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Antibody-secreting plasma cells are nonrecirculatory and lodge in splenic red pulp, lymph node medullary cords, and bone marrow. The factors that regulate plasma cell localization are poorly defined. Here we demonstrate that, compared with their B cell precursors, plasma cells exhibit increased chemotactic sensitivity to the CXCR4 ligand CXCL12. At the same time, they downregulate CXCR5 and CCR7 and have reduced responsiveness to the B and T zone chemokines CXCL13, CCL19, and CCL21. We demonstrate that CXCL12 is expressed within splenic red pulp and lymph node medullary cords as well as in bone marrow. In chimeric mice reconstituted with CXCR4-deficient fetal liver cells, plasma cells are mislocalized in the spleen, found in elevated numbers in blood, and fail to accumulate normally in the bone marrow. Our findings indicate that as B cells differentiate into plasma cells they undergo a coordinated change in chemokine responsiveness that regulates their movements in secondary lymphoid organs and promotes lodgment within the bone marrow.

An important role has emerged for chemokines in regulating the distribution of progenitor cells during hematopoietic cell development. As well as recruiting cells, chemokines promote cell retention and cytokine expression. Furthermore, chemokines have been found to have an inductive function in secondary lymphoid organ development.

Chemokines play well established roles as attractants of naïve and effector T cells. New studies indicate that chemokines also have roles in regulating T cell differentiation. Blocking Gi protein-coupled receptor signaling by pertussis toxin as well as deficiencies in G alpha 12, chemokine receptor 2 (CCR2), CCR5, chemokine ligand 2 (CCL2, also known as monocyte chemoattractant protein 1, or MCP-1), CCL3 (macrophage inflammatory protein 1 alpha, or MIP-1 alpha) and CCL5 (RANTES) have all been found to have effects on the magnitude and cytokine polarity of the T cell response. Here we focus on findings in the CCL2-CCR2 and CCL3-CCR5 ligand-receptor systems. The roles of these molecules in regulating T cell fate include possible indirect effects on antigen-presenting cells and direct effects on differentiating T cells. Models to account for the action of chemokines and G protein-coupled receptor signals in regulating T cell differentiation are discussed.

The spontaneous mutant mouse strain, plt/plt, lacks the secondary lymphoid organ chemokine (SLC)-ser gene and has disrupted trafficking of T cells and dendritic cells (DCs) to lymphoid tissues. We demonstrate here that the gene for the related chemokine, Epstein-Barr virus-induced molecule-1 ligand chemokine (ELC), is also deleted in this immunodeficient mouse strain. Using a combination of approaches, including bone marrow reconstitution and double in situ hybridization, we show in wild-type mice that ELC is expressed by T zone stromal cells that also make SLC. Smaller amounts of ELC are made by DCs, predominantly of the CD8(+) phenotype. We propose that ELC- and SLC-expressing T zone stromal cells play a central role in bringing naive T cells and DCs together for the initiation of immune responses.

We describe a protein with the hallmarks of a chemokine, designated CXCL16, that is made by dendritic cells (DCs) in lymphoid organ T cell zones and by cells in the splenic red pulp. CXCL16 contains a transmembrane domain and both membrane-bound and soluble forms are produced. Naïve CD8 T cells, natural killer T cells and a subset of memory CD4 T cells bind CXCL16, and activated T cells migrated chemotactically to the soluble chemokine. By expression cloning, Bonzo (also known as STRL33 and TYMSTR) was identified as a CXCL16 receptor. CXCL16 may function in promoting interactions between DCs and CD8 T cells and in guiding T cell movements in the splenic red pulp. CXCL16 was also found in the thymic medulla and in some nonlymphoid tissues, indicating roles in thymocyte development and effector T cell trafficking.

Follicular dendritic cells (FDCs), the best defined stromal cell subset within lymphoid follicles, play a critical role in presenting intact antigen to B lymphocytes. The discovery that many follicular stromal cells make B-lymphocyte chemoattractant (BLC), a CXC chemokine that attracts CXCR5+ cells, provides a basis for understanding how motile B cells come into contact with stationary FDCs. Here we review our work on BLC and discuss properties of BLC-expressing follicular stromal cells. We also review the properties of primary follicle and germinal center FDCs and suggest a model of FDC development that incorporates information about BLC expression. Finally, we consider how antigen recognition causes T and B lymphocytes to undergo changes in chemokine responsiveness that may help direct their movements into, or out of, lymphoid follicles.

