Limiting Dilution Cloning Research Articles

T cell lines recognizing the 70-kD protein of U1 small nuclear ribonucleoprotein (U1snRNP).

In sera of patients with mixed connective tissue disease (MCTD) high titres of IgG autoantibodies to U1snRNP-specific proteins (70 kD, A, C) are found, suggesting an antigen-driven and T-cell-dependent process. In order to establish U1snRNP-specific T cell lines we cultured under various culture conditions mononuclear cells from MCTD patients and healthy donors with a highly purified UsnRNP preparation from HeLa cells. Nine T cell lines were established by limiting dilution cloning from two MCTD patients and five T cell lines from a healthy individual. All T cell lines expressed the TCR alpha beta/CD3 complex. Surprisingly, most of the T cells lines exhibited the CD8 phenotype. Irrespective of this phenotype, all T cell lines showed a proliferative response to an N-terminal part (aa 51-195) of recombinant U1-specific 70-kD protein. One CD8+ T cell clone exhibited cytotoxic activity against an autologous B cell line pulsed with snRNP or recombinant fragments (aa 51-95 and aa 51-88). Interestingly, two T cell lines proliferated in response to four recombinant polypeptides representing different parts of the U1snRNP 70-kD protein. Since regions of sequence homology are distributed over the 70-kD molecule, it is suggested that conserved motifs may be recognized by the T cell lines.

Transgenic expression of HLA‐E single chain trimer protects porcine endothelial cells against human natural killer cell‐mediated cytotoxicity

The susceptibility of porcine endothelial cells (pEC) to human natural killer (NK) cells is related to the failure of human major histocompatibility complex (MHC)-specific killer inhibitory receptors to recognize porcine MHC class I molecules. The aims of this study were (i) to assess the protection of pEC against xenogeneic NK-mediated cytotoxicity afforded by the stable expression of HLA-E single chain trimers (SCT) composed of a canonical HLA-E binding peptide antigen, VMAPRTLIL, the mature human beta2-microglobulin, and the mature HLA-E heavy chain, and (ii) to test whether HLA-E expression on pEC and porcine lymphoblastoid cells affects the adhesion of human NK cells. Porcine EC lines expressing different levels of HLA-E SCT were generated by Ca(2)PO(4)-transfection followed by limiting dilution cloning. Surface expression of HLA-E was measured by flow cytometry. Susceptibility of transfected pEC lines against human NK cells was tested in (51)Cr-release cytotoxicity assays. Interactions between human NK cells and HLA-E positive pEC or porcine lymphoblastoid cells were further addressed in adhesion and conjugation assays. The level of protection of pEC from human NK-mediated cytotoxicity correlated with the intensity of surface HLA-E expression. Furthermore, the HLA-E SCT-mediated protection was specifically reversed by blocking the HLA-E specific NK inhibitory receptor CD94/NKG2A. HLA-E expression does neither affect the adhesion of human NK cells to pEC nor the heteroconjugate formation between human NK and porcine 13271.10 cells. Stable surface expression of HLA-E on pEC was achieved in the absence of extrinsic peptide pulsing and provided partial protection from human NK cytotoxicity. Though insufficient to inhibit xenogeneic NK cell reactivity completely, transgenic HLA-E expression on pig organs might contribute to a successful application of clinical xenotransplantation in combination with other protective strategies.

186: Superior antitumor in vitro responses of allogeneic matched sibling compared to autologous patient CD8 + T cells

Allogeneic cell therapy as a means to break immunotolerance to solid tumors is increasingly used for cancer treatment. To investigate cellular alloimmune responses in a human tumor model, primary cultures were established from renal cell carcinoma (RCC) tissues of 56patients. In three patients with stable RCC line and human leukocyte antigen (HLA)-identical sibling donor available, allogeneic and autologous RCC reactivities were compared using mixed lymphocyte/ tumor cell cultures (MLTC). Responding lymphocytes were exclusively CD8 + T cells, whereas CD4 + T cells or natural killer cells were never observed. Sibling MLTC populations showed higher proliferative and cytolytic antitumor responses compared with their autologous counterparts. The allo-MLTC responders originated from the CD8 + CD62L(high) + peripheral blood subpopulation containing naive precursor and central memory T cells. Limiting dilution cloning failed to establish CTL clones from autologous MLTCs or tumor-infiltrating lymphocytes. In contrast, a broad panel of RCC-reactive CTL clones was expanded from each allogeneic MLTC. These sibling CTL clones either recognized exclusively the original RCC tumor line or cross-reacted with nonmalignant kidney cells of patient origin. A minority of CTL clones also recognized patient-derived hematopoietic cells or other allogeneic tumor targets. The MHC-restricting alleles for RCC-reactive sibling CTL clones included HLA-A2, HLA-A3, HLA-A11, HLA-A24, and HLA-B7. In one sibling donor-RCC pair, strongly proliferative CD3 + CD16 + CD57 + CTL clones with non-HLA-restricted antitumor reactivity were established. Our results show superior tumor-reactive CD8 responses of matched allogeneic compared with autologous T cells. These data encourage the generation of antitumor T-cell products from HLA-identical siblings and their potential use in adoptive immunotherapy of metastatic RCC patients. (Cancer Res 2006; 66(23): 11447-54)

