CD99 Engagement Research Articles

Anti-CD99 Antibody Therapy Triggers Macrophage-Dependent Ewing Cell Death In Vitro and Myeloid Cell Recruitment In Vivo.

Ewing sarcoma is a rare tumor of the bone or soft tissues characterized by diffuse membranous staining for CD99. As this tumor remains incurable in the metastatic, relapsed, and refractory settings, we explored the downstream immune implications of targeting CD99. We discovered a human anti-CD99 antibody (NOA2) by phagemid panning and investigated NOA2 immune cell-mediated cytotoxicity in vitro and in vivo focusing on the myeloid cell compartment, given that M2 macrophages are present in human tumors and associated with a poor prognosis. NOA2 is capable of inducing immune effector cell-mediated Ewing death in vitro via engagement of macrophages. Mice with metastatic Ewing tumors, treated with NOA2, experience tumor growth arrest and an associated increase in intratumoral macrophages. Further, incubation of macrophages and Ewing cells with NOA2, in conjunction with anti-PILRα antibody blockade in vitro, results in the reactivation of previously dormant macrophages possibly due to interrupted binding of Ewing CD99 to macrophage PILRα. These studies are the first to demonstrate the role of human immune effector cells in anti-CD99-mediated Ewing tumor death. We propose that the engagement of CD99 by NOA2 results in the recruitment of intratumoral macrophages. In addition, interruption of the CD99:PILRα checkpoint axis may be a relevant therapeutic approach to activate tumor-associated macrophages.

Engagement of CD99 Activates Distinct Programs in Ewing Sarcoma and Macrophages.

Ewing sarcoma (EWS) is the second most common pediatric bone tumor. The EWS tumor microenvironment is largely recognized as immune-cold, with macrophages being the most abundant immune cells and their presence associated with worse patient prognosis. Expression of CD99 is a hallmark of EWS cells, and its targeting induces inhibition of EWS tumor growth through a poorly understood mechanism. In this study, we analyzed CD99 expression and functions on macrophages and investigated whether the concomitant targeting of CD99 on both tumor and macrophages could explain the inhibitory effect of this approach against EWS. Targeting CD99 on EWS cells downregulated expression of the "don't eat-me" CD47 molecule but increased levels of the "eat-me" phosphatidyl serine and calreticulin molecules on the outer leaflet of the tumor cell membrane, triggering phagocytosis and digestion of EWS cells by macrophages. In addition, CD99 ligation induced reprogramming of undifferentiated M0 macrophages and M2-like macrophages toward the inflammatory M1-like phenotype. These events resulted in the inhibition of EWS tumor growth. Thus, this study reveals what we believe to be a previously unrecognized function of CD99, which engenders a virtuous circle that delivers intrinsic cell death signals to EWS cells, favors tumor cell phagocytosis by macrophages, and promotes the expression of various molecules and cytokines, which are pro-inflammatory and usually associated with tumor regression. This raises the possibility that CD99 may be involved in boosting the antitumor activity of macrophages.

CD99 regulates CXCL12-induced chemotaxis of human plasma cells

Migration of plasma cells (PCs) is crucial for the control of PC survival and antibody production and is controlled by chemokines, most importantly by CXCL12. This study investigated the role of CD99 in CXCL12-induced PC migration. Among B cell subsets in the tonsils, CD99 expression was highest in PCs. CD99 expression increased during in vitro differentiation of germinal center B cells and was highest in PCs. CD99 engagement reduced chemotactic migration of PCs toward CXCL12 and reduced extracellular signal-regulated kinase (ERK) activation by CXCL12. An ERK inhibitor reduced CXCL12-mediated chemotactic migration, which suggests that ERK has a critical role in migration. CD99 engagement did not influence apoptosis, differentiation, or antibody secretion of PCs. We propose a novel role of CD99 in PCs that suppresses ERK activation and chemotactic migration of these cells.

Engagement of CD99 Reduces AP-1 Activity by Inducing BATF in the Human Multiple Myeloma Cell Line RPMI8226.

