Functional analysis of pokeweed mitogen-dependent cell interactions in murine spleen cells. I. Lack of B-cell mitogenicity and low frequency of effector helper T cells.

The nature of lymphocyte responses on addition of pokeweed mitogen (PWM) to normal murine spleen cells was studied in low cell density cultures. PWM, over a wide range of concentrations, stimulated proliferation in a set of cells roughly 10-fold smaller than the lymphocyte populations responding to either concanavalin A or lipopolysaccharide. PWM also induced a relatively small number of B lymphocytes in these cultures to mature to Ig-secreting plaque-forming cells (PFC).

The role of I-A/E molecules in B lymphocyte activation. I. Inhibition of lipopolysaccharide-induced responses by monoclonal antibodies.

A panel of 22 different monoclonal antibodies, including specificities against various antigenic clusters of I-A and I-E molecules, were probed over a wide range of concentrations for their ability to inhibit lipopolysaccharide-induced B lymphocyte proliferation and maturation to immunoglobulin-secreting plaque-forming cells (PFC). Most antibodies were competent to inhibit up to 80 to 100% of the response of appropriate target cells, although having little or no effect on irrelevant spleen cell cultures. Mixtures of either anti-I-A or anti-I-E specificities were more efficient inhibitors than individual antibodies, as shown by the average concentrations required for 50% inhibition (30 and 300 ng/ml, respectively).

A quantitative assay detecting small numbers of effector helper T cells, regardless of clonal specificity.

We have developed a new assay for quantitative detection of all helper T cells that can induce normal B lymphocytes to proliferation and Ig secretion. To establish the optimal assay conditions, we have used cloned T helper cells of defined specificities that had previously been shown to activate normal B lymphocytes expressing the specific antigen(s) on direct cellular interactions. As shown in this paper, 'irrelevant' B lymphocytes--that is, those that do not express either antigen or restriction elements recognized by the effector helper T cells--can also be induced in the presence of appropriate concentrations of pokeweed mitogen which are not mitogenic for the 'target' B lymphocytes.

The membrane receptor complex regulating induction and clonal expansion of normal murine B lymphocytes.

Induction of resting B lymphocytes results from the interaction of competent ligands or helper cells with "triggering receptors." Subsequent clonal expansion and performance are thought to be regulated by the interaction of selective growth or maturation factors with specific receptors on induced B cells. A set of membrane molecules of B lymphocytes, including IgM, IgD, IA, IE, lipopolysaccharide receptors, receptors for Fc and C3b, and other non-immunoglobulin structures recognized by some antiidiotypic antibodies, display ligand-induced relationships.

Functional and chemical characterization of B-cell growth factor produced by normal cloned T helper cells.

Media conditioned by clones of normal helper T cells exposed to appropriate antigen-presenting cells contain growth-promoting activity for B-cell blasts induced either by lipopolysaccharide or on direct interaction with competent helper cells. This B-cell growth factor (TH-BGAPet) is recovered on sodium doecyl sulphate polyacrylamide gel electrophoresis corresponding to mol. wt of 15,000-20,000 displays no mitogenicity for small, non-induced B lymphocytes and is completely devoid of the ability to activate immunoglobulin secretion in proliferating B cells.

T cell-dependent B cell activation.

T cell-dependent induction of small, resting B lymphocytes requires direct recognition of antigen and/or I-A/E molecules on the B cell surface by the inducing helper cell, and it does not require the participation of Ig receptors on the responding B cell. Triggering B cell receptors, therefore, are either the I-A/E molecules themselves, or other structures with complementarities on helper cell membranes that become available for productive interactions upon I-A/E recognition. It would appear that signal delivery by such triggering receptors can be regulated by a membrane complex of molecules, involving immunoglobulins, Class II MHC molecules and other classes of receptors, which in selective and distinct manners control the quantitative levels of expression and/or availability of the relevant structures.

B lymphocyte activation upon exclusive recognition of major histocompatibility antigens by T helper cells.

