Thymic Fibroblasts Research Articles

Expression of thymidylate synthase, orotate phosphoribosyltransferase and dihydropyrimidine dehydrogenase in thymic epithelial tumors

BackgroundIt remains unclear whether thymidylate synthase (TS), orotate phosphoribosyltransferase (OPRT) and dihydropyrimidine dehydrogenase (DPD) expressions are associated with the pathogenesis of thymic epithelial tumors. Therefore, we investigated the expression of TS, OPRT and DPD in thymic epithelial tumors. Patients and methodsFifty-six patients with thymic epithelial tumors were included in this study. Tumors sections were stained by immunohistochemistry for TS, OPRT, DPD, microvessel density (MVD) determined by CD34, and p53. We also conducted in vitro study of TS, OPRT and DPD expression using thymic carcinoma, thymic tumor and thymic fibroblast cell lines. ResultsTS, OPRT and DPD were expressed in 61%, 48% and 41%, respectively. High grade malignancy is significantly associated with higher expression of TS, OPRT and DPD in thymic epithelial tumors. These biomarkers were closely associated with p53 and MVD, and the overexpression of TS and DPD was a prognostic marker for predicting poor outcome in univariate analysis. Our in vitro study showed that marked overexpression of TS and OPRT was observed in thymic carcinoma cells, but not in thymic tumor cells, or thymic fibroblast cells. ConclusionsThe expression of TS, OPRT and DPD was closely related to the grade of malignancy in thymic epithelial tumors. A positive expression of TS, DPD and OPRT might be an important factor in predicting the effectiveness of 5-FU based chemotherapy in this disease.

Origin of Thymic Adipocytes in Aging: Incidental to Thymopoiesis or Instigator of Immunosenescence? (132.22)

Abstract Vishwa Deep Dixit, Yun-Hee Youm, Hyunwon Yang, Christo Venkov, Nancy R Manley, Eric Nielson, Todd Leff, Bolormaa Vandanmagsar By 50 years of age most of human thymus is replaced with adipocytes of unknown provenance and uncertain function. We investigated the lineage and mechanism of thymic adipocyte formation during aging and asked whether manipulating these pathways regulates thymic function. Using.We demonstrate that indelibly LacZ marked FoxN1 thymic epithelial cells transition to give rise to local tissue fibroblasts via the epithelial-mesenchymal transition(EMT)process. Importantly, we found an age-related increase in pro-EMT regulator FSP1 and pro-adipogenic transcription factor PPARγ in thymus. Consistent with these findings, a subset of FoxN1LacZ+ cells are FSP1+ with large unilocular lipid droplet, typical of adipocyte phenotype. Furthermore, aged FSP1.GFP reporter mice revealed that the sorted FSP1+ thymic fibroblasts lack TEC signatures and express PPARγ, indicative of adipogenic lineage commitment. Notably, overexpression of constitutive-active PPARγ in FSP1+ adipogenic lineage cells led to increased thymic adipogenesis and reduced thymopoiesis. Conversely, inhibition of EMT and thymoadipogenesis via anti-aging intervention, caloric restriction prevented thymic aging and immunosenescence. Future longevity studies using specific genetic manipulation of these pathways may provide a definitive answer to the puzzle of age-related thymic adiposity and involution.

Obesity induces premature thymic involution and restricts T cell repertoire diversity (36.47)

