InVivoMAb anti-human CD54 (ICAM-1)

Genes limiting T cell antitumor activity may serve as therapeutic targets. It has not been systematically studied whether there are regulators that uniquely or broadly contribute to T cell fitness. We perform genome-scale CRISPR-Cas9 knockout screens in primary CD8 T cells to uncover genes negatively impacting fitness upon three modes of stimulation: (1) intense, triggering activation-induced cell death (AICD); (2) acute, triggering expansion; (3) chronic, causing dysfunction. Besides established regulators, we uncover genes controlling T cell fitness either specifically or commonly upon differential stimulation. Dap5 ablation, ranking highly in all three screens, increases translation while enhancing tumor killing. Loss of Icam1-mediated homotypic T cell clustering amplifies cell expansion and effector functions after both acute and intense stimulation. Lastly, Ctbp1 inactivation induces functional T cell persistence exclusively upon chronic stimulation. Our results functionally annotate fitness regulators based on their unique or shared contribution to traits limiting T cell antitumor activity.

Genes limiting T cell antitumor activity may serve as therapeutic targets. It has not been systematically studied whether there are regulators that uniquely or broadly contribute to T cell fitness. We perform genome-scale CRISPR-Cas9 knockout screens in primary CD8 T cells to uncover genes negatively impacting fitness upon three modes of stimulation: (1) intense, triggering activation-induced cell death (AICD); (2) acute, triggering expansion; (3) chronic, causing dysfunction. Besides established regulators, we uncover genes controlling T cell fitness either specifically or commonly upon differential stimulation. Dap5 ablation, ranking highly in all three screens, increases translation while enhancing tumor killing. Loss of Icam1-mediated homotypic T cell clustering amplifies cell expansion and effector functions after both acute and intense stimulation. Lastly, Ctbp1 inactivation induces functional T cell persistence exclusively upon chronic stimulation. Our results functionally annotate fitness regulators based on their unique or shared contribution to traits limiting T cell antitumor activity.

Genes limiting T cell antitumor activity may serve as therapeutic targets. It has not been systematically studied whether there are regulators that uniquely or broadly contribute to T cell fitness. We perform genome-scale CRISPR-Cas9 knockout screens in primary CD8 T cells to uncover genes negatively impacting fitness upon three modes of stimulation: (1) intense, triggering activation-induced cell death (AICD); (2) acute, triggering expansion; (3) chronic, causing dysfunction. Besides established regulators, we uncover genes controlling T cell fitness either specifically or commonly upon differential stimulation. Dap5 ablation, ranking highly in all three screens, increases translation while enhancing tumor killing. Loss of Icam1-mediated homotypic T cell clustering amplifies cell expansion and effector functions after both acute and intense stimulation. Lastly, Ctbp1 inactivation induces functional T cell persistence exclusively upon chronic stimulation. Our results functionally annotate fitness regulators based on their unique or shared contribution to traits limiting T cell antitumor activity.

Genes limiting T cell antitumor activity may serve as therapeutic targets. It has not been systematically studied whether there are regulators that uniquely or broadly contribute to T cell fitness. We perform genome-scale CRISPR-Cas9 knockout screens in primary CD8 T cells to uncover genes negatively impacting fitness upon three modes of stimulation: (1) intense, triggering activation-induced cell death (AICD); (2) acute, triggering expansion; (3) chronic, causing dysfunction. Besides established regulators, we uncover genes controlling T cell fitness either specifically or commonly upon differential stimulation. Dap5 ablation, ranking highly in all three screens, increases translation while enhancing tumor killing. Loss of Icam1-mediated homotypic T cell clustering amplifies cell expansion and effector functions after both acute and intense stimulation. Lastly, Ctbp1 inactivation induces functional T cell persistence exclusively upon chronic stimulation. Our results functionally annotate fitness regulators based on their unique or shared contribution to traits limiting T cell antitumor activity.

