Stromal cell-derived factor-1 (SDF-1) is a key regulator of the behavior of normal and leukemic precursor-B (pre-B) cells. It is possible that inhibiting SDF-1-driven processes in pre-B acute lymphoblastic leukemia (ALL) may have therapeutic implications. In this study, we examined the ability of SDF-1 inhibitors to modulate pre-B ALL cell responses to SDF-1, including chemotaxis, migration into bone marrow stroma, and stromasupported survival and proliferation on human bone marrow stromal layers. The polyphemusin II-derived inhibitors, T140, TC140012, and T134, and the bicyclam AMD3100, effectively inhibited binding of the anti-CXCR4 monoclonal antibody 12G5 on the pre-B ALL cell line NALM6, with IC 50 values of 0.9, 0.9, 0.9, and 1.9 nM, respectively. Similar results were obtained with ALL samples. T140 (0.1 lM) and AMD3100 (1 lM) completely blocked SDF-1-induced chemotaxis and attenuated the migration of pre-B ALL cells into bone marrow stromal layers. AMD3100 and TC140012 at a concentration of 50 lM significantly inhibited stroma-dependent proliferation of six and four of the eight cases tested, respectively, without reducing the cell viability. In addition, AMD3100 and TC140012 enhanced the cytotoxic and antiproliferative effects of the cytotoxic agents vincristine and dexamethasone. The ability of SDF-1 inhibitors to modulate these biologically important functions of leukemic cells warrants further investigation.

CXCL12 and VCAM1 retain hematopoietic stem cells (HSCs) in the BM, but the factors mediating HSC egress from the BM to the blood are not known. The sphingosine-1-phosphate receptor 1 (S1P 1 ) is expressed on HSCs, and S1P facilitates the egress of committed hematopoietic progenitors from the BM into the blood. In the present study, we show that both the S1P gradient between the BM and the blood and the expression of S1P 1 are essential for optimal HSC mobilization by CXCR4 antagonists, including AMD3100, and for the trafficking of HSCs during steady-state hematopoiesis. We also demonstrate that the S1P 1 agonist SEW2871 increases AMD3100-induced HSC and progenitor cell mobilization.These results suggest that the combination of a CXCR4 antagonist and a S1P 1 agonist may prove to be sufficient for mobilizing HSCs in normal donors for transplantation purposes, potentially providing a single mobilization procedure and eliminating the need to expose normal donors to G-CSF with its associated side effects. (Blood. 2012;119(3):707-716)

The role of CXCL12 in the bone marrow (BM) homing and growth of B-cell progenitor acute lymphoblastic leukemia (ALL) has been established. However, the effect of modulating CXCL12/CXCR4 interactions on the retention of ALL cells within the supportive BM microenvironment and the expansion and dissemination of ALL cells in vivo has not been examined. We used mouse models of human childhood and murine leukemia and specific peptide and small molecule CXCR4 antagonists to examine the importance of CXCL12/CXCR4 in the development of leukemia in vivo. CXCR4 antagonists mobilized ALL cells into the peripheral blood (PB). Extended administration of CXCR4 antagonists to mice with leukemia resulted in a reduction in the number of leukemic cells in the PB and spleens of animals compared to control treated animals in three of the five cases tested. There was also a marked reduction in the dissemination of ALL cells to extramedullary sites including liver and kidney in all cases where this occurred. Considering the inhibitory effect of stromal layers on the activity of chemotherapeutic agents and the interactive effect of CXCL12 antagonists with chemotherapeutic agents in vitro, this raises the possibility of using these agents to potentiate the effects of current chemotherapy regimens.

SDF-1 and CXCR4 are an important chemokine ligand/receptor pair, which play a crucial role in numerous biological processes including hematopoiesis, cardiogenesis, vasculogenesis, neuronal development and immune cell trafficking. They have also been implicated in various pathological conditions such as cancer, infection with the human immunodeficiency virus (HIV) and various inflammatory conditions. Numerous pharmacological agents exist that can modulate SDF-1/CXCR4-induced responses both in vitro and in vivo. The usefulness of these agents in affecting the outcome of pathological conditions influenced by the SDF-1/CXCR4 axis is currently being investigated. Whilst some of these compounds have been shown to be safe and well tolerated in phase 1 clinical trials, the full repercussions of SDF-1/CXCR4 inhibition or stimulation on normal physiological functions are yet to be appreciated. Inhibition of the SDF-1/CXCR4 axis may have positive effects in regulating tumour metastasis and growth, however, this may also negate immunological responses through dysregulated lymphocyte trafficking and contribute to disruption of hematopoiesis. As with any therapy, the usefulness of this type of intervention will require a balance between its positive effect on the disease outcome and deleterious effects on normal physiological functions. A greater understanding of the role of SDF-1 and CXCR4 in the body will allow greater manipulation of this important biological axis to affect disease outcome. Greater knowledge of the SDF-1 interaction with its receptor and the structural elements within CXCR4 mediating the different signalling events, resulting in SDF-1-induced responses, will also enhance future drug design.

