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...