Localization, Shedding, Regulation and Function of Aminopeptidase N/CD13 on Fibroblast like Synoviocytes.

Aminopeptidase N/CD13 is highly expressed by fibroblast like synoviocytes (FLS) and may play a role in rheumatoid arthritis (RA). CD13 was previously detected in human synovial fluid where it was significantly increased in RA compared to osteoarthritis. In this study we found that CD13 in biological fluids (plasma, synovial fluid, FLS culture supernatant) is present as both a soluble molecule and on extracellular vesicles, including exosomes, as assessed by differential ultracentrifugation and density gradient separation. Having determined CD13 could be released as a soluble molecule from FLS, we examined potential mechanisms by which CD13 might be shed from the FLS membrane. The use of protease inhibitors revealed that CD13 is cleaved from the FLS surface by metalloproteinases. siRNA treatment of FLS revealed one of those proteases to be MMP14. We determined that pro-inflammatory cytokines (TNFα, IFNγ, IL-17) upregulated CD13 mRNA in FLS, which may contribute to the increased CD13 in RA synovium and synovial fluid. Inhibition of CD13 function by either inhibitors of enzymatic activity or anti-CD13 antibodies resulted in decreased growth and diminished migration of FLS. This suggests that CD13 may be involved in the pathogenic hyperplasia of RA FLS. This data expands potential roles for CD13 in the pathogenesis of RA.

Expression and function of aminopeptidase N/CD13 produced by fibroblast-like synoviocytes in rheumatoid arthritis: role of CD13 in chemotaxis of cytokine-activated T cells independent of enzymatic activity.

OBJECTIVE: Aminopeptidase N/CD13 (EC 3.4.11.2) is a metalloproteinase expressed by fibroblast-like synoviocytes (FLS). It has been suggested that CD13 can act chemotactically for T cells in rheumatoid arthritis (RA). We undertook this study to measure CD13 in vivo and in vitro in RA samples and to determine whether CD13 could play a role in the homing of T cells to the RA joint. METHODS: Interleukin-17-treated FLS were used to immunize mice, from which a novel anti-human CD13 monoclonal antibody (mAb), 591.1D7.34, was developed. The mAb 591.1D7.34 and a second anti-CD13 mAb, WM15, were used to develop a novel enzyme-linked immunosorbent assay (ELISA) for CD13, and CD13 enzymatic activity was measured in parallel. Chemotaxis of cytokine-activated T cells was measured by a chemotaxis-under-agarose assay. RESULTS: We detected substantial amounts of CD13 in synovial fluid (SF), sera, FLS lysates, and culture supernatants by ELISA, with a significant increase in CD13 in RA SF when compared to osteoarthritis SF. CD13 accounted for most but not all of the CD13-like enzymatic activity in SF. Recombinant human CD13 was chemotactic for cytokine-activated T cells through a G protein-coupled receptor and contributed to the chemotactic properties of SF independently of enzymatic activity. CONCLUSION: CD13 is released from FLS into culture supernatants and is found in SF. CD13 induces chemotaxis of cytokine-activated T cells, a T cell population similar to that found in RA synovium. These data suggest that CD13 could play an important role as a T cell chemoattractant, in a positive feedback loop that contributes to RA synovitis.

Synovial phenotypes in rheumatoid arthritis correlate with response to biologic therapeutics.

