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SUMMARYTransforming growth factor (TGF)-β plays crucial roles in differentiation of dendritic cells (DC). However, molecular mechanisms how TGF-β regulates DC differentiation remain largely unknown. Here, we show that selective repression of one of the TGF-β receptor-regulated SMADs (R-SMADs), SMAD3 directs conventional DC (cDC) differentiation, whereas maintenance of SMAD3 is indispensable for plasmacytoid DC (pDC) differentiation. Expression of SMAD3 was specifically downregulated in CD115+common DC progenitor (CDP), pre-cDCs and cDCs. SMAD3 deficient mice showed a significant reduction in pre-pDCs and pDCs with increased CDP, pre-cDCs and cDCs. SMAD3 upregulated the pDC-related genes: SPI-B, E2-2 and IKAROS, while it repressed FLT3 and the cDC-related genes: IRF4 and ID2. STAT3 and a SMAD transcriptional co-repressor, c-SKI repressed SMAD3 for cDC differentiation, whereas canonical SMAD-mediated TGF-β signalling maintained SMAD3 for pDC differentiation. Thus, SMAD3 is the pivotal determinant to bifurcate cDC and pDC differentiation in the steady-state condition.

Identification of a particular epitope on the domain 2 of human ICAM-1 led us to focus on its role in the treatment of rheumatoid arthritis (RA). Key observations from our previous xenotransplantation research included the generation of tolerogenic DCs, antigen-specific T-cell tolerance, and reduced production of inflammatory cytokines. The critically important point is the fact that it works initially on DC maturation. Ligation of this epitope with a recognizing antibody, MD-3, was also able to create a tolerogenic environment in RA in a manner sililar to that created by xenotransplantation. In this study, we noted that the disease progression, in terms of arthritis score and histopathology of joints, was significantly less severe in the MD-3-treated group than in the vehicle-treated group. Defective production of IL-6 and reduced proliferation of collagen-specific T cells were most remarkable laboratory findings. This type of ligation has a greater advantage over other types of therapeutics, in a sense that simple injection of this antibody inhibits antigen-specific T cell response. Due to the possibility of viral infection in this process, we regularly monitored cytomegalovirus reactivation status without detection of any viral gene replication. We are hoping that remarkable specializations that this interaction has, would be a promising target for therapeutic antibody in RA.

Antibody formation against therapeutic agents, such as tumor necrosis factor inhibitors and Factor VIII, that leads to treatment failure has become a major challenge in the treatment of rheumatoid arthritis and hemophilia. It is well known that anti-CD154 antibodies have the highest potential to inhibit these types of adverse immune responses. Nevertheless, the formation of thromboemboli is the major hurdle in the clinical application of these anti-CD154 blocking antibodies. For this, we attempted to derive an idea as to how this major complication can be eliminated. Consequently, we developed a novel anti-CD154 chimeric antibody, which was made by genetic modification of a portion of human IgG4 Fc. This antibody has an almost comparable antigen binding affinity to a previously developed 5C8 clone and near completely inhibited CD40-CD154 interaction and T cell-dependent B cell activation in vitro. Even under the condition, where we injected immune complexes comprised of RD-05 and CD154 antigen, the formation of thromboembolism was not seen in human FcγRIIA-transgenic mice, whereas the converse was exactly true in the case of 5C8 antibody. Notably, just two injections of RD-05 antibody was sufficient to inhibit the antibody formation against adalimumab during 3–4 months in cynomolgus macaques, in which adalimumab was repeatedly injected for 12 weeks. Based on these findings, we suggest that this RD-05 antibody can be applied to antibody-mediated autoimmune diseases, including systemic lupus erythematosus and immune thrombocytopenic purpura.

Identification of intrathymic eomesodermin+ (Eomes+) CD4 T cells creates a novel idea that there is more than one way for the generation of innate CD4 T cells. Promyelocytic leukemia zinc finger protein+ T cells and natural Th17 cells are known to be generated by sensing a high and persistent TCR strength, whereas this is not the case for Eomes+ CD4 T cells. These cells go through low-level signal during the entire maturation pathway, which subsequently leads to induction of high susceptibility to cytokine IL-4. This event seems to be a major determinant for the generation of this type of cell. These T cells are functionally equivalent to Th1 cells that are present in the periphery, and this event takes place both in transgenic and in wild-type mice. There is additional evidence that this type of Eomes+ innate CD4 T cell is also present in human cord blood.

CD99 is involved in many cellular events, such as the generation of Hodgkin and Reed-Sternberg cells, T cell co-stimulation, and leukocyte transendothelial migration. However, these studies have been limited to in vitro or in vivo experiments using CD99-deficient cell lines or anti-CD99 antibodies. In the present study, using CD99-deficient mice established by the exchangeable gene trap method, we investigated the physiologic function of murine CD99. In a B6 splenocytes → bm12 graft-versus-host disease model, wild-type cells were minimally lethal, whereas all mice that received CD99-deficient donor cells developed an early and more severe pathology. Graft-versus-host disease in these mice was associated with insufficient expansion of myeloid-derived suppressor cells. This was confirmed by experiments illustrating that the injection of wild-type donor cells depleted of Mac-1+ cells led to an almost identical disease course as the CD99-deficient donor system. Therefore, these results suggest that CD99 plays a crucial role in the attenuation of graft-versus-host disease by regulating the expansion of myeloid-derived suppressor cells.

