Aureobasidium pullulans culture supernatant significantly stimulates R-848-activated phagocytosis of PMA-induced THP-1 macrophages.

Toll-like receptors (TLRs), which recognize a wide range of microbial pathogens and pathogen-related products, play important roles in innate immunology. Macrophages have a variety of TLRs, and pathogen binding to TLR resulted in the activation of macrophages. R-848, an immune response modifier, is an analog of imidazoquinoline derivative and binds to an endosome-localized TLR to exert an anti-viral response on leukocytes. In the present study, we verified that co-treatment of R-848 with other TLR agonists would enhance immune response. The culture supernatant of Aureobasidium pullulans (A. pullulans, which contains predominantly soluble ß-glucan), which binds to cell membrane-localized TLR, and to C-type lectin receptor Dectin-1, was treated together with R-848 to THP-1 macrophages. Compared to R-848 treatment alone, co-treatment of R-848 with A. pullulans culture supernatant significantly augmented TNF-α and IL-12p40 cytokine expression. Next, we investigated whether or not apoptotic cell uptake would be increased by co-treatment of R-848 with A. pullulans culture supernatant. To detect engulfed apoptotic cells, we induced apoptosis in human lymphoma Jurkat cells by 5-fluorouracil and stained them with fluorescent dye 5(6)-carboxytetramethylrhodamine (TAMRA), whereas THP-1 macrophage was labeled with fluorescein isothiocyanate-anti-CD14 and determined the percentage increase in TAMRA-positive THP-1 macrophages by flow cytometric assay. Since R-848 or A. pullulans treatment alone stimulated THP-1 macrophages to induce phagocytosis, co-treatment of R-848 with A. pullulans culture supernatant significantly augmented phagocytosis of apoptotic Jurkat cells. These results suggest that the activation of several different innate immune receptor pathways may enhance the immune response of R-848 significantly.

The human CD10 lacking an N-glycan at Asn(628) is deficient in surface expression and neutral endopeptidase activity.

BACKGROUND: CD10, also known as neprilysin or enkephalinase exhibiting neutral endopeptidase (NEP) activity, is expressed by B-lineage hematopoietic cells as well as a variety of cells from normal tissues. It cleaves peptides such as cytokines to act for terminating inflammatory responses. Although CD10 molecules of the human pre-B-cell line NALM-6 have 6 consensus N-glycosylation sites, three of them are known to be N-glycosylated by X-ray crystallography. METHODS: In order to investigate the role of N-glycans in the full expression of NEP activity, we modified N-glycans by treatment of NALM6 cells with various glycosidases or alter each of the consensus N-glycosylation sites by generating site-directed mutagenesis and compared the NEP activities of the sugar-altered CD10 with those of intact CD10. RESULTS: CD10 of the human B-cell line NALM-6 was dominantly localized in raft microdomains and heterogeneously N-glycosylated. Although neither desialylation nor further degalactosylation caused defective NEP activity, removal of only a small part of N-glycans by treatment with glycopeptidase F under non-denaturing conditions decreased NEP activity completely. All of the three consensus sites of CD10 in HEK293 cells introduced with wild type-CD10 were confirmed to be N-glycosylated. Surface expression of N-glycan at Asn(628)-deleted CD10 by HEK293 cells was greatly decreased as well as it lost entire NEP activities. CONCLUSIONS: N-glycosylation at Asn(628) is essential not only for NEP activities, but also for surface expression. GENERAL SIGNIFICANCE: Quality control system does not allow dysfunctional ecto-type proteases to express on plasma membrane.

Lipid rafts enriched in monosialylGb5Cer carrying the stage-specific embryonic antigen-4 epitope are involved in development of mouse preimplantation embryos at cleavage stage.

