Application of affinity selection-mass spectrometry assays to purification and affinity-based screening of the chemokine receptor CXCR4.

Affinity selection-mass spectrometry (AS-MS) is a sensitive technology for identifying small molecules that bind to target proteins, and assays enabled by AS-MS can be used to delineate relative binding affinities of ligands for proteins. 'Indirect' AS-MS assays employ size-exclusion techniques to separate target-ligand complexes from unbound ligands, and target-associated ligands are then specifically detected by liquid chromatography mass spectrometry. We report how indirect AS-MS binding assays with known reference control compounds were used as guideposts for development of an optimized purification method for CXCR4, a G-protein coupled chemokine receptor, for which we sought novel antagonists. The CXCR4 purification method that was developed was amenable to scale-up and enabled the screening of purified recombinant human CXCR4 against a large combinatorial library of small molecules by high throughput indirect AS-MS. The screen resulted in the discovery of new ligands that competed off binding of reference compounds to CXCR4 in AS-MS binding assays and that antagonized SDF1α-triggered responses and CXCR4-mediated HIV1 viral uptake in cell-based assays. This report provides a methodological paradigm whereby indirect AS-MS-based ligand binding assays may be used to guide optimal integral membrane protein purification methods that enable downstream affinity selection-based applications such as high throughput AS-MS screens.

Isolation and identification of CXCR4-positive cells from human dental pulp cells.

INTRODUCTION: In previous studies, we found expression of stromal cell-derived factor-1α (SDF-1α)/CXC chemokine receptor 4 (CXCR4) in human dental pulp and the SDF-1α-CXCR4 axis might play a role in the recruitment of CXCR4-positive dental pulp cells (CXCR4(+) DPCs) toward the damaged sites. However, the specific function of CXCR4(+) DPCs in the injured dental pulp was still unknown. The purpose of this study was to isolate CXCR4(+) DPCs from dental pulp cells in vitro to pave the way for further study of their characteristics. METHODS: CXCR4(+) DPCs were isolated with magnetic-activated cell sorting (MACS). Freshly isolated CXCR4(+) DPCs were identified by immunohistochemistry with light microscopy or confocal microscopy. Then the phenotypes CXCR4, stromal cell surface marker-1 (STRO-1), CD146, and CD34 in 3 groups (ie, CXCR4(+) DPCs, CXCR4(-) DPCs, or non-sorted DPCs) were analyzed by flow cytometry after they were cultured and expanded in vitro. RESULTS: The results indicated the isolated subpopulation of DPCs was enriched with CXCR4(+) DPCs, and the positive rates of STRO-1 and CD146 in CXCR4(+) DPCs group were higher than CXCR4(-) DPCs or non-sorted DPCs groups (P < .05). There was no expression of CD34 in each group. CONCLUSIONS: We can isolate CXCR4(+) DPCs from DPCs with MACS and identify them by immunohistochemistry and flow cytometry.

CCR5 antagonists: a therapeutic option in HIV-1 perinatally infected children experiencing virologic failure?

OBJECTIVE: The risk of virologic failure and selection of resistant strains remains a challenge in HIV-1 perinatally infected children. HIV-1 coreceptor usage was determined in HAART-failing children followed in Necker Hospital (Paris, France) in order to estimate the proportion of these patients who may benefit from CCR5-antagonists therapy. METHODS: HIV-1 coreceptor usage was determined with the SVM(Geno2pheno10%) algorithm in 51 children with virologic failure after a median treatment exposure of 7.8 years. RESULTS: CXCR4-tropic strains were found in 31.4% of the patients. CXCR4 usage was associated with high HIV-1 DNA (P=0.01), old age (P=0.02), long ART cumulative exposure (P=0.006), and previous exposure to high number of different drugs (P=0.03) and ART combinations (P=0.03) in univariate analysis. Selection of resistant viruses and current exposure to a darunavir-based HAART tended to be more frequent in the CXCR4 group compared with the children infected with CCR5-tropic strains (P=0.06). In multivariate analysis, CXCR4 usage was exclusively correlated with HIV-1 DNA (P=0.03), which accurately reflects the cumulative exposure to viral replication over the whole duration of HIV infection. CONCLUSION: Two-thirds of HAART-failing children could benefit from CCR5 antagonists-based strategies, even in case of triple-class virologic failure. Such therapy should be discussed more appropriately at early stages of infection, when CCR5-tropic strains are most frequently isolated. However, before considering such strategies, further studies are needed to evaluate the efficacy and the tolerability of CCR5 antagonists in this pediatric population.

