Etienne Joly

Regional specification of rodent and human neurospheres

Neural precursor cells were isolated from various regions of the developing rat and human brain and grown in culture as aggregates termed neurospheres. We asked whether cells within human and rodent neurospheres are identical, or whether they have species specific characteristics or differences based on their region of origin. Under our culture conditions, rodent neurospheres isolated from the cortex (ctxNS) and striatum (strNS) grew faster than those from the mesencephalon (mesNS), but stopped growing after only eight to ten population doublings. In contrast, human neurospheres under identical culture conditions, continued to grow for over 40 population doublings. Following migration and differentiation of both rodent and human cultures, ctxNS and strNS generated high numbers of small neurons whereas mesNS generated small numbers of large neurons with many long fibres. Only very rare neurons from mesNS expressed dopaminergic markers, and thus may require further signals to fully mature. While the rat neurospheres generated high numbers of oligodendrocytes, very few were found to develop from human neurospheres from any region after a few weeks of passaging. FACS analysis revealed a unique population of smaller cells within human strNS and ctxNS, which appeared to be neuronal progenitors. However, large cells within neurospheres were capable of generating these small neuronal progenitors following further proliferation. Together, our data show that rat and human neurospheres have unique characteristics with regard to growth and differentiation, and that the majority of precursor cells within neurospheres are regionally specified to generate set numbers of neurons. These findings have important implications for understanding the nature of proliferating neural precursors isolated from the developing CNS, and their potential for brain repair.

Cutting Edge: CTLs Rapidly Capture Membrane Fragments from Target Cells in a TCR Signaling-Dependent Manner

Upon encounter of a CTL with a target cell carrying foreign Ags, the TCR internalizes with its ligand, the peptide-MHC class I complex. However, it is unclear how this can happen mechanistically because MHC molecules are anchored to the target cell’s surface via a transmembrane domain. By using antigenic peptides and lipids that were fluorescently labeled, we found that CTLs promptly capture target cell membranes together with the antigenic peptide as well as various other surface proteins. This efficient and specific capture process requires sustained TCR signaling. Our observations indicate that this process allows efficient acquisition of the Ag by CTL, which may in turn regulate lymphocyte activation or elimination.

The rat cim effect: TAP allele-dependent changes in a class I MHC anchor motif and evidence against C-terminal trimming of peptides in the ER.

Functional polymorphism in the rat peptide transporter associated with antigen processing (TAP) changes the peptide pool available for binding and presentation by a class I MHC allele, RT1.Aa. The peptide binding motif for RT1.Aa, determined by stabilization with synthetic peptides, included a strong preference for arginine at the peptide C terminus. Analysis of natural peptides bound to RT1.Aa by both pool sequencing and anhydrotrypsin chromatography revealed that TAP polymorphism determined the presence or absence of arginine as the peptide C-terminal residue. This result highlights the in vivo impact of TAP-peptide selectivity, and provides evidence against a high rate of generation of new C termini by protease activity in the endoplasmic reticulum.

Two Different, Highly Exposed, Bulged Structures for an Unusually Long Peptide Bound to Rat MHC Class I RT1-Aa

The rat MHC class Ia molecule RT1-Aa has the unusual capacity to bind long peptides ending in arginine, such as MTF-E, a thirteen-residue, maternally transmitted minor histocompatibility antigen. The antigenic structure of MTF-E was unpredictable due to its extraordinary length and two arginines that could serve as potential anchor residues. The crystal structure of RT1-Aa-MTF-E at 2.55 A shows that both peptide termini are anchored, as in other class I molecules, but the central residues in two independent pMHC complexes adopt completely different bulged conformations based on local environment. The MTF-E epitope is fully exposed within the putative T cell receptor (TCR) footprint. The flexibility demonstrated by the MTF-E structures illustrates how different TCRs may be raised against chemically identical, but structurally dissimilar, pMHC complexes.

Analysis of neural stem cells by flow cytometry: cellular differentiation modifies patterns of MHC expression.

Neural stem cells are currently considered very hopeful candidates for cell replacement therapy in neurodegenerative pathologies such as Parkinsons disease. Here we show that different cell types derived from neurospheres amplified in vitro can be identified by FACS analysis relying solely on physical parameters (FSC/SSC) or autofluorescence. Additionally, after treatment with a panel of inflammatory cytokines, neurospheres and their differentiated progeny were shown to express MHC antigens which could potentially cause transplant rejection. Astrocytes expressed the highest levels of MHC. Hence removing such cells prior to transplantation could potentially optimise transplant survival.

Neuronal cells are deficient in loading peptides onto MHC class I molecules.