BACKGROUND: Lipid rafts enriched in glycosphingolipids (GSLs), cholesterol and signaling molecules play an essential role not only for signal transduction started by ligand binding, but for intracellular events such as organization of actin, intracellular traffic and cell polarity, but their functions in cleavage division of preimplantation embryos are not well known. RESULTS: Here we show that monosialylGb5Cer (MSGb5Cer)-enriched raft domains are involved in development during the cleavage stage of mouse preimplantation embryos. MSGb5Cer preferentially localizes at the interfaces between blastomeres in mouse preimplantation embryos. Live-imaging analysis revealed that MSGb5Cer localizes in cleavage furrows during cytokinesis, and that by accumulating at the interfaces, it thickens them. Depletion of cholesterol from the cell membrane with methyl-beta-cyclodextrin (MbCD) reduced the expression of MSGb5Cer and stopped cleavage. Extensive accumulation of MSGb5Cer at the interfaces by cross-linking with anti-MSGb5Cer Mab (6E2) caused F-actin to aggregate at the interfaces and suppressed the localization of E-cadherin at the interfaces, which resulted in the cessation of cleavage. In addition, suppression of actin polymerization with cytochalasin D (CCD) decreased the accumulation of MSGb5Cer at the interfaces. In E-cadherin-targeted embryos, the MSGb5Cer-enriched raft membrane domains accumulated heterotopically. CONCLUSIONS: These results indicate that MSGb5Cer-enriched raft membrane domains participate in cytokinesis in a close cooperation with the cortical actin network and the distribution of E-cadherin.

GalNAcß1,3-linked paragloboside carries the epitope of a sperm maturation-related glycoprotein that is recognized by the monoclonal antibody MC121.

The functional maturation of spermatozoa during epididymal transit in mammals accompanies the changes in their plasma membrane due to the binding or removal of proteins or interactions with the proteases, glycosidases and glycosyltransferases present in the epididymis. In order to study the surface changes in spermatozoa during their maturation in the epididymis, we previously established several monoclonal antibodies against the 54kDa sialoglycoprotein of mouse cauda epididymal spermatozoa, which gradually increased the expression of antigenic determinants during epididymal transit. One of these monoclonal antibodies, MC121, reacted with mouse sperm glycoproteins on a polyvinylidene fluoride membrane after desialylation of the glycoproteins, and the treatment of the desialylated sperm glycoproteins with ß-N-acetylhexosaminidase greatly decreased the expression of the antigenic determinants. In addition to reacting with mouse cauda epididymal spermatozoa, MC121 reacted with human red blood cells (hRBCs). MC121 induced agglutination of sialidase-treated hRBCs and stained hRBCs fixed with formalin vapor much more heavily than it stained hRBCs fixed with methanol. The thin layer chromatography (TLC) immunostaining of the sialidase-treated lipids of hRBCs with MC121 suggested that the epitope-bearing molecule is a glycosphingolipids (GSL), and that MC121 reacts with a pentaose-GSL. Analysis of sialidase-treated GSLs by TLC-Blot-Matrix Assisted Laser Desorption Ionization Time-of-Flight mass spectrometry (MALDI TOF MS) revealed that the GSL bound by MC121 was [HexNAc][HexNAc+Hex][Hex][Hex]-Cer. The lipid band stained with mAb TH2, which is specific for a GSL, GalNAcß1-3Galß1-4GlcNAcß1-3Galß1-4Glcß1-ceramide. These results indicated that the epitope to which MC121 binds is present in a neolacto-series GSL, IV³GalNAcß-nLc4Cer² sequence.

Accelerated biosynthesis of neolacto-series glycosphingolipids in differentiated mouse embryonal carcinoma F9 cells detected by using dodecyl N-acetylglucosaminide as a saccharide primer.

Using dodecyl N-acetylglucosaminide (GlcNAc-C12) as a saccharide primer, we investigated the biosynthetic changes of neolacto-series glycosphingolipids (GSLs) in mouse embryonal carcinoma F9 cells during differentiation induced by retinoic acid plus dibutyryl cyclic AMP (RA/dbcAMP). In the differentiated cells, the glycosylation of GlcNAc-C12 was greatly enhanced. The sugar compositions of glycosylated primers were assigned as Hex-GlcNAc, [Hex](2)-GlcNAc, [Hex](2)[HexNAc]-GlcNAc, and [NeuAc][Hex]-GlcNAc by liquid chromatography-tandem mass spectrometry. The detection of augmented biosynthesis of endogenous sialylparagloboside indicated that [NeuAc][Hex]-GlcNAc was predicted to be the non-reducing end trisaccharide of sialylparagloboside. The transcription of B3gnt5, B4galt1, Ggta1, Fut4 and St3gal6, encoding glycosyltransferases involved in the neolacto-series glycosphingolipids biosynthesis, was increased, whereas that of Fut9 and St6galI was decreased after RA/dbcAMP treatment. Furthermore, the sialyltransferase activity of ST3GalVI sialylating paragloboside was enhanced with the increase in St3gal6 expression. Since most stage-specific embryonic antigen-1 (SSEA-1) active determinants are carried by glycoproteins in F9 cells, the changes in glycolipid metabolism do not seem to be closely related to loss of cell surface SSEA-1 expression upon F9 differentiation. These results indicate that RA/dbcAMP treatment activates the biosynthesis of neolacto-series GSL and enhances sialylation of paragloboside in F9 cells with down-regulation of Fut9 expression.

