银鲫精巢特异的Ly-6/uPAR相关蛋白的分子克隆及其特征分析

Ly-6/uPAR基因超家族(Ly-6 SF)成员广泛地存在于后生动物中, 开展该家族相关功能基因研究具有重要的意义。研究从银鲫(Carassius auratus gibelio)中鉴定到一个该家族新成员, cDNA全长为570 bp, 其中开放阅读框长度为300 bp, 编码99个氨基酸, 生物软件预测该蛋白含有一个LU结构域, 不含GPI锚信号序列, N端含有信号肽, 表明其可能为Ly-6基因超家族中分泌型蛋白。组织表达分析显示, 该基因只在银鲫精巢中特异表达, 且又是Ly-6基因超家族中一员, 因此将其命名为银鲫精巢特异的Ly-6/uPAR相关蛋白(Carassius auratus gibelio testis-specific Ly-6/uPAR related protein, 简称CagTslurp)。原位杂交结果显示, 该基因在银鲫精巢的精原细胞, 初级精母细胞以及次级精母细胞中表达, 精子细胞中存在少量的表达, 而在体细胞中不表达。这种精巢特异的表达模式, 暗示CagTslurp在银鲫精子发生中可能发挥了作用。       

Three-finger proteins from the Ly6/uPAR family: Functional diversity within one structural motif

The discovery in higher animals of proteins from the Ly6/uPAR family, which have structural homology with snake “three-finger” neurotoxins, has generated great interest in these molecules and their role in the functioning of the organism. These proteins have been found in the nervous, immune, endocrine, and reproductive systems of mammals. There are two types of the Ly6/uPAR proteins: those associated with the cell membrane by GPI-anchor and secreted ones. For some of them (Lynx1, SLURP-1, SLURP-2, Lypd6), as well as for snake α-neurotoxins, the target of action is nico- tinic acetylcholine receptors, which are widely represented in the central and peripheral nervous systems, and in many other tissues, including epithelial cells and the immune system. However, the targets of most proteins from the Ly6/uPAR family and the mechanism of their action remain unknown. This review presents data on the structural and functional properties of the Ly6/uPAR proteins, which reveal a variety of functions within a single structural motif.     

Structural and phylogenetic characterization of human SLURP-1, the first secreted mammalian member of the Ly-6/uPAR protein superfamily

Members of the Ly-6/uPAR protein family share one or several repeat units of the Ly-6/uPAR domain that is defined by a distinct disulfide bonding pattern between 8 or 10 cysteine residues. The Ly-6/uPAR protein family can be divided into two subfamilies. One comprises GPI-anchored glycoprotein receptors with 10 cysteine residues. The other subfamily includes the secreted single-domain snake and frog cytotoxins, and differs significantly in that its members generally possess only eight cysteines and no GPI-anchoring signal sequence. We report the purification and structural characterization of human SLURP-1 (secreted mammalian Ly-6/uPAR related protein 1) from blood and urine peptide libraries. SLURP-1 is encoded by the ARS (component B)-81/s locus, and appears to be the first mammalian member of the Ly-6/uPAR family lacking a GPI-anchoring signal sequence. A phylogenetic analysis based on the SLURP-1 primary protein structure revealed a closer relationship to the subfamily of cytotoxins. Since the SLURP-1 gene maps to the same chromosomal region as several members of the Ly-6/uPAR subfamily of glycoprotein receptors, it is suggested that both biologically distinct subfamilies might have co-evolved from local chromosomal duplication events.     

Cloning and expression of SOLD1 in ovine and caprine placenta,and their expected roles during the development of placentomes

Background  

The Ly-6 (Ly-6/uPAR) superfamily members share the Ly-6 domain defined by distinct disulfide bonding patterns between 8 or 10 cysteine residues. They comprise membrane- and secretory-type proteins. We recently reported the gene and protein characterization of the bovine secreted protein of Ly-6 domain 1 (SOLD1). Bovine SOLD1 is expressed in trophoblast mononucleate cells (TMCs) and is localized in the cotyledonary mesenchyme. Here, we compared the expression and functionality of SOLD1 among the ruminants. We examined mRNA expression by chorionic fibroblasts as a measure of one of the SOLD1 functions.     

