(668c) Effect of Patterning of ICAM-1 on the Upstream Migration of CD4+ T-Cells Under Shear Flow

Introduction: The leukocyte adhesion cascade is critical in leukocyte recruitment in response to pathogens, infections, and cancer treatments. The cascade consists of 4 stages: the initial capture and rolling of cells through selectin mediated interactions, firm adhesion to the endothelium later through integrins, migration, and transmigration. Recently it has been shown that some leukocyte cell types, including CD4+ T-cells, can migrate upstream under shear flow after arrest. CD4+ T-cells adhere to the adhesion molecules ICAM-1 and VCAM-1 through two different integrin receptors. It has been shown that CD4+ T-cells can migrate upstream on ICAM-1 but downstream on VCAM-1. By combining ICAM-1 and VCAM-1 on a surface, our laboratory previously showed that any amount of ICAM-1 supports upstream migration. The objective of this study was to determine if the patterning of ICAM-1 and VCAM-1 into stripes and patterns had any influence on the direction of T-cell migration.

Materials and Methods: Primary human CD4+ T-cells isolated from blood donations are activated with PHA, followed by Penn-Strep and Interluekin-2 for a week. Patterned PDMS stamps incubated with ICAM-1 and VCAM-1-Fc chimeras are stamped onto a UV activated PDMS coated glass slide. On patterned stamps, a small amount of Alexa-fluor 555 tagged Human IgG is added to visualize stamped patterns. The stamped area is blocked with Pluronic F-127 protein to prevent non-specific adhesion. Experiments are performed over 30 minutes in a flow chamber. Cells are tracked using ImageJ software and trajectories are analyzed in MATLAB. The Migration Index (MI), defined as the cell’s migration distance in the axis of flow divided by total migration distance, is calculated from the cell track. A negative value indicates net upstream migration, while a positive value indicates downstream migration. Surface protein concentrations were calculated using two methods: Elisa and fluorescent intensity using a confocal microscope. Fluorescent intensity was determined by imaging FITC tagged anti-ICAM-1 antibodies within the striped surfaces and on uniform surfaces of varying concentrations. Elisa experiments were performed HRP tagged antibody converting slow TMB to measure total chimera concentrations on uniform and patterned surfaces.

Results and Discussion: Primary CD4+ cells were seeded onto four types of patterned surfaces. Two PDMS stamp archetypes were utilized; Uniform stamps provide an even distribution of adhesion proteins on the slide and striped stamps print the adhesion proteins in stripes of 50, 100, or 200 μm width with the equivalent spacing between stripes. Experiments were performed an equal mass mixture of ICAM-1 and VCAM-1 with total 15μg/ml. 10μg/ml stamps without stripes were also included for comparison to previously published results. In addition, three flow conditions were chosen: static, a shear rate of 400 s^-1, and a shear rate of 800 s^-1. We show that on uniform surfaces, T-cells migrate upstream, consistent with previous results. As a control, no directional migration was seen on these surfaces under static conditions. Upstream migration increases with higher shear rates. On 50μm wide stripes oriented parallel to the flow, CD4+ T-cells migrate downstream under shear flow, rather than upstream as observed on the uniform surfaces. A significant portion of cells interact with or migrate along the edge of the stripe during migration. Widening the stripe to 100μm does not reestablish upstream migration, but rather led to migration that was not statistically different than random migration. While, most cells continue to interact with the edge of the stripe, whether the cell is at the edge or the center of stripe had no overall difference on the direction of migration on 100 μm stripes. On surfaces with 200μm stripes, upstream migration is re-established.

Conclusions: Confining CD4+ T-cells to stripes of 50 microns changes their observed direction of migration under shear flow. Rather migrating upstream as seen on uniform surfaces, T-cells migrate downstream in striped patterns of adhesion proteins. Increasing the width of the stamped stripes to 200μm reestablishes upstream migration, while the intermediate width of 100μm results in an intermediate migration index not statistically different from random migration.

Figure 1. Migration Index of CD4+ T-cells under shear flow on ICAM-1 & VCAM-1 mixed surfaces. Shown (left) each grouping (top to bottom) are different shear rates static, 400 s^-1, and 800 s^-1. Each grouping contains (top) uniform ICAM-1 and VCAM-1, (middle) 200μm stripes in direction of flow ICAM-1 and VCAM-1, (bottom middle) 100um stripes in direction of flow ICAM-1 and VCAM-1, (bottom) 50um stripes in direction of flow ICAM-1 and VCAM-1 surfaces. Negative value indicates upstream migration. Positive value indicates downstream migration. Mixed surfaces contain equal mass ratio of ICAM-1 to VCAM-1. All striped stamps incubated in total of 15μg/ml chimera proteins. Error bars are of S.E.M. with n = 3 or greater.

Figure 2. Scattergrams of cell tracks from single, sample experiments. Red tracks indicate upstream migration; blue tracks indicate downstream migration. Rows, from top to bottom, are uniform, 200μm horizontal, 100μm horizontal, and 50μm horizontal patterns. Columns, from left to right, are static, 400s^-1, and 800s^-1 flow conditions. All stamps incubated in total of 15μg/ml chimera proteins. Axis range from -400μm to 400μm in both the vertical and horizontal direction.

Figure 3. Examples of cells migrating on stripes. Dot and number represent the end location of each tacked cell. Flow is from left to right. Each image is from 400s^-1 flow condition with stamps incubated in 15μg/ml chimera proteins. A) 50μm stripes. Cells primarily migrate downstream. B) 200μm stripes. Cells primarily migrate upstream.