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Development of precytotoxic T cells in cyclosporine-suppressed mixed lymphocyte reactions
J W Hooton1, C L Miller, C D Helgason
1Department of Biochemistry, University of Alberta, Edmonton, Canada.
Abstract:
Cyclosporine (CsA) blocked the generation of cytolytic activity in a primary MLR of mouse spleen cells. As expected from the known mechanism of action of this drug, it also blocked the accumulation of IL-2 during the MLR. Addition of human rIL-2 did not overcome the inhibition of CTL generation, even when it was added daily to keep its level similar to that produced in a normal MLR. Daily addition was necessary, because the CsA-inhibited MLR consumed IL-2, either by utilization or degradation. The outcome of a 5-day MLR in the presence of CsA (CsA-MLR) depended on whether or not IL-2 was continuously present. In the presence of IL-2, there was no generation of CTL activity, probably because such cultures contained IL-2-dependent suppressive elements described previously. However, when day 5 CsA-MLR cells generated in the absence of IL-2 were washed and recultured with human rIL-2, there was a burst of CTL activity, with a more than 50-fold increase in alloantigen-specific cytotoxicity within 24 to 48 h. This increase is not explainable simply by the proliferation of existing effector CTL. The noncytotoxic cells produced in an MLR in the presence of CsA, and which can be rapidly activated to cytotoxic effector cells by IL-2, are termed "precursor-effector CTL" (peCTL). They could be detected by day 3 of a primary CsA-MLR culture. Their conversion to effector CTL by IL-2 was not inhibited by CsA. Exposure of peCTL to IL-4 also generated CTL activity, to a somewhat lesser degree than IL-2, but the IL-4-induced activation was inhibited by CsA, suggesting that it depended on the induction of another CsA-sensitive lymphokine. The intracellular levels of mRNA encoding the CTL-specific serine esterases CCP1 and CCP2 (granzymes B and C, respectively) increased rapidly during the IL-2-driven conversion of peCTL to effector CTL. This study demonstrates that in the presence of CsA precursors for CTL can accumulate, and that these can be rapidly converted to cytotoxic effector cells by IL-2.
Insights
Cyclosporine (CsA) prevents cytotoxic T lymphocyte (CTL) generation but allows precursor-effector CTL (peCTL) accumulation. These peCTL can be rapidly activated into effector CTL by interleukin-2 (IL-2), even in the presence of CsA.
Area of Science:
- Immunology
- Cellular immunology
- T cell activation
Background:
- Cyclosporine (CsA) is known to inhibit T cell activation and interleukin-2 (IL-2) production.
- The generation of cytolytic activity in mixed lymphocyte reactions (MLRs) is crucial for adaptive immunity.
- Understanding the precise mechanisms by which CsA affects T cell differentiation is important for immunosuppressive therapy.
Purpose of the Study:
- To investigate the effect of CsA on the generation of cytotoxic T lymphocytes (CTLs) in a primary mouse MLR.
- To determine if IL-2 can overcome CsA-induced inhibition of CTL generation.
- To characterize the nature of CsA-resistant T cell precursors and their activation.
Main Methods:
- Primary mouse spleen cell MLRs were conducted in the presence or absence of CsA.
- Interleukin-2 (IL-2) was added to cultures to assess its effect on CTL generation.
- Cells from CsA-treated MLRs were washed and recultured with IL-2 or IL-4 to evaluate activation potential.
- Intracellular mRNA levels for CTL-specific serine esterases (granzymes) were measured.
Main Results:
- CsA blocked the generation of cytolytic activity and IL-2 accumulation in MLRs.
- Exogenous IL-2 did not restore CTL generation in CsA-treated cultures due to IL-2 consumption.
- CsA-treated cultures generated non-cytotoxic precursor-effector CTL (peCTL) that could be rapidly converted to effector CTL by IL-2, independent of CsA.
- IL-4 also induced CTL activity from peCTL, but this activation was CsA-sensitive.
Conclusions:
- CsA permits the accumulation of CTL precursors (peCTL) that are resistant to CsA's inhibitory effects on their final differentiation.
- IL-2 can rapidly convert these peCTL into fully functional effector CTL, bypassing CsA's block on de novo CTL generation.
- The findings reveal a novel pathway for CTL activation that can occur despite CsA-induced immunosuppression.