Isolation of peripheral blood CD4+ T cells using RosetteSep™ and MACS™ for studies of DNA turnover by deuterium labeling

Tracking deuterium (H-2) incorporation into cellular DNA, after administration of (H2O)-H-2 or H-2(2)-glucose, is a recently developed, broadly applicable method for measuring in vivo cell proliferation and turnover that can be used safely in humans. This approach has been used to evaluate the turnover of T-cell subpopulations purified from the Peripheral blood of HIV-1-infected patients using fluorescence-activated cell sorting (FACS). A requirement for widespread adoption of this approach for medical decision-making and for use in larger clinical trials is a simple, reproducible, high-throughput method for isolation of highly purified CD4(+) T cells from peripheral blood. Here, we present a simple method, which does not require FACS, for isolating these cells in sufficient purity and yield for analysis of H-2 incorporation into DNA. When blood from HIV-1-infected patients was used. neither the depletion of unwanted cell lineages by erythrocyte crosslinking (RosetteSep(TM)) nor the enrichment of CD4(+) cells by immunomagnetic heads (MACS(TM)) individually resulted in sufficient purity. The successive application of the two techniques, however, permitted isolation of >95% pure CD4+ T cells in adequate yield (>10(6) cells/ 10 ml blood) from healthy donors and HIV-1-infected patients with CD4 counts between 300 and 700 cells/mul. Moreover, 2H incorporation into cellular DNA after administration of (H2O)-H-2 to HIV-1-infected patients was indistinguishable between CD4(+) T cells isolated by RosetteSep/MACS and FACS. Thus, both FACS and the new method isolate a similar mixture of long- and short-lived CD4(+) T cells. In practice, the RosetteSep/MACS method is simple, rapid, robust and capable of high throughput. (C) 2004 Elsevier B.V All rights reserved.