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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.

Unlike conventional T cells, innate CD8 T cells develop a memory-like phenotype in the thymus and immediately respond upon antigen stimulation, similar to memory T cells. The development of innate CD8 T cells in the thymus is known to require IL-4, which upregulates Eomesodermin (Eomes). These features are similar to that of virtual memory CD8 T cells and IL-4-induced memory-like CD8 T cells generated in the peripheral tissues. However, the relationship between these cell types has not been clearly documented. In the present study, IL-4-induced memory-like CD8 T cells generated in the peripheral tissues were compared with innate CD8 T cells in terms of phenotype and function. When an IL-4/anti-IL-4 antibody complex (IL-4C) was injected into C57BL/6 mice daily for 7 days, the Eomes(hi)CXCR3 (+) CD8 T cell population was markedly increased in the peripheral lymphoid organs and blood. These cells were generated from naive CD8 T cells or accumulated via the expansion of pre-existing CD44(hi)CXCR3 (+) CD8 T cells. Initially, the majority of these CXCR3 (+) CD8 T cells expressed low levels of CD44, which was followed by the conversion to the CD44(hi) phenotype. This conversion was associated with the acquisition of enhanced effector function. After discontinuation of IL-4C treatment, Eomes expression levels gradually decreased in CXCR3 (+) CD8 T cells. Taken together, the results of this study demonstrate that IL-4-induced memory-like CD8 T cells generated in the peripheral lymphoid tissues are phenotypically and functionally similar to the innate CD8 T cells generated in the thymus.

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.

Background To understand humoral responses elicited after xenotransplantation, we compared the induction of anti-non-Gal antibodies vs. anti-Gal antibodies in non-human primates (NHPs) after intraportal porcine islet transplantation (PITX). Methods Anti-Gal and anti-non-Gal IgGs were analyzed in serial plasma samples of NHP recipients after PITX by enzyme-linked immunosorbent assay (ELISA) using synthetic Gal and by flow cytometry using α-1,3-galactosyltransferase gene knockout (GTKO) porcine endothelial cells, respectively. Anti-non-Gal IgG was detected in some recipients after PITX. The specificity of anti-non-Gal IgG was investigated by two-dimensional electrophoresis of the protein extract from GTKO porcine endothelial cells, Western blot analysis of recipient pre- and post-PITX plasma, and MALDI-TOF/TOF mass spectrometry, revealing albumin, a non-glycosylated protein in the serum supplement of the islets solution, as a putative antigen for anti-non-Gal IgG. The binding of IgG antibodies to human albumin (HA), bovine albumin (BA), porcine albumin (PA), and Gal was compared by ELISA in pre- and post-PITX plasma samples of 30 NHP recipients subjected to intraportal PITX, which were grouped according to the use of CD40-CD154 blockade and sirolimus. Results One of the immunoblot-matched spots was identified as BA by mass spectrometry. By ELISA, the plasma used in the immunoblot analysis revealed strong IgG binding to BA and PA, but not to HA. Anti-PA, anti-BA, and anti-Gal antibodies in NHP recipients 1 month after PITX were detected in 5 (100%), 3 (60%), and 5 (100%), respectively, of the 5 recipients receiving various immunosuppression (IS) without CD40-CD154 blockade (group I) and in 0 (0%), 0 (0%), and 4 (16%), respectively, of the 25 recipients receiving IS with CD40-CD154 blockade and sirolimus (group II). This finding revealed significant differences between the groups (P

Objective Stem cell-like memory T (Tscm) cells are long-lived memory T cells that have multipotent capacity to differentiate into different subsets. However, the role of Tscm cells in autoimmune diseases remains unclear. Here, we performed phenotypic studies to identify Tscm cells in patients experiencing systemic lupus erythematosus (SLE). Methods CD4+ and CD8+ Tscm cells were identified in SLE patients and healthy controls (HCs). In in vitro culture systems, CD4+ Tscm cells were induced to differentiate into subsets of T cells, including follicular helper T (Tfh) cells, and cytokine production patterns were assessed after stimulation. After confirming induction of transcription factors for Tfh cells, the capacity of CD4+ Tscm-derived Tfh cells to help B cells was analyzed by measuring antibody secretion. Results The percentages of CD4+ and CD8+ Tscm cells among the naive CD4+/CD8+ or total CD4+ T cell populations were significantly higher in SLE patients than in HCs. Stimulated Tscm cells from SLE patients could replenish themselves and differentiate into other T lymphocyte subsets, including Tfh cells upon stimulation with T cell receptor. Production of T cell factor 1, which is an inducer of Tfh, was also increased. The differentiated Tfh cells increased antibody production by autologous B cells. Conclusion Taken together, these findings suggest that Tscm cells play a role in the pathogenesis of SLE by maintaining Tfh cells.