Interrelationships of the “Ly-6 complex” antigens

Competitive binding studies and immunoprecipitation experiments define at least five distinct epitopes encoded by Ly-6-linked genes—Ly-6A.2, Ly-6B.2, Ly-6C.2, Ly-6D.2, and ThB. Ly-6A.2, a 33 kd protein, and Ly-6D.2 are closely overlapping epitopes that can be distinguished by their unique thymus reactions of 10–20% or >90%, respectively. Similarly, the Ly-6C.2 antigen present on a 14 kd moiety loosely overlaps the Ly-6B.2 antigen. Ly-6C.2 and Ly-6B,2 antigens are distinct from Ly-6A.2 and Ly-6B.2, however. ThB is a 16–18 kd antigen which is not associated on the cell surface with any other Ly-6 antigens. In addition, independently derived antibodies made to the Ly-6C.2 antigen detect an identical epitope, as do antibodies to Ly-6A.2 and Ly-6B.2. These results imply the existence of a single antigenic site on each of these molecules.     

SAMP14, a novel,acrosomal membrane-associated,glycosylphosphatidylinositol-anchored member of the Ly-6/urokinase-type plasminogen activator receptor superfamily with a role in sperm-egg interaction

We report a new member of the Ly-6/urokinase-type plasminogen activator receptor (uPAR) superfamily of receptors, SAMP14, which is retained on the inner acrosomal membrane of the human spermatozoan following the acrosome reaction and may play a role in fertilization. The SAMP14 sequence predicted a glycosylphosphatidylinositol (GPI)-anchored protein with a signal peptide, a transmembrane domain near the carboxyl terminus, and a putative transamidase cleavage site in the proprotein. Attachment of SAMP14 to the membrane by a lipid anchor was confirmed by its sensitivity to phosphatidylinositol phospholipase C. SAMP14 has a single functional domain similar to the Ly-6 and urokinase plasminogen activator receptor superfamily of proteins, and the gene mapped to 19q13.33, near the PLAUR locus for uPAR at 19q13.2. Northern and dot blotting showed that SAMP14 expression was testis-specific. Indirect immunofluorescence and immunoelectron microscopy with antisera to purified recombinant SAMP14 localized the protein to outer and inner acrosomal membranes as well as the acrosomal matrix of ejaculated human sperm. Acrosome-reacted sperm demonstrated SAMP14 immunofluorescence, indicating its retention on the inner acrosomal membrane following the acrosome reaction. However, SAMP14 localized to the entire sperm when unwashed swim-up sperm from the ejaculate were stained, indicating that some SAMP14 is loosely associated with the plasma membrane. Antibodies against recombinant SAMP14 inhibited both the binding and the fusion of human sperm to zona free hamster eggs, suggesting that SAMP14 may have a role in sperm-egg interaction. SAMP14 represents a GPI-anchored putative receptor in the Ly-6/uPAR family that is exposed on the inner acrosomal membrane after the acrosome reaction.     

The glycosyl phosphatidylinositol anchor is critical for Ly-6A/E-mediated T cell activation.

Ly-6E, a glycosyl phosphatidylinositol (GPI)-anchored murine alloantigen that can activate T cells upon antibody cross-linking, has been converted into an integral membrane protein by gene fusion. This fusion product, designated Ly-6EDb, was characterized in transiently transfected COS cells and demonstrated to be an integral cell surface membrane protein. Furthermore, the fusion antigen can be expressed on the surface of the BW5147 class "E" mutant cell line, which only expresses integral membrane proteins but not GPI-anchored proteins. The capability of this fusion antigen to activate T cells was examined by gene transfer studies in D10G4.1, a type 2 T cell helper clones. When transfected into D10 cells, the GPI-anchored Ly-6E antigen, as well as the endogenous GPI-anchored Ly-6A antigen, can initiate T cell activation upon antibody cross-linking. In contrast, the transmembrane anchored Ly-6EDb antigen was unable to mediate T cell activation. Our results demonstrate that the GPI-anchor is critical to Ly-6A/E-mediated T cell activation.     