Lymphoid follicles are B-cell-rich compartments of lymphoid organs that function as sites of B-cell antigen encounter and differentiation. CXC chemokine receptor-5 (CXCR5) is required for B-cell migration to splenic follicles, but the requirements for homing to B-cell areas in lymph nodes remain to be defined. Here we show that lymph nodes contain two types of B-cell-rich compartment: follicles containing follicular dendritic cells, and areas lacking such cells. Using gene-targeted mice, we establish that B-lymphocyte chemoattractant (BLC/BCA1) and its receptor, CXCR5, are needed for B-cell homing to follicles in lymph nodes as well as in spleen. We also find that BLC is required for the development of most lymph nodes and Peyer's patches. In addition to mediating chemoattraction, BLC induces B cells to up-regulate membrane lymphotoxin alpha1beta2, a cytokine that promotes follicular dendritic cell development and BLC expression, establishing a positive feedback loop that is likely to be important in follicle development and homeostasis. In germinal centres the feedback loop is overridden, with B-cell lymphotoxin alpha1beta2 expression being induced by a mechanism independent of BLC.

CXCR5, the receptor for B lymphocyte chemoattractant (BLC), is required for normal development of Peyer's patches, inguinal lymph nodes, and splenic follicles. To test the in vivo activity of BLC in isolation of other lymphoid organizers, transgenic mice were generated expressing BLC in the pancreatic islets. In addition to attracting B cells, BLC expression led to development of lymph node-like structures that contained B and T cell zones, high endothelial venules, stromal cells, and the chemokine SLC. Development of these features was strongly dependent on B lymphocytes and on lymphotoxin alpha1beta2 and could be reversed by blocking lymphotoxin alpha1beta2. These findings establish that BLC is sufficient to activate a pathway of events leading to formation of organized lymphoid tissue.

Ag-mediated changes in B lymphocyte migration are important for normal immune function, yet the mechanisms by which these changes occur are poorly defined. Because chemokines direct many lymphocyte movements, molecules that regulate signaling by G protein-coupled chemokine receptors are likely to participate in Ag receptor-induced changes in cell migration. In this study, we have investigated the expression pattern and activity in murine B cells of members of the regulators of G protein signaling (RGS) family of molecules. We present the sequence of mouse RGS1 and describe a novel short isoform of RGS3 that we term RGS3s. Following in vivo activation by Ag, B cells rapidly up-regulate expression of RGS1 and RGS2 while simultaneously decreasing expression of RGS3 and RGS14. Anergic hen egg lysozyme autoantigen-binding B cells are also shown to have slightly elevated RGS1 and RGS2 expression. CD40 signaling, by contrast, fails to cause rapid up-regulation of RGS1 or RGS2. Using a transient transfection approach in a mature B cell line, 2PK3, we demonstrate that RGS1 and RGS3s are effective inhibitors of chemotaxis toward the lymphoid tissue chemokines stromal cell-derived factor-1, B lymphocyte chemoattractant, and EBV-induced molecule 1 ligand chemokine, whereas RGS2 has a minimal effect on migration to these chemokines. Together these findings support the conclusion that Ag-mediated changes in RGS molecule expression are part of the mechanism by which Ag receptor signaling regulates B cell migration within lymphoid tissues. The findings also suggest important roles for additional G protein-mediated events in B cell activation and tolerance.

Recent studies show that the chemokine receptor CCR7 helps T lymphocytes and dendritic cells to navigate into the T-cell zone of lymphoid organs. Furthermore, down-modulation of CCR7 redirects some activated T cells from these zones into other lymphoid tissue compartments.

Langerhans' cells migrating from contact-sensitized skin were found to up-regulate expression of macrophage-derived chemokine (MDC) during maturation into lymph node dendritic cells (DCs). Naïve T cells did not migrate toward MDC, but antigen-specific T cells rapidly acquired MDC responsiveness in vivo after a subcutaneous injection of antigen. In chemotaxis assays, maturing DCs attracted activated T cells more strongly than naïve T cells. These studies identified chemokine up-regulation as part of the Langerhans' cell maturation program to immunogenic T cell-zone DC. Preferential recruitment of activated T cells may be a mechanism used by maturing DCs to promote encounters with antigen-specific T cells.

