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.     

Identification of equine P-selectin glycoprotein ligand-1 (CD162)

P-selectin glycoprotein ligand-1 (PSGL-1, CD162) is a dimeric, mucin-like, transmembrane glycoprotein constitutively expressed on leukocytes. A high baseline level of P-selectin expression in circulating equine platelets suggests a primed state toward inflammation and thrombosis via P-selectin/PSGL-1 adhesion. To investigate the potential role of equine P-selectin in these events, we first identified the cDNA sequence of equine PSGL-1 (ePSGL-1) using degenerate PCR and RACE-PCR and then compared the predicted sequence with that of human PSGL-1 (hPSGL-1). ePSGL-1 protein subunit is predicted to be 43 kDa and composed of 420 amino acids with a predicted 18-amino-acid signal sequence showing 78% homology to hPSGL-1. Previously published work has shown that binding of P-selectin requires sulfation of at least one of three tyrosines and O-glycosylation of one threonine in the N-terminus of human PSGL-1. However, the corresponding domain in ePSGL-1, spanning residues 19–43, contains only one tyrosine in the vicinity of two threonines at positions 25 and 41. ePSGL-1 contains 14 threonine/serine-rich decameric repeats as compared to hPSGL-1 which contains 14–16 threonine-rich decameric repeats. The transmembrane and cytoplasmic domains display 91% and 74% homology to corresponding human PSGL-1 domains, respectively. In summary, there is 71% homology in comparing the open reading frame (ORF) of ePSGL-1 with that of hPSGL-1. The greatest homologies between species exist in the transmembrane domain and cytoplasmic tail while substantial differences exist in the extracellular domain.The nucleotide sequence data reported in this article has been submitted to GenBank and assigned the accession number AY298766.     

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.     

CD43 functions as a ligand for E-Selectin on activated T cells

E-selectin, an inducible cell adhesion molecule expressed on endothelial cells, mediates the rolling on endothelium of leukocytes expressing E-selectin ligands, such as neutrophils and activated T cells. Although previous studies using mice lacking P-selectin glycoprotein ligand-1 (PSGL-1) have indicated that PSGL-1 on Th1 cells functions as an E-selectin ligand, the molecular nature of E-selectin ligands other than PSGL-1 remains unknown. In this study, we show that a 130-kDa glycoprotein was precipitated by an E-selectin-IgG chimera from mouse Th1 cells. This protein was cleaved by O-sialoglycoprotein endopeptidase and required sialic acid for E-selectin binding. The mAb 1B11, which recognizes the 130-kDa glycoform of CD43, recognized the 130-kDa band in the E-selectin-IgG precipitate. In addition, immunoprecipitation of the E-selectin-IgG precipitate with 1B11 depleted the 130-kDa protein, further confirming its identity as CD43. CD43 was also precipitated with E-selectin-IgG from cultured human T cells. E-selectin-dependent cell rolling on CD43 was observed under flow conditions using a CD43-IgG chimera generated in Chinese hamster ovary cells expressing alpha-1,3-fucosyltransferase VII and a core 2 beta-1,6-N-acetylglucosaminyltransferase. These results suggest that CD43, when modified by a specific set of glycosyltranferases, can function as an E-selectin ligand and therefore potentially mediate activated T cell migration into inflamed sites.     

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.     

T cell-associated CD18 but not CD62L, ICAM-1, or PSGL-1 is required for the induction of chronic colitis