Purpose: Intercellular adhesion molecule-1 (ICAM-1, CD54) is an emerging therapeutic target for a variety of solid tumors including melanoma and anaplastic thyroid cancer (ATC). This study aims to develop an ICAM-1-targeted immuno-positron emission tomography (immunoPET) imaging strategy and assess its diagnostic value in melanoma and ATC models. Methods: Flow cytometry was used to screen ICAM-1-positive melanoma and ATC cell lines. Melanoma and ATC models were established using A375 cell line and THJ-16T cell line, respectively. An ICAM-1-specific monoclonal antibody (R6-5-D6) and a nonspecific human IgG were radiolabeled with ⁶⁴Cu and the diagnostic efficacies were interrogated in tumor-bearing mouse models. Biodistribution and fluorescent imaging studies were performed to confirm the specificity of the ICAM-1-targeted imaging probes. Results: ICAM-1 was strongly expressed on melanoma and advanced thyroid cancer cell lines. ⁶⁴Cu-NOTA-ICAM-1 immunoPET imaging efficiently delineated A375 melanomas with a peak tumor uptake of 21.28 ± 6.56 %ID/g (n = 5), significantly higher than that of ⁶⁴Cu-NOTA-IgG (10.63 ± 2.58 %ID/g, n = 3). Moreover, immunoPET imaging with ⁶⁴Cu-NOTA-ICAM-1 efficiently visualized subcutaneous and orthotopic ATCs with high clarity and contrast. Fluorescent imaging with IRDye 800CW-ICAM-1 also visualized orthotopic ATCs and the tumor uptake could be blocked by the ICAM-1 parental antibody R6-5-D6, indicating the high specificity of the developed probe. Finally, blocking with the human IgG prolonged the circulation of the ⁶⁴Cu-NOTA-ICAM-1 in R2G2 mice without compromising the tumor uptake. Conclusion: ICAM-1-targeted immunoPET imaging could characterize ICAM-1 expression in melanoma and ATC, which holds promise for optimizing ICAM-1-targeted therapies in the future.

Purpose: Intercellular adhesion molecule-1 (ICAM-1, CD54) is an emerging therapeutic target for a variety of solid tumors including melanoma and anaplastic thyroid cancer (ATC). This study aims to develop an ICAM-1-targeted immuno-positron emission tomography (immunoPET) imaging strategy and assess its diagnostic value in melanoma and ATC models. Methods: Flow cytometry was used to screen ICAM-1-positive melanoma and ATC cell lines. Melanoma and ATC models were established using A375 cell line and THJ-16T cell line, respectively. An ICAM-1-specific monoclonal antibody (R6-5-D6) and a nonspecific human IgG were radiolabeled with ⁶⁴Cu and the diagnostic efficacies were interrogated in tumor-bearing mouse models. Biodistribution and fluorescent imaging studies were performed to confirm the specificity of the ICAM-1-targeted imaging probes. Results: ICAM-1 was strongly expressed on melanoma and advanced thyroid cancer cell lines. ⁶⁴Cu-NOTA-ICAM-1 immunoPET imaging efficiently delineated A375 melanomas with a peak tumor uptake of 21.28 ± 6.56 %ID/g (n = 5), significantly higher than that of ⁶⁴Cu-NOTA-IgG (10.63 ± 2.58 %ID/g, n = 3). Moreover, immunoPET imaging with ⁶⁴Cu-NOTA-ICAM-1 efficiently visualized subcutaneous and orthotopic ATCs with high clarity and contrast. Fluorescent imaging with IRDye 800CW-ICAM-1 also visualized orthotopic ATCs and the tumor uptake could be blocked by the ICAM-1 parental antibody R6-5-D6, indicating the high specificity of the developed probe. Finally, blocking with the human IgG prolonged the circulation of the ⁶⁴Cu-NOTA-ICAM-1 in R2G2 mice without compromising the tumor uptake. Conclusion: ICAM-1-targeted immunoPET imaging could characterize ICAM-1 expression in melanoma and ATC, which holds promise for optimizing ICAM-1-targeted therapies in the future.

