OMIP‐080: 29‐Color flow cytometry panel for comprehensive evaluation of NK and T cells reconstitution after hematopoietic stem cells transplantation

Abstract

This 29-color panel was developed and optimized for the monitoring of NK cell and T cell reconstitution in peripheral blood of patients after HSCT. We considered major post-HSCT complications during the design, such as relapses, viral infections, and GvHD and identification of lymphocyte populations relevant to their resolution. The panel includes markers for all major NK cell and T cell subsets and analysis of their development and qualitative properties. In the NK cell compartment, we focus mainly on CD57 + NKG2C+ cells and the expression of activating (NKG2D, DNAM-1) and inhibitory receptors (NKG2A, TIGIT). Another priority is the characterization of T cell reconstitution; therefore, we included detection of CD4+ RTEs based on CD45RA, CD62L, CD95, and CD31 as a marker of thymus function. Besides that, we also analyze the emergence and properties of major T cell populations with a particular interest in CD8, Th1, ThCTL, and Treg subsets. Overall, the panel allows for comprehensive analysis of the reconstituting immune system and identification of potential markers of immune cell dysfunction.

Comparative Assessment of Cytometry by Time-of-Flight and Full Spectral Flow Cytometry Based on a 33-Color Antibody Panel

“…S1, S2). This panel encompasses major NK cell receptor classes such as natural cytotoxicity receptors (NCRs), C-type lectin like receptors (NKG2) and activation marker such as perforin, granzyme B and CD107a (Pegram et al, 2011;Mahnke et al, 2015;Vanikova et al, 2022;Kay et al, 2016). To ensure that a relevant comparison of the technologies could be made, we developed the identical panel for full spectral flow cytometry and mass cytometry encompassing the exact same antibodies.…”

Standardization of Workflow and Flow Cytometry Panels for Quantitative Expression Profiling of Surface Antigens on Blood Leukocyte Subsets: An HCDM CDMaps Initiative

“…Effector memory (EM) and terminal effector memory (TEMRA) stages contain further subsets of early, intermediate and late stages defined by CD28 expression (42) and correlate with the chronic carrier status of Cytomegalovirus and Epstein-Barr virus (46,47). Because expanding the knowledge of T cell subsets by polychromatic cytometry has led to identification of new functional subsets (48), subsets defined by checkpoint inhibitors (49) or tissue-specific subsets (28) at a fast pace, our simplified subset definitions may yield heterogeneous expression signals on some subsets containing finer subtypes, but the general description will be true and useful nonetheless. Detection of the PE signal from excitation with the 561 nm laser in the current study improved signal sensitivity because of decreased autofluorescence of leukocytes and reduced the data spread from decreased spillover of the FITC reagent, as expected (50).…”

Specific immune response to mRNA vaccines against COVID-19 in patients receiving allogeneic stem cell transplantation for myeloid malignancy was altered by immunosuppressive therapy

“…The recently published 29-colour panel of reagents OMIP-080 [14] was employed for the quantitative and qualitative analysis of the major immune cell populations in HSCT recipients at month 6 after HSCT. Flow cytometry measurement was performed using the BD Symphony A5 instrument equipped with five lasers.…”

“…Flow cytometry measurement was performed using the BD Symphony A5 instrument equipped with five lasers. Specification of antibodies, fluorochromes and instrument configuration was described previously and staining of cells was performed according to published protocol [14] . We evaluated frequencies of αβT cells, γδT cells, NK cells and NKT cells.…”