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IMMUNOBIOLOGY
T cells that emigrate from the thymus have primarily been
studied in vivo using fluorescent dye injection of the thymus. This study examined the properties of thymocytes that emigrate from cultured
thymic lobes in organ culture. Under these conditions, thymic emigrants
displayed the expected phenotype, that of mature thymocytes expressing
high levels of T-cell receptor (TCR- The thymus is the site of T-cell development.
Lymphoid precursors leave the bone marrow, enter the thymus, undergo a
complex series of gene rearrangements, positive and negative selection for T-cell receptor (TCR) specificity, proliferation, and
differentiation steps. When this program is complete, the T cell
emigrates from the thymus, beginning a pattern of recirculation from
blood to lymph to blood in search of its specific antigen in
peripheral tissues.
Little is known about the mechanism of emigration of cells from the
thymus and many questions remain regarding the markers and functional
properties of these emigrants. What is the trigger to emigrate? Where
do they go first? How long do they survive and what extrinsic trophic
signals maintain them? Are they homogeneous or are there subsets of
emigrants? Are they uniquely tolerizable to peripheral antigens? To
answer some of these questions, the most direct studies of the
properties of thymic emigrants have relied on the injection of
fluorescein isothiocyanate (FITC) into the thymus.1 FITC
is taken up by thymocytes, and cells leaving the thymus retain the dye
for the next few days and can be identified in peripheral sites based
on their fluorescence. Using flow microfluorimetry, other markers of
these cells can be assessed, and using cell sorting, their functional
properties can be examined.
A number of observations have been made regarding thymic emigrants
using the FITC method. The labeled cells have a relatively mature
single positive phenotype and no unique marker has been found that
distinguishes them from either mature thymocytes or peripheral T
cells.2 Functionally, the labeled cells are fully responsive to mitogens and alloantigens.3 However, other
methods suggest that recent emigrants secrete very little interleukin (IL)-2 compared to mature splenic T cells4 and that
emigrants are tolerized by alloantigen contact.5 If thymic
emigrants were anergized by contact with antigen, it could explain how
self-tolerance is established to those antigens that are not expressed
in the thymus.
One limitation of the method of thymus FITC injection is that
recirculating mature lymphocytes, and particularly those that have been
previously activated, continuously enter the thymus as shown in many
studies6-15 and would also be labeled. Many of the
FITC-labeled cells that leave the thymus and are detected peripherally
could therefore be mature recirculating T cells, rather than cells that
recently matured in the thymus. By one estimate, 30% of the cells that
are labeled this way and subsequently detected in spleen are mature
recirculating cells rather than cells that recently matured in the
thymus (P. Matzinger, oral communication, 1999). Thus, the
apparent functional maturity of thymic emigrants could be due to
contamination with mature, previously activated T cells, making it
desirable to develop another method of characterizing thymic emigrants.
In the present study, we developed a method for collecting thymocytes
that emigrate from the fetal thymus during organ culture in vitro, a
technique that avoids the problem of contamination with recirculating
mature T cells. We then examined the surface markers and functional
properties of these cells.
Mice
Fetal thymus organ culture
Fetal thymus lobes obtained from Tap Inhibitors of cell motility were tested on emigration by transferring
lobes (at day 12 of cultivation) to a well containing the inhibitor,
then 6 hours later emigrants were collected and characterized. Cytochalasin D (2 µM; Sigma, St Louis, MO)
inhibits microtubule assembly. Clostridium difficile toxin B
(50 ng/mL; Oravax, Cambridge, MA) glucosylates and thereby inhibits Rho
family small guanosine triphosphatases (GTPases). Pertussis toxin (50 ng/mL; Sigma) catalyzes adenosine diphosphate (ADP)-ribosylation of
some G proteins. Control wells contained dimethyl sulfoxide (DMSO;
0.5%) used to dissolve cytochalasin D.
