Thymocyte emigration is mediated by active movement away from stroma-derived factors (original) (raw)
Emigration of SP thymocytes and peripheral blood T cells from a thymic organoid or fragment is differentiation stage–specific and PTX-inhibitable. We have established an in vitro system in which bone marrow–derived stem/progenitor cells undergo T cell differentiation (6). The system consists of a coculture of murine thymic stroma and primitive hematopoietic cells on a three-dimensional grid. The system resembles T cell development in the thymus, with the production of immature DP CD4+CD8+ thymocytes and the ultimate emergence of mature SP CD3+CD4+CD8– and CD3+CD4–CD8+ T cells containing a high frequency of T cell receptor excision circles. The interval from stem/progenitor to SP thymocyte generation is 14 days. SP and DP thymocytes are not found within or outside the thymic organoid prior to this time, as shown in Figure 1a. At 14 days, SP CD4+ and CD8+ T cells were found at the periphery of the tissue-culture well (Figure 1b). SP thymocyte localization at the periphery was significantly reduced (P < 0.01, Student t test) when cocultures were exposed to PTX (Figure 1c). CD34+CD2– or TN CD3–CD4–CD8– cells were only detectable at the periphery of the culture up to 4–5 days after seeding the thymic stroma with primitive hematopoietic cells (Figure 1a). Primitive stem cells or TN thymocytes were not detected in significant numbers at the periphery of the culture after this time (Figure 1b).
Mature SP T cells migrate away from the thymic stroma/CD34+CD2– cell coculture in which they are generated. Purified human bone marrow–derived CD34+CD2– hematopoietic progenitor cells were plated onto confluent murine thymic stroma established on the three-dimensional matrix Cellfoam to form a thymic organoid. A sample of cells was harvested from inside and outside the thymic organoid under microscopic guidance at day 4 (a) and day 14 (b) and stained with anti-CD4 (FITC-labeled) and anti-CD8 (PERCP- or PE-labeled). The thymic organoid was also cultured in the presence of PTX (10 ng/ml) between day 7 and day 14, and cells were harvested and analyzed at day 14 (c). At day 14 of the coculture, cells began to appear at the edge of cultures away from the thymic stroma–coated Cellfoam. At day 14 the majority of cells from the periphery of the coculture were CD4+CD8– or CD4–CD8+, whereas the majority of cells within the Cellfoam were CD4+CD8+ (b). At day 14, in the culture containing the thymic organoid exposed to PTX, the majority of cells were detected in the grid and were either CD4+CD8+ and CD4+ CD8– or CD4–CD8– thymocytes. A very small proportion of CD4+CD8– and CD4–CD8– thymocytes was detected outside of the thymic organoid in these PTX-treated cultures (c). The percentage of cells falling into each immunophenotypic category is shown in the top right corner of each quadrant. *P < 0.05, **P < 0.01, Student t test.
To further substantiate the differentiation stage–specific emigration of thymocytes from the thymic organoid and its dependence on a PTX-inhibitable signal transduction pathway, purified subpopulations of human thymocytes were plated onto a thymic organoid containing stromal cells alone. The thymic organoid was placed on a polycarbonate membrane of a fixed, 3-μm pore size within the upper chamber of a transwell. Purified subpopulations of human or murine TN, DP, or SP thymocytes were then plated onto the stroma. Transmigration of thymocyte subpopulations from the grid was then quantitated. There were marked differences between the transmigration of different thymocyte subpopulations. Migration of immature DP thymocytes away from thymic stroma was minimal, with 4.1% ± 0.3% of input cells transmigrating (Figure 2a). In marked contrast, a significantly greater (P < 0.001, Student t test) proportion of SP thymocytes (CD4+ SP, 8.9% ± 1.0%; CD8+ SP, 13.4% ± 1.5%) transmigrated to the lower chamber of the transwell away from thymic stroma (Figure 2). Migration of SP thymocytes away from thymic stroma was significantly inhibited following exposure to PTX (CD4+ SP thymocytes, 2.9% ± 0.2%; CD8+ SP thymocytes, 2.2% ± 0.1%; P < 0.0001, Student t test). Migration of SP thymocytes was also abrogated when the gradient of thymic stromal chemokinetic factors was nullified by adding TSCM to both the upper and lower chambers of the transwell (Figure 2a). The transmigration of thymocyte subpopulations and fetal blood T cells was also studied in the absence of a thymic organoid (Figure 2a). Spontaneous migration of thymocyte and fetal blood T cell subpopulations was significantly less than directional movement away from the thymic organoid under these conditions.
