Flow cytometric identification of Tfh13 cells in mouse and human

Abstract

Anaphylaxis is a life-threatening allergic reaction caused by cross-linking of high-affinity IgE antibodies on the surface of mast cells and basophils. Understanding the cellular mechanisms that lead to high-affinity IgE production is required to develop better therapeutics for preventing this severe reaction. A recently discovered population of T follicular helper T_fh13 cells regulates the production of high-affinity IgE in mouse models of allergy and can also be found in patients with allergies with IgE antibodies against food or aeroallergens. Here we describe optimized protocols for identifying T_fh13 cells in both mice and humans.

Keywords: IL-13; IL-4; IL-5; IgE; T follicular helper cells; T(fh)13 cells; allergy; intracellular cytokine staining.

Figure 1.. Identification of polyclonal murine Tfh13 cells by PMA/ionomycin stimulation

Day 7 mediastinal lymph node (medLN) cells from intranasal (i.n.) immunization with Alternaria alternata and NP-OVA followed by in vitro PMA/ionomycin stimulation. (A) Gating strategy for murine Tfh13 cells. Histogram overlay of BCL-6 expression by naïve CD4⁺ T cells, non-Tfh cells, Tfh2 cells, Tfh13 cells, and fluorescence minus one (FMO) control. (B) IL-5 production by Tfh2 vs Tfh13 cells. (C) PD-1 and CXCR5 staining in non-immunized non-draining lymph node (ndLN), immunized Tfh cell-deficient medLN (Bcl6^f/fCd4^Cre), and FMO control for CXCR5 in immunized WT medLN (CXCR5 FMO). (D) IL-4 and IL-13 intracellular staining in immunized WT medLN without stimulation. Pre-gated on Tfh cells (live CD4⁺ TCRβ⁺ CD44⁺ PD-1⁺ CXCR5⁺). (E) IL-4 and IL-13 FMO and isotype control staining in immunized WT medLN with stimulation. Pre-gated on Tfh cells. Samples were acquired on a BD LSR II (BD Biosciences).

Figure 2.. Identification of polyclonal murine Tfh13 cells by anti-CD3/CD28 stimulation and antigen-specific Tfh13 cells by NP-OVA stimulation

Day 7 medLN from i.n. immunization with Alternaria alternata and NP-OVA following (A) anti-CD3/CD28 or (B) NP-OVA stimulation. (A) IL-4 and IL-13 intracellular staining in anti-CD3/CD28-stimulated or non-stimulated cells. Pre-gated on Tfh cells. (B) IL-4 and IL-13 intracellular staining in CD154− versus CD154+ NP-OVA-stimulated cells. Pre-gated on Tfh cells. CD154 helps enrich for antigen-specific Tfh13 cells during analysis. Samples were acquired on a CytoFLEX (Beckman Coulter).

Figure 3.. Enhanced cytokine production with stimulation in IMDM compared to RPMI

(A) Th2 cells were cultured in vitro for 3 days with plate-bound anti-CD3 (2 μg/ml), soluble anti-CD28 (2 μg/ml), human IL-2 (12.5 ng/ml), murine IL-4 (25 ng/ml), anti-IFN-γ (5 μg/ml), and anti-IL-12 (2 μg/ml). Th2 cells were re-plated on day 3 without anti-CD3 and rested for 2 additional days. On day 5 of the culture, Th2 cells were stimulated with PMA/ionomycin in complete RPMI versus IMDM. IL-4 and IL-13 intracellular staining is shown. Pre-gated on live CD4+ cells. (B) IL-4 and IL-13 intracellular staining in day 7 medLN cells from i.n. immunization with Alternaria alternata and NP-OVA. Cells were stimulated with PMA/ionomycin in complete RPMI versus IMDM. Pre-gated on Tfh cells (live CD4⁺ TCRβ⁺ CD44⁺ PD-1⁺ CXCR5⁺). Samples were acquired on a BD LSR II (BD Biosciences).

Figure 4.. Identification of polyclonal human Tfh13 cells

(A) Gating strategy for polyclonal human Tfh13 cells from an aeroallergen-sensitized patient after PMA/ionomycin stimulation. (B) IL-4 and IL-13 fluorescence minus one (FMO) and isotype control staining in cells from an allergic patient with stimulation. Pre-gated on circulating Tfh cells (live CD4⁺ CD3⁺ CD45RA^– CXCR5⁺). (C) IL-4 and IL-13 intracellular staining in an allergic patient and a healthy control following no stimulation or PMA/ionomycin stimulation. Pre-gated on circulating Tfh cells. (D) IFN-γ and IL-13 intracellular staining in an allergic patient following PMA/ionomycin stimulation. Pre-gated on circulating Tfh cells. Samples were acquired on a BD FACSymphony A5 (BD Biosciences).

Figure 5.. Identification of peanut-specific human Tfh13 cells

(A) Gating strategy for peanut-specific human Tfh13 cells from an allergic patient, with peanut stimulation or no stimulation. (B) IL-4 and IL-13 production by a healthy control, with peanut stimulation or no stimulation. Pre-gated on circulating Tfh cells (live CD4⁺ CD3⁺ CXCR5⁺). Samples were acquired on a BD LSRFortessa (BD Biosciences).