Preparation of cells
Total spleen cells and lymph node cells were prepared from mouse spleen and lymph node, respectively, by gentle disruption between 2 notched slide glasses. The single-cell suspensions were washed with PBS, and then red blood cells were lysed by treatment with ACK lysis buffer (0.15 M NH4Cl, 1.0 mM KHCO3, 0.1 mM EDTA) for 3 minutes. T cells were isolated from total spleen cells by a T-cell enrichment column (Collect-T, Biotex Lab, Alberta, Canada) according to the manufacturer's instructions. Dendritic cells were generated from mouse bone marrow. Briefly, total bone marrow cells (obtained from 2 femurs by flushing with PBS) were added to a 100-mm Petri dish in 10 mL cell culture medium supplemented with 800 U/mL recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF; PeproTech, Rocky Hill, NJ) and 400 U/mL recombinant murine IL-4 (PeproTech). Nonadherent cells were harvested the next day, supplemented with the cytokines, then added to 6-well plates at a density of 5 × 106/well in 5 mL medium and cultured. At day 3 and 4 from the initiation of the culture, nonadherent cells were discarded by a gentle shaking and fresh medium with cytokines added. Aggregates of dendritic cells were usually harvested at day 7, and then disssociated by gentle pipetting. Flow cytometric analysis The following mouse monoclonal antibodies were used: anti-CD4 (clone RM4-5), anti-CD8 (clone 53-6.7), anti-TCR / (clone
H57-597), anti-TCR / (clone GL3), anti-Thy1.2 (clone 53-2.1),
anti-CD44 (clone IM7), anti-CD69 (clone H1.2F3), anti-CD25 (clone 7D4), anti-LFA-1a (clone 2D7), anti-ICAM-1 (clone 3E2), anti-L-selectin (clone MEL-14), anti-4-integrin (clone R1-2),
anti-5-integrin (clone 5H10-27), anti-CD45 (clone
30F11.1), anti-HSA (clone M1/69), anti-CD28 (clone 37.51), anti-CTLA-4
(UC10-4F10-11), anti-H-Kb (clone AF6-88.5), and anti-CD16
/CD32 (clone 2.4G2). All of these antibodies were purchased from
Pharmingen (San Diego, CA).
For staining, cells were washed in a staining solution of PBS containing 5% FBS and 0.1% NaN3, and then resuspended in 50 µL staining solution containing 0.5 µg rat monoclonal antibody 2.4G2 and 10% normal mouse serum to reduce nonspecific binding of antibodies to Fc receptor. Cells were incubated for 10 minutes on ice, mixed with 50 µL prediluted antibody solution that contained 2 kinds of antibodies, and then incubated for 20 minutes on ice. Unbound antibodies were removed by washing the cells twice with staining buffer. If biotinylated antibody was used, the cell pellet obtained from the second wash was resuspended in 50 µL phycoerythrin (PE)- or FITC-conjugated avidin, incubated for 10 minutes on ice, and then washed twice with staining buffer. After staining, cells were fixed in 1% p-formaldehyde in PBS and flow cytometric analysis was performed on a FACStar Plus (Becton Dickinson Immunocytometry System, Mountain View, CA). Dead cells were gated out by their low forward angle light scatter intensity. In most analysis, 10 000 cells were scored. Mitogenic stimulation For phorbol myristate acetate (PMA) and ionophore A23187 stimulation, thymic emigrants (2 × 105/well) or purified adult splenic T cells (2 × 105/well) were cultured for 3 days, respectively, in 96-well U-bottom tissue culture plate in the presence of PMA (100 ng/mL), A23187 (1 µg/mL), or both. Cells were pulsed with tritiated thymidine (0.5 µCi/well) for the last 6 hours. For anti-CD3 monoclonal antibody stimulation, thymic emigrants (2 × 105/well) or purified adult splenic T cells (2 × 105/well) were mixed with irradiated (1000 R) syngeneic bone marrow-derived dendritic cells (2 × 103-5 × 104/well), cultured for 3 days, respectively, in 96-well U-bottom tissue culture plate in the presence of 1.0 µg/mL anti-CD3 monoclonal antibody (clone
145-2C11, PharMingen). Cells were pulsed with tritiated thymidine (0.5 µCi/well) for the last 6 hours.