We have recently shown that MHC class II-dependent thymocyte–thymocyte (T–T) interaction successfully generates CD4+ T cells (T–T CD4+ T cells), and that T–T CD4+ T cells expressing promyelocytic leukemia zinc finger protein (PLZF) show an innate property both in mice and humans. In this article, we report that the thymic T–T interaction is essential for the conversion of CD8+ T cells into innate phenotype in the physiological condition. CD8+ T cells developed in the presence of PLZF+ CD4+ T cells showed marked upregulation of eomesodermin (Eomes), activation/memory phenotype, and rapid production of IFN-γ on ex vivo stimulation. Their development was highly dependent on the PLZF expression in T–T CD4+ T cells and the IL-4 secreted by PLZF+ T–T CD4+ T cells. The same events may take place in humans, as a substantial number of Eomes expressing innate CD8+ T cells were found in human fetal thymi and spleens. It suggests that PLZF+ T–T CD4+ T cells in combination with Eomes+ CD8+ T cells might actively participate in the innate immune response against various pathogens, particularly in human perinatal period.

Recently, a novel CD4(+) T-cell developmental pathway was reported that generates thymocyte-thymocyte (T-T) CD4(+) T cells. We established a mouse system (CIITA(tg)CIITApIV(-/-)) where thymic positive selection occurred only by major histocompatibility complex (MHC) class II(+) thymocytes. T-T CD4(+) T cells selected via MHC class II-dependent T-T interaction are comprised of PLZF-negative and innate PLZF-positive populations. Until recently, the functional role of the PLZF-negative population was unclear. In this study, we demonstrate that naïve T-T CD4(+) T cells provide B-cell help to a level comparable with that of naïve conventional CD4(+) T cells. Considering the absence of PLZF expression in naïve T-T CD4(+) T cells, these results suggest that PLZF-negative naïve T-T CD4(+) T cells are functionally equivalent to conventional naïve CD4(+) T cells in terms of B-cell help.

Human thymocytes, unlike mouse thymocytes, express major histocompatibility complex (MHC) class II molecules on their surface, especially during the fetal and perinatal stages. Based on this observation, we previously identified a novel developmental pathway for the generation of CD4+ T cells via interactions between MHC class II–expressing thymocytes (thymocyte–thymocyte [T–T] interactions) with a transgenic mouse system. However, the developmental dissection of this T–T interaction in humans has not been possible because of the lack of known cellular molecules specific for T–T CD4+ T cells. We show that promyelocytic leukemia zinc finger protein (PLZF) is a useful marker for the identification of T–T CD4+ T cells. With this analysis, we determined that a substantial number of fetal thymocytes and splenocytes express PLZF and acquire innate characteristics during their development in humans. Although these characteristics are quite similar to invariant NKT (iNKT) cells, they clearly differ from iNKT cells in that they have a diverse T cell receptor repertoire and are restricted by MHC class II molecules. These findings define a novel human CD4+ T cell subset that develops via an MHC class II–dependent T–T interaction.

Unlike conventional T cells, innate-like T cells such as natural killer (NK) T cells are selected by homotypic T-cell interactions. Recently, a few reports have shown that T-T CD4(+) T cells can be generated in a similar manner to that for NKT cells. These two types of cells share common functional properties such as rapid response to antigenic encounters and the potential for a panoply of cytokine secretion. However, T-T CD4(+) T cells differ from NKT cells in that they are restricted by highly polymorphic major histocompatibility complex (MHC) II molecules and have a diverse T-cell receptor repertoire. Additional example of T-T interactions was recently reported in which peripheral T cells re-circulate to the thymus and participate in the thymocyte selection process. In this review, we dissect the cellular mechanisms underlying the production of T-T CD4(+) and NKT cells, with particular emphasis on the differences between these two T-cell prototypes. Finally, we propose that T-T CD4(+) T cells serve two major functions: one as an acute-phase reactant against viral infection and the other is the generation of anti-ergotypic CD4(+) T cells for regulatory purposes. All of these features make it possible to create a diverse set of functional cells through MHC class II-restricted T-T interactions.

PLZF-expressing invariant natural killer T cells and CD4 T cells are unique subsets of innate T cells. Both are selected via thymocyte-thymocyte interaction, and they contribute to the generation of activated/memory-like CD4 and CD8 T cells in the thymus via the production of IL-4. Here, we investigated whether PLZF(+) innate T cells also affect the development and function of Foxp3(+) regulatory CD4 T cells. Flow cytometry analysis of the thymus and spleen from both CIITA transgenic C57BL/6 and wild-type BALB/c mice, which have abundant PLZF(+) CD4 T cells and invariant natural killer T cells, respectively, revealed that Foxp3(+) T cells in these mice exhibited a CD103(+) activated/memory-like phenotype. The frequency of CD103(+) regulatory T cells was considerably decreased in PLZF(+) cell-deficient CIITA(Tg)Plzf(lu/lu) and BALB/c.CD1d(-/-) mice as well as in an IL-4-deficient background, such as in CIITA(Tg)IL-4(-/-) and BALB/c.lL-4(-/-) mice, indicating that the acquisition of an activated/memory-like phenotype was dependent on PLZF(+) innate T cells and IL-4. Using fetal thymic organ culture, we further demonstrated that IL-4 in concert with TGF-β enhanced the acquisition of the activated/memory-like phenotype of regulatory T cells. In functional aspects, the activated/memory-like phenotype of Treg cells was directly related to their suppressive function; regulatory T cells of CIITA(Tg)PIV(-/-) mice more efficiently suppressed ovalbumin-induced allergic airway inflammation compared with their counterparts from wild-type mice. All of these findings suggest that PLZF(+) innate T cells also augmented the generation of activated/memory-like regulation via IL-4 production.