Virally infected neurons avoid destruction by cytotoxic T lymphocytes (CTLs) by failing to express major histocompatibility complex (MHC) class I molecules. Like neurons in vivo and in primary culture, the OBL21 neuronal cell line expressed barely detectable levels of MHC class I molecules. This correlated with very low levels of mRNAs for the MHC class I heavy chains (alpha C). OBL21 cells also fail to provide MHC class I molecules with the peptides necessary for their efficient assembly and transport to the cell surface. This function can be restored by treatment with interferon-gamma (IFN-gamma). The mRNA for peptide transporters HAM1 and HAM2 was not detectable in OBL21 neuronal cells, but was induced by IFN-gamma treatment. Hence, the ability of neurons to evade CTL-mediated killing results from expression at low levels of the MHC class I alpha C, the peptide transporters HAM1 and HAM2, and possibly other genes of the peptide-loading machinery.

Proteolipidic composition of exosomes changes during reticulocyte maturation.

During the orchestrated process leading to mature erythrocytes, reticulocytes must synthesize large amounts of hemoglobin, while eliminating numerous cellular components. Exosomes are small secreted vesicles that play an important role in this process of specific elimination. To understand the mechanisms of proteolipidic sorting leading to their biogenesis, we have explored changes in the composition of exosomes released by reticulocytes during their differentiation, in parallel to their physical properties. By combining proteomic and lipidomic approaches, we found dramatic alterations in the composition of the exosomes retrieved over the course of a 7-day in vitro differentiation protocol. Our data support a previously proposed model, whereby in reticulocytes the biogenesis of exosomes involves several distinct mechanisms for the preferential recruitment of particular proteins and lipids and suggest that the respective prominence of those pathways changes over the course of the differentiation process.

Active trans‐synaptic capture of membrane fragments by natural killer cells

Prior to delivery of a lethal hit, NK cells form an immunological synapse to scan the target cells and engage their activatory and inhibitory receptors. Using freshly isolated NK cells, IL‐2‐activated polyclonal NK bulk or the NKL cell line, we report here that early during this recognition process, human NK cells actively capture target cell membrane fragments. This novel NK cell function occurs via the immunological synapse, is controlled by Src kinase, ATP, Ca2+ and PKC and involves rearrangements of the actin cytoskeleton. Furthermore, this process is down‐regulated bysignals emanating from inhibitory NK receptors recognizing protective MHC class I alleles.

Capture of Target Cell Membrane Components via Trogocytosis Is Triggered by a Selected Set of Surface Molecules on T or B Cells

Key events of T and B cell biology are regulated through direct interaction with APC or target cells. Trogocytosis is a process whereby CD4+ T, CD8+ T, and B cells capture their specific membrane-bound Ag through the acquisition of plasma membrane fragments from their cellular targets. With the aim of investigating whether the ability to trigger trogocytosis was a selective property of Ag receptors, we set up an assay that allowed us to test the ability of many different cell surface molecules to trigger trogocytosis. On the basis of the analysis of a series of surface molecules on CD4+ T, CD8+ T, and B cells, we conclude that a set of cell type-specific surface determinants, including but not limited to Ag receptors, do trigger trogocytosis. On T cells, these determinants include components of the TCR/CD3 as well as that of coreceptors and of several costimulatory molecules. On B cells, we identified only the BCR and MHC molecules as potentials triggers of trogocytosis. Remarkably, latrunculin, which prevents actin polymerization, impaired trogocytosis by T cells, but not by B cells. This was true even when the same Abs were used to trigger trogocytosis in T or B cells. Altogether, our results indicate that although trogocytosis is performed by all hemopoietic cells tested thus far, both the receptors and the mechanisms involved can differ depending on the lineage of the cell acquiring membrane materials from other cells. This could therefore account for the different biological consequences of Ag capture via trogocytosis proposed for different types of cells.

T cell activation correlates with an increased proportion of antigen among the materials acquired from target cells

We have investigated the density of peptides required to elicit different biological responses in cytotoxic T lymphocytes (CTL), including trogocytosis (i.e., the phenomenon whereby the lymphocytes actively capture fragments of plasma membrane from those cells with which they establish an immune synapse). We have used two separate mouse models of CTL recognising defined peptides presented by MHC class I molecules. In both systems, triggering of cytotoxicity and capture of membrane components reached saturation with low densities of ligand. On the other hand, down‐modulation of cell‐surface levels of TCR, induction of IFN‐γ production and detection of peptide captured required much higher ligand densities. Interestingly, fratricide (i.e., killing between CTL sharing the same specificity), a mechanism proposed to account for CTL exhaustion, was detected only at antigen concentrations still well above that second threshold leading to full blown activation. Taken together, our results show that the different thresholds that govern the elicitation of different CTL functions correlate with different proportions of antigen among the target cell components being captured via trogocytosis.