Andrew L. Glasebrook

Murine T Lymphocyte Clones with Distinct Immunological Functions

The study of cell-mediated immune responses has been facilitated by the development of model systems such as unidirectional mixed leukocyte culture (MLC) (Cerottini & Brunner 1974, Hayry et al. 1972, Wagner et al. 1973) which permit manipulation of the reacting cells in vitro. In most instances, the MLC is characterized by cell proliferation culminating in the development of cytolytic T lymphocytes (CTL) reactive toward the immunizing antigen. While antigenic stimulation induces cional expansion of reactive lymphocytes, the magnitude of the response is regulated by circuits of interacting T cell subsets exhibiting specific and/or non-specific helper or suppressor functions. Some of these interactions are mediated by soluble factors, others involve the expression of certain membrane proteins at the cell surface. Humoral responses are also characterized by the involvement of different cellular subsets which play diverse and highly specialized roles. Efforts to dissect the immune response and to characterize the nature of

T-cell lines which cooperate in generation of specific cytolytic activity

THE unidirectional mixed leukocyte culture (MLC) is characterised by cell proliferation and by the development of cytolytic T lymphocytes (CTL)1–3. In murine MLC, the generation of CTL seems to involve proliferation of at least two sub-populations of responding T cells—T cells of the Ly-2+3+ phenotype, which differentiate into CTL, and cells of the Ly-1+ phenotype, which seem to ‘help’ or ‘amplify’ the CTL re-sponse4–6. The mechanism by which ‘amplifier cells’ augment the CTL response is unclear; recent reports have shown, however, that CTL precursors present in populations of long-term MLC or of immune spleen cells7,8 can respond to supernatant fluid obtained from allogeneic MLC or mitogen-stimulated spleen cell cultures in the absence of specific antigen. Several groups using such conditioned medium have reported the successful long-term culture of normal and antigen-specific immune T cells in the absence of alloantigen stimuli9–11. Whereas amplifier supernatant fluids seem to substitute functionally for a T-cell subpopulation (Ly-1+?) in the absence of alloantigen, there is only indirect evidence that amplifier cells can be activated by alloantigen to release one or several soluble factors that influence the differentiation or proliferation ofCTL12–14. We have used modifications of techniques previously described for the long-term culture of immune T cells to isolate several cloned lines of MLC-reactive T cells, including a non-cytolytic T-cell line which, when co-cultured with alloantigen, permits another cell line to proliferate and express specific cytolytic activity.

Stimulator requirements for primed alloreactive T cells: Macrophages and dendritic cells activate T cells across all genetic disparities

The cellular requirements for stimulating primed alloreactive T cells have been investigated. In vitro-primed secondary alloreactive cells, long-term lines, and Ly 1+2- noncytolytic clones which reacted with allo-H-2K, D, or Mls (M locus) antigens were tested. The data indicated that a specialized antigen-presenting cell such as a macrophage or a dendritic cell was required for stimulating primed alloreactive cells across all the genetic disparities tested. B and T lymphocytes were ineffective stimulators. The stimulator requirement for secondary and Ly 1+2- clone responses was heterogeneous, since both macrophages and dendritic cells were effective stimulators. Thus, the allostimulator requirement for inducing proliferation and mediator secretion by the primed T-cell populations closely paralleled the requirement for stimulating unprimed populations. The only exception found was the peritoneal washout population, which did not stimulate a primary response but did stimulate secondary responses. The failure of peritoneal macrophages to stimulate a primary response was shown to be due to an inhibitory pathway which did not occur when the responding population was alloantigen primed.

Functional Murine T-Cell Clones

Many important immunological phenomena can be characterized only in operational terms, and this leads often to circular reasoning. For example, antigens are identified and characterized in terms of the biological responses, antibody formation or cell-mediated reactions, that are induced by the antigens. However, antibodies and reactive cells can be identified only on the basis of their reactivity with the immunizing antigen. This rather unsatisfactory situation results in large part from the heterogeneity of cellular and molecular processes that are involved in immune responses as well as the very large number of different kinds of antibodies that can be produced. It is possible to identify and quantify reactions due to rare cells or molecules present at extremely low frequency among other, similar cells and molecules. However, it is difficult to obtain sufficient numbers of specifically reactive cells or antibody molecules to be able to characterize such cells or molecules biochemically or structurally in sufficient detail for independent definition of the basis for their immunological reactivity.