Case Study: LTE Module vs. PBMC Assay
The MIMIC™ System reproduces both T cell and B cell responses. The availability of an in vitro culture system that provides a reliable readout of antigen-induced antibody responses is crucial for understanding the immunobiology of human B cell activation and differentiation. Current methods for the study of recall human antibody responses in vitro rely, in large part, on techniques that were developed thirty or more years ago, such as PBMC assays. The generation of naïve B cell responses in vitro has been quite challenging; positive results have only been achieved with highly modified culture systems that include the use of exogenous factors during the assay to polyclonally stimulate B cell proliferation and antibody production. The development of the novel MIMIC™ in vitro assay systems that supports the induction of primary human B cell responses in a physiologically relevant manner provides predictive insight into the biology of this lymphocyte population and serves as a valuable tool for assessing the immunogenicity of vaccines and other therapeutics prior to animal testing.
LTE Module Compared to PBMC Assays
VaxDesign is 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, DCs, and FDCs. The LTE module provides a more reliable method for inducing robust B cell activity against naïve and recall antigens than is possible with total PBMC.
The results demonstrate a clear advantage of the LTE module 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 module 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 module over unfractionated PBMC in generating influenza-specific antibody responses. Under peak conditions, the LTE module 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.
We observe similar enhancements of Ag-specific responses in the LTE module culture as compared to PBMCs for naïve antigens such as MSP-1 from malaria and rPA from bacillus anthracis as shown below. We have also added DCs to the PBMC culture, and still do not observe Ag-specific T cell responses.
