Functional human T lymphocyte development from cord blood CD34+ cells in nonobese diabetic/Shi-scid,IL-2 receptor gamma null mice

An experimental model for human T lymphocyte development from hemopoietic stem cells is necessary to study the complex processes of T cell differentiation in vivo. In this study, we report a newly developed nonobese diabetic (NOD)/Shi-scid, IL-2Rgamma null (NOD/SCID/gamma(c)(null)) mouse model for human T lymphopoiesis. When these mice were transplanted with human cord blood CD34(+) cells, the mice reproductively developed human T cells in their thymus and migrated into peripheral lymphoid organs. Furthermore, these T cells bear polyclonal TCR-alphabeta, and respond not only to mitogenic stimuli, such as PHA and IL-2, but to allogenic human cells. These results indicate that functional human T lymphocytes can be reconstituted from CD34(+) cells in NOD/SCID/gamma(c)(null) mice. This newly developed mouse model is expected to become a useful tool for the analysis of human T lymphopoiesis and immune response, and an animal model for studying T lymphotropic viral infections, such as HIV.     

RAG-2-deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement.

We have generated mice that carry a germline mutation in which a large portion of the RAG-2 coding region is deleted. Homozygous mutants are viable but fail to produce mature B or T lymphocytes. Very immature lymphoid cells were present in primary lymphoid organs of mutant animals as defined by surface marker analyses and Abelson murine leukemia virus (A-MuLV) transformation assays. However, these cells did not rearrange their immunoglobulin or T cell receptor loci. Lack of V(D)J recombination activity in mutant pre-B cell lines could be restored by introduction of a functional RAG-2 expression vector. Therefore, loss of RAG-2 function in vivo results in total inability to initiate V(D)J rearrangement, leading to a novel severe combined immune deficient (SCID) phenotype. Because the SCID phenotype was the only obvious abnormality detected in RAG-2 mutant mice, RAG-2 function and V(D)J recombinase activity, per se, are not required for development of cells other than lymphocytes.     

Intrahepatic transplantation of CD34+ cord blood stem cells into newborn and adult NOD/SCID mice induce differential organ engraftment

In vivo studies concerning the function of human hematopoietic stem cells (HSC) are limited by relatively low levels of engraftment and the failure of the engrafted HSC preparations to differentiate into functional immune cells after systemic application. In the present paper we describe the effect of intrahepatically transplanted CD34⁺ cells from cord blood into the liver of newborn or adult NOD/SCID mice on organ engraftment and differentiation.Analyzing the short and long term time dependency of human cell recruitment into mouse organs after cell transplantation in the liver of newborn and adult NOD/SCID mice by RT-PCR and FACS analysis, a significantly high engraftment was found after transplantation into liver of newborn NOD/SCID mice compared to adult mice, with the highest level of 35% human cells in bone marrow and 4.9% human cells in spleen at day 70. These human cells showed CD19 B-cell, CD34 and CD38 hematopoietic and CD33 myeloid cell differentiation, but lacked any T-cell differentiation. HSC transplantation into liver of adult NOD/SCID mice resulted in minor recruitment of human cells from mouse liver to other mouse organs. The results indicate the usefulness of the intrahepatic application route into the liver of newborn NOD/SCID mice for the investigation of hematopoietic differentiation potential of CD34⁺ cord blood stem cell preparations.     

Lymphoid bone marrow cultures can reconstitute heterogeneous B and T cell-dependent responses in severe combined immunodeficient mice

Long-term lymphoid bone marrow cultures (LBMC) produce B lymphocytes and their precursors for several months in vitro. To assess their differentiative potential and determine their capacity to function as immune effectors, cells from the cultures were transplanted into mice with severe combined immune deficiency disease (SCID). SCID mice are deficient in T and B lymphocytes and are serum immunoglobulin (Ig) negative, but grafts of normal lymphoid precursors can expand and differentiate in them, thereby restoring immunocompetence. The results of these studies indicate that cells from LBMC are able to reconstitute splenic B lymphocytes in the SCID mice. Upon in vivo transfer, LBMC cells secreted Ig that displayed isotype distribution and a pattern of heterogeneity comparable with normal BALB/c mice, as determined by two-dimensional gel electrophoresis. The transplanted LBMC cells were functional, because reconstituted mice could respond to immunization with the T-independent antigen TNP-Ficoll. The results also indicate that cultured cells could reconstitute T cell activity in SCID mice. Splenocytes from approximately one-third of the recipients could generate a cytotoxic response to alloantigens after 5 days of sensitization in a mixed lymphocyte culture, and all reconstituted SCID mice could respond to immunization with the T cell-dependent antigen TNP-BSA. These results demonstrate that B cells, as well as T cell activity, are present in LBMC-reconstituted SCID mice, and show that LBMC cells have the capacity to mediate an immune response.     

