Functional ramifications for the loss of P-selectin expression on hematopoietic and leukemic stem cells

Hematopoiesis is a tightly regulated biological process that relies upon complicated interactions between blood cells and their microenvironment to preserve the homeostatic balance of long-term hematopoietic stem cells (LT-HSCs), short-term HSCs (ST-HSCs), multipotent progenitors (MPPs), and differentiated cells. Adhesion molecules like P-selectin (encoded by the Selp gene) are essential to hematopoiesis, and their dysregulation has been linked to leukemogenesis. Like HSCs, leukemic stem cells (LSCs) depend upon their microenvironments for survival and propagation. P-selectin plays a crucial role in Philadelphia chromosome-positive (Ph(+)) chronic myeloid leukemia (CML). In this paper, we show that cells deficient in P-selectin expression can repopulate the marrow more efficiently than wild type controls. This results from an increase in HSC self-renewal rather than alternative possibilities like increased homing velocity or cell cycle defects. We also show that P-selectin expression on LT-HSCs, but not ST-HSCs and MPPs, increases with aging. In the absence of P-selectin expression, mice at 6 months of age possess increased levels of short-term HSCs and multipotent progenitors. By 11 months of age, there is a shift towards increased levels of long-term HSCs. Recipients of BCR-ABL-transduced bone marrow cells from P-selectin-deficient donors develop a more aggressive CML, with increased percentages of LSCs and progenitors. Taken together, our data reveal that P-selectin expression on HSCs and LSCs has important functional ramifications for both hematopoiesis and leukemogenesis, which is most likely attributable to an intrinsic effect on stem cell self-renewal.     

P-selectin glycoprotein ligand 1 promotes T cell lymphoma development and dissemination

P-selectin glycoprotein ligand-1 (PSGL-1) is a membrane-bound glycoprotein expressed in lymphoid and myeloid cells. It is a ligand of P-, E- and L-selectin and is involved in T cell trafficking and homing to lymphoid tissues, among other functions. PSGL-1 expression has been implicated in different lymphoid malignancies, so here we aimed to evaluate the involvement of PSGL-1 in T cell lymphomagenesis and dissemination. PSGL-1 was highly expressed at the surface of human and mouse T cell leukemia and lymphoma cell lines. To assess its impact on T cell malignancies, we stably expressed human PSGL-1 (hPSGL-1) in a mouse thymic lymphoma cell line, which expresses low levels of endogenous PSGL-1 at the cell surface. hPSGL-1-expressing lymphoma cells developed subcutaneous tumors in athymic nude mice recipients faster than control empty vector or parental cells. Moreover, the kidneys, lungs and liver of tumor-bearing mice were infiltrated by hPSGL-1-expressing malignant T cells. To evaluate the role of PSGL-1 in lymphoma cell dissemination, we injected intravenously control and hPSGL-1-expressing lymphoma cells in athymic mice. Strikingly, PSGL-1 expression facilitated disease infiltration of the kidneys, as determined by histological analysis and anti-CD3 immunohistochemistry. Together, these results indicate that PSGL-1 expression promotes T cell lymphoma development and dissemination to different organs.     

Functional role of P-selectin glycoprotein ligand 1/P-selectin interaction in the generation of tolerogenic dendritic cells

Dendritic cells (DCs) have a key role in both the generation of the immune response and the induction of tolerance to self-Ags. In this work, the possible role of P-selectin glycoprotein ligand 1 (PSGL-1) on the tolerogenic activity of human DCs was explored. We found that the engagement of PSGL-1 by P-selectin on DCs induced the expression of c-Fos, IDO, IL-10, and TGF-beta genes. Remarkably, stimulation of DCs through PSGL-1 with P-selectin enhanced their capability to generate CD4(+)CD25(+)Foxp3(+) regulatory T cells, which expressed high levels of TGF-beta1 mRNA, synthesized IL-10, and suppressed the proliferation of autologous CD4(+)CD25(-) T cells. Accordingly, we found that DCs from PSGL-1(-/-) mice expressed higher levels of MHC class II molecules, and exhibited an enhanced immunogenicity compared with wild-type mice. In addition, the percentage of CD4(+)CD25(+)Foxp3(+) regulatory T cells in the thymus of PSGL-1-deficient animals was significantly reduced. Our data reveal an unexpected role of PSGL-1 on the tolerogenic function of DCs, and the regulation of the immune response.     

