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.

Conflict of interest statement

The authors have no conflict of interest to declare.

© 2021 The Authors. Cytometry Part A published by Wiley Periodicals LLC on behalf of International Society for Advancement of Cytometry.

Figures

FIGURE 1

Manual gating strategy and algorithmic analysis. (A) Before the identification of individual subsets, debris, doublets and dead cells were removed. (B) Cell lineages were identified based on the expression of the following phenotypes: CD56+ (NK cells), CD56−CD3+ γδTCR+ (γδT cells), CD3+γδTCR‐CD56+ T cells, CD3+γδTCR−CD56−CD4+ (T helper cells) and CD3+γδTCR−CD56−CD8+ (T cytotoxic cells). (C) CD4+ T cells were delineated into CD25+FoxP3+ Tregs and non‐Treg cells. Activation of Tregs was evaluated based on the expression of CD39. Furthermore, expression of PD1, TIGIT, and Tim3 identifying highly suppressive Tregs was evaluated. Cells in the non‐Treg gate were divided into individual T helper subsets based on the combination of chemokine receptors. These cells were first gated for perforin+CD27−CTL cells and perforin− cells. The perforin− cells were gated for CCR10+ CCR4+ CCR6+ Th22 cells, CCR10+ CCR4+CCR6− ThGM‐CSF, CCR10−CCR4+CCR6− Th2 cells, CCR10−CCR4+CCR6+ Th17 cells, CCR10‐CCR4‐CCR6+CXCR3− Th9 cells and CCR10−CCR4−CCR6+/−CXCR3+ Th1 cells. Thymic function was evaluated on CD4+ gated cell via identification of CD45RA+CD62L+CD95−CD31+ cells. (D) Gated CD8+ cells were subtyped into individual memory subsets based on CD45RA and CD62L and expression of inhibitory receptors, activation markers and perforin was evaluated. Inhibiting receptors are only weakly expressed on T cells from healthy donors, and therefore we show a comparison between healthy and patients after HSCT. (E) Gating of γδT cells based on CD57−NKG2A+ cytokine‐producing cells, CD57+NKG2A− terminally differentiated cytotoxic cells and CD57−NKG2A− with intermediate phenotype. Expression of perforin and TIGIT on these cells is depicted using a histogram. (F) NK cells were subtyped based on the expression of CD56 and CD16, and a comparison of a healthy donor and post‐HSCT patient is shown. CD56hiCD16‐ cells represent less differentiated cytokine‐producing cells characterized by low expression perforin and high expression of CD27, whereas CD56loCD16+ cells express high levels of perforin and lack NKG2A and CD27. These cells include a subset of CD57+NKG2C+ memory‐like NK cells. (G) CD56+ T cells can be divided into three main lineages based on CD4 and CD8. The CD4+ cells are enriched with cytotoxic perforin expressing cells mostly terminally differentiated, as evident by expression of CD57. The CD8+ cell can be split into two subsets based on the expression of NKG2C. These subsets show distinct differentiation status, as evident by the expression patterns of CD45RA and CD62L. (H) Identification of individual cell types was performed on gated live single cells using the Embedding Guided by Self‐Organizing Map (EmbedSOM) algorithm [20]. Expression pattern of markers on individual cell subsets is show in Figure S8. FSC, forward‐scatter; SSC, side‐scatter [Color figure can be viewed at wileyonlinelibrary.com]