CD99 signaling is crucial to a diverse range of biological functions including survival and proliferation. CD99 engagement is reported to augment activator protein-1 (AP-1) activity through mitogen-activated protein (MAP) kinase pathways in a T-lymphoblastic lymphoma cell line Jurkat and in breast cancer cell lines. In this study, we report that CD99 differentially regulated AP-1 activity in the human myeloma cell line RPMI8226. CD99 was highly expressed and the CD99 engagement led to activation of the MAP kinases, but suppressed AP-1 activity by inducing the expression of basic leucine zipper transcription factor, ATF-like (BATF), a negative regulator of AP-1 in RPMI8226 cells. By contrast, engagement of CD99 enhanced AP-1 activity and did not change the BATF expression in Jurkat cells. CD99 engagement reduced the proliferation of RPMI8226 cells and expression of cyclin 1 and 3. Overall, these results suggest novel CD99 functions in RPMI8226 cells.

CD99 signals through soluble adenylyl cyclase to regulate leukocyte transendothelial migration (146.6)

Transendothelial migration (TEM) is the process by which leukocytes extravasate out of the vasculature into the interstitium and is one the key steps of the inflammatory response. PECAM and CD99 are two critical proteins at the endothelial cell border that function sequentially to regulate TEM (PECAM upstream of CD99). These proteins partially reside in a novel, intracellular endothelial cell compartment, known as the Lateral Border Recycling Compartment (LBRC), which supplies the extravasating leukocyte with unligated proteins and membrane surface area to facilitate its movement across the endothelium, in a process called targeted recycling (TR). While much is known regarding the function of PECAM during TEM, very little is understood regarding how CD99 signals. The C‐terminal tail of CD99 is short (39AA) and contains no known signaling motifs. Blocking homophilic interaction of leukocyte CD99 with endothelial cell CD99 arrests leukocytes part way through the junctions; cross‐linking endothelial CD99 overcomes this block. Here we show that engagement of leukocyte CD99 with endothelial cell CD99 (or crosslinking CD99 on endothelial cells to mimic this) activates PKA via a soluble form of adenylyl cyclase, ADCY10 (sAC). Blocking either PKA or sAC, but not transmembrane adenylyl cyclase (tmAC), recapitulates the phenotype of CD99 arrest on TEM and also prevents crosslinking‐mediated restoration of CD99 blockade. Activation of PKA restores TR of LBRC membrane to the arrested leukocytes to overcome the CD99 blockade. Current research is focused on identifying the mediators downstream of PKA as well as the signaling events taking place between the cytoplasmic domain of CD99 and sAC.This research was supported by F30 HL116100‐01A1 to RLW and R21 HL102519, R01 HL046849, and R37 HL064774 to WAM.Grant Funding Source: NIH NRSA F30 HL116100‐01A1

CD99 ligation induces intercellular cell adhesion molecule-1 expression and secretion in human gingival fibroblasts

ObjectiveTo examine CD99 expression and its functional role in ICAM-1 induction in human gingival fibroblasts (HGFs) and human gingival epithelial cells (HGECs) by activating cells with anti-CD99 monoclonal antibody, MT99/3. BackgroundEngagement of CD99 with agonistic antibodies has been shown to regulate immune responses, cell adhesion and migration, and cell death in several studies. Particularly, this engagement results in transendothelial migration of leukocytes mediated by intercellular adhesion molecule-1 (ICAM-1) induction in endothelial cells. MethodsTotal mRNA and protein were isolated from HGFs and HGECs for analyses of CD99 and ICAM-1 expression. Surface expression of CD99 and ICAM-1 was analysed by flow cytometry, and the detection of soluble ICAM-1 was assayed by immunoprecipitation and ELISA. ResultsCD99 surface expression was constitutive on HGFs to a greater extent than that on HGECs. CD99 ligation with MT99/3 induced ICAM-1 mRNA expression in HGFs, but not in HGECs. Interestingly, CD99 ligation led to an increased level of soluble ICAM-1 detected in culture supernatant, whereas interleukin-1β (IL-1β) treatment induced expression of membrane-bound ICAM-1. Furthermore, ICAM-1 induction by CD99 engagement was demonstrated to involve the activation of the p50 subunit of nuclear factor-kappaB (NF-κB), extracellular signal-regulated kinase, and p46 c-Jun N-terminal kinase that differed from that by IL-1β treatment. ConclusionOur study has shown the involvement of CD99 ligation in the up-regulation of ICAM-1 expression and its secretion in gingival fibroblasts, which may be essential for better understanding of the pathogenesis of periodontal disease.