BACKGROUND Pig islet xenotransplantation is a promising alternative to allogeneic transplantation. However, the wide immunologic barrier between pigs and primates limits the long-term survival of the graft. MD-3, a novel monoclonal antibody (mAb) that recognizes a particular epitope of human ICAM-1, can render T cells tolerant to a xenograft by arresting dendritic cell maturation. We report the long-term survival of adult wild-type pig islets and successful retransplantation in nonhuman primates using a protocol comprising induction with MD-3 mAb and maintenance with anti-CD154 mAb and sirolimus. METHODS Eleven rhesus monkeys were assigned to three groups. Group 1 (n = 4) involved treatment with MD-3 induction, short-term (

Background Intraportal islet transplantation (ITx) causes instant blood-mediated inflammatory reaction (IBMIR), resulting in an early loss of transplanted islets. Porcine islets, transplanted intraportally into nonhuman primates (NHPs), induce complement activation, contributing to the development of IBMIR; however, the exact mechanism is not clear. Methods Complement activation were compared after incubation of purified adult porcine islets in 20% human serum with or without complement inhibitors, C1 esterase inhibitor (C1E-inh), anti-factor B, and purified human factor H. Intraportal porcine ITx was performed in diabetic NHPs to which cobra venom factor (CVF), factor H, or none of complement inhibitor was administered during the peritransplant period. The extent of complement activation and function of islet grafts were monitored after ITx. Results The incubation of porcine islets with human serum resulted in generation of C3a, C4d, and factor Bb in the fluid phase. However, the generation of C3a after incubation was suppressed by anti-factor B or factor H, but not by C1E-inh. Moreover, in NHPs with porcine ITx, the administration of CVF or factor H ameliorated the increase in plasma C3a and factor Bb levels, as well as early release of porcine C-peptide after ITx. Furthermore, the functional survival of islet grafts was prolonged in the recipients of the CVF group compared to those in the control group. Conclusions The alternative complement pathway contributes to the development of IBMIR and the early loss of grafts in NHPs with porcine ITx. Complement inhibition during the peritransplant period may be beneficial for the survival of islet grafts.

Varieties of transforming growth factor-β (TGF-β) antagonists have been developed to intervene with excessive TGF-β signalling activity in cancer. Activin receptor-like kinase5 (ALK5) inhibitors antagonize TGF-β signalling by blocking TGF-β receptor-activated Smad (R-Smad) phosphorylation. Here we report the novel mechanisms how ALK5 inhibitors exert a therapeutic effect on a mouse B16 melanoma model. Oral treatment with a novel ALK5 inhibitor, EW-7197 (2.5 mg/kg daily) or a representative ALK5 inhibitor, LY-2157299 (75 mg/kg bid) suppressed the progression of melanoma with enhanced cytotoxic T-lymphocyte (CTL) responses. Notably, ALK5 inhibitors not only blocked R-Smad phosphorylation, but also induced ubiquitin-mediated degradation of the common Smad, Smad4 mainly in CD8(+) T cells in melanoma-bearing mice. Accordingly, T-cell-specific deletion of Smad4 was sufficient to suppress the progression of melanoma. We further identified eomesodermin (Eomes), the T-box transcription factor regulating CTL functions, as a specific target repressed by TGF-β via Smad4 and Smad3 in CD8(+) T cells. Thus, ALK5 inhibition enhances anti-melanoma CTL responses through ubiquitin-mediated degradation of Smad4 in addition to the direct inhibitory effect on R-Smad phosphorylation.

Langerhans cell histiocytosis (LCH) is the collective designation for a group of proliferative disorders of antigen-presenting cells in the epidermis. Over the past several decades, the etiology of LCH has been a controversial issue, particularly with respect to the pathologic process, that is, whether it is a neoplastic or inflammatory process. Recently, it was reported that the JL1 epitope, which encompasses the nonglycosylation site of CD43, is only exposed in the precursor stages of hematopoietic cells or in neoplastic conditions. We sought to investigate the possible utility of the JL1 monoclonal antibody as a diagnostic marker of LCH. In this study, we compared the staining characteristics of antibodies against the JL1 epitope with those of langerin and CD1a, which are widely used for the diagnosis of LCH. We found substantial differences in the staining patterns of these markers. The JL1 epitope could be bound by antibodies in cases of LCH and Langerhans cell (LC) sarcoma. In non-neoplastic lesions, JL1-positive LCs were found only in dermatitis, reflecting the immaturity of LCs in inflamed skin. However, anti-langerin antibodies were able to identify any form of LC, including those in normal skin, dermatitis, dermatopathic lymphadenopathy, and LCH. On the basis of these findings, we propose that the anti-JL1 antibody is a specific marker of immaturity, a feature that is shared in neoplastic LCs, and can be useful in the diagnosis of LCH.

