Cognate recognition of the endothelium induces HY-specific CD8+ T-lymphocyte transendothelial migration (diapedesis) in vivo by Federica M. Marelli-Berg, Martha J. James, John Dangerfield, Julian Dyson, Maggie Millrain, Diane Scott, Elizabeth Simpson, Sussan Nourshargh, and Robert I. Lechler Blood Volume 103(8):3111-3116 April 15, 2004 ©2004 by American Society of Hematology

Effect of antigen presentation by ECs on transendothelial migration of CD8+ T cells. 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 (H2k) (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 (H2k, 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-Db–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 (H2b) or from H2-congenic male B10.BR mice (H2k, 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 (H2b, P < .01) and male B10.BR EC monolayer (H2k, P < .03), at all the time points. (E-F) Adhesion of Calcein am–labeled polyclonal HY-specific, H2-Db–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 (H2b) or from congenic male B10.BR mice (H2k). In some experiments, H2b-expressing, male-derived EC monolayers were treated with an anti–H2-Db 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. Federica M. Marelli-Berg et al. Blood 2004;103:3111-3116 ©2004 by American Society of Hematology

Peritoneal membrane infiltration by HY-specific T cells in IFN-γ–treated male, but not female, mice. Peritoneal membrane infiltration by HY-specific T cells in IFN-γ–treated male, but not female, mice. (A) Male and female C57BL/6 mice were injected intraperitoneally with 600 U IFN-γ. This resulted in the selective up-regulation of MHC class I in the peritoneal cavity, as assessed by staining of various tissue samples (obtained 72 hours after IFN-γ injection) with a FITC-conjugated anti–H2-Db mAb (0.5 μg/mL). (B) At 2 days after IFN-γ injection, mice received an intravenous injection of 107 PKH26-labeled Db-restricted HY-specific T cells. The following day, mice were killed, and the presence of fluorescently labeled cells in the liver, kidney, lung, and peritoneal membrane was assessed by wide-field fluorescence microscopy. In both panels A and B, to minimize the effect of arbitrary choice of field, 10 × magnifications are shown. Tissue infiltration was quantified by randomly selecting ten 40 ×-magnified fields and assessing the number of fluorescent cells in each field. (C) The mean and SD of the total number of T cells observed in 10 randomly selected fields in each mouse from at least 3 animals (which gives at least 30 fields analyzed for each experimental group). *P is significant in comparison with female recipients (P = .002). Federica M. Marelli-Berg et al. Blood 2004;103:3111-3116 ©2004 by American Society of Hematology

Detection of T cells in the peritoneal lavage. Detection of T cells in the peritoneal lavage. The presence of T cells in the peritoneal lavage is detectable IFN-γ–treated male, but not female, mice. Male and female C57/BL6 mice were injected intraperitoneally with 600 U IFN-γ or PBS. Two days later, mice received an intravenous (IV) injection of 107 PKH26-labeled HY-specific T cells. The following day, mice were killed, and the presence of fluorescently labeled cells in the peritoneal lavage (A-B,E-F) and lymph nodes (from IFN-γ–treated mice; C,G) and spleen (from IFN-γ–treated mice; D,H) was assessed by flow cytometry. To facilitate visualization, cells were double stained with a FITC-conjugated anti-CD8 antibody. The mean and SD of the percentage of labeled cells recovered from the peritoneal lavage of at least 3 animals (n) are shown under the relevant panels. *P is significant versus female mice (P < .02). Federica M. Marelli-Berg et al. Blood 2004;103:3111-3116 ©2004 by American Society of Hematology

Cognate recognition of ECs enhances antigen-specific T-cell diapedesis in vivo. Cognate recognition of ECs enhances antigen-specific T-cell diapedesis in vivo. Cognate recognition of endothelial cells enhances antigen-specific T-cell diapedesis in vivo. Male C57BL/6 (H2b; ▪), congenic male B10.BR (H2k, ▦), or BALB/C and CBA/Ca (H2d, H2k; □) mice were treated with intrascrotal administration of IFN-γ (600 U in 400 μL saline). After 72 hours, fluorescently labeled HY-specific T cells (“Materials and methods”) were injected intravenously after surgical exteriorization of the cremaster muscle and T-cell interactions with venular walls were visualized and quantified by intravital microscopy for up to 40 minutes after injection of the cells (“Materials and methods”). Results are from 3 to 5 mice per group, and significant differences between the H2b in comparison with the H2k and H2d or the H2-congenic H2k strains of mice are shown by asterisk (*P < .05). (A) T-cell firm adhesion. (B) Extravasation. Federica M. Marelli-Berg et al. Blood 2004;103:3111-3116 ©2004 by American Society of Hematology