Current humanized mouse models for studying human immunology and HIV-1 immuno-pathogenesis

A robust animal model for “hypothesis-testing/mechanistic” research in human immunology and immuno-pathology should meet the following criteria. First, it has well-studied hemato-lymphoid organs and target cells similar to those of humans. Second, the human pathogens establish infection and lead to relevant diseases. Third, it is genetically inbred and can be manipulated via genetic, immunological and pharmacological means. Many human-tropic pathogens such as HIV-1 fail to infect murine cells due to the blocks at multiple steps of their life cycle. The mouse with a reconstituted human immune system and other human target organs is a good candidate. A number of human-mouse chimeric models with human immune cells have been developed in the past 20 years, but most with only limited success due to the selective engraftment of xeno-reactive human T cells in hu-PBL-SCID mice or the lack of significant human immune responses in the SCID-hu Thy/Liv mouse. This review summarizes the current understanding of HIV-1 immuno-pathogenesis in human patients and in SIV-infected primate models. It also reviews the recent progress in the development of humanized mouse models with a functional human immune system, especially the recent progress in the immunodeficient mice that carry a defective gammaC gene. NOD/SCID/gammaC^−/− (NOG or NSG) or the Rag2^−/−gammaC^−/− double knockout (DKO) mice, which lack NK as well as T and B cells (NTB-null mice), have been used to reconstitute a functional human immune system in central and peripheral lymphoid organs with human CD34⁺ HSC. These NTB-hu HSC humanized models have been used to investigate HIV-1 infection, immuno-pathogenesis and therapeutic interventions. Such models, with further improvements, will contribute to study human immunology, human-tropic pathogens as well as human stem cell biology in the tissue development and function in vivo.     

Human B cell growth and differentiation in the spleen of immunodeficient mice

Human mAbs (HumAbs) have therapeutic potential against infectious diseases and cancer. Heretofore, their production has been hampered by ethical constraints preventing the isolation of Ag-specific activated B cells by in vivo immunization. Alternatively, severe combined immune deficient (SCID) mice, transplanted i.p. with human (Hu)-PBLs, allow the in vivo stimulation of human Ab responses without the usual constraints. Unfortunately, human B cells only represent a minor fraction of the surviving graft, they are scattered all over the animal body, and thus are hard to isolate for subsequent immortalization procedures. To prevent this dispersion and to provide the human B cells with a niche for expansion and maturation, SCID mice were engrafted with Hu-PBL directly into the spleen. Simultaneously endogenous murine NK cell activity was depleted by treatment with an anti-mouse IL-2 receptor beta-chain Ab. During engraftment, human B lymphocytes became activated, divided intensely, and differentiated into plasmacytoid cells. In vivo exposure to a recall Ag after cell transfer induced expansion of Ag-specific B cell clones. One week after inoculation, human B cells were abundant in the spleen and could easily be recovered for fusion with a heteromyeloma line. This resulted in the formation of stable hybridoma cell lines that secreted Ag-specific HumAbs. Thus transplantation of human lymphoid cells in the spleens of immune deficient mice represents a model for the study of human T cell-dependent B cell activation and proves to be an excellent tool for the successful production of HumAbs.     

Fate and functions of human adult lymphoid cells in immunodeficient mice

Laboratory models enabling to study in vivo human leukocyte functions have been developed. Most of the models consist of human immunocytes transferred to mice homozygous for the scid mutation. Mice with additional immunodeficient-prone genetic background or with immunodeficiency-induced conditioning have also been used. Human grafts mainly consisted of human immune cells in suspension injected intraperitoneally, or in pieces of human organs containing immunocytes implanted subcutaneously. Cells in suspension could be easily manipulated in vitro before transfer to the animal, but disseminated within the mouse body. In opposition, human cells mostly remained within implantation areas of animals given human organ pieces. This favorizes cell interactions and helps for cell recovery after their in vivo passage. Moreover, the diversity of antibodies in animals transplanted with human lymphoid organ pieces appeared broader than that of mice transferred with lymphocytes in suspension. Spontaneous recall antibody and autoantibody productions have been generally observed in animals transferred with cells from donors with such antibodies. In vivo boosting of recall antibody by antigen has been most successful, but such a manipulation inconstantly boosted autoantibodies. Primary human T and B cell responses were difficult to obtain in xenochimeric animals, and success has been generally obtained by optimizing human immune response parameters, such as antigen presentation.     