SummaryDespite numerous reports on MHC class II expression by T cells from a wide spectrum of mammalian species including humans, the biological relevance of this phenomenon has never been tested with appropriately designed animal models. To address this issue, we developed mouse models in which immature thymocytes are the only positively selecting antigen-presenting cells in the thymus. In these mice, CD4+ T cells were generated with the appropriate maturation phenotype and showed a diverse repertoire of TCR Vβs. The CD4+ T cells were functionally competent, mediating effective allogeneic responses that involved polyclonal TCR Vβs. These results suggest that the thymocyte-thymocyte (T-T) interaction operates as an independent pathway for CD4+ T cell selection in the thymi of species with MHC II-positive thymocytes. This T-T interaction appears to be the basis for the generation of donor MHC-restricted CD4+ T cells in xenogeneic hosts.

Ultrastructural and immunohistochemical studies of 4 groups of cells-(human embryonic stem cells (hES), embryoid bodies (EB), and spontaneously and retinoic acid (RA)-induced differentiating cells)-were carried out to investigate their detailed phenotype. Immunohistochemically, the EB cells showed strong immunoreactivity for CD34, CD117, and nestin. Differentiating cells expressed pancytokertin, vimentin, CD31, CD56, GFAP, nestin, and NeuN as well as CD34, and c-Kit. However, synaptophysin and neurofilaments were not present in these same differentiating cells. Transmission electron microscopy showed that hES and EB cells were very similar to germ cells or cells of the inner cell mass. Spontaneously and RA-induced differentiating cells exhibited epithelial, mesenchymal, endodermal, and neuronal phenotypes. The perikarya of the neuronal cells had rich RERs (Nissl substance) and long cytoplasmic processes filled with numerous neural tubules. However, neither synaptic junctions nor synaptic vesicles were developed. In our study, RA treatment with brain-derived growth factor and TGFalpha in neuron differentiation medium induced not only neuronal differentiation but also pluripotential differentiation. Full neuronal differentiation did not occur after 2 weeks in culture, as no synaptic junctions and synaptic vesicles developed.

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.

Earlier report showed that expression of a splice variant of CD99 transmembrane protein increases invasive ability of human breast cancer cells. Cell motility was also significantly enhanced by the CD99 splice variant expression. In an effort to identify the cellular components that mediate a signal transduction pathway triggered by the CD99 splice variant, known signal path inhibitors were examined for their effects on the motility of the CD99 splice variant-transfected MDA-MB-231 breast cancer cells. Phenylarsine oxide, an inhibitor of phosphatase specific for focal adhesion kinase, and PP1, an inhibitor of src kinase family, significantly suppressed motility of the cells. Among different types of src transfectant clones generated, kinase-negative mutant src transfectant cells were 80% less motile than the mock cells transfected with an empty-vector, while v-src and c-src transfectants exhibited cell motility levels at or slightly above the mock transfectant. These results suggest that src and focal adhesion kinase mediate the intracellular signaling pathway of a CD99 splice variant for the induction of motility of human breast cancer cells.

In diagnostic pathology and immunocytochemical research, immunohistochemical techniques using the streptavidin-biotin-peroxidase system have played an extremely valuable role. This system, based on the high affinity of streptavidin for biotin, may, however, provoke false positive results because of endogenous streptavidin-binding sites in human tissues. With the advent of the antigen retrieval procedure and signal amplification method, this problem can be serious enough to cause mistakes in interpreting immunohistochemical staining results. Therefore, we examined the distribution of endogenous biotin-like molecules in various human tissues and the influence of various antigen retrieval procedures with or without signal amplification using biotinylated tyramine to reveal these biotin-like activities. We observed that endogenous biotin-like molecules were present in a wide range of tissues, and their activity was markedly enhanced by employing antigen retrieval procedures or signal amplification. Furthermore, the extent to which the activity of endogenous biotin-like activities was enhanced depended on the kinds of antigen retrieval procedures and signal amplification employed. Pressure cooking and tyramine amplification with microwave heating showed the highest activities. These results show that the antigen retrieval procedures and signal amplification with tyramine can enhance the activity of endogenous biotin or biotin-like molecules as well as antigenicity, which can be a pitfall in the interpretation of immunohistochemical data.

