Establishment of transplantable porcine tumor cell lines derived from MHC- inbred miniature swine

Cho, Patricia S.; Lo, Diana P.; Wikiel, Krzysztof J.; Rowland, Haley; Coburn, Rebecca C.; McMorrow, Isabel M.; Goodrich, Jennifer; Arn, J S; Billiter, Robert A.; Houser, Stuart L.; Shimizu, Akira; Yang, Yong‐Guang; Sachs, David H.; Huang, Christene A.

doi:10.1182/blood-2007-02-074450

Cited by 22 publications

(23 citation statements)

References 51 publications

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“…However, these sequences differ from eastern sequences by only one or two amino acids and of the seven new amino acid substitutions found among northwestern sequences only one substitution is located within a putative peptide interaction site. Rejection of transmissible tumours in experimental models can occur owing to only minor changes in MHC antigens or minor histocompatibility antigens (Sanford et al 1973;Cho et al 2007). However, given that Tasmanian devils with DFTD do not always have identical MHC Class I sequences to the tumour, the limited number of additional amino acid substitutions identified among northwestern devils may not affect DFTD transmission.…”

Section: Discussionmentioning

confidence: 99%

MHC gene copy number variation in Tasmanian devils: implications for the spread of a contagious cancer

et al. 2010

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Tasmanian devils face extinction owing to the emergence of a contagious cancer. Devil facial tumour disease (DFTD) is a clonal cancer spread owing to a lack of major histocompatibility complex (MHC) barriers in Tasmanian devil populations. We present a comprehensive screen of MHC diversity in devils and identify 25 MHC types and 53 novel sequences, but conclude that overall levels of MHC diversity at the sequence level are low. The majority of MHC Class I variation can be explained by allelic copy number variation with two to seven sequence variants identified per individual. MHC sequences are divided into two distinct groups based on sequence similarity. DFTD cells and most devils have sequences from both groups. Twenty per cent of individuals have a restricted MHC repertoire and contain only group I or only group II sequences. Counterintuitively, we postulate that the immune system of individuals with a restricted MHC repertoire may recognize foreign MHC antigens on the surface of the DFTD cell. The implication of these results for management of DFTD and this endangered species are discussed.

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Section: Discussionmentioning

confidence: 99%

MHC gene copy number variation in Tasmanian devils: implications for the spread of a contagious cancer

et al. 2010

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“…Therefore a CD80- or CD86-expressing porcine antigen presenting cell line is required in order to assess the binding function of the soluble porcine CTLA-4 in vitro . Using FACS analysis with hamster anti-mouse CD80 mAb (clone 16-10A1) (BioLegend, Cat#104703) a porcine CD80-expressing B-cell lymphoma cell line (LCL13271-5E4) was successfully screened from our available potential porcine B-cell lymphoma cell lines [10]. As shown in Figure 5A and 5B, FACS binding analysis demonstrated that both biotinylated glycosylated and non- N -glycosylated soluble porcine CTLA-4 bound to the porcine CD80-expressing B-cell lymphoma cell line LCL13271-5E4 in a dose-dependent manner.…”

Section: Resultsmentioning

confidence: 99%

“…Porcine B-cell lymphoma tumor cell lines available in our laboratory [10] were cultured in T75 culture flasks (Corning, Cat# 10-126-31) with 1x RPMI 1640 media supplemented with 12% fetal bovine serum, 10 mM hepes ( N -2-hydroxyethylpiperazine-N-2-ethanesulfonic acid), 1x nonessential amino acids, 1mM sodium pyruvate, 2 mM glutamine, and 2.5 × 10 −5 M 2-mercaptoethanol. Cultures were incubated at 37°C with 5% CO 2 until a cell density of 1.0 × 10 6 cells/mL was achieved.…”

