Complete arrest from pro- to pre-B cells in a case of B cell-negative severe combined immunodeficiency (SCID) without recombinase activating gene (RAG) mutations

The B-cell lineage in a patient with B-cell-negative severe combined immunodeficiency (SCID) was analysed by using antisurrogate light chain (SL) MoAbs. Peripheral CD3+ T cells and CD19+ B cells were absent in the patient. The common gamma (γc) chain was expressed normally on the patient's peripheral NK cells and his peripheral mononuclear cells did not possess any mutations in recombinase activating gene (RAG)-1, 2. Normal levels of expression of Ku70 and Ku80 protein were found by Western blot analysis. The patient did, however, display an increase in fibroblast sensitivity to irradiation. Furthermore, flow cytometric analyses of bone marrow cells showed that surface IgM and cytoplasmic µ positive cells were absent and that CD19+ B cells were composed of only CD34+ terminal deoxynucleotidyl transferase (TdT)+ SL+ pro-B cells. The complete arrest of pro- to pre-B cell development in the SCID patient's bone marrow suggests that some genes involved in V(D)J recombination, excepting the RAG gene, may play a causative role in the immunodeficiency

Visualization and targeted disruption of protein interactions in living cells

Protein–protein interactions are the basis of all processes in living cells, but most studies of these interactions rely on biochemical in vitro assays. Here we present a simple and versatile fluorescent-three-hybrid (F3H) strategy to visualize and target protein–protein interactions. A high-affinity nanobody anchors a GFP-fusion protein of interest at a defined cellular structure and the enrichment of red-labelled interacting proteins is measured at these sites. With this approach, we visualize the p53–HDM2 interaction in living cells and directly monitor the disruption of this interaction by Nutlin 3, a drug developed to boost p53 activity in cancer therapy. We further use this approach to develop a cell-permeable vector that releases a highly specific peptide disrupting the p53 and HDM2 interaction. The availability of multiple anchor sites and the simple optical readout of this nanobody-based capture assay enable systematic and versatile analyses of protein–protein interactions in practically any cell type and species