A microfabricated scaffold induces the spheroid formation of human bone marrow-derived mesenchymal progenitor cells and promotes efficient adipogenic differentiation.

Here, we report the highly efficient in vitro differentiation of human bone marrow-derived mesenchymal stem/progenitor cells (MPCs) using a novel nanotechnology-based culture plate, nanoculture plate(®) (NCP). The NCP contains uneven microfabrications with diameters of ∼2-3 µm arranged in a honeycomb pattern on its culture surface, which is devoid of animal-derived protein sources. When human MPCs were subjected to three-dimensional (3D) culture using an NCP, they rapidly formed adhesive spheroids. We showed that adipogenic differentiation in NCP-mediated 3D cultures led to more rapid accumulation of triglycerides than that in two-dimensional cultures. During adipogenesis in 3D cultures, the rapid and intense induction of adipocyte-specific gene expressions, such as peroxisome proliferator-activated receptor γ (PPAR-γ), CCAAT-enhancer-binding protein α (C/EBP-α), adipocyte protein 2 (aP2), and adiponectin was observed. Together, these results indicate that this 3D culture system is suitable for the differentiation of human MPCs into adipogenic lineage, and could be applicable to adipose tissue engineering under xeno-free condition.

Neuroblastoma cells can be classified according to glycosphingolipid expression profiles identified by liquid chromatography-tandem mass spectrometry.

It is hoped that the gangliosides contained in neuroblastomas (NBs) can be used as outcome predictors. We used liquid chromatography-tandem mass spectrometry (LC-MS) to analyze the gangliosides expressed in 11 NB cell lines. LC-MS analysis detected a number of gangliosides, including acetylated forms, with significantly higher sensitivity than conventional high-performance thin-layer chromatography analysis, and the results revealed that the expression profiles of the gangliosides GD1a, GD2, and acetylated GD2 differed according to the NB cell line. Hierarchical clustering based on the ganglioside expression profiles obtained by LC-MS analysis revealed that the NB cell lines could be classified into three types according to their expression of these three gangliosides: A-type characterized by high expression of GD1a and low or no expression of GD2/acetylated GD2, B-type characterized by low or no expression of GD1a and high expression of GD2/acetylated GD2, and AB-type characterized by expression of both GD1a and GD2/acetylated GD2. Interestingly, all three MYCN non-amplified cell lines were classified into the A-type. The classification was found to be correlated with mRNA expression of ganglioside synthase and neural-differentiation-related genes. The results of this study indicate that LC-MS analysis is useful as a tool for glycosphingolipid research on malignancies.

Differential effects of BAFF on B cell precursor acute lymphoblastic leukemia and Burkitt lymphoma.

B cell-activating factor belonging to the tumor necrosis factor superfamily (BAFF) is a crucial factor for B cell development and is involved in the survival of malignant B cells, but its effect on B cell precursors (BCPs) remains unclear. We investigated BCP acute lymphoblastic leukemia (-ALL) cells for BAFF receptor (-R) expression and compared the effect of BAFF on BCP-ALL cells and Burkitt lymphoma (BL) cells. Expression of BAFF-R was detected in some cell lines and some clinical specimens of both BL and BCP-ALL. BAFF acted on both BL and BCP-ALL cells and promoted proliferation by both. BAFF also inhibited apoptosis by BL cells induced by cross-linking of cell surface molecules and anticancer drugs, but failed to inhibit apoptosis by BCP-ALL cells. BAFF induced prompt and obvious activation of the NF-kappaB signaling pathway in BL cells, but only weak and delayed activation of the pathway in BCP-ALL cells. The results of this study indicate that some BCP-ALL cells and some BL cells express BAFF-R, but that the effects of BAFF on BCP-ALL cells are different from its effects on mature B cell malignancies.

Kinetics and effect of integrin expression on human CD34(+) cells during murine leukemia virus-derived retroviral transduction with recombinant fibronectin for stem cell gene therapy.