The activation of CD99 inhibits cell-extracellular matrix adhesion by suppressing β1integrin affinity

CD99 is known to be involved in the regulation of cell-cell adhesion. However, it remains unclear whether CD99 controls cell-extracellular matrix adhesion. In this study, the effects of CD99 activation on cell-extracellular matrix adhesion were investigated. It was found that engagement of CD99 with the stimulating antibody YG32 downregulated the adhesion of MCF-7 cells to fibronectin, laminin and collagen IV in a dose-dependent manner. The CD99 effect on cell-ECM adhesion was inhibited by overexpression of the dominant negative form of CD99 or CD99 siRNA transfection. Treatment of cells with Mn(2+) or by β(1) integrin-stimulating antibody restored the inhibitory effect of CD99 on cell-ECM adhesion. Cross-linking CD99 inactivated β(1) integrin through conformational change. CD99 activation caused dephosphorylation at Tyr-397 in FAK, which was restored by the β(1) stimulating antibody. Taken together, these results provide the first evidence that CD99 inhibits cell-extracellular matrix adhesion by suppressing β(1) integrin affinity. [BMB reports 2012; 45(3): 159-164].

Association of CD99 short and long forms with MHC class I, MHC class II and tetraspanin CD81 and recruitment into immunological synapses

BackgroundCD99, a leukocyte surface glycoprotein, is broadly expressed in many cell types. On the cell surface, CD99 is expressed as two distinct isoforms, a long form and a short form. CD99 has been demonstrated to play a key role in several biological processes, including the regulation of T cell activation. However, the molecular mechanisms by which CD99 participates in such processes are unclear. As CD99 contains a short cytoplasmic tail, it is unlikely that CD99 itself takes part in its multi-functions. Association of CD99 with other membrane proteins has been suggested to be necessary for exerting its functions.ResultsIn this study, we analyzed the association of CD99 with other cell surface molecules involved in T cell activation. We demonstrate the association of MHC class I, MHC class II and tetraspanin CD81 with CD99 molecules on the cell surface. Association of CD99 with its partners was observed for both isoforms. In addition, we determined that CD99 is a lipid raft-associated membrane protein and is recruited into the immunologic synapse during T cell activation. The implication of CD99 on T cell activation was investigated. Inhibition of anti-CD3 induced T cell proliferation by an anti-CD99 monoclonal antibody was observed.ConclusionsWe provide evidence that CD99 directly interact and form the complex with the MHC class I and II, and tetraspanin CD81, and is functionally linked to the formation of the immunologic synapse. Upon T cell activation, CD99 engagement can inhibit T cell proliferation. We speculate that the CD99-MHC-CD81 complex is a tetraspanin web that plays an important role in T cell activation.

Abstract 671: Triggering of CD99 induces apoptosis of Ewing's sarcoma cells through activation of p53 functions

