Polysialic Acid in the Immune System.

Polysialic acid (polySia) is a highly regulated polymer of sialic acid (Sia) with such potent biophysical characteristics that when expressed drastically influences the interaction properties of cells. Although much of what is known of polySia in mammals has been elucidated from the study of its role in the central nervous system (CNS), polySia is also expressed in other tissues, including the immune system where it presents dynamic changes during differentiation, maturation, and activation of different types of immune cells of the innate and adaptive response, being involved in key regulatory mechanisms. At least six polySia protein carriers (CCR7, ESL-1, NCAM, NRP2, ST8Sia 2, and ST8Sia 4) are expressed in different types of immune cells, but there is still much to be explored in regard not only to the regulatory mechanisms that determine their expression and the structure of polySia chains but also to the identification of the cis- and trans- ligands of polySia that establish signaling networks. This review summarizes the current knowledge on polySia in the immune system, addressing its biosynthesis, its tools for identification and structural characterization, and its functional roles and therapeutic implications.

Polysialic acid is expressed in human naïve CD4+ T cells and is involved in modulating activation.

The activation of human naïve CD4+ T cells, responsible for orchestrating the immune response, has been reported to cause increased de novo sialylation and overexpression of the genes coding for polysialyltransferases ST8SiaII and ST8SiaIV, suggesting the potential of CD4+ T cells to synthesize polysialic acid (PSA), a type of glycosylation not previously described in these cells. PSA has been found as a post-translational modification in a limited number of mammalian proteins, having a very relevant role in modulating interactions due to its characteristic biophysical properties. In this work, we confirm that human CD4+ T cells express both polysialyltransferases and synthesize PSA, as assessed with the anti-PSA monoclonal antibody (mAb) 12E3. The expression of PSA in resting cells was found restricted to a cell subpopulation (PSA+), that after anti-CD3/anti-CD28 mAbs mediated activation, increased in percentage and mean fluorescence intensity (MFI) expression. Additionally, through ST8SIAII and ST8SIAIV-silencing and by measuring the mRNA of IL-2, IL-2R and IFN-γ, we show that PSA is involved in modulating the activation response of CD4+ T cells.

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

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.

Activation of human naïve Th cells increases surface expression of GD3 and induces neoexpression of GD2 that colocalize with TCR clusters.

CD4+ T helper lymphocytes (Th) orchestrate the immune response after their activation by antigen-presenting cells. Activation of naïve Th cells is reported to generate the reduction in surface epitopes of sialic acid (Sia) in α2,3 and α2,6 linkages. In this work, we report that in spite of this glycophenotype, anti-CD3/anti-CD28-activated purified human naïve Th cells show a significant increase in surface Sia, as assessed by metabolic labeling, compared with resting naïve Th cells, suggesting an increased flux of Sia toward Siaα2,8 glycoconjugates. To understand this increase as a result of ganglioside up-regulation, we observed that very early after activation, human naïve Th cells show an increased expression in surface GD3 and neoexpression of surface GD2 gangliosides, the latter clustering with the T cell receptor (TCR). Also, we report that in contrast to GM2/GD2 synthase null mice, lentiviral vector-mediated silencing of the GM2/GD2 synthase in activated human naïve Th cells reduced efficient TCR clustering and downstream signaling, as assessed by proliferation assays and IL-2 and IL-2R expression, pointing to an important role of this enzyme in activation of human naive Th cells.