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A major obstacle hampering the therapeutic application of regulatory T (Treg) cells is the lack of suitable extracellular markers, which complicates their identification/isolation. Treg cells are normally isolated via CD25 (IL-2Ra) targeting, but this protein is also expressed by activated CD4

This translational study represents the first human demonstration of the efficacy of blood glutamate grabbers in the treatment of patients with stroke, paving the way for the development of a promising novel protective therapy. Ann Neurol 2018;84:260-273.

BackgroundExcitatory amino acid transporter 2 (EAAT2) plays a pivotal role in glutamate clearance in the adult brain, thereby preventing excitotoxic effects. Considering the high efficacy of EAAT2 for glutamate uptake, we hypothesized that the expression of this transporter in mesenchymal stem cells (MSCs) for systemic administration could yield a cell-based glutamate-grabbing therapy, combining the intrinsic properties of these cells with excitotoxic protection.MethodsTo address this hypothesis, EAAT2-encoding cDNA was introduced into MSCs and human embryonic kidney 293 cells (HEK cells) as the control cell line. EAAT2 expression and functionality were evaluated by in vitro assays. Blood glutamate-grabbing activity was tested in healthy and ischemic rat models treated with 3 × 106 and 9 × 106 cells/animal.FindingsThe expression of EAAT2 in both cell types conferred the expected glutamate-grabbing activity in in vitro and in vivo studies. The functional improvement observed in ischemic rats treated with EAAT2–HEK at low dose, confirmed that this effect was indeed mediated by the glutamate-grabbing activity associated with EAAT2 functionality. Unexpectedly, both cell doses of non-transfected MSCs induced higher protection than transfected EAAT2–MSCs by another mechanism independent of the glutamate-grabbing capacity.InterpretationAlthough the transfection procedure most likely interferes with some of the intrinsic protective mechanisms of mesenchymal cells, the results show that the induced expression of EAAT2 in cells represents a novel alternative to mitigate the excitotoxic effects of glutamate and paves the way to combine this strategy with current cell therapies for cerebral ischemia.

IL-12 is a cytokine that stimulates the expression of CD26, a T cell- and raft-associated ectopeptidase. IL-12 also enhances the interaction between CD26 and CD45RO, which removes the phosphatase CD45RO from raft microdomains. Since Janus kinases are known CD45 substrates, our hypothesis was that this relocation of CD45RO in nonraft areas of the membrane could be important to switch off the signaling via cytokine receptors, e.g., the IL-12 receptor (IL-12R). Accordingly, both IL-12R and CD45RO should be equally positioned in the cell membrane upon IL-12R ligation. However, there were no data available on the membrane distribution of IL-12R on human T cells. Working with phytohemagglutinin (PHA) lymphoblasts, we tried to fill that gap. The high-affinity IL-12R is made of two chains: IL-12Rbeta1 and IL-12Rbeta2. Using flow cytometry, Western blot and confocal microscopy, we obtained data suggesting that IL-12Rbeta1 is mainly associated to phospholipid-rich membrane areas, a location even enhanced upon IL-12 incubation of PHA blasts. Instead, IL-12Rbeta2 is found more segregated into membrane rafts, which could explain why two IL-12-triggered events, T-cell proliferation and ERK1/2 activation, are both methyl-beta-cyclodextrin-sensitive events. Ligation of IL-12R with IL-12 seems to induce a partial enrichment of IL-12Rbeta2 in phospholipid-rich areas, where according to our data IL-12Rbeta1 is already present. Therefore, although new data will be required, the present results support the initial hypothesis.

CD26 displays variable levels between effector (TH 17 ≫ TH 1 > TH 2 > Treg) and naïve/memory (memory > naïve) CD4 + T lymphocytes. Besides, IL-6/IL − 6R is associated with TH 17 -differentiation and asthma severity. Allergic/atopic asthma (AA) is dominated by TH 2 responses, while TH 17 immunity might either modulate the TH 2 -dependent inflammation in AA or be an important mechanism boosting non-allergic asthma (NAA). Therefore, in this work we have compared the expression of CD26 and CD126 (IL-6Rα) in lymphocytes from different groups of donors: allergic (AA) and non-allergic (NAA) asthma, rhinitis, and healthy subjects. For this purpose, flow cytometry, haematological/biochemical, and in vitro proliferation assays were performed. Our results show a strong CD26-CD126 correlation and an over-representation of CD26 − subsets with a highly-differentiated effector phenotype in AA (CD4 + CD26 −/low T cells) and NAA (CD4 − CD26 − γδ-T cells). In addition, we found that circulating levels of CD26 (sCD26) were reduced in both AA and NAA, while loss of CD126 expression on different leukocytes correlated with higher disease severity. Finally, selective inhibition of CD26-mRNA translation led to enhanced T cell proliferation in vitro . These findings support that CD26 down-modulation could play a role in facilitating the expansion of highly-differentiated effector T cell subsets in asthma.

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