Epstein-Barr virus (EBV)-encoded latent membrane protein-1 (LMP1) is highly expressed in Hodgkin and Reed-Sternberg (H-RS) cells from patients with EBV-associated Hodgkin disease. It was previously demonstrated that CD99 can be negatively regulated by LMP1 at the transcriptional level, and the decreased expression of CD99 in a B lymphocyte cell line generates H-RS-like cells. In this study, detailed dissection of the CD99 promoter region was performed to search regulatory factor(s) involved in the expression of the gene. Using various mutant constructs containing deletions in the promoter region, it was revealed that the maximal promoter activity was retained on 5'-deletion to the position -137 from the transcriptional initiation site. Despite the presence of multiple putative Sp1-binding sites in the promoter region, the site located at -95 contributes heavily as a positive cis-acting element to its basal promoter activity. However, on examination of the involvement of the positive-acting Sp1-binding site of the promoter for the repressive activity of LMP1, it appeared to be dispensable. Instead, the repressive effect was mapped to the nuclear factor (NF)-kappaB activation domains in the cytoplasmic carboxyl terminus of LMP1 despite the absence of the NF-kappaB consensus sequences in the CD99 promoter region. Furthermore, the decreased CD99 promoter activity by LMP1 was markedly restored when NF-kappaB activity was inhibited. Taken together, these data suggest that Sp1 activates, whereas LMP1 represses, transcription from the CD99 promoter through the NF-kappaB signaling pathway, and they might aid in the understanding of the molecular mechanisms of viral pathogenesis in EBV-positive Hodgkin disease. (Blood. 2001;97:3596-3604)

Recently we reported that the down-regulation of CD99 (Mic2) is a primary requirement for the generation of Hodgkin's and Reed-Sternberg (H-RS) cells seen in Hodgkin's disease. In this study, we provide evidence that the down-regulation of CD99 is induced by high expression of Epstein-Barr virus (EBV) latent membrane protein 1 (LMP-1), which is highly expressed in H-RS cells of EBV-associated Hodgkin's disease. To investigate the effect of LMP-1 on the expression of CD99 in vitro, we established a stable cell line by transfecting an SV40-early promoter driven-LMP-1 expression construct into a neoplastic lymphoblastoid B cell line, IM9, in which the level of endogenous LMP-1 expression is almost negligible. In this cell line, the overexpression of LMP-1 led to the down-regulation of CD99 and the acquisition of morphological and functional characteristics of H-RS cells indistinguishable from those in lymph nodes of Hodgkin's disease patients and in CD99-deficient B cells. In addition, induced LMP-1 expression in an EBV-negative B cell clone, BJAB, directly caused the down-regulation of surface CD99 expression. Northern and Western analysis data, showing that overexpression of LMP-1 negatively influenced the expression of CD99, were supported by experiments in which a CD99 promoter-driven luciferase promoter reporter construct transfected into 293T cells was down-regulated when LMP-1 was coexpressed. Therefore, our data strongly suggest that the EBV LMP-1 protein plays a pivotal role in the down-regulation of CD99 via transcriptional regulation, which leads to the generation of the H-RS cells. (Blood. 2000;95:294-300)

In a significant proportion of cases, anencephaly is associated with thymic enlargement, suggesting a possibility that anencephalic fetuses have a functional disturbance in thymocyte differentiation and development. In this report, we demonstrated that CD99 expression was consistently reduced in cortical thymocytes of all anencephalic fetuses. In addition, the CD99-dependent aggregation of immature cortical thymocytes was almost completely impaired and apoptosis of thymocytes was markedly reduced in several cases. These results are in agreement with previous findings that CD99 regulates the aggregation and apoptosis of various types of cells. These data strongly suggest that functional disturbance of thymocytes and thymic hyperplasia are related to the reduced expression of CD99 molecule in anencephalic fetuses.

