OMIP‐030: Characterization of human T cell subsets via surface markers

The present panel was optimized to quantify the relative frequency of the majority of the major T cell subsets currently described within human peripheral blood mononuclear cells (PBMCs) via the use of surface markers (Table 1). This includes all CD4+ T subsets that received a T helper—nomenclature to date and Tregs. Furthermore, a surrogate staining strategy for the identification of mNKT/MAIT cells without the need of Vα7.2 is proposed. The panel has been validated for fresh and cryopreserved PBMCs. Other tissue types have not been tested. Activated CD4+ T cells can differentiate into distinct lineages, commonly referred to as T helper (Th) cells, with distinct phenotype and function 1. Through the production of a specific blend of cytokines, Th subsets can orchestrate ongoing immune responses. Although Th subsets are ultimately defined by the transcription factors they express, the use of surface markers as surrogates facilitates their investigation. In particular, the expression of different combinations of chemokine receptors can be used to identify distinct Th subsets 2. However, some authors utilize staining panels with slightly different gating hierarchies to investigate similar CD4+ T cell subsets, which can complicate the comparison of studies. The aim here was to establish a staining panel that allows the identification of all well described CD4+ T helper subsets and Tregs in a single staining. Due to the significant heterogeneity, complexity and flexibility of T cell subsets, a single staining panel cannot encompass all T cell subsets. Therefore, we focused on the simultaneous identification of those T cell subsets that received a T helper—nomenclature to date (Table 2). By staining live cells for CD3, CD4, CD8, CD20, CD45RA, and CD197 (CCR7), one can differentiate and enumerate naïve (CD45RA+ CD197+), effector (CD45RA+ CD197−), central memory (TCM, CD45RA− CD197+), and effector memory (TEM, CD45RA− CD197−) CD4+ and CD8+ T cells 2. Additionally, the combination of CD25 and CD127 allows the enumeration of CD4+ regulatory T cells (Tregs, CD25+ CD127low) by surface markers 3. Furthermore, by inclusion of the chemokine receptors CCR10, CD183 (CXCR3), CD185 (CXCR5), CD194 (CCR4), and CD196 (CCR6), we could successfully differentiate and enumerate all CD4+ T subsets that received a Th-nomenclature to date. In particular, these are TFH (CD185+), Th1 (CD183+), Th2 (CCR10− CD194+ CD196−), Th9 (CD194− CD196+), Th17 (CCR10− CD194+ CD196+), Th22 (CCR10+ CD194+ CD196+) T cells, and GM-CSF+ Th cells (CCR10+ CD194+) that produce GM-CSF 4 and IL-3 5. The gating scheme is depicted in Figure 1 and described in detail in the Supporting Information. Example staining and gating. Cryopreserved PBMCs from healthy human donors were thawed, rested over night, and stained as outlined in the online protocol. The gating strategy for the identification of T cell subsets is shown. Besides conventional T cells, human PBMCs contain several innate like T cell populations 6. Most prominent in humans are Mucosal Associated Invariant T cells, also called MAIT 7 or mNKT 8 cells, that express an invariant TCRα chain, which in human utilizes Vα7.2/Jα33, that recognizes the MHC class I homolog MR1. The standard approach to identify mNKT/MAIT cells by flow cytometry is the combination of CD8, Vα7.2, and CD161 7, 9. However, we noticed that our staining panel allows the identification of mNKT/MAIT cells via chemokine receptors just with the addition of CD161. Therefore, we propose here an alternative strategy to identify mNKT/MAIT cells without the need of Vα7.2, as those T cells that are CD4− CD8+ CD127+ CD45RA− CD161+ CD194− CD196+ CD197− (Fig. 1 and Supporting Information Figs. 3G and 3H). We also aimed to gain additional functional information on T cells. Therefore, the activation markers CD38 was included in the panel. Additionally, the nuclear protein Ki67 marks cells in the active phases of the cell cycle (G1, S, G2, and mitosis) and can be used as proliferation marker 10. In summary, the panel outlined here allows for the enumeration of all CD4+ T subsets that received a Th-nomenclature to date, including Tregs, via surface markers. Furthermore, a surrogate staining strategy for mNKT/MAIT cells without the need of Vα7.2 is outlined. This OMIP is similar to OMIP-013, −015, −017, −018, and −021 in its objective to enumerate various human T cells subset in PBMCs. Additional Supporting Information may be found in the online version of this article. Supporting Information Supporting Information Supporting Information Supporting Information Supporting Information Supporting Information Supporting Information Supporting Information Supporting Information Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.