Immune cell doublets were distinguished from single cells or other multiple aggregates based on nuclear aspect ratios (≤ 0.5) and two-cell DNA content. Schematic diagram shows the aspect ratio (AR) as the ratio of the minor axis and major axis of a best-fit ellipse that encapsulates a T cell/macrophage conjugate. Scale bars, 10 μm.

(A) Brightfield and fluorescence images of T cell/macrophage conjugates stained with CD3 on the T cell and nuclear dye (DRAQ5) on both T cell and APC were used to identify the area of the IS. The blue transparent mask depicts the IS region derived from 2 independent functions. Left panel: Using the nuclear image the valley function creates a region centered at the dimmest pixel between the 2 nuclei (pink) in a conjugate. Right panel: Using the CD3 image (red) the interface function creates a region in the T cell image at the point of contact between the two conjugated cells. (B) Multispectral images of CD3 (red) and Lck (green) proteins of single T cell conjugates. At initial times and without a Ca⁺⁺ trigger, both Lck and CD3 cap away from the immune interface (white arrow). With a Ca⁺⁺ trigger, both proteins rapidly translocate to the IS (grey arrow) of a T cell conjugate. Co-localization of CD3 and Lck (yellow) in the composite images, obtained 60 s after adding Ca⁺⁺, reveals the development of a stable IS. Asterisks indicate the location of the APC in the conjugates. Scale bars, 10 μm. (C) Graphs show the mean (± s.d.) percent of T cell/macrophage conjugates with (i) Lck and (ii) CD3 polarized to the IS. Statistical analysis was performed on data from 3 independent experiments using the Student's t test.

Conjugates containing single T cells were selected from the doublet population using the CD3 image of the T cell. Conjugates with high CD3 AR (∼1) were defined as conjugates of one T cell and one APC. Multiple T cells involved in immune cell clusters have high CD3 area and low CD3 AR (lower panel; blue arrow). The blue mask identifies the region of CD3 staining. Scale bar, 10 μm.

At initial times and in the absence of Ca⁺⁺, there are fewer T cell/APC conjugates and only < 4% of those contain Lck at the IS (left plots). However, 6 min post Ca⁺⁺ trigger, both the total numbers of T cell/APC conjugates and the % with Lck translocated to the IS significantly increase, whether the IS is defined within the population of conjugates by the (A) Valley or (B) Interface functions.