In mice with a diverse B cell repertoire, hen egg lysozyme (HEL) autoantigen-binding B cells are excluded from follicles and eliminated in 3 days. To explore the roles of competitor B cells and of T cells in this mechanism of self-tolerance, HEL-specific B cells were transferred into mice containing HEL and deficient in endogenous B cells (muMT), T cells (TCR-/-), or B and T cells (RAG1-/-). Previous studies suggested a dual requirement for B cell receptor (BCR) engagement and competition in HEL autoantigen-binding B cell elimination, but interpretation of these experiments has been confounded by the possible failure to independently regulate autoantigen concentration and competitor B cell frequency. In experiments in this study, we have fixed one variable, HEL concentration, while varying the second, the presence or absence of other B cells. By this approach, we find that follicular exclusion and rapid elimination of autoreactive B cells require BCR engagement plus competition with other B cells, rather than BCR engagement alone. We also find, by transfers into T cell-deficient mice, that T cells are not required for this peripheral tolerance mechanism. Unexpectedly, in mice lacking both T cells and competitor B cells (RAG1-/-), transferred HEL-binding cells survive less well than in mice just lacking competitor B cells. These results suggest T cells can enhance autoreactive B cell survival. Enhanced survival of autoreactive B cells, due to the presence of T cells and the lack of competitor B cells, might contribute to the elevated frequency of autoimmunity in B cell-deficient individuals.

Secondary lymphoid tissue organogenesis requires tumor necrosis factor (TNF) and lymphotoxin alpha (LTalpha). The role of TNF in B cell positioning and formation of follicular structure was studied by comparing the location of newly produced naive recirculating and antigen-stimulated B cells in TNF-/- and TNF/LTalpha-/- mice. By creating radiation bone marrow chimeras from wild-type and TNF-/- mice, formation of normal splenic B cell follicles was shown to depend on TNF production by radiation-sensitive cells of hemopoietic origin. Reciprocal adoptive transfers of mature B cells between wild-type and knockout mice indicated that normal follicular tropism of recirculating naive B cells occurs independently of TNF derived from the recipient spleen. Moreover, soluble TNF receptor-IgG fusion protein administered in vivo failed to prevent B cell localization to the follicle or the germinal center reaction. Normal T zone tropism was observed when antigen-stimulated B cells were transferred into TNF-/- recipients, but not into TNF/LTalpha-/- recipients. This result appeared to account for the defect in isotype switching observed in intact TNF/LTalpha-/- mice because TNF/LTalpha-/- B cells, when stimulated in vitro, switched isotypes normally. Thus, TNF is necessary for creating the permissive environment for B cell movement and function, but is not itself responsible for these processes.

A B lymphocyte hyperactivity syndrome resembling systemic lupus erythematosus characterizes mice lacking the src-family kinase Lyn. Lyn is not required to initiate B cell antigen receptor (BCR) signaling but is an essential inhibitory component. lyn-/- B cells have a delayed but increased calcium flux and exaggerated negative selection responses in the presence of antigen and spontaneous hyperactivity in the absence of antigen. As in invertebrates, genetic effects of loci with only one functional allele can be used to analyze signaling networks in mice, demonstrating that negative regulation of the BCR is a complex quantitative trait in which Lyn, the coreceptor CD22, and the tyrosine phosphatase SHP-1 are each limiting elements. The biochemical basis of this complex trait involves a pathway requiring Lyn to phosphorylate CD22 and recruit SHP-1 to the CD22/BCR complex.

Secondary lymphoid organs (spleen, lymph nodes and Peyer's patches) are divided into compartments, such as B-cell zones (follicles) and T-cell zones, which provide specialized environments for specific steps of the immune response. Migration of lymphocyte subsets into these compartments is essential for normal immune function, yet the molecular cues guiding this cellular traffic are poorly defined. Chemokines constitute a family of chemotactic cytokines that have been shown to direct the migration of leukocytes during inflammation and which may be involved in the constitutive homing of lymphocytes into follicles and T-cell zones. Here we describe a novel chemokine, B-lymphocyte chemoattractant (BLC), that is strongly expressed in the follicles of Peyer's patches, the spleen and lymph nodes. BLC strongly attracts B lymphocytes while promoting migration of only small numbers of T cells and macrophages, and therefore is the first chemokine to be identified that is selective towards B cells. An orphan chemokine receptor, Burkitt's lymphoma receptor 1 (BLR-1), has been found to be required for B-cell migration into lymphoid follicles. We show that BLC stimulates calcium influx into, and chemotaxis of, cells transfected with BLR-1. Our results indicate that BLC functions as a BLR-1 ligand and may guide B lymphocytes to follicles in secondary lymphoid organs.

