T lymphocytes are key regulators that play a central role in controlling the outcome of any immune process. The activation of these T lymphocytes depends upon the recognition of antigenic peptides by the T cell receptor (TCR), modified by a complex microenvironment of modulating interactions. These interactions occur through a wide array of T cell surface molecules that are ligated by molecules on the surface of nearby cells, or by soluble factors including cytokines. These cytokines are critical messengers that help to mediate the development of a coordinated immune response. The profile of cytokines that a clone of T lymphocytes produces can greatly impact on the outcome of any immune reaction. They may help to determine whether an animal effectively eliminates an infectious agent or succumbs and whether a transplanted organ is accepted or rejected.
This thesis investigated the signals that are important for T lymphocyte activation as opposed to tolerance. Specifically, it looked at whether signals provided by varying the strength of TCR signalling, or by the ligation of different coactivating receptors, determined the cytokine production and ultimate function of a population of T cells. This was explored in a culture system that uses immobilized monoclonal antibodies (mAbs) against CD3S and a variety of other T cell surface markers to activate freshly isolated murine T lymphocytes with a high cloning efficiency. This system has the advantage that both immobilized mAbs and soluble factors such as cytokines can be added or excluded to provide a precisely regulated environment for activation. Accessory cells are not needed, avoiding the possibility of further, poorly defined signals interfering with the behaviour of the T cells being studied. This system permits the study of either purified populations of T cells or of single T cells, the latter further eliminating the possibility of unrecognized influences from small, unidentified groups of cells within a larger population.
This work showed that mAbs against CD4, CD8, CD11a and CD28 all increased the proliferation of T lymphocytes in cultures that contained exogenous IL-2. When exogenous IL-2 was not added, and the T cells were required to make sufficient IL-2 for their own proliferation, the anti-CD4 mAb behaved quite differently from the other coactivating mAbs studied, reducing rather than increasing proliferation. These findings suggested that the ligation of CD4 might reduce the ability of T cells to produce the important proliferative cytokine interleukin-2 (IL-2).
Further experiments investigated whether coactivation of T lymphocytes with mAbs against CD4 led to a distinct pattern of cytokine expression that might help to determine the cells' subsequent behaviour. It was shown that whereas mAbs against several other T cell surface markers increased the synthesis of a range of cytokines, mAbs against CD4 reduced the production of most cytokines. Despite this, anti-CD4 mAb increased the synthesis of IL-4 more than did any of the other mAbs tested.
IL-4 is the key type 2 cytokine and is important in the development of humoral immune responses. Its production by T lymphocytes can be amplified by exogenous IL-4, but the origin of the initial IL-4 to prime this process has been unclear. Single cell cultures, using this culture system, provided an ideal environment in which to determine whether the ligation of surface CD4 could induce T lymphocytes to produce IL-4 without exogenous IL- 4 or other complicating signals. Typically 10-40% of single CD4+ T cells developed into IL- 4 producing clones if they were coactivated with anti-CD4 mAb, whereas 0-6% of single CD4+ T cells coactivated with anti-CD 11 a or anti-CD28 mAbs produced IL-4. CD4+ T cells of naϊve (CD44low or CD62Lhigh) phenotype were more likely to develop into IL-4 producing clones than were CD4+ cells of activated (CD44low or CD62Lhigh) phenotype.
The work in this thesis demonstrated that the ligation of T cell surface molecules could influence the results of T cell activation in a mAb-based system. It showed for the first time that the ligation of CD4 could directly promote the synthesis of IL-4 by T lymphocytes of naive phenotype, independently of exogenous IL-4. It seems reasonable to speculate that differences in the type of antigen presenting cell (APC), or differences in the expression of molecules on the surface of an APC, could powerfully influence the predominant cytokines that are produced after T cell activation.