Hypoxia Increases Gefitinib-Resistant Lung Cancer Stem Cells through the Activation of Insulin-Like Growth Factor 1 Receptor

Accumulating evidence indicates that a small population of cancer stem cells (CSCs) is involved in intrinsic resistance to cancer treatment. The hypoxic microenvironment is an important stem cell niche that promotes the persistence of CSCs in tumors. Our aim here was to elucidate the role of hypoxia and CSCs in the resistance to gefitinib in non-small cell lung cancer (NSCLC) with activating epidermal growth factor receptor (EGFR) mutation. NSCLC cell lines, PC9 and HCC827, which express the EGFR exon 19 deletion mutations, were exposed to high concentration of gefitinib under normoxic or hypoxic conditions. Seven days after gefitinib exposure, a small fraction of viable cells were detected, and these were referred to as “gefitinib-resistant persisters” (GRPs). CD133, Oct4, Sox2, Nanog, CXCR4, and ALDH1A1–all genes involved in stemness–were highly expressed in GRPs in PC9 and HCC827 cells, and PC9 GRPs exhibited a high potential for tumorigenicity in vivo. The expression of insulin-like growth factor 1 (IGF1) was also upregulated and IGF1 receptor (IGF1R) was activated on GRPs. Importantly, hypoxic exposure significantly increased sphere formation, reflecting the self-renewal capability, and the population of CD133- and Oct4-positive GRPs. Additionally, hypoxia upregulated IGF1 expression through hypoxia-inducible factor 1α (HIF1α), and markedly promoted the activation of IGF1R on GRPs. Knockdown of IGF1 expression significantly reduced phosphorylated IGF1R-expressing GRPs under hypoxic conditions. Finally, inhibition of HIF1α or IGF1R by specific inhibitors significantly decreased the population of CD133- and Oct4-positive GRPs, which were increased by hypoxia in PC9 and HCC827 cells. Collectively, these findings suggest that hypoxia increased the population of lung CSCs resistant to gefitinib in EGFR mutation-positive NSCLC by activating IGF1R. Targeting the IGF1R pathway may be a promising strategy for overcoming gefitinib resistance in EGFR mutation-positive NSCLC induced by lung CSCs and microenvironment factors such as tumor hypoxia.     

Krüppel‐like factor 4 regulates stemness and mesenchymal properties of colorectal cancer stem cells through the TGF‐β1/Smad/snail pathway

Krüppel‐like factor 4 (KLF4) was closely associated with epithelial‐mesenchymal transition and stemness in colorectal cancer stem cells (CSCs)‐enriched spheroid cells. Nonetheless, the underlying molecular mechanism is unclear. This study showed that KLF4 overexpression was accompanied with stemness and mesenchymal features in Lgr5⁺CD44⁺EpCAM⁺ colorectal CSCs. KLF4 knockdown suppressed stemness, mesenchymal features and activation of the TGF‐β1 pathway, whereas enforced KLF4 overexpression activated TGF‐β1, phosphorylation of Smad 2/3 and Snail expression, and restored stemness and mesenchymal phenotypes. Furthermore, TGF‐β1 pathway inhibition invalidated KLF4‐facilitated stemness and mesenchymal features without affecting KLF4 expression. The data from the current study are the first to demonstrate that KLF4 maintains stemness and mesenchymal properties through the TGF‐β1/Smad/Snail pathway in Lgr5⁺CD44⁺EpCAM⁺ colorectal CSCs.     

MicroRNA expression profile of colon cancer stem-like cells in HT29 adenocarcinoma cell line

Increasing evidence has suggested cancer stem cells (CSCs) are considered to be responsible for cancer formation, recurrence, and metastasis. Recently, many studies have also revealed that microRNAs (miRNAs) strongly implicate in regulating self renewal and tumorigenicity of CSCs in human cancers. However, with respect to colon cancer, the role of miRNAs in stemness maintenance and tumorigenicity of CSCs still remains to be unknown. In the present study, we isolated a population of colon CSCs expressing a CD133 surface phenotype from human HT29 colonic adenocarcinoma cell line by Flow Cytometry Cell Sorting. The CD133⁺ cells possess a greater tumor sphere-forming efficiency in vitro and higher tumorigenic potential in vivo. Furthermore, the CD133⁺ cells are endowed with stem/progenitor cells-like property including expression of “stemness” genes involved in Wnt2, BMI1, Oct3/4, Notch1, C-myc and other genes as well as self-renewal and differentiation capacity. Moreover, we investigated the miRNA expression profile of colon CSCs using miRNA array. Consequently, we identified a colon CSCs miRNA signature comprising 11 overexpressed and 8 underexpressed miRNAs, such as miR-429, miR-155, and miR-320d, some of which may be involved in regulation of stem cell differentiation. Our results suggest that miRNAs might play important roles in stemness maintenance of colon CSCs, and analysis of specific miRNA expression signatures may contribute to potential cancer therapy.     

