Functional comparison of PBMCs isolated by Cell Preparation Tubes (CPT) vs. Lymphoprep Tubes

Chen, Han; Schürch, Christian M.; Noble, Kevin; Kim, Kenneth; Krutzik, Peter O.; O'Donnell, Erika; Tuig, Jason Vander; Nolan, Garry P.; McIlwain, David R.

doi:10.1186/s12865-020-00345-0

Cited by 36 publications

(24 citation statements)

References 14 publications

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“…For human Peripheral Blood Mononuclear Cells (PBMC) transwell assays, we seeded BT549 (WT or STAT1 KO ) in a 24-well plate (10,000 cells per well) and treated cells with DMSO or reversine for 96 hours. We isolated PBMCs from Buffycoat derived from healthy volunteers using the Ficoll-Paque method ( 70 ). For the transwell assay, we next seeded 2×10 6 human PBMCs on top of a filter membrane of a transwell insert (6.5 mm Transwell with 3.0 μm pore, Corning).…”

Section: Methodsmentioning

confidence: 99%

Cancer tolerance to chromosomal instability is driven by Stat1 inactivation in vivo

Schubert

Hong

Jilderda

et al. 2021

Preprint

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Chromosomal instability is a hallmark of cancer, but also an instigator of aneuploidy-induced stress, reducing cellular fitness. To better understand how cells with CIN adjust to aneuploidy and adopt a malignant fate in vivo, we performed a genome-wide mutagenesis screen in mice. We find that specifically aneuploid tumors inactivate Stat1 signaling in combination with increased Myc activity. By contrast, loss of p53 is common, but not enriched in CIN tumors. Validation in another tissue type confirmed that CIN promotes immune cell infiltration, which is alleviated by Stat1 loss combined with Myc activation, but not with p53 inactivation, or Myc activation alone. Importantly, we find that this mechanism is preserved in human aneuploid cancers. We conclude that aneuploid cancers inactivate Stat1 signaling to circumvent immune surveillance.

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Section: Methodsmentioning

confidence: 99%

Cancer tolerance to chromosomal instability is driven by Stat1 inactivation in vivo

Schubert

Hong

Jilderda

et al. 2021

Preprint

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“…Due to the limited viability of PBMCs in culture, PBMCs must be processed and cryopreserved [23]. However, it has been well established that significant cell loss occurs during cryopreservation and thawing, most likely as a result of freezing-induced stress and cell loss associated with additional washing and aspiration steps [24].…”

Section: Discussionmentioning

confidence: 99%

Assessment of chicken peripheral blood mononuclear cells isolated from freshly drawn blood versus 24 h refrigerated blood

et al. 2021

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Background and Aim: The peripheral blood mononuclear cell (PBMC) is an excellent cell source for in vitro studies, particularly those involving immunology. The aim of this study was to determine the quality and quantity of chicken PBMCs isolated from freshly drawn blood as well as blood that had been chilled for 24 h. In addition, the survival of PBMCs cultured in medium was investigated. Materials and Methods: Blood samples were collected from 12 Betong and 12 Leghorn chickens. Hemograms were analyzed. Density gradient centrifugation was used to isolate PBMCs. PBMCs (2×106 cells/mL) were cultured in a culture medium and incubated in a CO2 incubator for 5 consecutive days. The number of viable cells was determined using the trypan blue dye exclusion method. Results: Blood samples were obtained from healthy chickens. There was no statistically significant difference in the total amount of PBMC between fresh and refrigerated blood samples from both chicken breeds. The viability of PBMCs isolated from fresh blood (95%) was significantly greater than blood refrigerated for 24 h (90-92%) in both breeds. Furthermore, the viability of PBMCs isolated from both blood samples decreased significantly over time, from 90-95% to 60-65%. Conclusion: The total number of PBMC in fresh and refrigerated blood was not significantly different. Fresh blood-derived PBMCs had significantly higher viability than 24 h refrigerated blood PBMCs. Furthermore, the viability of PBMCs decreased significantly over time.

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“…Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats from consenting healthy donors (Sanquin Blood Supply Services, The Netherlands) and from these, CD14 + monocytes were isolated by magnetic bead separation, as previously described. 36,45 Monocyte-derived macrophage polarization…”

Section: Isolation Of Peripheral Blood Lymphocytes and Monocytesmentioning

confidence: 99%

Oncolytic Adenovirus ORCA-010 Activates Proinflammatory Myeloid Cells and Facilitates T Cell Recruitment and Activation by PD-1 Blockade in Melanoma

et al. 2021

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Immune checkpoint inhibitors have advanced the treatment of melanoma. Nevertheless, a majority of patients are resistant, or develop resistance, to immune checkpoint blockade, which may be related to prevailing immune suppression by myeloid regulatory cells in the tumor microenvironment (TME). ORCA-010 is a novel oncolytic adenovirus that selectively replicates in, and lyses, cancer cells. We previously showed that ORCA-010 can activate melanoma-exposed conventional dendritic cells (cDCs). To study the effect of ORCA-010 on melanoma-conditioned macrophage development, we used an in vitro co-culture model of human monocytes with melanoma cell lines. We observed a selective survival and polarization of monocytes into M2-like macrophages (CD14 + CD80-CD163 +) in co-cultures with cell lines that expressed macrophage colony-stimulating factor. Oncolysis of these melanoma cell lines, effected by ORCA-010, activated the resulting macrophages and converted them to a more proinflammatory state, evidenced by higher levels of PD-L1, CD80, and CD86 and an enhanced capacity to prime allogenic T cells and induce a type-1 T cell response. To assess the effect of ORCA-010 on myeloid subset distribution and activation in vivo, ORCA-010 was intratumorally injected and tested for T cell activation and recruitment in the human adenovirus nonpermissive B16-OVA mouse melanoma model. While systemic PD-1 blockade in this model in itself did not modulate myeloid or T cell subset distribution and activation, when it was preceded by i.t. injection of ORCA-010, this induced an increased rate and activation state of CD8a + cDC1, both in the TME and in the spleen. Observed increased rates of activated CD8 + T cells, expressing CD69 and PD-1, were related to both increased CD8a + cDC1 rates and M1/M2 shifts in tumor and spleen. In conclusion, the myeloid modulatory properties of ORCA-010 in melanoma, resulting in recruitment and activation of T cells, could enhance the antitumor efficacy of PD-1 blockade.

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Functional comparison of PBMCs isolated by Cell Preparation Tubes (CPT) vs. Lymphoprep Tubes

Cited by 36 publications

References 14 publications

Cancer tolerance to chromosomal instability is driven by Stat1 inactivation in vivo

Cancer tolerance to chromosomal instability is driven by Stat1 inactivation in vivo

Assessment of chicken peripheral blood mononuclear cells isolated from freshly drawn blood versus 24 h refrigerated blood

Oncolytic Adenovirus ORCA-010 Activates Proinflammatory Myeloid Cells and Facilitates T Cell Recruitment and Activation by PD-1 Blockade in Melanoma

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