P-selectin glycoprotein ligand 1 is not required for the development of experimental autoimmune encephalomyelitis in SJL and C57BL/6 mice

In multiple sclerosis and in its animal model experimental autoimmune encephalomyelitis (EAE), inflammatory cells migrate across the endothelial blood-brain barrier and gain access to the CNS. The involvement of P-selectin glycoprotein ligand 1 (PSGL-1) and of its major endothelial ligand P-selectin in this process have been controversial. In this study we demonstrate that although encephalitogenic T cells express functional PSGL-1, which can bind to soluble and immobilize P-selectin if presented in high concentrations, PSGL-1 is not involved T cell interaction with P-selectin expressing brain endothelial cells in vitro. Furthermore, neither anti-PSGL-1 Abs nor the lack of PSGL-1 in PSGL-1-deficient mice inhibits the recruitment of inflammatory cells across the blood-brain barrier or the development of clinical EAE. Taken together, our findings demonstrate that PSGL-1 is not required for the pathogenesis of EAE.     

PI3K is involved in β1 integrin clustering by PSGL-1 and promotes β1 integrin-mediated Jurkat cell adhesion to fibronectin

P-selectin glycoprotein ligand-1 (PSGL-1) is involved in the initial step of lymphocyte homing by interacting with P- or E-selectins expressed on activated endothelium cells. Besides, it also functions as a receptor to induce signals that increase integrin affinity to ligands and mediate cell adhesion to endothelium. Integrin is required for the second step of lymphocyte homing, whose activation has been reported tightly regulated by inside-out signals triggered by chemokines or the shear-stress generated during lymphocyte rolling on endothelium. However, the relationship between PSGL-1-triggered signals and integrin activation is not clear. In this study, we demonstrated that PSGL-1 ligation induces β1 integrin-mediated adhesion to fibronectin via regulation of both β1 subunit clustering and conformation changes. Phosphoinositide 3-kinase (PI3K) is required for PSGL-1-induced β1 integrin clustering which ultimately regulates β1 integrin-mediated Jurkat cell adhesion to fibronectin. However, PI3K is not involved in the conformation changes or increases in the total expression of β1 integrin. Taken together, we found a novel signal pathway, PSGL-1-PI3K-β1 integrin, demonstrating the cooperation between initial adhesion and subsequent arrest and stable adhesion.     

PSGL-1 regulates the migration and proliferation of CD8(+) T cells under homeostatic conditions

P-selectin glycoprotein ligand-1 (PSGL-1), a heavily glycosylated sialomucin expressed on most leukocytes, has dual function as a selectin ligand for leukocyte rolling on vascular selectins expressed in inflammation and as a facilitator of resting T cell homing into lymphoid organs. In this article, we document disturbances in T cell homeostasis present in PSGL-1(null) mice. Naive CD4(+) and CD8(+) T cell frequencies were profoundly reduced in blood, whereas T cell numbers in lymph nodes and spleen were at or near normal levels. Although PSGL-1(null) T cells were less efficient at entering lymph nodes, they also remained in lymph nodes longer than PSGL-1(+/+) T cells, suggesting that PSGL-1 supports T cell egress. In addition, PSGL-1(null) CD8(+) T cell proliferation was observed under steady-state conditions and PSGL-1(null) CD8(+) T cells were found to be hyperresponsive to homeostatic cytokines IL-2, IL-4, and IL-15. Despite these disturbances in T cell homeostasis, PSGL-1(null) mice exhibited a normal acute response (day 8) to lymphocytic choriomeningitis virus infection but generated an increased frequency of memory T cells (day 40). Our observations demonstrate a novel pleiotropic influence of PSGL-1 deficiency on several aspects of T cell homeostasis that would not have been anticipated based on the mild phenotype of PSGL-1(null) mice. These potentially offsetting effects presumably account for the near-normal cellularity seen in lymph nodes of PSGL-1(null) mice.     