Drug resistance and coreceptor usage in HIV type 1 subtype C-infected children initiating or failing highly active antiretroviral therapy in South Africa.

HIV-1 drug resistance monitoring in resource-poor settings is crucial due to limited drug alternatives. Recent reports of the increased prevalence of CXCR4 usage in subtype C infections may have implications for CCR5 antagonists in therapy. We investigated the prevalence of drug resistance mutations and CXCR4 coreceptor utilization of viruses from HIV-1 subtype C-infected children. Fifty-one children with virological failure during highly active antiretroviral therapy (HAART) and 43 HAART-naive children were recruited. Drug resistance genotyping and coreceptor utilization assessment by phenotypic and genotypic methods were performed. At least one significant drug resistance mutation was present in 85.4% of HAART-failing children. Thymidine analogue mutations (TAMs) were detected in 58.5% of HAART-failing children and 39.0% had ≥3 TAMs. CXCR4 (X4) or dual (R5X4)/mixed (R5, X4) (D/M)-tropic viruses were found in 54.3% of HAART-failing and 9.4% of HAART-naive children (p<0.0001); however, the HAART-failing children were significantly older (p<0.0001). In multivariate logistic regression, significant predictors of CXCR4 usage included antiretroviral treatment, older age, and lower percent CD4(+) T cell counts. The majority of genotypic prediction tools had low sensitivity (≤65.0%) and high specificity (≥87.5%) for predicting CXCR4 usage. Extensive drug resistance, including the high percentage of TAMs found, may compromise future drug choices for children, highlighting the need for improved treatment monitoring and adherence counseling. Additionally, the increased prevalence of X4/D/M viruses in HAART-failing children suggests limited use of CCR5 antagonists in salvage therapy. Enhanced genotypic prediction tools are needed as current tools are not sensitive enough for predicting CXCR4 usage.

Isolation and biological characterization of non-B HIV type 1 from Kenya.

The isolation and characterization of primary strains of human immunodeficiency virus (HIV) is a vital tool for assessing properties of viruses replicating in HIV-infected subjects. HIV-1 isolation was carried out from 30 HIV-1-infected patients from a Comprehensive Care Clinic (CCC) after informed consent. Virus was successfully isolated from 9 out of the 30 samples investigated. Seven of the isolates were from drug-naive patients while two were from patients on antiretroviral drugs. The isolates were biologically phenotyped through measurement of the syncytium-inducing capacity in MT2 cells. Six of the isolates exhibited syncytia induction (SI) associated with CXCR4 coreceptor usage while three of the isolates were non-syncytia-inducing (NSI) isolates associated with CCR5 coreceptor usage. In addition, the replication capacity of the isolates was further determined in established cell line CD4(+) C8166. Indirect immunofluorescence assay was used to check the antigen expression on the cells as a supplementary test. HIV-1 isolation success was 70% (7/10) and 20% (2/20) in naive and drug-experienced patients, respectively. The majority of the viral isolates obtained (6/9) were of the SI phenotype, though SI virus strains are rare among non-B subtypes. A significant correlation between virus isolation success and viral load was established. Coreceptor use data for heavily treatment-experienced patients with limited treatment options are scanty and this is the group with perhaps the most urgent need of novel antiretroviral agents.

High-level production, solubilization and purification of synthetic human GPCR chemokine receptors CCR5, CCR3, CXCR4 and CX3CR1.

