notion that hESC-derived CD34(high)CD43(low) cells are formed in HZs and are the only population containing multipotent hematopoietic precursor cells. Differentiation to T cells sequentially passes through the physiological intermediates: CD34(+)CD7(+) T/NK committed, CD7(+)CD4(+)CD8(-) immature single positive, CD4(+)CD8(+) double positive, and finally CD3(+)CD1(-)CD27(+) mature T cell stages. TCRalphabeta(+) and TCRgammadelta(+) T cells are generated. Mature T cells are polyclonal, proliferate, and secrete cytokines in response to mitogens. This protocol for the de novo generation of T cells from hESC could be clinically and scientifically relevant.

Functionally mature CD4 and CD8 TCRalphabeta cells are generated in OP9-DL1 cultures from human CD34+ hematopoietic cells.

Human CD34(+) hematopoietic precursor cells cultured on delta-like ligand 1 expressing OP9 (OP9-DL1) stromal cells differentiate to T lineage cells. The nature of the T cells generated in these cultures has not been studied in detail. Since these cultures do not contain thymic epithelial cells which are the main cell type mediating positive selection in vivo, generation of conventional helper CD4(+) and cytotoxic CD8(+) TCRalphabeta cells is not expected. Phenotypically mature CD27(+)CD1(-) TCRgammadelta as well as TCRalphabeta cells were generated in OP9-DL1 cultures. CD8 and few mature CD4 single-positive TCRalphabeta cells were observed. Mature CD8 single-positive cells consisted of two subpopulations: one expressing mainly CD8alphabeta and one expressing CD8alphaalpha dimers. TCRalphabeta CD8alphaalpha and TCRgammadelta cells both expressed the IL2Rbeta receptor constitutively and proliferated on IL-15, a characteristic of unconventional T cells. CD8alphabeta(+) and CD4(+) TCRalphabeta cells were unresponsive to IL-15, but could be expanded upon TCR stimulation as mature CD8alphabeta(+) and CD4(+) T cells. These T cells had the characteristics of conventional T cells: CD4(+) cells expressed ThPOK, CD40L, and high levels of IL-2 and IL-4; CD8(+) cells expressed Eomes, Runx3, and high levels of granzyme, perforin, and IFN-gamma. Induction of murine or human MHC class I expression on OP9-DL1 cells had no influence on the differentiation of mature CD8(+) cells. Similarly, the presence of dendritic cells was not required for the generation of mature CD4(+) or CD8(+) T cells. These data suggest that positive selection of these cells is induced by interaction between T precursor cells.

Thymic selection generates a large T cell pool recognizing a self-peptide in humans.

The low frequency of self-peptide-specific T cells in the human preimmune repertoire has so far precluded their direct evaluation. Here, we report an unexpected high frequency of T cells specific for the self-antigen Melan-A/MART-1 in CD8 single-positive thymocytes from human histocompatibility leukocyte antigen-A2 healthy individuals, which is maintained in the peripheral blood of newborns and adults. Postthymic replicative history of Melan-A/MART-1-specific CD8 T cells was independently assessed by quantifying T cell receptor excision circles and telomere length ex vivo. We provide direct evidence that the large T cell pool specific for the self-antigen Melan-A/MART-1 is mostly generated by thymic output of a high number of precursors. This represents the only known naive self-peptide-specific T cell repertoire directly accessible in humans.

Ly49E-dependent inhibition of natural killer cells by urokinase plasminogen activator.

The Ly49 natural killer (NK)-cell receptor family comprises both activating and inhibitory members, which recognize major histocompatibility complex (MHC) class I or MHC class I-related molecules and are involved in target recognition. As previously shown, the Ly49E receptor fails to bind to a variety of soluble or cell-bound MHC class I molecules, indicating that its ligand is not an MHC class I molecule. Using BWZ.36 reporter cells, we demonstrate triggering of Ly49E by the completely distinct, non-MHC-related protein urokinase plasminogen activator (uPA). uPA is known to be secreted by a variety of cells, including epithelial and hematopoietic cells, and levels are up-regulated during tissue remodeling, infections, and tumorigenesis. Here we show that addition of uPA to Ly49E-positive adult and fetal NK cells inhibits interferon-gamma secretion and reduces their cytotoxic potential, respectively. These uPA-mediated effects are Ly49E-dependent, as they are reversed by addition of anti-Ly49E monoclonal antibody and by down-regulation of Ly49E expression using RNA interference. Our results suggest that uPA, besides its established role in fibrinolysis, tissue remodeling, and tumor metastasis, could be involved in NK cell-mediated immune surveillance and tumor escape.

Rho GTPase Cdc42 is essential for human T-cell development.