Requirement for CD44 in homing and engraftment of BCR-ABL-expressing leukemic stem cells

In individuals with chronic myeloid leukemia (CML) treated by autologous hematopoietic stem cell (HSC) transplantation, malignant progenitors in the graft contribute to leukemic relapse, but the mechanisms of homing and engraftment of leukemic CML stem cells are unknown. Here we show that CD44 expression is increased on mouse stem-progenitor cells expressing BCR-ABL and that CD44 contributes functional E-selectin ligands. In a mouse retroviral transplantation model of CML, BCR-ABL1-transduced progenitors from CD44-mutant donors are defective in homing to recipient marrow, resulting in decreased engraftment and impaired induction of CML-like myeloproliferative disease. By contrast, CD44-deficient stem cells transduced with empty retrovirus engraft as efficiently as do wild-type HSCs. CD44 is dispensable for induction of acute B-lymphoblastic leukemia by BCR-ABL, indicating that CD44 is specifically required on leukemic cells that initiate CML. The requirement for donor CD44 is bypassed by direct intrafemoral injection of BCR-ABL1-transduced CD44-deficient stem cells or by coexpression of human CD44. Antibody to CD44 attenuates induction of CML-like leukemia in recipients. These results show that BCR-ABL-expressing leukemic stem cells depend to a greater extent on CD44 for homing and engraftment than do normal HSCs, and argue that CD44 blockade may be beneficial in autologous transplantation in CML.     

differential L-selectin binding activities of human hematopoietic cell L-selectin ligands, HCELL and PSGL-1

Expression of L-selectin on human hematopoietic cells (HC) is associated with a higher proliferative activity and a more rapid engraftment after hematopoietic stem cell transplantation. Two L-selectin ligands are expressed on human HCs, P-selectin glycoprotein ligand-1 (PSGL-1) and a specialized glycoform of CD44 (hematopoietic cell E- and L-selectin ligand, HCELL). Although the structural biochemistry of HCELL and PSGL-1 is well characterized, the relative capacity of these molecules to mediate L-selectin-dependent adhesion has not been explored. In this study, we examined under shear stress conditions L-selectin-dependent leukocyte adhesive interactions mediated by HCELL and PSGL-1, both as naturally expressed on human HC membranes and as purified molecules. By utilizing both Stamper-Woodruff and parallel-plate flow chamber assays, we found that HCELL displayed a 5-fold greater capacity to support L-selectin-dependent leukocyte adherence across a broad range of shear stresses compared with that of PSGL-1. Moreover, L-selectin-mediated leukocyte binding to immunopurified HCELL was consistently >5-fold higher than leukocyte binding to equivalent amounts of PSGL-1. Taken together, these data indicate that HCELL is a more avid L-selectin ligand than PSGL-1 and may be the preferential mediator of L-selectin-dependent adhesive interactions among human HCs in the bone marrow.     

P-selectin glycoprotein ligand 1 is a ligand for L-selectin on neutrophils, monocytes, and CD34+ hematopoietic progenitor cells

Selectins play a critical role in initiating leukocyte binding to vascular endothelium. In addition, in vitro experiments have shown that neutrophils use L-selectin to roll on adherent neutrophils, suggesting that they express a nonvascular L-selectin ligand. Using a L- selectin/IgM heavy chain (mu) chimeric protein as an immunocytological probe, we show here that L-selectin can bind to neutrophils, monocytes, CD34+ hematopoietic progenitors, and HL-60 and KG-1 myeloid cells. The interaction between L-selectin and leukocytes was protease sensitive and calcium dependent, and abolished by cell treatment with neuraminidase, chlorate, or O-sialoglycoprotein endopeptidase. These results revealed common features between leukocyte L-selectin ligand and the mucin-like P-selectin glycoprotein ligand 1 (PSGL-1), which mediates neutrophil rolling on P- and E-selectin. The possibility that PSGL-1 could be a ligand for L-selectin was further supported by the ability of P-selectin/mu chimera to inhibit L-selectin/mu binding to leukocytes and by the complete inhibition of both selectin interactions with myeloid cells treated with mocarhagin, a cobra venom metalloproteinase that cleaves the amino terminus of PSGL-1 at Tyr-51. Finally, the abrogation of L- and P-selectin binding to myeloid cells treated with a polyclonal antibody, raised against a peptide corresponding to the amino acid residues 42-56 of PSGL-1, indicated that L- and P-selectin interact with a domain located at the amino- terminal end of PSGL-1. The ability of the anti-PSGL-1 mAb PL-1 to inhibit L- and P-selectin binding to KG-1 cells further supported that possibility. Thus, apart from being involved in neutrophil rolling on P- and E-selectin, PSGL-1 also plays a critical role in mediating neutrophil attachment to adherent neutrophils. Interaction between L- selectin and PSGL-1 may be of major importance for increasing leukocyte recruitment at inflammatory sites.     