Preferential homing of naive lymphocytes to secondary lymphoid organs is thought to involve the action of chemokines, yet no chemokine has been shown to have either the expression pattern or the activities required to mediate this process. Here we show that a chemokine represented in the EST database, secondary lymphoid-tissue chemokine (SLC), is expressed in the high endothelial venules of lymph nodes and Peyer's patches, in the T cell areas of spleen, lymph nodes, and Peyer's patches, and in the lymphatic endothelium of multiple organs. SLC is a highly efficacious chemoattractant for lymphocytes with preferential activity toward naive T cells. Moreover, SLC induces firm adhesion of naive T lymphocytes via beta2 integrin binding to the counter receptor, intercellular adhesion molecule-1, a necessary step for lymphocyte recruitment. SLC is the first chemokine demonstrated to have the characteristics required to mediate homing of lymphocytes to secondary lymphoid organs. In addition, the expression of SLC in lymphatic endothelium suggests that the migration of lymphocytes from tissues into efferent lymphatics may be an active process mediated by this molecule.

It is not known how immunogenic versus tolerogenic cellular responses are signaled by receptors such as the B cell antigen receptor (BCR). Here we compare BCR signaling in naive cells that respond positively to foreign antigen and self-tolerant cells that respond negatively to self-antigen. In naive cells, foreign antigen triggered a large biphasic calcium response and activated nuclear signals through NF-AT, NF-kappa B, JNK, and ERK/pp90rsk. In tolerant B cells, self-antigen stimulated low calcium oscillations and activated NF-AT and ERK/pp90rsk but not NF-kappa B or JNK. Self-reactive B cells lacking the phosphatase CD45 did not exhibit calcium oscillations or ERK/pp90rsk activation, nor did they repond negatively to self-antigen. These data reveal striking biochemical differences in BCR signaling to the nucleus during positive selection by foreign antigens and negative selection by self-antigens.

The recent discovery of a receptor needed for lymphocyte migration into lymphoid follicles indicates that multiple chemoattractive gradients allow lymphocytes to navigate to specialized niches in lymphoid organs.

The need to eliminate autoreactive B cells must be checked against the need for a diverse B-cell repertoire. Protein tyrosine phosphatases SHP1 and CD45 act antagonistically within B cells to set the threshold level of antigen-receptor engagement required for B-cell elimination. The fate of B cells binding weak autoantigens is also regulated by interclonal competition. In the presence of a normal diverse repertoire of competitor B cells, the autoantigen-binding cells are excluded from follicles in spleen and lymph nodes and undergo rapid cell death. In the absence of interclonal competition, the autoreactive cells enter the follicular microenvironment and survive. A model in which B cells compete for access to limiting follicular niches in order to survive is discussed.

Elimination of self-reactive B cells must be balanced against the need for B-cell diversity for antibody responses to pathogens. To analyse factors that determine the extent of B-cell negative selection, we crossed CD45-deficient mice with mice carrying immunoglobulin transgenes specific for hen egg lysozyme (HEL). CD45 positively regulates antigen-receptor signalling and CD45-deficient HEL-specific B cells gave diminished signalling in response to HEL. Significantly, few mature CD45-/- B cells accumulated, despite normal immature B-cell production. Circulating HEL autoantigen mediates negative selection of mature CD45+/+ HEL-binding B cells but, in striking contrast, the autoantigen positively selected CD45-/- HEL-binding B cells, promoting their accumulation as long-lived IgD(hi) cells. These findings are consistent with a signal-threshold model for B-cell selection and demonstrate that changes in antigen receptor signalling can cause high-affinity self-reactive B cells to be actively retained instead of eliminated, thus revealing a potential mechanism for inherited susceptibility to autoimmune disease.

