Figure 3.
The Durability of Interaction of Human T Cell Subsets with Spatially Limiting Stimulatory Spots Does Not Correlate with the Arrest Coefficient
(A) Schematic of the micro-contact printing procedure for making stimulatory spots that emulate the spatially limiting and distributed nature of antigen presentation in lymph nodes with pervasive adhesion ligands and homeostatic chemokines. Anti-CD3ε is adsorbed on to the polydimethylsiloxane (PDMS) cast with patterned indentations. The PDMS is then stamped to transfer some of the adsorbed protein onto the coverglass. The entire surface is then coated sequentially with the chemokine CCL21 and ICAM1. See Experimental Procedures for details.
(B) Calcium influx (in black), arrest (in green), and spreading (in blue) of motile human naive CD8 T cells on the stimulatory spots (in red) captured by temporally aligning tracks of cells as they reach and arrest on the spots. Average values from 20 cells based on such a virtual synchronization are shown.
(C) Activation status of cells after 30 hr of interaction with the stimulatory spots presenting varying amounts of Okt3 and 9.3 antibodies. See also Figure S1.
(D) Proliferation of naive CD8 T cells from 3 donors after 72 hr of interaction with the stimulatory spots presenting varying amounts of Okt3 and 9.3 antibodies. The division index is the average number of divisions for all cells present.
(E) Illustration for the calculation of the half-life of interaction with stimulatory spots. T cells are introduced to find and arrest onto the spots. Live-cell imaging commences when at least ∼50% of the spots are occupied by arrested cells. The cells found to be initially arrested on the spots are tracked through the time lapse, and the percentage of cells remaining on the same spots is tallied. Natural logarithmic transformation of the percentage of remaining cells provides a curve with a very good linear fit. The half-life of interaction is calculated by the formula ln(2)/slope according to first-order kinetics.
(F) Co-stimulation by the 9.3 antibody did not influence the half-life of interaction with stimulatory spots.
(G) A larger percentage of memory CD8 T cells (in cyan) leave the spot onto which they had initially arrested, reflecting a shorter half-life of interaction. It is to be noted that memory cells, or, for that matter, naive cells, that leave a spot typically engage and attach onto another neighboring spot. However, such re-engagements are disregarded in this “survival” analysis.
(H–J) Half-life of interaction of specified T cell subsets upon attachment. The type of T cell subset examined is denoted at the bottom and in the category names of the dot plots (human CD8s in H, human CD4s in I, and murine CD8s in J). Human naive T cells have an appreciably longer half-life of interaction compared with the memory counterparts. Mean values of plotted data points are given as red horizontal bars. Each half-life measurement shown here came from different donors.
(K) Relationship between the half-life of interaction on stimulatory spots and the positional stability index on a continuous stimulatory surface among human T cell subsets. Mean values from all the donors examined are plotted.