Ly-6 superfamily members Ly-6A/E,Ly-6C,and Ly-6I recognize two potential ligands expressed by B lymphocytes

Most hemopoietic cells express one or more members of the Ly-6 supergene family of small glycosylphosphatidylinositol-linked proteins. Although levels of Ly-6 proteins vary with stages of differentiation and activation, their function largely remains unknown. To ascertain whether ligands for Ly-6 proteins exist, chimeric proteins were constructed in which Ly-6E, Ly-6C, and Ly-6I were fused to the murine IgM heavy chain. These chimeras specifically stained both developing and mature B lymphocytes, as assessed by flow cytometry. Analysis of variants of the CH27 B cell lymphoma revealed that Ly-6A/E and Ly-6I recognized different molecules. CH27 cells with low levels of Ly-6A/E ligand activity also lost expression of CD22, and cells transfected with CD22 gained the ability to bind the Ly-6A/E chimera and, to a lesser extent, the Ly-6C and Ly-6I chimeric proteins. As many mature B cells coexpress Ly-6A/E and CD22, the function of Ly-6 molecules may be to associate with other membrane proteins, possibly concentrating these ligands in lipid rafts, rather than acting directly as cell:cell adhesion molecules.     

Demonstration of phosphatidylinositol anchors on Ly-6 molecules by specific phospholipase C digestion and gel electrophoresis in octylglucoside

Release by phosphatidylinositol-specific phospholipase C is frequently provided as evidence for membrane anchorage of a protein through phosphatidylinositol. Demonstration that the enzyme removes a lipophilic moiety from the protein is stronger evidence, and is presented here for members of the Ly-6 family of lymphocyte antigens: Ly-6A, C and E. Treatment of these molecules with the enzyme greatly increased their electrophoretic mobilities on polyacrylamide gels containing nonionic detergent. Furthermore, the mobilities of the digested, but not native Ly-6 molecules, were independent of detergent. This analytical method was applied to pure antigen, radiolabelled immunoprecipitate, or immunochemically detected Ly-6 antigens on blots.     

Definition of new alloantigens encoded by genes in the Ly-6 complex

Three alloantigens encoded by Ly-6-linked genes are defined by monoclonal antibodies. The Ly-27.2 antigen is defined by antibody 5075-19.1, Ly-28.2 by 5075-3.6, -12.1, -16.10 and by 5095-16.6. The strain distribution pattern of these antibodies is the same and identical with Ly-6.2. However the tissue distribution of these antigens is unique and distinguishes these antigens from the Ly-6.2 antigen or any known antigen encoded by Ly-6-linked genes. Ly-27.2 is present on all thymocytes, T cells, and B cells but is absent from bone marrow cells, whereas Ly-28.2 is absent from most thymocytes and is present on a subpopulation of T cells and B cells but is found on 60–70% of bone marrow cells. No recombination between the Ly-6/Ly-27/Ly-28 loci was found in linkage studies using 41 recombinant inbred strains and 57 backcross mice and indicates very close linkage of these genes. In addition, close linkage to 24 minor histocompatibility genes was excluded using the Bailey HW bilineal congenic mice. The data presented indicate that either the Ly-6 complex is composed of a family of tightly linked genes or the antigens are the products of a single gene that undergoes extensive modification during differentiation.     

