PubMed · 11094410
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A Thiel, A Radbruch. 1999-10-26. Antigen-specific cytometry.. https://doi.org/10.1186/ar7
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Cells that express the NG2 proteoglycan will spread on surfaces coated with monoclonal antibodies against this membrane-spanning protein. On surfaces coated with the N143 monoclonal antibody, this cell spreading occurs by extension of lamellipodia, suggesting that activation of the small GTPase rac is involved in the observed morphological change. Support for this hypothesis comes from the finding of increased levels of GTP-bound rac in cells spreading on N143-coated surfaces. Furthermore, lamellipodia extension is blocked by transfection of cells with the dominant negative rac construct N17rac, but not by transfection with N17cdc42. Formation of lamellipodia on the N143-coated surfaces is also inhibited by transfection of the dominant negative CasdeltaSD construct. This result implicates p130cas as an additional functional player in NG2-mediated cell spreading.
The production of edible vaccines in transgenic plants and plant cell culture may be improved through a better understanding of antigen processing and assembly. The hepatitis B surface antigen (HBsAg) was chosen for study because it undergoes substantial and complex post-translational modifications, which are necessary for its immunogenicity. This antigen was expressed in soybean (Glycine max L. Merr. cv Williams 82) and tobacco NT1 (Nicotiana tabacum L.) cell suspension cultures, and HBsAg production in batch culture was characterized. The plant-derived antigen consisted predominantly of disulfide cross-linked HBsAg protein (p24(s)) dimers, which were all membrane associated. Similar to yeast, the plant-expressed HBsAg was retained intracellularly. The maximal HBsAg titers were obtained with soybean suspension cultures (20-22 mg/L) with titers in tobacco cultures being approximately 10-fold lower. For soybean cells, electron microscopy and immunolocalization demonstrated that all the HBsAg was localized to the endoplasmic reticulum (ER) and provoked dilation and proliferation of the ER network. Sucrose gradient analysis of crude extracts showed that HBsAg had a complex size distribution uncharacteristic of the antigen's normal structure of uniform 22-nm virus-like particles. The extent of authentic epitope formation was assessed by comparing total p24(s) synthesized to that reactive by polyclonal and monoclonal immunoassays. Depending on culture age, between 40% and 100% of total p24(s) was polyclonal antibody reactive whereas between 6% and 37% was recognized by a commercial monoclonal antibody assay. Possible strategies to increase HBsAg production and improve post-translational processing are discussed.
The importance of the enzyme-linked immunosorbent spot (ELISPOT) assay as a tool for studying immune responses in vitro is becoming increasingly apparent. However, there remains a need for enhanced sensitivity for the detection of low frequency antigen-specific T cell responses. We reasoned that the addition of a combination of the cytokines interleukin (IL)-7 and IL-15 would selectively increase interferon-gamma (IFN-gamma) production from antigen-stimulated CD4+ and CD8+ effector memory T cells. Freshly isolated or cryopreserved peripheral blood mononuclear cells (PBMC) from four healthy donors were analysed by ELISPOT for the frequency of purified protein derivative (PPD)-specific CD4+ T cells or cytomegalovirus (CMV) peptide-specific CD8+ T cells. Addition of IL-7 and IL-15 increased the number of PPD-specific CD4+ T cells up to 2.4-fold in fresh PBMC and up to 18-fold in cryopreserved PBMC. The cytokines also increased the number of CMV peptide-specific CD8+ T cells in fresh PBMC up to 7.5-fold. No additional increases were seen when antibodies to co-stimulatory molecules CD28 and CD49d were applied together with the cytokine combination. These data demonstrate that the sensitivity of the ELISPOT assay may be significantly augmented by addition of the cytokines IL-7 and IL-15 to antigen-stimulated cells. This method will be particularly useful for the assessment of antigen-stimulated cytokine production by T cells in cryopreserved biological specimens.