The CH-296 recombinant fragment of human fibronectin is essential for murine leukemia virus (MLV)-derived retroviral transduction of CD34(+) cells for the purpose of stem cell gene therapy. Although the major effect of CH-296 is colocalization of the MLV-derived retrovirus and target cells at specific adhesion domains of CH-296 mediated by integrins expressed on CD34(+) cells, the precise roles of the integrins are unclear. We examined the kinetics of integrin expression on CD34(+) cells during the course of MLV-derived retrovirus-mediated gene transduction with CH-296. Flow cytometry revealed that the levels of both very late activation protein (VLA)-4 and VLA-5 on CD34(+) cells freshly isolated from cord blood were insufficient for effective MLV-derived retroviral transduction. However, increases were achieved during culture for preinduction and MLV-derived retrovirus-mediated gene transduction in the presence of a cocktail of cytokines. In addition, we confirmed by using specific antibodies that inhibition of the cell adhesion mediated by the integrins significantly reduced transduction efficiency, indicating that integrin expression is indeed important for CH-296-based MLV-derived retroviral transduction. Only a few cytokines are capable of inducing integrin expression, and stem cell factor plus thrombopoietin was found to be the minimal combination that was sufficient for effective transduction of an MLV-derived retrovirus based on CH-296. Our findings should be useful for improving the culture conditions for CH-296-based MLV-derived retroviral transduction in stem cell gene therapy.

The expression of granulysin in systemic anaplastic large cell lymphoma in childhood.

The expression of granulysin, a cytolytic protein produced by activated T and NK cells, has been revealed to be correlated with the prognosis of some adult cancer patients. By examination on various childhood lymphoma tissues, we found that granulysin level was especially high in systemic anaplastic large cell lymphoma (ALCL) cases, whereas no close correlation with the expression of CD96, a marker for activated T and NK cells, was observed. We further demonstrated that both ALCL cells in biopsy specimens and cell lines established from ALCL express granulysin, indicating some correlation of granulysin with biological features of ALCL.

EWS/ETS regulates the expression of the Dickkopf family in Ewing family tumor cells.

BACKGROUND: The Dickkopf (DKK) family comprises a set of proteins that function as regulators of Wnt/beta-catenin signaling and has a crucial role in development. Recent studies have revealed the involvement of this family in tumorigenesis, however their role in tumorigenesis is still remained unclear. METHODOLOGY/PRINCIPAL FINDINGS: We found increased expression of DKK2 but decreased expression of DKK1 in Ewing family tumor (EFT) cells. We showed that EFT-specific EWS/ETS fusion proteins enhance the DKK2 promoter activity, but not DKK1 promoter activity, via ets binding sites (EBSs) in the 5' upstream region. EWS/ETS-mediated transactivation of the promoter was suppressed by the deletion and mutation of EBSs located upstream of the DKK2 gene. Interestingly, the inducible expression of EWS/ETS resulted in the strong induction of DKK2 expression and inhibition of DKK1 expression in human primary mesenchymal progenitor cells that are thought to be a candidate of cell origin of EFT. In addition, using an EFT cell line SK-ES1 cells, we also demonstrated that the expression of DKK1 and DKK2 is mutually exclusive, and the ectopic expression of DKK1, but not DKK2, resulted in the suppression of tumor growth in immuno-deficient mice. CONCLUSIONS/SIGNIFICANCE: Our results suggested that DKK2 could not functionally substitute for DKK1 tumor-suppressive effect in EFT. Given the mutually exclusive expression of DKK1 and DKK2, EWS/ETS regulates the transcription of the DKK family, and the EWS/ETS-mediated DKK2 up-regulation could affect the tumorigenicity of EFT in an indirect manner.

Ex vivo expanded cord blood CD4 T lymphocytes exhibit a distinct expression profile of cytokine-related genes from those of peripheral blood origin.

