Ronald B. Corley

Lymphocyte stimulation in vitro: Generation of cytotoxic effector lymphocytes using subcellular fractions of a lymphoid cell line☆

Abstract Subcellular fractions, isolated from the lymphoid cell line IM-1, are capable of stimulating a weak proliferative response in allogeneic lymphocytes. They also stimulate the generation of cytotoxic effector lymphocytes. The proliferative response to subcellular fractions, as measured by 3H-thymidine incorporation, is only one-fourth to one-sixth as great as that to intact IM-1 cells, suggesting that a component(s) synthesized during the mixed lymphocyte reaction (MLR), or a short-lived cellular constituent, may be responsible for the ability of intact cells to stimulate a lymphocyte proliferative response. This component appears to be lacking or in limiting quantity in subcellular fractions, including the soluble fractions. In contrast to the decreased proliferative response to subcellular fractions, the cytotoxic capacity of the stimulated lymphocytes is comparable to that after stimulation by intact IM-1 cells. The data demonstrate that, in this system, cytotoxic effector lymphocytes can be generated in the absence of the extensive proliferative response normally observed in the MLR. The antigenic stimulus responsible for the generation of cytotoxic effector cells appears to reside on intracellular components as well as on plasma membrane. In these reactions, specificity is shown by the failure of the cytotoxic cells to release 51Cr from autologous target cells. In fact, reactivity of lymphocytes stimulated by subcellular fractions is more specific than the reactivity of cells stimulated by intact IM-1 as judged by their lytic capacity for another target cell, RPMI 4265.

T cell help in cytotoxic T lymphocyte responses. Role of the I region in helper cell induction.

We have studied the in vivo induction of T helper (TH) cells that participate in the generation of cytotoxic T (Tc) lymphocytes. Helper activity was measured by the ability of the cells to help resting thymic Tc cell precursors develop into effector Tc cells in vitro. Direct injection of allogeneic spleen cells into the footpads of mice led to the generation of alloantigen-specific helper cells in the draining popliteal lymph nodes within 4 to 6 days. Helper activity was mediated by nylon-wool-nonadherent Lyt-1+ T lymphocytes; some activity was associated with Lyt-1,2+ cells.The genetic requirements for both the induction and restimulation of C3H anti-H-2d TH cells were investigated using cells from H-2k/H-2d recombinant mice as in vivo immunogens and in vitro stimulators. Evidence is presented that shows in a direct assay that TH cells themselves are specific for I region-coded determinants. Thus, disparity at the left side of the H-2 complex (K to I-E) but not at H-2K alone was necessary and sufficient to induce and reactivate TH cells. Proliferation in mixed lymphocyte culture was measured in combinations in which TH cells were not detectable, supporting the idea that proliferation cannot be strictly considered a measurement of helper cells.

Regulation of Growth and Differentiation in B Cell Clones and Hybrids

Analysis of the molecular events which occur during specific B cell activation or inactivation has been hampered by the rare frequency of antigen-specific B cells, as well as by the heterogeneity of B cell subpopulations and stages of differentiation. An initial approach to the solution of this dilemma is to enrich for antigen-specific lymphocytes. The purification of B cells of a single hapten-specificity has, indeed, been possible for a number of years (1). However, such enrichment procedures do not overcome the heterogeneity which exists in B cells of a single specificity. The ability to clone hapten-specific B cells and/or hybridize them to an appropriate tumor cell would resolve many of these difficulties, assuming that the “normal” B cell phenotype can be retained. In analogy to the technology of T cell cloning, which is abundantly apparent at the present meeting, clones of hapten specific B cells are now being prepared. In contrast to T cell cloning, however, it is not yet possible to repeatedly stimulate B cells with specific antigen in the absence of terminal differentiation to antibody secreting cells. The present report describes the current state of the art with regard to B cell cloning technology to yield functional short-term hapten-specific clones as well as long-term hapten-specific inducible hybrids. We will focus on the kinds of B cell clones which we are able to grow and detect in terms of differential function and will begin an analysis of the regulation of the growth and/or differentiation of such clones by antigen, mitogenic growth potentiators, and cellderived factors. Finally, the use of cell hybridization technology will be described. This yielded several B cell tumor hybrids, one of which has been studied in detail. The regulation of the growth of such a hybrid by T cell products will be introduced.