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.

Optimization of a flow cytometry-based protocol for detection and phenotypic characterization of multipotent mesenchymal stromal cells from human bone marrow.

BACKGROUND: To study the biology of rare bone marrow (BM) multipotent mesenchymal stromal cells (MSCs), recognized protocols are needed. Colony-forming unit-fibroblast (CFU-F) assays have historically been used for the enumeration of MSCs. However, the need to isolate and further analyze MSCs requires new strategies based on cell surface markers. The purpose of this work was to verify the phenotype of BM MSCs in vivo and to develop flow cytometry-based methods for their evaluation. METHODS: Pre-enrichment with D7-FIB-conjugated microbeads, cell sorting for CD45low D7-FIB+ LNGFR+ cells, and CFU-F assay were used to confirm the phenotype of BM MSCs in vivo. Further phenotypic characterization of MSCs was performed using three-color flow cytometry following pre-enrichment or by direct four-color flow cytometry. The sensitivity of direct flow cytometry/rare event analysis for the accurate enumeration of MSCs was validated using 85 samples from patients with neoplastic BM diseases. RESULTS: In normal BM, a significant correlation was found between the frequencies of CFU-Fs and CD45low D7-FIB+ LNGFR+ cells (n = 19, R = 0.719, P = 0.001). Following cell sorting, 15% of these cells were clonogenic. The same cells were enriched using LNGFR-based positive selection, CD45/Glycophorin A-based depletion, or plastic adherence. CD45low D7-FIB+ LNGFR+ cells expressed classic makers of cultured MSCs CD73/SH3 and CD105/SH2 and markers of stromal reticular cells CD106/VCAM and alkaline phosphatase. Novel markers were identified including leukemia inhibitory factor receptor and gp130. CD45low D7-FIB+ LNGFR+ cells were increased fourfold in the floating fat fraction of normal BM aspirates. Their frequency was decreased in chronic lymphocytic leukemia (threefold, n = 13, P = 0.049) and chronic myelogenous leukemia (ninefold, n = 11, P = 0.001) compared with that in age-matched controls (n = 26 and n = 31, respectively). CONCLUSIONS: This study demonstrates the usefulness of flow cytometry-based methods for the detection, enumeration and further phenotypic analysis of BM MSCs. These findings have broad applications for the future evaluation of BM MSCs in health and disease.

Interleukin-7 deficiency in rheumatoid arthritis: consequences for therapy-induced lymphopenia.

We previously demonstrated prolonged, profound CD4+ T-lymphopenia in rheumatoid arthritis (RA) patients following lymphocyte-depleting therapy. Poor reconstitution could result either from reduced de novo T-cell production through the thymus or from poor peripheral expansion of residual T-cells. Interleukin-7 (IL-7) is known to stimulate the thymus to produce new T-cells and to allow circulating mature T-cells to expand, thereby playing a critical role in T-cell homeostasis. In the present study we demonstrated reduced levels of circulating IL-7 in a cross-section of RA patients. IL-7 production by bone marrow stromal cell cultures was also compromised in RA. To investigate whether such an IL-7 deficiency could account for the prolonged lymphopenia observed in RA following therapeutic lymphodepletion, we compared RA patients and patients with solid cancers treated with high-dose chemotherapy and autologous progenitor cell rescue. Chemotherapy rendered all patients similarly lymphopenic, but this was sustained in RA patients at 12 months, as compared with the reconstitution that occurred in cancer patients by 3-4 months. Both cohorts produced naive T-cells containing T-cell receptor excision circles. The main distinguishing feature between the groups was a failure to expand peripheral T-cells in RA, particularly memory cells during the first 3 months after treatment. Most importantly, there was no increase in serum IL-7 levels in RA, as compared with a fourfold rise in non-RA control individuals at the time of lymphopenia. Our data therefore suggest that RA patients are relatively IL-7 deficient and that this deficiency is likely to be an important contributing factor to poor early T-cell reconstitution in RA following therapeutic lymphodepletion. Furthermore, in RA patients with stable, well controlled disease, IL-7 levels were positively correlated with the T-cell receptor excision circle content of CD4+ T-cells, demonstrating a direct effect of IL-7 on thymic activity in this cohort.

Isolation and characterization of bone marrow multipotential mesenchymal progenitor cells.

OBJECTIVE: There is an increased interest in rheumatology in mesenchymal progenitor/stem cells (MPCs) and their roles in rheumatic diseases, but little is known about the phenotype of these cells in vivo. The aim of this study was to isolate and characterize human bone marrow (BM) MPCs. METHODS: Fluorescence microscopy was used to identify putative MPCs among adherent BM cells. To purify them, a positive selection with antifibroblast microbeads was used, combined with fluorescence-activated cell sorting (FACS) for microbead+,CD45(low) cells. A more detailed phenotype of these cells was determined using 4-color flow cytometry, and standard chondrogenic, osteogenic, and adipogenic assays were used to investigate their differentiation potentials. RESULTS: Putative MPCs microscopically identified as large, fibroblast-like, D7-FIB+ cells were purified using positive selection with D7-FIB-conjugated (antifibroblast) microbeads followed by FACS for specifically bound microbead+,CD45(low) cells. These cells represented 0.01% of mononuclear cells in the BM. They were uniformly positive for CD105, LNGFR, HLA-DR, CD10, CD13, CD90, STRO-1, and bone morphogenetic protein receptor type IA (BMPRIA) and were negative for CD14, CD34, CD117, and CD133. Only cells with this phenotype could proliferate and produce adherent cell monolayers capable of chondrogenic, osteogenic, and adipogenic differentiation. D7-FIB- cells in the BM lacked any MPC activity. Uncultured skin fibroblasts had a phenotype similar to that of BM MPCs, but were negative for LNGFR, STRO-1, HLA-DR, and BMPRIA. CONCLUSION: This study shows the distinct phenotype, morphology, and method of isolation of BM MPCs. The findings may have implications for defining the physiologic roles of MPCs in arthritis, bone diseases, and joint regeneration.