Abstract Yun-Hee Youm, Hyunwon Yang, Bolormaa Vandanmagsar, Jennifer Rood, K. Ganesh Kumar, Andrew A. Butler and Vishwa Deep Dixit Obesity increases risk of infections and cancer, suggesting adverse effects on immune-surveillance. Here we report that obesity compromises the mechanisms regulating T cell generation via inducing premature thymic involution. Diet-induced obesity reduced the total number of thymocytes and significantly increased apoptosis in developing T cell populations. Obesity accelerated the age-related reduction of naïve T cells and decreased T cell receptor excision circle bearing peripheral T cells, an index of recently generated T cells from thymus. Defects in thymopoiesis in obese mice were due to a reduction in the lymphoid progenitor pool and deterioration in the thymic stromal microenvironment. Interestingly, obesity also increased the frequency of thymic fibroblasts and reduced the number of thymic epithelial cells (TECs). Furthermore, the TECs of DIO mice displayed reduced expression of IL-7 and auto immune regulator (Aire) mRNA. Obesity induced via melanocortin 4 receptor (Mc4r) deficiency also constricted the T cell receptor repertoire diversity, recapitulating the thymic defects observed with diet-induced obesity. In humans, progressive adiposity with or without type-2-diabetes also compromised thymic output. Collectively, these findings establish that obesity constricts T cell diversity by accelerating age-related thymic involution.

Flt3 ligand–receptor interaction is important for maintenance of early thymic progenitor numbers in steady‐state thymopoiesis

T-cell production throughout life depends on efficient colonization and intrathymic expansion of BM-derived hematopoietic precursors. After irradiation-induced thymic damage, thymic recovery is facilitated by Flt3 ligand (FL), expressed by perivascular fibroblasts surrounding the thymic entry site of Flt3 receptor-positive progenitor cells. Whether intrathymic FL-Flt3 interactions play a role in steady-state replenishment of T cells remains unknown. Here, using competitive BM transplantation studies and fetal thymic organ cultures we demonstrated the continued numerical advantage of Flt3+ intrathymic T-cell precursors. Sub-kidney capsule thymic transplantation experiments, in which WT and FL-/- thymic lobes were grafted into FL-/- recipients, revealed that FL expression by the thymic microenvironment plays a role in steady-state thymopoiesis. The deficiency of the most immature thymic T-cell precursors correlated to upregulation of FL by thymic MTS15+ fibroblasts, suggesting that the number of Flt3+ progenitor cells may regulate the thymic expression of this cytokine. Together, these results show that FL expression by thymic stromal fibroblasts interacting with Flt3+ T-cell progenitors is important for the physiological maintenance of early T-cell development.

Inhibition of Thymic Adipogenesis by Caloric Restriction Is Coupled with Reduction in Age-Related Thymic Involution

Aging of thymus is characterized by reduction in naive T cell output together with progressive replacement of lymphostromal thymic zones with adipocytes. Determining how calorie restriction (CR), a prolongevity metabolic intervention, regulates thymic aging may allow identification of relevant mechanisms to prevent immunosenescence. Using a mouse model of chronic CR, we found that a reduction in age-related thymic adipogenic mechanism is coupled with maintenance of thymic function. The CR increased cellular density in the thymic cortex and medulla and preserved the epithelial signatures. Interestingly, CR prevented the age-related increase in epithelial-mesenchymal transition (EMT) regulators, FoxC2, and fibroblast-specific protein-1 (FSP-1), together with reduction in lipid-laden thymic fibroblasts. Additionally, CR specifically blocked the age-related elevation of thymic proadipogenic master regulator, peroxisome proliferator activated receptor gamma (PPARgamma), and its upstream activator xanthine-oxidoreductase (XOR). Furthermore, we found that specific inhibition of PPARgamma in thymic stromal cells prevented their adipogenic transformation in an XOR-dependent mechanism. Activation of PPARgamma-driven adipogenesis in OP9-DL1 stromal cells compromised their ability to support T cell development. Conversely, CR-induced reduction in EMT and thymic adipogenesis were coupled with elevated thymic output. Compared with 26-mo-old ad libitum fed mice, the T cells derived from age-matched CR animals displayed greater proliferation and higher IL-2 expression. Furthermore, CR prevented the deterioration of the peripheral TCR repertoire diversity in older animals. Collectively, our findings demonstrate that reducing proadipogenic signaling in thymus via CR may promote thymopoiesis during aging.