INTRODUCTION: Rheumatoid arthritis (RA) is a complex and clinically heterogeneous autoimmune disease. Currently, the relationship between pathogenic molecular drivers of disease in RA and therapeutic response is poorly understood. METHODS: We analyzed synovial tissue samples from two RA cohorts of 49 and 20 patients using a combination of global gene expression, histologic and cellular analyses, and analysis of gene expression data from two further publicly available RA cohorts. To identify candidate serum biomarkers that correspond to differential synovial biology and clinical response to targeted therapies, we performed pre-treatment biomarker analysis compared with therapeutic outcome at week 24 in serum samples from 198 patients from the ADACTA (ADalimumab ACTemrA) phase 4 trial of tocilizumab (anti-IL-6R) monotherapy versus adalimumab (anti-TNFα) monotherapy. RESULTS: We documented evidence for four major phenotypes of RA synovium - lymphoid, myeloid, low inflammatory, and fibroid - each with distinct underlying gene expression signatures. We observed that baseline synovial myeloid, but not lymphoid, gene signature expression was higher in patients with good compared with poor European league against rheumatism (EULAR) clinical response to anti-TNFα therapy at week 16 (P =0.011). We observed that high baseline serum soluble intercellular adhesion molecule 1 (sICAM1), associated with the myeloid phenotype, and high serum C-X-C motif chemokine 13 (CXCL13), associated with the lymphoid phenotype, had differential relationships with clinical response to anti-TNFα compared with anti-IL6R treatment. sICAM1-high/CXCL13-low patients showed the highest week 24 American College of Rheumatology (ACR) 50 response rate to anti-TNFα treatment as compared with sICAM1-low/CXCL13-high patients (42% versus 13%, respectively, P =0.05) while anti-IL-6R patients showed the opposite relationship with these biomarker subgroups (ACR50 20% versus 69%, P =0.004). CONCLUSIONS: These data demonstrate that underlying molecular and cellular heterogeneity in RA impacts clinical outcome to therapies targeting different biological pathways, with patients with the myeloid phenotype exhibiting the most robust response to anti-TNFα. These data suggest a path to identify and validate serum biomarkers that predict response to targeted therapies in rheumatoid arthritis and possibly other autoimmune diseases. TRIAL REGISTRATION: ClinicalTrials.gov NCT01119859

The roles of IFN-γ versus IL-17 in pathogenic effects of human Th17 cells on synovial fibroblasts.

OBJECTIVES: Th17 cells, while indispensable in host defense, may play pathogenic roles in many autoimmune diseases, including rheumatoid arthritis (RA). However, the mechanisms by which human Th17 cells drive autoimmunity have not been fully defined. We assessed the potential of the human Th17 CD4 T cell subset to induce expression of cell-cell interaction molecules and inflammatory mediators by fibroblast-like synoviocytes (FLS), and the roles of IFN-γ and IL-17 in these interactions. METHODS: Th1 or Th17 cells were induced from healthy adult donor CD4 T cells and were co-cultured with FLS for 48 h with/without neutralization of IFN-γ, IL-17A, or both. Alternatively, FLS were treated only with IFN-γ or IL-17 for 48 h. FLS expression of CD40, CD54, and MHC-II, as well as IL-6 and IL-8 secretion, were assessed by surface staining followed by flow cytometry and ELISA, respectively. RESULTS: Both Th1 and Th17 cells secreted IL-17 as well as IFN-γ, although IFN-γ production was much greater from Th1 cells. FLS expression of CD40, CD54, and MHC-II significantly increased upon co-culture with Th1 cells, while Th17 cells increased only the percentage of FLS that were CD54+. Both T cell subsets induced IL-6 and IL-8 secretion by RA FLS. Neutralization of IL-17A did not reduce FLS expression of CD40, MHC-II, or CD54, but did inhibit IL-6 and IL-8 secretion. Although IFN-γ was a weak inducer of IL-6 secretion and significantly inhibited IL-8 secretion from FLS when used as a single stimulus, neutralization of IFN-γ inhibited the secretion of both cytokines in Th17/FLS co-cultures with RA but not OA FLS. CONCLUSION: FLS cell-cell interaction molecules and soluble inflammatory mediators are differentially regulated by IFN-γ and IL-17. The effects of IFN-γ may depend in part on the particular milieu of other co-existing cytokines and its potential to induce cell-cell interactions. The potential benefit of therapeutic neutralization of either IL-17 or IFN-γ could depend on the relative proportions of these cytokines in the synovial compartment of an RA patient.

Sensitivity and resistance to regulation by IL-4 during Th17 maturation.

Th17 cells are highly pathogenic in a variety of immune-mediated diseases, and a thorough understanding of the mechanisms of cytokine-mediated suppression of Th17 cells has great therapeutic potential. In this article, we characterize the regulation of both in vitro- and in vivo-derived Th17 cells by IL-4. We demonstrate that IL-4 suppresses reactivation of committed Th17 cells, even in the presence of TGF-ß, IL-6, and IL-23. Downregulation of IL-17 by IL-4 is dependent on STAT6 and mediated by inhibition of STAT3 binding at the Il17a promoter. Although Th1 cytokines were shown to induce IFN-γ expression by Th17 cells, IL-4 does not induce a Th2 phenotype in Th17 cells. Suppression by IL-4 is stable and long-lived when applied to immature Th17 cells, but cells that have undergone multiple rounds of stimulation, either in vivo during a Th17-mediated inflammatory disease, or in vitro, become resistant to suppression by IL-4 and lose the ability to signal through IL-4R. Thus, although IL-4 is a potent suppressor of the Th17 genetic program at early stages after differentiation, prolonged stimulation renders Th17 cells impervious to regulatory cytokines.