Retention of human bone marrow-derived cells in murine lungs following bleomycin-induced lung injury

We studied the capacity of adult human bone marrow-derived cells (BMDC) to incorporate into distal lung of immunodeficient mice following lung injury. Immunodeficient NOD/SCID and NOD/SCID/beta(2) microglobulin (beta(2)M)(null) mice were administered bleomycin (bleo) or saline intranasally. One, 2, 3 and 4 days after bleo or saline, human BMDC labeled with CellTracker Green CMFDA (5-chloromethylfluorescein diacetate) were infused intravenously. Retention of CMFDA(+) cells was maximal when delivered 4 days after bleo treatment. Seven days after bleo, <0.005% of enzymatically dispersed lung cells from NOD/SCID mice were CMFDA(+), which increased 10- to 100-fold in NOD/SCID/beta(2)M(null) mice. Preincubation of BMDC with Diprotin A, a reversible inhibitor of CD26 peptidase activity that enhances the stromal-derived factor-1 (SDF-1/CXCL12)/CXCR4 axis, resulted in a 30% increase in the percentage of CMFDA(+) cells retained in the lung. These data indicate that human BMDC can be identified in lungs of mice following injury, albeit at low levels, and this may be modestly enhanced by manipulation of the SDF-1/CXCR4 axis. Given the overall low number of human cells detected, methods to increase homing and retention of adult BMDC, and consideration of other stem cell populations, will likely be required to facilitate engraftment in the treatment of lung injury.     

Human natural regulatory T cell development, suppressive function, and postthymic maturation in a humanized mouse model

CD4(+) regulatory T cells (Tregs) control adaptive immune responses and promote self-tolerance. Various humanized mouse models have been developed in efforts to reproduce and study a human immune system. However, in models that require T cell differentiation in the recipient murine thymus, only low numbers of T cells populate the peripheral immune systems. T cells are positively selected by mouse MHC and therefore do not function well in an HLA-restricted manner. In contrast, cotransplantation of human fetal thymus/liver and i.v. injection of CD34(+) cells from the same donor achieves multilineage human lymphohematopoietic reconstitution, including dendritic cells and formation of secondary lymphoid organs, in NOD/SCID mice. Strong Ag-specific immune responses and homeostatic expansion of human T cells that are dependent on peripheral human APCs occur. We now demonstrate that FOXP3(+)Helios(+) "natural" Tregs develop normally in human fetal thymic grafts and are present in peripheral blood, spleen, and lymph nodes of these humanized mice. Humanized mice exhibit normal reversal of CD45 isoform expression in association with thymic egress, postthymic "naive" to "activated" phenotypic conversion, and suppressive function. These studies demonstrate the utility of this humanized mouse model for the study of human Treg ontogeny, immunobiology and therapy.     

Intra-bone marrow transplantation of human CD34+ cells into NOD/LtSz-scid IL-2rγnull mice permits multilineage engraftment without previous irradiation

Background aimsNon-irradiated immunodeficient recipients provide the best physiologic setting for revealing hematopoietic stem cell (HSC) functions after xenotransplantion. An approach that efficiently permits the detection of human hematopoietic repopulating cells in non-irradiated recipients is therefore highly desired.MethodsWe compared side-by-side the ability to reconstitute hematopoiesis via intra-bone marrow transplantation (IBMT) in three commonly used mouse strains avoiding previous irradiation.ResultsNon-irradiated NOD/SCID and NOD/SCID (β2m?/? mouse strains prevent engraftment even after IBMT. In contrast, combining the robustness of the NOD/SCID IL-2Rγ?/? recipient with the sensitivity of IBMT facilitates the detection, without previous host irradiation, of human SCID-repopulating cells 10 weeks after transplantation. The level of chimerism averaged 14% and multilineage engraftment (lymphoid dominant) was observed consistently in all mice. Analysis of injected and non-injected bones, spleen and peripheral blood demonstrated that engrafting cells were capable of in vivo migration and expansion.ConclusionsCombining the robustness of the NOD/SCID IL-2Rγ?/? mouse strain with the sensitivity of IBMT strongly facilitates long-term multilineage engraftment and migration for human CD34⁺ cells without the need for previous irradiation.     

