PGRL is a major CD81-associated protein on lymphocytes and distinguishes a new family of cell surface proteins.

CD81 exerts a range of interesting effects on T cells including early thymocyte differentiation, LFA-1 activation, and provision of costimulation. To better understand the mechanisms by which CD81 influences T cell function we evaluated CD81 molecular complexes on T cells. The most prominent CD81-associated cell surface protein on thymocytes as well as a number of T cell and B cell lines has an apparent molecular mass of 75 kDa. The 75-kDa protein was purified and analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry followed by postsource-decay profiling. p75 is a novel type I transmembrane protein of the Ig superfamily which is most similar to FPRP. We cloned and sequenced both human and mouse PG regulatory-like protein (PGRL) and characterized mouse PGRL expression in both lymphocytes and nonlymphoid tissues. The discovery of PGRL allows for the clustering of a small family of related proteins including PGRL, FPRP, V7/CD101, and IGSF3. Expression constructs containing various domains of PGRL with an epitope tag were coexpressed with CD81 and used to determine that the interaction of CD81 with PGRL requires the membrane distal Ig3-Ig4 domains of PGRL. Although it remains to be determined whether PGRL possesses PG regulatory functions, transwell chamber experiments show that PGs and CD81 coordinately regulate T cell motility.

Anti-CD81 activates LFA-1 on T cells and promotes T cell-B cell collaboration.

CD81 is expressed on human T cells at all stages of development. CD81 is physically associated with CD4 and CD8 and antibodies against CD81 generate signals which influence thymocyte adhesion and proliferation. Here we evaluate the function of CD81 on mature T cells. We employ a system in which B cells present superantigen to autologous T cells and find that anti-CD81 promotes T cell-B cell collaboration. Anti-CD81 induces T cell-B cell adhesion of peripheral blood lymphocytes which is partially mediated by LFA-1. CD81 engagement promotes LFA-1-dependent T cell activation, IL-2 production and proliferation. The antibody 5A6 was uniquely potent in exerting these effects compared to another antibody to CD81 or to antibodies that react with other tetraspanins expressed on T cells, anti-CD53 or anti-CD82. CD81-derived signals rapidly induce high-avidity LFA-1 as measured by cell binding to recombinant ICAM-3-coated fluorescent microspheres or by cell adhesion to ICAM-3-coated plastic. 5A6 activation of LFA-1 does not expose the high-affinity conformation epitope recognized by monoclonal antibody 24.

Differential expression of murine CD81 highlighted by new anti-mouse CD81 monoclonal antibodies.

We describe the use of a soluble CD81-Fc fusion protein to screen for novel monoclonal antibody (MAb) reactive with the extracellular loops of murine CD81 (TAPA-1). Two such MAbs, Eat1 and Eat2 (for Extracellular Anti-TAPA1), were used to assess the expression and function of CD81 on murine lymphocytes. Although CD81 is expressed uniformly on all human lymphocytes, murine CD81 was found to be expressed at much higher levels on resting B cells than on resting T cells. This was particularly evident when staining with the new MAbs, Eat1 and Eat2. The molecule is also functionally active on B cells, as Eat1 and Eat2 induce homotypic adhesion of B lymphocytes. Stimulated B cells undergo early apoptotic events in the presence of Eat2, as shown by binding of Annexin V-fluorescein isothiocyanate (FITC). Polyclonal activation of murine T cells also induces higher level CD81 expression, and many immortalized murine T-cell lines express high levels of the protein. In contrast to human CD81, which is expressed equally on all thymocytes, murine CD81 is induced during thymic development, being expressed at high levels on CD4+CD8+ thymocytes, in contrast to other subsets of thymocytes. Finally, murine dendritic cells, splenic macrophages, and non-killer (NK) cells all express high levels of CD81. We conclude that CD81 is differentially expressed in the murine immune system, and is involved in regulating the adhesion and activation of murine B cells.

Sequences and expression of six new members of the tetraspanin/TM4SF family.

Tetraspanins (or TM4SF) are expressed in a wide variety of species and regulate cell adhesion, migration, proliferation and differentiation. We have identified and sequenced six new members of the tetraspanin family, called Tspan-1-6, from human cDNA. Amino acid sequence analysis of the Tspans highlights conserved residues which may be critical to tetraspanin structure and function. The Tspans are differentially expressed in human tissues.

CD81 (TAPA-1): a molecule involved in signal transduction and cell adhesion in the immune system.