The induction and perpetuation of chronic colitis are thought to involve a complex set of adhesive interactions between T cells and endothelial cells located on the vasculature within secondary lymphoid tissue and the intestine. The objective of this study was to assess the roles of T cell-associated CD18, CD62L (L-selectin), ICAM-1, and P-selectin glycoprotein ligand-1 (PSGL-1) in the induction of chronic colitis in mice. CD4(+)CD25(-) T cells derived from either wild-type (WT), CD18-deficient [CD18 knockout (KO)], CD62L KO, ICAM-1 KO, or PSGL-1 KO mice were adoptively transferred into recombinase activating gene-1 (RAG-1)-deficient mice (RAG KO mice) to assess the potential of these T cells to induce chronic colitis. At 8-10 wk following T cell transfer, we observed moderate to severe colitis as assessed by increases in colon weight-to-length ratios and by blinded histopathological analysis. In contrast, we found that transfer of CD18 KO T cells into RAG KO recipients resulted in the significant attenuation of colonic inflammation in these mice. Furthermore, we observed fewer infiltrating CD4(+) T cells in the colonic lamina propria in the CD18 KO-->RAG KO group compared with the WT-->RAG KO group. Finally, message levels of colonic TNF-alpha, IL-1beta, and IFN-gamma were significantly reduced in CD18 KO-->RAG KO mice compared with colitic control animals. We conclude that T cell-associated CD18, but not CD62L, ICAM-1, or PSGL-1, is required for the development of chronic colitis.     

Human P-selectin glycoprotein ligand-1 (PSGL-1) interacts with the skin-associated chemokine CCL27 via sulfated tyrosines at the PSGL-1 amino terminus

P-selectin glycoprotein ligand-1 (PSGL-1), a sialomucin expressed on leukocytes, is a major ligand for P-selectin and mediates leukocyte rolling on the endothelium. Here we show that human PSGL-1 interacts with CCL27 (CTACK/ILC/ESkine), a skin-associated chemokine that attracts skin-homing T lymphocytes. A recombinant soluble form of PSGL-1 (rPSGL-Ig) preferentially bound CCL27 among several chemokines tested. This interaction was abrogated by arylsulfatase treatment of rPSGL-Ig, suggesting that sulfated tyrosines play a critical role. In contrast, removal of either N-glycans or O-glycans by glycosidase treatment of rPSGL-Ig did not affect the interaction. The binding of CCL27 to a recombinant PSGL-1 synthesized in the presence of a sulfation inhibitor was lower than that produced in normal medium. Moreover, mutation of the tyrosines at the amino terminus of PSGL-1 to phenylalanine abolished the binding, further supporting the role of sulfated tyrosines in the CCL27-PSGL-1 interaction. Functionally, rPSGL-Ig reduced the chemotaxis of L1.2 cells expressing CCR10, the receptor for CCL27. In addition, the expression of human PSGL-1 on CCR10-expressing L1.2 cells resulted in reduced chemotaxis to CCL27. These findings suggest a role for PSGL-1 in regulating chemokine-mediated responses, in addition to its role as a selectin ligand.     

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.     

Expression of Epstein-Barr virus nuclear antigen-1 induces B cell neoplasia in transgenic mice.

The Epstein-Barr virus (EBV) nuclear antigen-1 (EBNA-1) is a pleiotropic protein which has been characterized extensively both biochemically and functionally. It is the only one of the identified latent protein-encoding genes to be consistently expressed in viral-associated endemic Burkitt's lymphoma cells. As such, it is the only candidate viral protein to possibly perform a maintenance function in the tumour pathology. Despite this, no oncogenic activity has been attributed to the protein in tissue culture assays. The experiments described here were initiated to explore the activity of the protein in B cells in vivo. EBNA-1 transgenic mice were generated with transgene expression directed to the B cell compartment using the mouse Ig heavy chain intron enhancer. Transgene expression was demonstrated in the lymphoid tissues of mice of two independent lines. Transgenic positive mice of both lines succumb to B cell lymphoma. The B cell tumours are monoclonal, frequently of follicular centre cell origin and remarkably similar to those induced by transgenic c-myc expression. These results demonstrate that EBNA-1 is oncogenic in vivo and suggest that the gene product may play a direct role in the pathogenesis of Burkitt's lymphoma and possibly other EBV-associated malignancies.     

Structural basis for apoptosis inhibition by Epstein-Barr virus BHRF1

Epstein-Barr virus (EBV) is associated with human malignancies, especially those affecting the B cell compartment such as Burkitt lymphoma. The virally encoded homolog of the mammalian pro-survival protein Bcl-2, BHRF1 contributes to viral infectivity and lymphomagenesis. In addition to the pro-apoptotic BH3-only protein Bim, its key target in lymphoid cells, BHRF1 also binds a selective sub-set of pro-apoptotic proteins (Bid, Puma, Bak) expressed by host cells. A consequence of BHRF1 expression is marked resistance to a range of cytotoxic agents and in particular, we show that its expression renders a mouse model of Burkitt lymphoma untreatable. As current small organic antagonists of Bcl-2 do not target BHRF1, the structures of it in complex with Bim or Bak shown here will be useful to guide efforts to target BHRF1 in EBV-associated malignancies, which are usually associated with poor clinical outcomes.     

