Long-term in vivo survival of receptor-modified syngeneic T cells in patients with human immunodeficiency virus infection.

To study human immunodeficiency virus (HIV)-specific cellular immunity in vivo, we transferred syngeneic lymphocytes after ex vivo expansion and transduction with a chimeric receptor gene (CD4/CD3-zeta) between identical twins discordant for HIV infection. Single and multiple infusions of 10(10) genetically modified CD8(+) T cells resulted in peak fractions in the circulation of approximately 10(4) to 10(5) modified cells/10(6) mononuclear cells at 24 to 48 hours, followed by 2- to 3-log declines by 8 weeks. In an effort to provide longer high-level persistence of the transferred cells and possibly enhance anti-HIV activity, we administered a second series of infusions in which both CD4(+ )and CD8(+) T cells were engineered to express the chimeric receptor and were costimulated ex vivo with beads coated with anti-CD3 and anti-CD28. Sustained fractions of approximately 10(3) to 10(4) modified cells/10(6) total CD4(+) or CD8(+) cells persisted for at least 1 year. Assessment of in vivo trafficking of the transferred cells by lymphoid tissue biopsies revealed the presence of modified cells in proportions equivalent to or below those in the circulation. The cell infusions were well tolerated and were not associated with substantive immunologic or virologic changes. Thus, adoptive transfer of genetically modified HIV-antigen-specific T cells was safe. Sustained survival in the circulation was achieved when modified CD4(+ )and CD8(+) T cells were infused together after ex vivo costimulation, indicating the important role played by antigen-specific CD4(+) T cells in providing "help" to cytotoxic effectors. (Blood. 2000;96:467-474)

The mode and duration of anti-CD28 costimulation determine resistance to infection by macrophage-tropic strains of human immunodeficiency virus type 1 in vitro.

We have investigated the ability of anti-CD28 antibody costimulation to induce resistance to macrophage (M)-tropic strains of human immunodeficiency virus type 1 (HIV-1) in vitro. Our results confirm the observations of Levine et al. (15) that stimulation of CD4 T cells with anti-CD3/anti-CD28 antibodies coimmobilized on magnetic beads renders the cells resistant to infection by M-tropic strains of HIV-1. The resistance was strongest when the beads were left in the cultures throughout the experiment. In contrast, stimulation of CD4 T cells with the same antibodies immobilized on the surface of plastic culture dishes failed to induce resistance and resulted in high levels of p24 production. This was true even if the cells were passaged continuously on freshly coated plates. If the beads were removed after initial stimulation, p24 production increased over time and produced a result intermediate to the other forms of stimulation. For beads-in, beads-out, and one-time plate stimulated cultures, resistance to infection correlated with down-regulation of CCR5 expression at the cell surface and with increased production of beta-chemokines. However, cultures of CD4 T cells continuously passaged on anti-CD3/anti-CD28-coated plates produced large amounts of p24 despite decreased levels of CCR5 expression and increasing production of beta-chemokines. Expression of the T-cell activation markers CD25 and CD69 and production of gamma interferon further supported the differences in plate versus bead stimulation. Our results explain the apparent contradiction between the ability of anti-CD28 antibody costimulation to induce resistance to HIV infection when presented on magnetic beads and the increased ability to recover virus from the cells of HIV-positive donors who are on highly active antiretroviral therapy when cells are stimulated by anti-CD3/anti-CD28 immobilized on plastic dishes.

Escherichia coli 5'-nucleotidase: purification, properties and its release by osmotic shock.

Escherichia coli 5'-nucleotidase was purified to apparent homogeneity as judged by polyacrylamide gel electrophoresis. Its molecular weight was estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, by gel exclusion chromatography, and by membrane filtration; values of 66 000, 48 500, and 15 000 to 30 000, respectively, were obtained. The enzyme was completely released from bacteria by osmotic shock treatment. The apparently anomalous behaviour of 5'-nucleotidase in terms of the molecular sieving hypothesis for the release of enzymes by osmotic shock proposed by Smith & Wyatt (1974) and extended by Broad & Smith (1979) is discussed.