CD81 (TAPA-1) is a widely expressed cell-surface protein involved in an astonishing variety of biologic responses. It has been cloned independently several times for different functional effects and is reported to influence adhesion, morphology, activation, proliferation, and differentiation of B, T, and other cells. On B cells CD81 is part of a complex with CD21, CD19, and Leu13. This complex reduces the threshold for B cell activation via the B cell receptor by bridging Ag specific recognition and CD21-mediated complement recognition. Similarly on T cells CD81 associates with CD4 and CD8 and provides a costimulatory signal with CD3. In fetal thymic organ culture, mAb to CD81 block maturation of CD4-CD8- thymocytes, and expression of CD81 on CHO cells endows those cells with the ability to support T cell maturation. However, CD81-deficient mice express normal numbers and subsets of T cells. These mice do exhibit diminished antibody responses to protein antigens. CD81 is also physically and functionally associated with several integrins. Anti-CD81 can activate integrin alpha 4 beta 1 (VLA-4) on B cells, facilitating their adhesion to tonsilar interfollicular stroma. Similarly, anti-CD81 can activate alpha L beta 2 (LFA-1) on human thymocytes. CD81 can also affect cognate B-T cell interactions because anti-CD81 increases IL-4 synthesis by T cells responding to antigen presented by B cells but not by monocytes. The tetraspanin superfamily (or TM4SF) includes CD81, CD9, CD37, CD53, CD63, CD82, CD151, and an increasing number of additional proteins. Like CD81, several tetraspanins are involved in cell adhesion, motility, and metastasis, as well as cell activation and signal transduction.

The tetraspanin superfamily: molecular facilitators.

A legacy of molecular evolution is the formation of gene families encoding proteins that often serve related functions. One such family gaining recent attention is the tetraspanin superfamily, whose membership has grown to nearly 20 known genes since its discovery in 1990. All encode cell-surface proteins that span the membrane four times, forming two extracellular loops. Some of these genes are found in organisms as primitive as schistosomes and nematodes. Alternately known as the transmembrane 4 (TM4) superfamily or the TM4SF, 4TM, or tetraspan family, we propose here that the name tetraspanins be used for the purpose of standardization. What do the tetraspanins do? Awaiting definitive functional studies, we can only put together pieces of a puzzle that has been built by raising antibodies against these proteins and looking at their distribution, associations, and functions. A brief overview indicates that some tetraspanins are found in virtually all tissues (CD81, CD82, CD9, CD63), whereas others are highly restricted, such as CD37 (B cells) or CD53 (lymphoid and myeloid cells). Many of these proteins have a flair for promiscuous associations with other molecules, including lineage-specific proteins, integrins, and other tetraspanins. In terms of function, they are involved in diverse processes such as cell activation and proliferation, adhesion and motility, differentiation, and cancer. We propose that these functions may all relate to their ability to act as "molecular facilitators," grouping specific cell-surface proteins and thus increasing the formation and stability of functional signaling complexes.

CD81 expressed on human thymocytes mediates integrin activation and interleukin 2-dependent proliferation.

Lymphocyte recognition of antigen by the antigen-specific T cell receptor (TCR) and coreceptor complexes rapidly alters the cell's adhesive properties facilitating high avidity cell-ligand interactions necessary for lymphocyte development and function. Here, we report the expression of CD81 (target of antiproliferative antigen [TAPA]-1) on human thymocytes and the physical association of CD81 with CD4 and CD8 T cell coreceptors. Antibody ligation of CD81 on thymocytes promotes the rapid induction of integrin-mediated cell-cell adhesion via lymphocyte function-associated molecule-1 (LFA-1). Cross-linking CD81 is also shown to be costimulatory with signaling through the TCR/CD3 complex inducing interleukin 2-dependent thymocyte proliferation. These data suggest that a CD81-mediated pathway in thymocytes is involved in the regulation of both cell adhesion and activation.

EBV induces proliferation of immature human thymocytes in an IL-2-mediated response.

The receptor for EBV, CD21 is expressed on a population of immature human thymocytes and facilitates infection of these cells by EBV. Thymocytes infected by EBV become responsive to exogenous rIL-2- or CD2-mediated stimulation in vitro. To address whether such costimulation may be provided by thymic presenting cells and to study the cellular effects of EBV infection, the present work utilizes thymocyte cultures containing autologous thymic presenting cells. In the presence of these presenting cells, EBV induces proliferation of thymocytes. EBV infection promotes the formation of adhesions between two populations of cells in an APC responder fashion, and separation of these two populations abrogates the proliferative response to EBV. The response is mediated by IL-2 because Ab blocking of the IL-2R inhibits proliferation as does cyclosporin A. EBV promotes an expansion in the number of CD4+8+ thymocytes, and the proliferating population is vulnerable to TCR/CD3-generated signals, indicating that the responding cells are phenotypically and functionally immature. Finally, addition of exogenous IL-2 to EBV-exposed thymocytes promotes a second wave of proliferation. Phenotypic characterization of the EBV-induced, IL-2-responding cells shows them to express reduced levels of CD1 and a transitional CD4(high)8(low) phenotype. These data characterize the cellular response to EBV infection in thymocytes and may offer insight into EBV-associated T lineage malignancies and autoimmune disorders.