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.     

A protective role for human IL-10-expressing CD4+ T cells in colitis

IL-10 is an immunoregulatory cytokine expressed by numerous cell types. Studies in mice confirm that different IL-10-expressing cell subsets contribute differentially to disease phenotypes. However, little is known about the relationship between cell- or tissue-specific IL-10 expression and disease susceptibility in humans. In this study, we used the previously described human (h)IL10BAC transgenic model to examine the role of hIL-10 in maintaining intestinal homeostasis. Genomically controlled hIL-10 expression rescued Il10(-/-) mice from Helicobacter-induced colitis and was associated with control of proinflammatory cytokine expression and Th17 cell accumulation in gut tissues. Resistance to colitis was associated with an accumulation of hIL-10-expressing CD4(+)Foxp3(+) regulatory T cells specifically within the lamina propria but not other secondary lymphoid tissues. Cotransfer of CD4(+)CD45RB(lo) cells from Il10(-/-)/hIL10BAC mice rescued Rag1(-/-) mice from colitis, further suggesting that CD4(+) T cells represent a protective source of hIL-10 in the colon. In concordance with an enhanced capacity to express IL-10, CD4(+)CD44(+) T cells isolated from the lamina propria exhibited lower levels of the repressive histone mark H3K27Me3 and higher levels of the permissive histone mark acetylated histone H3 in both the human and mouse IL10 locus compared with the spleen. These results provide experimental evidence verifying the importance of T cell-derived hIL-10 expression in controlling inflammation within the colonic mucosa. We also provide molecular evidence suggesting the tissue microenvironment influences IL-10 expression patterns and chromatin structure in the human (and mouse) IL10 locus.     

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.     

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.     

Demonstration of a CD3+ lymphocyte subset in the epidermis of athymic nude mice. Evidence for T cell receptor diversity.

The existence of CD3/TCR-bearing lymphocytes in athymic and thymectomized chimeric mice implies that T cell maturation can occur in the absence of a thymus. Considering the possibility that the epidermis may be one of the organs providing T cell educating stimuli, we attempted to characterize the Thy-1+ epidermal lymphocyte population of athymic mice. Immunohistologic studies of epidermal sheets revealed (1) that Thy-1+ epidermal cells of C57BL/6 nu/nu mice are CD5-, CD4-, and predominantly CD8-, and (2) that a minor subset of these cells displays anti-CD3 epsilon reactivity. Although these CD3+ epidermal cells could hardly be detected at 6 wk of age, they comprised approximately 2% of all Thy-1+ epidermal cells in 12-mo-old athymic mice. Most of these CD3+ cells expressed TCR-gamma/delta, but TCR-alpha/beta+ cells were also present. TCR-gamma/delta+ epidermal T cells of athymic mice preferentially expressed TCR V gamma 2, V gamma 4, and V gamma 5 specificities rather than TCR V gamma 3 as found on DETC of euthymic mice. Using mitogenic stimuli, we have succeeded in establishing cell lines and clones from BALB/c nu/nu and C57BL/6 nu/nu epidermis. Their marker profile corresponds to that seen on resident CD3+ epidermal cells, as well as on a very small subset of CD3+ splenic and lymph node lymphocytes of athymic mice. The ontogenetic relationship, if any, between the epidermal and lymphoid CD3+, CD5-, CD4-, CD8- cells, has yet to be clarified. Cell lines/clones representative of resident CD3+ epidermal cells of nu/nu mice should provide a useful tool in the elucidation of homing patterns and functional properties of extrathymically matured T cells.     