Chemokine receptors belong to a class of integral membrane G-protein coupled receptors (GPCRs) and are responsible for transmitting signals from the extracellular environment. However, the structural changes in the receptor, connecting ligand binding to G-protein activation, remain elusive for most GPCRs due to the difficulty to produce them for structural and functional studies. We here report high-level production in E.coli of 4 human GPCRs, namely chemokine receptors (hCRs) CCR5, CCR3, CXCR4 and CX3CR1 that are directly involved in HIV-1 infection, asthma and cancer metastasis. The synthetic genes of CCR5, CCR3, CXCR4 and CX3CR1 were synthesized using a two-step assembly/amplification PCR method and inserted into two different kinds of expression systems. After systematic screening of growth conditions and host strains, TB medium was selected for expression of pEXP-hCRs. The low copy number pBAD-DEST49 plasmid, with a moderately strong promoter tightly regulated by L-arabinose, proved helpful for reducing toxicity of expressed membrane proteins. The synthetic Trx-hCR fusion genes in the pBAD-DEST49 vector were expressed at high levels in the Top10 strain. After a systematic screen of 96 detergents, the zwitterionic detergents of the Fos-choline series (FC9-FC16) emerged as the most effective for isolation of the hCRs. The FC14 was selected both for solubilization from bacterial lysates and for stabilization of the Trx-hCRs during purification. Thus, the FC-14 solubilized Trx-hCRs could be purified using size exclusion chromatography as monomers and dimers with the correct apparent MW and their alpha-helical content determined by circular dichroism. The identity of two of the expressed hCRs (CCR3 and CCR5) was confirmed using immunoblots using specific monoclonal antibodies. After optimization of expression systems and detergent-mediated purification procedures, we achieved large-scale, high-level production of 4 human GPCR chemokine receptor in a two-step purification, yielding milligram quantities of CCR5, CCR3, CXCR4 and CX3CR1 for biochemical, biophysical and structural analysis.

In vitro reconstitution and preparative purification of complexes between the chemokine receptor CXCR4 and its ligands SDF-1alpha, gp120-CD4 and AMD3100.

CXCR4 belongs to the family of G protein-coupled receptors and mediates the various developmental and regulatory effects of the chemokine SDF-1alpha. In addition, CXCR4 acts as a co-receptor along with CD4 for the HIV-1 viral glycoprotein gp120. Recently, there has also been a small molecule described that antagonizes both SDF-1 and gp120 binding to CXCR4. The structural and mechanistic basis for this dual recognition ability of CXCR4 is unknown largely due to the technical challenges of biochemically producing the components of the various complexes. We expressed the human CXCR4 receptor using a modified baculovirus expression vector that facilitates a single step antibody affinity purification of CXCR4 to >80% purity from Hi5 cells. The recombinant receptor undergoes N-linked glycosylation, tyrosine sulfation and is recognized by the 12G5 conformation specific antibody against human CXCR4. We are able to purify CXCR4 alone as well as complexed with its endogenous ligand SDF-1, its viral ligand gp120, and a small molecule antagonist AMD3100 by ion-exchange chromatography. We anticipate that the expression and purification scheme described in this paper will facilitate structure-function studies aimed at elucidating the molecular basis for CXCR4 recognition of its endogenous chemokine and viral ligands.

Solubilization, stabilization, and purification of chemokine receptors using biosensor technology.

Establishing solubilization conditions for membrane-associated receptors is often a tedious empirical process. Here we describe a novel application of SPR biosensor technology to screen solubilization conditions automatically and to assess receptor activity directly. We focus on two chemokine receptors, CXCR4 and CCR5, which are important in HIV cell invasion. The autosampler in Biacore 3000 permitted whole cells expressing C-terminally tagged receptors to be automatically lysed under a given solubilization condition and the lysates to be injected over an antibody surface. The total amount of solubilized receptor could be quantitated from the antibody capture level, whereas the amount of active receptor could be quantitated using a subsequent injection of conformationally sensitive antibody or protein. Using this approach, we identified detergent/lipid/buffer combinations that enhanced and maintained receptor activity. We also used the biosensor to demonstrate CD4-dependent binding of gp120 to solubilized CCR5 and to develop affinity chromatography-based purification methods that increased receptor activity more than 300%. Together, these results illustrate the benefits of using the biosensor as a tool for isolating functional membrane receptors and for analyzing ligand/receptor interactions.

Heterogeneous populations of bone marrow stem cells--are we spotting on the same cells from the different angles?

Accumulated evidence suggests that in addition to hematopoietic stem cells (HSC), bone marrow (BM) also harbors endothelial stem cells (ESC), mesenchymal stem cells (MSC), multipotential adult progenitor cells (MAPC), pluripotent stem cells (PCS) as well as tissue committed stem cells (TCSC) recently identified by us. In this review we discuss the similarities and differences between these cell populations. Furthermore, we will present the hypothesis that all of these versatile BM derived stem cells are in fact different subpopulations of TCSC. These cells accumulate in bone marrow during ontogenesis and being a mobile population of cells are released from BM into peripheral blood after tissue injury to regenerate damaged organs. Furthermore, since BM is a "hideout" for TCSC, their presence in preparations of bone marrow derived mononuclear cells should be considered before experimental evidence is interpreted simply as trans-differentiation or plasticity of HSC. Finally, our observation that the number of TCSC accumulate in the bone marrow of young animals and their numbers decrease during senescence provides a new insight into aging and may explain why the regeneration processes becomes less effective in older individuals.