With an increase in the importance of umbilical cord blood (CB) as an alternative source of haematopoietic progenitors for allogenic transplantation, donor lymphocyte infusion (DLI) with donor CB-derived activated CD4(+) T cells in the unrelated CB transplantation setting is expected to be of increased usefulness as a direct approach for improving post-transplant immune function. To clarify the characteristics of activated CD4(+) T cells derived from CB, we investigated their mRNA expression profiles and compared them with those of peripheral blood (PB)-derived activated CD4(+) T cells. Based on the results of a DNA microarray analysis and quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR), a relatively high level of forkhead box protein 3 (Foxp3) gene expression and a relatively low level of interleukin (IL)-17 gene expression were revealed to be significant features of the gene expression profile of CB-derived activated CD4(+) T cells. Flow cytometric analysis further revealed protein expression of Foxp3 in a portion of CB-derived activated CD4(+) T cells. The low level of retinoic acid receptor-related orphan receptor gamma isoform t (RORgamma t) gene expression in CB-derived activated CD4(+) T cells was speculated to be responsible for the low level of IL-17 gene expression. Our data indicate a difference in gene expression between CD4(+) T cells from CB and those from PB. The findings of Foxp3 expression, a characteristic of regulatory T cells, and a low level of IL-17 gene expression suggest that CB-derived CD4(+) T cells may be a more appropriate source for DLI.

Human osteoblasts support hematopoietic cell development in vitro.

BACKGROUND/AIM: Although osteoblasts are thought to be the major component of the hematopoietic stem cell niche in the bone marrow microenvironment, the role of osteoblasts in hematopoiesis is still unclear. The ability of human osteoblasts to support early hematopoiesis was investigated. METHODS AND RESULTS: Human CD34+ bone marrow cells cultured on human osteoblasts were capable of surviving without addition of cytokines and differentiated into myeloid cells with slight proliferation. The results of immunohistochemical experiments suggested activation of FAK and AKT in hematopoietic cells attached to osteoblasts. When stem cell factor, Flt3-L, and IL-3 were added to the coculture system, each cytokine distinctively enhanced proliferation and differentiation of CD34+ bone marrow cells. CONCLUSION: The results suggest that human osteoblasts have the ability to support hematopoietic cell development in vitro.

B-cell-activating factor inhibits CD20-mediated and B-cell receptor-mediated apoptosis in human B cells.

B-cell-activating factor (BAFF) is a survival and maturation factor for B cells belonging to the tumour necrosis factor superfamily. Among three identified functional receptors, the BAFF receptor (BAFF-R) is thought to be responsible for the effect of BAFF on B cells though details of how remain unclear. We determined that a hairy-cell leukaemia line, MLMA, expressed a relatively high level of BAFF-R and was susceptible to apoptosis mediated by either CD20 or B-cell antigen receptor (BCR). Using MLMA cells as an in vitro model of mature B cells, we found that treatment with BAFF could inhibit apoptosis mediated by both CD20 and BCR. We also observed, using immunoblot analysis and microarray analysis, that BAFF treatment induced activation of nuclear factor-kappaB2 following elevation of the expression level of Bcl-2, which may be involved in the molecular mechanism of BAFF-mediated inhibition of apoptosis. Interestingly, BAFF treatment was also found to induce the expression of a series of genes, such as that for CD40, related to cell survival, suggesting the involvement of a multiple mechanism in the BAFF-mediated anti-apoptotic effect. MLMA cells should provide a model for investigating the molecular basis of the effect of BAFF on B cells in vitro and will help to elucidate how B cells survive in the immune system in which BAFF-mediated signalling is involved.

Inducible expression of chimeric EWS/ETS proteins confers Ewing's family tumor-like phenotypes to human mesenchymal progenitor cells.

Ewing's family tumor (EFT) is a rare pediatric tumor of unclear origin that occurs in bone and soft tissue. Specific chromosomal translocations found in EFT cause EWS to fuse to a subset of ets transcription factor genes (ETS), generating chimeric EWS/ETS proteins. These proteins are believed to play a crucial role in the onset and progression of EFT. However, the mechanisms responsible for the EWS/ETS-mediated onset remain unclear. Here we report the establishment of a tetracycline-controlled EWS/ETS-inducible system in human bone marrow-derived mesenchymal progenitor cells (MPCs). Ectopic expression of both EWS/FLI1 and EWS/ERG proteins resulted in a dramatic change of morphology, i.e., from a mesenchymal spindle shape to a small round-to-polygonal cell, one of the characteristics of EFT. EWS/ETS also induced immunophenotypic changes in MPCs, including the disappearance of the mesenchyme-positive markers CD10 and CD13 and the up-regulation of the EFT-positive markers CD54, CD99, CD117, and CD271. Furthermore, a prominent shift from the gene expression profile of MPCs to that of EFT was observed in the presence of EWS/ETS. Together with the observation that EWS/ETS enhances the ability of cells to invade Matrigel, these results suggest that EWS/ETS proteins contribute to alterations of cellular features and confer an EFT-like phenotype to human MPCs.