A polymorphism in the interleukin-4 receptor affects the ability of interleukin-4 to regulate Th17 cells: a possible immunoregulatory mechanism for genetic control of the severity of rheumatoid arthritis.

INTRODUCTION: Rheumatoid arthritis (RA) is now suspected to be driven by pathogenic Th17 cells that secrete interleukin (IL)-17 and can be regulated by IL-4. A single-nucleotide polymorphism (SNP), I50V, in the coding region of the human IL-4 receptor (IL-4R) is associated with rapid development of erosive disease in RA. The present study was undertaken to determine whether this SNP renders the IL-4R less able to transduce signals that regulate IL-17 production. METHODS: Peripheral blood mononuclear cells were activated under Th17-stimulating conditions in the presence or absence of IL-4, and IL-17 production was measured by both enzyme-linked immunosorbent assay (ELISA) and flow cytometry. Serum IL-17 was also measured by ELISA. Paired comparisons were performed using the two-tailed t-test. IL-4 receptor gene alleles were determined by polymerase chain reaction. RESULTS: In healthy individuals, IL-4 significantly inhibited IL-17 production by cells from subjects with the I/I genotype (P = 0.0079) and the I/V genotype (P = 0.013), but not the V/V genotype (P > 0.05). In a cross-sectional sample of patients with established RA, the magnitude of the in vitro effect of IL-4 was lower and was not associated with a specific IL-4R allele. Serum IL-17 levels were higher in RA patients than in healthy individuals, as was the percentage of CD4+ cells that produced IL-17. CONCLUSIONS: These results indicate that an inherited polymorphism of the IL-4R controls the ability of the human immune system to regulate the magnitude of IL-17 production. However, in established RA, this pattern may be altered, possibly due to secondary effects of both RA itself as well as immunomodulatory medications. Ineffective control of Th17 immune responses is a potential mechanism to explain why IL-4R is an important severity gene in RA, but this issue will require careful study of a cohort of new-onset RA patients.

Regulation of pathogenic IL-17 responses in collagen-induced arthritis: roles of endogenous interferon-gamma and IL-4.

INTRODUCTION: Interleukin (IL)-17 plays an important role in the pathogenesis of rheumatoid arthritis and the mouse model collagen-induced arthritis (CIA). Interferon(IFN)-gamma and IL-4 have been shown to suppress Th17 development in vitro, but their potential immunoregulatory roles in vivo are uncertain. The goals of this study were to determine the relationship between Th17 responses and disease severity in CIA and to assess regulation of IL-17 by endogenous IFN-gamma and IL-4. METHODS: DBA1/LacJ mice were immunized with type II collagen in complete Freund's adjuvant (CFA) to induce arthritis, and treated with neutralizing antibody to IFN-gamma and/or IL-4. Systemic IL-17, IFN-gamma, and IL-4 were measured in serum. At the peak of disease, cytokine production was measured by ELISA of supernatants from spleen, lymph node and paw cultures. Paws were also scored for histologic severity of arthritis. RESULTS: Joint inflammation was associated with a higher ratio of systemic IL-17/IFN-gamma. Neutralization of IFN-gamma accelerated the course of CIA and was associated with increased IL-17 levels in the serum and joints. The IFN-gamma/IL-4/IL-17 responses in the lymphoid organ were distinct from such responses in the joints. Neutralization of IL-4 led to increased arthritis only in the absence of IFN-gamma and was associated with increased bone and cartilage damage without an increase in the levels of IL-17. CONCLUSIONS: IL-4 and IFN-gamma both play protective roles in CIA, but through different mechanisms. Our data suggests that the absolute level of IL-17 is not the only determinant of joint inflammation. Instead, the balance of Th1, Th2 and Th17 cytokines control the immune events leading to joint inflammation.

Interactions of T cells with fibroblast-like synoviocytes: role of the B7 family costimulatory ligand B7-H3.