Incorporation of adult organ-derived endothelial cells into tumor blood vessel

In this study, we attempted to assess the incorporable potential of vascular endothelial cells derived from adult organ blood vessels into tumor blood vessels. Two kinds of adult organ-derived vascular endothelial cells, human aorta endothelial cells (HAEC) and umbilical vein endothelial cells (HUVEC), were administered into murine tumors inoculated to SCID mice. Many human blood vessel networks were visualized in the murine tumors. These cells in solid tumor not only survived and proliferated, but also incorporated into tumor endothelium. These results suggest that adult organ-derived vascular endothelial cells possess the potential to form the neovascular network in various tissues such as vascular endothelial progenitor-like cells in vivo. We propose that these cells can be regarded as a congenic (autologous) vector for vascular regeneration cell therapy and tumor vascular targeting gene therapy.     

Primary HIV-1 infection of human CD4+ T cells passaged into SCID mice leads to selection of chronically infected cells through a massive fas-mediated autocrine suicide of uninfected cells

We have recently shown that a human CD4+ T cell line (CEM-SS) acquires the permissiveness to M-tropic strains and primary isolates of HIV-1 after transplantation into SCID mice. This permissiveness was associated with the acquisition of a memory (CD45RO+) phenotype as well as of a functional CCR5 coreceptor. In this study, we have used this model for invest-igating in vivo the relationships between HIV-1 infection, apoptosis and T cell differentiation. When an in vivo HIV-1 infection was performed, the CEM cell tumors grew to a lower extent than the uninfected controls. CEM cells explanted from uninfected SCID mice (ex vivo CEM) underwent a significant level of spontaneous apoptosis and proved to be CD45RO+, Fas+ and Fas-L+, while Bcl-2 expression was significantly reduced as compared to the parental cells. Acute HIV-1 infection markedly increased apoptosis of uninfected ex vivo CEM cells, through a Fas/Fas-L-mediated autocrine suicide/fratricide, while parental cells did not undergo apoptosis following viral infection. The susceptibility to apoptosis of ex vivo CEM cells infected with the NSI strain of HIV-1, was progressively lost during culture, in parallel with the loss of Fas-L and marked changes in the Bcl-2 cellular distribution. On the whole, these results are strongly reminiscent of a series of events possibly occurring during HIV-1 infection. After an initial depletion of bystander CD4+ memory T cells during acute infection, latently or chronically infected CD4+ T lymphocytes are progressively selected and are protected against spontaneous apoptosis through the development of an efficient survival program. Studies with human cells passaged into SCID mice may offer new opportunities for an in vivo investigation of the mechanisms involved in HIV-1 infection and CD4+ T cell depletion.     

Humanized Rag1-/- γc-/- mice support multilineage hematopoiesis and are susceptible to HIV-1 infection via systemic and vaginal routes

Several new immunodeficient mouse models for human cell engraftment have recently been introduced that include the Rag2(-/-)γc(-/-), NOD/SCID, NOD/SCIDγc(-/-) and NOD/SCIDβ2m(-/-) strains. Transplantation of these mice with CD34(+) human hematopoietic stem cells leads to prolonged engraftment, multilineage hematopoiesis and the capacity to generate human immune responses against a variety of antigens. However, the various mouse strains used and different methods of engrafting human cells are beginning to illustrate strain specific variations in engraftment levels, duration and longevity of mouse life span. In these proof-of-concept studies we evaluated the Balb/c-Rag1(-/-)γ(-/-) strain for engraftment by human fetal liver derived CD34(+) hematopoietic cells using the same protocol found to be effective for Balb/c-Rag2(-/-)γc(-/-) mice. We demonstrate that these mice can be efficiently engrafted and show multilineage human hematopoiesis with human cells populating different lymphoid organs. Generation of human cells continues beyond a year and production of human immunoglobulins is noted. Infection with HIV-1 leads to chronic viremia with a resultant CD4 T cell loss. To mimic the predominant sexual viral transmission, we challenged humanized Rag1(-/-)γc(-/-) mice with HIV-1 via vaginal route which also resulted in chronic viremia and helper T cell loss. Thus these mice can be further exploited for studying human pathogens that infect the human hematopoietic system in an in vivo setting.     