The chemokine stromal-derived factor-1A (SDF-1A) regulates leukemic cell motility and proliferation; however, the importance of these functions in the growth and dissemination of leukemia is unclear. We examined SDF-1A-mediated responses of cells from 27 cases of acute lymphoblastic leukemia (ALL). Although cells from the majority of cases showed chemotactic and proliferative responses to SDF-1A, a subset of cases did not undergo chemotaxis in response to SDF-1A, while still demonstrating dependence on SDF-1A for proliferation in stroma-supported cultures. This chemotactic defect was associated with an absence of phosphorylation ofp38 mitogen-activated protein kinase (MAPK) induced by SDF-1A, and of SDF-1A-induced augmentation of B 1 integrinmediated adhesion. Signaling through phosphoinositide 3-kinase and MEK was not affected. No correlation was observed between CXCR4 expression and chemotactic function, in vitro migration into bone marrow stromal layers, and engraftment of leukemic cells in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. This study suggests that signaling through p38 MAPK is required for ALL cell chemotaxis but not for proliferation, and that the loss of a chemotactic response to SDF-1A does not impede engraftment in NOD/SCID mice. (Cancer Res 2005; 65(8): 3290-8)

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Viral phenotype, tropism, coreceptor usage, and envelope gene diversity were examined in blood isolates collected from 27 individuals at different stages of human immunodeficiency virus type 1 (HIV-1) disease and tissue derived isolates from 10 individuals with AIDS. The majority (89%) of blood and all tissue HIV-1 isolates from all stages of infection were non-syncytium inducing and macrophage (M) tropic. Tropism and productive infection by HIV isolates in both monocytes and monocyte-derived macrophages (MDM) increased in advanced disease (HIV tropism for monocytes, 1 of 6 from categories I and II versus 11 of 21 [P = 0.05] from category IV and II [CD4 < 250]; and high-level replication in MDM, 1 of 6 from categories I and II versus 16 of 21 from categories IV and II [P = 0.015]). There was a high level of replication of blood and tissue isolates in T lymphocytes without restriction at any stage. Overall, the level of replication in MDM was 5- to 10-fold greater than in monocytes, with restriction in the latter occurring mainly at entry and later stages of replication. Only three blood isolates were identified as syncytium inducing, and all had a dualtropic phenotype. There was a significant increase of HIV envelope gene diversity, as shown by a heteroduplex mobility assay, in advanced disease; this may partly underlie the increase of HIV replication in MDM. Unlike blood isolates (even those from patients with advanced disease), tissue isolates displayed greater similarities (90%) in productive infection between MDM and monocytes. The majority (87%) of all isolates, including those from patients with advanced disease, used CCR5, and only 5 of 37 isolates showed expanded coreceptor usage. These results indicate that in the late stage of disease with increasing viral load and diversity, CCR5 utilization and M-tropism persist in blood and tissue and the replicative ability in macrophages increases. This suggests that these characteristics are advantageous to HIV and are important to disease progression.

We have used humanized mice, in which human immune cells differentiate de novo from transplanted cord blood progenitor cells, to study the human immune responses to infection with Mycobacterium bovis bacillus Calmette-Guérin and Mycobacterium tuberculosis. Granulomas with a core containing giant cells, human CD68+ macrophages, and high bacilli numbers surrounded by a layer of CD3 + T cells and a fibrotic response encapsulating the lesions were observed in livers and lungs from bacillus CalmetteGuérin-infected humanized mice but not in nonhumanized infected controls. Paradoxically, humanized mice contained higher mycobacterial numbers in organs than nonhumanized controls. The enhancement of bacterial load was mediated by human CD4 + cells and associated to an increased expression of Programmed Death-1 protein and CD57 on T cells, molecules associated with inhibition and senescence. The lesions from mice depleted of CD4 + cells were scarcer, minimal, and irregular compared with those from mice depleted of CD8 + cells or nondepleted controls. Granulomas of bacillus Calmette-Guérin-infected humanized mice administered with a TNF-neutralizing TNF receptor fusion molecule preserved their structure, but contained higher levels of intracellular bacilli. Extended necrosis was observed in granulomas from M. tuberculosisbut not bacillus Calmette-Guérin-infected humanized mice. Our data indicate that humanized mice can be used as a model to study the formation and maintenance of human granuloma in tuberculosis and other infectious or noninfectious diseases.A pproximately one third of the human population is infected by M. tuberculosis, of whom 5 to 15% develop active tuberculosis (TB) during their lifetime (1). Mouse models have contributed to our understanding of the pathogenesis and immunology of TB. However, the relative hierarchy of immune-mediated killing mechanisms in humans is still unclear. Moreover, the mouse model fails to form granulomas that reproduce those seen in humans (2). Typical mouse lesions lack central necrosis and are less organized than TB lesions found in immune-competent humans. On the contrary, experimental studies of human immunity to mycobacterial infections are limited by obvious practical and ethical restrictions.The host counters mycobacterial infections primarily via CD4 +