Selection of T-cell receptors with a recurrent CDR3ß peptide-contact motif within the repertoire of alloreactive CD8(+) T cells.

Peptide/MHC complexes recognized by alloreactive T lymphocytes (TLs) have been identified, but their contribution to in vivo allo-rejection is not known. We previously characterized the peptide pBM1, highly represented among endogenous H-2K(b) (K(b) )-associated peptides and critically required to induce full activation of H-2(k) monoclonal CD8(+) TLs expressing the cognate TCR-BM3.3. Here, we asked whether a pBM1/K(b) -specific TL subset could be detected within a polyclonal TL population rejecting allogeneic cells in vivo. We show that the proportion of pBM1/K(b) -binding CD8(+) TLs increased from <0.04% in naïve mice to 3% of activated CD44(+) CD8(+) TLs in H-2(k) mice rejecting K(b) -expressing cells. Among these, TCR-Vß2 usage was greatly enriched, and 75% of them shared a TCR-Vß2 CDR3ß motif with the prototype TCR-BM3.3. Fewer than 5% of K(b) -reactive CD44(+) CD8(+) TLs not binding pBM1/K(b) displayed this CDR3ß motif. We found that the recurrent CDR3ß motif of pBM1/K(b) -binding TLs was assembled from distinct V/D/J recombination events, suggesting that it is recruited upon immunization for its optimal TCR-peptide/MHC fit. Thus, a CDR3ß motif generated by a process akin to "convergent recombination" accounts for a sizable fraction of the alloreactive anti-K(b) TCR repertoire.

Flow cytometric analysis of intracellular phosphoproteins in human monocytes.

BACKGROUND: Using antibodies against intracellular phosphoproteins, flow cytometry can be used to monitor simultaneously multiple signaling pathways. Here, we tested a recently released procedure to analyze phosphorylation events in human monocytes upon different types of stimulation. METHODS: Whole blood was treated by lipopolysaccharide (LPS) or granulocyte-macrophage colony-stimulating factor (GM-CSF), then cells were labeled by antibodies recognizing cell surface and cytosolic proteins. Human monocytes were identified by a CD14 - CD45 staining and three phosphorylated proteins such as AKT, ERK-1/2, and STAT5, were simultaneously detected by multicolor phosphoflow analysis. RESULTS: By this rapid method, we are able to detect directly from a blood sample several signaling events in human monocytes where LPS stimulation induces preferentially ERK-1/2 phosphorylation where as GM-CSF stimulation induces STAT5 phosphorylation. CONCLUSIONS: This procedure provides a simultaneous measurement of multiple activated signaling molecules using a simplified and rapid protocol. © 2015 International Clinical Cytometry Society.

Automation of a phospho-STAT5 staining procedure for flow cytometry for application in drug discovery.

Drug discovery often requires the screening of compound libraries on tissue cultured cells. Some major targets in drug discovery belong to signal transduction pathways, and PerFix EXPOSE* allows easy flow cytometry phospho assays. We thus investigated the possibility to further simplify and automate this assay, to allow the direct screening of drugs targeting signaling pathways. We show here the sensitivity of this fully automated assay on human growth hormone (hGH)-driven JAK/STAT5-activated IM-9 cells, and we discuss the throughput of this system, which is compatible with medium-throughput drug screening. Because the kit works directly on whole blood samples, ex-vivo assays are also possible with this approach, which could allow for the screening of drugs under more physiological conditions.