Primary and secondary mixed lymphocyte response (MLR) For primary MLR, thymic emigrants (2 × 105/well) or purified adult splenic T cells (2 × 105/well) were mixed with irradiated (1000 R) syngeneic or allogeneic bone marrow-derived dendritic cells (2 × 103-5 × 104/well), respectively, and cultured for 3 days in 96-well U-bottom tissue culture plate. Cells were pulsed with tritiated thymidine (0.5 µCi/well) for the last 6 hours.For secondary MLR, thymic emigrants (3 × 106/well) or purified adult splenic T cells (3 × 106/well) were first mixed with irradiated (1000 R) syngeneic or allogeneic bone marrow-derived dendritic cells (2 × 106/well), respectively, in wells of 24-well tissue culture plate, and cultured for 2 days. Viable cells were separated by Ficoll-Hypaque centrifugation, counted, and plated to wells of 96-well U-bottom plates at a density of 1 × 105/well, and then rested for 1 day. Secondary stimulation was induced by adding irradiated (1000 R) syngeneic or allogeneic bone marrow-derived dendritic cells (2 × 103-5 × 104/well), respectively, to each primarily stimulated group of cells. The cultures were incubated for 3 days, and were pulsed with tritiated thymidine (0.5 µCi/well) for the last 6 hours. Graft-versus-host (GVH) reaction Thymic emigrants (2-4 × 106/recipient) generated from C57BL/6 (H-2b) were injected intravenously into syngeneic severe combined immunodeficiency (SCID) mice or allogeneic CB-17 (H-2d) SCID mice that were treated with anti-asialoGM1 monoclonal antibody and then irradiated with 2.5 Gy. For comparison, unfractionated C57BL/6 lymph node cells (0.5-2 × 106/recipient) and purified splenic T cells (0.5-2 × 106/recipient) were also injected intravenously into the same-treated SCID recipients, respectively. Mortality was monitored every day and body weight was monitored twice a week initially then once a week thereafter. At day 49 after transfer, blood was drawn and hematocrits were performed, after which recipients were killed and skin and liver sections examined for signs of GVH disease (GVHD) by a veterinary pathologist. Thymic emigrants repopulating the lymphoid organs of syngeneic or allogeneic SCID recipients were analyzed by examining the expression of T-cell markers such as CD3, CD4, and CD8, and donor marker H-2Kb. Two complete experiments were performed (on different dates) using in vivo transfers of thymic emigrants.
Thymic organ culture for generating emigrants To examine cells that emigrate from the thymus during organ culture, a method was devised for collecting thymic emigrants. Previous methods of culturing the embryonic thymus have shown that placing the lobe at the interface of air and medium is much superior for promoting T-cell development compared to submerging the organ.16 The air interface optimizes the availability of O217 because the explanted organ is disconnected from normal vasculature. Previous methods of organ culture generally supported the thymus on a nucleopore membrane that in turn was supported by a gelfoam sponge floating on medium. Cells that emigrate from the thymus in this culture system cannot be easily separated from those that remain in the thymus. We modified this system by placing the thymic lobe on a fine nylon mesh that in turn rested on a cylinder careful adjustment of the medium height maintained the thymic lobe at the interface of medium and air. Cells that emigrate from the lobe pass freely through the nylon mesh and fall to the bottom
of the tissue culture well.
The rate of emigration from cultured thymic lobes is shown in Figure 1. We have analyzed thymic emigrants at various times of organ culture, but the subsequent studies were performed following 12 days of culture for the sake of consistency, and because we occasionally found some immature cells "leaked" at earlier time points. Over the following 18 hours, the number of recovered emigrants numbered approximately 0.5% of the total number of cells in the thymus. Phenotype of thymic emigrants Analysis of these thymic emigrants by flow microfluorimetry revealed characteristics of the most mature thymocytes as indicated in Figure 2 by the criteria of staining with CD4, CD8, TCR and TCR. In the lineage, the most
mature thymocytes express high levels of TCR and are single
positive for CD4 or CD8, whereas their predecessors express low to
intermediate levels of TCR and express both CD4 and CD8. Thus,
among emigrants, the proportion of single-positive CD4 cells was
29.1%, compared to 10% within the thymus, and the proportion of
single-positive CD8 cells was 51.6% in emigrants, compared to 28.2%
within the thymus, whereas double-positive cells accounted for only 6%
in emigrants, compared to 36.7% within the thymus. Likewise the
proportion of high TCR cells was 63.7% (up to 91% in other
experiments) in emigrants compared to 11.2% within the thymusnote
the percentages written in Figure 2 refer to both intermediate and high
TCR. The TCR cells also emigrated from the thymus,
accounting for 13.1% of emigrants. Overall, the emigrant population
was primarily comprised of mature thymocytes that had completed
development and selection. This method of organ culture, therefore,
appeared to generate the types of cells that normally exit the thymus
in an intact mouse.
