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Defects of T-cell (TC) proliferation and in TC-receptor (TCR) signaling have been demonstrated in several autoimmune diseases. The detailed mechanisms governing activation and proliferation of activated TC, however, are still not completely known. Here, we will show that under certain conditions human peripheral blood (PB) TC, once activated by anti-CD3 monoclonal antibody (mab) in vitro, fail to respond to a subsequent re-stimulation via the TCR. This unresponsiveness is caused at the transcriptional level by an impaired production of IL-2, and this defect is temporary.
PB mononuclear cells (MC) from healthy donors were pre-stimulated (PS) by anti-CD3PS for 96 h following restimulation by IOT-3, IL-2, or IL-15, as well as other mitogens. In control experiments (NS), PBMC were cultered in medium alone for the first 4 days. Restimulation of both populations was also performed in the presence of freshly isolated monocytes (MO). Furthermore, after the first incubation T-cells and MO from PS and NS cultures were seperated by magnetic beads and incubated for re-stimulation in a criss-cross design. Surface immunophenotype of both, TC and MO were analysed by flow cytometry. Cytokine production was determined by rtPCR and intracellular signaling protein content of TC in PS and NS cultures were compared by western blotting.
PBMC pre-incubated for 96 h with medium alone showed a good proliferation to subsequent stimulation with anti-CD3 mab, whereas IL-2 induced only little proliferation. Unresponsive TC fail to produce IL-2 as demonstrated at transcription level by rt-PCR. In contrast, PS cells responded only minimally to subsequent stimulation with anti-CD3, but the addition of IL-2 induced a strong proliferation, comparable to IL-15. Both, PS and NS TC responded well to re-stimulation by PHA, whereas Con A induced proliferation mainly of NS cells and thus had similar effects as anti-CD3. In the presence of 10% freshly isolated MO PS cells were able to respond significantly to subsequent TCR challenge. But the addition of MO from NS cultures to PS-TC did not fully restore proliferation. Interestingly, when cells were allowed to rest for 168 h, the responsiveness of PS lymphocytes was restored. Surprisingly, immunoblots revealed that PS cells had a higher intracellular content of ζ-chain and p56lck. Both, PS TC and MO show higher expression of different activation associated surface molecules (HLA-DR, CD25, CD69, and costimulatory molecules).
Our results show a mechanism leading to a temporary unresponsiveness to TCR ligation of preactivated TC although adaequate costimulatory support seems available. The rate limiting events for IL-2 production can be overcome by bypassing the TCR via mitogens or addition of freshly isolated MO. Although we have not been able to fully define the rate limiting events we have been able to exclude various possibilities. TC pre-activated via the TCR can continue to produce and express a variety of molecules such as IFN-γ, IL-2R, and cell surface molecules. Thus, their effector function in G1-phase, but not their progression into a mitotic cell cycle seems to be sustained.