Tumor-infiltrating lymphocytes (TILs), found in patients with advanced pancreatic ductal adenocarcinoma (PDAC), are shown to correlate with overall survival (OS) rate. Although majority of TILs consist of CD8⁺/CD4⁺ T cells, the presence of NK cells and their role in the pathogenesis of PDAC remains elusive. We performed comprehensive analyses of TIL, PBMC, and autologous tumor cells from 80 enrolled resectable PDAC patients to comprehend the NK cell defects within PDAC. Extremely low frequencies of NK cells (<0.5%) were found within PDAC tumors, which was attributable not to the low expression of tumor chemokines, but to the lack of chemokine receptor, CXCR2. Forced expression of CXCR2 in patients' NK cells rendered them capable of trafficking into PDAC. Furthermore, NK cells exhibited impaired cell-mediated killing of autologous PDAC cells, primarily due to insufficient ligation of NKG2D and DNAM-1, and failed to proliferate within the hypoxic tumor microenvironment. Importantly, these defects could be overcome by ex-vivo stimulation of NK cells from such patients. Importantly, when the proliferative capacity of NK cells in vitro was used to stratify patients on the basis of cell expansion, patients whose NK cells proliferated <250-fold experienced significantly lower DFS and OS than those with ≥250-fold. Ex-vivo activation of NK cells restored tumor trafficking and reactivity, hence provided a therapeutic modality while their fold expansion could be a potentially significant prognostic indicator of OS and DFS in such patients.

Tumor-infiltrating lymphocytes (TILs), found in patients with advanced pancreatic ductal adenocarcinoma (PDAC), are shown to correlate with overall survival (OS) rate. Although majority of TILs consist of CD8⁺/CD4⁺ T cells, the presence of NK cells and their role in the pathogenesis of PDAC remains elusive. We performed comprehensive analyses of TIL, PBMC, and autologous tumor cells from 80 enrolled resectable PDAC patients to comprehend the NK cell defects within PDAC. Extremely low frequencies of NK cells (<0.5%) were found within PDAC tumors, which was attributable not to the low expression of tumor chemokines, but to the lack of chemokine receptor, CXCR2. Forced expression of CXCR2 in patients' NK cells rendered them capable of trafficking into PDAC. Furthermore, NK cells exhibited impaired cell-mediated killing of autologous PDAC cells, primarily due to insufficient ligation of NKG2D and DNAM-1, and failed to proliferate within the hypoxic tumor microenvironment. Importantly, these defects could be overcome by ex-vivo stimulation of NK cells from such patients. Importantly, when the proliferative capacity of NK cells in vitro was used to stratify patients on the basis of cell expansion, patients whose NK cells proliferated <250-fold experienced significantly lower DFS and OS than those with ≥250-fold. Ex-vivo activation of NK cells restored tumor trafficking and reactivity, hence provided a therapeutic modality while their fold expansion could be a potentially significant prognostic indicator of OS and DFS in such patients.

Cortactin is an actin-binding molecule that regulates various cellular processes requiring actin dynamics. We recently described cortactin-deficient mice and despite its pivotal role for actin remodeling in vitro, these mice are surprisingly healthy. Analyzing cortactin functions in endothelium under inflammatory conditions, we found that cortactin is required for endothelial barrier functions and leukocyte extravasation in vivo. Importantly, these effects were not regulated by defective actin dynamics but instead by a failure to activate the small GTPases Rap1 and RhoG in endothelial cells. Defective RhoG signaling led to reduced ICAM-1 clustering that supported the interaction with leukocytes. These clusters originally seen as rings surrounding adherent leukocytes actually represented in many cases ICAM-1 containing protrusions as they were described before as docking structures. Thus, cortactin is essential for the formation of endothelial docking structures as well as for leukocyte adhesion and extravasation.