Definitive hematopoiesis initiates through a committed erythromyeloid progenitor in the zebrafish embryo

Shifting sites of blood cell production during development is common across widely divergent phyla. In zebrafish, like other vertebrates, hematopoietic development has been roughly divided into two waves, termed primitive and definitive. Primitive hematopoiesis is characterized by the generation of embryonic erythrocytes in the intermediate cell mass and a distinct population of macrophages that arises from cephalic mesoderm. Based on previous gene expression studies, definitive hematopoiesis has been suggested to begin with the generation of presumptive hematopoietic stem cells (HSCs) along the dorsal aorta that express c-myb and runx1. Here we show, using a combination of gene expression analyses, prospective isolation approaches, transplantation, and in vivo lineage-tracing experiments, that definitive hematopoiesis initiates through committed erythromyeloid progenitors (EMPs) in the posterior blood island (PBI) that arise independently of HSCs. EMPs isolated by coexpression of fluorescent transgenes driven by the lmo2 and gata1 promoters exhibit an immature, blastic morphology and express only erythroid and myeloid genes. Transplanted EMPs home to the PBI, show limited proliferative potential, and do not seed subsequent hematopoietic sites such as the thymus or pronephros. In vivo fate-mapping studies similarly demonstrate that EMPs possess only transient proliferative potential, with differentiated progeny remaining largely within caudal hematopoietic tissue. Additional fate mapping of mesodermal derivatives in mid-somitogenesis embryos suggests that EMPs are born directly in the PBI. These studies provide phenotypic and functional analyses of the first hematopoietic progenitors in the zebrafish embryo and demonstrate that definitive hematopoiesis proceeds through two distinct waves during embryonic development.     

Identification of an immunomodulating metalloprotease of Pseudomonas aeruginosa (IMPa)

Phagocytosis by neutrophils is the essential step in fighting Pseudomonas infections. The first step in neutrophil recruitment to the site infection is the interaction of P-selectin (on endothelial cells) with P-selectin glycoprotein ligand-1 (PSGL-1) on neutrophils. Pseudomonas aeruginosa secretes various proteases that degrade proteins that are essential for host defence, such as elastase and alkaline protease. Here we identify PA0572 of P. aeruginosa as an inhibitor of PSGL-1 and named this secreted hypothetical protease immunomodulating metalloprotease of P. aeruginosa or IMPa. Proteolytic activity was confirmed by cleavage of recombinant and cell-surface expressed PSGL-1. Functional inhibition was demonstrated by impaired PSGL-1-mediated rolling of IMPa-treated neutrophils under flow conditions. Next to PSGL-1, IMPa targets CD43 and CD44 that are also involved in leucocyte homing. These data indicate that IMPa prevents neutrophil extravasation and thereby protects P. aeruginosa from neutrophil attack.     

Dimerization of P-Selectin Glycoprotein Ligand-1 (PSGL-1) Required for Optimal Recognition of P-Selectin

Interactions between P-selectin, expressed on endothelial cells and activated platelets, and its leukocyte ligand, a homodimer termed P-selectin glycoprotein ligand-1 (PSGL-1), mediate the earliest adhesive events during an inflammatory response. To investigate whether dimerization of PSGL-1 is essential for functional interactions with P-selectin, a mutant form of PSGL-1 was generated in which the conserved membrane proximal cysteine was mutated to alanine (designated C320A). Western blotting under both denaturing and native conditions of the C320A PSGL-1 mutant isolated from stably transfected cells revealed expression of only a monomeric form of PSGL-1. In contrast to cells cotransfected with α1-3 fucosyltransferase-VII (FucT-VII) plus PSGL-1, K562 cells expressing FucT-VII plus C320A failed to bind COS cells transfected with P-selectin in a low shear adhesion assay, or to roll on CHO cells transfected with P-selectin under conditions of physiologic flow. In addition, C320A transfectants failed to bind chimeric P-selectin fusion proteins. Both PSGL-1 and C320A were uniformly distributed on the surface of transfected K562 cells. Thus, dimerization of PSGL-1 through the single, conserved, extracellular cysteine is essential for functional recognition of P-selectin.     

