Effect of antigen presentation by ECs on transendothelial migration of CD8⁺ T cells. Antigen presentation by ECs selectively enhances transendothelial migration of CD8⁺ T cells. (A-B) The C6 CD8⁺ T-cell clone (5 × 10⁵ cells per well) was seeded onto IFN-γ–treated EC monolayers derived from either male (•) or female (○) CBA/Ca mice (H2^k) (A). In some experiments, the male CBA/Ca-derived EC monolayer was treated with an anti–H2-Kk mAb (HB25, 1μg/mL, open triangles), for 30 minutes at room temperature, and then washed thoroughly prior to seeding the T lymphocytes. In parallel experiments (B), a CD4⁺ ovalbumin-specific H2-Ab–restricted DO11.10 TCR-transgenic T-cell line (of irrelevant antigen specificity) was seeded onto duplicate EC monolayers. T-cell migration was monitored for the following 26 hours and is expressed as a percentage of migrated T cells at the specified time points. The average percentage of migrated T cells at the specified time points in 3 experiments with similar design is shown. Standard error bars are shown. ^*P is significant versus control female CBA/Ca EC monolayer (H2^k, P < .003) at the time points between 5 and 26 hours. ^**P is significant for inhibition compared with male CBA/Ca EC monolayer in the absence of anti–H2-Kk mAb (P < .04) at the time points between 5 and 26 hours. (C) A polyclonal HY-specific, H2-D^b–restricted CD8⁺ T-cell line (5 × 105 cells per well) was seeded onto IFN-γ–treated EC monolayers derived from either female (•) or male (○) C57BL6 mice (H2^b) or from H2-congenic male B10.BR mice (H2^k, empty □). (D) A DO11.10 TCR-transgenic T-cell line was seeded onto the duplicate EC monolayers. T-cell migration was monitored and expressed as described in “Lymphocyte adhesion and transmigration assays.” The average percentage of migrated T cells at the specified time points in 3 experiments with similar design is shown. Standard error bars are shown. ^*P is significant versus control female C57BL/6 EC monolayer (H2^b, P < .01) and male B10.BR EC monolayer (H2^k, P < .03), at all the time points. (E-F) Adhesion of Calcein am–labeled polyclonal HY-specific, H2-D^b–restricted CD8⁺ T-cell line and DO11.10 TCR-transgenic T-cell line (5 × 10⁴ per well), respectively, to IFN-γ–treated EC monolayers (2 × 10⁴ per well) derived from either female or male C57BL/6 mice (H2^b) or from congenic male B10.BR mice (H2^k). In some experiments, H2^b-expressing, male-derived EC monolayers were treated with an anti–H2-D^b mAb (clone 28-14-8, 1 μg/mL). Adhesion was measured by fluorimetry, and the average percentage of T-cell adhesion in 3 experiments with similar design is shown. Standard error bars are shown.