Activated integrins identify functional antigen-specific CD8⁺T cells within minutes after antigen stimulation

Abstract

SignificanceAssessing antigen-specific T cells is crucial for our understanding of immune reactions against pathogens and tumors, and for evaluating immunotherapies in patients. Existing techniques to evaluate the functionality of T lymphocytes all rely on de novo expression of proteins, typically intracellular cytokines, and therefore require elaborated protocols and reagents. We have established a simple flow cytometry-based method to assess the functionality of CD8+ T cells by identifying immediate changes in the conformation and valency of cell surface integrins that occur within minutes following antigenic stimulation. Because of its robustness, sensitivity, and broad applicability, the assay can be rapidly implemented for the measurement and isolation of functional T cells for basic research and in the clinical setting.

Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition)

“…Therefore, short stimulation times should be preferred; for instance, cytokines and rapid activation markers (e.g., CD154, CD137, CD69) typically require only 5–12 h of stimulation before their levels are measurable intracellularly, on cell surfaces or in culture supernatants, ensuring minimal manipulation . Furthermore, β2‐integrin activation on activated T cells occurs even within minutes .…”

“…A common drawback of these techniques is that they all rely on upregulation or de novo synthesis of the read‐out markers, e.g., activation markers or cytokines, and therefore, require at least several hours of stimulation. Recently, a new approach for rapid identification of activated CD8 + T cells has been introduced, based on immediate changes of surface integrins that occur within minutes following antigen stimulation . The authors made use of the fact that resting antigen‐experienced T cells express high levels of membrane‐bound β2‐integrins .…”

Integrin activation enables simultaneous and sensitive detection of functional virus-specific CD4+ and CD8+ T cells

“…Antigen-speci c β 2 -integrin activation on CD8 + T cells can be visualized by either mICAM-1 or m24 Ab staining In our previous work, antigen-speci c CD8 + T cells were detected with mICAM-1 complexes (without EDTA) [5]. We therefore tested if staining of activated CD8 + T cells with m24 Ab would be comparable to that obtained with mICAM-1, and to which extent EDTA in uences detection.…”

“…We have previously shown that antigen-speci c CD8 + T cells are successfully detected by staining of activated β 2 -integrins with uorescent mICAM-1 [5]. We therefore investigated whether mICAM-1 binding could also be used as a marker for detection of antigen-speci c CD4 + T cells.…”

“…We observed that binding of mICAM-1 to b 2 -integrins on CD8 + T cells identi es functional effectors that will later degranulate and produce effector cytokines. In addition to being a quick read-out (approximately after 4 to 20 min stimulation in most cases) based on the use of a single detecting reagent, the assay preserves cell viability and can be used for the enrichment of antigen-responding CD8 + T cells for immediate or post in vitro expansion analysis [5,6].…”