The molecular and cellular basis of T-lymphocyte activation remains a central question in immunology. The growth of already proliferating T cells is known to depend on T-cell growth factor (TCGF), a physiological mitogen. Noncycling T lymphocytes, however, are not sensitive to TCGF. They require a short contact with mitogenic lectins, such as concanavalin A (Con A) or leukoagglutinin to bind and respond to TCGF, and will thereafter maintain exponential growth for long periods provided that TCGF is not limiting. While the induction of TCGF reactivity results from the direct contact of Con A with resting T cells, the lectin-dependent production of TCGF is known to involve two cell types, both present in mouse spleen. One consists of I-A-negative cells, most of which are Thy-1-positive T lymphocytes, and the other consists of I-A-positive, immunoglobulin-negative, Thy-1-negative cells, most of which are macrophages. The nature of the respective contributions of the two cell types, and in particular the cellular origin of TCGF, has not yet been established. We have now established the I-A-negative population as the source of TCGF and show here that macrophages are required to supply a 20,000-molecular weight factor, chemically and functionally distinct from TCGF, which supports the production of TCGF by the I-A-negative cells.

The spleen of adult antigen-free mice contains a sizable proportion (5-15%) of activated cells in all lymphocyte sets, as marked by the membrane expression of immunoglobulins, L3T4 and Lyt-2 antigens. The frequency of activated cells is very high in early post-natal life, and reaches adult levels by 6 weeks of age when it is comparable to that observed in healthy unmanipulated mice raised in conventional conditions. The effector B cell compartment is quantitatively similar in antigen-free mice and specific pathogen-free mice, but the former is deficient in isotype diversification, since IgG- and IgA-secreting cells are drastically reduced. The effector T cell compartment is slightly reduced in number, but is equally competent in providing help or suppression of syngeneic B cells. The results indicate the existence of a compartment of the immune system displaying autonomous self-determined activity which is predominant early in life. This compartment, physically localized to the spleen, appears to be distinct from an antigen-dependent compartment which is essential for the development of peripheral lymphoid organs draining sites of "natural" environmental immunization.

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The "natural" T-cell activity in normal unimmunized mice was studied. By double-parameter fluorescence-activated cell sorter analysis, it was found that 5-10% of all splenic Lyt-2+ and L3T4+ lymphocytes are large, of which more than half are in mitotic cycle. In contrast with small resting cells of the same phenotype, activated (large) T cells isolated from normal mice are functional effector cells: L3T4+ large cells induce normal B lymphocytes into proliferation and antibody secretion, while large Lyt-2+ cells efficiently suppress B-lymphocyte responses, No effector cell cytolytic activity could be detected among naturally activated T cells. The significance of these findings for the internal activity in the normal immune system is discussed.Much attention has been given in the past to natural antibodies, from both the theoretical (1) and the clinical points of view (2, 3). Astonishingly, the counterpart on the T-cell compartment of such natural activity of B lymphocytes has not been described thus far. The evaluation of current perspectives of an integrated immune system, which implies some degree of autonomous activity (4, 5), led us to investigate the presence of activated T cells in normal animals. The T-cell equivalents of "natural" plasma cells would be effector cells: helper (TH), suppressor (Ts), or cytotoxic T lymphocytes (CTL). The availability of assay systems that selectively detect such effector functions regardless of clonal specificities (6, 7) makes it possible to investigate the presence of those cells. Furthermore, lymphocytes differentiate both in bone marrow and thymus to a resting state of small immunocompetent cells (8,9), allowing the identification of activated cells in the periphery by their presence in the mitotic cycle. We report here that roughly 10o of all T lymphocytes in normal unimmunized mice are large blast cells, of which at least some exert effector functionsnamely, helping or suppressing B lymphocytes. MATERIALS AND METHODSMice. BALB/c mice, kept in conventional specific pathogen-free conditions, were used at ages between 8-12 weeks.Antibodies and Reagents. The following monoclonal antibodies were used with rabbit complement in cytotoxic treatments: rat IgMK, anti-Thy-1, J1J, a kind gift of J. Sprent (Scripps Clinic and Research Foundation, La Jolla, CA); mouse IgMK, anti-Lyt-2.2, HO-2.2 (10); rat IgGK, anti-L3T4, GK-1.5 (11); and mouse IgMK, anti-I-Ab/d, B17-263 (12).These were used as ascitic fluids or culture supernatants at the appropriate dilutions. For immunofluorescence studies, the following biotin-conjugated monoclonal antibodies were used to identify the various lymphocyte classes: rat IgGK, anti-A chain, R33-60 (13); rat IgGK, anti-Lyt-2, 53-6.72 (14); and rat IgGK, anti-L3T4, H-129-19.69 (15). These were used after ion-exchange column purification from culture super- Cell Cultures and Assays. All cultures were set in 0.2-ml flat-bottom microtiter plates as described (16). Growth of purified small and large T lymphocytes was assayed in cultures of 105 cell...

Growth factors contained in Con A(concanavalin A)-conditioned media (CM) maintain exponential growth in T cell blasts derived from mitogen stimulation of spleen cells with Con A, phytohemagglutinin, lentil lectin and pokeweed mitogen (PWM), as well as from mixed lymphocyte reactions to H-2D or I-C-encoded determinants and from non-H-2 Mls locus-controlled reactions. Such growth factors are strictly T cell blast-specific, inasmuch as they do not stimulate resting, small T or B lymphocytes nor B cell blasts generated by lipopolysaccharide or lipoprotein activation. The responsiveness of T cell blasts to CM appears to be the result of the availability on the cell surface of an acceptor site for growth factors which is not expressed in resting cells, because T cell blasts, but not small spleen cells, absorb out the growth-promoting activity contained in CM. Furthermore, lectins such as Con A and PWM interfere with the blast surface membrane in such a way that they inhibit the response to growth factors. Finally, there is no detectable allotypic or isotypic restriction in the activity of Con A-CM on a variety of target T cell blasts.