Cortactin is an actin-binding molecule that regulates various cellular processes requiring actin dynamics. We recently described cortactin-deficient mice and despite its pivotal role for actin remodeling in vitro, these mice are surprisingly healthy. Analyzing cortactin functions in endothelium under inflammatory conditions, we found that cortactin is required for endothelial barrier functions and leukocyte extravasation in vivo. Importantly, these effects were not regulated by defective actin dynamics but instead by a failure to activate the small GTPases Rap1 and RhoG in endothelial cells. Defective RhoG signaling led to reduced ICAM-1 clustering that supported the interaction with leukocytes. These clusters originally seen as rings surrounding adherent leukocytes actually represented in many cases ICAM-1 containing protrusions as they were described before as docking structures. Thus, cortactin is essential for the formation of endothelial docking structures as well as for leukocyte adhesion and extravasation.

Cortactin is an actin-binding molecule that regulates various cellular processes requiring actin dynamics. We recently described cortactin-deficient mice and despite its pivotal role for actin remodeling in vitro, these mice are surprisingly healthy. Analyzing cortactin functions in endothelium under inflammatory conditions, we found that cortactin is required for endothelial barrier functions and leukocyte extravasation in vivo. Importantly, these effects were not regulated by defective actin dynamics but instead by a failure to activate the small GTPases Rap1 and RhoG in endothelial cells. Defective RhoG signaling led to reduced ICAM-1 clustering that supported the interaction with leukocytes. These clusters originally seen as rings surrounding adherent leukocytes actually represented in many cases ICAM-1 containing protrusions as they were described before as docking structures. Thus, cortactin is essential for the formation of endothelial docking structures as well as for leukocyte adhesion and extravasation.

Cortactin is an actin-binding molecule that regulates various cellular processes requiring actin dynamics. We recently described cortactin-deficient mice and despite its pivotal role for actin remodeling in vitro, these mice are surprisingly healthy. Analyzing cortactin functions in endothelium under inflammatory conditions, we found that cortactin is required for endothelial barrier functions and leukocyte extravasation in vivo. Importantly, these effects were not regulated by defective actin dynamics but instead by a failure to activate the small GTPases Rap1 and RhoG in endothelial cells. Defective RhoG signaling led to reduced ICAM-1 clustering that supported the interaction with leukocytes. These clusters originally seen as rings surrounding adherent leukocytes actually represented in many cases ICAM-1 containing protrusions as they were described before as docking structures. Thus, cortactin is essential for the formation of endothelial docking structures as well as for leukocyte adhesion and extravasation.

Cortactin is an actin-binding molecule that regulates various cellular processes requiring actin dynamics. We recently described cortactin-deficient mice and despite its pivotal role for actin remodeling in vitro, these mice are surprisingly healthy. Analyzing cortactin functions in endothelium under inflammatory conditions, we found that cortactin is required for endothelial barrier functions and leukocyte extravasation in vivo. Importantly, these effects were not regulated by defective actin dynamics but instead by a failure to activate the small GTPases Rap1 and RhoG in endothelial cells. Defective RhoG signaling led to reduced ICAM-1 clustering that supported the interaction with leukocytes. These clusters originally seen as rings surrounding adherent leukocytes actually represented in many cases ICAM-1 containing protrusions as they were described before as docking structures. Thus, cortactin is essential for the formation of endothelial docking structures as well as for leukocyte adhesion and extravasation.

With antigen stimulation, naïve CD4+ T cells differentiate to several effector or memory cell populations, and cytokines contribute to differentiation outcome. Several proteins on these cells receive costimulatory signals, but a systematic comparison of their differential effects on naïve T cell differentiation has not been conducted. Two costimulatory proteins, CD28 and ICAM-1, resident on human naïve CD4+ T cells were compared for participation in differentiation. Under controlled conditions, and with no added cytokines, costimulation through either CD3+CD28 or CD3+CAM-1 induced differentiation to T effector and T memory cells. In contrast, costimulation through CD3+ICAM-1 induced differentiation to Treg cells whereas costimulation through CD3+CD28 did not.

