Modulating cell surface immunoreactivity by metabolic induction of unnatural carbohydrate antigens  George A Lemieux, Carolyn R Bertozzi  Chemistry & Biology  Volume 8, Issue 3, Pages 265-275 (March 2001) DOI: 10.1016/S1074-5521(01)00008-4

Scheme 1 A strategy for inducing immune recognition of cell surfaces based on unnatural sialic acid biosynthesis. Rabbits are immunized with unnatural sialic acids conjugated to keyhole limpet hemocyanin (3). Cells are induced to express SiaLev (2) as a component of their cell surface glycoconjugates by treatment with the metabolic precursor acetylated ManLev (1). Sera from the immunized rabbits are highly reactive only with SiaLev-expressing cells, while cells expressing natural sialosides are not reactive with the sera. Chemistry & Biology 2001 8, 265-275DOI: (10.1016/S1074-5521(01)00008-4)

Fig. 1 Synthesis of SiaLev–KLH (3). (a) (i) MeOH, TFA; (ii) acetyl chloride, HCl; (iii) AgClO4, allyl alcohol, 4 Å molecular sieves: 56% over three steps. (b) Di-tert-butyl dicarbonate, DMAP, TEA, THF: 57%. (c) NaOMe/MeOH: 93%. (d) (i) 10% TFA in CH2Cl2; (ii) NaOMe, 8, methanol: 40% over two steps. (e) (i) NaOH; (ii) 2-aminoethanethiol hydrochloride, 254 nm light: 60% over two steps. (f) (i) thiophosgene, Na2CO3, 1:1 H2O/CHCl3; (ii) carrier protein (KLH or BSA), 300 mM NaHCO3, pH 9: 60–70% of available lysines were conjugated. Chemistry & Biology 2001 8, 265-275DOI: (10.1016/S1074-5521(01)00008-4)

Fig. 2 Reactivity of rabbit antisera with SiaLev as determined by ELISA. (a) Reactivity of whole sera with SiaLev and SiaLacNAc. (b) Titer of sera from different rabbits following each antigen boost. Titer is defined as the dilution of serum which yields an optical density of half saturation in a set of serial dilutions such as that shown in (a). Chemistry & Biology 2001 8, 265-275DOI: (10.1016/S1074-5521(01)00008-4)

Fig. 3 Surface reactivity of live cells with SiaLev antisera assessed by flow cytometry. (a) Characteristic cell surface reactivity of cells determined by flow cytometry. Red: HeLa cells cultured with 30 μM acetylated ManLev (Ac4ManLev) for 3 days, reacted with SiaLev antiserum, and RG-16-FITC (monoclonal mouse anti-rabbit Ig-FITC). Dark blue: HeLa cells cultured with 30 μM acetylated ManLev for 3 days, reacted with pre-immune serum from the same rabbit that produced the SiaLev antiserum, and RG-16-FITC. Light blue: HeLa cells cultured without ManLev, reacted with whole SiaLev antisera, and RG-16-FITC. (b) Cell surface reactivity of Jurkat cells cultured with different doses of acetylated ManLev (0–30 μM) and reacted with serial dilutions of sera (1:600–1:19 200). Reported are the mean fluorescence intensities, expressed in arbitrary units (AU), of populations of 104 cells. Error bars represent the high and low values of duplicate experiments. Chemistry & Biology 2001 8, 265-275DOI: (10.1016/S1074-5521(01)00008-4)

Fig. 4 SiaLev antiserum reactivity with cells treated with different sialic acid biosynthetic precursors. Mean fluorescence intensities were determined by flow cytometry analysis of HeLa cells cultured for 3 days with 30 μM acetylated ManNAc (green), ManPent (red), ManLev (blue), or no biosynthetic precursor (black). The cells were then washed and stained with sera from different immunized rabbits (R) collected after each antigen boost (B), followed by treatment with RG-16-FITC. Error bars represent the high and low values of duplicate experiments. Chemistry & Biology 2001 8, 265-275DOI: (10.1016/S1074-5521(01)00008-4)

Fig. 5 Intracellular localization of SiaLev in fixed and permeabilized HeLa cells. (a) Cells cultured with 30 μM acetylated ManLev, stained with affinity-purified SiaLev antiserum, goat anti-rabbit IgG (H+L)-Alexa®Fluor™-488, and DAPI. (b) Cells cultured without acetylated ManLev, otherwise treated as in panel a. (c) Cells cultured with 30 μM acetylated ManNAc but without acetylated ManLev, otherwise treated as in panel a. (d) Cells cultured with 30 μM acetylated ManLev, treated with non-specific rabbit IgG, goat anti-rabbit IgG (H+L)-Alexa®Fluor™-488, and DAPI. Images shown are single 2 μm thick optical sections at 1000× magnification in a plane through the center of the nuclei. Scale bars represent 10 μm. Chemistry & Biology 2001 8, 265-275DOI: (10.1016/S1074-5521(01)00008-4)

Fig. 6 Antibody-dependent, complement-mediated lysis of SiaLev-expressing cells. (a) Jurkat cells were cultured with 20 μM acetylated ManLev (Ac4ManLev), 20 μM acetylated ManNAc (Ac4ManNAc), or with no supplement, then treated with: SiaLev antiserum and 2.5% rabbit serum complement (dark blue), non-specific rabbit IgG and 2.5% rabbit serum complement (red), 2.5% rabbit serum complement only (green), or SiaLev antiserum and 2.5% heat-inactivated rabbit serum complement (light blue). Percent cytolysis was measured as described in Section 4. (b) Jurkat cells were cultured with varying concentrations of acetylated ManLev (0.03–30 μM) over 3 days, then treated with either SiaLev antiserum and 2.5% rabbit serum complement or non-specific rabbit IgG and 2.5% rabbit serum complement. Values for percent cytolysis in both panels a and b are the average of triplicate experiments±S.D. Chemistry & Biology 2001 8, 265-275DOI: (10.1016/S1074-5521(01)00008-4)