(a) Emigration of thymocyte and peripheral blood T cells from the thymic organoid. Purified human (Hu) CD4+ and CD8+ SP thymocytes, DP CD4+CD8+ thymocytes, and fetal blood T cells were plated onto a grid coated with thymic stroma in the upper chamber of a transwell. Emigration from the upper chamber into the lower chamber was quantitated (black bars). Cells in the upper chamber were also pretreated with PTX (light gray bars), or TSCM was added to the upper and lower chambers before the addition of thymocytes (dark gray bars). Thymocyte populations or fetal blood T cells were also plated into the upper chamber in the absence of thymic stroma (white bars). The percentage of cells emigrating under each condition was assessed in three independent experiments, and the mean migration with SE bars is shown. (b) Emigration of thymocytes and peripheral blood T cells from murine (Mu) thymic fragments. Murine thymic fragments were placed in the upper chamber of a transwell. Emigration of TN (CD3–CD4–CD8–), DP (CD4+CD8+), and SP (CD4+ and CD8+) thymocytes from the thymic fragment into the lower chamber was quantitated (black bars). Thymic fragments in the upper chamber were also pretreated with PTX (light gray bars), murine TSCM was added to the upper and lower chambers prior to the addition of the fragment (dark gray bars), or thymic fragments were pretreated with paraformaldehyde (PF) (white bars). The percentage of cells emigrating under each condition was assessed in three independent experiments. The mean migration with SE bars is shown.
Emigration of thymocytes from the thymus was also examined in a system that more closely resembles the physiological setting. Freshly prepared murine thymic fragments were placed on a polycarbonate membrane of a fixed, 3-μm pore size within the upper chamber of a transwell.
Emigration of TN, SP, and DP thymocytes from the thymic fragment into the lower chamber was then quantitated by cell counting and flow cytometry. Migration of both CD4+ and CD8+ SP thymocytes away from the thymic fragment was significantly greater than that for immature TN (P < 0.0001, Student t test) and DP thymocyte subpopulations (P < 0.0001, Student t test) (Figure 2b). Similar to the thymic organoid, emigration of SP thymocytes from the thymic fragment was inhibitable by exposure to PTX or by abrogation of the gradient of thymic stroma–derived chemokinetic factors by the addition of TSCM to both upper and lower chambers of the transwell. Pretreatment of thymic fragments with paraformaldehyde significantly inhibited emigration of SP thymocytes, thus confirming that movement of SP thymocytes away from the thymic gradient was an active process and not simply passive movement of cells through the pores of the transwell membrane.
Emigration of SP thymocytes and peripheral blood T cells is dependent on a gradient of thymic stromal chemokinetic factor(s). As demonstrated above, the addition of TSCM to both the upper and lower chambers of the transwell system involving both thymic organoid and thymic fragment abrogated differentiation stage–specific emigration of both SP thymocytes and fetal blood T cells. We therefore postulated that movement of SP thymocytes and T cells was dependent on a gradient of a thymic stromal chemokinetic factor or factors generated in the upper chamber by the thymic organoid or fragment.
To further examine the dependence of SP thymocyte and fetal blood T cell emigration on a gradient of thymic stroma–derived chemokinetic factor(s), a checkerboard analysis of thymocyte and fetal blood T cell migration to serial dilutions of TSCM was performed (Figure 3). The transmigration of thymocyte and peripheral blood T cell subpopulations in response to positive and negative gradients of serial dilutions of TSCM was assessed using a checkerboard analysis of cell migration. TN and DP thymocytes showed negligible chemotactic and fugetactic responses to TSCM that was undiluted or diluted 1:10 or 1:100 in serum-free medium (data not shown). Fetal peripheral blood T cells demonstrated a peak chemotactic response to 1:10 TSCM under which conditions 6.3% ± 0.7% of cells transmigrated. In contrast, 9.3% ± 0.8% of input fetal T cells moved away from undiluted TSCM (Figure 3). Directional responses both toward and away from TSCM were significantly greater than chemokinesis (random movement) seen at either dilution of TSCM in the absence of a gradient. Furthermore, preincubation of fetal blood T cells in TSCM prior to exposure of these cells to gradients of the known chemoattractant SDF-1 did not effect cell movement toward or away from the chemokine (data not shown). Therefore, these data support the view that gradients of TSCM induce directional movement of fetal blood T cells rather than blocking cellular movement.