Axin expression in thymic stromal cells contributes to an age-related increase in thymic adiposity and is associated with reduced thymopoiesis independently of ghrelin signaling

The adipocytes are the predominant cell types that constitute the bulk of the thymic microenvironment by the fifth decade of life in healthy humans. An age-related increase in thymic adiposity is associated with reduced thymopoiesis and compromised immune surveillance in the elderly. However, the mechanisms regulating the generation of intrathymic adipocytes during aging remain to be elucidated. Here, we report that the CD45- thymic stromal cells (TSCs) are amenable to adipogenesis. We identified that the Wnt inhibitor axin is expressed in the lymphoid as well as stromal cells of the thymus with increased expression in CD45- TSCs of older mice. Knockdown of axin by RNA interference in CD45- primary TSCs led to a marked reduction in adipogenesis with significantly lower expression of adipogenic transcripts peroxisome proliferator-activated receptor 2 (PPAR), adipocyte fatty acid-binding protein (aP2), and perilipin. Age-related elevated axin expression was increased specifically in thymic fibroblasts and medullary thymic epithelial cells (TECs) but not in the cortical TEC or CD45+ cells. Consistent with a role of axin in promoting thymic adipogenesis, axin expression was also colocalized with lipid-expressing adipogenic cells in aging thymus. The prolongevity intervention, caloric restriction (CR), prevented the age-related increase in axin and the adipogenic cell in the thymus together with increase in thymic output. We have recently demonstrated that CR induces ghrelin, which can partially reverse thymic involution. Here, we show that axin expression is not affected by ablation of ghrelin receptors in aging mice, suggesting a ghrelin-independent mechanism for regulation of axin. Our data are consistent with the hypothesis that blocking the specific proadipogenic signals in the thymus may complement the present approaches to rejuvenate thymic function during aging.

Bone Marrow in Aging: Changes? Yes; Clinical Malfunction? Not So Clear.

Like every organ system, the bone marrow undergoes changes with age. The most readily apparent change is a decline in marrow cellularity. The percentage of marrow space occupied by hematopoietic tissue goes from 40–60% in young adults to 20–40% in older people, with the remaining space being taken up by fat. It is unclear whether the expansion of marrow fat with age is a cause or an effect of aging. A similar change happens in the thymus much earlier in adulthood. At about age 30, thymic fibroblasts become fat-laden and the expansion of fatty tissue essentially replaces the cellular elements of thymopoiesis. In late life, this thymic inactivity is manifested by a reduction in naïve T cells and a compromise in cellular immunity. It is unclear whether the processes in the marrow and the thymus are qualitatively similar. Bone remodeling and osteoporosis can also lead to a decrease in trabecular bone in the marrow. Several age-related changes have been noted in hematopoietic cells, including shortening of telomeres, accumulation of mitochondrial DNA mutations, skewed X-inactivation, and others. However, in the absence of disease, sufficient stem cell proliferation and maturation is preserved so that people are capable of living their entire life span, up to 120 years, without running out of blood cells. Indeed, even when a person is given stem cells from another person, presumably in numbers far less than were present in the host before myeloablation, the transferred stem cells appear to be capable of maintaining normal hematopoiesis throughout the lifespan of their adoptive host. Normal blood counts are maintained throughout life. Physiologic compensations may be necessary to achieve this end. A longitudinal study of older people with normal hemoglobin levels over a period of 30 years showed that people increase serum levels of erythropoietin over time to maintain normalcy. Older people also seem to increase their neutrophil count to a lower extent and somewhat more slowly in response to pharmacological G-CSF than younger people, though such an intervention is generally done in people with underlying disease. Qualitative neutrophil defects in older people may include impaired phagocytosis and respiratory burst response to soluble signals. Enhanced in vitro platelet aggregation in older people does not have a clear in vivo functional correlate.