Neutrophil extravasation and the regulation of vascular permeability require dynamic actin rearrangements in the endothelium. In this study, we analyzed in vivo whether these processes require the function of the actin nucleation-promoting factor cortactin. Basal vascular permeability for high molecular weight substances was enhanced in cortactin-deficient mice. Despite this leakiness, neutrophil extravasation in the tumor necrosis factor-stimulated cremaster was inhibited by the loss of cortactin. The permeability defect was caused by reduced levels of activated Rap1 (Ras-related protein 1) in endothelial cells and could be rescued by activating Rap1 via the guanosine triphosphatase (GTPase) exchange factor EPAC (exchange protein directly activated by cAMP). The defect in neutrophil extravasation was caused by enhanced rolling velocity and reduced adhesion in postcapillary venules. Impaired rolling interactions were linked to contributions of beta(2)-integrin ligands, and firm adhesion was compromised by reduced ICAM-1 (intercellular adhesion molecule 1) clustering around neutrophils. A signaling process known to be critical for the formation of ICAM-1-enriched contact areas and for transendothelial migration, the ICAM-1-mediated activation of the GTPase RhoG was blocked in cortactin-deficient endothelial cells. Our results represent the first physiological evidence that cortactin is crucial for orchestrating the molecular events leading to proper endothelial barrier function and leukocyte recruitment in vivo.

Induction of antigen-specific T cell tolerance would aid treatment of diverse immunological disorders and help prevent allograft rejection and graft versus host disease. In this study, we establish a method of inducing antigen-specific T cell tolerance in situ in diabetic humanized mice and Rhesus monkeys receiving porcine islet xenografts. Antigen-specific T cell tolerance is induced by administration of an antibody ligating a particular epitope on ICAM-1 (intercellular adhesion molecule 1). Antibody-mediated ligation of ICAM-1 on dendritic cells (DCs) led to the arrest of DCs in a semimature stage in vitro and in vivo. Ablation of DCs from mice completely abrogated anti-ICAM-1-induced antigen-specific T cell tolerance. T cell responses to unrelated antigens remained unaffected. In situ induction of DC-mediated T cell tolerance using this method may represent a potent therapeutic tool for preventing graft rejection.

Induction of antigen-specific T cell tolerance would aid treatment of diverse immunological disorders and help prevent allograft rejection and graft versus host disease. In this study, we establish a method of inducing antigen-specific T cell tolerance in situ in diabetic humanized mice and Rhesus monkeys receiving porcine islet xenografts. Antigen-specific T cell tolerance is induced by administration of an antibody ligating a particular epitope on ICAM-1 (intercellular adhesion molecule 1). Antibody-mediated ligation of ICAM-1 on dendritic cells (DCs) led to the arrest of DCs in a semimature stage in vitro and in vivo. Ablation of DCs from mice completely abrogated anti-ICAM-1-induced antigen-specific T cell tolerance. T cell responses to unrelated antigens remained unaffected. In situ induction of DC-mediated T cell tolerance using this method may represent a potent therapeutic tool for preventing graft rejection.

With antigen stimulation, naïve CD4+ T cells differentiate to several effector or memory cell populations, and cytokines contribute to differentiation outcome. Several proteins on these cells receive costimulatory signals, but a systematic comparison of their differential effects on naïve T cell differentiation has not been conducted. Two costimulatory proteins, CD28 and ICAM-1, resident on human naïve CD4+ T cells were compared for participation in differentiation. Under controlled conditions, and with no added cytokines, costimulation through either CD3+CD28 or CD3+CAM-1 induced differentiation to T effector and T memory cells. In contrast, costimulation through CD3+ICAM-1 induced differentiation to Treg cells whereas costimulation through CD3+CD28 did not.