Superior AntitumorIn vitroResponses of Allogeneic Matched Sibling Compared with Autologous Patient CD8+ T Cells

Allogeneic cell therapy as a means to break immunotolerance to solid tumors is increasingly used for cancer treatment. To investigate cellular alloimmune responses in a human tumor model, primary cultures were established from renal cell carcinoma (RCC) tissues of 56 patients. In three patients with stable RCC line and human leukocyte antigen (HLA)-identical sibling donor available, allogeneic and autologous RCC reactivities were compared using mixed lymphocyte/tumor cell cultures (MLTC). Responding lymphocytes were exclusively CD8(+) T cells, whereas CD4(+) T cells or natural killer cells were never observed. Sibling MLTC populations showed higher proliferative and cytolytic antitumor responses compared with their autologous counterparts. The allo-MLTC responders originated from the CD8(+) CD62L(high)(+) peripheral blood subpopulation containing naive precursor and central memory T cells. Limiting dilution cloning failed to establish CTL clones from autologous MLTCs or tumor-infiltrating lymphocytes. In contrast, a broad panel of RCC-reactive CTL clones was expanded from each allogeneic MLTC. These sibling CTL clones either recognized exclusively the original RCC tumor line or cross-reacted with nonmalignant kidney cells of patient origin. A minority of CTL clones also recognized patient-derived hematopoietic cells or other allogeneic tumor targets. The MHC-restricting alleles for RCC-reactive sibling CTL clones included HLA-A2, HLA-A3, HLA-A11, HLA-A24, and HLA-B7. In one sibling donor-RCC pair, strongly proliferative CD3(+)CD16(+)CD57(+) CTL clones with non-HLA-restricted antitumor reactivity were established. Our results show superior tumor-reactive CD8 responses of matched allogeneic compared with autologous T cells. These data encourage the generation of antitumor T-cell products from HLA-identical siblings and their potential use in adoptive immunotherapy of metastatic RCC patients.

Phenotypic variation during cloning procedures: Analysis of the growth behavior of clonal cell lines

The production of recombinant protein from mammalian cells is a key feature of the biotechnology industry. However, the generation of recombinant mammalian cell lines is still largely performed on an empirical basis and there are many potential areas for enhancement. We have shown previously that despite two rounds of limiting dilution cloning (LDC) of recombinant cell lines, there remained a high degree of heterogeneity in the resulting cell lines. We suggested that a rapid phenotypic drift occurred with these cells. It was unclear if this was a consequence of the added burden of production of a recombinant protein, the selection procedures, or merely an inherent feature of cell growth in culture. To address this, we have subjected untransfected (parental) cells to three successive rounds of LDC and monitored the growth properties of the resultant cells. The results show that despite repeated rounds of cloning, it was not possible to obtain phenotypically similar cell lines. We also demonstrated that this phenotypic drift is not due to gross changes in the protein p27, a key regulators of the cell cycle. Although cells with a range of growth properties were observed even after three rounds of cloning, the variation in growth patterns between cell lines decreased after cloning. Hence, we suggest that by cloning it may be possible to generate untransfected cells, which have particular growth properties. Starting with a well-defined population of parental cells may aid in the subsequent generation of tranfectants with desired growth properties.