PATE gene clusters code for multiple, secreted TFP/Ly-6/uPAR proteins that are expressed in reproductive and neuron-rich tissues and possess neuromodulatory activity

We report here syntenic loci in humans and mice incorporating gene clusters coding for secreted proteins each comprising 10 cysteine residues. These conform to three-fingered protein/Ly-6/urokinase-type plasminogen activator receptor (uPAR) domains that shape three-fingered proteins (TFPs). The founding gene is PATE, expressed primarily in prostate and less in testis. We have identified additional human PATE-like genes (PATE-M, PATE-DJ, and PATE-B) that co-localize with the PATE locus, code for novel secreted PATE-like proteins, and show selective expression in prostate and/or testis. Anti-PATE-B-specific antibodies demonstrated the presence of PATE-B in the region of the sperm acrosome and at high levels on malignant prostatic epithelial cells. The syntenic mouse Pate-like locus encompasses 14 active genes coding for secreted proteins, which are all, except for Pate-P and Pate-Q, expressed primarily in prostate and/or testis. Pate-P and Pate-Q are expressed solely in placental tissue. Castration up-regulates prostate expression of mouse Pate-B and Pate-E, whereas testosterone ablates this induced expression. The sequence similarity between TFP/Ly-6/uPAR proteins that modulate activity of nicotinic acetylcholine receptors and the PATE (Pate)-like proteins stimulated us to see whether these proteins possess analogous activity. Pharmacological studies showed significant modulation of the nicotinic acetylcholines by the PATE-B, Pate-C, and Pate-P proteins. In concert with these findings, certain PATE (Pate)-like genes were extensively expressed in neuron-rich tissues. Taken together, our findings indicate that in addition to participation of the PATE (Pate)-like genes in functions related to fertility and reproduction, some of them likely act as important modulators of neural transmission.     

Phenotypic changes induced by interferon in resting T cells: major enhancement of Ly-6 antigen expression

The murine Ly-6 locus controls multiple cell surface antigenic specificities with distinct cellular and tissue distributions. Although the functions of Ly-6 antigens are unknown, several of these antigens represent interesting markers of T cell differentiation and activation. In this work we used a panel of monoclonal antibodies (MAb) in conjunction with flow cytofluorometry (FCF) analysis to investigate the effect of interferon (IFN) on the surface representation of T cell-associated Ly-6 antigens. It was found that in vitro treatment of purified T cells from both C57Bl/6 (Ly-6.2) and BALB/c (Ly-6.1) mice with 10 to 10(4) U IFN-alpha/beta/ml results in a dose-dependent enhancement of Ly-6 antigen expression. This effect was already detectable after 12 to 18 hr and culminated after 48 hr of incubation. Both frequencies and brightness of Ly-6 bearing cells were increased. The most dramatic shifts were observed for the Ly-6A, D, and E antigens, which were augmented by eightfold to 20-fold upon exposure to 10(4) U IFN alpha/beta/ml. Expression of Ly-6C antigens was enhanced by fourfold to sixfold under the same conditions. Immunochemical analyses and use of metabolic inhibitors additionally demonstrated that such IFN-alpha/beta-induced phenotypic alterations of T cells reflect augmented de novo biosynthesis of Ly-6 molecules. Comparison of purified IFN-alpha and IFN-beta revealed that both are equally active in influencing Ly-6. IFN-gamma also enhanced Ly-6 expression but less efficiently than IFN-alpha/beta. Additional experiments were carried out to determine the selectivity of IFN-alpha/beta action on T cell phenotype. These studies demonstrated that IFN-induced Ly-6 enhancement occurs without emergence of interleukin 2 or transferrin receptors. Expression of H-2 and beta 2m antigens, previously known to be sensitive to IFN, was increased but to a much lesser extent than Ly-6. Most other cell surface antigens examined were minimally affected by IFN-alpha/beta with the exception of Ly-11 and Ly-23. Augmentation of these latter markers was lower than for Ly-6 antigens, however. Therefore the Ly-6 locus appears to be preferentially activated by IFN-alpha/beta in resting T cells. Additional exploration of this phenomenon should provide insight into both the biological significance of Ly-6 antigens and the mechanism(s) by which IFN affect T cell functions.     

