Flow cytometric analysis of lymphoid organs

Subsequently, identified aberrant phenotypes attributed to the immune system can be further characterized by application of detailed hypothesis-driven analyses of lymphoid organs. We have established a series of standardized secondary examinations that comprise flow cytometric analyses of primary (bone marrow, thymus) and secondary (spleen, lymph nodes) lymphoid organs, and allow identification of a broad range of cellular parameters: T cell subsets in the thymus undergoing different maturational stages; hematopoietic stem cells, lineage progenitors and developing B cells in the bone marrow; a vast variety of subpopulations derived from lymphocytes and myeloid cells in spleen and lymph nodes. 

Immune challenge

In many cases, phenotypic screening based on the surface maker signature alone is not able to estimate the severity of the immunologic defect. Hypothesis-driven in vivo challenges, like infection with intracellular bacterium Listeria monocytogenes, provide detailed insights into the quality of innate and adaptive immune functions. Listeria has been widely used as laboratory mouse infection models with predictable response patterns. It induces an inflammatory response that substantially restricts bacterial growth and is essential for early survival of infected mice. Assessment of bacteria loads within three days after Listeria infection represent a sensitive readout for the competence of innate immunity. Competent adaptive immune system is detectable around day seven post infection. The Listeria-specific CD8⁺ T cells are promoted to reach peak expansion and secrete effector cytokines, which are crucial for bacterial clearance. In order to evaluate long-term protective immunity governed by T cell-mediated immune responses, mice are first immunized with a low dose of Listeria monocytogenes followed by a secondary rechallenge with a high-dose infection within the memory phase several months later. Antigen-specific CD8⁺ T cells can be analysed in more detail via utilization of the MHC-I multimer technique. Moreover, additional characterization of effector and memory surface markers as well as cytokine production can be examined in response to restimulation of specific antigenic peptides.