Fibroblast-like synoviocytes (FLS) and T cells can activate each other in vitro, and in vivo interactions between these cells may be important in rheumatoid arthritis (RA), yet FLS lack significant expression of CD28 ligands. We sought to identify molecules homologous to CD28 ligands that are strongly expressed by FLS, and documented strong B7-H3 expression on FLS and by fibroblasts of other tissues, which was unaffected by a variety of cytokines. Western blot analysis of FLS lysates showed predominant expression of the larger, four Ig-like domain isoform of B7-H3. Immunohistological sections of RA synovial tissue showed strong staining for B7-H3 on FLS. Cells expressing B7-H3 were distinct from but in close proximity to cells that expressed CD45, CD20, and CD3. Confocal microscopy of FLS and T cell cocultures showed localization of B7-H3 in the region of the T cell-FLS contact point, but distinct from the localization of T cell CD11a/CD18 (LFA-1) and FLS CD54 (ICAM-1). Reduction of B7-H3 expression on FLS by RNA interference affected interactions of FLS with resting T cells or cytokine-activated T cells. Resting T cells showed increased production of TNF-alpha, IFN-gamma, and IL-2, whereas cytokine-activated T cells showed reduced cytokine production relative to control. However, cytokine production by T cells activated through their TCR was not notably altered by knock down of B7-H3. These observations suggest that B7-H3 may be important for the interactions between FLS and T cells in RA, as well as other diseases, and the outcome of such interactions depends on the activation state of the T cell.

Molecular interactions between T cells and fibroblast-like synoviocytes: role of membrane tumor necrosis factor-alpha on cytokine-activated T cells.

The mechanism of fibroblast-like synoviocyte (FLS) transformation into an inflammatory phenotype in rheumatoid arthritis (RA) is not fully understood. FLS interactions with invading leukocytes, particularly T cells, are thought to be a critical component of this pathological process. Resting T cells and T cells activated through the T-cell receptor have previously been shown to induce inflammatory cytokine production by FLS. More recently, a distinct population of T cells has been identified in RA synovium that phenotypically resembles cytokine-activated T (Tck) cells. Using time lapse microscopy, the interactions of resting, superantigen-activated, and cytokine-activated T cells with FLS were visualized. Rapid and robust adhesion of Tck and superantigen-activated T cells to FLS was observed that resulted in flattening of the T cells and a crawling movement on the FLS surface. Tck also readily activated FLS to produce interleukin IL-6 and IL-8 in a cell contact-dependent manner that was enhanced by exogenous IL-17. Although LFA-1 and ICAM-1 co-localized at the Tck-FLS synapse, blocking the LFA-1/ICAM-1 interaction did not substantially inhibit Tck effector function. However, antibody blocking of membrane tumor necrosis factor (TNF)-alpha on the Tck surface did inhibit FLS cytokine production, thus illustrating a novel mechanism for involvement of TNF-alpha in cell-cell interactions in RA synovium and for the effectiveness of TNF-alpha blockade in the treatment of RA.

Presentation of arthritogenic peptide to antigen-specific T cells by fibroblast-like synoviocytes.

OBJECTIVE: To assess the ability of rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) to function as antigen-presenting cells (APCs) for arthritogenic autoantigens found within inflamed joint tissues. METHODS: Human class II major histocompatibility complex (MHC)-typed FLS were used as APCs for murine class II MHC-restricted CD4 T cell hybridomas. Interferon-gamma (IFNgamma)-treated, antigen-loaded FLS were cocultured with T cell hybridomas specific for immunodominant portions of human cartilage gp-39 (HC gp-39) or human type II collagen (CII). T cell hybridoma activation was measured by enzyme-linked immunosorbent assay of culture supernatants for interleukin-2. Both synthetic peptide and synovial fluid (SF) were used as sources of antigen. APC function in cocultures was inhibited by using blocking antibodies to human class II MHC, CD54, or CD58, or to murine CD4, CD11a, or CD2. RESULTS: Human FLS could present peptides from the autoantigens HC gp-39 and human CII to antigen-specific MHC-restricted T cell hybridomas. This response required pretreatment of FLS with IFNgamma, showed MHC restriction, and was dependent on human class II MHC and murine CD4 for effective antigen presentation. Furthermore, FLS were able to extract and present antigens found within human SF to both the HC gp-39 and human CII T cell hybridomas in an IFNgamma-dependent and MHC-restricted manner. CONCLUSION: RA FLS can function as APCs and are able to present peptides derived from autoantigens found within joint tissues to activated T cells in vitro. In the context of inflamed synovial tissues, FLS may be an important and hitherto overlooked subset of APCs that could contribute to autoreactive immune responses.

