Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy

Summary

We describe a standard antibody staining protocol for use in microscopy to determine the membrane expression and localization of gangliosides in resting and activated human naïve CD4⁺ T cells. Also described are real-time PCR experiments using <40,000 cells that do not require additional low input RNA kits.

Abstract

The methods described herein for activation of naïve CD4⁺ T cells in suspension and their adherence in coverslips for confocal microscopy analysis allow the spatial localization and visualization of gangliosides involved in CD4⁺ T cell activation, that complement expression profiling experiments such as flow cytometry, western blotting or real-time PCR. The quantification of ganglioside expression through flow cytometry and their cellular localization through microscopy can be obtained by the use of anti-ganglioside antibodies with high affinity and specificity. Nonetheless, an adequate handling of cells in suspension involves the treatment of culture plates to promote the necessary adherence required for fluorescence or confocal microscopy acquisition. In this work, we describe a protocol for determining GD3 and GD2 ganglioside expression and colocalization with the TCR during naïve CD4⁺ T cell activation. Also, real-time PCR experiments using <40,000 cells are described for the determination of the GD3 and GM2/GD2 synthase genes, demonstrating that gene analysis experiments can be performed with a low number of cells and without the need of additional low input RNA kits.

Introduction

The CD4⁺ T cells orchestrate the immune response through their effector functions after activation by antigen presenting cells¹. The study of the cellular mechanisms that are modulated during activation allows insight into a basic process of immune function. However, the study of naïve CD4⁺ T cells can be complicated because they represent a very small population of cells in the blood periphery².

Through fluorescence microscopy several reports have studied the localization of different molecules involved in CD4⁺ T cell activation, mainly proteins associated to the plasma membrane³. The gangliosides are sialic acid containing glycosphingolipids and although they have been extensively studied in nerve cells where they are abundant, other cells such as immune cells also express gangliosides with biologically relevant functions^4,5. We previously reported that during activation of human naïve CD4⁺ T cells there is an upregulation of the α2,8 sialyltransferase ST8Sia 1 (GD3 synthase) and the GM2/GD2 synthase, that induce the significant surface neoexpression of GD2 and the upregulation of GD3 ganglioside⁶. Further study of GD3, GD2 and other gangliosides in immune cells is necessary to complement a protein-based partial view of immune function.

Commonly, the study of ganglioside expression is based on techniques such as Thin Layer Chromatography (TLC)⁷, but this technique does not allow the spatial localization of gangliosides at the plasma membrane or in subcellular compartments, limiting biological analysis.

In this work, we describe a protocol for the antibody-mediated identification and localization of GD3 and GD2 gangliosides in human naïve CD4⁺ T cells and PBMCs after anti-CD3/anti-CD28 activation. With this protocol it is also possible to analyze the gene and molecular expression of gangliosides in a low number of cells in suspension, with acquisition of high quality images⁶, considering the small size of lymphocytes (9 µm).

Protocol

Peripheral blood from healthy male donors was obtained with informed consent and approval of the Bioethics Committee of the Centro de Investigaciòn en Dinámica Celular- Universidad Autònoma del Estado de Morelos. 1. Isolation and Activation of Human Naïve CD4+ T Cells Collect 2 ml of peripheral blood derived from healthy human donors through informed consent and dilute with 2 ml of sterile PBS-EDTA (1x Phosphate Buffered Saline (PBS), 2 mM EDTA, pH 7….

Representative Results

The protocol described in this manuscript renders a good quality of cultured and adhered resting and activated human naïve CD4+ T cells (Figure 1). The activated CD4+ T cells show the characteristic proliferative profile (Figure 1B) in comparison to the resting condition (Figure 1A). The CD25 late activation marker is useful to evaluate the efficient activation at 72 hr observed by confocal microscopy (F…

Discussion

The described protocol can be used to localize gangliosides or proteins in cell suspensions of CD4⁺ T cells or other immune cells (e.g., PMBCs, Figure 5) starting from a small number of cells. Because of the small size of T cells and non-adherent properties, the acquisition of fluorescence microscopic images results in poor information or low quality if the cells are not correctly adhered.

This protocol combined with a good quality confocal microscopy analy…

Materials

Advanced RPMI 1640	Thermo Fisher Scientific	12633-012	Suplemented with 3% FBS
mouse anti human CD3 antibody	eBioscience	16-0037-81	OKT3 clone
mouse anti human CD28 antibody	ebioscience	16-0288-81	CD28.6 clone
mouse anti human GD3  antibody	Abcam	ab11779	R24 clone
mouse anti human GD2 antibody	Santa Cruz Biotechnology	sc-53831	14G2a clone
FITC-conjugated anti-mouse IgG3 antibody	Abcam	ab97259
Alexa Fluor 488 conjugated antimouse	Thermo Fisher Scieni¡tific	A-21131
IgG2a antibody
Alexa Fluor 647 conjugated antimouse	Thermo Fisher Scieni¡tific	A-21241
IgG2a antibody
Hoechst 333258	Sigma-Aldrich	861405
Ficoll Paque-Plus	GE	17-1440-02
Trizol Reagent	Invitrogen	15596-026
Naive CD4+ T Cell Isolation Kit II, human	MACS Miltenyi Biotec	130-094-131
BD FACSAria II	BD Biosciences		Sorting
Nunc Lab-tek chamber slide system	Sigma-Aldrich	C7182
Olympus FV 1000 Laser Confocal Microscope (Olympus)	Olympus		Upright BX61WI and IX81
Forward sequence primer GD3 synthase	5´-GAGCGTTCAGGAAACAAATGG- 3´		Ref. 7
Reverse sequence primer GD3 synthase	5´-CCTGTGGGAAGAGAGAGTAAG-3´		Ref. 7
Forward sequence primer GM2/GD2 synthase	5´-CAACACAGCAGACACAGTCC-3´		Ref. 7
Reverse sequence primer GM2/GD2 synthase	5´-GTGGCAATCGTGACTAGAGC-3´		Ref. 7