Cellular proliferation is an essential feature of the adaptive immune response. The introduction of the division tracking dye carboxyfluorescein diacetate succinimidyl ester (CFSE) has made it possible to monitor the number of cell divisions during proliferation and to examine the relationship between proliferation and differentiation. Although qualitative examination of CFSE data may be useful, substantially more information about division and death rates can be extracted from quantitative CFSE time-series experiments. Quantitative methods can reveal in detail how lymphocyte proliferation and survival are regulated and altered by signals such as those received from co-stimulatory molecules, drugs and genetic polymorphisms. In this protocol, we present a detailed method for examining time-series data using graphical and computer-based procedures available to all experimenters.

Cells in the brain express unusually low levels of antigens encoded by the major histocompatibility complex (MHC). This is somewhat surprising as class I (H-2) and class II (Ia) MHC antigens have critical roles in immune responses. The activation of T lymphocytes is associated with the enhanced expression of these antigens and this effect is mediated by a specific T-cell lymphokine, gamma-interferon (IFN-gamma). Here we show that IFN-gamma induces a dramatic increase in the expression of H-2 antigens on the cells of the brain. After exposure to IFN-gamma in vitro, all surviving cells, including most astrocytes, oligodendrocytes, microglia and at least some neurones, express H-2 antigens. Direct injection of IFN-gamma into the brains of mice indicated that H-2 antigens were also induced in vivo. Furthermore, IFN-gamma induced Ia antigens on a subpopulation of astrocytes. The induction of H-2 antigens by IFN-gamma may render brain cells competent to initiate and participate in immune reactions and may therefore contribute to both immunoprotective and immunopathological responses in the brain.

The labeling kinetics of 5 dendritic cell (DC) subtypes within the lymphoid organs of healthy laboratory mice during continuous administration of bromodeoxyuridine (BrdU) was determined to investigate developmental relationships and determine turnover rates. Individual DC subtypes behaved as products of separate developmental streams, at least as far back as their dividing precursors. The rate of labeling varied with the lymphoid organ and the DC subtype. Labeling was faster overall in spleen and mesenteric lymph nodes (LNs) and slower in thymus and skin-draining LNs. The CD8+ DC subtype displayed the most rapid turnover, with a uniformly short (3-day) lifespan in spleen but with distinct short-lived and longer-lived subgroups in thymus. All the skin-derived DCs in LNs showed delayed and slow BrdU labeling, indicating a long overall lifespan; however, this was shown to reflect a long residence time in skin rather than a long-duration presenting antigen in the draining LN. Epidermal-derived Langerhans DCs displayed longer BrdU labeling lag and slower overall turnover than the dermal-derived DCs, and the movement of fluorescent Langerhans DC from skin to LN was slower than that of dermal DCs following skin painting with a fluorescent dye. However, once they arrived in lymphoid organs, all DCs present in healthy, uninfected mice displayed a rapid turnover, and this turnover was even faster after antigenic or microbial product stimulation.