The spliceosomal autoantigen heterogeneous nuclear ribonucleoprotein A2 (hnRNP-A2) is a major T cell autoantigen in patients with systemic lupus erythematosus

A hallmark of systemic lupus erythematosus (SLE) is the appearance of autoantibodies to nuclear antigens, including autoantibodies directed to the heterogeneous nuclear ribonucleoprotein A2 (hnRNP-A2), which occur in 20% to 30% of SLE patients as well as in animal models of this disease. To investigate the underlying cellular reactivity and to gain further insight into the nature and potential pathogenic role of this autoimmune response we characterized the T cell reactivity against hnRNP-A2 in patients with SLE in comparison to healthy controls. Cellular proliferation of peripheral blood T cells to hnRNP-A2 was determined by [3H]thymidine incorporation and T cell clones (TCCs) specific for hnRNP-A2 were grown by limiting dilution cloning; IFNγ, IL-4 and IL-10 in culture supernatants were measured by ELISA. Bioactivity of culture supernatants was determined by incubation of anti-CD3/anti-CD28 stimulated peripheral blood CD4+ T cells with supernatants of TCCs. Stimulation assays performed with peripheral blood mononuclear cells of 35 SLE patients and 21 healthy controls revealed pronounced proliferative responses in 66% of SLE patients and in 24% of the controls, which were significantly higher in SLE patients (p < 0.00002). Furthermore, hnRNP-A2 specific TCCs generated from SLE patients (n = 22) contained a relatively high proportion of CD8+ clones and mostly lacked CD28 expression, in contrast to TCCs derived from healthy controls (n = 12). All CD4+ TCCs of patients and all control TCCs secreted IFNγ and no IL-4. In contrast, CD8+ TCCs of patients secreted very little IFNγ, while production of IL-10 did not significantly differ from other T cell subsets. Interestingly, all CD8+ clones producing IL-10 in large excess over IFNγ lacked expression of CD28. Functional assays showed a stimulatory effect of the supernatants derived from these CD8+CD28- hnRNP-A2 specific TCCs that was similar to that of CD4+CD28+ clones. Taken together, the pronounced peripheral T cell reactivity to hnRNP-A2 observed in the majority of SLE patients and the distinct phenotype of patient-derived CD8+ TCCs suggest a role for these T cells in the pathogenesis of SLE.

979. Clonal Expansion of a CD8+ T Cell Clone after Retroviral Transduction of Mature T Lymphocytes with the IL-15 Gene

Top of pageAbstract We have characterized a primary human T cell line, designated LC15, which has apparently been immortalized after transduction with an MSCV-based retroviral vector encoding the T cell growth factor, IL-15. This was an unexpected finding because, in previously reported animal and human studies, the target cells most at risk for insertional mutagenesis events were primitive progenitor cells. LC15 was generated by activating peripheral blood lymphocytes with OKT3 and IL-2. Subsequently, the cells were transduced with a retroviral vector encoding an IL-15 gene. Long-term culture of these cells demonstrated logarithmic growth for more than 365 days and constitutive expression of IL-15. In order to evaluate the LC15 cells for clonality, we utilized a 5'RACE technique to determine T cell receptor |[beta]|-chain usage; LC15 cells express only V|[beta]|13s.3 and all ofthe sequences contained identical D- and J-regions. Spectral karyotyping revealed a clonal abnormality with 4 translocations: 44, X, der(X)t(X;16)(p22.3;p10), der(13;16)(q10;p10), der(14;21)(q10;q10), |[minus]|16,+22,i(22)(q10). This result confirmed that LC15 is a clone and rules out chromosomal rearrangement as a mechanism for dysregulated growth of the cell line because all of the chromosomal breaks occurred at centromeres. We performed Southern blot analysis and found that the vector was intact and that the cells contained four vector integration sites. Phenotypically, the cells evolved into a pure population of CD8 lymphocytes with an effector memory phenotype (CD45 RA-, CD45RO+, and CD62L-). LC15 cells exhibited heterogeneous staining for CD5, CD25, and CD38, suggesting that multiple levels of activation existed within the population. Presumably, constitutive IL-15 expression contributes to the growth and maintenance of the cell line, however proliferation of the LC15 cell line could not be blocked by an anti-IL-15 monoclonal antibody. The cell line could not be grown by limiting dilution cloning and could not be established in immunodeficient mice, thus its malignant potential remains uncertain. Telomere length and telomerase activity were also examined. After 2 months in culture, the telomeres were approximately 4 kb in length. After 12 months in culture, the telomeres were approximately 2.3 kb. Telomerase was constitutively active in these cells in contrast to normal lymphocytes in which telomerase is upregulated by stimulation. We believe that this is the first report describing retroviral vector-induced transformation of a mature T lymphocyte. We have transduced a total of 20 different human T cell cultures with the IL-15 retroviral vector and LC15 represents the only incidence of clonal expansion; we hypothesize that this cell line is the result of an unusual and infrequent retroviral vector integration event. Current efforts are directed towards identifying the genomic integration sites in LC15 and evaluating the expression of genes in proximity of these sites. Potentially, this may reveal new insights into the genes that regulate T lymphocyte growth and survival. Furthermore, investigation of this cell line may identify genetic alterations involved in the transformation of mature T lymphocytes.

