Clonally expanded memory CD8+ T cells accumulate in atherosclerotic plaques and are pro-atherogenic in aged mice

Development of a Spectral Flow Cytometry Analysis Pipeline for High-Dimensional Immune Cell Characterization

“…At minimum, this means that CD44 and CD62L will be used to differentiate these three populations. 17 Sometimes additional markers like markers for T cell exhaustion (i.e. PD-1) and chemokine receptor markers like C-C Chemokine Receptor 5 (CCR5) will be used to determine more nuanced phenotypes of these three populations.…”

“…Using these four additional markers after isolating the CD8 + cell population limits the decision of which markers to choose for the first two antibodies, which are typically CD44 and CD62L, followed by the phenotypic markers PD-1 and CCR5. 17 However, with spectral flow cytometry, there may be an additional 14 or more markers after isolating the CD8 + population. In this situation, one may still decide to drill down into naïve, effector memory, and central memory cells in a biaxial manner first with CD44 and CD62L.…”

“…All combinations are not typically considered because prior knowledge informs the gating strategy order. 17,[20][21][22][23] Spectral flow cytometry allows for additional antibodies to be used within the same panel even up to 50 colors in a single panel. 9,24,24 By expanding the number of additional markers (i.e., CD49d, CXCR3, CCR5, CXCR6, CD44, CD27, TCRY, CD28, PD1, LAG3, KLRG1, CD62L, MHCII, MHCI, TOX, and B220), the complexity for gating strategies increases substantially.…”

“…In conventional flow cytometry, antibody panels incorporating a live/dead stain typically include between 6-12 antibodies and are now able to scale up to 28 antibodies with optimization. 17 As an example, a typical strategy for analyzing CD8 + T cells would involve gating on live, CD45 + CD3e + CD8a + cells. 17 After this, surface markers such as CD62L, CD44, PD1, Tox, CD27, and CD28 may be used to further categorize distinct subsets of CD8 + T cells.…”

“…17 As an example, a typical strategy for analyzing CD8 + T cells would involve gating on live, CD45 + CD3e + CD8a + cells. 17 After this, surface markers such as CD62L, CD44, PD1, Tox, CD27, and CD28 may be used to further categorize distinct subsets of CD8 + T cells. 17,18 Conventional flow cytometry gating strategies examine two markers at a time to subset cells.…”