Abstract CD99 is a transmembrane 32 KDa protein, whose expression is constantly associated to Ewing's Sarcomas Family of Tumors (EWSFT), a class of child bone tumors with particular poor prognosis. The monoclonal antibody 0662, raised against CD99, triggers massive and rapid cell death of Ewing Sarcoma's cells in vitro and in vivo, through a non-canonical, non caspase-dependent apoptosis. To elucidate the underlying molecular mechanism, we evaluated by phosphor- and micro-arrays specific pathways triggered by CD99 engagement in 6647 Ewing Sarcoma cell line. Despite its role in physiological and cancer scenery has not been yet fully elucidated, CD99 appears to be sufficient to modulate many important processes involved in: I) adhesion; II) migration and metastasis; III) cell-cycle regulation and cell signaling; IV) apoptosis and death receptor signaling. These data are consistent with our previous analysis of 0662 effects in vivo and in vitro: anti-CD99 monoclonal antibody triggers, indeed, homotypic aggregation and a rapid death response of ES cells, thus resulting in growth inhibition and reduced metastatic/colony-forming potential. As pointed out by array studies and subsequent western blotting validations, upon 0662 treatment, the oncosuppressor protein p53 appears to be readily activated by phosphorilation on serine 15. We therefore evaluated p53 and MDM2 activation and sub-cellular localization upon CD99 engagement both in p53 WT (LAP35 and WE68) or mutated (6647 and TC-71) cell lines: WT (LAP35 and WE68) or mis-sense point mutated p53 (6647: S241F) cell lines display a higher sensitivity to 0662 if compared to p53 truncated cell lines (TC71). Consistently we demonstrated that whereas 0662 readily induces p53 canonical targets (p21, BAX) in p53 expressing cell lines, no increase in p21 levels is detectable in TC71 p53TRUNC cells. We also demonstrated that, upon 0662 treatment, decreased or delocalized MDM2 levels may account both for p53 activation and sustained IGF-IR signaling, since both p53 and IGF1-Rß are targeted by the ubiquitine ligase for degradation. Since most Ewing Sarcoma patients display unmodified p53 status, its functional inactivation -e.g. through MDM2/MDM4 overexpression- may account for gained tumor aggressiveness, or progressed disease. Nonetheless, involvement of TP53 might give reason for higher effectiveness we reported against local tumors and metastases in the combined employment of CD99 monoclonal antibodies with Doxorubicin and may support 0662 mAb treatment in association with other drugs targeting e.g. the p53 regulators MDM2-MDM4. (grants from EU project Eurobonet and Italian Association for Cancer Research) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 671.

CD99 signal regulates the proliferation of multiple myeloma cell lines, RPMI8226. (131.10)

Abstract CD99 is a 32-kDa cell surface protein that is variously expressed on hematopoietic cells and hematopoietic-derived cancer cells. CD99 is highly expressed in some multiple myeloma cells including RPMI8226. In this study, we show that engagement of CD99 by agonistic anti-CD99 monoclonal antibody (mAb), YG32, inhibited proliferation of RPMI8226 multiple myeloma cells. However, YG32 mAb did not induce the cell death of RPMI 8226 cells. We carried out the microarray analysis to investigate the expression of genes involved in growth retardation by CD99 engagement. In microarray data, RPMI8226 cells engaged by anti-CD99 mAb had reduced expression of cyclin D1 and D2. Expression of CDKN2A gene, which codes cell cycle inhibitors p16INK4A and p19ARF, were induced by anti-CD99 mAb. These results suggest that CD99 signal regulates the proliferation of multiple myeloma cells by modulating proliferative and anti-prolifearative genes. The study was supported by National Research Foundation (NRF) grant of the Korean government A (R13-2008-023-01003)

Identification of cyclophilin A as a CD99-binding protein by yeast two-hybrid screening

CD99 is a 32 kDa surface glycoprotein, which is involved in the migration of leukocytes and the transport of ganglioside GM1 and transmembrane proteins. To identify signaling mechanisms triggered by CD99 engagement, a LexA-based yeast two-hybrid system was utilized to identify proteins interacting with the cytoplasmic domains of CD99. In seven positive clones, we attempted to ascertain whether cyclophilin A (CypA) was involved in CD99-mediated signaling, since CypA had been implicated as a signaling regulator for kinases and phosphatases. The interaction between CD99 and CypA was confirmed by co-immunoprecipitation and confocal immunofluorescence studies. Interestingly, the amounts of CypA associated with CD99 increased upon CD99 engagement. We prepared an expression plasmid by inserting CypA cDNA into pEGFP, in order to visualize cellular CypA. In HeLa or HEK 293T cells transfected with the pEGFP-CypA plasmid, GFP-tagged CypA was diffusely present in the cytoplasm of untreated cells. However, CypA-GFP moved to the cell periphery and membrane blebbing, and became colocalized with CD99 upon CD99 engagement. These results suggest that CypA may be either a signaling mediator or a signaling regulator for CD99.