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.

Previously, we developed a JL1 mouse monoclonal antibody that specifically recognizes the leukemic cells of T, B, and myeloid lineages, but not the peripheral blood cells and pluripotent hematopoietic stem cells. Here, we identified that JL1 mAb recognized a specific epitope of human CD43 and validated its potential as an anti-leukemic targeting agent. After the comprehensive screening of JL1 Ag in the human thymocyte cDNA library, multiple fusion gene constructs encoding human CD43 were generated to identify its specific epitope to JL1 antibody. JL1 antibody interacted with a developmentally regulated and non-glycosylated epitope of the human CD43 extracellular domain (AA 73-81, EGSPLWTSI). In an in vivo leukemia model using NOD/SCID mice injected with CCRF-CEM7 cells, JL1 antibody induced effective cytotoxicity in tumor cells and prolonged survival (p < 0.05). Saporin conjugation to JL1 antibody effectively depleted tumor cells in in vitro cytotoxic assays and also prolonged survival in a leukemic mouse model (p < 0.001). These preclinical results further support the therapeutic potential of the JL1 antibody in the management of acute leukemia.

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.

Administration of an ICAM-1–specific antibody arrests dendritic cells in a semi-immature state and facilitates antigen-specific T cell tolerance to islet allografts in humanized mice and Rhesus monkeys., 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.

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.

The detection of papillary microcarcinomas of the thyroid is increasing due to frequent use of ultrasound and fine-needle aspiration biopsy. Although most of the papillary microcarcinomas remain quiescent and follow an indolent clinical course, some behave aggressively and metastasize early, giving rise to clinically significant disease. There have been few studies concerning factors predictive of lymph node metastasis in papillary microcarcinomas. We analyzed the expression of S100A4, cyclin D1, p27 and MUC1, the presence of the BRAF V600E mutation and the clinicopathological features of the tumors, including patient age, tumor size (>or=5 vs

Despite considerable progress over the last decade, Hodgkin lymphoma (HL) remains a malignant tumor of unknown pathogenesis. The role of Epstein-Barr virus (EBV) and the activation of transcription factor NF-κB have been noted in the tumorigenesis of classic HL. Recently, a distinctive interaction between CD99 and the EBV viral latent membrane protein 1 (LMP1) was observed in the process of the malignant transformation of EBV-infected B cells. It was previously reported that CD99 expression is negatively regulated by LMP1, subsequently generating cells with Hodgkin and Reed-Sternberg (HRS) cell phenotype. Interestingly, several pieces of evidence have been recently revealed the presence of mutual regulation between CD99 and LMP1, which may assist in determining HL pathogenesis. In this review, the roles of CD99 and LMP1 in the generation of the HRS cell, a tumor cell of HL, will be discussed. We also suggest that CD99 and LMP1 interaction in an in vivo system may provide new insight into the understanding of the molecular mechanism of HL.

A microfluidic colorimetric sensor array was developed for detection and identification of various antioxidants. The sensor was fabricated by a photolithographic method, and consists of an array of printed cross-responsive indicators. The microfluidic design also incorporates pre-activation spots to allow printing of chemically incompatible components separately. Separately printed oxidizer allowed an oxidation of adjacent redox indicators only when aqueous sample was added to the sensor cartridge. Antioxidants were primarily detected by measuring the extent of inhibition of this oxidation reaction. Using this flow-based technique, a clear differentiation of 8 different antioxidants and 4 different teas has been demonstrated with 98.5% sensitivity.

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.

