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

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.

Biology of GD2 ganglioside: implications for cancer immunotherapy

“…Melanocytes, the pigmental cells of the skin, have also been shown to express GD2, in line with their neuroectodermic origin ( Svennerholm et al, 1994 ). Noteworthy, expression of GD2 has been detected on T cells activated artificially or in disease ( Hersey and Jamal, 1989 ; Villanueva-Cabello et al, 2015 ; Villanueva-Cabello and Martinez-Duncker, 2016 ). This may have an impact on GD2-targeting therapies that aim to engage the immune system, as T cells may be targeted as well, with particular relevance for GD2 CAR-T cells.…”