Detection of HBsAg mutants

HBsAg screening is carried out routinely to detect hepatitis B virus (HBV) infection. The immunoassays used employ capture antibodies often having specificity for epitopes present on the antigenic (a) determinant of the HBsAg. Loss of detection may occur due to mutations within and/or outside of the a determinant that affect conformational epitope recognition or HBsAg secretion or expression. Most of the mutations associated with immune escape occur within the second loop of the a determinant. In order to detect these HBsAg mutants, antibodies to subdominant regions within the a determinant or outside of the HBsAg may be required, and this has been the focus of many recent studies. Any changes to immunoassay formulations should also address the possible effect of HBV genotypic polymorphisms on assay specificity and sensitivity. HBsAg mutants may also be identified through nucleic acid detection of HBV in serum. Various molecular analysis methods have been developed to provide specific and sensitive detection of HBsAg mutants, including sequencing, limiting dilution cloning PCR (LDC-PCR), gap ligase chain reaction (gLCR), and real time PCR. Sequencing the HBsAg coding region provides specific information on the nucleotide sequence; however, it is relatively insensitive for the detection of minority quasispecies. Other nucleic acid methods offer greater sensitivity for the detection of point mutations. To improve immunoassays, further research will be required to increase detection sensitivity and specificity. Ultimately, a better understanding of the structure of antibody-bound HBsAg will help identify the immunological targets required for the accurate detection of HBsAg in blood.

Isolation and Characterization of Umbilical Cord Blood-Derived Multipotent Stem Cells Arising from Adherent CD45+/CD34+ Cell Subset.

Introduction: Using a cell separation medium (PrepaCyte-MLPC, BioE, Inc.) and plastic adherence, umbilical cord blood (UCB)-derived multipotent stem cells were isolated, expanded, and characterized. These stem cells, termed multi-lineage progenitor cells (MLPCs), are the only such cell type from any source demonstrated to be CD45+/CD34+ upon initial isolation.Methods: PrepaCyte-MLPC, an antibody-based cell separation medium, is added to an UCB unit (American Red Cross Cord Blood Program). Following homo- and heterophilic aggregation of undesired cell populations and subsequent sedimentation to gravity, the supernatant containing stem cells is expressed. After overnight incubation in a T-flask in MSCGM (Cambrex, Inc.), non-adherent cells are washed, leaving adherent cells to expand in culture. As MLPC colonies are observed, cells are further enriched by detachment (PBS/0.1% EGTA) and transfer to a new T-flask.Results: MLPCs were isolated and expanded from 3 of 10 UCB units. Cells were initially positive for CD45, CD34, CD133, SSEA-3, SSEA-4, and several MSC markers. Several days into culture, expression of HSC/ESC markers was lost. MSC marker expression was maintained; cells remain positive for CD9, CD13, CD29, CD44, CD73, CD90, and CD105. These MLPC lines have been cultured through at least 16 passages with no apparent impact on expansion or differentiation potential. Cell lines have been successfully differentiated into cell types representative of each embryonic layer (i.e., adipocytes, osteocytes, myocytes, hepatocytes, and endothelial and neural cells). Additional experiments and limited-dilution cloning are underway, and further studies (cytogentic/molecular and animal teratoma studies) are planned.Conclusions: UCB-derived MLPCs were isolated as a rare population of adherent, initially CD45+/CD34+ cells with lymphoid morphology. Following successful expansion in culture, multi-potency was demonstrated. MLPCs may serve a role in bone marrow transplantation (for malignant and non-malignant disease) and regenerative medicine; clinical studies are in an early phase of development.

Blood‐brain barrier‐specific properties of a human adult brain endothelial cell line

Establishment of a human model of the blood-brain barrier has proven to be a difficult goal. To accomplish this, normal human brain endothelial cells were transduced by lentiviral vectors incorporating human telomerase or SV40 T antigen. Among the many stable immortalized clones obtained by sequential limiting dilution cloning of the transduced cells, one was selected for expression of normal endothelial markers, including CD31, VE cadherin, and von Willebrand factor. This cell line, termed hCMEC/D3, showed a stable normal karyotype, maintained contact-inhibited monolayers in tissue culture, exhibited robust proliferation in response to endothelial growth factors, and formed capillary tubes in matrix but no colonies in soft agar. hCMEC/D3 cells expressed telomerase and grew indefinitely without phenotypic dedifferentiation. These cells expressed chemokine receptors, up-regulated adhesion molecules in response to inflammatory cytokines, and demonstrated blood-brain barrier characteristics, including tight junctional proteins and the capacity to actively exclude drugs. hCMEC/D3 are excellent candidates for studies of blood-brain barrier function, the responses of brain endothelium to inflammatory and infectious stimuli, and the interaction of brain endothelium with lymphocytes or tumor cells. Thus, hCMEC/D3 represents the first stable, fully characterized, well-differentiated human brain endothelial cell line and should serve as a widely usable research tool.