Section: Methodsmentioning

confidence: 99%

Expression and purification of soluble porcine CTLA-4 in yeast Pichia pastoris

Peraino¹,

Zhang²,

Hermanrud³

et al. 2012

Protein Expression and Purification

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Co-stimulation blockade can be used to modulate the immune response for induction of organ transplantation tolerance, treatment of autoimmune disease as well as cancer treatment. Cytotoxic T-Lymphocyte Antigen-4 (CTLA-4), also known as CD152, is an important co-stimulatory molecule which serves as a negative regulator for T cell proliferation and differentiation. CTLA-4/CD28-CD80/CD86 pathway is a critical co-stimulatory pathway for adaptive immune response. T cell activation through the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for CD80 and CD86. MGH MHC-defined miniature swine provide a unique large animal model useful for preclinical studies of transplantation tolerance and immune regulation. In this study, we have expressed the codon-optimized soluble porcine CTLA-4 in the yeast Pichia pastoris system. The secreted porcine CTLA-4 was captured using Ni-Sepharose 6 fast flow resin and further purified using strong anion exchange resin Poros 50HQ. Glycosylation analysis using PNGase F demonstrated the N-linked glycosylation on Pichia pastoris expressed soluble porcine CTLA-4. To improve the expression level and facilitate the downstream purification we mutated the two potential N-linked glycosylation sites with non-polarized alanines by site-directed mutagenesis. Removal of the two N-glycosylation sites significantly improved the production level from ~2 mg/L to ~8 mg/L. Biotinylated glycosylated and non-N-glycosylated soluble porcine CTLA-4 both bind to a porcine CD80-expressing B-cell lymphoma cell line (KD = 13 nM) and competitively inhibit the binding of an anti-CD80 monoclonal antibody. The availability of soluble porcine CTLA-4, especially the non-N-glycosylated CTLA-4, will provide a very valuable tool for assessing co-stimulatory blockade treatment for translational studies in the clinically relevant porcine model.

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“…Porcine IL-2 fusion toxins were eluted using 40 mM imidazole. Western blot analysis, FACS analysis, FACS competition/blocking analysis and K D determination were all performed as previously described (Peraino et al, 2012) using LCL13271 cells (Cho et al, 2007). The Ontak ® -like monovalent human IL-2 fusion toxin (DT390-hIL-2) used as a control for our in vitro assay was constructed, expressed and purified exactly same as the monovalent porcine IL-2 fusion toxin.…”

Section: Methodsmentioning

confidence: 99%

Development of a diphtheria toxin-based recombinant porcine IL-2 fusion toxin for depleting porcine CD25+ cells

Peraino

Schenk

et al. 2013

Journal of Immunological Methods

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Regulatory T cells (Tregs) have been widely recognized as crucial players in controlling immune responses. Because their major role is to ensure that the immune system is not over reactive, Tregs have been the focus of multiple research studies including those investigating transplantation tolerance, autoimmunity and cancer treatment. On their surface Tregs constitutively express CD25, a high affinity receptor for the cytokine interleukin-2 (IL-2). The reagents constructed in this study were generated by genetically linking porcine IL-2 to the truncated diphtheria toxin (DT390). This reagent functions by first binding to the cell surface via the porcine IL-2/porcine CD25 interaction then the DT390 domain facilitates internalization followed by inhibition of protein synthesis resulting in cell death. Four versions of the porcine IL-2 fusion toxin were designed in an interest to find the most effective isoform: 1) monovalent glycosylated porcine IL-2 fusion toxin (Gly); 2) monovalent non-N-glycosylated porcine IL-2 fusion toxin (NonGly); 3) bivalent glycosylated porcine IL-2 fusion toxin (Bi-Gly); 4) bivalent non-N-glycosylated porcine IL-2 fusion toxin (Bi-NonGly). Using a porcine CD25+ B cell lymphoma cell line (LCL13271) in vitro analysis of the fusion toxins’ ability to inhibit protein synthesis demonstrated that the Bi-NonGly fusion toxin is the most efficient reagent. These in vitro results are consistent with binding affinity as the Bi-NonGly fusion toxin binds strongest to CD25 on the same LCL13271 cells. The Bi-Gly fusion toxin significantly prolonged the survival (p=0.028) of tumor-bearing NOD/SCID IL-2 receptor γ−/− (NSG) mice injected with LCL13271 cells compared with untreated controls. This recombinant protein has great potential to function as a useful tool for in vivo depletion of porcine CD25+ cells for studying immune regulation.

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Establishment of transplantable porcine tumor cell lines derived from MHC- inbred miniature swine

Cited by 22 publications

References 51 publications

MHC gene copy number variation in Tasmanian devils: implications for the spread of a contagious cancer

MHC gene copy number variation in Tasmanian devils: implications for the spread of a contagious cancer

Expression and purification of soluble porcine CTLA-4 in yeast Pichia pastoris

Development of a diphtheria toxin-based recombinant porcine IL-2 fusion toxin for depleting porcine CD25+ cells

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