The Lymphotoxin Pathway Regulates Aire-Independent Expression of Ectopic Genes and Chemokines in Thymic Stromal Cells

Medullary thymic epithelial cells (mTEC) play an important and unique role in central tolerance, expressing tissue-restricted Ags (TRA) which delete thymocytes autoreactive to peripheral organs. Since deficiencies in this cell type or activity can lead to devastating autoimmune diseases, it is important to understand the factors which regulate mTEC differentiation and function. Lymphotoxin (LT) ligands and the LTbetaR have been recently shown to be important regulators of mTEC biology; however, the precise role of this pathway in the thymus is not clear. In this study, we have investigated the impact of this signaling pathway in greater detail, focusing not only on mTEC but also on other thymic stromal cell subsets. LTbetaR expression was found in all TEC subsets, but the highest levels were detected in MTS-15(+) thymic fibroblasts. Rather than directing the expression of the autoimmune regulator Aire in mTEC, we found LTbetaR signals were important for TRA expression in a distinct population of mTEC characterized by low levels of MHC class II (mTEC(low)), as well as maintenance of MTS-15(+) fibroblasts. In addition, thymic stromal cell subsets from LT-deficient mice exhibit defects in chemokine production similar to that found in peripheral lymphoid organs of Lta(-/-) and Ltbr(-/-) mice. Thus, we propose a broader role for LTalpha1beta2-LTbetaR signaling in the maintenance of the thymic microenvironments, specifically by regulating TRA and chemokine expression in mTEC(low) for efficient induction of central tolerance.

Melanocortin signaling via the MC4 receptor regulates T cell function

The central melanocortin pathway is critical for regulating food intake and energy expenditure. However, precise role of melanocortins and MC4 receptor in regulation of thymic function remains to be elucidated. Here we demonstrate that MC4R are highly expressed in thymus and lymphoid organs. Compared to wild type control mice, genetic deletion of MC4R led to reduced cellularity and loss of cortico‐medullary junctions suggesting increased thymic involution. Interestingly, the absence of MC4R mediated signals caused a marked increase in lipid bearing thymic fibroblasts and reduction in medullary thymic epithelial cells with reduced thymic output. Analysis of lymphoid progenitors revealed significant reduction in LSK cells in bone marrow and ETP cells in thymus. Using complementarity determining region 3 (CDR3) length polymorphism analysis we next demonstrated that absence of MC4R causes a severe contraction of peripheral T cell receptor diversity suggesting reduced immune‐surveillance. Collectively, these data demonstrates a novel role of melanocortin ‐MC4R pathway in coupling metabolic and immune system. In addition, our findings suggest that specific Mc4R agonists may serve as an important target for rejuvenating thymic function.

A Unique Thymic Fibroblast Population Revealed by the Monoclonal Antibody MTS-15

T cell differentiation in the thymus is dependent upon signals from thymic stromal cells. Most studies into the nature of these signals have focused only on the support provided by the thymic epithelium, but there is an emerging view that other stromal cells such as mesenchymal fibroblasts may also be involved. Study of the latter has been hindered by a lack of appropriate markers, particularly those allowing their isolation. In this study, we describe a new surface marker of thymic stroma, MTS-15, and demonstrate its specificity for fibroblasts and a subset of endothelial cells. Coculture experiments showed that the determinant could be transferred between cells. Extensive biochemical analysis demonstrated that the Ag bound by MTS-15 was the glycosphingolipid Forssman determinant, consistent with the distribution observed. Transcriptional analysis of purified MTS-15(+) thymic fibroblasts revealed a unique expression profile for a number of chemokines and growth factors important to thymocyte and epithelial cell development. In a model of cyclophosphamide-induced thymic involution and regeneration, fibroblasts were found to expand extensively and express growth factors important to epithelial proliferation and increased T cell production just before thymic regeneration. Overall, this study identifies a useful marker of thymic fibroblasts and highlights this subpopulation as a key player in thymic function by virtue of their support of both thymocytes and epithelial cells.