In Figures 3 through 6 (summarized in Table
1), the surface markers on thymic
emigrants were examined more extensively. These analyses focus on the
Most markers showed the same level of expression on emigrants as on
mature intrathymic or splenic T cells. These markers include CD44
(Figure 3), LFA-1
Several markers that were examined are more highly expressed on intrathymic cells than on splenic T cells. Thus, it would be possible for emigrants to either express the high thymic level or the low peripheral level. Whereas thymic emigrants more resembled intrathymic cells for their high expression of ICAM-1 (Figure 4), they more resembled splenic T cells for their low expression of CD69 (Figure 3). Emigrants showed intermediate expression (between intrathymic cells and splenic T cells) for Thy1 (Figure 3) and HSA (Figure 5). The most unique marker detected on thymic emigrants was CTLA-4 (Figure
5), which was virtually undetectable on peripheral T cells and only
slightly visible on intrathymic cells. Although emigrants expressed a
low level of CTLA-4 (relative to other markers), the level was similar
to that on activated peripheral T cells (Figure
6). Thus, the level of CTLA-4 on
emigrants could be sufficiently high to deliver similar signals to that
in mature T cells, in which it performs a largely inhibitory
role.18
It is important to point out that expression of many of these markers is surprisingly heterogeneous on thymic emigrants. For example, L-selectin (Figure 4) or HSA (Figure 5) expression on emigrants ranged from brightly positive cells to negative cells. This may indicate that different subsets of cells emerge from the thymus with commitments to different functions and migration patterns. Thus, thymic emigrants expressing high L-selectin levels may be already committed to home to lymph nodes, whereas those with low levels may home elsewhere. Emigration from the thymus following positive selection The phenotype of thymic emigrants of the lineage indicated
they had completed positive selection (they were single positive for
CD4 and CD8 and expressed high levels of TCR). To determine how
rapidly emigration occurred after positive selection, we modified the
organ culture such that positive selection was induced synchronously, then examined how rapidly mature emigrants emerged. Our approach took
advantage of the block in positive selection of CD8 cells that occurs
in the thymus from Tap / mice, which is due to the
instability of the class I major histocompatibility complex
(MHC)/peptide complex that is required for positive selection of CD8
cells. Synchronous positive selection of CD8 cells can be induced in
cultures of Tap/ thymus organs by adding
2m,19 which stabilizes class I molecules (presumably together with extracellular peptides present in the culture). As shown in Figure 7, within 1 day following addition of 2m to these cultures, mature
(high TCR) CD8 cells appeared both within the thymus (rising from
0.9% to 5.5%) and among emigrants (rising from 3.1% to 9.0%). Thus,
emigration can occur within a day of positive selection.
Inhibitors of cell motility block emigration For mature thymocytes to emigrate from the thymus, an active motility process would seemingly be involved. This assumption was verified using several types of inhibitors of lymphocyte motility, which dramatically blocked the emigration process during a 6-hour exposure to the drugs. As shown in Figure 8, cytochalasin D blocked emigration of mature thymocytes by over 98%. Inhibition of G proteins via pertussis toxin blocked emigration by over 84%. Inhibition of the Rho family of small GTPases by C difficile toxin B blocked emigration by over 86%. These inhibitors blocked the emigration of high TCR
cells, rather than reducing the surface expression of TCR,
because the expression of TCR on intrathymic cells was not
reduced (not shown). We then compared histologic sections of these
treated lobes, which suggested a possible route of emigration. As shown
in Figure 9, comparison of normal versus
drug-inhibited lobes showed differences in the lymphocyte content of
radially oriented cords that could represent tracks of emigrating
mature thymocytes. In normal lobes, these tracks could represent a
string of outwardly migrating thymocytes, whereas in cytochalasin D- or
Clostridium toxin-treated lobes, fewer cells were observed within these cords. Pertussis toxin treatment on the other hand induced
apparent "congestion" within the cords. However, these are static
pictures of a dynamic process and other interpretations are possible.
The control sections also indicate the integrity of the capsule in
lobes maturing in these culture conditions, arguing against a passive
"leak" of cells and supporting our view that cells that emerge from
these organs do so by an active emigration process.