CD99-Like 2 (CD99L2) is a Type I glycoprotein expressed on leukocytes and endothelial cells as well as other cell types. It is related to CD99, although it shows only 38% sequence identity. CD99L2 has been shown to play a role in leukocyte extravasation in mice under various inflammatory conditions using anti-CD99L2 antibodies and, in one case by targeted deletion of CD99L2. We report here studies on an independently made CD99L2 “knockout mouse” that extend our knowledge of the role of CD99L2 in inflammation. CD99L2 deficiency did not affect the total or relative numbers of circulating leukocyte subsets, red blood cells, or platelets. Neither did CD99L2 deficiency affect the expression of ICAM-1, PECAM, or CD99 on endothelial cells. Mice lacking CD99L2 had a defective inflammatory response in the thioglycollate peritonitis model with a greater than 80% block in neutrophil infiltration and a nearly complete block in monocyte emigration into the peritoneal cavity measured 16 hours after the inflammatory challenge. The mice will be a useful resource to study the role of CD99L2 in various acute and chronic inflammatory diseases.

Memory-like CD8+ T cells expressing eomesodermin are a subset of innate T cells initially identified in a number of genetically modified mice, and also exist in wild mice and human. The acquisition of memory phenotype and function by these T cells is dependent on IL–4 produced by PLZF+ innate T cells; however, their physiologic function is still not known. Here we found that these IL-4-induced innate CD8+ T cells are critical for accelerating the control of chronic virus infection. In CIITA-transgenic mice, which have a substantial population of IL-4-induced innate CD8+ T cells, this population facilitated rapid control of viremia and induction of functional anti-viral T-cell responses during infection with chronic form of lymphocytic choriomeningitis virus. Characteristically, anti-viral innate CD8+ T cells accumulated sufficiently during early phase of infection. They produced a robust amount of IFN-γ and TNF-α with enhanced expression of a degranulation marker. Furthermore, this finding was confirmed in wild-type mice. Taken together, the results from our study show that innate CD8+ T cells works as an early defense mechanism against chronic viral infection., Author Summary Over the course of viral infection there may be a limited time period during which the host system can eliminate the virus. When viruses are not eliminated within this period of time, virus can establish persistent infection. Here, we show that IL-4-induced innate CD8+ T cells are able to effectively control chronic virus infection. Innate T cells are heterogeneous population of T cells that acquire effector/memory phenotype as a result of their maturation process in thymus, unlike conventional T cells that differentiate into memory cells after antigen encounter in periphery. Previous data suggest that innate T cells might serve as a first-line of defense against certain bacterial pathogens. IL-4-induced innate CD8+ T cells are a unique subset of innate T cells that were recently identified in both mouse and human. We found that IL-4-induced innate CD8+ T cells immediately accumulated after viral infection and produced a robust amount of effector cytokines. Thereby, IL-4-induced innate CD8+ T cells provide an effective barrier to the establishment of persistent infection via effective virus control during the early phase of viral infection. Collectively our data show that IL-4-induced innate CD8+ T cells works as an early defense mechanism against chronic viral infection.

Papillary thyroid cancers often occur as multiple foci. Multifocal cancers have been considered to have a poor prognosis because they are thought to be the consequence of intrathyroidal spread of the papillary cancer. However, to the authors' knowledge there has been little investigation into whether multifocal thyroid papillary carcinomas arise from the intrathyroidal spread of a single carcinoma or from independent primary tumors. To answer this question, the BRAF(V600E) mutational status of individual tumor foci was examined. This approach was justified because in the Korean population a high proportion (65%) of papillary carcinomas harbor the BRAF mutation.DNA was isolated from paraffin-embedded tissue samples of multifocal papillary thyroid carcinoma and the BRAF exon 15 was amplified by the polymerase chain reaction (PCR). The PCR product was digested with restriction endonuclease TspRI to test for the presence of the BRAF(V600E) (T1799A) mutation.In all, 140 cancers from 61 patients diagnosed with multifocal papillary carcinoma were examined. The BRAF mutation was found in all the individual cancers in 29 (47.5%) of the patients (all-positive group) and the mutation was absent in all the individual cancers in 8 (13.1%) patients (all-negative group). However, in 24 (39.3%) patients, some of the individual cancers contained the BRAF mutation, whereas others did not (mixed group).At least 39.3% of the multifocal papillary cancers in the Korean population that were examined could be attributed to independently arising papillary cancers rather than to intrathyroidal spread of single cancers.

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.