Development and characterization of cancer stem cell-based tumoroids as an osteosarcoma model

Three-dimensional (3D) cancer tumor models are becoming vital approaches for high-throughput drug screening, drug targeting, development of novel theranostic systems, and personalized medicine. Yet, it is becoming more evident that the tumor progression and metastasis is fueled by a subpopulation of stem-like cells within the tumor that are also called cancer stem cells (CSCs). This study aimed to develop a tumoroid model using CSCs. For this purpose CD133⁺ cells were isolated from SaOS-2 osteosarcoma cell line with magnetic-activated cell sorting. To evaluate tumoroid formation ability, the cells were incubated in different cell numbers in agar gels produced by 3D Petri Dish® method. Subsequently, CD133⁺ cells and CD133⁻ cells were co-cultured to investigate CD133⁺ cell localization in tumoroids. The characterization of tumoroids was performed using Live&Dead staining, immunohistochemistry, and quantitative polymerase chain reaction analysis. The results showed that, CD133⁺, CD133⁻ and SaOS-2 cells were all able to form 3D tumoroids regardless of the initial cell number, but, while 72 hr were needed for CD133⁺ cells to self-assemble, 24 hr were enough for CD133⁻ and SaOS-2 cells. CD133⁺ cells were located within tumoroids randomly with high cell viability. Finally, when compared to two-dimensional (2D) cultures, there were 5.88, 4.14, 6.95, and 1.68-fold higher messenger RNA expressions for Sox2, OCT3/4, Nanog, and Nestin, respectively, in CD133⁺ cells that were cultured within 3D tumoroids, showing longer maintenance of stem cell phenotype in 3D, that can allow more relevant screening and targeting efficiency in pharmaceutical testing. It was concluded that CSC-based tumoroids are propitious as 3D tumor models to fill the gap between conventional 2D in vitro culture and in vivo animal experiments for cancer research.     

CD133<Superscript>+</Superscript>CD44<Superscript>+</Superscript> subgroups may be human small intestinal stem cells

The identification and separation of small intestinal epithelial stem cells are still on the preliminary stage. In this study, we planned to utilize immunohistochemistry, fluorescence-activated cell sorting (FACS) and RT-PCR to investigate the possibility of CD133 and CD44 as markers of human small intestinal epithelial stem cells. The expressions of CD133, CD44 and Lgr5 were studied by immunohistochemistry. Four subgroups of CD133⁺CD44⁺, CD133⁺CD44⁻, CD133⁻CD44⁺, CD133⁻CD44⁻ were sorted out through FACS and the expression level of Lgr5 gene was measured by RT-PCR and polyacrylamide gel electropheresis (PAGE) with sliver stained. Ten cases of samples were available for analyzing. By immunohistochemical staining, few cells with positive expressions of CD133, CD44 and Lgr5 were distributed in the bottom of crypts with the expression locations somewhat overlapped. The average percentage of CD133⁺CD44⁺ cells was 0.0580 ± 0.0403%, while the corresponding contents of CD133⁺CD44⁻ cells, CD133⁻CD44⁺ cells and CD133⁻CD44⁻ cells were 0.4000 ± 0.1225%, 0.7000 ± 0.2646% and 76.5600 ± 3.5529% respectively. Ten times of positive expressions of Lgr5 were detected in the CD133⁺CD44⁺ groups, while 9/10, 8/10 and 4/10 times for CD133⁺CD44⁻, CD133⁻CD44⁺ and CD133⁻CD44⁻ subgroups respectively. With the help of Quantityone 4.62 software, the densities of corresponding place to Lgr5 and reference gene were obtained. The density ratios of corresponding place to Lgr5 to reference gene were significant difference between subgroups (P < 0.001). By means of LSD method, the density ratios in CD133⁺CD44⁺ subgroups had statistical differences from the other subgroups (P < 0.05). We concluded CD133⁺CD44⁺ cells may be human small intestinal epithelial stem cells, which need further researches to confirm.     

