To understand the mechanisms that underlie the function of the immune system, immunologists have searched for methods to trace individual lymphocytes in their migration to lymphoid organs and in their subsequent interactions with other cells. In previous studies, radioactive markers were the method of choice to label lymphocytes, using autoradiographic techniques to trace them in tissues (1–3). These elaborate procedures have become obsolete with the development of fluorescent labels: powerful new techniques such as fluorescence microscopy and flow cytometry have made it relatively easy to follow, quantify, and phenotype adoptively transferred lymphocytes ex vivo (4–6). Currently, many fluorescent dyes exist, each with its specific features and (dis)advantages (7): some dyes have the ability to bind DNA and thus stain the nucleus (e.g., Hoechst 33342), some are highly lipophilic and localize in the cell membrane (e.g., 1,1′dioctadecyl- 3,3,3′-tetramethylindocarbocyanine perchlorate and PKH26), and others are lipophilic enough to cross the cell membrane and localize in the cytoplasm (e.g., fluorescein isothiocyanate [FITC] and 5-[and-6]-carboxyfluorescein diacetate, succinimidyl ester [CFSE]). Most of the labels that have been considered suitable for lymphocyte proliferation and migration studies are characterized by their nontoxicity, high fluorescence, and persistent staining: labels such as PKH26, calcein acetoxymethyl ester (Calcein), 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF), and CFSE have been shown to adequately label lymphocytes, and labeled cells have been shown to localize in peripheral lymphoid organs after in vivo transfer (6, 8, 9). Nonetheless, it remains obscure whether the applied labels leave the migratory behavior of cells unaltered.
Other methods to trace cells after in vivo transfer without the necessity of cellular labeling depend on the use of karyotypes or allelic differences between congenic mouse strains that can be traced with monoclonal antibodies. A widely used example of the latter are the murine allotypes Ly5.1 and Ly5.2 of the CD45 molecule (10, 11), which have been applied in adoptive transfer studies (12) and bone marrow transplantation (13). Using this system, donor cells require minimal handling before transfer, thus decreasing potential negative effects on the behavior of the cells.
With the aim of investigating the extent to which cell handling and labeling can affect in vivo lymphocyte migration, we combined Ly5 congenic mice with cell labeling in short-term homing assays. Therefore, labeled and unlabeled lymphocytes were transferred simultaneously into the same recipient. Because both populations of cells originated from the same donor, they were intrinsically identical and differed only in the absence or presence of a cellular label. Any interfering effect of a label of interest could thus be directly monitored because there was an internal control for the effect of each label. This approach offers new insights into the effect of (non-)fluorescent dyes on cell migration in vivo.
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Although fluorescent dyes are widely used to track lymphocyte migration in vivo, this study shows that cell labeling can severely affect the migratory behavior of lymphocytes. When used according to standard protocols and applied in short-term homing assays, the fluorescent labels Calcein, BCECF, CMFDA, CFSE, and FITC displayed a strong negative effect on B-cell migration to lymph nodes. These data clearly indicate that fluorescent dyes are not inert and can actually interfere with cellular functions, which is supported by in vitro data (20–22). It should be noted that the concentrations of the fluorescent dyes used in this study were not excessively high and were in most cases even below the generally recommended concentrations. For instance, CFSE was used at a concentration of 0.5 μM in the present study, whereas others have used this label up to 5 to 10 μM (6, 20, 23, 24). It could be anticipated that such high concentrations might result in even stronger side effects than those described here and could even affect other cell types than B cells. In addition, it is conceivable that fewer side effects of these labels can be achieved by lowering the temperature during the labeling procedure, because we also found negative effects of biotinylating cells when labeling at 37°C but not at room temperature (Fig. 3B). However, such alterations to the conventional protocols will most likely have unfavorable effects on the labeling efficiency because the labeling process depends on the activity of intracellular enzymes. Therefore, we suggest that labeling cells at room temperature with the nonfluorescent, extracellular label biotin is the most appropriate method to label lymphocytes for homing assays because it did not show any negative effects on in vivo migration (Fig. 3B), as has been reported previously for additional in vitro assays (25, 26).
