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,§
Departments of
*
Pathology,
Internal Medicine, and
Anatomy, Seoul National University College of Medicine, Seoul, Korea; and
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Institute of Allergy and Clinical Immunology, Seoul National University, Seoul, Korea
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß
complex is quantitatively regulated, and the differentiation of
immature T cells includes an increase in steady-state levels of nascent
TCR ß complexes (7). However, despite the vast amount of
information available on the positive selection process, the identities
of the molecules that modulate the positive selection and those that
mediate death by lack of recognition are unknown. CD99 (Mic2) is a cell surface glycoprotein with a molecular mass of 32 kDa, and its encoding gene has been localized to the pseudoautosomal regions of both human X and Y chromosomes (8). CD99 is highly expressed on human cortical thymocytes and is involved in homotypic aggregation of CD4+8+ thymocytes (9). We showed previously that CD99-dependent induction of cell adhesion in T cells, as well as in the neoplastic B cell line IM-9, is mediated through the LFA-1/ICAM-1 pathway (10). Although the CD99 expression pattern and the mechanism of its involvement in cell adhesion have been characterized in part, the biologic relevance of its high level expression on human cortical thymocytes is unclear. It has been shown that treatment of CD4+8+ thymocytes with mAb to CD99 (engagement) induces phosphatidylserine movement and exposure at the surface of the immature thymocytes (11), resulting in the apoptosis of these cells (12). The unique anatomical expression and the induction of cell adhesion and death by engagement of CD99 in immature cortical thymocytes led us to the assumption that CD99 might participate in the cell maturation process, during which cell death and selection occur.
In this paper, we investigate the functional role of CD99 in the process of T cell development. We show that treatment of immature human thymocytes with the anti-CD99 mAb DN16 induces rapid cell aggregation as well as up-regulation of the surface expression of TCR and MHC class I and II molecules via accelerated intracellular transport. On the basis of our findings, we suggest that CD99 is a cell surface modulator protein that might influence positive selection by enhancing the efficiency of TCR-MHC interaction.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Anti-human CD99 mAb (DN16) was obtained from hybridoma clones developed in this laboratory. The anti-CD99 mAb 12E7, which recognizes an epitope distinct from that of DN16, was a generous gift from P. N. Goodfellow (Smithkline Beecham, Essex, U.K.). The hybridoma clone that produces mAb A1G3 (reactive with human medullary thymocytes) was obtained from American Type Culture Collection (ATCC, Manassas, VA). Anti-TCR-FITC, anti-CD4-FITC, anti-CD8-PE, and an unconjugated form of anti-TCR Abs were purchased from Becton Dickinson (San Jose, CA). FITC-conjugated anti-MHC class I and II Abs and the unconjugated forms of anti-MHC class I and II Abs were all purchased from Biosource (Camarillo, CA).
Cell purification
Human thymic tissues were obtained from donors undergoing cardiac surgery. Prior permission was granted from the parents of patients who required partial thymectomy for clear exposure of the heart. After incubation with A1G3 on ice for 30 min, TCRlow DP thymocytes were purified by negative panning on plates coated with goat anti-mouse Ig Ab.
Suspension culture
Thymocytes (2 x 106 cells/ml) were cultured in 24-well tissue culture plates that contained RPMI 1640 medium supplemented with 10% FCS in the presence of DN16 (10 µg/ml) or 12E7 (10 µg/ml) and secondary Ab raised against mouse Ig (50 µg/ml).
Northern blot analysis
Total RNA was prepared from Ab-engaged thymocytes with use of the guanidine thiocyanate-cesium chloride method (13). RNAs were separated by electrophoresis through a 1.3% agarose gel containing 2.2 M formaldehyde and then transferred to nylon membrane filters. The filters were hybridized at 42°C overnight with the appropriate DNA probes. The final wash of the filters was at 55 to 65°C in 0.2 x SSC. Cß region of TCR-ß, HLA-B7, DRß, and ß-actin genes were used as probes for detection of TCR, MHC class I, MHC class II, and actin transcripts, respectively.
Fluorescent staining of cell surface and intracellular molecules and flow cytometric analysis
After engagement with Abs (see above under Suspension culture), thymocytes were washed three times with 1% FCS-1 x PBS, incubated on ice for 30 min with normal mouse serum to block the nonspecific Ab binding of cells, stained directly with FITC-conjugated mAbs, and analyzed on FACScan (Becton Dickinson). To gate the viable cells, thymocytes stained for surface expression of TCR were washed, suspended in 400 µl of 1% FCS-1 x PBS containing 10 µg/ml of propidium iodide (PI; Sigma, St. Louis, MO), and analyzed on flow cytometer. For cytoplasmic staining of the TCR and MHC class I and II molecules, cells were first incubated with unconjugated TCR mAb, MHC class I mAb, or MHC class II mAb. The cells were then fixed with 1% paraformaldehyde in PBS at room temperature and permeabilized with 1% saponin (Sigma), which was included in all subsequent staining and washing steps. The fixed cells were washed, stained with FITC-conjugated mAbs, and were analyzed on a FACScan.