Neutrophil extravasation and the regulation of vascular permeability require dynamic actin rearrangements in the endothelium. In this study, we analyzed in vivo whether these processes require the function of the actin nucleation-promoting factor cortactin. Basal vascular permeability for high molecular weight substances was enhanced in cortactin-deficient mice. Despite this leakiness, neutrophil extravasation in the tumor necrosis factor-stimulated cremaster was inhibited by the loss of cortactin. The permeability defect was caused by reduced levels of activated Rap1 (Ras-related protein 1) in endothelial cells and could be rescued by activating Rap1 via the guanosine triphosphatase (GTPase) exchange factor EPAC (exchange protein directly activated by cAMP). The defect in neutrophil extravasation was caused by enhanced rolling velocity and reduced adhesion in postcapillary venules. Impaired rolling interactions were linked to contributions of beta(2)-integrin ligands, and firm adhesion was compromised by reduced ICAM-1 (intercellular adhesion molecule 1) clustering around neutrophils. A signaling process known to be critical for the formation of ICAM-1-enriched contact areas and for transendothelial migration, the ICAM-1-mediated activation of the GTPase RhoG was blocked in cortactin-deficient endothelial cells. Our results represent the first physiological evidence that cortactin is crucial for orchestrating the molecular events leading to proper endothelial barrier function and leukocyte recruitment in vivo.

With antigen stimulation, naïve CD4+ T cells differentiate to several effector or memory cell populations, and cytokines contribute to differentiation outcome. Several proteins on these cells receive costimulatory signals, but a systematic comparison of their differential effects on naïve T cell differentiation has not been conducted. Two costimulatory proteins, CD28 and ICAM-1, resident on human naïve CD4+ T cells were compared for participation in differentiation. Under controlled conditions, and with no added cytokines, costimulation through either CD3+CD28 or CD3+CAM-1 induced differentiation to T effector and T memory cells. In contrast, costimulation through CD3+ICAM-1 induced differentiation to Treg cells whereas costimulation through CD3+CD28 did not.

Neutrophil extravasation and the regulation of vascular permeability require dynamic actin rearrangements in the endothelium. In this study, we analyzed in vivo whether these processes require the function of the actin nucleation-promoting factor cortactin. Basal vascular permeability for high molecular weight substances was enhanced in cortactin-deficient mice. Despite this leakiness, neutrophil extravasation in the tumor necrosis factor-stimulated cremaster was inhibited by the loss of cortactin. The permeability defect was caused by reduced levels of activated Rap1 (Ras-related protein 1) in endothelial cells and could be rescued by activating Rap1 via the guanosine triphosphatase (GTPase) exchange factor EPAC (exchange protein directly activated by cAMP). The defect in neutrophil extravasation was caused by enhanced rolling velocity and reduced adhesion in postcapillary venules. Impaired rolling interactions were linked to contributions of beta(2)-integrin ligands, and firm adhesion was compromised by reduced ICAM-1 (intercellular adhesion molecule 1) clustering around neutrophils. A signaling process known to be critical for the formation of ICAM-1-enriched contact areas and for transendothelial migration, the ICAM-1-mediated activation of the GTPase RhoG was blocked in cortactin-deficient endothelial cells. Our results represent the first physiological evidence that cortactin is crucial for orchestrating the molecular events leading to proper endothelial barrier function and leukocyte recruitment in vivo.

Neutrophil extravasation and the regulation of vascular permeability require dynamic actin rearrangements in the endothelium. In this study, we analyzed in vivo whether these processes require the function of the actin nucleation-promoting factor cortactin. Basal vascular permeability for high molecular weight substances was enhanced in cortactin-deficient mice. Despite this leakiness, neutrophil extravasation in the tumor necrosis factor-stimulated cremaster was inhibited by the loss of cortactin. The permeability defect was caused by reduced levels of activated Rap1 (Ras-related protein 1) in endothelial cells and could be rescued by activating Rap1 via the guanosine triphosphatase (GTPase) exchange factor EPAC (exchange protein directly activated by cAMP). The defect in neutrophil extravasation was caused by enhanced rolling velocity and reduced adhesion in postcapillary venules. Impaired rolling interactions were linked to contributions of beta(2)-integrin ligands, and firm adhesion was compromised by reduced ICAM-1 (intercellular adhesion molecule 1) clustering around neutrophils. A signaling process known to be critical for the formation of ICAM-1-enriched contact areas and for transendothelial migration, the ICAM-1-mediated activation of the GTPase RhoG was blocked in cortactin-deficient endothelial cells. Our results represent the first physiological evidence that cortactin is crucial for orchestrating the molecular events leading to proper endothelial barrier function and leukocyte recruitment in vivo.