Direct real-time observation of E- and P-selectin-mediated rolling on cutaneous lymphocyte-associated antigen immobilized on Western blots

Human memory T cells associated with cutaneous inflammatory responses are characterized by their expression of cutaneous lymphocyte-associated Ag (CLA), a carbohydrate determinant differentially expressed on P-selectin glycoprotein ligand-1 (PSGL-1). Although expression of the CLA epitope on PSGL-1 (CLA(+) PSGL-1) by memory T cells is associated with acquisition of E-selectin ligand activity, it is not known whether CLA(+) PSGL-1, itself, is a ligand for E-selectin on human T cells or whether other glycoproteins, with or without CLA modification, support E-selectin-dependent rolling in shear flow. To address this issue, we developed a method for real-time analysis of functional adhesive interactions between selectin-bearing cells in shear flow with leukocyte ligands resolved by SDS-PAGE and immobilized on standard Western blots. The results of these studies provide direct evidence that CLA(+) PSGL-1 is a functional ligand for both E- and P-selectin, confirm that the P-selectin ligand activity of PSGL-1 is independent of CLA modification, and identify a distinct, non-PSGL-1 E-selectin ligand on CLA-positive human memory T cells.     

Functional analysis of the combined role of the O-linked branching enzyme core 2 beta1-6-N-glucosaminyltransferase and dimerization of P-selectin glycoprotein ligand-1 in rolling on P-selectin

Leukocyte P-selectin glycoprotein ligand-1 (PSGL-1) is expressed as a homodimer and mediates leukocyte rolling through interactions with endothelial P-selectin. Previous studies have shown that PSGL-1 must be properly modified by specific glycosyltransferases including alpha1,3-fucosyltransferase-VII, core 2 beta1-6-N-glucosaminyltransferase (C2GlcNAcT-I), one or more alpha2,3-sialytransferases, and a tyrosulfotransferase. In addition, dimerization of PSGL-1 through its sole extracellular cysteine (Cys(320)) is essential for rolling on P-selectin under shear conditions. In this report, we measured the contributions of both C2GlcNAcT-I glycosylation and dimerization of PSGL-1 to adhesive bonds formed during tethering and rolling of transfected cell lines on purified P-selectin. Tethering to P-selectin under flow increased with dimerization compared with cells expressing monomeric PSGL-1 (referred to as C320A). The rolling defects (decreased cellular accumulation, PSGL-1/P-selectin bond strengths and tethering rates, and increased velocities and skip distance) demonstrated by transfectants expressing monomeric PSGL-1 could be overcome by increasing the substrate P-selectin site density and by overexpressing C2GlcNAcT-I in C320A transfectants. Two molecular weight variants of PSGL-1 were isolated from cell lines transfected with PSGL-1, C320A, and/or C2GlcNAcT-I cDNAs, and these differences in electrophoretic mobility appeared to correlate with C2GlcNAcT-I expression. C320A transfectants expressing low molecular weight PSGL-1 had lower C2GlcNAcT-I levels (measured by reactivity to core 2 specific linkage antibody, CHO-131) and compromised rolling on P-selectin (regardless of site density) compared with C320A cells with high levels of C2GlcNAcT-I and high molecular weight PSGL-1. Both C2GlcNAcT-I glycosylation and PSGL-1 dimerization increased the rate of tethering to P-selectin under flow, whereas C2GlcNAcT-I levels primarily influenced tether bond strength.     

Characterization of glycoprotein ligands for P-selectin on a human small cell lung cancer cell line NCI-H345.

P-selectin (CD62P) is a cell adhesion molecule expressed on stimulated endothelial cells and on activated platelets. It interacts with PSGL-1 (P-selectin glycoprotein ligand-1; CD162) on leukocytes and mediates recruitment of leukocytes during inflammation. P-selectin also binds to several types of cancer cells in vitro and facilitates growth and metastasis of colon carcinoma in vivo. Here we show that P-selectin, but not E-selectin, binds to NCI-H345 cells, a cell line derived from a human small cell lung cancer. EDTA or P7 (a leukocyte adhesion blocking mAb to P-selectin), but not PL5 (a leukocyte adhesion blocking mAb to PSGL-1), can inhibit this binding. P-selectin affinity chromatography can precipitate a approximately 110-kDa major band and a approximately 220-kDa minor band from [3H]-glucosamine-labeled NCI-H345 cells. No expression of PSGL-1 protein and mRNA can be detected in NCI-H345 cells. Taken together, these results suggest that NCI-H345 cells express glycoprotein ligands for P-selectin that are distinct from leukocyte PSGL-1.     