Interleukin-17 as a molecular target in immune-mediated arthritis: immunoregulatory properties of genetically modified murine dendritic cells that secrete interleukin-4.

OBJECTIVE: Our previous studies have shown that murine dendritic cells (DCs) genetically modified to express interleukin-4 (IL-4) reduce the incidence and severity of murine collagen-induced arthritis. The present studies were performed to assess the immunoregulatory mechanisms underlying this response, by assessing the effects of IL-4 DCs on cytokine production by subsets of T helper cells. METHODS: Male DBA mice ages 6-8 weeks old were immunized with type II collagen. Splenic T cells obtained during the initiation phase and the end stage of arthritis were cultured with IL-4 DCs or untransduced DCs in the presence of collagen rechallenge. Interferon-gamma (IFNgamma) and IL-17 responses were measured. Antibodies to IL-4, IL-12, and IL-23, and recombinant IL-4, IL-12, and IL-23 were used to further study the regulation of T cell cytokine production by IL-4 DCs. RESULTS: Splenic T cells obtained during the initiation phase of arthritis produced less IL-17 when cultured in the presence of IL-4 DCs, despite their production of increased quantities of other proinflammatory cytokines (IFNgamma and tumor necrosis factor). T cell IL-17 production after collagen rechallenge was not inhibited by a lack of IL-23, since IL-4-mediated suppression of IL-17 was not reconstituted by IL-23, an otherwise potent inducer of IL-17 production by T cells. Although IL-4 DCs can produce increased quantities of IL-12 and IFNgamma, suppression of IL-17 production by IL-4 DCs was independent of both. While IL-17 production by T cells obtained during the initiation phase of arthritis was regulated by IL-4 DCs, IL-17 production by T cells obtained during end-stage arthritis was not altered. CONCLUSION: Our data suggest that IL-4 DCs exert a therapeutic effect on collagen-induced arthritis by targeting IL-17. IL-17 suppression by IL-4 DCs is robust and is not reversed by IL-23. Timing might be important in IL-17-targeted therapy, since IL-17 production by T cells obtained during end-stage arthritis did not respond to suppression by IL-4 DCs.

Immunocompetent properties of human osteoblasts: interactions with T lymphocytes.

UNLABELLED: We sought to determine whether osteoblasts (OBs) can serve as accessory cells (ACs) for T-cell activation and whether T cells directly activate OB production of IL-6, using primary human OBs (NHOst), the transformed fetal osteoblast line hFOB1.19, and an osteosarcoma line SaOS-2. Robust, bidirectional activating interactions were shown using each of these three human ostoblast lines. INTRODUCTION: Osteoblasts (OBs) could come into contact with lymphocytes during inflammatory joint destruction and fracture repair. MATERIALS AND METHODS: We used several in vitro assays to assess the ability of T cells and OBs to interact in the generation of immune and inflammatory responses. RESULTS: By flow cytometry, three OB cell lines all were found to express ligands for T-cell co-stimulation. The integrin ligand CD54/ICAM-1 was constitutively expressed by hFOB1.19 and NHOst and was upregulated on SaOS-2 by IFN-gamma. MHC Class II was upregulated on all three lines by IFN-gamma. CD166/ALCAM, a ligand of the T-cell molecule CD6, was constitutively expressed on all three lines. A second putative CD6 ligand designated 3A11 was expressed on hFOB1.19 and NHOst, but not consistently on SaOS-2. The ectoenzyme CD26 (dipeptidyl peptidase IV) was expressed on hFOB1.19 and NHOst, but not on SaOS-2. All three cell lines presented superantigen to T cells, especially after treatment with IFN-gamma. Superantigen presentation was inhibited by antibodies to the leukocyte integrin CD11a/CD18 (LFA-1), MHC Class II, and CD54/ICAM-1. T cells, particularly when cytokine activated for 7 days before co-culture, stimulated all three osteoblast lines to produce interleukin (IL)-6, and this effect was boosted when IL-17 was added to the co-cultures with either resting T cells or cytokine-activated T cells. CONCLUSIONS: Bidirectional activating interactions are readily shown between human T cells and several types of human OBs. The expression by OBs of ligands for the T cell-specific molecule CD6, as well as other molecules involved in immune interactions, strongly suggests that such in vitro interactions are representative of physiologic or pathologic events that occur in vivo.

Expression and characterization of a novel CD6 ligand in cells derived from joint and epithelial tissues.