Molecular mechanisms of CD99-induced caspase-independent cell death and cell-cell adhesion in Ewing's sarcoma cells: actin and zyxin as key intracellular mediators.

CD99 is a unique 32-kDa cell surface molecule with broad cellular expression but still poorly understood biological functions. In cancer cells, CD99 is highly expressed in virtually all Ewing's sarcoma (ES). Engagement of CD99 induces fast homotypic aggregation of ES cells and caspase-independent apoptosis. In this study, we analysed signal transduction after CD99 engagement on ES cells. Findings obtained with selective inhibitors indicated that only actin cytoskeleton integrity was essential for cell-cell adhesion and apoptosis of ES cells. Indeed, CD99 stimulation induced actin repolymerization, further supporting the role of cytoskeleton in CD99 signaling. Gene expression profiling of ES cells after CD99 engagement showed modulation in the expression of 32 genes. Among the pool of upregulated genes reported to be involved in cell adhesion, we chose to analyse the role of zyxin, a cytoplasmic adherens junction protein found to play a role in the regulation of the actin cytoskeleton. Overexpression of zyxin after CD99 ligation was confirmed by real-time PCR and Western blot. Treatment of ES cells with zyxin antisense oligonucleotides inhibited CD99-induced cell aggregation and apoptosis, suggesting a functional role for this protein. Therefore, our findings indicate that CD99 functions occur through reorganization of cytoskeleton and identify actin and zyxin as the early signaling events driven by CD99 engagement.

Proteolytic regulation of Forkhead transcription factor FOXO3a by caspase-3-like proteases.

Forkhead family transcription factors are critical regulators of cell cycle progression and apoptosis in hematopoietic cells. Here, we show that FOXO3a (also known as FKHRL1) is a new substrate of caspase-3-like proteases during apoptosis in T lymphocytes. FOXO3a was cleaved in vivo by caspases in leukemic Jurkat cells following engagement of Fas (CD95) receptor, staurosporine, and etoposide treatment, but not following engagement of CD99, a caspase-independent cell death inducer. Caspase-mediated cleavage of FOXO3a was also observed in CD4+ peripheral T cells subjected to activation-induced cell death. The expression of the death adapter FADD and caspase-8 was required for Fas-induced FOXO3a cleavage, but activation of survival pathways by overexpression of FLICE-inhibitory protein or phorbol myristate acetate treatment prevented it. FOXO3a was cleaved in vitro by caspase-3-like proteases at the consensus sequence DELD304A, releasing the N-terminal DNA-binding domain of FOXO3a from its C-terminal transactivating domain. Whereas full-length FOXO3a enhanced Forkhead response element-dependent transcription and apoptosis in Jurkat cells, both fragments were inactive to promote gene activation and cell death. In contrast, a caspase-resistant FOXO3a mutant exhibited enhanced transcriptional and proapoptotic activities. Together, these results indicate that the proteolytic cleavage of FOXO3a by caspases may represent a novel regulatory mechanism of FOXO3a activity during death receptors signaling.

Engagement of CD99 triggers the exocytic transport of ganglioside GM1 and the reorganization of actin cytoskeleton

We studied the role of lipid rafts and actin cytoskeleton in CD99-mediated signaling to elucidate the mechanism of protein transport upon CD99 engagement. CD99 engagement in Jurkat cells elicited the exocytic transport of GM1 as well as several surface molecules closely related with CD99 functions. In addition, CD99 molecules were rapidly incorporated into lipid rafts and appeared to rearrange the actin cytoskeleton upon CD99 stimulation. Association of CD99 with actin cytoskeleton was inhibited by methyl-β-cyclodextrin, while CD99-mediated GM1 clustering was inhibited by cytochalasin D. Therefore, we suggest that CD99 may play a role in the vesicular transport of transmembrane proteins and lipid rafts from the intracellular location to the cell surface, possibly by effecting actin cytoskeleton reorganization.