CD44 and CD24 cannot act as cancer stem cell markers in human lung adenocarcinoma cell line A549

Cancer stem cells (CSCs) are subpopulations of tumor cells that are responsible for tumor initiation, maintenance and metastasis. Recent studies suggested that lung cancer arises from CSCs. In this study, the expression of potential CSC markers in cell line A549 was evaluated. We applied flow cytometry to assess the expression of putative stem cell markers, including aldehyde dehydrogenase 1 (ALDH1), CD24, CD44, CD133 and ABCG2. Cells were then sorted according to the expression of CD44 and CD24 markers by fluorescence-activated cell sorting (FACS) Aria II and characterized using their clonogenic and sphere-forming capacity. A549 cells expressed the CSC markers CD44 and CD24 at 68.16% and 54.46%, respectively. The expression of the putative CSC marker ALDH1 was 4.20%, whereas the expression of ABCG2 and CD133 was 0.93%. Double-positive CD44/133 populations were rare. CD44⁺/24⁺ and CD44⁺/CD24^?/low subpopulations respectively exhibited 64% and 27.92% expression. The colony-forming potentials in the CD44⁺/CD24⁺ and CD44⁺/CD24^?/low subpopulations were 84.37 ± 2.86% and 90 ± 3.06%, respectively, while the parental A549 cells yielded 56.65 ± 2.33% using the colony-formation assay. Both isolated subpopulations formed spheres in serumfree medium supplemented with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). CD44 and CD24 cannot be considered potential markers for isolating lung CSCs in cell line A549, but further investigation using in vivo assays is required.     

Simultaneous color‐coded imaging to distinguish cancer “stem‐like” and non‐stem cells in the same tumor

In this study, we demonstrate that the differential behavior, including malignancy and chemosensitivity, of cancer stem‐like and non‐stem cells can be simultaneously distinguished in the same tumor in real time by color‐coded imaging. CD133⁺ Huh‐7 human hepatocellular carcinoma (HCC) cells were considered as cancer stem‐like cells (CSCs), and CD133^? Huh‐7 cells were considered as non‐stem cancer cells (NSCCs). CD133⁺ cells were isolated by magnetic bead sorting after Huh‐7 cells were genetically labeled with green fluorescent protein (GFP) or red fluorescent protein (RFP). In this scheme, CD133⁺ cells were labeled with GFP and CD133^? cells were labeled with RFP. CSCs had higher proliferative potential compared to NSCCs in vitro. The same number of GFP CSCs and the RFP NSCCs were mixed and injected subcutaneously or in the spleen of nude mice. CSCs were highly tumorigenic and metastatic as well as highly resistant to chemotherapy in vivo compared to NSCCs. The ability to specifically distinguish stem‐like cancer cells in vivo in real time provides a visual target for prevention of metastasis and drug resistance. J. Cell. Biochem. 111: 1035–1041, 2010. © 2010 Wiley‐Liss, Inc.     

B7H1 Expression and Epithelial-To-Mesenchymal Transition Phenotypes on Colorectal Cancer Stem-Like Cells

Cancer stem cells (CSCs) can invade and metastasize by epithelial-to-mesenchymal transition (EMT). However, how they escape immune surveillance is unclear. B7H1 is crucial negative co-stimulatory molecule but little information about whether it works in CSCs. Therefore, we determined the expression of B7H1 and EMT-associated markers in colorectal cancer stem-like cells to investigate a possible immunoevasion way of CSCs. We enriched CD133⁺ colorectal cancer cells which manifested the CSCs-like properties such as higher levels of other stem cell markers Oct-4 and Sox-2, tumor sphere forming ability and more tumorigenic in NOD/SCID mice. These CD133⁺ cells possess EMT gene expression profile including higher level of Snail, Twist, vimentin, fibronectin and lower level of E-cadherin. Moreover, CD133⁺ cells in both cell line and colorectal cancer tissues expressed high level of negative co-stimulate molecule B7H1. Furthermore, some B7H1⁺ cancer cells also showed the characteristic of EMT, indicating EMT cells could escape immune attack during metastasis. B7H1 expression and EMT phenotypes on CSCs indicates a possible immunoevasion way.     

