Improving Predictability of Human Immune Response with the LTE

We are pursuing novel 2D and 3D assays to provide a more suitable environment for the induction of naïve antigen-specific B cell and T cell responses in an in vitro system. We have established a relatively straightforward, standard well-format method for testing the response of human immune cells, including B cells, T cells and DCs.  We have shown that the LTE provides a reliable method for inducing robust B cell activity against recall antigens than is possible with total PBMC.  We are now looking at T cell responses.  We are also developing methods to apply the LTE to naive responses.

Click the thumbnails to view larger, more detailed versions of the charts.

The results demonstrate a clear advantage of the LTE system over unfractionated PBMC in generating lymphocyte responses against the recall antigen, tetanus toxoid (TT). While there was only a slight increase in the frequency of dividing T (Fig a) and B (Fig b) cells in cultures that received TT for 7 days, the number of true antigen-specific B cells, as assessed by ELISPOT assay, increased approximately 6-fold in the same assay wells. In contrast, the addition of antigen to total PBMC triggered little or no increase in the frequency of responding CD3+ T cells or CD19+ B cells (Figures a and b, respectively), nor was there any increase in the number of specific B cells detected by ELISPOT (Fig. c).

To evaluate the generalized utility of the LTE for supporting in vitro B cell responses, we performed similar assays as described above, but used influenza as the antigen. With this approach, cytokine-derived DC were cultured overnight with no antigen, a trivalent influenza vaccine (Fluzone™) or heat-killed (HK) influenza virus strain A/New Caledonia/20/99 (A/NC).

After 7 days, the total number of influenza-specific B cells was calculated by ELISPOT assay using plate-bound HK A/NC as the target antigen. The chart above demonstrates a clear advantage of the LTE over unfractionated PBMC in generating specific antibody responses. Under peak conditions the LTE cultures generated an approximately 10-fold higher influenza-specific B cell response than unfractionated PBMC cultures from the same donor. The advantage of using Fluzone™ to generate more robust influenza-specific B cell activity might be related to the adjuvant effects of this vaccine formulation on DC activation/maturation and subsequent induction of influenza-specific T cells.