Rapid and Semi-Automated Screening of Multiple Samples for Antigen-Specific T Lymphocytes

Abstract 4839

Functional characterizations of T lymphocytes are performed to gain understanding on their contribution in certain immunological situations, to monitor the course of diseases, and to track therapeutic interventions. Meanwhile the flow cytometric analysis of antigen-specific T cells examined for intracellular cytokine production and expression of activation markers after a short-term in vitro antigenic challenge is a well-established method for research applications. Despite the advantages of this approach to qualify samples on a single cell level and by multiple parameters, the broad use of this analysis for immune monitoring purposes is hampered. Screening of a lot of samples is time-consuming, requires many manual handling steps, and operator experience in flow cytometric analysis of stimulated T cell samples. To overcome these hurdles, we worked out a complete strategy to rapidly study cytokine and activation marker profiles in antigen-specific T cells of multiple samples by a semi-automated process.

For the simultaneous analysis of multiple samples we examined for the T cell stimulation an antigen pre-coated 96-strip-well culture system. This flexible and ready-to-use format provides the opportunity to screen either for a single antigen or in parallel for up to twelve antigen specificities by combining 8-well-strips possessing different antigens. The coated antigens consist of pools of overlapping 15-mer peptides derived from a single viral protein of cytomegalovirus, Epstein-Barr-Virus, or adenovirus. The peptide pools have been designed for activation of the specific CD4⁺ as well as CD8⁺ T cells. They are solubilized and thereby accessible for T cell stimulation after addition of a cell sample, e.g. peripheral blood mononuclear cells, suspended in culture medium into the antigen-coated well. After a stimulation period of six hours the induced T cell response is comparable to the activation with a conventional lyophilized and reconstituted peptide pool. To reduce the time and work load for cell harvesting, fixation, permeabilization, and staining, we developed a protocol and reagents to allow a rapid and easy-to-handle intracellular staining procedure. Compared to conventional staining protocols, all steps are executed in the 96-strip-well culture plate, i.e. cell harvesting is dispensable. Without any washing step, cells are fixed and stained with defined reagent cocktails containing antibodies to identify virus-specific CD3⁺ CD4⁺ CD154⁺ and/or CD3⁺ CD8⁺ T cells and various Anti-cytokine-fluorochrome conjugates to evaluate the cytokine pattern. With these modifications, we drastically diminished the overall processing time for the staining of up to 96 samples to only 50 minutes. Furthermore, we integrated an automated flow cytometric analysis process. This includes the possibility to measure the samples in the 96-strip-well plates hands-free using pre-defined experiment settings and acquisition templates. We also applied an automated gating strategy for the data analysis. Finally, a report summarizes the results of the T cell response against several viral proteins for all samples tested, e.g. frequencies of cytokine⁺ CD154⁺ CD4⁺ and cytokine⁺ CD8⁺ T cell subsets are indicated. Hands-on time for the multi-sample acquisition and analysis is only minimal and the standardized reagents/protocol and sample analysis process decrease inter- and intra-assay variations.

In summary, with our newly developed tools and protocols for in vitro T cell stimulation, staining of activation markers as well as intracellular cytokines, and automated flow cytometric analysis we have set up a fast and convenient procedure to routinely monitor antigen-specific T cell responses.