Glutathione metabolism is essential for self-renewal and chemoresistance of pancreatic cancer stem cells

BACKGROUNDCellular metabolism regulates stemness in health and disease.  A reduced redox state is essential for self-renewal of normal and cancer stem cells (CSCs). However, while stem cells rely on glycolysis, different CSCs, including pancreatic CSCs, favor mitochondrial metabolism as their dominant energy-producing pathway. This suggests that powerful antioxidant networks must be in place to detoxify mitochondrial reactive oxygen species (ROS) and maintain stemness in oxidative CSCs. Since glutathione metabolism is critical for normal stem cell function and CSCs from breast, liver and gastric cancer show increased glutathione content, we hypothesized that pancreatic CSCs also rely on this pathway for ROS detoxification.AIMTo investigate the role of glutathione metabolism in pancreatic CSCs.METHODSPrimary pancreatic cancer cells of patient-derived xenografts (PDXs) were cultured in adherent or CSC-enriching sphere conditions to determine the role of glutathione metabolism in stemness. Real-time polymerase chain reaction (PCR) was used to validate RNAseq results involving glutathione metabolism genes in adherent vs spheres, as well as the expression of pluripotency-related genes following treatment. Public TCGA and GTEx RNAseq data from pancreatic cancer vs normal tissue samples were analyzed using the webserver GEPIA2. The glutathione-sensitive fluorescent probe monochlorobimane was used to determine glutathione content by fluorimetry or flow cytometry. Pharmacological inhibitors of glutathione synthesis and recycling [buthionine-sulfoximine (BSO) and 6-Aminonicotinamide (6-AN), respectively] were used to investigate the impact of glutathione depletion on CSC-enriched cultures. Staining with propidium iodide (cell cycle), Annexin-V (apoptosis) and CD133 (CSC content) were determined by flow cytometry. Self-renewal was assessed by sphere formation assay and response to gemcitabine treatment was used as a readout for chemoresistance.RESULTSAnalysis of our previously published RNAseq dataset E-MTAB-3808 revealed up-regulation of genes involved in the KEGG (Kyoto Encyclopedia of Genes and Genomes) Pathway Glutathione Metabolism in CSC-enriched cultures compared to their differentiated counterparts. Consistently, in pancreatic cancer patient samples the expression of most of these up-regulated genes positively correlated with a stemness signature defined by NANOG, KLF4, SOX2 and OCT4 expression (P < 10^-5). Moreover, 3 of the upregulated genes (MGST1, GPX8, GCCT) were associated with reduced disease-free survival in patients [Hazard ratio (HR) 2.2-2.5; P = 0.03-0.0054], suggesting a critical role for this pathway in pancreatic cancer progression. CSC-enriched sphere cultures also showed increased expression of different glutathione metabolism-related genes, as well as enhanced glutathione content in its reduced form (GSH). Glutathione depletion with BSO induced cell cycle arrest and apoptosis in spheres, and diminished the expression of stemness genes. Moreover, treatment with either BSO or the glutathione recycling inhibitor 6-AN inhibited self-renewal and the expression of the CSC marker CD133. GSH content in spheres positively correlated with intrinsic resistance to gemcitabine treatment in different PDXs r = 0.96, P = 5.8 × 10¹¹). Additionally, CD133⁺ cells accumulated GSH in response to gemcitabine, which was abrogated by BSO treatment (P < 0.05). Combined treatment with BSO and gemcitabine-induced apoptosis in CD133⁺ cells to levels comparable to CD133^- cells and significantly diminished self-renewal (P < 0.05), suggesting that chemoresistance of CSCs is partially dependent on GSH metabolism.CONCLUSIONOur data suggest that pancreatic CSCs depend on glutathione metabolism. Pharmacological targeting of this pathway showed that high GSH content is essential to maintain CSC functionality in terms of self-renewal and chemoresistance.     

