流式细胞仪对 LAG3 抗体的比较分析显示了不同的结合模式

IF 1.6 4区 医学 Q4 BIOCHEMICAL RESEARCH METHODS
Colin G. Graydon , Allison Balasko , Monika Kowatsch , Keith R. Fowke
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引用次数: 0

摘要

背景LAG3是一种具有新兴治疗用途的免疫检查点分子。本研究使用流式细胞术比较了健康供体的 T 细胞、NK 细胞、B 细胞、pDCs 和单核细胞的表面 LAG3 表达。本研究还比较了三种单克隆 LAG3 抗体和一种常用的多克隆 LAG3 抗体在健康供体的体外和 PHA-细胞以及 LAG3+ 和 LAG3- 细胞系上的表达情况。结果LAG3 在经典单核细胞和中间单核细胞上的表达量最高(分别为 25% 和 32%),而在 B 细胞、NK 细胞和 iNKT 细胞上的表达量几乎可以忽略不计。值得注意的是,多克隆抗体对所有细胞类型的染色比例都高于单克隆抗体,而单克隆抗体的染色模式彼此相似。使用 LAG3+ 和 LAG3- 细胞系进行的进一步研究表明,单克隆抗体 T47-530 比多克隆抗体具有更高的特异性和相似的敏感性。此外,单克隆和多克隆 LAG3 抗体之间的差异值得在设计未来的研究和解释过去的研究时加以考虑,这也可以解释文献中的差异。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Comparative analysis of LAG3 antibodies shows differential binding patterns by flow cytometry

Background

LAG3 is an immune checkpoint molecule with emerging therapeutic use. Expression of LAG3 is well studied on T cells, but the proportion of LAG3-expressing cells varies greatly by study and its comparative expression between non-T cells is lacking.

Methods/objectives

This study uses flow cytometry to compare surface LAG3 expression between T cells, NK cells, B cells, pDCs and monocytes of healthy donors. This study also compares three monoclonal LAG3 antibodies to a commonly used polyclonal LAG3 antibody on ex vivo and PHA-blasts from healthy donors and LAG3+ and LAG3- cell lines.

Results

LAG3 was most highly expressed on classical and intermediate monocytes (25 % and 32 %, respectively), while LAG3 expression on B cells, NK cells and iNKT cells was negligible. Notably, the polyclonal antibody stained a higher proportion of all cell types than the monoclonal antibodies, which had similar staining patterns to one another. Further study using LAG3+ and LAG3- cell lines showed greater specificity and similar sensitivity of the monoclonal antibody T47–530 than the polyclonal antibody.

Conclusion

Monocytes may represent an unappreciated source of LAG3 and target of LAG3 checkpoint inhibitors. Furthermore, the discrepancies between monoclonal and polyclonal LAG3 antibodies warrants consideration when designing future studies and interpreting past studies, and may explain discrepancies in the literature.

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来源期刊
CiteScore
4.10
自引率
0.00%
发文量
120
审稿时长
3 months
期刊介绍: The Journal of Immunological Methods is devoted to covering techniques for: (1) Quantitating and detecting antibodies and/or antigens. (2) Purifying immunoglobulins, lymphokines and other molecules of the immune system. (3) Isolating antigens and other substances important in immunological processes. (4) Labelling antigens and antibodies. (5) Localizing antigens and/or antibodies in tissues and cells. (6) Detecting, and fractionating immunocompetent cells. (7) Assaying for cellular immunity. (8) Documenting cell-cell interactions. (9) Initiating immunity and unresponsiveness. (10) Transplanting tissues. (11) Studying items closely related to immunity such as complement, reticuloendothelial system and others. (12) Molecular techniques for studying immune cells and their receptors. (13) Imaging of the immune system. (14) Methods for production or their fragments in eukaryotic and prokaryotic cells. In addition the journal will publish articles on novel methods for analysing the organization, structure and expression of genes for immunologically important molecules such as immunoglobulins, T cell receptors and accessory molecules involved in antigen recognition, processing and presentation. Submitted full length manuscripts should describe new methods of broad applicability to immunology and not simply the application of an established method to a particular substance - although papers describing such applications may be considered for publication as a short Technical Note. Review articles will also be published by the Journal of Immunological Methods. In general these manuscripts are by solicitation however anyone interested in submitting a review can contact the Reviews Editor and provide an outline of the proposed review.
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