Self-reactive B cells are eliminated in a series of checkpoints that are triggered by antigen binding. Recent reports have shown that in addition to the processes of elimination at the immature B-cell stage, B-cell anergy and regulation of T-cell help, self-reactive cells are also controlled by follicular competition, Fas-mediated elimination by T cells and censoring in the germinal centres. Each checkpoint operates at a threshold that reflects the need to maintain immune diversity at the same time as suppressing autoimmune disease. Analysis of the motheaten mutation has given a direct demonstration of how such thresholds can be modulated by genetic effects.

Anergic self-reactive B cells competing within a polyclonal B cell repertoire fail to migrate into primary follicles and die after 1-3 days residence in T cell zones. Transfer of anergic HEL-specific B cells to recipients lacking HEL autoantigen and continuous bromodeoxyuridine labeling in mixed bone marrow chimeras confirms that follicular exclusion and cell death in 1-3 days is not an intrinsic characteristic of anergic cells but results from competition with B cells bearing other specificities together with continued binding of autoantigen. When naive (nontolerant) HEL-specific B cells were transferred into mice expressing HEL autoantigen, they were also excluded from follicles and their lifespan was dramatically shortened, although they became activated to express CD86 (B7-2/B70). In the presence of helper T cells, activated B cells but not anergic B cells were rescued from death and formed large extrafollicular foci to autoantibody-secreting cells. Antigen-induced exclusion from follicles is therefore an independent process from anergy that prevents self-reactive B cells from recirculating in the long-lived repertoire and may foster interactions with T cells during immune responses. By contrast, anergy prevents self-reactive B cells from collaborating with helper T cells and secreting autoantibody while trapped in the T zone.

The normal migration route of B cells into follicular areas of spleen and lymph nodes is altered in the case of autoreactive cells that have bound self-antigen. To begin characterizing the molecular requirements for B cell migration into follicles, cells were treated with pertussis toxin (PTX), an inhibitor of signaling by many G protein-coupled chemokine receptors. Lymphocyte accumulation in the spleen is not inhibited by PTX and, therefore, the distribution of transferred cells was examined in this tissue. In contrast to untreated cells that localized predominantly in follicular areas within white pulp cords, PTX-treated B cells failed to enter white pulp areas altogether and accumulated in the splenic red pulp. T cells were also excluded from white pulp cords and in the case of both cell types, the adenosine diphosphate-ribosylating subunit of the toxin was required to block white pulp entry. These findings implicate a G protein-coupled receptor in lymphocyte migration into splenic white pulp cords. Exclusion of PTX-treated cells from all organized areas of secondary lymphoid tissues raises the possibility that the association observed between PTX treatment and predisposition to autoimmune disease results from inhibition of tolerance mechanisms that normally operate within secondary lymphoid tissues.

Motheaten viable (mev) mice are deficient in the cytosolic protein tyrosine phosphatase, PTP1C, and exhibit severe B cell immunodeficiency and autoantibody production. The role of PTP1C in B cell selection and function was analyzed by breeding immunoglobulin transgenes specific for a defined antigen, hen egg lysozyme, into mev mice. Antigen triggered a greater and more rapid elevation of intracellular calcium in PTP1C-deficient B cells, indicating that this phosphatase negatively regulates immunoglobulin signaling. Elimination of self-reactive B cells carrying this signal-enhancing mutation was triggered during their development by binding a lower valency form of self-antigen than is normally required. These findings establish that activation of distinct repertoire-censoring mechanisms depends on quantitative differences in antigen receptor signaling, whose thresholds are determined by negative regulation through PTP1C.

Antibodies recognising the products of alternatively spliced exons near the N-terminus of the leukocyte common antigen, CD45, have been widely used to distinguish populations of lymphocytes with different functional properties. These alternatively spliced regions contain a high content of serine and threonine residues (average 35%) and are heavily O-glycosylated. Despite evidence that the O-glycosylation contributes significantly to the antigenic character of this region of CD45, work with leukosialin and mucin glycoproteins leads to the prediction that the majority of epitopes in the N-terminal exons should be linear protein determinants. In this study the exons of CD45 were expressed in Escherichia coli as non-glycosylated proteins fused to glutathione S-transferase (GST). Fourteen out of 17 mAbs specific for human CD45R reacted with a fusion protein containing exons 4, 5 and 6 (ABC) of human CD45, and four out of six mAbs specific for rat CD45R reacted with an equivalent rat protein. mAbs recognising the product of rat exon B are reported for the first time. Kinetic analysis of MRC OX22 antibody binding to spleen CD45 and to GST fusion proteins showed that the carbohydrate affected the kinetics of binding of antibodies to the protein backbone. In conclusion, heterogeneity in the glycosylation of heavily O-glycosylated cell surface proteins can affect interactions of these proteins both directly through the carbohydrate and indirectly through effects on the protein backbone.