Construction of an expression system for the motor protein prestin in Chinese hamster ovary cells

The electromotility of outer hair cells (OHCs) is believed to be a major factor in cochlear amplification that enables the high sensitivity of hearing in mammals. This motility is thought to be based on voltage-dependent conformational changes of a motor protein embedded in the lateral wall of the OHC. In 2000, this motor protein was identified and termed prestin. To obtain knowledge on the function of prestin, research at the molecular level is necessary. For this purpose, a method of obtaining a large amount of prestin is required. In this study, an attempt was therefore made to construct an expression system for prestin. Prestin cDNA was introduced into Escherichia coli ( E. coli), insect cells and Chinese hamster ovary (CHO) cells, and the expression of prestin was examined by Western blotting. As CHO cells expressed prestin well, we generated prestin-expressing cell lines using CHO cells by limiting dilution cloning. The stable expression and the activity of prestin in generated cell lines were then confirmed. Finally, to obtain prestin from these cell lines efficiently, culture conditions of the cells were examined, and it was clarified that cells should be cultured in serum-free medium and harvested around 48 h after passage.

Cd8+ T-Cells Specifically Cytotoxic to Renal Cell Carcinoma (RCC) Cells Can Be Isolated from Patients with Regressing Metastatic Kidney Cancer after Allogeneic Nonmyeloablative Hematopoietic Cell Transplantation (NMHCT).

Graft-vs-tumor (GVT) effects following NMHCT induce disease regression in a subset of patients (pts) with advanced metastatic kidney cancer. At present, little is known about the antigens serving as tumor targets in responding pts. In an effort to characterize GVT effectors and their tumor antigens, we generated RCC cell lines to use as targets in cytotoxicity assays in three pts (one non-responder and two responders) undergoing a cyclophosphamide/fludarabine-based NMHCT from HLA matched siblings. Peripheral blood lymphocytes (PBL) were collected from pts at multiple time-points after transplantation and were expanded in-vitro with either irradiated patient (pre-transplant) PBL/EBV-LCL or autologous tumor cells. RCC pt #1 developed grade II skin GVHD on day 22 but did not have an objective tumor response and died from progressive tumor on day 203. In a Cr51 release assay, minor histocompatibility antigen specific (mHa) T-cell lines generated using pre-transplant pt PBL/EBV-LCL stimulators lysed 98% of pt CD40-ligand stimulated B cells (CD40L-B) but did not kill pt RCC cells. RCC pt #2 developed grade II skin GVHD on Day 51 and was noted to have regression of lung metastasis on day 183. Using PBL obtained during tumor regression, mHa- reactive T-cell lines were generated (pre-transplant pt PBL/EBV-LCL used as stimulators) that lysed 76% and 12% of pt EBV-LCL and autologous RCC tumor cells at a 20:1 E:T ratio. Following limiting dilution cloning, 6 CD8+ T cell clones were expanded that killed pt EBV-LCL (but not donor) including one MHC class-1 restricted T cell clone that lysed both pt EBV-LCL and pt tumor cells. RCC pt #3 developed grade III skin GVHD on day 120, had regression of lung metastasis on day 160, and survives more than 4 years after transplantation. PBL collected from this pt 40 months after transplant contained CD8+ T-cell populations that secreted IFN-g (0.9% by intracellular cytokine staining) when co-cultured with pt RCC cells but not after co-culture with pt CD40L-B cells. CD3+/CD8+ CTL were expanded from these PBL using pt RCC cells as stimulators; in vitro, these CTL killed the pt's RCC cells but did not kill pt fibroblasts or pt EBV-LCL (Figure A). Following co-culture with tumor, intracellular IFN-g staining combined with TCR Vb antibodies revealed 3 tumor reactive CD8+ T-cell populations (TCR Vb7+, TCRVb5.1+, and TCRVb non-staining); 25.2% of these CTL were TCRVb7+. IFN-g secretion by TCR Vb7+ T cells was blocked when tumor cells were pre-incubated with mAbs to CD8, pan MHC class I and HLA-A11 (Figure B) consistent with recognition of an HLA-11 restricted tumor antigen. Following limiting dilution cloning, several CD8+ T-cell clones (both TCR Vb7+ and TCR Vb non-staining) with RCC-specific cytotoxicity were identified. We conclude that donor T-cells with both broad alloreactivity and tumor specificity play a role in mediating GVT effects in RCC pts having disease regression following NMHCT. [Display omitted]