Function of emigrants in vitro The ability of thymic emigrants to proliferate in response to polyclonal activators was compared to that of mature peripheral T cells. As shown in Figure 10, the combination of PMA and ionophore elicited a response in emigrants that was similar in magnitude to that of splenic T cells. The combination of anti-CD3 and syngeneic dendritic cells also triggered a strong proliferative response in emigrants, although it was slightly lower than that of splenic T cells. The ability to recognize alloantigens was measured using the MLR. In a primary MLR (Table 2), emigrants mounted a vigorous proliferative response to allogeneic dendritic cells, although the magnitude was slightly lower than that of splenic T cells. In a secondary MLR (Table 3), proliferation of emigrants was virtually the same as splenic T cells. Thus, the functional capacities of emigrants were similar to those of mature peripheral T cells, at least regarding their proliferative response in vitro to dendritic cell stimulation. Hence, the CTLA-4 expression on emigrants (noted above) did not greatly impede proliferation of emigrants in vitro.
Function of emigrants in vivo To determine whether the thymic emigrants generated in this organ culture system could function in vivo, they were transferred into syngeneic or allogeneic SCID mice. As shown in Figure 11, repopulation of both lymph nodes and spleen was observed in either syngeneic or allogeneic hosts. Both CD4 and CD8 cells were found in syngeneic lymph node (panel 1) and spleen (panel 3). In allogeneic recipients, donor cells were observed in lymph node (panel 2) and spleen (Figure 12). Although CD4 cells repopulated both syngeneic and allogeneic recipients similarly, CD8 repopulation was much lower in allogeneic recipients (Figure 12).
Acute GVHD can be induced by very small numbers of mature peripheral T
cells when injected into a SCID mouse (that was previously sublethally
irradiated and treated with anti-asialoGm1). Thymic emigrants did not
elicit acute GVHD as shown in Figure
13. It was surprising, given the
vigorous proliferative response of thymic emigrants to alloantigens in
vitro (Tables 1 and 2), that they did not induce an acute GVHD even
when administered at 8 times the number of splenic T cells that would
elicit a lethal acute GVH syndrome. Skin and liver sections from 3 syngeneic and 3 allogeneic recipients were examined (blind) by a
veterinary pathologist who reported no indications of GVHD in
allogeneic recipients; in fact, syngeneic recipients showed more
inflammation and lymphocytic infiltration of both skin and liver. As
another possible indicator of T-cell activation in allogeneic hosts,
spleen cells were analyzed for expression of CD25 but it was not
detected. On the other hand, hematocrits did reflect a potential GVH
reaction; 2 of 3 allogeneic recipients (with 31, 34.5, 49.5) fell below
normal, whereas all 3 syngeneic recipients (with 43.5, 46, 48.5) were
normal and comparable to control mice similarly treated and injected
with syngeneic lymph node cells (with 40 and 47.5). This suggests that,
although in vivo allogeneic reactivity of emigrants is sufficiently
attenuated to protect from acute lethality, significant reactivity in
hematopoietic tissues may remain.
The properties of thymic emigrants were examined using a new method for collecting cells that emigrate from the fetal thymus during organ culture. Phenotypic analysis indicated that most of the cells that emigrate had a mature phenotype, in that they were single positive for CD4 or CD8 and expressed high levels of TCR. The profile of most surface markers coincided with that of mature splenic T cells, except that CTLA-4 was expressed at a higher level, similar to that on activated T cells. Emigration was induced within 24 hours after positive selection and was blocked by several different inhibitors of cell motility. Functional analysis in vitro indicated full proliferative responsiveness to alloantigens and polyclonal activators. Transferred in vivo, thymic emigrants repopulated lymphoid organs. Unlike mature peripheral T cells, thymic emigrants failed to induce acute GVHD in allogeneic SCID mice. It should be emphasized that this system may only relate to emigration from the fetal or newborn thymus, in that these emigrants correspond to postnatal day 8. A question of major theoretical interest regarding thymic emigrants is whether they are tolerized by peripheral self-antigens not expressed in the thymus.4,5,20 Thus, although clonal deletion of self-reactive cells would be effective for epitopes expressed in the thymus, a peripheral tolerance mechanism is presumably needed for epitopes not expressed in the thymus. One hypothesis that could account for peripheral tolerance would be that the thymic emigrant is rendered tolerant by antigen contact. It has previously been shown using the intrathymic injection of FITC to tag emigrants, that labeled cells found in the periphery are fully reactive against lectins and alloantigens in vitro.3 However, as noted above, it is unclear how many of the cells detected by this method are mature T cells that were passing through the thymus, rather than newly generated T cells. Hence, the