Techniques to improve the direct ex vivo detection of low frequency antigen‐specific CD8⁺ T cells with peptide‐major histocompatibility complex class I tetramers

Abstract

The ability to quantify and characterize antigen-specific CD8 1 T cells irrespective of functional readouts using fluorochrome-conjugated peptide-major histocompatibility complex class I (pMHCI) tetramers in conjunction with flow cytometry has transformed our understanding of cellular immune responses over the past decade. In the case of prevalent CD8 1 T cell populations that engage cognate pMHCI tetramers with high avidities, direct ex vivo identification and subsequent data interpretation is relatively straightforward. However, the accurate identification of low frequency antigen-specific CD8 1 T cell populations can be complicated, especially in situations where T cell receptor-mediated tetramer binding occurs at low avidities. Here, we highlight a few simple techniques that can be employed to improve the visual resolution, and hence the accurate quantification, of tetramer binding CD8 1 T cell populations by flow cytometry. These methodological modifications enhance signal intensity, especially in the case of specific CD8 1 T cell populations that bind cognate antigen with low avidities, minimize background noise, and enable improved discrimination of true pMHCI tetramer binding events from nonspecific uptake. ' 2008 International Society for Advancement of Cytometry Key terms CD8 1 T cell; peptide-major histocompatibility complex class I tetramer; polychromatic flow cytometry ADAPTIVE immunity is mediated by a complex network of cellular and molecular interactions that sense and respond to antigenic stimuli derived from dangerous entities. To deconvolute this system, it is important that the necessary tools are available to enable the accurate and reproducible measurement of antigen-specific cell populations directly ex vivo; however, it is equally important that these tools are used with an understanding of their limitations. One of the most significant advances in immunotechnology over the past few years has been the development of soluble recombinant peptide-major histocompatibility complex class I (pMHCI) multimers (1,2). These reagents bind stably to cognate T cell receptors (TCRs) expressed on the surface of antigen-specific CD8 1 T cells, despite the low affinity and rapid kinetics of monomeric TCR/pMHCI binding, and are internalized at physiological binding temperatures (3,4); thus, in fluorochrome-conjugated form, pMHCI multimers allow the visualization of cognate CD8 1 T cells by flow cytometry. In general, pathogen-specific CD8 1 T cell populations, which tend to express TCRs that bind cognate pMHCI with high affinities (5) and comprise a substantial proportion of the memory T cell pool, are easily identified with relatively basic flow cytometers and associated software. However, it is sometimes difficult to distinguish true antigen-specific CD8 1 T

β-Cell–Specific CD8 T Cell Phenotype in Type 1 Diabetes Reflects Chronic Autoantigen Exposure

“…Consistent with this notion, greater proportions of CD8 T cells with a stem cell memory phenotype (T SCM ; CD27 + CD45RO 2 CD95 + CCR7 + ) (28) were present in type 1 diabetic patients compared with healthy control subjects across all pooled b-cell specificities (P = 0.025), as well as individually within the autoreactive populations specific for PPI [15][16][17][18][19][20][21][22][23][24] (P = 0.05) and InsB [10][11][12][13][14][15][16][17][18] (P = 0.029). Moreover, single-marker analyses revealed that b-cell-specific CD8 T cells expressed higher frequencies of CD57 (P = 0.0002) and CD95 (P , 0.0001) in type 1 diabetic patients compared with healthy control subjects (Fig.…”

“…In particular, antigen-specific CD8 T cells can now be enumerated routinely by flow cytometry irrespective of functional outputs due to the advent of recombinant peptide-HLAI (pHLAI) proteins in various fluorochrome-tagged multimeric formats (10)(11)(12)(13). Moreover, developments in instrumentation and fluorochrome technology continue to expand the horizons of polychromatic flow cytometry (14,15), facilitating the identification of functionally distinct T cell subsets across a spectrum of phenotypic heterogeneity (16).…”

MHC-derived allopeptide activates TCR-biased CD8+ Tregs and suppresses organ rejection

“…This dual fluorochrome strategy, previously described for antigen-specific CD8 + effector T cells, allows the discrimination of signal and noise staining, as specific CD8 + Tregs will bind equally to each version of RT1.A a /Du51 tetramer, whereas random elements will not (26). Among double-positive stained events, nonpeptide-specific cells can be visualized and excluded, since they bind to a control tetramer bearing the same heavy-chain RT1.A a , but with an irrelevant associated peptide (Supplemental Figure 4C).…”

T-cell immune responses to Wilms tumor 1 protein in myelodysplasia responsive to immunosuppressive therapy

“…10,19,20 Flow cytometry for tetramer analysis Sample staining was performed in 50 L of phosphate-buffered saline (PBS)/1% fetal calf serum using 3 ϫ 10 6 PBMCs prestained with a violet amine-reactive dye (Aqua-blue or ViViD; Invitrogen) to eliminate dead cells from the final analysis. Fluorochrome-labeled HLA-A*0201 tetramers (1-2 g per test with respect to the peptide-major histocompatibility complex class I component) were added for 15 to 30 minutes at 37°C.…”