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.
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.
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 functions4,5. 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).
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
2. Adherence of Resting and Activated Naïve CD4+ T Cells
3. Collecting and Fixation of Resting and Activated Naïve CD4+ T Cells
4. Staining, Mounting and Visualization by Confocal Microscopy
5. Isolation of RNA
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 (Figure 1C). The CD69 marker is currently used as early activation marker by flow cytometry or microscopy. Adherence of resting and activated naïve CD4+ T cells to a coverslip coated with poly-L-lysine are useful to study the expression and localization of GD3 and TCR showed by a double labeling with anti GD3 and anti TCR antibodies (Figure 2). As we reported previously, activation is accompanied by remodeling of GD3 and GD2 gangliosides in the cells surface6. Additionally, gene expression quantification of the GD3 and GM2/GD2 synthases is performed from 4 x 104 cells (Figures 2 and 3). The neo-expression of the GD2 ganglioside and TCR colocalization is adequately observed by confocal microscopy, demonstrating a possible role of GD2 in TCR clustering during activation (Figure 3). Figure 4 shows the localization of the GD3 and GD2 ganglioside stained simultaneously in activated CD4+ T cells. Additionally, activated PBMCs were stained with anti GD3 and anti GD2 antibodies showing that this protocol can be used in other immune cell populations found in PMBCs, particularly GD2 that was found to be expressed in all the activated PMBCs (Figure 5).
Figure 1: Visualization of naïve CD4+ T cells after adherence and efficient anti CD3/anti-CD28 activation. (A) Resting CD4+ T cells at 72 hr post-activation attached on poly-L-lysine treated coverslip are observed by brightfield microscopy. (B) Activated CD4+ T cells at 72 hr attached on poly-L-lysine treated coverslip. (C) CD25 marker expression (red) and Hoechst 333258 stained nuclei (blue) of anti CD3/anti CD28 activated CD4+ T cells at 72 hr post-activation. Scale bar = 50 µm. Images were obtained by confocal microscopy with a 60X S/1.3 oil objective with 2X digital zoom. Please click here to view a larger version of this figure.
Figure 2: GD3 ganglioside and TCR stain and microscopy localization in resting and activated naïve CD4+ T cells at 72 h post-activation. Resting CD4+ T cells with (A) nuclei stained with Hoechst 333258, (B) GD3 ganglioside localization, (C) Merge of nuclei and GD3 ganglioside. Activated CD4+ T cells with (D) nuclei stained with Hoechst 333258, (E) GD3 ganglioside localization and (F) Merge of nuclei and GD3 ganglioside. (G) and (H) show TCR and GD3 ganglioside in resting naïve CD4+ T cells. (J and K) corresponds to TCR and GD3 ganglioside staining in activated CD4+ T cells. Confocal images of TCR (red) and GD3 ganglioside (green) localization in resting naïve CD4+ T cells (I) and anti CD3/CD28 Activated naïve CD4+ T cells 72 hr post-activation (L). The GD3, TCR and nuclei stain was assessed by confocal microscopy from one stack with a 60X S/1.3 oil objective with 2X digital zoom. (M) The graph shows the gene expression for GD3 synthase determined by real-time PCR expressed as Fold change from 4 x 104 resting (Rest) and activated (Act) naïve CD4+ T cells at 72 hr post-activation. Data are the mean ± S. D. from three independent donors. Please click here to view a larger version of this figure.
Figure 3: GD2 ganglioside and TCR stain and microscopy localization in resting and activated naïve CD4+T cells at 72 h post-activation. Resting CD4+ T cells with (A) nuclei stained with Hoechst 333258, (B) GD2 ganglioside localization, (C) Merge of nuclei and GD2 ganglioside. Activated CD4+ T cells with (D) nuclei stained with Hoechst 333258, (E) GD2 ganglioside localization and (F) Merge of nuclei and GD2 ganglioside. (G and H) show TCR and GD2 ganglioside in resting naïve CD4+ T cells. (J and K) corresponds to TCR and GD2 ganglioside staining in activated CD4+ T cells. Confocal images of TCR (red) and GD2 ganglioside (green) localization in resting naïve CD4+ T cells (I) and anti CD3/CD28 Activated naïve CD4+ T cells 72 hr post-activation (L). The GD2, TCR and nuclei stain was assessed by confocal microscopy from one stack with a 60X S/1.3 oil objective with 2X digital zoom. (M) The graph shows the gene expression for GM2/GD2 synthase determined by real-time PCR expressed as Fold change from 4 x 104 resting (Rest) and activated (Act) naïve CD4+ T cells at 72 hr post-activation. Data are the mean ± S. D. from three independent donors. Please click here to view a larger version of this figure.