CD43 plays both antiadhesive and proadhesive roles in neutrophil rolling in a context-dependent manner

As the first step in the recruitment of neutrophils into tissues, the cells become tethered to and roll on the vessel wall. These processes are mediated by interactions between the P- and E-selectins, expressed on the endothelial cells of the vessel wall, and their ligands, expressed on the neutrophils. Recently, we reported that CD43 on activated T cells functions as an E-selectin ligand and thereby mediates T cell migration to inflamed sites, in collaboration with P-selectin glycoprotein ligand-1 (PSGL-1), a major P- and E-selectin ligand. Here, we examined whether CD43 on neutrophils also functions as an E-selectin ligand. CD43 was precipitated with an E-selectin-IgG chimera from mouse bone marrow neutrophils. A CD43 deficiency diminished the E-selectin-binding activity of neutrophils when PSGL-1 was also deficient. Intravital microscopy showed that the CD43 deficiency significantly increased leukocyte rolling velocities in TNF-alpha-stimulated venules blocked with an anti-P-selectin mAb, where the rolling was mostly E-selectin dependent, when PSGL-1 was also absent. In contrast, in venules with trauma-induced inflammation, where the rolling was largely P-selectin dependent, the CD43 deficiency reduced leukocyte rolling velocities. Collectively, these observations suggest that CD43 generally serves as an antiadhesive molecule to attenuate neutrophil-endothelial interactions, but when E-selectin is expressed on endothelial cells, it also plays a proadhesive role as an E-selectin ligand.     

Characterization of T cell clones from an athymic mouse.

Although Thy-1+ lymphocytes have been observed in lymphoid tissues of athymic mice, attempts to analyze these cells on the clonal level have previously yielded only populations of CD4-CD8+ cytolytic T cells. Furthermore, studies of responses of these cells to various mitogenic stimuli have demonstrated significant defects in the ability of these cells to proliferate in culture. We report here on the cloning and maintenance in long term culture of T cells from an athymic mouse stimulated in vitro with allogeneic spleen cells. Of 10 Thy-1+ clones, 7 CD4+CD8- and 3 CD4-CD8+ Ag-specific cells were obtained. Among the CD4+ T cells, we observed a variety of specificities, including an autoreactive I-Aq specific clone, a minor lymphocyte stimulating determinant (Mls)-reactive clone, and five allo-I-Ad-specific CD4+ clones; a class II-specific CD4-CD8+ clone was also obtained. In addition, we observed two Thy-1-CD3+ clones (one of which is also CD4+ and expresses V beta 8) which are constitutively responsive to the lymphokines IL-2 and IL-4. Of 11 clones tested, 7 produce IL-2 and/or IL-4 lymphokines after stimulation through the TCR, whereas 4 do not, requiring exogenous lymphokines for optimal responses to Ag. Of 10 clones tested for IL-2R expression, 3 had notably low levels, correlating with low proliferative responses to IL-2. The results reveal the spectrum of T cells available to a mouse which is congenitally athymic and describe the heterogeneity of immune defects expressed in such cells at the clonal level.     

Toward a comprehensive quantitative proteome database: protein expression map of lymphoid neoplasms by 2-D DIGE and MS

Using 2-D DIGE, we constructed a quantitative 2-D database including 309 proteins corresponding to 389 protein spots across 42 lymphoid neoplasm cell lines. The proteins separated by 2-D PAGE were identified by MS and assigned to the expression data obtained by 2-D DIGE. The cell lines were categorized into four groups: those from Hodgkin's lymphoma (HL) (4 cell lines), B cell malignancies (19 cell lines), T cell malignancies (16 cell lines), and natural killer (NK) cell malignancies (3 cell lines). We characterized the proteins in the database by classifying them according to their expression level. We found 28 proteins with more than a 2-fold difference between the cell line groups. We also noted the proteins that allowed multidimensional separation to be achieved (1) between HL cells and other cells, (2) between the cells derived from B cells, T cells and NK cells, and (3) between HL cells and anaplastic large cell lymphoma cells. Decision tree classification identified five proteins that could be used to classify the 42 cell lines according to differentiation. These results suggest that the quantitative 2-D database using 2-D DIGE will be a useful resource for studying the mechanisms underlying the differentiation phenotypes of lymphoid neoplasms.