Confocal analysis
For immunofluorescence labeling, incubated cells were fixed in
paraformaldehyde, permeabilized with 1% saponin, and incubated with
FITC-conjugated Abs. Confocal analyses were performed with a 600 MRC
equipped with an Argon/Krypton laser (Bio-Rad Labs, Hercules, CA).
Green fluorescence was detected at 
> 515 nm after excitation at
488 nm.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We examined the effect of CD99 engagement on the surface
expression of TCR, MHC class I, CD4, and CD8 molecules, since these are
considered to be the major molecules involved in thymic positive
selection. The expression patterns of TCR and MHC class I molecules on
untreated thymocytes vary greatly from low to high. When thymocytes
were incubated with anti-CD99 Abs (DN16 or 12E7), the surface
expression of TCR and MHC class I molecules increased dramatically, as
compared with an anti-CD1a Ab-treated control group. Expression of
CD4 and CD8 remained unchanged under both conditions (Fig. 1
A). The up-regulation
of TCR and MHC class I surface expression, when thymocytes were engaged
with anti-CD99, occurred in a dose-dependent manner. A linear
relation was evident between the degree of surface expression and the
amount of Ab used within the 100 ng/ml to 10 µg/ml range. The maximal
effect occurred at an anti-CD99 Ab concentration of 10 µg/ml
(Fig. 2).
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B) in terms of TCR
expression and cell viability. There was no identifiable difference in
the percentage of dead cell population (R1) between DN16 mAb-treated
and control Ab-treated cells. Gating on the PI-negative viable
population (R2) clearly demonstrated that the TCR up-regulation was due
to the specific effect of CD99 engagement rather than the
apoptosis-related up-regulation of surface molecules. However, 1.3% of
immature thymocytes remained unchanged in their TCR expression even
after CD99 engagement (R3). Cellular mechanism for the up-regulation of TCR and MHC class I molecules
The increased surface expression of TCR and MHC class I molecules
was observed as early as 10 min after the CD99 engagement. To identify
the cellular mechanism involved in the up-regulation of TCR and MHC
class I surface expression, we tested whether de novo mRNA synthesis or
subcellular distribution of already synthesized proteins was altered
upon engagement of CD99. We found no evidence of quantitative changes
in the mRNA synthesis of TCR or MHC class I mRNA after CD99 engagement,
as compared with control Ab-treated samples (Fig. 3A). We next performed
flow cytometric analysis to compare the kinetics of cytosolic and
cell-surface expression of TCR and MHC class I protein complexes. As
shown in Figure 3B, unengaged thymocytes expressed high
levels of intracellular TCR molecules but were relatively low in their
level of surface expression. With CD99 engagement, the surface
expression of TCR molecules increased markedly as the cytoplasmic
concentration of TCR declined, suggesting that accelerated cellular
transport might be a major mechanism for the up-regulation of TCR
molecules on the cell surfaces. This result indicates that, although
30% of unengaged immature thymocytes show no expression of TCR at
their cell surface (in Fig. 1B), a vast majority of
them have a considerable amount of TCR molecules in their cytosolic
compartment. In fact, only a minimal fraction of immature thymocytes
(R3 in Fig. 1B, 1.3%) constitutes cell population lacking
internal TCR molecules, and this is a population in which
TCR-rearrangement was not completed yet.
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A). The fluorescence
intensity of TCR molecules at the plasma membrane increased with a
concomitant decrease in intracellular fluorescence in response to
anti-CD99 Ab treatment (Fig. 4B). The change in
subcellular localization of MHC class I molecules when cells were
engaged with anti-CD99 Ab was almost identical to that of TCR
molecules, except that a more condensed localization of MHC class I
molecules occurred at cell-cell contact sites (Fig. 4, C and
D).
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We reported previously that MHC class II molecules are present on
a significant fraction (10–30%) of fetal thymocytes throughout their
developmental progression to mature T cells (15) and that there is
actual T-T interaction during thymocyte development (3). Therefore, we
examined the CD99-mediated up-regulation of MHC class II molecules on
the surface of cultured thymocytes. MHC class II expression on
unengaged thymocytes is on the average less than 30% but varies
depending on the thymus sample. We found that CD99 engagement also
induced the up-regulation of MHC class II molecules on thymocyte
surfaces (Fig. 5A,
left). However, the results of this experiment are
partly in conflict with the data obtained for TCR and MHC class I
molecules, in that the MHC class II-negative population, though small,
still existed even after cells were incubated with maximal dose of
anti-CD99 Ab.
