Human B cells express Neuron/Glial 2 antigen. Possible implications for health and autoimmune disease of the central nervous system.
Creators: name: GANDOGLIA, ILARIA
Publisher: name: Universit� degli studi di Genova
Year: 2020

description: Neuron/Glial 2 antigen (NG2) is a membrane proteoglycan expressed in many cellular types. In central nervous system (CNS), NG2 is expressed on oligodendrocyte precursor cells and on pericytes. NG2 is involved in cellular adhesion mechanisms, but its role is not completely clear. We showed that in the animal model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE), NG2 is also expressed on immune cells like T cells, monocytes and dendritic cells (DCs) and that its expression on immune cells significantly influences the course of EAE, with NG2KO mice displaying a milder disease. In this study, we have assessed the expression of NG2 on human immune cells and its possible role in their activation in healthy donors (HD) and in patients affected by autoimmune diseases of CNS (MS and neuromyelitis optica (NMO)). 
Monitoring of adaptive and innate cell immune populations for their expression of NG2 was performed by flow cytometry of B-, T- and natural killer cell subsets validating the use of a monoclonal anti-human NG2 antibody (clone 148.3). Our results on three HD blood samples indicated that while T cells do not express NG2, and NK cells express it to a very minor extent, 12.1 ? 7.6% CD19+ B cells are NG2+. Accordingly, we have furthered our analysis to single B cell subsets, and show that about 25% B memory cells were NG2+, while NG2+ B regulatory and B mature cells are present in lesser amount (8%). 
We showed that the frequency of NG2+ cells did not differ between HD (N=16) and patients with MS (N=16). Moreover, we investigated NG2 expression on B cells isolated from 2 patients affected with NMO, a rare predominantly B-cell-mediated autoimmune disease of CNS. In each of the two patients we observed a drastic increase in percentage of NG2+ B cells in the total B-cell population and on the different B-cell subsets as compared to HD or MS patients. 
To evaluate a possible role of NG2 in immune cells activation, we studied NG2 expression upon in vitro activation of B cells. Interestingly, NG2 expression decreased upon activation of total CD19+ B cells. To assess if activation of B cells is associated with changes in NG2 expression, we stimulated separately sorted NG2+ and NG2- B cells. We demonstrated a mild decrease in NG2 expression on NG2+ sorted cells, whereas some NG2+ cells were observed in the NG2- sorted cells, indicating a possible correlation between cell-activation and NG2 expression. Moreover, we investigate NG2 expression on B cells collected form MS patients treated with alemtuzumab, an anti-CD52 monoclonal antibody associated with development of secondary B-cell-mediated autoimmune disease in 30% of patients. We, therefore, postulated the possibility that the autoimmunity risk might be associated with changes in NG2 expression on B cells and/or changes in frequency of NG2-expressing B cells. Accordingly, we investigated the impact of anti-CD52 treatment on NG2 expression on different B-cell subsets at times when secondary autoimmunity generally develops, in 7 treated patients. Notably, we observed that the treatment was associated to a significant decrease of B memory cells but we did not show any significant difference in frequency of NG2+ cells indicating that NG2+ cells are probably not involved in autoimmune adverse reactions related to the treatment.
In conclusion our data show for the first time that NG2 is expressed on human immune cells and in particular on B memory cells which play a crucial role in MS pathogenesis. The results suggest that NG2 expression fluctuates with B-cell activation, but further studies are necessary to better understand the possible role of NG2 on human immune cells.