Specific reactions between purified HIV-1 particles and CD4+ cell membrane fragments in a cell-free system of virus fusion or entry.

The initial step of human immunodeficiency virus type 1 (HIV-1) infection has been studied by Env-mediated fusion or entry assays with appropriate cells expressing CD4 or CXCR4/CCR5 receptors in cultures, where many factors underlying cellular activities likely regulate the fusion/entry efficiency. Here we attempted to develop a more simplified in vitro cell-free fusion/entry reaction that mimics HIV-1 infection in cultures. Membrane fragments of target cells and intact infectious HIV-1 particles were purified, mixed and incubated. The core p24 protein was released from the purified virions and detected by ELISA without detergents in the supernatant of the reaction mixtures. This release reaction proceeded temperature-dependently and in a dose-dependent manner between the virion and membrane fractions, and was specific for HIV-1 Env and CD4. Env-deleted or VSV-G-pseudotyped HIV-1 released little p24, if any. Pretreatment of the membrane fragments with anti-CD4 antibodies inhibited the p24 induction from both X4-tropic and R5-tropic HIV-1. Furthermore, X4 but not R5 HIV-1 reacted with the membrane prepared from intrinsically CXCR4-positive HeLa-CD4 cells, whereas both viruses reacted with that prepared from CCR5-transduced HeLa-CD4 cells, indicating that this cell-free reaction mimics coreceptor usage of HIV-1 infection. Therefore, a potent entry inhibitor of X4 HIV-1, SDF-1alpha, blocked the release from X4 but not R5 HIV-1. Inversely, a weak entry inhibitor of R5 HIV-1, MIP-1beta, partially affected only the release from R5 HIV-1. These results suggest that this cell-free reaction system provides a useful tool to study biochemical fusion/entry mechanisms of HIV-1 and its inhibitors.

CXCR4 heterogeneity in primary cells: possible role of ubiquitination.

The chemokine receptor CXCR4 is a primary coreceptor for the HIV-1 virus. The predicted molecular weight (MW) of glycosylated CXCR4 is 45-47 kDa. However, immunoblots of whole cell lysates from human lymphocytes, monocytes, macrophages, and the Jurkat T-lymphocyte line revealed multiple MW isoforms of CXCR4. Three of the bands could be precipitated by anti-CXCR4 monoclonal antibodies (101 and 47 kDa) or coprecipitated with CD4 (62 kDa). Expression of these isoforms was enhanced by infection with a recombinant vaccinia virus encoding CXCR4. In immunoblots of two-dimensional gels, antiubiquitin antibodies reacted with the 62-kDa CXCR4 species from monocytes subsequent to coprecipitation with anti-CD4 antibodies. Culturing of monocytes and lymphocytes with lactacystin enhanced the amount of the 101-kDa CXCR4 isoform in immunoblots by three- to sevenfold. In lymphocytes, lactacystin also increased cell-surface expression of CXCR4, which correlated with enhanced fusion with HIV-1 envelope-expressing cells. Similar increases in the intensity of the 101-kDa isoform were seen after treatment with the lysosomal inhibitors monensin and ammonium chloride. Antiubiquitin antibodies reacted with multiple proteins above 62 kDa, which were precipitated with anti-CXCR4 antibodies. Our data indicate that ubiquitination may contribute to CXCR4 heterogeneity and suggest roles for proteasomes and lysosomes in the constitutive turnover of CXCR4 in primary human cells.

Chemokine receptor expression profile of eosinophils at inflamed tissue sites: Decreased CCR3 and increased CXCR4 expression by lung eosinophils.