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A, dotted lines). As was the case with both
TCR and MHC class I molecules, increased MHC class II surface
expression after CD99 engagement was also associated with the
concomitant reduction of intracellular staining (Fig. 5A,
solid lines). Northern analysis showed no change in MHC class II mRNA
expression upon engagement of thymocytes with anti-CD99 Ab (Fig. 5B). After CD99 engagement, accumulation of MHC class
II molecules became evident on the cell surface and was concentrated at
cell-cell contact regions as determined by confocal microscopy (Fig. 6, A and B).
This analysis constitutes a direct visual demonstration that, upon CD99
engagement, MHC class II molecules migrate from an internal reservoir
to the cell surface region where cell-cell contacts occur.
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Immature DP thymocytes are phenotypically heterogeneous with
respect to the expression level of TCR (16, 17, 18) and can be subdivided
into TCRlow and TCRhigh cell populations.
Because the responses of TCRlow and TCRhigh
cells to various stimuli can differ (19, 20, 21), we sought to determine
what subpopulation of thymocytes showed up-regulated expression of TCR
and MHC class I and II after binding of anti-CD99 mAb. For separate
enrichment of the TCRlow and TCRhigh cells, DP
thymocytes were applied to a panning plate after binding with A1G3 Ab,
which was reported to recognize medullary thymocytes and to be
associated with functional maturity of the thymocyte (22). Our results
revealed that the A1G3 recognition Ag was expressed in most
TCRhigh DP cells as well as in a mature medullary
population of thymocytes (Fig. 7, dotted
lines). Therefore, we conclude that A1G3 mAb is a powerful experimental
tool that can distinguish between the two subpopulations of DP
thymocytes.
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, solid lines). MHC class II molecules were expressed
normally only on the surface of A1G3- TCRlow
thymocytes (Fig. 7, dotted lines), and this is also the major cell
population in which CD99-mediated up-regulation of MHC class II
molecules occurs (Fig. 7, solid lines). The TCRhigh cells
were completely devoid of MHC class II molecules on their surfaces, and
the CD99-dependent changes in the localization of MHC class II
molecules were negligible. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß
) and fully assembled (ß
) TCR
complexes exit the ER and transit through the Golgi system. The export
of the TCR-CD3 complex from ER to Golgi is slow, requiring at least
3 h for the processing of half the complexes from the endonuclease
H-sensitive to the endonuclease H-resistant form (reviewed in 23 .
Therefore, considering the time required for transport from the
cytosolic compartment to the cell surface, it is not likely that
up-regulated surface expression results from de novo synthesis and
assembly of TCR molecules. A similar hypothesis may also apply to the CD99-dependent surface expression of MHC class I and II molecules. MHC class I molecules assemble and achieve their native conformation within 2 to 15 min of synthesis (24). Thereafter, the half-time taken for MHC class I proteins to egress from the ER varies between 20 and 55 min, depending on the haplotype of the molecule (25). Newly synthesized MHC class II molecules accumulate in the MIIC compartment after 4 h of synthesis (26), and MHC class II-containing vesicles concentrated in the MIIC move toward the plasma membrane within the 5-min period (27).
Experiments performed in this laboratory support the idea that the molecular signals generated by CD99 engagement induce actin polymerization, which leads to the mobilization of cell surface molecules. First, TCR up-regulation mediated by CD99 engagement was inhibited by treating thymocytes with inhibitors of actin polymerization (cytochalasin B and E; unpublished data). Second, the treatment of C3 exoenzyme, which inhibits Rho-mediated actin polymerization, completely abolished CD99-induced cell aggregation (unpublished data). The hypothesis that CD99 engagement induces actin polymerization is supported further by independent reports. For example, the transport of MHC class II molecules from lysosomal structures to the plasma membrane involves microtubules, as nocodazole and colcemid (microtubule blockers) inhibit the movement of MHC class II molecule-containing vesicles (27). In addition, the close interrelationship of actin- and microtubule-based systems has been demonstrated in organelle movement (28) and fusion of endosomal carrier vesicles (29). Finally, linker proteins (for example, p150Glued) have been shown to reside between microtubules and actin filament (30, 31). Taken together, these data suggest that Ab engagement of CD99 can lead to either activation of cytoskeletal components or activation of linker proteins between microtubule- and actin-based cytoskeletal systems, thereby inducing accelerated mobilization of specific Ags to the cell surface. Our data generated from flow cytometric analysis and confocal microscopic examination lend firm support to the idea that up-regulated expression via CD99 engagement results from accelerated intracytoplasmic transport rather than de novo synthesis of these molecules. The unique distribution of TCR and MHC molecules, which are densely concentrated at cell-cell contact sites after CD99 engagement, is able to increase the number of proper contacts formed between these molecules.