A novel idea that there is an alternative pathway for intrathymic generation of mature CD4+ T cells came from the observation that immature thymocytes were also able to express MHC class II on their cell surfaces during fetal thymic development [1]. This idea was subsequently confirmed both by in vitro reaggregate thymic organ culture (RTOC) [2] and in vivo plck-CIITAtg mouse system [3, 4] where MHC class II molecules are designed to be expressed only on T cells. These CD4 T cells selected via T-T interaction has been, therefore, referred to as T-T CD4+ T cells. Consequently, this novel pathway creates very intriguing and important innate T cell subsets; PLZF+ T-T CD4 T cells [5], Eomes+ innate CD8 T cells [6] and, last but not at the least, Eomes+ natural Th1 cells [7]. Of these three different types of innate T cells, PLZF+ T-T CD4 T cells are developed via T-T interaction in thymus, whereas both nTh1 and Eomes+ innate CD8 T cells are generated via bystander effect in the presence of Promyelocytic leukemia zinc-finger protein (PLZF)-positive T-T CD4 T cells [6, 7]. PLZF+ T-T CD4 T cells shared several functional characteristics with iNKT cells such as rapid production of both IL-4 and IFN-γ upon TCR stimulation, and the unique dependence on the SLAM-SAP signaling pathway. However, PLZF+ T-T CD4 T cells and iNKT cells are really rather different in one key aspect; that PLZF+ T-T CD4 T cells have extreme TCR diversity as was in conventional CD4 T cells [5]. IL-4 produced by PLZF+ T-T CD4 T or NKT cells induces the generation of Eomes+ innate CD8 and nTh1 cells [6, 7]. Eomes+ innate CD8 and nTh1 cells are in fact extraordinarily susceptible to IL-4 and have much higher number of IL-4 receptor in their precursor cells which were produced right after positive selection. Eomes+ innate CD8 T cells produced large amounts of IFN-γ and TNF-α within a few hours upon TCR stimulation. The functional significance of this cell population was further highlighted by their ability to rapidly clear viremia during chronic lymphocytic choriomeningitis virus (LCMV) infection (unpublished observations). Remarkable specialization that nTh1 cells have is that a low affinity/avidity interaction selects this type of T cells during thymic positive selection. A series of events underlies the generation of Eomes+ nTh1 cells; low affinity/avidity interaction between the TCR and MHC peptide, increased susceptibility to common γ-chain (γc) cytokines (in this case, IL-4), concomitant expression of IL-4Rα and Eomes, and, finally, dramatic upregulation of anti-apoptotic protein Bcl-2 for their survival. This mechanism appears to work contrary to other types of innate cells, such as iNKT cells, nTh17 cells, and PLZF+ T-T CD4 T cells, which receive high levels of signals from the interaction between the MHC/peptide and the appropriate TCR (Figure ​(Figure1).1). All of these events dramatically illustrate the fact that there are inverse relationship between signal strength received by T cells and γc cytokine susceptibility. T-T interaction and subsequent generation of PLZF+ T-T CD4 T cells seem to exist in wide range of mammalians, and generation of three types of innate T cells via T-T interaction takes place in human system as well [5–7]. Especially, we discovered that both Eomes+ CD4 and CD8 T cells exist not only in cord blood cells but also in adult human peripheral blood mononuclear cells (unpublished observations). Figure 1 A hierarchy of TCR affinity/avidity of developing T cells Based on a variety of consideration, we propose that the biological relevance of T-T interaction is the generation of early effector T cells against diverse foreign pathogens, particularly viruses. From an evolutionary point of view, the limited TCR diversity of mouse innate T-cell subsets (e.g. iNKT cells, H2-M3-specific T cells and MR1-specific T cells) more serves to defend against bacteria, whereas nTh1 cells and Eomes+ innate CD8 T cells with their extremely diverse TCR repertoire would be more effective in defending against viral infections. Of note, nTh1 cells express stem-like memory T cell markers (CD45R0−, CCR7+, CD45RA+, CD62L+, CD27+, CD28+, IL-2Rβ+ and CXCR3+) particularly in human. Considering the fact that human thymus starts to involute relatively early in their life, stem-like peripheral Eomes+ T cells with their potential homeostatic proliferation might maintain a competent anti-viral defense even long after complete thymic involution. Since nTh1 cells have gone through low affinity/avidity interaction during entire thymocyte maturation pathway, they have relatively low chances to see their self-antigens in the periphery to initiate autoimmunity. In summary, T-T interaction is the real basis for the development of three different types (PLZF+ CD4 T, and Eomes+ CD4 and CD8 T cells) of innate T cells both in mouse and human immune system. We again would like to emphasize the possibility that Eomes+ nTh1 cells with innate-like properties but with diverse TCR repertoire might play a critical role to defend against extremely diverse exogenous pathogens, particularly viruses.

Despite the fact that Hodgkin’s and Reed-Sternberg (H-RS) cells are morphological hallmarks of Hodgkin’s disease (HD), the nature of H-RS cells still remains to be resolved. Here we report that downregulation of CD99 (Mic2) leads to the generation of cells with an H-RS phenotype. IM9 and BJAB B-cell lines that were transfected with an antisense CD99 expression construct showed the morphological and immunological characteristics of H-RS cells such as multinuclearity, expression of CD15, decreased expression of major histocompatibility complex (MHC) class I and CD45RB, and deregulated secretion of cytokines. The reduced expression of CD99 was also confirmed in H-RS cells of patient’s lymph nodes and three HD-derived cell lines, L428, KM-H2, and HDLM-2. Moreover, features characteristic of H-RS cells were completely abolished by forced expression of CD99 and by a constitutively active form of Rac, which functions downstream of CD99. We suggest that CD99 molecules play a crucial role in regulating functions and morphology of cells through a Rac-Rho signaling pathway and that the loss of CD99 expression is a significant molecular event to generate H-RS cells.

Despite the fact that Hodgkin’s and Reed-Sternberg (H-RS) cells are morphological hallmarks of Hodgkin’s disease (HD), the nature of H-RS cells still remains to be resolved. Here we report that downregulation of CD99 (Mic2) leads to the generation of cells with an H-RS phenotype. IM9 and BJAB B-cell lines that were transfected with an antisense CD99 expression construct showed the morphological and immunological characteristics of H-RS cells such as multinuclearity, expression of CD15, decreased expression of major histocompatibility complex (MHC) class I and CD45RB, and deregulated secretion of cytokines. The reduced expression of CD99 was also confirmed in H-RS cells of patient’s lymph nodes and three HD-derived cell lines, L428, KM-H2, and HDLM-2. Moreover, features characteristic of H-RS cells were completely abolished by forced expression of CD99 and by a constitutively active form of Rac, which functions downstream of CD99. We suggest that CD99 molecules play a crucial role in regulating functions and morphology of cells through a Rac-Rho signaling pathway and that the loss of CD99 expression is a significant molecular event to generate H-RS cells.