To investigate the level at which the fluorescent dyes inhibit the migration of lymphocytes to lymph nodes, in vitro experiments were performed that allowed examination of the individual steps of the migration process. The HEV-binding assay clearly showed that CFSE labeling does not interfere with the L-selectin–dependent binding of (B) lymphocytes to their natural ligands on HEVs (Fig. 5). Other labels were not tested, but because it has been conclusively shown that FITC has no negative effect on this process (14), we assumed that the effect of the different labels on in vivo lymphocyte migration has its origin on a different level than on the L-selectin–dependent interaction with the endothelium.
The consecutive steps in lymphocyte migration involve the chemokine-dependent arrest of the rolling lymphocytes and their subsequent transmigration. To mimic this process, transwell assays were performed, and these studies clearly showed that none of the applied labels interfered with the chemotactic ability of T or B cells (Fig. 4). Nonetheless, it should be recognized that this approach did not provide an accurate representation of the in vivo situation, particularly due to the lack of adhesion molecules. Addition of natural ligands to the transwell, such as intracellular adhesion molecule 1 (27), could make this assay more sensitive and might reveal yet unobserved differences in the process of transmigration.
Despite the fact that these in vitro studies did not identify a mechanism responsible for the decreased lymphocyte migration, B cells appeared to be mostly affected in their homing capacity. However, Calcein did show some stimulatory effect on T cells in vivo (Fig. 2A) and in vitro (Fig. 4). Because it has been shown that B cells are more brightly labeled than T cells when using Calcein (8) or FITC (4), this does signify that B cells can respond differently from T cells to fluorescent dyes. It is conceivable that these labels interfere with B-cell–specific molecules that are directly or indirectly related to HEV-dependent migration. A potential candidate could be the B-cell–specific chemokine receptor CXCR5, because changes to its sensitivity for its ligand BLC can have direct effects on entry into lymphoid organs (16). Alternatively, a Calcein-specific effect on the T-cell chemokine receptor CCR7 or its signaling pathways could be the reason for the slight increase in T-cell migration.
Further, it should be noted that decreased numbers of CFSE- and CMFDA-labeled B cells were also found in spleen and blood, although this decrease was less apparent than observed in lymph nodes (Fig. 2). This would argue that CFSE- and CMFDA-labeled B cells are not only less capable of entering lymph nodes but may be even more prone to cell death upon transfer. For the dyes needing intracellular modification, this may be the result of the formation of toxic byproducts such as formaldehyde and acetic acid during hydrolysis of the acetoxymethyl and acetate esters of these dyes (28).
To avoid the use of intrusive cellular labels, we also compared the migratory behavior of the congenic Ly5.1-Ly5.2 system, but to our surprise these genetically almost identical mice did show differences in lymphocyte distribution upon in vivo transfer (Fig. 6B). This could be due to a different activation state of the transferred lymphocytes because this approach required that the two populations of injected cells originate from distinct donors. However, we corrected for this by using donor mice that were matched for sex, age, and health status, and the same differences could be observed in all experiments. Therefore, it well may be that in these experiments depletion from the circulation rather than disturbed migration was the cause of these effects because the unequal distribution was found not only found in lymph nodes but also in blood and spleen. Although functional differences between congenic mouse strains would not be expected due to their genetic resemblance, it has been shown that the small differences between these Ly5 allotypes can cause differences in in vitro immune responses (29) and can even decrease long-term donor engraftment upon congenic bone marrow transplantation (30). These findings indicate that even B6-Ly5.1 and B6-Ly5.2 mice are functionally more distinct than would be expected from the small genetic difference in the Ly5 gene only.
In conclusion, our data show that adoptive transfer experiments can be negatively influenced by the method chosen to trace the transferred cells. It will be important to find out whether these and other labels can also affect other cellular functions or different cell types. Taken together, it is clear that only labeling of cells with biotin does not interfere with the lymphocyte's physiology or migratory capacities. Biotin labeling is fast and easy but does require additional stainings for detection. When direct fluorescent labeling is required to follow cells in vivo, e.g., in proliferation studies or with intravital microscopy and biotin can not be used, one should be aware of negative side effects of labeling on cell function.