Ly-6A.2 and Ly-6E.1 molecules are antithetical and identical to MALA-1

Rat monoclonal antibodies YE3/19.1, defining the murine-activated lymphocyte antigen MALA-1, and D7, detecting an Ly-6 locus-controlled antigen, bound highly purified Ly-6E.1. On western blots of lymphocyte surface proteins which had been solubilized and electrophoretically separated in octylglucoside, they detected bands which comigrated with Ly-6A.2 or Ly-6E.1 antigens. On cells or in an immunoassay they blocked alloantibodies against Ly-6A.2 or Ly-6E.1. The tissue distribution of MALA-1 also correlated with Ly-6A.2 or Ly-6E.1. Upon octylglucoside or sodium dodecyl sulfate-polyacrylamide gel electrophoresis, these antigens displayed similar sizes. Thus, Ly-6A.2 and Ly-6E.1 are most likely products of alternate alleles. Electrophoretic analysis showed a similar size and charge for Ly-6A.2, Ly-6B.2, Ly-613.2, and Ly-27.2. Ly-6C.2 and Ly-28.2 appeared to be identical, and were similar in size to Ly-6A.2, but they differed in charge and in intrachain disulfide constraints. Since Ly-613.2 and Ly-27.2 may represent the same or different epitopes on the Ly-6A.2 molecule, the previously postulated five Ly-6-like antigens that were thought to be separable on the basis of tissue distribution, may represent no more than three separate proteins which can be assigned to one of two distinct categories by electrophoretic mobility in gels containing octylglucoside.     

Studies of the mouse Ly-6 alloantigen system

The discovery of several monoclonal antibodies provided the impetus to revisit the Ly-6 group of antigens. Our serological data point to the existence of at least five separate Ly-6 antigens. They are distinguished by the patterns of their tissue expression as (1) the classical Ly-6 alloantigen of peripheral lymphocytes (Ly-m6.2A), (2) a bone marrow cell-restricted antigen (Ly-m6.2B), (3) an antigen shared by bone marrow cells and peripheral lymphocytes (Lym6.2C, possibly identical with H9/25),(4) an antigen expressed on bone marrow cells, thymocytes, and peripheral lymphocytes (Ly-m6.2D), and (5) an antigen occurring exclusively on lymphoblasts (Ly-m6.IE, similar to Ala-1). ThB is a sixth distinct antigen of the group. The assumption that separate antigens exist is supported by distinctive distribution patterns in normal and neoplastic tissues. The genes controlling Ly-6 antigens are closely linked, as they are transmitted as two haplotypes only. One incidence of a crossover within the Ly-6 region was observed: the Ly-6B.2 alloantigen was expressed in NZB mice, which type Ly-6.1 for other Ly-6 specificities.     

Treatment of resting T lymphocytes with interferon-alpha/beta augments their proliferative response to activation signals delivered through their surface Ly-6 antigen

Although the exact significance of Ly-6 antigens is unknown, recent evidence suggests they may provide an important alternative pathway for murine T-cell activation. Thus, Shevach et al. (1986, Fed. Proc. 45, 1131) discovered that cross-linking of Ly-6 antigens on the cell surface acts in concert with phorbol myristate acetate to trigger mitogenesis in T cells. Previously, we reported that surface expression of Ly-6 antigens on T cells is markedly increased following exposure to interferon-alpha/beta (IFN-alpha/beta). The purpose of the present work was to determine the effect of IFN-induced Ly-6 enhancement on Ly-6-mediated T-cell stimulation. Purified T cells were incubated in vitro for 1-27 hr with various doses (10-10(4) units/ml) of IFN-alpha/beta. This was found to result in various degrees of augmentation of the proliferative responses of these T cells to stimulation through their Ly-6 antigen. Surprisingly, while maximal enhancement of Ly-6 expression occurred only after the longest pulses with the highest IFN concentrations, treatment with as little as 100 units IFN/ml for 12 hr was sufficient to induce a dramatic (25-fold) and nearly maximal enhancement of proliferation. This high sensitivity to IFN-alpha/beta of the Ly-6 pathway of T-cell activation led us to speculate that this pathway may play a role in the immunomodulatory activities of IFN-alpha/beta.     