With antigen stimulation, naive CD4+ T cells differentiate to several effector or memory cell populations, and cytokines contribute to differentiation outcome. Several proteins on these cells receive costimulatory signals, but a systematic comparison of their differential effects on naive T cell differentiation has not been conducted. Two costimulatory proteins, CD28 and ICAM-1, resident on human naive CD4+ T cells were compared for participation in differentiation. Under controlled conditions, and with no added cytokines, costimulation through either CD3+CD28 or CD3+CAM-1 induced differentiation to T effector and T memory cells. In contrast, costimulation through CD3+ICAM-1 induced differentiation to Treg cells whereas costimulation through CD3+CD28 did not.

Neutrophil extravasation and the regulation of vascular permeability require dynamic actin rearrangements in the endothelium. In this study, we analyzed in vivo whether these processes require the function of the actin nucleation-promoting factor cortactin. Basal vascular permeability for high molecular weight substances was enhanced in cortactin-deficient mice. Despite this leakiness, neutrophil extravasation in the tumor necrosis factor-stimulated cremaster was inhibited by the loss of cortactin. The permeability defect was caused by reduced levels of activated Rap1 (Ras-related protein 1) in endothelial cells and could be rescued by activating Rap1 via the guanosine triphosphatase (GTPase) exchange factor EPAC (exchange protein directly activated by cAMP). The defect in neutrophil extravasation was caused by enhanced rolling velocity and reduced adhesion in postcapillary venules. Impaired rolling interactions were linked to contributions of beta(2)-integrin ligands, and firm adhesion was compromised by reduced ICAM-1 (intercellular adhesion molecule 1) clustering around neutrophils. A signaling process known to be critical for the formation of ICAM-1-enriched contact areas and for transendothelial migration, the ICAM-1-mediated activation of the GTPase RhoG was blocked in cortactin-deficient endothelial cells. Our results represent the first physiological evidence that cortactin is crucial for orchestrating the molecular events leading to proper endothelial barrier function and leukocyte recruitment in vivo.

Leukocytes extravasate from the blood in response to physiologic or pathologic demands by means of complementary ligand interactions between leukocytes and endothelial cells. The multistep model of leukocyte extravasation involves an initial transient interaction ("rolling" adhesion), followed by secondary (firm) adhesion. We recently showed that binding of CD44 on activated T lymphocytes to endothelial hyaluronan (HA) mediates a primary adhesive interaction under shear stress, permitting extravasation at sites of inflammation. The mechanism for subsequent firm adhesion has not been elucidated. Here we demonstrate that the integrin VLA-4 is used in secondary adhesion after CD44-mediated primary adhesion of human and mouse T cells in vitro, and by mouse T cells in an in vivo model. We show that clonal cell lines and polyclonally activated normal T cells roll under physiologic shear forces on hyaluronate and require VCAM-1, but not ICAM-1, as ligand for subsequent firm adhesion. This firm adhesion is also VLA-4 dependent, as shown by antibody inhibition. Moreover, in vivo short-term homing experiments in a model dependent on CD44 and HA demonstrate that superantigen-activated T cells require VLA-4, but not LFA-1, for entry into an inflamed peritoneal site. Thus, extravasation of activated T cells initiated by CD44 binding to HA depends upon VLA-4-mediated firm adhesion, which may explain the frequent association of these adhesion receptors with diverse chronic inflammatory processes.