In vivo biotinylation of the major histocompatibility complex (MHC) class II/peptide complex by coexpression of BirA enzyme for the generation of MHC class II/tetramers

Success in generation of major histocompatibility complex (MHC) tetramer relies on application of a key technique, biotinylation of MHC molecule specifically on a single lysine residue using the BirA enzyme. However, in vitro biotinylation of MHC-BSP ( BirA enzyme substrate peptide) fusion protein using BirA enzyme is laborious and is prone to losses of target proteins to unacceptable levels. To circumvent this problem, an in vivo biotinylation strategy was developed where the BirA enzyme was coexpressed with target protein, HLA-DR2BSP/MBP, in an insect cell expression system. Bacterial BirA enzyme expressed in Drosophila melanogaster 2 (D. Mel-2) cell lines was biologically functional and was able to biotinylate secretary target protein (on specific lysine residue present on the BSP tag). Biotinylation efficiency was maximized by providing exogenous d-biotin in the culture medium and optimization of the expression vector ratios for cotransfection. By limiting dilution cloning, a clone was identified where the expressed DR2BSP/MBP protein was completely biotinylated. DR2BSP/MBP protein expressed and purified from such a clone was ready to be tetramerized with streptavidin to be used for staining antigen-specific T cells.

In vitro generation and life span extension of human papillomavirus type 16-specific, healthy donor-derived CTL clones.

Human papillomavirus (HPV) type 16 infection is strongly associated with the development of cervical carcinoma (CxCa) in women. The HPV16-derived oncoproteins E6 and E7, responsible for both onset and maintenance of malignant transformation, are expressed constitutively in CxCa cells and represent tumor-associated Ags. As a result, E6 and E7 constitute potential targets for adoptive CTL-mediated immunotherapy of CxCa. However, the availability to date of well-characterized HPV16-specific, CxCa-reactive human CTLs is extremely limited. The current study describes the in vitro generation and isolation of HPV16 E7-specific, CxCa-reactive human CTL clones from low-frequency healthy donor-derived CD8beta-positive precursors. For this purpose, an in vitro CTL induction protocol was used involving mature monocyte-derived dendritic cells as stimulator cells loaded with an HLA-A2.1-restricted, E7(11-20)-derived high-affinity altered peptide ligand. A double tetramer-guided isolation procedure and subsequent limiting-dilution cloning resulted in Ag-specific CTL clones. Stringent CTL characterization clearly indicated Ag-specific, HLA-A2.1-restricted reactivity against different HPV16-transformed CxCa cell lines. To allow expansion of E7(11-20)-specific CTL clones to numbers required for prolonged in vitro as well as in vivo application, their life span was significantly extended by ectopic expression of human telomerase reverse transcriptase. Collectively, our results show that optimized CTL induction and stringent CTL selection procedures, followed by human telomerase reverse transcriptase-mediated life span extension will allow continued availability of low-frequency HPV16-specific, CxCa-reactive human CTL clones. This may enhance the prospects of HPV16-specific adoptive CTL immunotherapy in CxCa patients.

Methyl-CpG Binding Domain Protein 2 Represses Transcription from Hypermethylated π-Class Glutathione S-Transferase Gene Promoters in Hepatocellular Carcinoma Cells

