The lymphocytosis-promoting factor of Bordetella pertussis is a potent mitogen for murine lymphocytes in vitro. The stimulatory response was not the result of specific antigen stimulation. Spleen and lymph node cells were responsive, whereas normal thymocytes were unresponsive. However, DNA replication was induced in cortisone-resistant thymocytes by lymphocytosis-promoting factor (LPF). Bone marrow cells were not stimulated by LPF.

A B S T R A C T The purified lymphocytosis promoting factor (LPF) from Bordetella pertussis was found to be a potent mitogen for peripheral blood lymphocytes (PBL) PBL from most patients with chronic lymphatic leukemia and lymphosarcoma cell leukemia were even less responsive to LPF than to phytohemagglutinin, whereas PBL from patients with lymphosarcoma usually responded to both mitogens. It can be inferred from the results of experiments with both normal and leukemic cells that LPF, which is a murine thymtusderived (T)-cell mitogen, is also a T-cell mitogen for human PBL. The exact cell requirement and mode of action, however, are as yet unknown.

In an accompanying communication (1) we described the in vitro mitogenic effect of the lymphocytosis-promoting factor (LPF) I of phase I Bordetella pertussis on murine lymphocytes. It was found that LPF caused proliferation of spleen and lymph node cells of unsensitized mice, but had no effect on bone marrow cells. The majority of thymocytes were not stimulated by LPF, but the population of cortisone-resistant thymocytes was reactive.In this paper we present the results of experiments designed to delineate the nature of the lymphocyte population which responds to LPF, and the requirement for accessory cells in the attainment of an optimum mitogenic effect. Materials and MethodsMice.

The nature of the helper lymphocytes in lymphocytosis-promoting factor (LPF)-induced proliferation was explored. Removal of macrophages from adherent splenocytes by either carbonyl-iron incubation or passage through Sephadex G-10 columns did not affect their synergistic function. Nor did cytolysis with Thy-1.2 antiserum and complement. The helper cells were found to be surface immunoglobulin-positive (sIg+) because they are retained by anti-Ig columns, susceptible to lysis by rabbit anti-mouse immunoglobulin and complement, and occurred in the sIg+ fractions of splenocytes after separation on the fluorescence-activated cell sorter. Further delineation of the surface markers on helper cells showed that complement receptors are not the determining marker for synergistic function. The requirement for B-helper cells in the stimulation of T lymphocytes by LPF is unique for a mouse of T-cell mitogen.

Cytotoxic effector lymphocytes were induced in cultures of mouse spleen or lymph node cells by lymphocytosis promoting factor (LPF). The LPF-activated cytotoxic cells: (a) were not generated unless proliferation occurred; (b) sedimented in the lighter density fraction of a bovine serum albumin gradient; (c) were large, blast-like cells; and (d) were lysed by Thy-1.2 antiserum plus complement and, therefore, were T cells. Neither LPF alone nor supernates from stimulated cultures were cytotoxic. Unlike the situation with concanavalin A and phytohemagglutinin P, LPF-stimulated cytotoxic effector lymphocytes required no further addition of mitogen for maximal cytotoxicity. The effector cells displayed specificity, destroying only allogeneic but not syngeneic normal cells; in the case of tumor cells, both allogeneic and syngeneic cells werelysed in the absence of added mitogen. The reason for differentiated cytotoxicity toward syngeneic tumor and normal cells is not clear but may have some relevance to in vivo tumor rejection initiated by Bordetella pertussis.