Characterization of a 70-kDa, EBV gp350/220-binding protein on HSB-2 T cells.

EBV binds and infects HSB-2 T cells via a receptor distinct from CD21. To further study this novel EBV receptor, we expressed the first 470 amino acids of the EBV-gp350/220 using the baculovirus expression system. The recombinant gp350/220(1-470) has a m.w. of 95 kDa, reacts with anti-gp350/220 Abs, and binds CD21 in ELISA. Radiolabeled gp350/220(1-470) binds both HSB-2 and Raji cells. The gp350/220(1-470) protein also inhibits EBV binding to both HSB-2 and Raji, detected by flow cytometry. Lysates of HSB-2 cells compete with CD21 for binding to gp350/220(1-470), suggesting that the two receptors bind related epitopes on the recombinant protein. Scatchard analysis reveals that gp350/220(1-470) binds to 34,000 high affinity sites/HSB-2 cell (Kd = 0.92 x 10(-8) M) compared with the 97,000 high affinity sites bound/Raji cell (Kd = 1.78 x 10(-8) M). Utilizing a gp350/220(1-470)-affinity matrix, we identify a 70-kDa (55-kDa nonreduced) protein on the surfaces of 125I-labeled HSB-2 cells. Binding of this protein to the matrix is inhibited by anti-gp350/220 Ab 72A1. In summary, we characterize a novel EBV-binding molecule on HSB-2 cells, compare its reactivity with gp350/220 to that of CD21, and provide evidence of a gp350/220-reactive, 70-kDa protein on the surfaces of HSB-2 cells. In view of previous evidence of HSB-2 infectivity by EBV, we propose that the 70 kDa protein represents the novel EBV receptor.

CD1+ human thymocytes proliferate in response to superantigen staphylococcal enterotoxin B1.

Exposure of human thymocytes to superantigens results in the deletion of thymocytes expressing specific TCR-V beta genes. The factors that contribute to this deletion may relate to the inherent nature of the T cell at a given stage of development. In this paper, we demonstrate that CD1+ human cortical thymocytes are capable of proliferating in response to a bacterial superantigen (staphylococcal enterotoxin B (SEB)) in the presence of autologous CD2-/low thymic APCs. Phenotypic analysis of the responding populations revealed that the majority of the CD1+ cells were CD4+CD8low or CD8+CD4low cells. The response is triggered by low concentrations of SEB, requires the participation of the TCR and IL-2R molecules, and is inhibited by cyclosporin A. Thymocytes that express specific V beta genes are expanded, which results in an engagement profile that parallels that found in PBLs. Additionally, four V beta-chains that have not been reported previously are shown to engage SEB. Once stimulated, the thymocytes failed to respond to additional SEB; however, they could be induced to proliferative with IL-2, which suggests that these expanded populations had become anergic. These data represent the first demonstration of a human cortical thymocyte subpopulation that responds to superantigen by proliferation and subsequent anergy.

Characterization of the EBV/C3d receptor on the human Jurkat T cell line: evidence for a novel transcript.

EBV is a human herpes virus that causes mononucleosis and is associated with various tumors. EBV infects cells via the CR2 that was previously thought to be expressed only on the surface of B cells and certain epithelial cells. Recent findings in our laboratory and those of others, however, have shown that the EBV receptor is also present on T cells. Our study shows that Jurkat human T cells have a molecule that reacts with both anti-CR2 antibodies and the third component of complement, C3. Furthermore, the data indicate that this molecule binds EBV detected by incubation with biotin-conjugated virus and streptavidin phycoerythrin. Viral binding is specific, as it is inhibited by nonconjugated virus, with anti-CR2 antibodies, and with an antibody reactive with the glycoprotein (gp350) that EBV uses to bind CR2. In addition, EBV variably infects Jurkat cells as demonstrated by the presence of transcripts of Epstein Barr nuclear Ag (EBNA-1) using the polymerase chain reaction. Immunoprecipitation experiments with anti-CR2 antibodies and SDS-PAGE analysis reveal a protein with an apparent molecular mass of 155 kDa which is higher than the one seen in B cells. The size of this molecule is reduced to 119 kDa upon endoglycosidase F treatment. Northern blot analysis of Jurkat poly(A)+ RNA shows a transcript of 4.7 kb upon probing with the B cell CR2 cDNA. This size is consistent with that of B cell CR2 mRNA. Two cDNA clones were identified upon screening of a Jurkat cell cDNA library with the B cell CR2 cDNA. One of the clones possesses an identical nucleotide sequence to the one corresponding to B cell CR2, whereas the other represents a differentially spliced transcript which lacks the exon 8b of B cell CR2. Analysis of Jurkat and Raji mRNA by PCR demonstrated the presence of this novel splice variant in both cell lines.