Identification and expression of a novel member of Ly-6 superfamily in zebrafish <Emphasis Type="Italic">Denio rerio</Emphasis>

Ly-6 superfamily members are present in many metazoans and are divided into two groups: secreted proteins and glycosylphosphatidyl inositol (GPI)-anchored membrane proteins. They both contain one or more conserved domain identified as Ly-6/uPAR (LU) domain and play key roles in cellular adhesion and signaling. Here, we identify a novel member, lymphocyte antigen-6 epidermis (lye), of Ly-6 superfamily in zebrafish. In silico analyses revealed that lye codes for a predicted GPI-anchored membrane protein containing a conserved LU domain and 10 position-specific conserved cysteines typical of known Ly-6 proteins. Whole mount in situ hybridization showed that lye is predominantly expressed in epidermis. We thus named the gene lye, highlighting it is expressed in epidermis. Lye exhibits a dynamic expression pattern during development, which is initially expressed in enveloping layer at gastrula stage, then expressed in epidermis at later stages. It is also expressed in olfactory placode at 24 h post-fertilization. Subsequently, epidermal expression of lye becomes weaker gradually, whereas the expression in pharyngeal arch and pectoral fin increases at 2 and 3 days post-fertilization. Our study lays a foundation for further investigation of lye roles in early developmental stages.     

Expression of an unusual T cell receptor (TCR)-V beta repertoire by Ly-6C+ subpopulations of CD4+ and/or CD8+ thymocytes. Evidence for a developmental relationship between Ly-6C+ thymocytes and CD4-CD8-TCR-alpha beta+ thymocytes.

A novel thymocyte subpopulation expressing an unusual TCR repertoire was identified by high surface expression of the Ly-6C Ag. Ly-6C+ thymocytes were distributed among all four CD4/CD8 thymocyte subsets, and represented a readily identifiable subpopulation within each one. Ly-6C+ thymocytes express TCR-alpha beta, arise late in ontogeny, and appear in the CD4/CD8 developmental pathway after birth in a sequence that resembles that followed by conventional Ly-6C- cells during fetal ontogeny. Most interestingly, adult Ly-6C+ thymocytes express an unusual TCR-V beta repertoire that is identical to that expressed by CD4-CD8-TCR-alpha beta+ thymocytes in its overexpression of TCR-V beta 8 and in its expression of some potentially autoreactive TCR-V beta specificities. This unusual TCR-V beta repertoire was even expressed by Ly-6C+ thymocytes contained within the CD4+ CD8- 'single positive' thymocyte subset. Thus, expression of this unusual TCR-V beta repertoire is not limited to CD4-CD8-thymocytes, and is unlikely to be a consequence of their double negative phenotype. Rather, we think that Ly-6C+TCR-alpha beta+ thymocytes and CD4-CD8-TCR-alpha beta+ are developmentally interrelated, a conclusion supported by several lines of evidence including the selective failure of both Ly-6C+ and CD4-CD8-TCR-alpha beta+ thymocyte subsets to appear in TCR-beta transgenic mice. In contrast, peripheral Ly-6C+ T cells are developmentally distinct from Ly-6C+ thymocytes in that peripheral Ly-6C+ T cells expressed a conventional TCR-V beta repertoire and developed normally in TCR-beta transgenic mice in which Ly-6C+ thymocytes failed to arise. We conclude that: 1) expression of a skewed TCR-V beta repertoire is a characteristic of Ly-6C+TCR-alpha beta+ thymocytes as well as CD4-CD8-TCR-alpha beta+ thymocytes, and is not unique to thymocytes expressing neither CD4 nor CD8 accessory molecules; and 2) Ly-6C+ thymocytes are developmentally linked to CD4-CD8-TCR-alpha beta+ thymocytes, but not to Ly-6C+ peripheral T cells. We suggest that Ly-6C+TCR-alpha beta+ thymocytes are not the developmental precursors of Ly-6C+ peripheral T cells, but rather may be the developmental precursors of CD4-CD8-TCR-alpha beta+ thymocytes.     