antigen responsiveness previously detected could have been attributable to the presence of recirculating mature T cells, rather than true emigrants. Our results addressing functional reactivity of thymic emigrants clearly show that, on one hand, emigrants tested in vitro show vigorous proliferative responses against polyclonal activators and alloantigens in primary and secondary MLRs induced by dendritic cell stimulators. Our results, thus, confirm the conclusions reached using the FITC method3 that showed emigrants were equal to mature peripheral T cells in their ability to proliferate to lectins and lyse allogeneic target cells. This reactivity would argue against a simple peripheral tolerance model in which the emigrant is killed or anergized by antigen. On the other hand, emigrants did not induce acute GVHD in SCID mice in vivo, as do fully mature T cells from spleen or lymph node. There are a number of possible explanations for the difference in emigrant functions measured in vivo versus in vitro. (1) Emigrants may be prone to tolerance induction in vivo, perhaps based on their expression of CTLA-4, a primarily negative regulator of T-cell activation,18 or other mechanisms such as preferentially encountering nonprofessional antigen-presenting cells (APCs).21 (2) They may produce a spectrum of cytokines that is less injurious to the host. Thus, although not actually rendered tolerant, their impact would be less severe than mature T cells. Consistent with this explanation, using the FITC method, labeled cells produced more IL-4 and less IL-2 than mature peripheral cells.22,23 (3) Perhaps their migratory pattern does not take them to sites of major GVH injury, such as the intestine or liver. There was no sign of GVHD in liver or skin of allogeneic recipients, although several individuals showed depressed hematocrits that could represent GVHD in hematopoietic tissue. Thus, although our findings show that the emigrant does not acutely harm the allogeneic host, it remains to be shown whether this is actually based on a tolerance mechanism. Acute GVHD has been induced by both the HSA-positive and HSA-negative fractions of CD4+ thymocytes from adult thymus,24 which seemingly predicts that emigrants would also have the potential to induce acute GVHD. One explanation of the differences from our findings, showing no acute GVH induction by emigrants, is that the adult thymus contains substantial numbers of recirculating mature T cells, even small numbers of which might be capable of inducing acute GVHD. Another explanation is that the embryonic thymocytes used in our studies have different properties from their adult counterparts. However, the same study also observed that unfractionated newborn thymocytes induced acute GVHD. This would seem to indicate a difference in the GVHD model used in those studies compared to ours, because in unfractionated thymus from adults25,26 or embryos (data not shown), we have not observed induction of acute GVHD. That study induced acute GVHD by injecting C3H thymocytes into sublethally irradiated AKR mice (a barrier of minor histocompatibility and minor lymphocyte stimulating antigens [MLS]). Our studies used C57BL/6 thymic emigrants transferred to CB.17 SCID mice (a barrier of MHC, minor histocompatibility, and MLS antigens) that had been sublethally irradiated and treated with anti-asialoGM1. Fewer CD8 cells were detected in allogeneic recipients than syngeneic recipients, whereas the numbers of CD4 cells were similar in both syngeneic and allogeneic recipients. Perhaps this reflects a requirement for CD8 emigrants to obtain survival signals from the correct MHC in the periphery. On the other hand, expansion of cell numbers must occur after transfer of emigrants, perhaps through stimulation with environmental antigens, and CD8 emigrants may fail to recognize foreign antigens in the allogeneic host and thus fail to expand. The surface markers on emigrants generated using our method are
consistent with markers detected using the in vivo
methods,2,27 both methods concluding that mature cells
emigrate from the thymus. In our study, heterogeneity of thymic
emigrants was noted particularly for expression of L-selectin and HSA.
This suggests that subsets develop in the thymus and will have
different properties after they leave. For example, L-selectin promotes
exit of lymphocytes from the blood and had previously been found on all
spleen and lymph node cells labeled by the FITC method.27
Our study partly confirms this, in that more emigrants expressed
L-selectin than did their mature intrathymic counterparts;
nevertheless, 20% of the mature There were several differences in emigrants previously described
in vivo2 versus our in vitro method. The CD4/CD8 ratio was
high in vivo and low in vitro, and we detected quite a few (13.1%)
The mechanism of thymic emigration has not been elucidated and this culture system can be used to study this process. Three inhibitors that blocked the emigration process, cytochalasin D, pertussis toxin, and C difficile toxin B, suggest possible mechanisms of emigration as follows. Emigrants expressed high levels of the |
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Abstract
Introduction
