Efficient adenoviral vector transduction of human hematopoietic SCID-repopulating and long-term culture-initiating cells.

This article presents our studies on the adenoviral transduction efficiency, level of transgene expression, cell cycle status, and multilineage reconstitution ability of human CD34+ hematopoietic cells transduced under proliferating and survival growth conditions. Bone marrow and umbilical cord blood CD34+ cells were cultured in serum-free medium under survival conditions with thrombopoietin (Tpo) alone, or under proliferating conditions with Tpo, c-Kit ligand (KL), and Flt3 ligand (FL). Adenoviral vectors carrying the enhanced green fluorescent protein (EGFP) gene under the control of the PGK-1 promoter were used to transduce CD34+ cells. Approximately 10% of CD34+ cells were EGFP+ under both culture conditions. In contrast, up to 50% of CD34+CD38- cells were EGFP+, whereas a maximum of 8% of CD34+CD38(high) cells were EGFP+ (p < 0.001). Both colony-forming unit cells (CFU-C) and 5-week long-term culture-initiating cells (LTC-ICs) were efficiently transduced. Under survival conditions, a substantial fraction of transduced CD34+ cells remained quiescent. The nondividing CD34+EGFP+ cells contained LTC-ICs capable of reconstituting longterm culture for as long as 10 weeks. CD34+EGFP+ cells also retained the ability to engraft and multilineage-reconstitute NOD/SCID mice. These observations demonstrate that primitive human hematopoietic progenitor cells can be efficiently transduced by adenoviral vectors.

Mutations in the MHC class II binding domains of staphylococcal enterotoxin A differentially affect T cell receptor Vbeta specificity.

C-terminal residues of staphylococcal enterotoxin A (SEA), including H187, D225, and D227, are involved in moderate affinity binding to MHC class II beta-chain, whereas N-terminal residues, including F47, are involved in low affinity binding to MHC class II alpha-chain. The effect of alanine substitutions at residues D227 or F47 on induction of T cell proliferation and the expansion of specific TCR Vbeta families was determined. SEA wild type specifically activated T cells expressing Vbeta1, Vbeta5.2, Vbeta6, Vbeta7, Vbeta9, Vbeta18, and Vbeta22. Although SEA-D227A exhibited substantially reduced mitogenicity compared with SEA wild type, it expanded the same Vbeta-bearing T cells, except those expressing Vbeta1. By contrast, SEA-F47A, which was slightly less mitogenic than SEA wild type, induced expansion only of T cells expressing Vbeta6, Vbeta7, and to a lesser extent Vbeta22. Therefore, specific mutations affecting either MHC class II alpha or beta binding sites differentially affect the Vbeta specificity of this superantigen. The lack of expansion in four of seven Vbeta families by SEA-F47A suggests that the class II alpha binding site may position SEA on the MHC class II molecules in an appropriate conformation for interaction with certain Vbeta elements.

Characterization of two distinct MHC class II binding sites in the superantigen staphylococcal enterotoxin A.

Bacterial superantigens (SAgs) are potent activators of T lymphocytes and play a pathophysiological role in Gram-positive septic shock and food poisoning. To characterize potential MHC class II binding sites of the bacterial SAg staphylococcal enterotoxin (SE) A, we performed alanine substitution mutagenesis throughout the C-terminus and at selected sites in the N-terminal domain. Four amino acids in the C-terminus were shown to be involved in MHC class II binding. Three of these amino acids, H225, D227 and H187, had a major influence on MHC class II binding and appeared to be involved in coordination of a Zn2+ ion. Alanine substitution of H225 and D227 resulted in a 1000-fold reduction in MHC class II affinity. Mutation at F47, which is equivalent to the F44 previously shown to be central in the MHC class II binding site of the SAg, SEB, resulted in a 10-fold reduction in MHC class II affinity. The combination of these mutations in the N- and C-terminal sites resulted in a profound loss of activity. The perturbation of MHC class II binding in the various mutants was accompanied by a corresponding loss of ability to induce MHC class II-dependent T cell proliferation and cytotoxicity. All of the SEA mutants were expressed as Fab-SEA fusion proteins and found to retain an intact T cell receptor (TCR) epitope, as determined in a mAb targeted MHC class II-independent T cell cytotoxicity assay.(ABSTRACT TRUNCATED AT 250 WORDS)

A recombinant C-terminal fragment of staphylococcal enterotoxin A binds to human MHC class II products but does not activate T cells.