L-selectin and SDF-1 enhance the migration of mouse and human cardiac mesoangioblasts

Efficient delivery of stem cells to heart regions is still a major problem for cell therapy. Here, we report experiments aimed to improve migration of mouse and human cardiac mesoangioblasts to the damaged heart. Cardiac mesoangioblasts were induced to transmigrate through the endothelium by factors released by cardiomyocytes or cytokines, among which stromal-derived factor 1 (SDF-1) was the most potent. Cardiac mesoangioblasts were also delivered into the left ventricular (LV) chamber of mice after coronary artery ligation (CAL), and their in vivo homing to the damaged heart was found to be quite modest. Pretreatment of cardiac mesoangioblasts with SDF-1 or transient expression of L-selectin induced a two- to three-fold increase in their transmigration and homing to the damaged heart. Therefore, combined pretreatment with SDF-1 and L-selectin generated modified cardiac mesoangioblasts, 50% of which, after injection into the LV chamber of mice early after CAL, home directly to the damaged free wall of the heart. Finally, modified mouse cardiac mesoangioblasts, injected into the LV chamber regenerate a larger surface of the ventricle in long-term experiments in comparison with their control counterparts. This study defines the requirements for efficient homing of cardiac mesoangioblasts to the damaged heart and offers a new potent tool to optimize efficiency of future cell therapy protocols for cardiovascular diseases.     

FGF23 ameliorates ischemia-reperfusion induced acute kidney injury via modulation of endothelial progenitor cells: targeting SDF-1/CXCR4 signaling

The levels of fibroblast growth factor 23 (FGF23) rapidly increases after acute kidney injury (AKI). However, the role of FGF23 in AKI is still unclear. Here, we observe that pretreatment with FGF23 protein into ischemia-reperfusion induced AKI mice ameliorates kidney injury by promoting renal tubular regeneration, proliferation, vascular repair, and attenuating tubular damage. In vitro assays demonstrate that SDF-1 induces upregulation of its receptor CXCR4 in endothelial progenitor cells (EPCs) via a non-canonical NF-κB signaling pathway. FGF23 crosstalks with the SDF-1/CXCR4 signaling and abrogates SDF-1-induced EPC senescence and migration, but not angiogenesis, in a Klotho-independent manner. The downregulated pro-angiogenic IL-6, IL-8, and VEGF-A expressions after SDF-1 infusion are rescued after adding FGF23. Diminished therapeutic ability of SDF-1-treated EPCs is counteracted by FGF23 in a SCID mouse in vivo AKI model. Together, these data highlight a revolutionary and important role that FGF23 plays in the nephroprotection of IR-AKI.Subject terms: Extracellular signalling molecules, DNA methylation, Acute kidney injury, Experimental models of disease     

Anti-CCR7 therapy exerts a potent anti-tumor activity in a xenograft model of human mantle cell lymphoma

Background

The chemokine receptor CCR7 mediates lymphoid dissemination of many cancers, including lymphomas and epithelial carcinomas, thus representing an attractive therapeutic target. Previous results have highlighted the potential of the anti-CCR7 monoclonal antibodies to inhibit migration in transwell assays. The present study aimed to evaluate the in vivo therapeutic efficacy of an anti-CCR7 antibody in a xenografted human mantle cell lymphoma model.

Methods

NOD/SCID mice were either subcutaneously or intravenously inoculated with Granta-519 cells, a human cell line derived from a leukemic mantle cell lymphoma. The anti-CCR7 mAb treatment (3 × 200 μg) was started on day 2 or 7 to target lymphoma cells in either a peri-implantation or a post-implantation stage, respectively.

Results

The anti-CCR7 therapy significantly delayed the tumor appearance and also reduced the volumes of tumors in the subcutaneous model. Moreover, an increased number of apoptotic tumor cells was detected in mice treated with the anti-CCR7 mAb compared to the untreated animals. In addition, significantly reduced number of Granta-519 cells migrated from subcutaneous tumors to distant lymphoid organs, such as bone marrow and spleen in the anti-CCR7 treated mice. In the intravenous models, the anti-CCR7 mAb drastically increased survival of the mice. Accordingly, dissemination and infiltration of tumor cells in lymphoid and non-lymphoid organs, including lungs and central nervous system, was almost abrogated.

Conclusions

The anti-CCR7 mAb exerts a potent anti-tumor activity and might represent an interesting therapeutic alternative to conventional therapies.