Figure 4: Double stain with anti-GD3 and anti-GD2 antibodies for GD3 and GD2 gangliosides localization. (A) GD3 ganglioside identified with R24 anti GD3 antibody in activated CD4+ T cells shows intracellular and membrane localization. (B) GD2 ganglioside identified with 14G2a anti GD2 antibody in activated CD4+ T cells shows only plasma membrane localization. (C) Brightfield microscopy from activated CD4+ T cells. The GD3 and GD2 stain was assessed by confocal microscopy from one stack with a 60X S/1.3 oil objective with 2X digital zoom. Please click here to view a larger version of this figure.
Figure 5: GD3 and GD2 ganglioside localization in activated PBMCs. (A) GD2 ganglioside was detected with 14G2a anti GD2 antibody in anti CD3/anti CD28 72 hr post-activated PBMCs overlay with brightfield microscopy, (B) GD2 ganglioside expression and (C) GD2 ganglioside and nuclei merge. (D) GD3 ganglioside stained with R24 anti GD3 antibody overlay with brightfield, (E) GD3 ganglioside expression and (F) GD3 ganglioside and nuclei merge. The GD3, GD2 and nuclei stain was assessed by confocal microscopy from one stack with a 60X S/1.3 oil objective with 2X digital zoom. Scale bar = 45 µm. Please click here to view a larger version of this figure.
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 analysis is a key advantage over the use of techniques such as thin layer chromatography for detection of gangliosides because it reveals the spatial localization and dynamics of gangliosides during treatment (e.g., T cell activation), leading to acquisition of biologically relevant data11,12,13. Also, this protocol allows the evaluation of ganglioside expression starting from a small number of CD4+ T cells (2 x 104) that is useful for optimizing the number of replicates or different assays6.
The careful treatment during washes and antibody staining after adhesion is crucial to maintain cell number and integrity. Because of the weak binding of CD4+ T cells to the coverslip, it is also very important to carefully add and remove solutions; otherwise a reduction in cell number and increased fluorescent debris will be encountered. When assessed through confocal microscopy, the immunostaining of gangliosides confers additional information such as spatial localization or co-localization with other molecules in the plasma membrane or intracellular compartments. Subcellular localization requires the use of subcellular markers14,15. In addition, fresh and not fixed CD4+ T cells need to be used for staining when determining ganglioside expression restricted to the cell surface. Although fixation of CD4+ T cells helps preserve the sample for at least 5 days, it carries the risk of permeabilization and intracellular staining.
However, because of the structural similarity between gangliosides, it is always advisable to take into account the specificity of antibodies used. Several of the commercially available anti-ganglioside monoclonal antibodies can be used and for some of them the specificity has been probed by ELISA, TLC, etc.16,17,18.
Previous works demonstrate that it is possible to obtain information regarding localization and expression of GD3 and GD2 gangliosides from 2 x 104 cells/per condition6. Also, this protocol describes the capability of performing gene profiling experiments from 4 x 104 cells, allowing optimization of silencing experiments such as those based in lentiviral particles6.
The authors have nothing to disclose.
We are grateful to Dr. José Luis Daniotti for comments. We thank the assistance to Dr. J. Arturo Pimentel and the Laboratorio Nacional de Microscopìa Avanzada-UNAM for acquisition of confocal images. I.M.-D. and T.M.V.C. were supported by grants 157634 and 253596 from Consejo Nacional de Ciencia y Tecnologìa (CONACYT), Sociedad Latinoamericana de Glicobiologìa, A.C. T.M.V.-C. is recipient of a scholarship (245192) from CONACYT. We also thank the support of the Red Temática Glicociencia en Salud – CONACYT (253596).
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 |