BACKGROUND: To date, most studies dealing with eosinophil chemokine receptors have used eosinophils isolated from peripheral blood. During the movement of eosinophils from the peripheral blood to inflamed tissue sites, microenvironmental signals might alter their expression of chemokine receptors. However, little is known about the profile of expression of chemokine receptors by eosinophils at inflamed tissue sites in human beings. OBJECTIVE: The purpose of this study was to determine whether eosinophils that have migrated into inflamed tissues exhibit a profile of chemokine receptor expression that is qualitatively and/or quantitatively different from that of eosinophils in peripheral locations. METHODS: We studied simultaneously the expression and function of chemokine receptors in eosinophils in both bronchoalveolar lavage fluid (BALF) and peripheral blood specimens of 7 patients with eosinophilic lung diseases. RESULTS: De novo expression of CCR2, CCR4, and CCR5 was not detected at either the protein or the mRNA level. However, surface expression of CCR3 was decreased and CXCR4 was conversely increased with statistical significance in BALF eosinophils. Moreover, the changes in CCR3 and CXCR4 expression were reflected in the altered migratory response to their ligands. On the other hand, the levels of CXCR1, CXCR2, CXCR3, and CCR1 were virtually unchanged in BALF eosinophils, and these receptors did not have functional significance. CONCLUSION: Eosinophils at inflamed tissue sites exhibited an expression profile qualitatively similar to that in peripheral locations, except for decreased CCR3 and increased CXCR4 expression. Our results suggest that CCR3 is primarily and CXCR4 is cooperatively involved in eosinophil accumulation at inflamed tissue sites.

AMD3100, a potent and specific antagonist of the stromal cell-derived factor-1 chemokine receptor CXCR4, inhibits autoimmune joint inflammation in IFN-gamma receptor-deficient mice.

Autoimmune collagen-induced arthritis (CIA) in IFN-gammaR-deficient DBA/1 mice was shown to be reduced in severity by treatment with the bicyclam derivative AMD3100, a specific antagonist of the interaction between the chemokine stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4. The beneficial effect of the CXCR4 antagonist was demonstrable when treatment was initiated between the time of immunization and appearance of the first symptoms. Treatment also reduced the delayed-type hypersensitivity response to the autoantigen, collagen type II. These observations are indicative of an action on a late event in the pathogenesis, such as chemokine-mediated attraction of leukocytes toward joint tissues. The notion of SDF-1 involvement was further supported by the observation that exogenous SDF-1 injected in periarthritic tissue elicited an inflammatory response that could be inhibited by AMD3100. The majority of leukocytes harvested from inflamed joints of mice with CIA were found to be Mac-1(+) and CXCR4(+), and AMD3100 was demonstrated to interfere specifically with chemotaxis and Ca(2+) mobilization induced in vitro by SDF-1 on Mac-1(+)/CXCR4(+) splenocytes. We conclude that SDF-1 plays a central role in the pathogenesis of murine CIA, by attracting Mac-1(+)/CXCR4(+) cells to the inflamed joints.

A CD14-independent LPS receptor cluster.

Bacterial lipopolysaccharide (LPS), the major structural component of the outer wall of Gram-negative bacteria, is a potent initiator of an inflammatory response and serves as an indicator of bacterial infection. Although CD14 has been identified as the main LPS receptor, accumulating evidence has suggested the possible existence of other functional receptor(s). In this study, using affinity chromatography, peptide mass fingerprinting and fluorescence resonance energy transfer, we have identified four new proteins that form an activation cluster after LPS ligation and are involved in LPS signal transduction. Here we present evidence that implicates heat shock proteins 70 and 90, chemokine receptor 4 and growth differentiation factor 5 as the main mediators of activation by bacterial lipopolysaccharide.

Solubilization of the chemokine receptor CXCR4.

The chemokine receptor CXCR4 was solubilized from the human T-cell line CEM by using the detergent n-dodecyl-beta-maltoside (DDM) and cholesteryl hemisuccinate ester (CHS). Binding studies with (125)I-SDF-1alpha revealed a dissociation constant of 5.33 nM and a receptor density (B(max)) of 2.68 pmol/mg in CEM membranes at 4 degrees C. The affinity of solubilized CXCR4 for SDF-1alpha was identical to membrane-bound CXCR4. Binding of gp120 to solubilized CXCR4 was demonstrated by coprecipitation of gp120 with anti-CXCR4 antibodies.

Characterization of recombinant human CXCR4 in insect cells: role of extracellular domains and N-glycosylation in ligand binding.