This study has investigated whether exclusive recognition of I-A or I-E molecules on the B cell surface by T helper cells is sufficient to activate resting B cells. Lines and clones of long-term-cultured T helper cells with specificity for I-A or I-E antigens have been derived from mixed lymphocyte cultures between spleen cells from major histocompatibility complex (MHC)-congenic mouse strains. These cells were tested for helper activity and proved competent to induce resting B lymphocytes expressing the specific MHC antigens to polyclonal expansion and maturation to Ig secretion.

Differential requirements for activation and growth of unprimed cytotoxic and helper T lymphocytes.

The requirements for activation and growth of T lymphocytes capable of mediating either cytolytic activity or help to B lymphocytes were studied in unprimed splenic T cell populations. The selectivity of expression of Lyt-2 antigens, the reactivity to soluble concanavalin A (Con A), to partially purified interleukin 2 (IL 2, T cell growth factor[s]) and to lectin-pulsed macrophages (M phi) were used in this analysis. Lectin-dependent cytotoxicity assays and a novel method that allows for the detection of all effector helper cells, regardless of their clonal specificities, were used for the functional identification of the responding T cells.

Activation of helper T cells for B lymphocytes in primary mixed lymphocyte cultures.

Normal B10.BR (H-2k, C57B1/6 background) spleen cells, enriched in primary mixed lymphocyte culture (MLC) for antigens of C3H/Tif mice (H-2k, C3H background), induced normal C3H/Tif but not B10.BR B lymphocytes to proliferate and produce Ig.

Distinct helper activities control growth or maturation of B lymphocytes.

A clone (C-11) of C3H/HeJ Lyt-1+2-T cells with specificity for "minor" antigens of C3H/Tif has been isolated which, in contrast to other similarly derived clones, did not activate polyclonal plaque-forming cell (PFC) responses in T cell-depleted "target" spleen cells. This clone, however, showed unaltered proliferative responses to the naturally occurring antigen(s) on presenting cells, and strongly synergized with regular helper clones in the induction of PFC responses. Further analysis demonstrated that C-11 cells are competent to stimulate extensive "target" B cell proliferation, but lack the ability to produce (or participate in the production of) maturation factors for activated B cells.

Differential macrophage requirements for T helper cell and T helper cell-induced B lymphocyte proliferation.

Major histocompatibility complex-restricted helper T cell clones against "minor" antigens expressed on B cell and macrophage surfaces, when confronted with appropriate T cell-depleted spleen cells, are induced to proliferation and, in turn, activate "target-responder" B cells to polyclonal growth and maturation. Irradiation of helper cell populations, however, demonstrates that their effector functions (and B lymphocyte responses) are independent of proliferative activity. Adherent cell depletion on Sephadex G10 columns, while completely abrogating helper T cell proliferation, does not abolish helper cell-induced B cell responses, demonstrating a remarkable quantitative difference in macrophage requirements for the growth of these two cell types.

Ontogenic development of B cell reactivities to cooperative cell signals: dissociation between proliferation and antibody secretion.

Cloned helper T cell lines, with specificity for "minor" antigens expressed on all B lymphocyte (and macrophage) surfaces were used to study the postnatal development of B cell reactivity to helper T cell cooperative signals. Such helper T cell populations, upon physiologic interactions with "target" B lymphocytes, activate them polyclonally, allowing for the analysis of functional ontogeny independent of V gene repertoires. Due to the high frequency of responding cells, these methods also offer a high level of sensitivity and permit measurement of B cell proliferation in the absence of antibody production.

MHC restriction of male-antigen-specific T helper cells collaborating in antibody responses.

By priming female C57BL/6 mice with syngeneic male spleen cells and enriching inguinal and paraaortic lymph node cells in long-term culture (LTC) by repeated restimulations, H-Y-specific T helper cells can be produced. In response to male spleen cells carrying I-Ab antigens these cells activate "antigen-expressing" B cells to secrete polyclonal antibody. Before the end of the second week in LTC it was impossible to detect any helper activity.

Clonal analysis of the specificity of alloreactive cells: "dominance" of E beta reactive clones.

The present experiments analyze the specificity of cells proliferating in murine mixed lymphocyte cultures (MLC). Lymphocytes from the B10.A(5R) strain, enriched by repeated restimulations with B10.