Heavy water labeling of DNA for measurement of cell proliferation and recruitment during primary murine lymph node responses against model antigens

Abstract

Lymph node (LN) responses to antigens involve inflammatory lymphocyte recruitment and proliferation of rare antigen-specific precursors; the relative contributions of these processes have not been well quantified. The popliteal LN assay (PLNA), used for immunotoxicity screening, measures LN swelling as a surrogate of antigen-specific immunity, but nonspecific irritants cause false-positive results. Quantification of proliferating cells may improve specificity, but commonly-used biosynthetic labels (e.g., BrdU) have limitations. In vivo labeling with heavy water ( 2H 2O) is nontoxic and 2H incorporation into the DNA of dividing cells highly consistent, even in apoptotic microenvironments such as the thymus. Here, we have used continuous 2H 2O labeling and GC/MS analysis to quantify the cumulative fraction of recently divided cells ( f) in draining LN of mice. Priming of BALB/c mice with model antigens (KLH, DNCB) increased both LN cell counts and f in responding lymphocyte subsets, whereas lymphocyte recruitment to LN by irritants (IFA, DMSO) increased cell counts but had little effect on f. Thus, antigen-driven proliferation (possibly including a bystander component) was reflected in f, whereas LN cellularity was primarily increased by recruitment. Cell counts responded differentially to changes in Ag dose and immunization with IFA, whereas f was unaffected by these variables. GC/MS analysis of 2H 2O-labeled lymphocyte DNA affords sensitive, precise measurements of fractional lymphoproliferation. Dissection of proliferation and cell recruitment by this approach may be useful for preclinical in vivo screening of novel adjuvants and immunomodulatory agents, for studying their mechanism of action, and for immunotoxicity screening in the PLNA.