Checkerboard analysis of migratory responses of fetal T cells to TSCM. Numbers shown represent mean number of cells ± SEM, from three independent experiments, that migrated through a polycarbonate membrane with a 5-μm pore size into the lower chamber of a transwell. Human fetal blood T cells were counted in the lower chamber 3 hours after the introduction of 5 × 104 cells into the upper chamber, and human TSCM was added at the indicated dilutions in the upper chamber (above diagonal), creating a negative gradient, or the lower chamber (below diagonal), creating a positive gradient. Squares along the diagonal represent cell migration in the presence of TSCM in the absence of a gradient and correspond to chemokinesis or random movement. ND, not determined.
SP thymocytes also demonstrated a maximal chemotactic response to 1:10 TSCM (8.8% ± 1.1% of input cells) and moved away from undiluted TSCM (12.5% ± 1.5% of input cells) (data not shown). These data further supported the postulate that differentiation stage–specific movement of thymocyte populations away from thymic stromal chemokinetic factors was gradient-dependent.
Thymic stroma–derived SDF-1 contributes to thymocyte emigration from the thymic organoid and thymic fragment and is mediated via the chemokine receptor CXCR4. We have demonstrated previously that mature human T cells actively move away from the chemokine SDF-1, a mechanism we termed fugetaxis or chemofugetaxis (7). Furthermore, since hemodynamic effects or chemotactic recruitment postulated for the native thymus could not be present to explain emigration from the thymic organoid or fragment, we assessed whether SP thymocytes may undergo fugetaxis or active movement away from SDF-1 generated by thymic stroma. The feasibility of this phenomenon was demonstrated in semisolid cultures in which SDF-1 was added at differing concentrations at specific locations in the culture. Distinct movement characteristics and directions were noted at low concentrations (movement toward SDF-1: chemotaxis) and high concentration (movement away from SDF-1: fugetaxis) (see supplemental data, www.jci.org/cgi/content/full/109/8/1101/DC1). Directional movement of four representative cells was documented for each of the three video clips using image analysis (Figure 4). This study demonstrates that the cells move consistently toward SDF-1 at a peak concentration of 100 ng/ml at an approximate speed of 8.4 μm/min and away from SDF-1 at a peak concentration of 10 μg/ml at 6.8 μm/min. These data provided support for active repulsion of mature T cells from high concentrations of SDF-1 in the thymus as a possible mechanism for T cell emigration from the thymus.
Quantitation of T cell movement depicted in time-lapse digital video microscopy. Quantitation of human T cell movement during each video clip (see supplemental data, www.jci.org/cgi/content/full/109/8/1101/DC1). The speed and direction of movement was plotted for each of four representative cells in each of the video clips: left, medium alone; middle, SDF-1 at a peak concentration of 100 ng/ml; right, SDF-1 at a peak concentration of 10 μg/ml. The position of each cell at the beginning of each video clip and the movement away from that point are plotted. The paths of each cell were then overlaid. The direction of the gradient and the scale are shown for each condition. Cells migrate toward SDF-1 in the middle plot and away from SDF-1 in the right plot.
We next assessed whether thymic stroma produced high concentrations of SDF-1. TSCM was prepared from confluent cultures of human thymic stroma grown on Cellfoam. Ion exchange chromatography was performed and fractions collected for assessment of biological activity and biochemical analysis. One peak of fugetactic activity was noted in fraction 9, which was inhibitable by neutralizing Ab to CXCR4, the receptor for SDF-1 (P < 0.001, Student t test). In addition, a second peak of activity was noted in fraction 4. Movement of T cells away from fraction 4 was inhibited by anti-CXCR4, but this did not reach statistical significance (P < 0.1, Student t test) (Figure 5). Both fractions inducing fugetaxis were inhibitable by PTX. Western blot analysis demonstrated SDF-1 in fraction 9. The approximate concentration of SDF-1 in TSCM was estimated from the Western blot at 1.3 μg/ml when compared with the control blot for recombinant SDF-1 containing 100 ng of the chemokine. Fraction 4 did not immunoblot with anti–SDF-1. This fraction was not apparent in bone marrow stroma-conditioned medium (data not shown). These data suggest that a specific fraction of TSCM contained a high concentration of SDF-1 that could elicit active movement of mature T cells away from it. This migratory response to the fraction containing SDF-1 was inhibitable by pretreatment of T cells with an mAb that blocks the chemokine-binding site on its sole receptor, CXCR4.
