CD28 stimulation lowers the threshold for TCR triggering. Calcium mobilization in normal Indo-1–loaded T cells, before and after TCR stimulation with anti-CD3 followed by cross-linking with polyclonal rabbit antimouse Ig antibody (RAM). The plots show intracellular calcium concentrations (ratio of bound:free Indo-1) over a 240-second time period. The white line represents the median calcium concentration at each 10-second time slice. In (A), a suboptimal level of anti-CD3 was determined that, with cross-linking in the presence of U937-M3P cells, gave poor median calcium mobilization. Costimulation through CD28 by addition of a specific activating antibody at the same time as anti-CD3 markedly increased the number of activated T cells (B) not seen using anti-CD28 alone (D). Enhanced T-cell activation was also seen when CD28 stimulation was provided by CD80⁺U937-CD80 cells instead of U937-M3Ps (C). U937-CD80 cells, in the absence of anti-CD3, also failed to trigger calcium mobilization (data not shown).