Augmentation of LDL receptor activities on lymphocytes by interleukin-2 and anti-CD3 antibody: A flow cytometric analysis

Suzuki, Kunihiro; Hara, Masako; Kitani, Atsushi; Harigai, Masayoshi; Norioka, Kenichi; Kondo, Kazuo; Hirata, F.; Sakata, Nobuhiro; Kawakami, Makoto; Kawagoe, Mikio; Nakamura, Haruo

doi:10.1016/0005-2760(90)90164-s

Cited by 21 publications

(18 citation statements)

References 24 publications

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“…LDLR expression and LDL uptake into human lymphocyte subsets have consistently been shown to be enhanced in Ag-or mitogenstimulated cells (14)(15)(16)(17). In our previous studies, we confirmed the generality of this phenomenon by using the bovine ApoB-100 Ab, 3E8.1 (2,12), to show the marked increase in bovine LDL binding to LDLRs to support rapid proliferation of activated lymphocytes in FCS-containing medium.…”

Section: Ldlr and Apob Binding Increase Substantially On Resting Macrsupporting

confidence: 69%

Apolipoprotein B Binding Domains: Evidence That They Are Cell-Penetrating Peptides That Efficiently Deliver Antigenic Peptide for Cross-Presentation of Cytotoxic T Cells

Sakamoto

Rosenberg

2011

The Journal of Immunology

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Low-density lipoproteins (LDLs) are a good source of cholesterol, which is important in cellular homeostasis and production of steroids. Apolipoprotein B-100 (ApoB-100), the sole protein component of LDL, is known to bind to cell surface LDL receptor (LDLR) or cell surface-bound proteoglycans and to be internalized into cells. We found that APCs, consisting of macrophages and dendritic cells, upregulate LDLR on culture in vitro without obvious stimulation. In contrast, T cell populations only upregulate LDLR on activation. Thus, we strategized that tagging immunogens to ApoB-100 might be a useful means to target Ag to APCs. We generated fusion proteins consisting of receptor binding sites in ApoB-100, coupled to OVA peptide (ApoB-OVA), as Ag delivery vehicles and demonstrated that this novel delivery method successfully cross-presented OVA peptides in eliciting CTL responses. Surprisingly, internalization of ApoB-OVA peptide occurred via cell surface proteoglycans rather than LDLRs, consistent with evidence that structural elements of ApoB-100 indicate it to have cell-penetrating peptide properties. Finally, we used this strategy to assess therapeutic vaccination in a tumor setting. OVA-expressing EL-4 tumors grew progressively in mice immunized with ApoB-100 alone but regressed in mice immunized with ApoB-OVA fusion protein, coinciding with development of OVA-specific CTLs. Thus, to our knowledge, this is the first article to describe the cell-penetrating properties of a conserved human origin cell penetrating peptide that may be harnessed as a novel vaccination strategy as well as a therapeutics delivery device.

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Section: Ldlr and Apob Binding Increase Substantially On Resting Macrsupporting

confidence: 69%

Apolipoprotein B Binding Domains: Evidence That They Are Cell-Penetrating Peptides That Efficiently Deliver Antigenic Peptide for Cross-Presentation of Cytotoxic T Cells

Sakamoto

Rosenberg

2011

The Journal of Immunology

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“…Suzuki et al used rIL-2 and anti-CD3 monoclonal antibodies to stimulate lymphocytes for proliferation (17). Recently, an anti-CD3/CD28 monoclonal antibody with functional properties that allow easy stimulation of T-lymphocytes with high specificity became available (18).…”

Section: Introductionmentioning

confidence: 99%

A novel method for determining functional LDL receptor activity in familial hypercholesterolemia: Application of the CD3/CD28 assay in lymphocytes