Fugetactic activity is present in two fractions of human TSCM prepared on a cation column, one with undetectable SDF-1 and a second containing SDF-1. A 1:10 dilution of column fractions (Fxn 1–10) derived from an SP cation-ion-exchange column eluted with a linear gradient of NaCl concentration (0.1–1 M) was used in experiments. The fugetactic activity was determined using a standard transwell assay system. Each fraction was loaded into the upper chamber of the transwell alone, and the migration of CD4+ CD45RO T cells away from the fraction was quantitated as described above. Fractions 4 and 9 contained fugetactic activity for CD4+ CD45RO T cells. The fugetactic activity of fraction 9 alone was inhibitable if T cells were preincubated with anti-CXCR4. The control fugetactic activity of 1 μg/ml SDF-1 (SDF/m) and medium alone (m/m) was also tested. Each data point represents the mean and SEM of three independent experiments. Fractions of TSCM prepared on a cation-exchange column were analyzed by Western blot with a polyclonal goat anti-human SDF-1 Ab (R&D Systems Inc., Minneapolis, Minnesota, USA) and a peroxidase-conjugated secondary Ab. Only fraction 9 and control recombinant human (rHu) SDF-1 (100 ng) generated a positive band of approximately 8 kDa (inset). *P < 0.1, **P < 0.001, Student t test.
Three experimental approaches were then used to determine the role that active movement of thymocyte populations away from SDF-1 played in thymic emigration: the quantitation of CXCR4 expression on thymocyte subpopulations, the study of the effects of CXCR4 blockade, or the abrogation of an SDF-1 gradient on thymocyte migratory responses from the thymic organoid or fragment and the effect of inhibitors known to inhibit components of the signal transduction signaling movement toward or away from SDF-1.
All subpopulations of peripheral blood T cells and thymocytes were shown to express CXCR4 at high levels, as determined by flow cytometry (Table 1). Our data confirm the finding of CXCR4 on both immature DP and mature SP thymocyte populations, as well as mature peripheral blood T cell subpopulations.
CXCR4 expression of thymocyte and peripheral blood T cell subpopulations
We then examined the role of the SDF-1/CXCR4 axis in the differentiation stage–specific emigration of SP thymocytes from the thymic organoid and thymic fragment. Purified subpopulations of human thymocytes were applied to the thymic organoid, and emigration of these cells was then quantitated as described above. Thymocyte subpopulations were exposed to anti-CXCR4 prior to addition onto the thymic organoid (Figure 6a). Anti-CXCR4 was shown to significantly inhibit the active movement of CD4+ SP (P < 0.05, Student t test) and CD8+ SP (P < 0.001, Student t test) thymocytes from the grid, as compared with control conditions in which thymocyte or peripheral blood T cell subpopulations were added to the upper chamber of the transwell containing the thymic organoid. Furthermore, when recombinant SDF-1 at a final concentration of 1 μg/ml was added to the lower chamber of the transwell, emigration of SP thymocytes and peripheral blood T cells was also inhibited (Figure 6a). This concentration of SDF-1 was chosen because we had demonstrated previously by Western blot analysis that the concentration of SDF-1 in TSCM was approximately 1.3 μg/ml. In this way we demonstrated that emigration of SP thymocytes was dependent on both the presence of SDF-1 receptor, CXCR4, on thymocyte subpopulations and the presence of a gradient of SDF-1.
(a) CXCR4 and SDF-1 and the emigration of SP thymocytes from thymic organoid. Purified human thymocyte subpopulations, and peripheral blood (PB) T cells were plated directly onto a three-dimensional grid coated with thymic stroma in the upper chamber of a transwell. Emigration from the grid and upper chamber into the lower chamber was quantitated (black bars). Cells in the upper chamber were pretreated with anti-CXCR4 (white bars) or SDF-1 (1 μg/ml) was added to the lower chamber of the transwell (gray bars) prior to the addition of purified thymocyte populations. The percentage of cells emigrating under each of the conditions was assessed in three independent experiments, and the mean migration with SE bars is shown. (b) Inhibitor profile for emigration of SP thymocytes from murine thymic tissue fragment. The 2-mm3 murine thymic tissue fragments were cultured on a 3-μm-pore transwell membrane. The number of SP thymocytes emigrating from each tissue fragment was quantitated. Inhibitors were added to the well for the second period of 3 hours. The number and phenotype of thymocytes emigrating from each thymic tissue fragment were quantitated and compared directly with the emigrants from the first 3-hour time period. The data are expressed as the percentage of inhibition of SP thymocyte emigration from the thymic fragment in the presence of putative inhibitors of this process. Thymic fragments were treated with PTX, isotype IgG, anti-CXCR4 Ab, wortmannin, genistein, 8-Br-cGMP, or 8-Br-cAMP. The data represent the mean ± SEM for three experiments.