A structural model for the location of the Rodgers and the Chido antigenic determinants and their correlation with the human complement component C4A/C4B isotypes

In this study, the relative mass of the Ly-6A.2 antigen was shown to be 12 000–14 000, in contrast to initial studies which showed the relative mass to be 33 000. Using polymorphic Ly-6-specific antibodies, the 33 000 molecules could be immunoprecipitated from surface-iodinated thymocytes of Ly-6A.2⁺, Ly-6A.2^– strains and a Ly-6A.2^– mutant cell line BW(Thy-1^–e). This clearly demonstrated that 33 000 molecules were not associated with the Ly-6 polymorphism. By contrast, when biosynthetically labeled Ly-6A.2⁺ spleen cell lysates were analyzed, the major species immunoprecipitated by the polymorphic Ly-6A.2-specific antibody was 12000–14000, although a minor 33 000 species were also evident. The Ly-6A-specific antibody D7 which detects a monomorphic epitope on the Ly-6A molecule could immunoprecipilate the 12000–14000 molecules from surface-labeled cells. By contrast, the Ly-6A.2-specific antibodies detecting the polymorphic Ly-6A.2 determinant could not, though the reasons for this difference are not clear. Thus 12 000–14 000 molecules were only immunoprecipitated from Ly-6A.2⁺ cells, whereas 33 000 molecules were precipitated from both Ly-6A.2⁺ cells and Ly-6A.2^– cells. These findings suggest that the 33 000 molecules immunoprecipitated by 5041-24.2 are most likely to be an unrelated protein, possibly cross-reactive with some Ly-6A.2 antibodies.     

Characterization and differentiation of CD4-, CD8- thymocytes sorted with the Ly-24 marker

Heterogeneity within the CD4-, CD8- thymocyte population was explored by flow microfluorometry on thymocytes from 6-wk-old female C57BL/6 mice. Double negative cells were obtained by twice killing thymocytes with anti-CD4 and anti-CD8 antibodies. The resultant population lacked CD4, CD8, and Ig on cell surfaces; it contained cells bearing Ly-24 (27%), Ly-6C (6%), and Ly-5 (B220) detected by 6B2 (6%). These markers are the same as those characteristic of lpr/lpr T cells; they also are found on bone marrow cells. In order to investigate the maturational pathway of CD4-, CD8- thymocytes, such cells were cultured in vitro for 6 days with phorbol myristate acetate + calcium ionophore. There was a marked increase in cells bearing Ly-24 with time; by 6 days essentially all bore Ly-24. A lesser increase (to 15%) in 6B2 + Thy-1 positive cells was observed. Small numbers of cells bearing CD4 and/or CD8 also were found after 6 days in vitro. In additional studies, CD4-, CD8- cells were first sorted with respect to Ly-24 and then cultured with phorbol myristate acetate + calcium ionophore. Ly-24+ cells proliferated vigorously and formed clusters whereas Ly-24- cells did not. The former gave rise to large numbers of CD4+, CD8+ cells; the latter exhibited little differentiation. These studies demonstrate substantial heterogeneity within the CD4-, CD8- thymocyte population. Use of the markers Ly-24, Ly6C, and 6B2 allows a subdivision of such progenitor thymocytes. Different stages of maturation as well as possible lineages of cells may be investigated by combining such hemopoietic cell surface markers with in vitro culture.