Induction of lytic pathways in T cell clones derived from wild-type or protein tyrosine kinase Fyn mutant mice.

The OVA-reactive CD4 + Thl clones and alloreactive CD8 + clones derived from wild-type or fyn -l- mice serve as model systems which have allowed us to investigate several aspects of the molecular events associated with T cell-mediated cytotoxicity, including 1) the differential utilization of two distinct cytolytic pathways by CD4 + Thl clones and CDB8 + CTL, 2) a comparison of the pathways of lysis induced by stimulation of the TCR or by alternative stimuli, 3) the requirement of Fyn for derivation of antigen-specific T-cell clones having properties of CD4 + Thl and CD8 + CTL cells 4) the differential requirement of Fyn in the induction of responses by TCR and the alternative stimuli. Stimulation through the TCR, either by APC bearing relevant antigen or by immobilized anti-CD3 mAb, resulted in comparable levels of target cell lysis by clones from both wild-type and fyn -l- mice. These clones also utilize the Fas pathway to lyse target cells. Thus, Fyn does not appear to be required for expression of the Fas pathway when triggered through the TCR. In contrast, lysis of target cells by T-cell clones lacking Fyn was deficient when stimulated through Thy-l or Ly-6C (using mAb) or with Con A or phorbol ester as compared to clones derived from wild-type mice. The basis for the defect in response to stimulation through the GPI-linked molecules appears to be a signaling defect which affects all of the functional responses we measured, while the defect in response to Con A stimulation appears to affect lysis but not lymphokine production. Thus, Fyn expression is selectively required for efficient activation of the Fas pathway of lysis through Thy-l, Ly-6C, and by Con A or phorbol ester in these T-cell clones. CD8 + clones derived from fyn -l- mutant mice, like clones derived from wild-type mice, display antigen-specific lysis, and appear to express perforin message and perforin protein. A Ca ++ -dependent (presumably perforin/exocytosis) component and Fas component of lysis was detected in CD8 + clones derived from fyn -l- mutant mice. Thus, Fyn is not required for expression of these components of antigen specific lysis by CD8 + alloreactive CTL clones. It appears that CD8 + clones that use multiple lytic mechanisms may selectively employ the perforin or Fas-based pathway depending on properties of the target cell or the stimulus. If selective employment of distinct lytic pathways also occurs in vivo, it will be important to understand the regulation of these events in order to effectively mainpulate host responses. Collectively, these results demonstrate that normal Fyn expression is not required for differentiation of antigen-specific CD4 + IL-2 producing Thl clones or CD8 + alloreactive CTL clones in vitro. Furthermore, Fyn is not required for the induction of antigen-specific functional responses of these cloned T cells in vitro. There is a selective requirement for Fyn expression for the activation of these T-cell clones through the GPI-linked cell surface molecules Thy-1 and Ly-6C. This defect was evident in the capacity of the mAb against Thy-1 and Ly-6 to induce distinct functional responses including cytolysis, lymphokine production, and proliferation. Other defects were also observed in the capacity of the Fyn deficient clones to lyse target cells in response to stimulation with Con A or PMA. Unlike the defect in response to stimulation through Thy-l and Ly-6C, the defect observed with Con A affected lysis but not lymphokine production. Similarly, phorbol ester alone induced suboptimal lysis in clones derived from fyn -l- mice, but phorbol ester in combination with calcium ionophore induced normal levels of lymphokine production or lysis by these cells. Thus, there may be multiple defects in signaling pathways leading to cell-mediated lysis as a result of the lack of expression of PTK Fyn.