Detecting variable (V), diversity (D) and joining (J) gene segment recombination using a two-colour fluorescence system.

BACKGROUND: Diversity of immunoglobulins and the T cell antigen receptors is achieved via the recombination activating gene (RAG)-mediated rearrangement of variable (V), diversity (D) and joining (J) gene segments, and this underpins the efficient recognition of a seemingly limitless array of antigens. Analysis of V(D)J recombination activity is typically performed using extrachromosomal recombination substrates that are recovered from transfected cells and selected using bacterial transformation. We have developed a two-colour fluorescence-based system that simplifies detection of both deletion and inversion joining events mediated by RAG proteins. RESULTS: This system employs two fluorescent reporter genes that differentially mark unrearranged substrates and those that have undergone RAG-mediated deletion or inversion events. The recombination products bear the hallmarks of true V(D)J recombination and activity can be detected using fluorescence microscopy or flow cytometry. Recombination events can be detected without the need for cytotoxic selection of recombination products and the system allows analysis of recombination activity using substrates integrated into the genome. CONCLUSIONS: This system will be useful in the analysis and exploitation of the V(D)J recombination machinery and suggests that similar approaches could be used to replace expression of one gene with another during lymphocyte development.

Proteolytic activation of the cytotoxic phenotype during human NK cell development.

NK cells induce apoptosis in target cells via the perforin-mediated delivery of granzyme molecules. Cytotoxic human NK cells can be generated by IL-15-mediated differentiation of CD34(+) cells in vitro and these cultures have been used extensively to analyze the development of the NK cell surface phenotype. We have used NK cell differentiation in vitro together with protease-deficient human NK cells to analyze the acquisition of the cytotoxic phenotype. Granzymes are synthesized as inactive zymogens and are proteolytically activated by the cysteine protease cathepsin C. Cathepsin C is also synthesized as a zymogen and activated by proteolysis. We show that human NK cells generated in vitro undergo granule exocytosis and induce the caspase cascade in target cells. IL-15 and stem cell factor (IL-15 plus SCF) induced the expression of the granzyme B and perforin genes and the activation of cathepsin C and granzyme B zymogens. Perforin activation is also mediated by a cysteine protease and IL-15 plus SCF-mediated differentiation was accompanied by perforin processing. However, cathepsin C-deficient human NK cells revealed that perforin processing could occur in the absence of cathepsin C activity. The combination of IL-15 plus SCF is therefore sufficient to coordinate the development of the NK cell surface phenotype with the expression and proteolytic activation of the cytotoxic machinery, reflecting the central role of IL-15 in NK cell development.

A family with Papillon-Lefevre syndrome reveals a requirement for cathepsin C in granzyme B activation and NK cell cytolytic activity.

Activation of granzyme B, a key cytolytic effector molecule of natural killer (NK) cells, requires removal of an N-terminal pro-domain. In mice, cathepsin C is required for granzyme processing and normal NK cell cytolytic function, whereas in patients with Papillon-Lefèvre syndrome (PLS), loss-of-function mutations in cathepsin C do not affect lymphokine activated killer (LAK) cell function. Here we demonstrate that resting PLS NK cells do have a cytolytic defect and fail to induce the caspase cascade in target cells. NK cells from these patients contain inactive granzyme B, indicating that cathepsin C is required for granzyme B activation in unstimulated human NK cells. However, in vitro activation of PLS NK cells with interleukin-2 restores cytolytic function and granzyme B activity by a cathepsin C-independent mechanism. This is the first documented example of a human mutation affecting granzyme B activity and highlights the importance of cathepsin C in human NK cell function.

AC133+ G0 cells from cord blood show a high incidence of long-term culture-initiating cells and a capacity for more than 100 million-fold amplification of colony-forming cells in vitro.

AC133+ cells may provide an alternative to CD34+ cells as a target for cell expansion and gene therapy protocols. We examined the differences in proliferative potential between cord blood selected for AC133 or CD34 in serum-free, stroma cell-free culture for up to 30 weeks. Because most hemopoietic stem cells reside within the G0/G1 phase of the cell cycle, we combined enrichment according to AC133 or CD34 expression with G0 position in the cell cycle to identify populations enriched for putative stem cells. Our results show that AC133+ G0 cells demonstrated a long-term culture-initiating cell incidence of 1 in 4.2 cells, had a colony-forming cell incidence of 1 in 2.8 cells, were capable of producing 660 million-fold expansion of nucleated cells and 120 million-fold expansion of colony-forming units-granulocyte-macrophage over a period of 30 weeks, and were consistently superior to CD34+ G0 cells according to these parameters. Furthermore, we have shown that AC133+CD34- cells have the ability to generate CD34+ cells in culture, which suggests that at least some AC133+ cells are ancestral to CD34+ cells. We conclude that AC133 isolation provides a better means of selection for primitive hemopoietic cells than CD34 and that, in combination with isolation according to G0 phase of the cell cycle, AC133 isolation identifies a highly enriched population of putative stem cells.