CD6 is a T cell surface glycoprotein that plays an important role in interactions of thymocytes with thymic epithelial cells and in mature T cell interactions with selected nonprofessional tissue APCs. We describe a novel CD6 ligand (CD6L) 3A11 Ag that is distinct from the known CD6L (CD166). The 3A11 protein is expressed on cells derived from human thymus, skin, synovium, and cartilage, and its expression is enhanced by IFN-gamma. mAbs directed against the 3A11 Ag and CD166 exhibit distinct patterns of binding to a panel of cell lines. Confocal microscopy shows that both CD166 and the 3A11 Ag are expressed at the cell surface, and that these proteins colocalize. The 3A11 Ag has a molecular mass of 130 kDa and is immunoprecipitated using either mAb 3A11 or soluble CD6-Ig fusion protein. mAbs directed against individual CD6L were less potent than was soluble CD6-Ig fusion protein in reducing adhesion of T cells to adherent 3A11-positive epithelial cells in vitro, suggesting that these Abs recognize epitopes on the 3A11 Ag and CD166 that are distinct from CD6 binding sites. Finally, transfection of epithelial cells with CD166-specific small interfering RNAs significantly decreased CD166 expression without alteration in 3A11 Ag levels, and thus confirmed that these two CD6L are distinct. Taken together, our data identifies a novel 130-kDa CD6L that may mediate interactions of synovial and epithelial cells with T lymphocytes.

Xenogeneic cells and superantigen induce human T-cell activation in the absence of T-cell recognition of xenoantigen.

The extent of interaction between human T-lymphocytes and xenogeneic antigen-presenting cells (APCs) is species-dependent. A successful interaction requires high-affinity receptor-ligand pairing across species and recognition of antigens presented by foreign major histocompatibility complex (MHC). A deficient human T-lymphocyte response to xenogeneic cells is likely the result of a defect in these interactions. However, the requirements for a T-cell response to superantigen (SAg) may differ from those of other T-cell responses. Using irradiated murine splenocytes, which are believed to be incapable of eliciting human T-cell responses, and porcine aortic endothelial cells (PAECs) as the APC populations, we studied the human T-lymphocyte response to antigens presented by these cells. Direct proliferation of human T-lymphocytes to SAg presented by murine APCs was demonstrated; it was blocked by anti-human LFA-1 and anti-murine MHC class II but not by anti-human MHC class II. PAECs also presented SAg to human T-cells, generating a proliferative response greater than the primary response to porcine xenoantigen. Culture of human T-cells with murine splenocytes or PAECs and SAg Staphylococcus enterotoxin A (SEA) for 7 days primed human T-cells to proliferate in a secondary culture in response to autologous APCs. This autologous secondary response was human MHC class II-dependent and was inhibited by anti-human LFA-1, anti-human CD2, and anti-human CD98. Surprisingly, both of these responses were also blocked by anti-SEA, suggesting that despite vigorous washing, a small amount of functionally important SAg was carried over from primary to secondary culture, probably bound to the surface of T-cells. Xenogeneic APCs, even those that fail to stimulate human T-cells directly, can serve as APCs for primary human T-cell responses. After such interactions T-cells can develop secondary responses in autologous interactions based on retention of minute amounts of SAg. Such interactions may have important implications for xenotransplantation.

CD6: expression during development, apoptosis and selection of human and mouse thymocytes.

CD6, a 130-kDa surface glycoprotein, is expressed primarily on cells of T lineage. A co-stimulatory role for CD6 in mature T cells has been shown, but the function of CD6 during thymocyte development is unknown. Since CD6 ligands are expressed on thymic epithelium, their interactions with CD6 could be important in thymic selection. In this report we show that CD6 is developmentally regulated in human and mouse thymocytes, and further demonstrate that increase in the level of CD6 expression correlates with expression of the selection marker CD69. We also show that activation via CD2 induces CD6 expression on mature human thymocytes and on a subset of immature human thymocytes that are resistant to apoptosis. In human and mouse thymocytes that express heterogeneous TCR, CD6 increases occur as double-positive thymocytes are selected to a single-positive stage. In contrast, in thymocytes from TCR transgenic mice, CD6 is barely increased following selection, suggesting that as functional avidity increases, requirements for CD6-dependent co-stimulation decrease. Taken together, these results indicate that during thymic development CD6-dependent signals may contribute both to thymocyte survival, and to the overall functional avidity of selection in both man and mouse.