The cDNA of the human CXCR4/fusin was isolated from a human HeLa cell cDNA library by PCR and functionally expressed in Sf9 insect cells. The recombinant receptor was found to bind its natural ligand SDF-1alpha with an affinity comparable to that of the native receptor. Sequence-specific antibodies against each of the four extracellular domains were generated and used to investigate the interactions between the different domains of the receptor and the ligand. Each of the four antibodies was found to be able to inhibit ligand binding. CXCR4 was shown to be a glycoprotein. The role of N-glycosylation of CXCR4 in ligand binding was investigated in the insect cells overexpressed with recombinant CXCR4. Two potential N-glycosylation sites (Asn-11 and Asn-176) were either singly or doubly mutated to a leucine residue. Both single mutant receptors exhibited a significant decrease in ligand binding activity and affinity. The double mutant receptor showed little binding activity. Our data suggest that all of the extracellular domains are involved in ligand-receptor interactions and that N-glycosylation is required to maintain high-affinity ligand binding.

Cutting edge: CXCR4-Lo: molecular cloning and functional expression of a novel human CXCR4 splice variant.

Human CXCR4 is a specific receptor for the CXC chemokine stromal cell-derived factor-1 (SDF-1) and a coreceptor for T cell line tropic strains of HIV-1. Genetic knockouts of CXCR4 and SDF-1 have delineated their critical role during embryonic cardiogenesis, leukopoiesis, and vasculogenesis. Herein, we used bioinformatics and differential strategies like isoform-specific RT-PCR and Northern blots to identify and clone a novel unspliced isoform of human CXCR4, termed CXCR4-Lo. CXCR4-Lo corresponds to a larger approximately 4. 0-kb mRNA transcript and differs from the known human CXCR4 by the first 9 aa in the functionally important NH2-terminal extracellular domain of the receptor. CXCR4-Lo-transfected rat basophil leukemia-2H3 cells responded to SDF-1 with a transient rise of intracellular Ca2+ concentration and by undergoing chemotaxis. Expression of CXCR4-Lo is noteworthy, as it may have differential affinity as a coreceptor for HIV strains in comparison with CXCR4. Furthermore, CXCR4-Lo may also provide a functional backup to CXCR4 during embryogenesis.

Cloning of two chemokine receptor homologs (CXC-R4 and CC-R7) in rainbow trout Oncorhynchus mykiss.

Two rainbow trout chemokine receptors have been sequenced, with homology to CXC-R4 and CC-R7 molecules. The CXC-R4 sequence consisted of 1681 nucleotides, which translated into a mature protein of 357 amino acids, with 80.7% similarity to human CXC-R4. The CC-R7 sequence consisted of 2287 nucleotides, which translated into a 368-amino acid mature protein with 64.5% similarity to human CC-R7. Both sequences contained seven hydrophobic regions, representing the seven transmembrane domains (TM) typical of G-protein-coupled receptors. Extracellular cysteines, transmembrane prolines, and the DRY motif immediately following TM3 were conserved. Phylogenetic tree analysis revealed a tight clustering of trout CXC-R4 with CXC-R3-5 genes. Trout CC-R7 clustered with CC-R6-7 and CXC-R1-2. Reverse transcriptase-polymerase chain reaction analysis demonstrated a wide tissue distribution of CXC-R4 and CC-R7 message in trout, being present in head-kidney leukocytes, blood, gill, brain, spleen, and liver.

Pertussis toxin-sensitive signal controls the trafficking of thymocytes across the corticomedullary junction in the thymus.

We investigated a role of chemokines in thymocyte trafficking. Genes encoding stromal cell-derived factor-1 and its receptor CXCR4 were detected in the cortex by in situ hybridization. Early immigrant cells did not express CXCR4, whereas their descendant CD44+CD25+CD4-CD8- cells did. CXCR4 expression was down-modulated when CD4+CD8+ double-positive cells became CD4+CD8- or CD4-CD8+ single-positive (SP) cells. Positively selected CD69+CD3intermediate cells gained CCR4, of which ligand, thymus activation-regulated chemokine, was expressed in the medulla. At the next developmental stage, CD69-CD3high cells lost CCR4 but gained CCR7. These results suggest that thymocytes use different chemokines along with their development. Blockade of chemokine receptor-mediated signaling by pertussis toxin perturbed the normal distribution of SP cells and resulted in the accumulation of SP cells in the cortex. Thus, a pertussis toxin-sensitive event controls the trafficking of SP cells across the corticomedullary junction.