HUlip is a human homologue of a C. elegans gene, unc-33, that is developmentally regulated during maturation of the nervous system. HUlip is highly expressed only in the fetal brain and spinal cord, and is undetected in the adult brain. The purpose of this study was to investigate the pattern of hUlip expression in the developing human brain and nervous system tumors. Ten human brains at different developmental stages and 118 cases of nervous system tumor tissues were examined by immunohistochemistry. Twelve related tumor cell lines were also analyzed by northern blotting and immunoblotting. HUlip was expressed in late fetal and early postnatal brains; strongly in the neurons of the brain stem, basal ganglia/thalamus, and dentate gyrus of the hippocampus, and relatively weakly in the cerebral and cerebellar cortex. Among tumors, hUlip expression was easily detected in tumor cells undergoing neuronal differentiation such as ganglioneuroblastomas and ganglioneuromas. Furthermore, hUlip immunoreactivity was also found in various brain tumors showing neuronal differentiation: central neurocytomas (6 of 6 cases were positive), medulloblastomas (5/11), atypical teratoid rhabdoid tumor (1/1) and gangliogliomas (4/7). Some astrocytic tumors also showed weak positivity: astrocytomas (1 of 5 cases), anaplastic astrocytomas (2/5), and glioblastomas (3/11). Subependymal giant cell astrocytomas and subependymomas, which are of controversial histogenetic origin, showed strong hUlip immunoreactivity. The results of this study indicate that the expression of hUlip protein is distinctly restricted to the late fetal and early postnatal periods of human nervous system development and to certain subsets of nervous system tumors. The exact function of hUlip needs to be further clarified; yet the results of our study strongly imply that hUlip function is important in human nervous system development and its aberrant expression in various types of nervous system tumors suggests a role of hUlip as an oncofetal neural antigen.

Although NKT cells has been known to exert protective roles in the development of autoimmune diseases, the functional roles of NKT cells in the downstream events of antibody-induced joint inflammation remain unknown. Thus, we explored the functional roles of NKT cells in antibody-induced arthritis using the K/BxN serum transfer model. NKT cell-deficient mice were resistant to the development of arthritis, and wild-type mice administrated with alpha-galactosyl ceramide, a potent NKT cell activator, aggravated arthritis. In CD1d-/- mice, transforming growth factor (TGF)-beta1 was found to be elevated in joint tissues, and the blockade of TGF-beta1 using neutralizing monoclonal antibodies restored arthritis. The administration of recombinant TGF-beta1 into C57BL/6 mice reduced joint inflammation. Moreover, the adoptive transfer of NKT cells into CD1d-/- mice restored arthritis and reduced TGF-beta1 production. In vitro assay demonstrated that interleukin (IL)-4 and interferon (IFN)-gamma were involved in suppressing TGF-beta1 production in joint cells. The adoptive transfer of NKT cells from IL-4-/- or IFN-gamma-/- mice did not reverse arthritis and TGF-beta1 production in CD1d-/- mice. In conclusion, NKT cells producing IL-4 and IFN-gamma play a role in immune complex-induced joint inflammation by regulating TGF-beta1.

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 have previously reported the identification of a unique thymocyte-specific surface molecule, JL1, which was detected using the monoclonal antibody (mAb), anti-JL1. Interestingly, JL1 was shown to be expressed in most leukemias, irrespective of their immunophenotype, and subpopulations of normal bone marrow (BM) mononuclear cells (MNCs). Here we investigated the potential usefulness of the anti-JL1 mAb as a therapeutic tool for leukemia. We demonstrated that the proliferation of cultured human leukemia cells was dramatically inhibited in vitro by anti-JL1 mAb conjugated with the polypeptide toxin, gelonin, but not by gelonin alone. We then systematically investigated the reactivity of the anti-JL1 mAb against normal human tissues to evaluate possible side effects along with various hematopoietic and nonhematopoietic tumor cell lines. All of 33 types of normal tissues except thymus and subpopulation of BM MNCs were clearly devoid of JL1 expression. Among tumor cell lines, all the nonhematopoietic cell lines tested were negative for JL1 expression, while some hematopoietic cell lines contained JL1 antigen. Collectively, the results showed the cytotoxic effects of anti-JL1-based immunotoxin against JL1-positive leukemic cells, sparing most normal tissues other than thymocytes and some BM MNCs. Therefore, we strongly suggest that gelonin-conjugated anti-JL1 mAb immunotoxin could be developed as a potential immunotherapeutic agent in the treatment of various types of JL1-positive acute leukemias.

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.

