Regulatory T cells reversibly suppress cytotoxic T cell function independent of effector differentiation
Click on images to view the movies.
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B cell entry into a LN via high endothelial venules. Directly after intravenous injection, dual-labeled, unpulsed B cells (red with purple/blue center) appear in the microcirculation of the popliteal LN (blood plasma is faintly labeled in red) and undergo adhesive interactions with the postcapillary endothelium, prior to extravasation into the LN T cell area, where primed CTLs (green) are vigorously migrating. Second harmonic signals from collagen fibers are shown in blue. Time in minutes and seconds. Scale bar = 15 µm |
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CTLs undergo short-lasting interactions with Ag-free control B cells. This recording was obtained from the same preparation as Video S1, but 1.5 hours later. Many B cells (purple) have now entered the LN T cell area. CTLs are interacting only briefly with unpulsed B cells (highlighted by yellow circles). Second harmonic signals from collagen fibers are shown in blue. Time in minutes and seconds. Scale bar = 15 µm |
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Ag-dependent CTL contacts to B cells induce loss of motility, followed by change in red and blue fluorescence emission. Two interactions are highlighted in this movie. The first one, circled in white, is characterized by relatively slow co-migration of a CTL (green) and a B cell (purple) and, after 34 min, is terminated by detachment of the CTL. The B cell is rapidly engaged by another CTL (42 min). The B cell stops to migrate shortly thereafter and, starting at 52 min, undergoes a sudden color change, indicative of loss of structural integrity. The second conjugate, circled in yellow, is characterized by a much more vigorous initial phase of co-migration, which is followed by an abrupt migratory arrest of the conjugate and, eventually, formation of a large red bleb concomitant with whitening of the B cell center. At the end of the video blue circles highlight four other B cells that have already undergone the phenotypic changes characteristic for killed B cells. Note that all B cell-CTL conjugates are monogamous, and the migrating B cells are always leading the way. Second harmonic signals from collagen fibers are shown in blue. Time in minutes and seconds. Scale bar = 15 µm |
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Fluorescent material from lysed B cells becomes concentrated in LN phagocytes. 13 hours after their intravenous injection only very few viable antigen-pulsed B cells (purple) are found in the tumor-draining, CTL-containing LNs. Instead, red and blue fluorescent dyes, are concentrated in clustered vesicular structures, which likely represent phagolysosomes of macrophages or dendritic cells that have ingested killed B cells or taken up free fluorescent dye released from dead cells. The arrows in the upper left quadrant indicates a CTL (green) in the process of mitosis. Second harmonic signals from collagen fibers are shown in blue. Time in minutes and seconds. Scale bar = 15 µm |
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Regulated CTLs are inefficient at killing Ag-pulsed B cells. This movie was taken from a region within a tumor-draining LN that is comparable to that shown in Movie S3, but in this case Treg cells (which are non-fluorescent) are present. Interactions between regulated CTLs (green) and B cells (purple) rarely lead to signs of B cell apoptosis. Because of the high motility of the conjugates (highlighted by circles) and the long duration of most interactions, most of the observations are terminated by the conjugates leaving the recorded region of the LN. In the remaining cases, however, detachment of the CTLs from viable B cells is the most frequent outcome. Second harmonic signals from collagen fibers are shown in blue. Time in minutes and seconds. Scale bar = 15 µm |
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Clusters of regulated CTLs in tumor-draining LNs. On day 6 after tumor-injection, CTLs (green) are occasionally found in clusters, which likely reflect ongoing interactions with non-fluorescent antigen-presenting dendritic cells. This suggests that the presence of Treg cells does not abrogate the formation of tight conjugates between CD8+ T cells and dendritic cells. Second harmonic signals from collagen fibers are shown in blue. Time in minutes and seconds. Scale bar = 15 µm |
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Encounters of primed Treg cells with CTLs in LNs are predominantly short lasting. On day 6 after tumor-injection, activated ds-Red+ Treg cells (red) interact only infrequently and mostly briefly with primed EGFP+ CTLs (green) in the LN T cell area. A rare instance of longer-lasting (~20 min) Treg-CTL co-localization is highlighted by a yellow circle. The cells do not appear to form a stable contact zone, suggesting that both may interact with another cell, possibly a non-fluorescent antigen-presenting dendritic cell. Treg cells are not observed to interfere with contact formation between CTLs and Ag-pulsed B cells (blue). Second harmonic signals from collagen fibers are shown in blue. Time in minutes and seconds. Scale bar = 15 µm |