Binding of staphylococcal enterotoxin A (SEA) to MHC class II encoded proteins is a prerequisite for its subsequent activation of a large fraction of T lymphocytes through interaction with variable segments of the TCR-beta chain. We cloned SEA in Escherichia coli and produced four recombinant fragments covering both the N- and C-terminal regions. These fragments were used to analyze the interaction between SEA and the human MHC class II products. A C-terminal fragment of SEA, representing amino acids 107-233 bound to HLA-DR and HLA-DP but did not activate T cells. The three other fragments (amino acids 1-125, 1-179 and 126-233) neither bound to MHC class II Ag nor activated T cells. SEA apparently bind to HLA-DR and HLA-DP through its C-terminal part, whereas T cell activation is dependent on additional parts of the protein.

Tumour-growth suppression in nude mice by a murine monoclonal antibody: factors hampering successful therapy.

The murine MAb C215 has been shown to mediate ADMMC in vitro and to have a tumour-growth-suppressive effect on xenografted COLO 205 human colocarcinoma cells in nude mice. To overcome the limitations of MAb therapy, it is necessary to understand the underlying mechanisms of tumour-growth suppression. In the present work, we have used C215 to define the importance of different parameters involved in tumour therapy with murine IgG2a antibodies. The results show that there exists a period of roughly 2 days after inoculation into animals during which the tumour cells are sensitive to an inhibitory antibody-mediated effect. After this initial period, the in-vivo sensitivity of tumour cells to antibody-mediated inhibition is much reduced. Tumour cells can remain "dormant" and, despite ongoing antibody treatment, develop into tumours with a reduced growth rate, which is not caused by outgrowth of antigen-deficient tumour cells. Finally, a pronounced dependence of antibody-mediated tumour suppression on antibody dose was observed.

Human major histocompatibility complex class II-negative colon carcinoma cells present staphylococcal superantigens to cytotoxic T lymphocytes: evidence for a novel enterotoxin receptor.

The staphylococcal enterotoxins (SE) bind to major histocompatibility complex (MHC) class II molecules on target cells and activate T cells expressing particular T cell receptor V beta sequences. In this report we demonstrate that SE bind to the MHC class II- SW620, Colo320DM and WiDr human colon carcinoma cell lines and direct cytotoxic T lymphocytes (CTL) to mediate strong target cell killing. Flow cytometry analysis, immunoprecipitation and Northern blotting experiments failed to demonstrate any surface expression of HLA-DR, HLA-DP and HLA-DQ isotypes on the SW620 colon carcinoma cell line, whereas abundant expression of these isotypes was seen on Raji cells, SEB and SEC1 were efficiently presented at picomolar concentration by the MHC class II- colon carcinoma cells and MHC class II+ Raji cells, whereas SEA and SED were preferentially presented on the MHC class II+ Raji cells. An anti-HLA-DR monoclonal antibody inhibited SEB-induced CTL targeting to Raji, but did not influence the killing of SW620 cells. Our data suggests the existence of functionally active SE-binding structures on human colon carcinoma cells which are distinct from the conventional MHC class II molecules. The possibility that these putative new SE receptors play a role in the enterotoxin action of SE must be considered.

Magnetic carbohydrate nanoparticles for affinity cell separation.

Magnetically responsive nanoparticles were prepared from enzymatically hydrolysed starch and magnetite. Two different monoclonal antibodies were covalently coupled to the particles. The antibody-coupled particles were in the size range of 100-300 nm and had an iron content of about 60%. Using 100 micrograms of magnetic particles (coupled with monoclonal mouse anti-rat Ig kappa light chain antibody) a very high depletion of surface Ig positive cells (mostly B-cells) from one million rat peripheral blood mononuclear cells could be achieved. The separation efficiency was evaluated by flow cytofluorometric analysis. This technique permits the detection of a small number of surface Ig positive cells among 10,000 negative cells.

Evidence for the existence of W3/13+ monocytes in the rat.

Rat blood mononuclear cells were studied with W3/13, OX19, and W3/25 monoclonal antibodies in a dual staining procedure. Cells recognized as W3/13+ OX19- W3/25+ showed a high light scatter pattern were plastic adherent and exhibited spreading, and were identified as monocytes when stained with May-Grunwald-Giemsa. This evidence suggests that at least a subpopulation of monocytes/macrophages are W3/13+.