We have demonstrated previously a distinct inhibitor profile for active movement of T cells away from SDF-1 as compared with movement toward the chemokine for purified subpopulations of mature T cells (7). Movement both toward and away from SDF-1 was inhibitable by the phosphoinositide (PI) 3-kinase inhibitor wortmannin. Movement away from SDF-1 was significantly less sensitive to the tyrosine kinase inhibitors genistein and herbamycin than was movement toward the chemokine. The contrary was evident for the cAMP agonist 8-Br-cAMP: movement of T cells away from SDF-1 was significantly more sensitive to inhibition than was movement toward the chemokine. We therefore examined the broader inhibitor profile of the movement of SP thymocytes away from thymic fragments in order to determine whether this was similar to that seen for T cells moving away from SDF-1. Thymic fragments were pretreated with inhibitors including anti-CXCR4, wortmannin, genistein, 8-Br-cGMP, and 8-Br-cAMP, and SP thymocyte emigration was subsequently quantitated using the assay system described above. Emigration of SP thymocytes from murine thymic fragments was inhibitable by pretreatment of the thymic fragment with anti-CXCR4 (12G5) mAb (53.5% ± 6.5% inhibition), wortmannin (70.2% ± 2.6% inhibition), and 8-Br-cAMP (46.4% ± 8.6% inhibition) (Figure 6b). In contrast, emigration of SP thymocytes from the thymic fragment was not significantly inhibited by pretreatment with the tyrosine kinase inhibitor genistein (P = 0.4, Student t test), or the cyclic nucleotide agonist 8-Br-cGMP (P = 0.2, Student t test) (Figure 6b). Therefore, the inhibitor sensitivity profile for thymic emigration in vitro resembled that which we had demonstrated previously for the active movement of mature T cells away from SDF-1. These data support the hypothesis that SDF-1 generated by thymic stroma might elicit emigration of maturing T cells in the thymus in vivo.
Differentiation stage–specific movement of human fetal thymocyte and T cell subpopulations away from recombinant SDF-1. We quantitated the transmigration of purified human fetal thymocyte and peripheral blood T cell subpopulations in response to positive and negative gradients of SDF-1 using a standard checkerboard analysis of chemotaxis. DP and TN thymocytes demonstrated minimal migratory responses toward or away from SDF-1 at peak concentrations of 10 ng/ml, 100 ng/ml, 1 μg/ml, or 10 μg/ml (Figure 7a).
Checkerboard analysis of migratory responses of fetal thymocyte and T cells to SDF-1. Sorted human fetal DP thymocytes (a) or fetal blood CD4+ T cells (b) were introduced into the upper chamber of a transwell. Numbers shown represent mean number of cells ± SEM, from three independent experiments, that migrated through a polycarbonate membrane with a 5-μm pore size into the lower chamber of a transwell. Cells were counted in the lower chamber 3 hours after the introduction of 5 × 104 cells into the upper chamber, and SDF-1 was added at the indicated concentrations in the upper chamber (above diagonal), creating a negative gradient, or the lower chamber (below diagonal), creating a positive gradient.
In contrast, SP thymocytes demonstrated a peak chemotactic response to SDF-1 at a concentration of 100 ng/ml, under which conditions 9.8% ± 1.3% of cells transmigrated. In addition, 10.9% ± 2.5% of SP thymocytes moved away from a concentration of 10 μg/ml (data not shown). Fetal peripheral blood T cell subpopulations demonstrated a bidirectional response to SDF-1, with 15.3% ± 1.4% of input cells migrating toward a peak SDF-1 concentration of 100 ng/ml and 7.9% ± 0.9% and 14.6% ± 1.6% of cells migrating away from SDF-1 at a concentration of 1 μg/ml and 10 μg/ml, respectively (Figure 7b).