Abstract. We describe a novel approach to study tyrosine-phosphorylated (PY) integrins in cells transformed by virally encoded tyrosine kinases. We have synthesized a peptide (PY/~ peptide) that represents a portion of the cytoplasmic domain of the/~1 integrin subunit and is phosphorylated on the tyrosine residue known to be the target of oncogenic tyrosine kinases. Antibodies prepared against the PY/3~ peptide, after removal of cross-reacting antibodies by absorption and affinity purification, recognized the PYfll peptide and the tyrosine-phosphorylated form of the intact/~ subunit, but did not bind the nonphosphorylated/~ peptide, the nonphosphorylated/~ subunit or other unrelated tyrosine-phosphorylated proteins. The anti-PY/~I antibodies labeled the podosomes of Rous sarcoma virus-transformed fibroblasts, but did not detectably stain nontransformed fibroblasts. The localization of the tyrosine phosphorylated ~ subunit appeared distinct from that of the/~1 subunit. Adhesion plaques were stained by the anti-/~l subunit antibodies in Rous sarcoma virus-transformed fibroblasts plated on fibronectin, whereas neither podosomes nor adhesion plaques were labeled on vitronectin or on uncoated plates. Anti-phosphotyrosine antibodies labeled podosomes, adhesion plaques and cell-cell boundaries regardless of the substratum. One of the SH2 domains of the p85 subunit of phosphatidylinositol-3-kinase bound to the PY~I peptide, but not to the nonphosphorylated ~1 cytoplasmic peptide. Other SH2 domains did not bind to the PY~1 peptide. These results show that the phosphorylated form of the ~ integrin subunit is detected in a different subcellular localization than the nonphosphorylated form and suggest that the phosphorylation on tyrosine of the ~ subunit cytoplasmic domain may affect cellular signaling pathways. INTEGRINS are a family of transmembrane proteins composed of an c~ and/3 subunit. Many of the integrins are receptors for extracellular matrix proteins and mediate cell adhesion to matrices (for reviews, see ' Ruoslahti, 1991;Hynes, 1992). The//~ integrin subunit combines with several c~ subunits to form receptors for extracelhlar matrix proteins such as fibronectin, laminin, and collagens. The cytoplasmic domains of the integrins are thought to bind cytoskeletal and other intraceilular components. The ~1 integrin subunit has been shown to interact with talin ) and ce-actinin (Otey et al., 1990(Otey et al., , 1993 in vitro. Furthermore, the cytoplasmic tail is necessary and sufficient for ~1 integrins to localize in adhesion plaques (Solowska et al., 1989; l_aFlamme et al., 1992). Cell transformation by vitally encoded tyrosine kinases has been shown to cause increased phosphorylation on tyrosine of the ~1 subunit cytoplasmic domain (Hirst et al., 1986). The transforming protein of Rous sarcoma virus (RSV),I p60 v~'c, is a cytoplasmic tyrosine kinase with constitutive activity . Fibroblasts transformed by RSV express high levels of p60v-s% which causes extensive protein tyrosine phosphorylation of cellular proteins ...

The conformational flexibility of methyl α-cellobioside in water and dimethyl sulfoxide solutions was investigated by 1D 1H,H T-ROESY experiments. In combination with molecular dynamics simulations, effective proton−proton distances could be derived using experimentally determined cross-relaxation rates. An anti-ψ-conformational state was present in both solvents confirming a previous flexibility hypothesis at this torsion angle. In water solution, an anti-φ-conformational state was also detected and quantified. These results show that already at the disaccharide level a large flexibility is present at the glycosidic linkage. In addition to the syn-conformation which is present to ∼93% for the title compound in water solution, the minor anti-φ- and anti-ψ-conformational states are populated to ∼2% and ∼5%, respectively.

A number of ligands which bind to T cell surfaces were screened on murine spleen cells for their functional ability to induce either of the two necessary steps in the process of T cell triggering: (a) expression of growth receptors by T lymphocytes and (b) production of T cell growth factors in cultures containing both T cells and accessory cells. Among four lectins tested, concanavalin A was found to induce both responses but is primarily a “step 2” ligand, in particular at low concentrations; leucoagglutinin was the most potent “step 1” ligand with very limited “step 2” activity; wheat germ agglutinin and soybean lectin were inactive. Direct mitogenicity of the lectin to normal spleen cell cultures was limited by the poorest of these two functional properties. A number of rabbit anti‐lymphocyte antisera, anti‐Thy‐1 alloantisera, anti‐H‐2 and anti‐Ia monoclonal antibodies, as well as complexes of rabbit IgG, were all found to be devoid of “step 1” activity, and consequently, non‐ mitogenic for spleen cells. In contrast, a rabbit anti‐mouse brain antiserum, although also devoid of mitogenicity, was found to be capable of “step 1” induction. These results suggest that not all rearrangements of T cell surface membrane components upon binding of a ligand result in the functional expression of growth receptors. This appears to be the result of selective interactions with specific sites which include structures binding to concanavalin A, leucoagglutinin and rabbit anti‐mouse brain antibodies.