Two different approaches to follow clones of B lymphocytes in a diverse preimmune repertoire reveal a new process for eliminating self-reactive cells in the periphery which depends on competition between cells with different specificities. A key feature of this censoring mechanism is the selective exclusion of self-antigen-binding B cells from the normal migration route into lymphoid follicles, resulting in their premature death. This is a striking example of homeostasis by cellular competition for limiting niches and may explain the paradoxical association between immunodeficiency and autoimmunity.

Many proteins have been predicted to contain domains with immunoglobulin-like folds and hence to be members of the immunoglobulin superfamily (IgSF). However, several members lack the Cys residues capable of forming the disulphide bond that forms a characteristic bridge between the beta sheets in the Ig fold, e.g. domain 1 of the lymphocyte antigen CD2. The assignment of beta strands in CD2 by sequence analysis was tested by attempting to introduce a disulphide bridge between the beta sheets by mutating two residues in the relevant positions to Cys. Mutant, soluble forms of CD2 were expressed in Chinese hamster ovary cell lines and amino acid sequencing showed that a disulphide bond was formed as predicted, but not in the control where one Cys residue was misplaced by four residues. Evidence that both mutated molecules folded correctly is given by the indistinguishable binding of three monoclonal antibodies recognising different epitopes on CD2. The 3-D structure of rat CD2 domain 1 has been determined by NMR spectroscopy and X-ray crystallography, confirming the predictions from the sequence. Applications of this method of insertion of disulphide residues for probing protein structures are discussed, together with other structures of IgSF domains lacking the typical inter-sheet disulphide bond.

The adhesion interaction between the immunoglobulin superfamily molecules CD2 and CD58 (lymphocyte function-associated antigen 3) plays an important role in T cell and natural killer cell interaction with various antigen-presenting and target cells. Determination of the solution structure of rat CD2 domain 1 has allowed a model of human CD2 domain 1 to be generated, and a series of mutants based on this model have been made. Residues of domain 1 of human CD2 predicted to be solvent exposed were substituted with the equivalent residues present in the rat CD2 molecule. The ability of these mutants to mediate rosetting with human and sheep erythrocytes was studied. Results show that the binding site of CD2 for both human and sheep CD58 maps to the beta sheet containing beta strands CC'C"F and G. Residues K34 and E36 in beta strand C, R48 and K49 in beta strand C', and K91 and N92 in the loop connecting beta strands F and G are shown to be critical in the interaction. The data support the proposition that the interaction between CD2 and CD58 involves the major beta sheet face of CD2.

CD2, CD48 and CD58 are structurally similar cell adhesion-molecules forming a subset of the immunoglobulin superfamily (IgSF). In humans CD58 is a ligand for CD2 while in mice CD2 binds CD48. We constructed a soluble chimeric molecule comprising the extracellular portion of rat CD48 and domains 3 and 4 of rat CD4 (sCD48-CD4) and used it to examine whether CD2 is a ligand for CD48 in rats. sCD48-CD4-coated polystyrene Dynabeads formed rosettes on rat CD2-transfected COS-7 cells, and this rosetting was blocked by anti-CD2 (OX34) and anti-CD48 (OX45) monoclonal antibodies. We used sucrose-gradient ultracentrifugation to show that sCD48-CD4 binds, in solution, to soluble forms of rat CD2 including the single NH2-terminal IgSF domain of rat CD2 expressed in bacteria. The upper limit of the affinity of the rat CD48-CD2 interaction is 4 x 10(5) M-1, lower than the published affinity of human CD2 for CD58. These results show that rat CD48 binds CD2 on its NH2-terminal IgSF domain with a low affinity and that binding is independent of glycosylation.