Induction of antigen-specific T cell tolerance would aid treatment of diverse immunological disorders and help prevent allograft rejection and graft versus host disease. In this study, we establish a method of inducing antigen-specific T cell tolerance in situ in diabetic humanized mice and Rhesus monkeys receiving porcine islet xenografts. Antigen-specific T cell tolerance is induced by administration of an antibody ligating a particular epitope on ICAM-1 (intercellular adhesion molecule 1). Antibody-mediated ligation of ICAM-1 on dendritic cells (DCs) led to the arrest of DCs in a semimature stage in vitro and in vivo. Ablation of DCs from mice completely abrogated anti-ICAM-1-induced antigen-specific T cell tolerance. T cell responses to unrelated antigens remained unaffected. In situ induction of DC-mediated T cell tolerance using this method may represent a potent therapeutic tool for preventing graft rejection.

Despite a long-standing hypothesis that chronic graft-versus-host disease (cGVHD) is an autoimmune disorder, most mouse models of cGVHD have been developed on the assumption that donor T cells are essential for its development. Here we show that cGVHD may be caused by autoreactive host T cells in mice that have been lethally irradiated and grafted with T-cell-depleted allogeneic bone marrow cells. In this chimera, host T cells derived from radioresistant intrathymic T-cell precursors caused dermal fibrosis and periportal inflammation, without the requirement for donor T cells. The lack of host DCs within the thymus after high-dose irradiation allowed autoreactive host T cells to escape thymic negative selection. Moreover, the homeostatic expansion of these T cells may augment their autoreactivity. These findings indicate that host T-cell-mediated cGVHD is an autoimmune process that occurs following the grafting of T-cell-depleted BM cells into hosts with functioning thymuses. We propose, based on the present data, that host T-cell-dependent autoimmunity is a potential mechanism by which cGVHD is induced.

Here, we show that the interaction between two membrane proteins, the mouse homologue of CD99 (designated D4) and its ligand, paired immunoglobulin-like type 2 receptor (PILR), is one of the major mechanisms of thymocyte apoptosis. Using the polymeric fusion protein of PILR and IgG1 (PILR-Ig), we demonstrated that D4 ligation in the absence of T cell receptor (TCR) engagement leads to the induction of apoptosis, mainly at the double-positive stage of thymocytes. This was further confirmed by a blocking study in which blocking the interaction between D4 and PILR by soluble D4 protein led to reduced apoptosis in the fetal thymic organ culture with wild type and TCRalpha(-/-) mice. Furthermore, the dissection of intracellular signaling pathway demonstrated that D4 cross-linking led to caspase activation without any change in mitochondrial membrane potential. Based on these data, we propose a mechanism for thymocyte depletion in which the interaction between D4 and PILR delivers an active signal.

The authors describe a newly characterized murine monoclonal antibody to the human leukocyte surface antigen, SHL-1. The antigen belongs to the leukocyte common antigen (LCA) family, and its molecular weight is about 180,000 daltons, which is similar to that of some previously characterized LCAs. The SHL-1 antigen is resistant to conventional tissue-fixation and embedding procedures. This antibody can therefore be used in the immunohistochemical staining of paraffin-embedded tissue sections. Wide screening with a sufficient number of both fresh and routinely processed paraffin-embedded tissues was done with indirect immunoperoxidase technique. With this procedure, SHL-1 labeled the majority of normal leukocytes and hematopoietic malignancies. Some B-cell malignancies were not stained with this antibody. The non-hematologic malignancies posing diagnostic problems of differentiation from lymphomas or leukemias were completely negative to SHL-1. The immunoreactivity to SHL-1 of samples from 24 leukemic patients and 15 human tumor cell lines was determined by the immunofluorescence method. Of 24 leukemic preparations, 23 were strongly reactive to this antibody. One case of B-cell leukemia did not react with SHL-1. No immunoreactivity was demonstrated in non-hematopoietic tumor cell lines. The overall reaction pattern of SHL-1 proved its usefulness in both diagnostic and research practice in hematological disorders. This antibody detected cell surface antigens of the T cell series more effectively than those of the B-cell series in terms of the positive number of cells and mean fluorescence intensity.

The CD99 gene encodes two distinct transmembrane proteins by alternative splicing of its transcript. To examine the effects of two CD99 isoforms on the invasive phenotypes of breast cancer cells, MDA-MB-231 and MCF-7 human breast cancer cell lines were stably transfected with CD99 cDNAs encoding the major wild-type form (type I) or a minor splice variant (type II). As a result, expression of CD99 type II, but not type I, markedly elevated the motility, binding to fibronectin, MMP-9 expression, and invasiveness of MDA-MB-231 and MCF-7 breast cancer cells. In MDA-MB-435 breast cancer cells expressing both CD99 type I and type II, invasion-related cellular activities were inhibited by the transfection of small interfering RNA (siRNA) targeted to CD99 type II. Meanwhile, CD99 type II-induced MMP-9 expression in MDA-MB-231 cells was shown to be mediated by the binding of AP-1 factors to the MMP-9 gene promoter. Gel shift assay revealed that ligation of CD99 type II with antibody resulted in the binding of JunD to the AP-1 site of the MMP-9 promoter region. Initiation of CD99 type II signaling by antibody ligation increased expression of JunD and FosB AP-1 factors, along with phosphorylation of Src, Akt, p38 MAPK, ERK, and JNK. Knockdown of JunD and FosB by siRNA transfection abolished the positive effects of CD99 type II on the motility and MMP-9 expression of MDA-MB-231 cells. Increased expression of JunD and FosB as well as elevated cell motility and MMP-9 expression by CD99 type II ligation were also abrogated by inhibitors, dominant-negative forms, and siRNAs for Akt1, ERK1/2, and JNK1 but not for p38 MAPK. These results suggest that expression of a splice variant of CD99 contributes to the invasive ability of human breast cancer cells by up-regulating AP-1-mediated gene expression through the Akt-dependent ERK and JNK signaling pathways.