Expression of the CD7 Ligand K-12 in Human Thymic Epithelial Cells: Regulation by IFN-?

CD7 is an immunoglobulin superfamily molecule expressed on T, NK, and pre-B lymphocytes. Previous studies have demonstrated a role for CD7 in T- and NK-cell activation and cytokine production. Recently, an epithelial cell secreted protein, K12, was identified as a CD7 ligand. Although CD7 is expressed intrathymically, it is not known if K12 is produced in human thymus. To determine roles that K12 might play in the human thymus, we analyzed expression of K12 in human thymocytes, thymic epithelial cells (TE), and thymic fibroblasts. We found that recombinant human K12 bound strongly to soluble hCD7, with a Keq of 37.6x10(-9) M, and this interaction was inhibited by a novel antihuman K12 monoclonal antibody (K12-A1). K12 mRNA was detected by RT-PCR and northern analysis in human TE and thymic fibroblasts, but not in human thymocytes. Expression of K12 in TE cells was upregulated by IFN-gamma. Taken together, these data demonstrated that K12 is produced by human TE cells and thymic fibroblasts, and is regulated in thymus by IFN-gamma. These data suggest a role for thymic microenvironment-produced K12 in regulation of thymocyte signaling and cytokine release, particularly in the setting of thymus pathology where IFN-gamma is upregulated such as myasthenia gravis.

Fibroblasts inhibit the production of interleukin-12p70 by murine dendritic cells.

Interleukin-12 p70 (IL-12p70) is a key cytokine produced by dendritic cells (DC) able to drive the development of T helper type 1 (Th1) lymphocytes. We showed that thymic and other fibroblasts strongly inhibit IL-12p70 production by splenic DC stimulated by lipopolysaccharide plus either anti-CD40 or interferon-gamma (IFN-gamma) and by purified splenic DC stimulated by Pansorbin plus IFN-gamma. This IL-12p70 inhibitory activity is secreted in the conditioned medium of primary fibroblasts and fibroblast cell lines but not by haematopoietic cell lines. As IL-10 was the unique factor able to inhibit IL-12p70 produced by cultured splenic DC, we showed that a neutralizing antibody to IL-10 did not suppress the IL-12p70 inhibitory activity of thymic fibroblast-conditioned medium (FCM). This FCM potently inhibits the maturation and expression of major histocompatibility complex class II and co-stimulatory molecules induced by stimulation of spleen-derived DC. While thymic FCM suppressed the IL-12p70 expression by stimulated spleen-derived DC, tumour necrosis factor-alpha production is not affected. This inhibitory activity is able to down-regulate the IL-12p35 subunit transcription and expression, resulting in the impaired assembly of IL-12p70 heterodimer. As fibroblasts are present in the tissue microenvironment and are active players in the establishment of an immune response, the nature and role of the fibroblastic inhibitory activity remain to be established.

Analysis of thymic stromal cell populations using flow cytometry

The complexity of the lymphostromal interplay that is essential to αβT-cell development is reflected by the heterogeneity of both lymphocytes and thymic stromal cells. While panels of monoclonal antibodies have described many of the cellular components of these microenvironments, the means to quantify stromal cell subsets using flow cytometry remains poorly defined. This study refines and compares various stromal cell isolation procedures and determines the effects of various digestion enzymes on important surface molecules. Three- and four-color flow cytometry is used to correlate established and novel stromal cell markers to define thymic fibroblasts, epithelium and a unique subset of thymic endothelium that express MHC class II. This work provides a basis for the purification of thymic stromal cells for further phenotypic, functional and genetic analysis.

Studies on the role of IL-7 presentation by mesenchymal fibroblasts during early thymocyte development.