Indoleamine 2,3-dioxygenase (IDO) is overexpressed in many different types of tumor and is associated with activation of FOXP3+ regulatory T cells (Treg cells) and downregulation of cytotoxic cellular immunity in the tumor microenvironment. It has been suggested that IDO inhibitors can be utilized as an effective therapeutic agent against human cancers. However, the expression of IDO and its association with tumor-infiltrating lymphocytes (TILs) remain unclear in papillary thyroid microcarcinoma (PTMC).Immunohistochemical staining for IDO expression was performed on 124 PTMC samples. TIL subsets (CD3+, CD8+, and FOXP3+ T cells) were counted in serial sections. The relationships between the expression of IDO and infiltration of TIL subsets, as well as the relationships between these immunomodulating factors and clinicopathologic parameters of PTMCs, were analyzed.There was a significant correlation between IDO expression and reduced CD3+ TIL and increased FOXP3+ TIL. IDO expression was found in 31% of PTMC and was associated with aggressive clinicopathologic features of the tumor such as extrathyroidal extension (ETE) and multifocality. High infiltration of FOXP3+ Treg cells in the tumor was associated with lymph node metastasis, ETE, and multifocality. Furthermore, high FOXP3/CD8+ ratio was associated with multifocality and lymph node metastasis, and high FOXP3+/CD3+ ratio was related to ETE and multifocality. In multivariate analyses, IDO expression was found to be an independent predictive factor for ETE and tumor multifocality.IDO expression and infiltration of FOXP3+ Treg cells were closely related to each other and were associated with aggressive features of PTMC, suggesting that disruption of antitumor immunity by IDO expression, and thus, infiltration of FOXP3+ Treg cells may contribute to tumor progression in PTMC.

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)

We have designed the gene delivery carrier targeted to Molt 4 cells, human leukemia T cells, using monoclonal antibody against leukemia-specific JL1 antigen, anti-JL1 antibody, as a targeting moiety. Anti-JL1 antibody has been proven to bind to JL1 antigen and subsequently be internalized into Molt 4 cells, demonstrating that anti-JL1 antibody has the potential as a targeting ligand for leukemia-specific gene transfer. Anti-JL1 antibody was modified with the heterobifunctional crosslinker, PDPH, at carbohydrate sites and conjugated to thiolated poly- l -lysine (PLL) via disulfide bridges. The composition and antigen binding affinity of antibody–PLL conjugates were analyzed by the amino acid analysis and the flow cytometry, respectively. Antibody–PLL conjugates neutralized pSV-β-galactosidase plasmid DNA at 5:1 weight ratio and condensed into about 200–300-nm complexes. DNA/antibody–PLL complexes were effectively internalized into Molt 4 cells after 4 h incubation at 37°C and showed significantly higher in vitro transfection efficiency than DNA/PLL complexes and DNA/Lipofectin™ formulation due to the targeting effect of receptor-mediated endocytosis induced by anti-JL1 antibody.

CM1 (centrocyte/-blast marker 1) defined by a mAb developed against concanavalin-A activated PBMC, is expressed specifically on some tonsillar germinal center (GC) B cells. In single flow cytometric analysis, the bone marrow did not express these molecules nor did the PBMC or the thymocytes. The peripheral B lymphocytes showed more than 90% positive, while the peripheral T lymphocytes showed approximately 60% positive at 48 h after activation by PMA/ionomycin, respectively. A western blot analysis and an immunoprecipitation for CM1 showed a band at 70 kDa. Cross-linking of CM1 with anti-CM1 mAb induced apoptosis of the GC B cells (CD38(+)IgD(-)). Immunohistochemical staining revealed that the CM1 molecule is distributed over the entire area except the proximal dark zone of the tonsillar germinal centers. These results suggest that the CM1 molecule might be involved in differentiation of the germinal center B cells as one of the novel centrocyte markers.

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.

Abnormality of the cyclin D1/cdk4/p16INK4a/pRb pathway during tumorigenesis has recently been reported. Hepatoblastoma is a rare malignant liver tumor of childhood, but underlying abnormalities of cell-cycle regulating protein remain to be elucidated. The expression of cyclin D1, cdk4, p16 and retinoblastoma gene product (pRb) was studied by immunohistochemistry in 17 paraffin-embedded tissues consisting of both tumor and corresponding non-neoplastic tissues. Tumor tissues showed overexpression of cyclin D1 (13/17, 76%) and cdk4 (15/17, 88%). Eleven cases showed co-overexpression of both cyclin D1 and cdk4. No abnormal p16 or pRb expression was noted. In the group with a high score (+4) for cyclin D1 expression, a positive correlation with tumor recurrence was noted (P = 0.043). These data suggest that overexpressed cyclin D1 and cdk4 protein might play an important role in the tumorigenesis of hepatoblastoma and that in the group with high cyclin D1 expression, tumor recurrence may be more frequent.

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.