During the pathogenesis of human hepatocellular carcinoma (HCC), the CpG island encompassing the pi-class glutathione S-transferase gene (GSTP1) becomes hypermethylated. Repression of transcription accompanying CpG island hypermethylation has been proposed to be mediated by methyl-CpG binding domain (MBD) proteins. We report here that inhibition of transcription from hypermethylated GSTP1 promoters in Hep3B HCC cells, which fail to express GSTP1 mRNA or GSTP1 polypeptides, appears to be mediated by MBD2. Treatment of Hep3B cells with 5-azadeoxycytidine (5-aza-dC), a methyltransferase inhibitor, activated GSTP1 expression, whereas treatment with trichostatin A, a histone deacetylase inhibitor, had little effect. To more precisely assess the contribution of the pattern of GSTP1 CpG island methylation on GSTP1 mRNA expression, Hep3B cells were treated for 72 h with 5-aza-dC and then subjected to limiting dilution cloning. Bisulfite sequencing was used to map the methylation patterns of the GSTP1 promoter region in GSTP1-expressing and -non-expressing clones. In the clone that expressed GSTP1 mRNA determined by Northern blot analysis and quantitative reverse transcriptase (RT)-PCR, widespread demethylation of at least one GSTP1 allele was evident. Chromatin immunoprecipitation experiments revealed the presence of MBD2, but not Sp1, at the GSTP1 promoter in Hep3B cells. In contrast, Sp1 was detected at the GSTP1 promoter in a GSTP1-expressing Hep3B 5-aza-dC subclone. To test whether MBD2 might be responsible for the inhibition of GSTP1 transcription from hypermethylated GSTP1 promoters, siRNAs were used to reduce MBD2 polypeptide levels in Hep3B cells. SssI-catalyzed methylation of GSTP1 promoter sequences resulted in diminished luciferase reporter activity after transfection into Hep3B cells. However, when hypermethylated GSTP1 promoter sequences were transfected into Hep3B cells that had been treated with siRNA-targeting MBD2 mRNA, no repression of luciferase reporter expression was evident. These findings implicate MBD2 in the repression of GSTP1 expression associated with GSTP1 CpG island hypermethylation in HCC cells.

Selective expansion of fibroblast subpopulations from pulmonary artery adventitia in response to hypoxia.

Proliferation of fibroblasts contributes to the adventitial thickening observed during the development of hypoxia-induced pulmonary hypertension. However, whether all or only specific subpopulations of fibroblasts proliferate during this process is unknown. Because lung, skin, and gingiva contain multiple fibroblast subpopulations, we hypothesized that the pulmonary artery (PA) adventitia of neonatal calves is composed of multiple fibroblast subpopulations and that only selective subpopulations expand under chronic hypoxic conditions. Fibroblast subpopulations were isolated from PA adventitia of control calves using limited dilution cloning techniques. These subpopulations exhibited marked differences in morphology, actin expression, and serum-stimulated growth. Only select fibroblast subpopulations demonstrated the ability to proliferate in response to hypoxia. Fibroblast subpopulations were similarly isolated from calves exposed to hypoxia (14 days). With regard to morphology, actin expression, and serum-stimulated growth of subpopulations, there were no obvious differences in fibroblast subpopulations between the hypoxic and the control calves. However, the number of fibroblast subpopulations with about a twofold increase in hypoxia-induced DNA synthesis was significantly greater in the hypoxic calves (26%) compared with control calves (10%). We conclude that the bovine PA adventitia comprises numerous phenotypically and biochemically distinct fibroblast subpopulations and that select subpopulations expand in response to chronic hypoxia.

Activation of Natural Killer Cells and Macrophages by Porcine Endothelial Cells Augments Specific T-Cell Xenoresponse

The rejection of xenografts is characterized by infiltration of monocytes and natural killer (NK) cells into the graft, suggesting an important role for the innate immune system in xenorecognition. In this study, purified human NK or T cells were cocultured with porcine endothelial cells, and cytokines were analyzed by ELISA and intracellular FACS. We demonstrated a vigorous human anti-porcine xenoresponse that was associated with a strong T-cell proliferation against porcine endothelial cells. Limiting dilution cloning and T-cell receptor (TCR) Vbeta gene usage revealed a low number of xenoreactive T-cell precursors. We demonstrated that xenogeneic porcine but not allogeneic human endothelial cells induced the early production of interferon (IFN)-gamma by human NK cells but not by CD3+ T cells. Porcine xenoantigen-induced IFN-gamma production was only partially dependent on IL-12. Blocking IL-12 with neutralizing antibodies or by depletion of human macrophages partially decreased IFN-gamma production by CD56+ NK cells. Three-color flow cytometry revealed that IL-12 was produced through a species-specific activation of human macrophages by porcine endothelial cells. Our results indicate that the direct activation of NK cells and macrophages by porcine endothelial cells provides a unique pathway of xenorecognition that augments downstream specific T-cell immunity and represents a powerful effector mechanism in xenograft rejection.

Tissue-specific peptides influence human T cell repertoire to porcine xenoantigens.