T cell precursor development depends upon poorly defined interactions between thymocyte precursors and stromal cells involving cell surface molecules, extracellular matrix (ECM) components and soluble growth factors. To determine whether presentation of soluble factors by ECM is involved in early T cell development, we analyzed expression of ECM components in individual thymic stromal subsets and investigated their ability to present ECM-associated IL-7, a factor known to be important during early thymocyte development. We show that MHC class II(+) thymic epithelium and fibroblasts - essential requirements for development of CD4(-)8(-)precursors - both show surface expression of ECM components such as fibronectin and heparan sulfate. Use of biotinylated IL-7 protein indicates that both cell types bind IL-7, while enzymatic disruption of specific ECM components indicates that IL-7 presentation by both cell types is dependent upon heparan sulfate. However, disruption of IL-7 presentation specifically on fibroblasts does not affect their ability to contribute to T cell development. Collectively, these data suggest that while ECM-mediated presentation of IL-7 may be a general function of thymic stromal cells during thymocyte development, heparan sulfate-mediated IL-7 presentation specifically by fibroblasts is not essential and that the specific requirement for fibroblasts in early development involves additional undefined interactions.

The role of mesenchyme in thymus development.

We have reviewed the evidence that thymic mesenchymal cells and their progeny thymic fibroblasts play an important role in early T-cell development. Although it is possible that mesenchyme plays an inductive role in thymic epithelial morphogenesis, we have presented evidence to suggest that there is a direct effect of mesenchyme and fibroblasts on T-cell development. Moreover the association of these cell types with an ECM raises the possibility that the latter might be important in integrin and/or cytokine presentation especially during the CD4(-)8- phase of T-cell development.

Cellular and matrix interactions during the development of T lymphocytes.

The thymus contains an extensive extracellular matrix. Although thymocytes express integrins capable of binding to matrix molecules, the functional significance of the matrix for T cell development is uncertain. We have shown that the matrix is associated with thymic fibroblasts which are required for the CD44+ CD25+ stage of double negative (CD4-8-) thymocyte development. The survival of cells at this stage is dependent on IL-7 and we propose that the role of fibroblasts is to present, via the matrix, IL-7 to developing T cells.

A Thymic Nurse Cell-Specific Monoclonal Antibody

A thymic epithelial cell line (tsTNC-1) that maintains the ability to selectively bind and internalize immature αβTCRloCD4+CD8+thymocytesin vitrowas used in the development of a monoclonal antibody that is specific to the cell surface of thymic nurse cells (TNCs) in the thymus. The rat monoclonal antibody ph91 showed specificity to cells of the subcapsular region of the thymic cortex. Upon mechanical dispersion of the thymusin vitro, ph91 recognized cells displaying the multicellular morphology unique to TNCs. Ph91 staining was not detected on fresh thymocytes, stromal cells of the inner thymic cortex, thymic medullary cells, B cells or fibroblasts. Ph91 recognized a 43-kDa protein on the surface of TNCs. Exposure of tsTNC-1 cells to ph91 in tissue culture significantly reduced the percentage of binding of the αβTCRloCD4+CD8+thymocyte subset previously shown to target TNCs. In organ culture, ph91 reduced the viability of developing thymocytes by 70%. The largest reduction was found in the αβTCR+CD4+CD8+thymocyte subset. These results represent the first report of a TNC-specific monoclonal antibody. Further, the antigen to which ph91 binds may play a role in the process of thymocyte binding and their subsequent internalization which is unique to TNCs and important to the T cell developmental process.

