Measuring lymphocyte proliferation, survival and differentiation using CFSE time-series data

Abstract

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.

Manipulation of B-cell responses with histone deacetylase inhibitors

“…We chose the soluble crosslinking anti-CD40 antibody 1c10 because when used in the presence of saturating IL-4, it induces typical proliferation, differentiation and isotype switching as membrane-bound CD40L 31,33 . B cells were cultured in varying concentrations of HDACi and daily quantitative measurements of viability, cell numbers and division progression determined by flow cytometry 29,31 . In cultures treated with vorinostat and panobinostat, cell viability as a proportion of total (Fig.…”

“…B cells were prepared, CFSE labelled and cultured as described 30,60 . Cell numbers were determined by reference to FACS calibration beads as described 29,31 . B cells were typically 495% B220 þ , CD19 þ , IgM þ , IgD þ as determined by flow cytometry.…”

“…T cells were activated with 10 mg ml À 1 plate-bound aCD3 clone 145-2C11 (eBioscience, San Diego, CA, USA) in the presence of 100 U ml À 1 saturating IL-2 (R&D Systems, Minneapolis, MN, USA). T cells were CFSE labelled and the analysis of cell survival and proliferation performed as described 29 .…”

“…Red blood cells were lysed and stained for subsets using antibodies against CD4, CD8, CD62L, B220, CD19, CD138. Total cell numbers were determined by reference to a known number of calibration beads as described 29 . Serum for antinuclear antibody analysis was incubated with pre-coated nuclear antigen plates (Cat#MK200, The Binding Site, Birmingham, AL, USA), and murine Ig detected with goat anti-mouse Ig (H þ L)-HRP (cat#1010-05 Southern Biotech Associates).…”

“…However, the direct functional effect of HDACi on the protective B-cell response and consequence for cancer patients undergoing HDACi therapy, how specific classes of HDACi regulate components of the antibody response, and analysis of newly emerging HDACi such as panobinostat has not yet been determined. Recently, quantitative systems have emerged as a powerful tool for dissecting components that contribute to lymphocyte responses in vitro [29][30][31][32] . The aim of this study was to dissect how these compounds affected the separate components of the protective antibody response and shed light on the possible immunological consequences for cancer patients post HDACi therapy.…”