CD77-dependent retrograde transport of CD19 to the nuclear membrane: Functional relationship between CD77 and CD19 during germinal center B-cell apoptosis

A region of the N-terminal extracellular domain of the B-cell restricted cell differentiation antigen, CD19, has high amino acid sequence similarity to the receptor binding subunit B of verotoxin 1 (VT), an Escherichia coli elaborated cytotoxin, which specifically binds to the cell surface glycolipid, globotriaosylceramide, also known as the germinal center (GC) B-cell differentiation antigen, CD77. We have previously provided evidence of the association of CD19 and CD77 on the cell surface and in CD19-mediated homotypic adhesion of the Daudi Burkitt Lymphoma cell line, one normal counterpart of which is a subset of GC B cells. Evidence for the role of CD77 in CD19-induced apoptosis is now presented. Initial cell surface distribution, antibody-induced redistribution, internalization, and intracellular routing of CD19 were studied by confocal microscopy, IF, and postembedding IEM in CD77^+ve and CD77^−ve cells to investigate the possible role of CD77 in CD19 internalization and signaling. Daudi Burkitt's lymphoma cells were used as CD77^+ve cells and as CD77^−ve cells, Daudi mutant VT500 cells, and Daudi cells treated with PPMP, an inhibitor of CD77 synthesis, were used. Antibody ligated CD19 surface redistribution, internalization, and subcellular distribution of internalized CD19 was found to be different in CD77^+ve and CD77^−ve cells. A delay in internalization of antibody-CD19 complex was observed in CD77^−ve cells. Internalized CD19 was targeted to the nuclear envelope in CD77^+ve cells in a manner similar to that reported for VT, but not in CD77^−ve cells. Internalization of CD77 by ligation with verotoxin prevented the internalization of ligated CD19. Induction of apoptosis following crosslinking of cell surface CD19 was greater in CD77^+ve cells than in CD77^−ve cells. The nuclear targeting of internalized CD19 and induction of apoptosis following CD19 crosslinking only in CD77^+ve cells indicates a role for CD77-dependent CD19 retrograde transport from the B cell surface via the ER to the nuclear envelope in CD19-mediated signal transduction for apoptosis. J. Cell. Physiol. 176:281–292, 1998. © 1998 Wiley-Liss, Inc.     

Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal and produce differentiated progeny¹. These properties allow CSCs to recapitulate the original tumor when injected into immunocompromised mice. CSCs within an epithelial malignancy were first described in breast cancer and found to display specific cell surface antigen expression (CD44⁺CD24^low/-)². Since then, CSCs have been identified in an increasing number of other human malignancies using CD44 and CD24 as well as a number of other surface antigens. Physiologic properties, including aldehyde dehydrogenase (ALDH) activity, have also been used to isolate CSCs from malignant tissues^3-5.Recently, we and others identified CSCs from pancreatic adenocarcinoma based on ALDH activity and the expression of the cell surface antigens CD44 and CD24, and CD133^6-8. These highly tumorigenic populations may or may not be overlapping and display other functions. We found that ALDH⁺ and CD44⁺CD24⁺ pancreatic CSCs are similarly tumorigenic, but ALDH⁺ cells are relatively more invasive⁸. In this protocol we describe a method to isolate viable pancreatic CSCs from low-passage human xenografts⁹. Xenografted tumors are harvested from mice and made into a single-cell suspension. Tissue debris and dead cells are separated from live cells and then stained using antibodies against CD44 and CD24 and using the ALDEFLUOR reagent, a fluorescent substrate of ALDH¹⁰. CSCs are then isolated by fluorescence activated cell sorting. Isolated CSCs can then be used for analytical or functional assays requiring viable cells.     

Co‐expression of CD133+/CD44+ in human colon cancer and liver metastasis

Although relatively good therapeutic results are achieved in non‐advanced cancer, the prognosis of the advanced colon cancer still remains poor, dependent on local or distant recurrence of the disease. One of the factors responsible for recurrence is supposed to be cancer stem cells (CSCs) or tumor‐initiating cells, which are a population of cancer cells with ability to perpetuate themselves through self‐renewal and to generate differentiated cells, thought to be responsible for tumor recurrence. This study globally approach the possible role of tissue‐derived stem cells in the initiation of colon cancer and its metastatic process in the liver. Fresh surgical specimens from colon cancer, non‐tumor tissue and liver metastasis were obtained directly from the operating room, examined, and immediately processed. CSCs were selected under serum‐free conditions and characterized by CD44 and CD133 expression levels. CD133⁺/CD44⁺ cell populations were then investigated in paraffin‐embedded tissues and circulating tumor cells isolated from peripheral blood of the same group of colon cancer patients. Our data demonstrate that metastatic properties of cell populations from blood and liver metastasis, differently from primitive tumors, seem to be strictly related to the phenotype CD133 positive and CD44 positive. J. Cell. Physiol. 228: 408–415, 2013. © 2012 Wiley Periodicals, Inc.     

