Figure 5. Activation of CD4⁺ T_EM by allogeneic EC is necessary to enhance CD8⁺ T_EM responses to the same EC.

(A and B) Addition of CD4⁺ T_EM to EC/CD8⁺ T_EM cocultures enhances CD8⁺ expansion as measured by CFSE dilution and flow cytometry at 7 days (A), but CD4⁺ T_EM do not enhance survival of CD8⁺ T_EM (B) as measured by viability. (C) Knockdown of CIITA expression by siRNA, to prevent CD4⁺ T_EM activation, inhibits CD4⁺ T_EM enhancement of CD8⁺ T_EM expansion at 7 days as measured by CFSE dilution and flow cytometric analysis at 7 days. Note that the inclusion of activated CD4⁺ T_EM increases the rounds of replication of CD8⁺ T_EM (inset boxes). Similar results were seen in three independent experiments. *P < 0.05, 2-tailed Student's t test. T_EM, effector memory T cell; CIITA, class II MHC transactivator; FVD, fixable viability dye; EC,endothelial cells.