The introduction of antigen retrieval (AR) techniques has dramatically improved the sensitivity of immunohistochemical detection of various antigens in formalin-fixed, paraffin-embedded tissues. The microwave-heating and pressure-cooking procedures are the most effective AR methods reported to date. Although extensive efforts have been made to optimize AR procedures using these two methods, previous studies have not led to a standard protocol applicable to all antibodies derived from different clones. In this study we have investigated the optimal AR buffer conditions for 29 antibodies that are in common use for diagnostic purposes in hospitals worldwide. Borate (pH 8.0) and Tris buffer (pH 9.5) yielded the highest retrieved antigen immunoreactivity against most antibodies as compared to other buffers tested. In addition, the microwave pressure-cooking gave better results than microwave-heating alone. Therefore, borate (pH 8.0) or Tris (pH 9.5) buffer used in conjunction with the pressure-cooking procedure is strongly recommended for standard routine use.

Despite the fact that major histocompatibility complex class II transactivator (CIITA) has been known to be involved in Th1/Th2 balance in addition to its major role as a master regulator for the expression of MHC class II genes, the exact role of CIITA in Th1/Th2 balance is still controversial. To investigate whether the Th1/Th2 balance could be modulated by T cell specific expression of CIITA, we generated CIITA-transgenic mice, in which the CIITA expression is controlled by the distal promoter of p56lck, resulting in constitutive expression of CIITA predominantly in peripheral T cells. Naive CD4+ T cells from CIITA-transgenic mice exhibited a low level of IFN-gamma secretion as well as impaired Th1 polarization in vitro, while IL-4 secretion was enhanced under Th2 condition. In addition, the development of experimental autoimmune encephalomyelitis (EAE), a prototype of Th1-mediated disease, was repressed in CIITA-transgenic mice. Resistance to EAE was correlated with reduced production of IFN-gamma in response to MOG35-55, while the proliferation of MOG35-55 -specific T cells was not affected in CIITA-transgenic mice. Together, these data demonstrate that overexpression of CIITA in T cells inhibits Th1 differentiation and function, suggesting that the expression of CIITA in T cells might play a role in the regulation of the Th1/Th2 balance during the T cell lineage commitment.

CD99 is a 32-kDa cell surface molecule present on thymocytes, peripheral T cells, many other hematopoietic stem cells and somatic cells were implicated in cell-cell adhesion and cell-activation phenomena. Two major subtypes have been identified so far, designated CD99 type I and type II. We have investigated the correlation between the degree of neural differentiation and the expression of CD99 subtypes in three differentially differentiated cell lines such as CADO-ES1, RD-ES, and SH-N-SY5Y, in order of differentiation. In addition, we induced differentiation of the RD-ES cell line by N(6),2'-dibutyryl-cAMP (db-cAMP). Six days after treatment with db-cAMP, RD-ES cell line has changed its morphology from uniform round cells to cells with neurites, and initially CD99 type II-overexpressed RD-ES cells showed significant down-regulation of CD99 type II, whereas CD99 type I expression remained constant. When RD- ES cells were transfected with the cDNA encoding for CD99 type I-green fluorescence protein (GFP) and type II-GFP, CD99 type II transfected RD-ES cell line remained unchanged with morphology of undifferentiated form. Our data suggest that CD99 type II acts as a negative regulator in the neural differentiation of precursor cells that might occur during nerve system development.

The CD99 antigen has been implicated in various cellular processes, including apoptosis in T cells. Previously, we reported two monoclonal antibodies that recognize different epitopes of the CD99 molecule, named DN16 and YG32. In this study, we investigated the role of each CD99 epitope in T cell apoptosis. Unlike the DN16 epitope, CD99 ligation via the YG32 epitope failed to induce T cell death. Surprisingly, however, the YG32 signal enhanced Fas-mediated apoptosis in Jurkat T cells. Augmentation of Fas-mediated apoptosis by YG32 ligation was inhibited by treatment with either of the caspase inhibitors z-VAD-fmk or z-IETD-fmk, and YG32 ligation appeared to induce Fas oligomerization. These results suggest that each CD99 epitope plays a distinct role in T cell biology, especially in T cell apoptosis.

Major histocompatibility complex (MHC) class II surface levels on thymocytes increase after CD99 ligation. The functional implication of the up-regulated MHC class II was assessed by engaging MHC class II on CD99-ligated cells. MHC class II engagement down-modulated surface levels of T cell receptor and MHC molecules, and inhibited apoptosis of CD99-ligated thymocytes and CEM tumor cells, antagonistic effects on the previously reported CD99 functions. The results were reproducible regardless of the order of ligation of MHC class II and CD99. We suggest that signaling via MHC class II on CD99-engaged cells might be involved in the thymic maturation process by damping CD99 ligation effects.