P-selectin glycoprotein ligand-1 and E-selectin ligand-1 are differentially modified by fucosyltransferases Fuc-TIV and Fuc-TVII in mouse neutrophils

P-selectin glycoprotein ligand-1 (PSGL-1) and E-selectin ligand-1 (ESL-1) are the two major selectin ligands on mouse neutrophils. Transfection experiments demonstrate that each ligand requires alpha1,3-fucosylation for selectin-binding. However, the relative contributions made by the two known myeloid alpha1, 3-fucosyltransferases Fuc-TVII or Fuc-TIV to this alpha1, 3-fucosylation are not yet clear. To address this issue, we have used mice deficient in Fuc-TIV and/or Fuc-TVII to examine how these enzymes generate selectin-binding glycoforms of PSGL-1 and ESL-1 in mouse neutrophils. Selectin binding was analyzed by affinity isolation experiments using recombinant, antibody-like forms of the respective endothelial selectins. We observe essentially normal binding of E- or P-selectin to PSGL-1 expressed by Fuc-TIV-deficient neutrophils but find that PSGL-1 expressed by Fuc-TVII-deficient neutrophils is not bound by E- or P-selectin. By contrast, E-selectin binds with normal efficiency to ESL-1 on Fuc-TVII-deficient neutrophils but exhibits an 80% reduction in its ability to bind ESL-1 isolated from Fuc-TIV-deficient neutrophils. The same specificity with which Fuc-TVII and Fuc-TIV generate selectin-binding forms of PSGL-1 and ESL-1 was found in transfection experiments with CHO-Pro(-)5 cells. In contrast, each fucosyltransferase alone could generate selectin-binding glycoforms of each of the two ligands in CHO-DUKX-B1 cells. Our data imply that in mouse neutrophils and their precursors, Fuc-TVII exclusively directs expression of PSGL-1 glycoforms bound with high affinity by P-selectin. By contrast, Fuc-TIV preferentially directs expression of ESL-1 glycoforms that exhibit high affinity for E-selectin. This substrate specificity can be mimicked in CHO-Pro(-)5 cells.     

Bending rigidities of cell surface molecules P-selectin and PSGL-1

P-selectin is a cell adhesion molecule expressed on activated endothelial cells and platelets. P-selectin glycoprotein ligand 1 (PSGL-1) is a mucin expressed on leukocytes. The interaction of P-selectin and PSGL-1 mediates leukocyte tethering to and rolling on the vascular surface, which are initiating events in inflammatory and thrombotic processes. In the hemodynamic environment of the circulation, P-selectin and PSGL-1 are subject to a wide range of forces, which can cause deformation. For P-selectin/PSGL-1 interaction to be physically possible, these molecules may need to project above much of the glycocalyx layers of the respective cell surfaces, suggesting that they are either longer than the thickness of glycocalyx or better able to support compression than the glycocalyx. As such, the mechanical properties of these molecules and their functional implications merit investigation. Here we report determination of the bending rigidities of P-selectin and PSGL-1 by analyzing their thermally excited curvature fluctuations, whose values are of the order of magnitude of 100 pN nm².     

CD43 collaborates with P-selectin glycoprotein ligand-1 to mediate E-selectin-dependent T cell migration into inflamed skin

Activated T cell migration into nonlymphoid tissues is initiated by the interactions of P- and E-selectin expressed on endothelial cells and their ligands on T cells. P-selectin glycoprotein ligand-1 (PSGL-1) has been the only E-selectin ligand demonstrated to function during the in vivo migration of activated T cells. We show in this study that CD43-deficient Th1 cells, like PSGL-1-deficient cells, exhibited reduced E-selectin-binding activity compared with wild-type cells. Th1 cells with a PSGL-1 and CD43 double deficiency showed even less E-selectin-binding activity. In migration assays in which adoptively transferred cells migrate to inflamed skin P- and E-selectin dependently, CD43 contributed significantly to PSGL-1-independent Th1 cell migration. In addition, in vivo activated T cells from the draining lymph nodes of sensitized mice deficient in PSGL-1 and/or CD43 showed significantly decreased E-selectin-binding activity and migration efficiency, with T cells from double-deficient mice showing the most profound decrease. Collectively, these results demonstrate that the CD43 expressed on activated T cells functions as an E-selectin ligand and thereby mediates T cell migration to inflamed sites, in collaboration with PSGL-1.