Leukocytes extravasate from the blood in response to physiologic or pathologic demands by means of complementary ligand interactions between leukocytes and endothelial cells. The multistep model of leukocyte extravasation involves an initial transient interaction ("rolling" adhesion), followed by secondary (firm) adhesion. We recently showed that binding of CD44 on activated T lymphocytes to endothelial hyaluronan (HA) mediates a primary adhesive interaction under shear stress, permitting extravasation at sites of inflammation. The mechanism for subsequent firm adhesion has not been elucidated. Here we demonstrate that the integrin VLA-4 is used in secondary adhesion after CD44-mediated primary adhesion of human and mouse T cells in vitro, and by mouse T cells in an in vivo model. We show that clonal cell lines and polyclonally activated normal T cells roll under physiologic shear forces on hyaluronate and require VCAM-1, but not ICAM-1, as ligand for subsequent firm adhesion. This firm adhesion is also VLA-4 dependent, as shown by antibody inhibition. Moreover, in vivo short-term homing experiments in a model dependent on CD44 and HA demonstrate that superantigen-activated T cells require VLA-4, but not LFA-1, for entry into an inflamed peritoneal site. Thus, extravasation of activated T cells initiated by CD44 binding to HA depends upon VLA-4-mediated firm adhesion, which may explain the frequent association of these adhesion receptors with diverse chronic inflammatory processes.

Intercellular adhesion molecule-1 (ICAM-1) is present on the endothelium and binds to one or more members of the CD11/CD18 family of leukocyte surface integrins. To assess the role of these molecules in mediating chemotaxis of neutrophils across the endothelium, an in vitro model consisting of monolayers of human umbilical vein endothelial cells (HUVEC) grown on amniotic connective tissue was used. Neutrophils placed on the apical sides of these cultures migrated across the endothelium in response to chemoattractants added basally. Monoclonal antibodies (MoAbs) to CD11a, CD11b, and CD18 on the neutrophils inhibited this migration by 52% +/- 11%, 29% +/- 19%, and 90% +/- 7%, respectively. An MoAb to ICAM-1 inhibited transendothelial chemotaxis of the leukocytes by 55% +/- 16%. Inhibition was mediated by binding of the MoAb to ICAM-1 on the HUVEC, rather than by any direct effect of the antibody on the neutrophils. When used in combination, MoAbs to CD11a and to CD11b inhibited migration in a nearly additive fashion. A similar additive effect was observed when MoAbs to CD11b and to ICAM-1 were used together. In contrast, MoAbs to CD11a and to ICAM-1 produced no more inhibition when used in combination than when added singly. These results show that ICAM-1, CD11a/CD18, and CD11b/CD18 all participate in controlling migration of neutrophils across endothelial monolayers in response to chemotactic agents.

Intercellular adhesion molecule-1 (ICAM-1) is present on the endothelium and binds to one or more members of the CD11/CD18 family of leukocyte surface integrins. To assess the role of these molecules in mediating chemotaxis of neutrophils across the endothelium, an in vitro model consisting of monolayers of human umbilical vein endothelial cells (HUVEC) grown on amniotic connective tissue was used. Neutrophils placed on the apical sides of these cultures migrated across the endothelium in response to chemoattractants added basally. Monoclonal antibodies (MoAbs) to CD11a, CD11b, and CD18 on the neutrophils inhibited this migration by 52% +/- 11%, 29% +/- 19%, and 90% +/- 7%, respectively. An MoAb to ICAM-1 inhibited transendothelial chemotaxis of the leukocytes by 55% +/- 16%. Inhibition was mediated by binding of the MoAb to ICAM-1 on the HUVEC, rather than by any direct effect of the antibody on the neutrophils. When used in combination, MoAbs to CD11a and to CD11b inhibited migration in a nearly additive fashion. A similar additive effect was observed when MoAbs to CD11b and to ICAM-1 were used together. In contrast, MoAbs to CD11a and to ICAM-1 produced no more inhibition when used in combination than when added singly. These results show that ICAM-1, CD11a/CD18, and CD11b/CD18 all participate in controlling migration of neutrophils across endothelial monolayers in response to chemotactic agents.