Leukosialin (CD43) is a major glycoprotein of T lymphocytes which has an extracellular domain of 45 nm in length that is heavily O-glycosylated. Monoclonal antibodies (mAb) to the extracellular domain of human leukosialin induce aggregation of T lymphocytes, monocytes and some cell lines that express leukosialin. The aggregation was reported in one case to be inducible by Fab fragments. In the present study, nine mAb specific for rat leukosialin were tested as inducers of thymocyte aggregation and all were effective. The level of aggregation was reduced by metabolic and cytoskeletal inhibitors, by removal of divalent cations and by reducing the temperature from 37 degrees C to 4 degrees C. The aggregation produced by mAb specific for certain epitopes was less sensitive to these inhibitors than aggregation induced by mAb to other epitopes. To examine the requirement for cross-linking, Fab fragments of four of the antibodies were tested and found to be inactive except for those derived from the OX75 mAb. However, OX75 Fab showed a tendency to dimerize, and monomeric OX75 Fab obtained directly after gel filtration was unable to induce aggregation. Thus induction of rat thymocyte aggregation by anti-leukosialin antibodies requires bivalent cross-linking and maximal aggregation is dependent on energy and an intact cytoskeleton. Mechanisms of antibody-induced aggregation are considered and it is proposed that in the case of leukosialin the antibodies may cross-link cells to overcome inherent repulsion between them and that subsequently other adhesion molecules complete the clustering process.

Large peptides expressed from cDNA fragments of a clone encoding the mite allergen Der p I were able to bind IgE and IgG in sera from allergic individuals. The binding was found for peptides from sequences throughout the molecule, with at least five regions, comprising residues 1-56, 53-99, 98-140, 166-194, and 188-222. The only limitation was that more than 30 amino acid residues were required for consistent binding. Each of seven sera examined showed a different profile of antibody binding to the peptides. For the most part the pattern of IgE and IgG binding to the peptides for each serum was similar, demonstrating a concordant repertoire. In 5/7 sera, however, IgG bound to some peptides which had little or no IgE binding activity, thus showing more diverse specificities. It is suggested that some divergence of repertoire can develop during the maturation of the B cell response.

The CD2 antigen is largely restricted to cells of the T-lymphocyte lineage and has been established as an important adhesion molecule in interactions between human T lymphocytes and accessory cells. In the adhesion reaction, CD2 on T cells binds to LFA-3 on other cells, with binding through domain 1 of CD2. CD2 can also be a target for the delivery of mitogenic signals to T lymphocytes cultured with combinations of anti-CD2 antibodies. Two predictions that are contradictory have been made for the structure of CD2 domain 1. One suggests an immunoglobulin (Ig) fold, on the basis of sequence patterns conserved in the Ig-superfamily (IgSF), whilst the other proposes a pattern of alternating alpha-helices and beta-strands, on the basis of secondary structure predictions. Thus CD2 domain 1 is an important test case for the validity of IgSF assignments based on sequence patterns. We report here the expression of domain 1 of rat CD2 in an Escherichia coli expression system and have determined a low-resolution solution structure by NMR spectroscopy.

Leukosialin (CD43) is a major glycoprotein of T lymphocytes whose extracellular domain of 224 amino acids contains on average one O-linked carbohydrate unit per three amino acids. This suggests an unfolded structure for the extracellular domain which has now been established to extend to a length of 45 nm by transmission electron microscopy following low angle rotary shadowing. The antigenicity of rat leukosialin has been studied using nine monoclonal antibodies (MAbs) whose binding is differentially affected by the cell type on which leukosialin is expressed and by the removal of sialic acid. From these observations it appears that the epitopes are affected by glycosylation, yet seven of the nine MAbs reacted clearly with the extracellular domain of leukosialian expressed in an unglycosylated form in Escherichia coli. The MAbs showing this positive reaction included three of the four antibodies whose epitopes were affected by neuraminidase treatment of leukosialin. It thus appears that linear protein epitopes are recognized and that some of these can be modified in the native structure by glycosylation. The positions of the antigenic determinants have been mapped by expressing fusion proteins of different lengths and the identity of one epitope was proven by the binding of two MAbs to an octapeptide expressed as a fusion protein. For three MAbs, the location of epitopes in the native protein was confirmed by electron microscopy of shadowed leukosialin--Fab complexes. Overall it is concluded that leukosialin is a major component at the periphery of the T lymphocyte and that despite its high level of glycosylation, protein determinants are exposed that could be ligands in cell interactions.