Assays PBMC proliferation assay

Proliferation assays performed with PBMC cultures from a number of healthy donors demonstrated that IRIV indeed elicited cell proliferation in all tested cultures to an extent variable from donor to donor (Fig. 1 A). Further proliferation assays with CD4+ T-cells or CD8-I- T-cells in coculture with CD 14+ cells demonstrated that IRIV induce CD4+ T-cell proliferation, whereas no proliferation of CD8+ T-cells could be observed (Fig. 1C). Dissection of the CD4+ T-cell population into CD4+CD45RO+ and CD4+CD45RA+ cell subsets pointed to CD4+CD45RO+ cells as proliferative responders (Fig. ID). Importantly, culture of cord blood mononuclear cells in presence of IRIV did not result in major cell proliferation (Fig. IB), underlining antigen experience as prerequisite for IRIV-induced cell proliferation. 

In addition to these simpler cell-based assays, more complex assays can be employed to measure the induction of specific T cell responses to haptens. It is possible to stimulate naive T cells to proliferate in vitro in the presence of antigen-presenting cells such as LCs.27-28 In addition, effector T cells (T cell clones) can be used to investigate T cell stimulation induced by haptens in the presence of the appropriate antigen-presenting cells.29 30 Another complex, but easy to use assay is based on the culture of purified human PBMC in the presence of potential reactive chemicals, and the measurement of proliferation or cytokine production.31-33... 

Cell proliferation was addressed by conventional H-thymidine incorporation assays. Briefly, PBMCs were cultured in the presence of IRIV and liposomes, and in absence of any stimuli. On day 5 of culture, cells were pulsed with H-thymidine for 18 hours, then harvested, and cell proliferation was determined by tracer incorporation measurement. 

An alternative to the traditional in vivo TDAR assay is the recently developed in vitro human lymphocyte activation (HuLA) assay using human peripheral blood mononuclear cells (PBMCs), which measures secondary influenza-specific responses. This assay is sensitive to, and can differentiate the responses of, a number of known immunosuppressive compounds with different mechanisms of action at concentrations within their respective therapeutic ranges. Various endpoints can be evaluated, including proliferation and flu antigen-specific antibody (IgM and lgG)-secreting cells (Collinge et al., 2010). 

Fig. 8.2. A. B. Ex vivo antigen-specific proliferation of peripheral blood mononuclear cells (PBMC) isolated on day 25 post-withdrawal of CLA from the diet. Pigs had been fed either a control diet (left) or a CLA-supplemented diet (right) for 72 days. PBMC were cultured for 5 days with spirochetal antigens (B. hyodysenteriae), viral antigens (pseudorabies virus a relative of herpes simplex virus) or both. Data represents the mean stimulation index of cells in a lymphocyte blastogenesis assay based on incorporation of methyl-[3H] thymidine. Pigs were immunized on days 0 and 20. Significant differences (P < 0.05) caused by the interaction between vaccine and diet are shown with the (yen) sign.

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