Human mesenchymal stromal cells (MSC) have been shown to dampen immune response and promote tissue repair, but the underlying mechanisms are still under investigation. Herein, we demonstrate that umbilical cord-derived MSC (UC-MSC) alter the phenotype and function of monocyte-derived dendritic cells (DC) through lactate-mediated metabolic reprogramming. UC-MSC can secrete large quantities of lactate and, when present during monocyte-to-DC differentiation, induce instead the acquisition of M2-macrophage features in terms of morphology, surface markers, migratory properties and antigen presentation capacity. Microarray expression profiling indicates that UC-MSC modify the expression of metabolic-related genes and induce a M2-macrophage expression signature. Importantly, monocyte-derived DC obtained in presence of UC-MSC, polarize naïve allogeneic CD4+ T-cells into Th2 cells. Treatment of UC-MSC with an inhibitor of lactate dehydrogenase strongly decreases lactate concentration in culture supernatant and abrogates the effect on monocyte-to-DC differentiation. Metabolic analysis further revealed that UC-MSC decrease oxidative phosphorylation in differentiating monocytes while strongly increasing the spare respiratory capacity proportional to the amount of secreted lactate. Because both MSC and monocytes are recruited in vivo at the site of tissue damage and inflammation, we propose the local increase of lactate concentration induced by UC-MSC and the consequent enrichment in M2-macrophage generation as a mechanism to achieve immunomodulation.

NK-cell resistance to transduction is a major technical hurdle for developing NK-cell immunotherapy. By using Baboon envelope pseudotyped lentiviral vectors (BaEV-LVs) encoding eGFP, we obtained a transduction rate of 23.0 ± 6.6% (mean ± SD) in freshly-isolated human NK-cells (FI-NK) and 83.4 ± 10.1% (mean ± SD) in NK-cells obtained from the NK-cell Activation and Expansion System (NKAES), with a sustained transgene expression for at least 21 days. BaEV-LVs outperformed Vesicular Stomatitis Virus type-G (VSV-G)-, RD114-and Measles Virus (MV)-pseudotyped LVs (p < 0.0001). mRNA expression of both BaEV receptors, ASCT1 and ASCT2, was detected in FI-NK and NKAES, with higher expression in NKAES. Transduction with BaEV-LVs encoding for CAR-CD22 resulted in robust CAR-expression on 38.3 ± 23.8% (mean ± SD) of NKAES cells, leading to specific killing of NK-resistant pre-BALL -RS4;11 cell line. Using a larger vector encoding a dual CD19/CD22-CAR, we were able to transduce and re-expand dual-CAR-expressing NKAES, even with lower viral titer. These dual-CAR-NK efficiently killed both CD19 KO-and CD22 KO-RS4;11 cells. Our results suggest that BaEV-LVs may efficiently enable NK-cell biological studies and translation of NK-cell-based immunotherapy to the clinic.

Parkinson disease (PD) is characterized by the preferential, but poorly understood, vulnerability to degeneration of midbrain dopaminergic (mDA) neurons in the ventral substantia nigra compacta (vSNc). These sensitive mDA neurons express Pitx3, a transcription factor that is critical for their survival during development. We used this dependence to identify, by flow cytometry and expression profiling, the negative regulator of G-protein signaling Rgs6 for its restricted expression in these neurons. In contrast to Pitx3−/− mDA neurons that die during fetal (vSNc) or post-natal (VTA) period, the vSNc mDA neurons of Rgs6 −/− mutant mice begin to exhibit unilateral signs of degeneration at around 6 months of age, and by one year cell loss is observed in a fraction of mice. Unilateral cell loss is accompanied by contralateral degenerating neurons that exhibit smaller cell size, altered morphology and reduced dendritic network. The degenerating neurons have low levels of tyrosine hydroxylase (TH) and decreased nuclear Pitx3; accordingly, expression of many Pitx3 target gene products is altered, including Vmat2, Bdnf, Aldh1a1 (Adh2) and Fgf10. These low TH neurons also express markers of increased dopamine signaling, namely increased DAT and phospho-Erk1/2 expression. The late onset degeneration may reflect the protective action of Rgs6 against excessive DA signaling throughout life. Rgs6-dependent protection is thus critical for adult survival and maintenance of the vSNc mDA neurons that are most affected in PD.

The homeodomain transcription factor Pitx3 is critical for the survival of midbrain dopaminergic (mDA) neurons. Pitx3-deficient mice exhibit severe but selective developmental loss of mDA neurons, with accompanying locomotor deficits resembling those seen in Parkinson's disease (PD) models. Here, we identify specific mDA cell subpopulations that are consistently spared in adult Pitx3-hypomorphic (aphakia) mice, demonstrating that Pitx3 is not indiscriminately required by all mDA neurons for their survival. In aphakia mice, virtually all surviving mDA neurons in the substantia nigra (SN) and the majority of neurons in the adjacent ventral tegmental area (VTA) also express calbindin-D28k, a calcium-binding protein previously associated with resistance to injury in PD and in animal models. Cell-mapping studies in wild-type mice revealed that Pitx3 is primarily expressed in the ventral SN, a region particularly susceptible to MPTP and other dopaminergic neurotoxins. Furthermore, Pitx3-expressing SN cells are preferentially lost following MPTP treatment. Finally, SN mDA neurons in Pitx3 hemizygous mice show increased sensitivity when exposed to MPTP. Thus, SN mDA neurons are represented by at least two distinct subpopulations including MPTP-resistant Pitx3-autonomous, calbindin-positive neurons, and calbindin-negative Pitx-3-dependent cells that display elevated vulnerability to toxic injury, and probably correspond to the subpopulation that degenerates in PD. Impairment of Pitx3-dependent pathways therefore increases vulnerability of mDA neurons to toxic injury. Together, these data suggest a novel link between Pitx3 function and the selective pattern of mDA cell loss observed in PD.