To induce proper immune responses, T lymphocytes require two types of stimuli, antigen-specific and costimulatory signals. Among costimulatory molecules, CD28-engagement promotes the survival and proliferation of both naive and memory T cells. In addition, it is now believed that Fas may play a role in T cell activation in the human system. It is, however, controversial whether Fas can act as a costimulatory signal in the murine system. Thus, we investigated fundamental differences in the capacity to induce proliferation of T cells between Fas and CD28 in mice. Fas-mediated T cell proliferation was observed only with a full mitogenic dose of anti-CD3 antibodies, whereas CD28 engagement was able to enhance T cell proliferation in the presence of a suboptimal level of anti-CD3 antibody. Furthermore, Fas-engaged T cells showed faster response in the upregulation of CD25 and CD69 expression than CD28-engaged ones. Here, we report that Fas might play a role in mature T cell activation in the mouse system through a different mechanism from that in CD28 costimulation.

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 Ca2+.

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.

ILK (beta1-integrin-linked protein kinase) is a recently identified 59-kDa serine/threonine protein kinase that interacts with the cytoplasmic domain of the beta1-integrin containing four ankyrin-like repeats. We have developed a polyclonal antibody against ILK and explored the ILK immunoreactivity in normal human cells and tissues. ILK was mainly expressed in cardiac muscle and skeletal muscles. Surprisingly, ILK expression was observed in Ewing's sarcoma (ES; 100%), primitive neuroectodermal tumour (PNET; 100%), medulloblastoma (100%), and neuroblastoma (33.3%), whereas other small round cell sarcomas were not stained by the anti-ILK antibody. These results suggest that ILK could be a novel marker for tumours with primitive neural differentiation. Our findings support the notion that ES is a tumour that is closely related to PNET and that both originate from the neuroectoderm. ILK may be a sensitive and specific immunohistochemical marker and useful for the positive identification of ES and PNET in formalin-fixed, paraffin-embedded tissue sections.

We previously demonstrated the expression of MHC class II molecules in a significant percentage of human fetal and postnatal thymocytes. These results, at that time, raised the question as to whether the MHC class II molecules on immature thymocytes could actively be involved in the selection of immature T cells. We have developed a human reaggregate culture system to address this issue. Surprisingly, despite the fact that thymic epithelial cells (TECs) have been shown to be a major selecting cell type of positive selection, we were clearly able to see the involvement of MHC class II+ thymocytes during selection process through T-T interaction. In addition, maturation to single positive (SP) cells occurred only in the presence of MHC class II molecules and immature thymocytes were found to be arrested at the double positive (DP) stage of differentiation by blocking of TCR recognition of MHC class II molecules. All these results strongly suggest that human MHC class II+ thymocytes actively participate in the selection of the TCR repertoire, for which TCR recognition of peptide/MHC class II may be an initial determining step.

Summary Two childhood cases of spinal cord ganglioglioma are described. Both cases showed a remarkable similarity in presentation, radiologically and morphologically. The first case was a 6 1/2-year-old boy who had ganglioglioma involving the entire length of the spinal cord. He has been suffering from a long span of scoliosis and gait disturbance since he was 1 year. The tumor contained multiple syrinx. The second case was a 3-year-old girl with ganglioglioma involving a large portion of the spinal cord from lower medulla to T3. This case also contained syringobulbia and syringomyelia in the multiple levels. She suffered from progressive motor weakness of all extremities. Owing to a good demarcation from normal spinal cord, the tumors could easily be removed by the microsurgical dissection through second stage operation in both cases. Histopathologically and ultrastructurally, the tumors were benign gangliogliomas. The symptoms improved a lot after surgery. Based on our two cases and literature review we confirmed that the spinal cord gangliogliomas are slowly growing tumors occurring in childhood or young adulthood. They often involve usually extensive level or entire length of the spinal cord and may be accompanied by scoliosis and gait disturbance when the tumor presents since infancy or early childhood as in our cases. They also often exhibit a cystic change of the tumor associated with large or small syringomyelia. Nearly total removal is possible due to a good demarcation from the normal spinal cord. Therefore, early diagnosis and surgical management are fundamental for the avoidance of unnecessary delay of surgery and a good result.

The down-regulation of surface expression of MHC class I molecules has recently been reported in the CD99-deficient lymphoblastoid B cell line displaying the characteristics of Hodgkin’s and Reed-Sternberg phenotype. Here, we demonstrate that the reduction of MHC class I molecules on the cell surface is primarily due to a defect in the transport from the Golgi complex to the plasma membrane. Loss of CD99 did not affect the steady-state expression levels of mRNA and protein of MHC class I molecules. In addition, the assembly of MHC class I molecules and the transport from the endoplasmic reticulum to the cis-Golgi occurred normally in the CD99-deficient cells, and no difference was detected between the CD99-deficient and the control cells in the pattern and degree of endocytosis. Instead, the CD99-deficient cells displayed the delayed transport of newly synthesized MHC class I molecules to the plasma membrane, thus causing accumulation of the molecules within the cells. The accumulated MHC class I molecules in the CD99-deficient cells were colocalized with α-mannosidase II and γ-adaptin in the Golgi compartment. These results suggest that CD99 may be associated with the post-Golgi trafficking machinery by regulating the transport to the plasma membrane rather than the endocytosis of surface MHC class I molecules, providing a novel mechanism of MHC class I down-regulation for immune escape.