Human CD8+ T cells elicit a vigorous response to allo- or xenogeneic MHC class I molecules. However, the influence of a given MHC-bound peptide to the responding allo- or xenoreactive T cell repertoire is not clear. In this study, we analyzed individual T cell responses to unique tissue epitopes presented on syngeneic porcine endothelial and lymphoblastoid cells by limiting dilution analysis and analyzed the responding T cell repertoire by T cell receptor beta (TCR Vbeta) chain spectrotyping. Both porcine endothelial and lymphoblastoid cells were able to elicit swine leukocyte antigen (SLA) class I restricted and peptide-dependent cytotoxic T lymphocyte (CTL) responses. The responding human CD8+ T cells showed a heterogenous but limited TCR Vbeta gene usage. Interestingly, although a large portion of the selected TCR Vbeta gene usage in response to endothelial and lymphoblastoid cells were shared (i.e., Vbeta-1, 2, 6.1, 13), unique Vbeta usage was noted in T cells that respond to either endothelial (Vbeta-5.3) or lymphoblastoid cells (Vbeta-5.1, 11), suggesting that porcine tissue-specific epitopes play a role in modulating the responding T cell repertoire. Limiting dilution cloning analysis revealed that a majority (89%) of the CTL clones stimulated by porcine endothelial cells recognized shared peptides presented by both endothelial cells and syngeneic lymphoblastoid cells. However, a significant portion (11%) of the CTL clones recognized unique peptides presented only in the context of SLA class I molecules on endothelial cells. These results provide evidence for the first time that tissue-specific peptides can directly influence T cell repertoire in response to the xenogeneic stimulus.

The Apolipoprotein E-dependent Low Density Lipoprotein Cholesteryl Ester Selective Uptake Pathway in Murine Adrenocortical Cells Involves Chondroitin Sulfate Proteoglycans and an α2-Macroglobulin Receptor

Cells acquire lipoprotein cholesterol by receptor-mediated endocytosis and selective uptake pathways. In the latter case, lipoprotein cholesteryl ester (CE) is transferred to the plasma membrane without endocytosis and degradation of the lipoprotein particle. Previous studies with Y1/E/tet/2/3 murine adrenocortical cells that were engineered to express apolipoprotein (apo) E demonstrated that apoE expression enhances low density lipoprotein (LDL) CE uptake by both selective and endocytic pathways. The present experiments test the hypothesis that apoE-dependent LDL CE selective uptake is mediated by scavenger receptor, class B, type I (SR-BI). Surprisingly, SR-BI expression was not detected in the Y1/E/tet/2/3 clone of Y1 adrenocortical cells, indicating the presence of a distinct apoE-dependent pathway for LDL CE selective uptake. ApoE-dependent LDL CE selective uptake in Y1/E/tet/2/3 cells was inhibited by receptor-associated protein and by activated alpha(2)-macroglobulin (alpha(2)M), suggesting the participation of the LDL receptor-related protein/alpha(2)M receptor. Reagents that inhibited proteoglycan synthesis or removed cell surface chondroitin sulfate proteoglycan completely blocked apoE-dependent LDL CE selective uptake. None of these reagents inhibited SR-BI-mediated LDL CE selective uptake in the Y1-BS1 clone of Y1 cells in which LDL CE selective uptake is mediated by SR-BI. We conclude that LDL CE selective uptake in adrenocortical cells occurs via SR-BI-independent and SR-BI-dependent pathways. The SR-BI-independent pathway is an apoE-dependent process that involves both chondroitin sulfate proteoglycans and an alpha(2)M receptor.

Characterization of the stability of recombinant protein production in the GS-NS0 expression system.

The GS-NS0 system is an important mammalian expression system used largely within industry for the high-level expression of recombinant proteins for therapeutic use. It is essential that the productivity of this system remains stable throughout culture expansion for the successful long-term production of recombinant proteins. Here we present a study of the stability of recombinant protein production from unamplified GS-NS0 cell lines over extended period of continuous culture. The cell lines used in this study were generated by the transfection of NS0 cells with DNA encoding for a secreted recombinant protein and by two subsequent rounds of limiting dilution cloning prior to analysis of stability. The stability of recombinant protein production was assessed at intervals over a period of 134 days using repeated batch culture in shake flasks. Heterogeneous stability was identified. The productivity of some clones remained consistent throughout 134 days of continuous culture. Others exhibit rapid and progressive loss of productivity. Analysis of the causal relationships underlying stability indicates that the initial transfectant determines the susceptibility to loss or retention of productivity. Selection of production clones on the basis of growth and productivity alone will not predict stability during long-term culture. Our research indicates that stable high-producing clones can readily be obtained from use of the GS-NS0 system in the absence of amplification but there may be molecular features of the original transfectants that could serve as very important predictive indicators of the stability of recombinant protein production.