The detergent-insoluble microdomains, rafts, can be used as an effective immunogen.

Detergent-insoluble microdomains, or rafts, act as a platform to transduce signals from the extracellular space into the cytoplasm. In the process of developing monoclonal antibodies against raft molecules for the purpose of studying the molecular mechanism of raft-mediated signaling, we observed the uniqueness and certain advantages of immunization with rafts. Simple subcutaneous injection of mice with a phosphate-buffered saline (PBS) suspension of rafts without mixing with Freund's adjuvant made it possible to increase the titer of antiserum reacting with raft components. Interestingly, injection of rafts prepared from certain specific cell lines induced monoglycolipid-specific antibodies. Furthermore, antibodies were produced by raft-immunization of even syngeneic mice. Our findings suggest that this phenomenon does not represent a breakdown of immunological self-tolerance, but typical immune reactions accompanying the class switch from IgM antibodies to IgG antibodies.

Preferential localization of SSEA-4 in interfaces between blastomeres of mouse preimplantaion embryos.

The monoclonal antibody 6E2 raised against the embryonal carcinoma cell line NCR-G3 had been shown to also react with human germ cells. Thin-layer chromatography (TLC) immunostaining revealed that 6E2 specifically reacts with sialosylglobopenta osylceramide (sialylGb5), which carries an epitope of stage-specific embryonic antigen-4 (SSEA-4), known as an important cell surface marker of embryogenesis. The immunostaining of mouse preimplantation embryos without fixation showed that the binding of 6E2 caused the clustering and consequent accumulation of sialylGb5 at the interface between blastomeres. These results suggest that SSEA-4 actively moves on the cell surface and readily accumulates between blastomeres after binding of 6E2.

Laminin binding protein, 34/67 laminin receptor, carries stage-specific embryonic antigen-4 epitope defined by monoclonal antibody Raft.2.

We previously produced monoclonal antibodies against the detergent-insoluble microdomain, i.e., the raft microdomain, of the human renal cancer cell line ACHN. Raft.2, one of these monoclonal antibodies, recognizes sialosyl globopentaosylceramide, which has the stage-specific embryonic antigen (SSEA)-4 epitope. Although the mouse embryonal carcinoma (EC) cell line F9 does not express SSEA-4, some F9 cells stained with Raft.2. Western analysis and matrix-assisted laser desorption ionization-time of flight mass spectrometry identified the Raft.2 binding molecule as laminin binding protein (LBP), i.e., 34/67 laminin receptor. Weak acid treatment or digestion with Clostridium perfringens sialidase reduced Raft.2 binding to LBP on nitrocellulose sheets and [(14)C]galactose was incorporated into LBP, indicating LBP to have a sialylated carbohydrate moiety. Subcellular localization analysis by sucrose density-gradient centrifugation and examination by confocal microscopy revealed LBP to be localized on the outer surface of the plasma membrane. An SSEA-4-positive human EC cell line, NCR-G3 cells, also expressed Raft.2-binding LBP.

Dietary bioflavonoids induce apoptosis in human leukemia cells.

Dietary bioflavonoids are secondary metabolites of plants that are known to have a variety of bio-effects, including anti-cancer activity. In this study, we examined the effects of flavonoids on the growth of human leukemia cells and found that certain flavonoids induce apoptosis in a variety of human leukemia cells. The apoptosis induced by bioflavonoids was dose-dependent and was accompanied by a disruption of the mitochondrial transmembrane potential and the activation of caspase. Our data suggests that dietary bioflavonoids may be useful chemotherapeutic reagents for leukemia patients.

Development of novel monoclonal antibody 4G8 against swine leukocyte antigen class I alpha chain.

A mouse monoclonal antibody (MAb) was generated against swine leukocyte antigen (SLA) class I alpha chain. A newly developed series of MAb clones that react with pan leukocytes were selected and tested by immuno-histochemistry using SLA class I alpha chain expressing Cos-7 cells. Among them, MAb 4G8 was characterized by the following features: (1) 4G8 reacted with Cos-7 cells transfected with SLA class I alpha chain from the d haplotype, (2) 4G8 recognized epitopes that were different from those of commercially available anti-SLA class I MAbs 74-11-10 and PT85A, and (3) 4G8 could be used to immunostain frozen sections of thymus, spleen, lymph node, kidney, and liver tissues with good results.