     

Monocyte-derived dendritic cells induce a house dust mite-specific Th2 allergic inflammation in the lung of humanized SCID mice: involvement of CCR7

In rodents, airway dendritic cells (DCs) capture inhaled Ag, undergo maturation, and migrate to the draining mediastinal lymph nodes (MLN) to initiate the Ag-specific T cell response. However, the role of human DCs in the pathogenesis of the Th2 cell-mediated disease asthma remains to be clarified. Here, by using SCID mice engrafted with T cells from either house dust mite (HDM)-allergic patients or healthy donors, we show that DCs pulsed with Der p 1, one of the major allergens of HDM, and injected intratracheally into naive animals migrated into the MLN. In the MLN, Der p 1-pulsed DCs from allergic patients induced the proliferation of IL-4-producing CD4(+) T cells, whereas those from healthy donors induced IFN-gamma-secreting cells. In reconstituted human PBMC-reconstituted SCID mice primed with pulsed DCs from allergic patients, repeated exposure to aerosols of HDM induced 1) a strong pulmonary inflammatory reaction rich in T cells and eosinophils, 2) an increase in IL-4 and IL-5 production in the lung lavage fluid, and 3) increased IgE production compared with that in mice primed with unpulsed DCs. All these effects were reduced following in vivo neutralization of the CCR7 ligand secondary lymphoid tissue chemokine. These data in human PBMC-reconstituted SCID mice show that monocyte-derived DCs might play a key role in the pathogenesis of the pulmonary allergic response by inducing Th2 effector function following migration to the MLN.     

microRNA-27b suppresses mouse MSC migration to the liver by targeting SDF-1αin vitro

The SDF-1/CXCR4 axis is critical for inducing stem cell mobilization into the circulation, for homing stem cells to the site of injury, and for stem cell participation in the regeneration of liver tissue. In this study, we have gained insight into the molecular mechanisms involved in regulating the expression of SDF-1α by miRNAs. Using microarray and bioinformatics approaches, we identified six miRNAs with differential expression in damaged liver tissue (21 days after liver injury) compared to normal C57BL/6 murine liver tissue and further confirmed these observations by qPCR; miR-23a, which was identified by other researchers, was also included for comparative purposes. We found that miR-23a, miR-27a and miR-27b expression was significantly lower in the damaged liver than in the normal liver (p<0.05). We further confirmed that miR-27b could directly interact with the 3'UTR of SDF-1α to suppress SDF-1α protein expression using a luciferase reporter assay and Western blot analysis. In addition, we found that the over-expression of miR-27b significantly reduced the directional migration of primary cultured CRCX4-positive murine mesenchymal stem cells (mMSCs) in vitro using a transwell assay. These results suggest that miR-27b may be a unique signature of the stem cell niche in the damaged mouse liver and that mir-27b can suppress the directional migration of mMSCs by down-regulating SDF-1α expression by binding directly to the SDF-1α 3'UTR.     

Annexin 1 modulates monocyte-endothelial cell interaction in vitro and cell migration in vivo in the human SCID mouse transplantation model

The effect of the glucocorticoid inducible protein annexin 1 (ANXA1) on the process of monocytic cell migration was studied using transfected U937 cells expressing variable protein levels. An antisense (AS) (36.4AS; approximately 50% less ANXA1) and a sense (S) clone (15S; overexpressing the bioactive 24-kDa fragment) together with the empty plasmid CMV clone were obtained and compared with wild-type U937 cells in various models of cell migration in vitro and in vivo. 15S-transfected U937 cells displayed a reduced (50%) degree of trans-endothelial migration in response to stromal cell-derived factor-1alpha (CXC chemokine ligand 12 (CXCL12)). In addition, the inhibitory role of endogenous ANXA1 on U937 cell migration in vitro was confirmed by the potentiating effect of a neutralizing anti-ANXA1 serum. Importantly, overexpression of ANXA1 in clone 15S inhibited the extent of cell migration into rheumatoid synovial grafts transplanted into SCID mice. ANXA1 inhibitory effects were not due to modifications in adhesion molecule or CXCL12 receptor (CXCR4) expression as shown by the similar amounts of surface molecules found in transfected and wild-type U937 cells. Likewise, an equal chemotactic response to CXCL12 in vitro excluded an intrinsic defect in cell motility in clones 15S and 36.4AS. These data strongly support the notion that ANXA1 critically interferes with a leukocyte endothelial step essential for U937 cell, and possibly monocyte, transmigration both in vitro and in vivo.