Characterization and epitope mapping of two monoclonal antibodies against human CD99.

CD99 plays a critical role in the diapedesis of monocytes, T cell differentiation, and the transport of MHC molecules. Engagement of CD99 by agonistic monoclonal antibodies has been reported to trigger multifactorial events including T cell activation as well as cell-cell adhesion during hematopoietic cell differentiation. In this study, to identify the functional domains participating in the cellular events, we mapped the epitopes of CD99, which are recognized by two agonistic CD99 monoclonal antibodies, DN16 and YG32. Using recombinant fusion proteins of GST with whole or parts of CD99, we found that both antibodies interact with CD99 molecules independently of sugar moieties. DN16 mAb detected a linear epitope located in the amino terminal region of CD99 while YG32 mAb bound another linear epitope in the center of the extracellular domain. To confirm that the identified epitopes of CD99 are actually recognized by the two mAbs, we showed the presence of physical interaction between the mAbs and the fusion proteins or synthetic peptides containing the corresponding epitopes using surface plasmon resonance analyses. The dissociation constants of DN16 and YG32 mAbs for the antigen were calculated as 1.27 x 10(-7) and 7.08 x 10(-9) M, respectively. These studies will help understand the functional domains and the subsequent signaling mechanism of CD99.

Differential activation of MAP kinase family members triggered by CD99 engagement

The molecular basis for the modulatory properties of CD99 is not well understood. Treatment of human Jurkat T lymphocytes with anti-CD99 antibody led to activation of three mitogen-activated protein kinase (MAPK) members, ERK, JNK, and p38 MAPK, along with homotypic aggregation. While phosphorylation of ERK and JNK was inhibited by the pretreatment of a PKC inhibitor, bisindolylmaleimide I, activation of p38 MAPK was upregulated by the same pretreatment. The signaling pathways to MAPKs by CD99 engagement were independent of PI-3 kinase, distinguishing from those by CD3 engagement. Among MAPKs, ERK pathway was essential for homotypic aggregation together with intracytoplasmic Ca 2+.

Engagement of CD99 Induces Apoptosis Through a Calcineurin-Independent Pathway in Ewing's Sarcoma Cells

Programmed cell death (PCD) is a prominent feature of the development of the immune and nervous systems. In both systems, widespread PCD occurs in primitive progenitor cells during development. In this study, we demonstrated that Ewing's sarcoma (ES) cells, undifferentiated neural precursors, underwent apoptosis upon engagement of CD99 with anti-CD99 monoclonal antibody. Apoptosis via CD99 occurred only in the undifferentiated state of ES cells, but not in differentiated ES cells. CD99-induced apoptosis in ES cells appeared to require de novo synthesis of RNA and protein as well as caspase activation. Cyclosporin A, known to be a potent inhibitor of both calcineurin activation and mitochondrial permeability transition pore opening, inhibited CD99-mediated apoptosis, whereas FK-506, a specific calcineurin inhibitor, did not, indicating the induction of CD99-mediated apoptosis through a calcineurin-independent pathway. Furthermore, the dying cells displayed the reduction of mitochondrial transmembrane potential (delta psi m). These results suggest that CD99 engagement induce CsA-inhibitable mitochondrial permeability transition pore opening, followed by a reduction of delta psi m and caspase activation, thereby leading to apoptosis. Based on these results, we suggest the possible involvement of CD99 in the apoptotic processes that occur during nervous system development and also its application in immunotherapeutic trials for ES cases.