The amount of class II in cells of the immune system is not static but can be up- or down-regulated often via intracellular transport activity (32, 33, 34) in response to a large number of external stimuli, including cytokines, cross-linking of surface Ags (for example, IgM, IgD, Lyb2, and B220), and mitogens (reviewed in 35 . Human peripheral T cells in a resting state can transcribe mRNA for class II molecules but normally do not express this Ag at their surfaces in a detectable amount until after activation (36). Results from the present study imply that the same is true for the TCRlow stage of developing thymocytes, because surface expression of MHC class II molecules that are confined to the cytoplasmic pool must await the delivery of molecular signals generated by the engagement of CD99.
Instances of CD99-induced up-regulation of TCR and MHC class I and II molecules have certain characteristics in common. One is that induced surface expression of these Ags appears to be differentially regulated depending on the developmental stage of thymocytes: higher surface expression was observed on immature thymocytes (A1G3- cells) than on more mature ones (A1G3+ cells). The biologic relevance of CD99-induced up-regulation of TCR and MHC molecules at the cell surface can be discussed along with current representative hypothetical views on positive selection. In view of the differential avidity model for T cell selection, the increased surface levels of TCR and MHC class I and II after CD99 engagement might provide a basis for the increased avidity of TCR-MHC interaction on developing thymocytes (37). Therefore, immature thymocyte whose TCRs i) are expressed at low density, ii) encounter peptide-MHC complexes for which they have low affinity, or iii) encounter cells with peptide-MHC expressed at low density can be rescued from death by the up-regulation of these molecules through CD99 signaling. Lanzavecchia et al. have suggested a serial triggering model where the key parameter is the absolute numbers of TCR engaged (38, 39). Therefore, unlike cells that express low amounts of TCR, cells expressing higher numbers of TCRs rapidly reach the number of engaged TCRs required for selection. In this regard, a larger percentage of cells may be rescued from the lack of recognition when CD99 signaling is operative.
We reported previously that MHC class II molecules are present on a significant fraction of fetal thymocytes, particularly during the last trimester of the gestational period (15) and that there is an actual presence of T-T interaction during thymocyte development (3). Given that CD99 engagement increases the expression density of TCR and MHC class I and II molecules on the surface of thymocytes, it is not surprising that even more opportunities exist for substantial T-T interaction once the signal through CD99 is delivered. Using morphologic visualization, we observed that TCR and MHC class I and II molecules, after CD99 engagement, were concentrated at the plasma membrane near cell-cell contacting sites, which suggests the possibility of increased TCR-MHC interaction during T-T interaction. Therefore, the increased T-T interaction may also have additive effects on the selecting event, as compared with the situation where only TECs are present as the selecting cell. The observation and hypotheses outlined above emphasize the role of CD99 engagement in the generation of the increased opportunities for positive selection. Therefore, CD99 can be defined as a biologic enhancer during the thymic education process, increasing the number of thymocytes that are positively selected.
A role for CD99 in apoptosis has been reported recently (12). When combined with our observations, one may postulate that CD99 has a dual contradictory function: one is to create more opportunities for positive selection by increasing TCR-MHC avidity and the other is to induce apoptosis of immature thymocytes. We do not know at present why ligation of the same molecule, CD99, can result in such dramatically different responses. We propose that, in the course of thymocyte development, CD99 engagement alone may not be sufficient to induce differentiation of DP thymocytes, which requires coengagement of TCR and other coinducer molecules provided by thymocytes and TECs. Therefore, the decision regarding the fate of the thymocyte can be made only at the CD99-induced TCRhigh immature stage. Thymocytes positively selected after proper TCR-MHC interaction might lead to the increased synthesis of transcripts and proteins for the TCR molecule and then differentiate to single positive (SP) cells (40). Otherwise, the thymocyte should be removed by apoptosis.
In summary, we suggest that molecular signaling through CD99 engagement is a basic requirement for the survival, maintenance, and maturation of thymocytes and is involved in the regulation of its contradictory dual functions.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Seong Hoe Park, Department of Pathology, Seoul National University College of Medicine, 28 Yongon-dong Chongno-gu, Seoul 110–799, Korea. E-mail address:
3 Abbreviations used in this paper: TEC, thymic epithelial cell; PI, propidium iodide; ER, endoplasmic reticulum; DP, double positive.