Tada

Kawashiri

Noguchi

et al. 2009

Clinica Chimica Acta

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Abbreviated title: An improved assay for LDL receptor activity using anti-CD3/CD28 beads Abbreviations: DiI, 3,3"--dioctadecylindocarbocyanin; FH, familial hypercholesterolemia; NARC-1, neural apoptosis-regulated convertase 1; FITC, fluorescein isothiocyanate; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; LDLR, low-density lipoprotein receptor; LPDS, lipoprotein deficient serum; rIL-2, recombinant interleukin-2; MF, mean fluorescence; TC, total cholesterol; CV, coefficient of variation. 3 AbstractBackground: The objective of this study was to develop a new and simple method for measuring low-density lipoprotein receptor (LDLR) activity using peripheral lymphocytes enabling us to clinically diagnose familial hypercholesterolemia (FH) and ascertain the involved mutations (such as K790X mutation), that might not be clearly detected in the conventional method.Methods: Our method comprised the following 2 features: First, we used anti-CD3/CD28 beads to stimulate T-lymphocytes to obtain a uniform fraction of lymphocytes and maximum up-regulation of LDLR. Second, we excluded the possibility of overestimation of lymphocyte signals bound only to its surface, by adding heparin to the cultured lymphocytes used for the LDLR assay.Results: Based on the genetic mutation, the FH subjects were divided into two groups, K790X, (n = 20) and P664L, (n = 5), and their LDLR activities was measured by this method, which was found to be 55.3 ± 8.9% and 63.9 ± 13.8%, respectively, of that of the control group (n = 15). In comparison, the LDLR activity was 86.1 ± 11.6% (K790X) and 73.3 ± 6.3% (P664L) of that of the control group when measured by the conventional method, indicating that impairment of LDLR function in FH K790X subjects was much more clearly differenciated with our method than with the conventional method (paired t-test, p < 0.0001). The levels of LDLR expression also showed similar tendencies, that is, 89.4 ± 13.2% (K790X) and 76.9 ± 17.4% (P664L) of that of the control group when measured by the conventional method, and 78.1 ± 9.7% (K790X) and 70.3 ± 26.5% (P664L) when measured by our new method. In addition, we confirmed that there was little influence of statin treatment on LDLR activity among the study subjects when our method was used.Conclusion: These results demonstrate that our new method is applicable for measuring LDLR 4 activity, even in subjects with an internally defective allele, and that T-lymphocytes of the FH K790X mutation possess characteristics of that allele.

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“…Different assays for the measurement of receptor activity on blood mononuclear cells have been developed ( 1,2, 6,12,18,[20][21][22]. In radioligand assays, radiolabelled LDL is taken up by the cell, and the nonprotein-bound radioactivity subsequently released to the medium is measured (2,12).…”

mentioning

confidence: 99%

Standardization of a flow cytometric method for measurement of low‐density lipoprotein receptor activity on blood mononuclear cells

et al. 1995

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Flow cytometric methods for measurement of low-density lipoprotein (LDL) receptor activity on peripheral blood mononuclear cells (PBMC) may be used to identify patients with familial hypercholesterolemia (FH). However, cellular LDL receptor activities measured in FH heterozygotes may overlap with those of healthy subjects. Analytical variation is probably responsible for some of this overlap. We have examined several technical details that may affect analytical variation. In each analysis, we included one standard and two control cell preparations. These were cells isolated firom three donors and stored in aliquots at -135°C. Use of standard cells reduced between-series analytical variation of the controls by approximately 50%. Preincubationconditions used to induce the maximum number of receptors, the concentration of fluorochrome 1,l'-dioctadecyl-3,3,3' ,3'-tetramethylindocarbocyanineperchlorate (Di1)-LDL, labelling time, and conditions during storage of labelled cells before flow cytometry were also examined in order to reduce analytical variation. Having standardized the assay, we found among 20 healthy subjects a median receptor activity of 100% vs. 51% among 26 patients who fulfilled clinical criteria for FH. However, four of the patients showed distinctly normal receptor activities, which may suggest either the presence of some other biochemical defect or that in vivo dysfunctional receptors may be measured as normal in some patients with our assay. o 1995 Wiley-Liss, I~C .

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Augmentation of LDL receptor activities on lymphocytes by interleukin-2 and anti-CD3 antibody: A flow cytometric analysis

Cited by 21 publications

References 24 publications

Apolipoprotein B Binding Domains: Evidence That They Are Cell-Penetrating Peptides That Efficiently Deliver Antigenic Peptide for Cross-Presentation of Cytotoxic T Cells

Apolipoprotein B Binding Domains: Evidence That They Are Cell-Penetrating Peptides That Efficiently Deliver Antigenic Peptide for Cross-Presentation of Cytotoxic T Cells

A novel method for determining functional LDL receptor activity in familial hypercholesterolemia: Application of the CD3/CD28 assay in lymphocytes

Standardization of a flow cytometric method for measurement of low‐density lipoprotein receptor activity on blood mononuclear cells

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