CD99 Engagement on Human Peripheral Blood T Cells Results in TCR/CD3-Dependent Cellular Activation and Allows for Th1-Restricted Cytokine Production

We have assessed the functional effect of CD99 engagement on resting human peripheral blood (PB) T cells. CD99, as detected by the mAb 3B2/TA8, is constitutively expressed on all PB T cells and becomes further up-regulated upon cellular activation. In this study we demonstrate that cross-linking of the CD99 molecule with the agonistic mAb 3B2/TA8 cooperates with suboptimal TCR/CD3 signals, but not with phorbol ester, ionomycin, or CD28 mAb stimulation, to induce proliferation of resting PB T cells. Comparable stimulatory effects were observed with the CD99 mAb 12E7. Characterization of the signaling pathways involved revealed that CD99 engagement leads to the elevation of intracellular Ca2+, which is dependent on the cell surface expression of the TCR/CD3 complex. No CD99 mAb-induced calcium mobilization was observed on TCR/CD3-modulated or TCR/CD3-negative T cells. To examine the impact of CD99 stimulation on subsequent cytokine production by T cells, we cross-linked CD99 molecules in the presence of a suboptimal TCR/CD3 trigger followed by determination of intracellular cytokine levels. Significantly, T cell lines as well as Th1 and Th0 clones synthesized TNF-alpha and IFN-gamma after this treatment. In contrast, Th2 clones were unable to produce IL-4 or IFN-gamma when stimulated in a similar fashion. We conclude that CD99 is a receptor that mediates TCR/CD3-dependent activation of resting PB T cells and specifically induces Th1-type cytokine production in polyclonally activated T cell lines, Th1 and Th0 clones.

Engagement of CD99 Induces Up-Regulation of TCR and MHC Class I and II Molecules on the Surface of Human Thymocytes

CD99 is a cell surface molecule involved in the aggregation of lymphocytes and apoptosis of immature cortical thymocytes. Despite its high level expression on immature cortical thymocytes, the functional roles of this molecule during thymic selection are only now being elucidated. Examination of the effects of CD99 engagement on the expression kinetics of the TCR and MHC class I and II molecules, which are involved primarily in thymic positive selection, revealed a marked up-regulation of these proteins on the surface of immature thymocytes. This increase was the result of accelerated mobilization of molecules stored in cytosolic compartments to the plasma membrane, rather than increased RNA and protein synthesis. Confocal microscopic analysis revealed the changes in subcellular distribution of these molecules. When CD99 was engaged, TCR and MHC class I and II molecules were concentrated at the plasma membrane, particularly at cell-cell contact sites. The TCRlow subpopulation of immature double positive thymocytes was much more responsive to CD99-mediated up-regulation than was the TCRhigh population. These findings suggest that CD99-dependent up-regulation may have possible implication in positive selection during thymocyte ontogeny.

CD99 (MIC2) regulates the LFA-1/ICAM-1-mediated adhesion of lymphocytes, and its gene encodes both positive and negative regulators of cellular adhesion.

Despite the fact that integrin-mediated lymphocyte adhesion is a crucial event for an appropriate immune response, little is known about the mechanisms that control the adhesion and deadhesion processes generated by the engagement of CD99 between various types of immune cells. Here we report that the CD99 gene encodes two distinct proteins with opposite functions in the LFA-1/intercellular adhesion molecule 1 (ICAM-1)-mediated cell adhesion process. The two forms of the CD99 protein are produced by alternative splicing of the CD99 gene transcript. The major form induced homotypic adhesion of the human B lymphoblastoid cell line IM-9, whereas the minor, truncated form inhibited the adhesion process. Activation of the major form of CD99 with anti-CD99 monoclonal antibodies induced rapid aggregation of IM-9 cells, which was blocked by the addition of mAbs to LFA-1 or intracellular adhesion molecule 1. Overexpression of the minor truncated form of CD99 markedly down-regulated the expression of LFA-1. The two forms of CD99 are differentially expressed in most human cells tested and are highly conserved in monkey. Taken together, these observations suggest that the two forms of CD99 function in vivo in both positive and negative regulation of LFA-1-mediated adhesion of lymphocytes during an immune response.