Received for publication January 15, 1998. Accepted for publication March 24, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß T-cell antigen receptor expression results from differential stability of nascent TCR proteins within the endoplasmic reticulum of immature and mature T cells. EMBO J. 13:4504.[Medline]
ß+ T cells: one population is deleted by ligation of ß TCR. Cell 58:1047.[Medline]
protein is an early response to TCR signaling that compensates for TCR- instability, improves TCR assembly, and parallels other indicators of positive selection. J. Exp. Med. 181:193.This article has been cited by other articles:
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  H.-J. Byun, I.-K. Hong, E. Kim, Y.-J. Jin, D.-I. Jeoung, J.-H. Hahn, Y.-M. Kim, S. H. Park, and H. Lee A Splice Variant of CD99 Increases Motility and MMP-9 Expression of Human Breast Cancer Cells through the AKT-, ERK-, and JNK-dependent AP-1 Activation Signaling Pathways J. Biol. Chem., November 17, 2006; 281(46): 34833 - 34847. [Abstract] [Full Text] [PDF]
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 A.-M. Imbert, G. Belaaloui, F. Bardin, C. Tonnelle, M. Lopez, and C. Chabannon CD99 expressed on human mobilized peripheral blood CD34+ cells is involved in transendothelial migration Blood, October 15, 2006; 108(8): 2578 - 2586. [Abstract] [Full Text] [PDF]
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 P. L. W. Yun, A. A. DeCarlo, and N. Hunter Gingipains of Porphyromonas gingivalis Modulate Leukocyte Adhesion Molecule Expression Induced in Human Endothelial Cells by Ligation of CD99 Infect. Immun., March 1, 2006; 74(3): 1661 - 1672. [Abstract] [Full Text] [PDF]
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 Y.-L. Choi, Y. H. Xuan, Y. K. Shin, S. W. Chae, M. C. Kook, R. H. Sung, S. J. Youn, J. W. Choi, and S. H. Kim An Immunohistochemical Study of the Expression of Adhesion Molecules in Gallbladder Lesions J. Histochem. Cytochem., May 1, 2004; 52(5): 591 - 602. [Abstract] [Full Text] [PDF]
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 J. A. Strauchen and L. K. Miller Lymphoid Progenitor Cells in Human Tonsils International Journal of Surgical Pathology, January 1, 2003; 11(1): 21 - 24. [Abstract] [PDF]
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I.-s. Lee, M. K. Kim, E. Y. Choi, A. Mehl, K. C. Jung, M. C. Gil, M. Rowe, and S. H. Park CD99 expression is positively regulated by Sp1 and is negatively regulated by Epstein-Barr virus latent membrane protein 1 through nuclear factor-{kappa}B Blood, June 1, 2001; 97(11): 3596 - 3604. [Abstract] [Full Text] [PDF]
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 H. W. Sohn, Y. K. Shin, I.-S. Lee, Y. M. Bae, Y. H. Suh, M. K. Kim, T. J. Kim, K. C. Jung, W. S. Park, C.-S. Park, et al. CD99 Regulates the Transport of MHC Class I Molecules from the Golgi Complex to the Cell Surface J. Immunol., January 15, 2001; 166(2): 787 - 794. [Abstract] [Full Text] [PDF]
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 K. Scotlandi, N. Baldini, V. Cerisano, M. C. Manara, S. Benini, M. Serra, P.-L. Lollini, P. Nanni, G. Nicoletti, G. Bernard, et al. CD99 Engagement: An Effective Therapeutic Strategy for Ewing Tumors Cancer Res., September 1, 2000; 60(18): 5134 - 5142. [Abstract] [Full Text]
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 H. W. Sohn, E. Y. Choi, S. H. Kim, I.-s. Lee, D. H. Chung, U. A Sung, D. H. Hwang, S. S. Cho, B. H. Jun, J. J. Jang, et al. Engagement of CD99 Induces Apoptosis Through a Calcineurin-Independent Pathway in Ewing's Sarcoma Cells Am. J. Pathol., December 1, 1998; 153(6): 1937 - 1945. [Abstract] [Full Text] [PDF]
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S. H. Kim, E. Y. Choi, Y. K. Shin, T. J. Kim, D. H. Chung, S. I. Chang, N. K. Kim, and S. H. Park Generation of Cells With Hodgkin's and Reed-Sternberg Phenotype Through Downregulation of CD99 (Mic2) Blood, December 1, 1998; 92(11): 4287 - 4295. [Abstract] [Full Text] [PDF]
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