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

TY - JOUR

T1 - Isolation of peripheral blood CD4(+) T cells using RosetteSep and MACS for studies of DNA turnover by deuterium labeling

AU - Busch, Robert

AU - Cesar, Denise

AU - Higuera-Alhino, Dana

AU - Gee, Tracy

AU - Hellerstein, Marc K

AU - McCune, Joseph M

PY - 2004/3

Y1 - 2004/3

N2 - Tracking deuterium ((2)H) incorporation into cellular DNA, after administration of (2)H(2)O or (2)H(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 (2)H incorporation into DNA. When blood from HIV-1-infected patients was used, neither the depletion of unwanted cell lineages by erythrocyte crosslinking (RosetteSep) nor the enrichment of CD4(+) cells by immunomagnetic beads (MACS) 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/microl. Moreover, (2)H incorporation into cellular DNA after administration of (2)H(2)O 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.

AB - Tracking deuterium ((2)H) incorporation into cellular DNA, after administration of (2)H(2)O or (2)H(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 (2)H incorporation into DNA. When blood from HIV-1-infected patients was used, neither the depletion of unwanted cell lineages by erythrocyte crosslinking (RosetteSep) nor the enrichment of CD4(+) cells by immunomagnetic beads (MACS) 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/microl. Moreover, (2)H incorporation into cellular DNA after administration of (2)H(2)O 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.

KW - Administration, Oral

KW - Adult

KW - CD4-Positive T-Lymphocytes

KW - DNA

KW - Deuterium

KW - Female

KW - Flow Cytometry

KW - HIV Infections

KW - HIV-1

KW - Humans

KW - Immunomagnetic Separation

KW - Isotope Labeling

KW - Male

KW - Middle Aged

KW - Rosette Formation

U2 - 10.1016/j.jim.2003.12.012

DO - 10.1016/j.jim.2003.12.012

M3 - Article

C2 - 15087225

SN - 0022-1759

VL - 286

SP - 97

EP - 109

JO - Journal of immunological methods

JF - Journal of immunological methods

IS - 1-2

ER -