CD34+CD38−lin− Cord Blood Cells Develop into Dendritic Cells in Human Thymic Stromal Monolayers and Thymic Nodules

Thymic dendritic cells (DCs) appear to have distinct biologic and functional properties compared with DCs in other tissues. Currently, little is known about human thymic DCs because they have been difficult to isolate and culture in vitro. Here, we report that human thymic stroma can support the development of primitive human hemopoietic stem cells into mature DCs without cytokine or serum supplementation. Coculture of CD34+CD38-lineage (lin)- and CD34+CD38+lin- umbilical cord blood cells with thymic stromal monolayers induced 43 +/- 17-fold and 32 +/- 16-fold expansions, respectively, of umbilical cord blood progenitors and also generated large numbers of cells with the morphologic, phenotypic, and functional characteristics of mature DCs. These cells expressed class I and class II MHC, CD1a, CD2, CD4, CD11c, CD40, CD45, CD80, CD83, and CD86 and were potent stimulators of allogeneic T cell activation. Primitive hemopoietic progenitors also developed into mature DCs in a novel tissue culture system of thymic nodules wherein thymic epithelial cells and fibroblasts were grown in nodular aggregates in vitro. These results demonstrate that human thymic stroma efficiently supports the development of CD34+CD38-lin- cord blood cells into mature DCs. In addition, the culture conditions described in this report are useful systems for studying the ontogeny of human DCs in thymic microenvironments.

Fibroblast dependency during early thymocyte development maps to the CD25+ CD44+ stage and involves interactions with fibroblast matrix molecules.

We have investigated the role of specific components of the thymic stroma during development of CD4-8-T cell precursors by separating and reaggregating precursor subsets with individual or combinations of stromal cells. We show that while the development of CD25+ 44+ precursors is dependent upon a combination of major histocompatibility complex (MHC) class II+ thymic epithelial cells and fibroblasts, their direct descendants, CD25+ 44- precursors, develop to the CD4+ 8+ stage in the presence of MHC class II+ thymic epithelial cells alone. Thus, CD25+ 44+ precursors are the last developmental stage to be dependent upon fibroblast support. In addition, while metabolically inactive, 1-ethyl-3-(3'-dimethylaminopropyl) carbodiimide (ECDI)-treated fibroblasts retain the ability to promote T cell development, prior treatment with hyaluronidase abrogates this effect, suggesting that fibroblast-associated extracellular matrix components are the key elements involved. In support of this, we show that fibroblasts are located in cortical regions of the thymus where T cell precursors are known to reside, and that these fibroblasts are associated with an extensive extracellular matrix not found on thymic epithelial cells. Finally, antibodies to alpha 4 integrin and CD44 interfere with the efficiency with which CD4+ 8+ cells are generated from CD25+ 44+ precursors in reaggregate cultures and also reduce the binding of the latter to 3T3 fibroblasts, suggesting these molecules play a role in bringing T cell precursors into contact with fibroblast-associated extracellular matrix.

Identification and characterization of a 100-kD ligand for CD6 on human thymic epithelial cells.

CD6 is a 130-kD glycoprotein expressed on the surface of thymocytes and peripheral blood T cells that is involved in TCR-mediated T cell activation. In thymus, CD6 mediates interactions between thymocytes and thymic epithelial (TE) cells. In indirect immunofluorescence assays, a recombinant CD6-immunoglobulin fusion protein (CD6-Rg) bound to cultured human TE cells and to thymic fibroblasts. CD6-Rg binding to TF and TE cells was trypsin sensitive, and 54 +/- 4% of binding was divalent cation dependent. By screening the blind panel of 479 monoclonal antibodies (mAbs) from the 5th International Workshop on Human Leukocyte Differentiation Antigens for expression on human TE cells and for the ability to block CD6-Rg binding to TE cells, we found one mAb (J4-81) that significantly inhibited the binding of CD6-Rg to TE cells (60 +/- 7% inhibition). A second mAb to the surface antigen identified by mAb J4-81, J3-119, enhanced the binding of CD6-Rg to TE cells by 48 +/- 5%. Using covalent cross-linking and trypsin digestion, we found that mAb J4-81 and CD6-Rg both bound to the same 100-kD glycoprotein (CD6L-100) on the surface of TE cells. These data demonstrate that a 100-kD glycoprotein on TE cells detected by mAb J4-81 is a ligand for CD6.