Anti-CD3-induced T-cell activation in vivo—I. Flow cytometric analysis of dose-responsive, time-dependent, and cyclosporin a-sensitive parameters of CD4+ and CD8+ cells from the draining lymph nodes of C57B1/6 mice

An in vivo model to assess the effects of chemicals on T-cell activation has been characterized and validated using the immunosuppressive drug, cyclosporin A (CsA). The dose response and kinetic effects of the hamster anti-mouse monoclonal antibody 145-2C11 (anti-CD3) on various parameters of T-cell activation were examined in cells from the draining popliteal and inguinal lymph nodes of C57Bl/6 mice. Parameters of anti-CD3-induced T-cell activation included 3H-TdR incorporation (+/- recombinant murine IL-2), and flow cytometric analysis of CD3 and IL-2 receptor (IL-2R) expression on CD4+ and CD8+ cells. Increases in the percentage of lymphocyte subsets in the S/G2M phase of the cell cycle and total cell recovery following anti-CD3 are also reported. Increased 3H-TdR incorporation was maximal over a dose range of 0.25-25 micrograms anti-CD3, while maximal increases in the percentage of CD4+ and CD8+ cycling occurred after a dose of 2.5 micrograms anti-CD3. At 24 h after anti-CD3 treatment, CD3 expression on both CD4+ and CD8+ cells was dose dependently down-modulated while IL-2R expression and IL-2-driven 3H-TdR incorporation were dose dependently increased. In addition, total cell recovery increased at 24 h and correlated with an increase in the percentage of B220+ cells present in the lymph nodes. There was a corresponding decrease in the percentage of Thy 1.2+, CD4+, and CD8+ cells. No increase in cycling of B220+ cells was observed, suggesting an influx of B220+ cells into the node rather than proliferation. Elevation in 3H-TdR incorporation occurred as early as 4 h after anti-CD3 treatment, while increases in the percentage of CD4+ and CD8+ cells cycling were not apparent until 24 h. At 48 h, the percentage of CD8+ cells cycling doubled while the percentage of CD4+ cells cycling remained constant. Down-modulation of CD3 expression on CD4+ and CD8+ cells was apparent as early as 1 h after treatment with less than 10% of CD4+ and CD8+ cells expressing CD3 by 12 h. Induction of IL-2R expression and IL-2-driven 3H-TdR incorporation was maximal at 12 h after anti-CD3. The immunosuppressive drug, CsA (25, 50, or 100 mg/kg, i.p.) decreased anti-CD3-induced 3H-TdR incorporation. Concurrently, anti-CD3-induced increases in the percentage of CD4+ and CD8+ cells cycling were inhibited by CsA. Likewise, IL-2 responsiveness and IL-2R expression on both T-cell subsets were inhibited by CsA.(ABSTRACT TRUNCATED AT 400 WORDS)