Cancer/testis antigens can be immunological targets in clonogenic CD133<Superscript>+</Superscript> melanoma cells

“Cancer stem cells” that resist conventional treatments may be a cause of therapeutic failure in melanoma. We report a subpopulation of clonogenic melanoma cells that are characterized by high prominin-1/CD133 expression in melanoma and melanoma cell lines. These cells have enhanced clonogenicity and self-renewal in vitro, and serve as a limited in vitro model for melanoma stem cells. In some cases clonogenic CD133⁺ melanoma cells show increased expression of some cancer/testis (CT) antigens. The expression of NY-ESO-1 in an HLA-A2 expressing cell line allowed CD133⁺ clonogenic melanoma cells to be targeted for killing in vitro by NY-ESO-1-specific CD8⁺ T-lymphocytes. Our in vitro findings raise the hypothesis that if melanoma stem cells express CT antigens in vivo that immune targeting of these antigens may be a viable clinical strategy for the adjuvant treatment of melanoma. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

miR-30 family promotes migratory and invasive abilities in CD133<Superscript>+</Superscript> pancreatic cancer stem-like cells

Pancreatic cancer is a deadly disease with a poor prognosis. Recently, miRNAs have been reported to be abnormally expressed in several cancers and play a role in cancer development and progression. However, the role of miRNA in cancer stem cells remains unclear. Therefore, our aim was to investigate the role of miRNA in the CD133⁺ pancreatic cancer cell line Capan-1M9 because CD133 is a putative marker of pancreatic cancer stem cells. Using miRNA microarray, we found that the expression level of the miR-30 family decreased in CD133 genetic knockdown shCD133 Capan-1M9 cells. We focused on miR-30a, -30b, and -30c in the miR-30 family and created pancreatic cancer cell sublines, each transfected with these miRNAs. High expression of miR-30a, -30b, or -30c had no effect on cell proliferation and sphere forming. In contrast, these sublines were resistant to gemcitabine, which is a standard anticancer drug for pancreatic cancer, and in addition, promoted migration and invasion. Moreover, mesenchymal markers were up-regulated by these miRNAs, suggesting that mesenchymal phenotype is associated with an increase in migration and invasion. Thus, our study demonstrated that high expression of the miR-30 family modulated by CD133 promotes migratory and invasive abilities in CD133⁺ pancreatic cancer cells. These findings suggest that targeted therapies to the miR-30 family contribute to the development of novel therapies for CD133⁺ pancreatic cancer stem cells.     

Down‐expression of miR‐154 suppresses tumourigenesis in CD133+ glioblastoma stem cells

Glioblastoma multiforme (GBM) is the most common and aggressive form of brain cancer. Evidences have suggested that CD133 is a marker for a subset of glioblastoma cancer stem cells. However, whether miRNA plays a critical role in CD133⁺ GBM is poorly understood. Here, we identified that miR‐154 was upregulated in CD133⁺ GBM cell lines. Knockdown of miR‐154 remarkably suppressed proliferation and migration of CD133⁺ GBM cells. Further study found that PRPS1 was a direct target of miR‐154 in CD133⁺ GBM cells. Overexpression of PRPS1 exhibited similar effects as miR‐154 knockdown in CD133⁺ GBMs. Our study identified miR‐154 as a previously unrecognized positive regulator of proliferation and migration in CD133⁺ GBM cells and a potentially therapeutic target of GBMs. Copyright © 2016 John Wiley & Sons, Ltd.     

Characterization of the natural immune response of rhesus monkey CD4+ve T cells to the bacterial antigen streptolysin O (SLO)

Abstract: Rhesus monkeys show a high proliferative T cell response to the bacterial exotoxin SLO without prior immunization. The present study was undertaken to characterize this naturally present SLO-responsiveness with particular emphasis on CD4^+ve reactive T cells. It is demonstrated that the frequency of SLO-reactive cells in the circulation ranges between 1 in 75 and 1 in 610 CD4^+ve T cells as determined with limiting dilution analysis. It is also shown that induction of a good proliferative response requires Mhc-DR matching between T cell and the antigen presenting cells (APC). Stable and DR-restricted SLO-specific CD4^+ve T cell lines were generated from CD8 depleted peripheral blood mononuclear cells (PBMC). The SLO-reactive CD4^+ve cell lines are tentatively characterized as Thl-like based on the predominant production of interferon-gamma (IFN-γ) over IL-4, although this seems contradicted by the IL-4 dependent growth of the lines.