BACKGROUND: Rho GTPases are involved in the regulation of many cell functions, including some related to the actin cytoskeleton. Different Rho GTPases have been shown to be important for T-cell development in mice. However, their role in human T-cell development has not yet been explored. DESIGN AND METHODS: We examined the expression and activation of Rho GTPases along different stages of T-cell development in the human thymus. Early stage human thymocytes were transduced with constitutively active and dominant negative mutants of different Rho GTPases to explore their role in human T-cell development, as analyzed in fetal thymus organ cultures. The use of these mutants as well as Rho GTPase-specific inhibitors allowed us to explore the role of GTPases in thymocyte migration. RESULTS: We found that the expression of several Rho GTPases is differently regulated during successive stages of T-cell development in man, suggesting a specific role in human thymopoiesis. In chimeric fetal thymus organ culture, T-cell development was not or only mildly affected by expression of dominant negative Rac1 and Rac2, but was severely impaired in the presence of dominant negative Cdc42, associated with enhanced apoptosis and reduced proliferation. Kinetic analysis revealed that Cdc42 is necessary in human T-cell development both before and after expression of the pre-T-cell receptor. Using inhibitors and retrovirally transferred mutants of the aforementioned Rho GTPases, we showed that only Rac1 is necessary for migration of different thymocyte subsets, including the early CD34(+) fraction, towards stromal cell-derived factor-1 alpha. Constitutively active mutants of Rac1, Rac2 and Cdc42 all impaired migration towards stromal cell-derived factor-1 alpha and T-cell development to different degrees. CONCLUSIONS: This is the first report on Rho GTPases in human T-cell development, showing the essential role of Cdc42. Our data suggest that enhanced apoptotic death and reduced proliferation rather than disturbed migration explains the decreased thymopoiesis induced by dominant negative Cdc42.

Epidemiological study of phylogenetic transmission clusters in a local HIV-1 epidemic reveals distinct differences between subtype B and non-B infections.

BACKGROUND: The number of HIV-1 infected individuals in the Western world continues to rise. More in-depth understanding of regional HIV-1 epidemics is necessary for the optimal design and adequate use of future prevention strategies. The use of a combination of phylogenetic analysis of HIV sequences, with data on patients' demographics, infection route, clinical information and laboratory results, will allow a better characterization of individuals responsible for local transmission. METHODS: Baseline HIV-1 pol sequences, obtained through routine drug-resistance testing, from 506 patients, newly diagnosed between 2001 and 2009, were used to construct phylogenetic trees and identify transmission-clusters. Patients' demographics, laboratory and clinical data, were retrieved anonymously. Statistical analysis was performed to identify subtype-specific and transmission-cluster-specific characteristics. RESULTS: Multivariate analysis showed significant differences between the 59.7% of individuals with subtype B infection and the 40.3% non-B infected individuals, with regard to route of transmission, origin, infection with Chlamydia (p = 0.01) and infection with Hepatitis C virus (p = 0.017). More and larger transmission-clusters were identified among the subtype B infections (p < 0.001). Overall, in multivariate analysis, clustering was significantly associated with Caucasian origin, infection through homosexual contact and younger age (all p < 0.001). Bivariate analysis additionally showed a correlation between clustering and syphilis (p < 0.001), higher CD4 counts (p = 0.002), Chlamydia infection (p = 0.013) and primary HIV (p = 0.017). CONCLUSIONS: Combination of phylogenetics with demographic information, laboratory and clinical data, revealed that HIV-1 subtype B infected Caucasian men-who-have-sex-with-men with high prevalence of sexually transmitted diseases, account for the majority of local HIV-transmissions. This finding elucidates observed epidemiological trends through molecular analysis, and justifies sustained focus in prevention on this high risk group.

Endothelial progenitor cells: identity defined?

In the past decade, researchers have gained important insights on the role of bone marrow (BM)-derived cells in adult neovascularization. A subset of BM-derived cells, called endothelial progenitor cells (EPCs), has been of particular interest, as these cells were suggested to home to sites of neovascularization and neoendothelialization and differentiate into endothelial cells (ECs) in situ, a process referred to as postnatal vasculogenesis. Therefore, EPCs were proposed as a potential regenerative tool for treating human vascular disease and a possible target to restrict vessel growth in tumour pathology. However, conflicting results have been reported in the field, and the identification, characterization, and exact role of EPCs in vascular biology is still a subject of much discussion. The focus of this review is on the controversial issues in the field of EPCs which are related to the lack of a unique EPC marker, identification challenges related to the paucity of EPCs in the circulation, and the important phenotypical and functional overlap between EPCs, haematopoietic cells and mature ECs. We also discuss our recent findings on the origin of endothelial outgrowth cells (EOCs), showing that this in vitro defined EC population does not originate from circulating CD133(+) cells or CD45(+) haematopoietic cells.