ABSTRACT+ AC133 + population was also enriched (sevenfold) in dendritic cell precursors, and the dendritic cells generated were functionally active in a mixed lymphocyte reaction assay. AC133 + cells should be useful in the study of cellular and molecular mechanisms regulating primitive hemopoietic cells.

IntroductionNatural killer (NK) cells are cytolytic lymphocytes whose function is to control infection and malignancy. Granule exocytosis delivers granzymes, a family of pro-apoptotic serine proteases, to target cells leading to the induction of caspasedependent and caspase-independent apoptosis. 1 Mouse granzyme B is required for the rapid induction of apoptosis in target cells. 2 However, other granzyme molecules contribute to the overall cytolytic phenotype, 3 with human cells expressing granzymes A, B, H, K, and M. 1,4 Activation of granzymes requires the removal of an N-terminal dipeptide. 5 Cathepsin C, a cysteine protease also known as dipeptidyl peptidase I (DPPI), has been implicated in granzyme A and B processing. 6,7 Cathepsin C-deficient mice fail to process granzymes A and B and hence lack both NK and T-cell-mediated cytolytic activity. 7 Human cathepsin C deficiency is the cause of Papillon-Lefèvre syndrome (PLS). [8][9][10] This is an extremely rare, autosomal recessive condition characterized by palmoplantar keratosis and severe periodontitis. In addition, skin infections and liver abscesses have been reported in PLS, 10,11 strongly suggesting an immunodeficiency component to the disease. However, in contrast to the mouse model, interleukin-2 (IL-2)-generated lymphokine activated killer (LAK) cell activity in these patients appears to be normal, and granzyme B is active in PLS LAK cells. 12 Here, we have tested the requirement for cathepsin C in the processing of granzyme B in unstimulated human NK cells from PLS patients. We describe an NK cell cytolytic defect and a failure of granzyme B activation. Study design PatientsPLS patients 1 and 2 are brother and sister from a consanguineous family, family 6 in Toomes et al. 8 Both patients are homozygous for a mutation changing glycine 277 of cathepsin C to a serine (G277S). This nomenclature is that used in the structural study 13 and refers to cathepsin C with the 24 amino-acid signal peptide removed. A third family member is unaffected by PLS and has one G277S allele and one wild-type allele and is referred to as the heterozygote. Blood samples from patients, the heterozygote, and healthy control donors were collected with informed consent and approval from the Leeds Teaching Hospital NHS Trust UK ethics committee. Functional studiesNK cells were phenotyped in whole blood using flow cytometry and isolated from peripheral blood mononuclear cells (PBMCs) using the NK isolation kit II (Miltenyi Biotec, Bergisch Gladbach, Germany). NK cells were used directly or cultured for 10 to 14 days with 50 units/mL human recombinant IL-2 (Sigma, St Louis, MO). Whole cell lysates were assayed for cathepsin C or granzyme B activity using the colorimetric substrates Gly-Phe-pNA (Sigma) and Ac-IEPD-pNA (Calbiochem, San Diego, CA), respectively, 14,15 or used in immunoblotting experiments. Aprotinin agarose binding was performed as described. 16,17 Killing and caspase activation assays were performed using flow cytometry, using CellTracker dye (Molecular Probes, Eug...