Recent advances in molecular genetics, especially the sequencing of human and mouse genomes, opens new prospects for deciphering the mechanisms of immunological homeostasis and immune responses. The ability to preserve tolerance to self and effectively eliminate pathogens and cells carrying altered or foreign antigens are highly complex immunological traits governed by the interaction of many genes. However, individual in vivo functions remain largely unknown for most of the genes – only about 20% of all human genes have a function attributed to them. As infections, cancer and autoimmune diseases are a major health problem worldwide, understanding the intricate genetic machinery involved in immune defence mechanisms is essential for the development of new therapeutic and vaccination strategies. Furthermore, a key prerequisite for success in this field is the availability of animal models for immunodeficiency-associated diseases (1-4).
Ultimate goal of the Immunology Screen at the German Mouse Clinic (GMC) is to identify mouse mutant lines, which can serve as representative models for specific human immunological diseases (chronic inflammatory diseases, autoimmune diseases, immune-deficiencies, defects in protective immunity). To achieve this primary goal, we have set forth as objectives the development of a high-throughput screening approach; continuous improvement by utilization of most up-to-date high-throughput technologies (for example, application of multiparameter FACS, multiplex bead array, especially to reduce the sample sizes needed for our tests) and automated data analysis; in-depth characterization of mouse lines with new immunological phenotypes in collaboration with the mouse providers; dependent on the phenotype, new tests and assays are included in our array of examinations (“secondary screen”).
Results
Screen status
Since the start of the GMC we have established a detailed primary screening protocol for the analysis of peripheral blood samples of mice for the following parameters: Plasma levels of IgM, IgG1, IgG2a, IgG2b, IgG3 and IgA are measured by a Bioplex bead array, while rheumatoid factor and anti-DNA antibodies are determined by standard indirect ELISAs. Leukocyte subpopulations are stained with fluorescence conjugated antibodies and identified by flow cytometry. The following cell surface markers are included in the screening for immunological parameters: CD3, CD4, CD8, CD19, CD25, CD103, B220, CD5, IgD, DX5, CD11b, Gr-1, ##TCR, CD45RA, MHC class II (strain dependent), Ly6C, CD44, CD62L; especially through combination of the different markers in 9-color FACS settings and through automated data analysis, the total number of parameters extracted from this screen currently exceeds 50 different subsets of immune cells. The Bioplex/ELISA protocols could be almost completely automated by pipetting-robots. Also for flow cytometry analysis, most of the procedures (except cell pelleting by centrifugation) could be automated including cell staining, sample acquisition (multiwell autosampler), and data analysis (using FlowJo software). Standard operation procedures (SOP) were precisely defined for all working steps, allowing very sensitive and highly reproducible measurements of this large amount of different immunological parameters. To our knowledge this is so far the most extensive high-throughput analysis of immunological parameters in the mouse. For validation of our SOP, we analyzed a variety of different mouse inbred strains (BALB/c, C57BL/6, C3H, 129) with the primary screening protocol. The datasets revealed that we are able to precisely reproduce known strain and sex dependent differences in distinct immunological parameters; in addition, we identified a substantial number of so far unknown strain and sex dependent differences (publication in preparation).
Since the development and regulation of antigen-specific immune responses involves a large number of different genes, measurement of specific antibodies and T cells after in vivo antigen challenge is a sensitive screen to obtain information regarding the functionality of the immune system in mutant mice. We, therefore, developed different types of antigen challenge tests: (1) Immunization with KLH and subsequent determination of KLH-specific antibodies of IgM and IgG subtypes. (2) Immunization with purified proteins from the intracellular bacterium Listeria monocytogenes together with immune-stimulatory CpG-DNA as adjuvant, and subsequent measurement of antigen-specific T cells by the use of MHC-multimer reagents (the necessary reagents are in the Busch laboratory, and SOP are defined). These assays are applicable for addressing specific questions during secondary screening.
Our laboratory is internationally recognized for its expertise in the analysis of immune-regulatory molecules involved in specific immune responses. Among the many collaborations with other institutes in Europe and the USA (5-12), the close collaborations with The Memorial Sloan Kettering Cancer Center (New York, Prof. Eric Pamer) and The Scripps Research Institute (San Diego, Prof. Luc Teyton) are of special relevance for further technical developments at the GMC.
Novel immunological phenotypes with relevance to human pathology
The Immunology Screen has so far analyzed more than 40 already established mouse mutant lines (MML), which were produced with the intention to represent models for various human pathological conditions. Most interestingly, for half of the MML we were able to assign new immunological phenotypes. These included changes in the frequencies of major cell lineages like T-, B-lymphocytes, NK cells, granulocytes, or altered levels of various immunoglobulin subclasses.
We also were able to perform in depth analysis of several newly-identified ENU-immunological mutants with altered T cell differentiation. These are promising candidates for further dissecting the intricate mechanisms of immune responses.
During our efforts to standardize immunological analyses in the mouse, we were able to uncover in collaboration with the group of Andreas Lengeling (GBF, Braunschweig) unexpected sex and strain differences in inbred strains infected with Listeria monocytogenes, that have not been described so far. It is well documented that sex-dependent factors affect susceptibility to infection, with most mouse models demonstrating higher resistance in females. We made the unexpected observation that infection with the intracellular bacterium Listeria monocytogenes showed an opposite pattern in several commonly used inbred mouse strains: female C57BL/6J, BALB/c, C3H/HeN, and CBA/J mice were significantly more susceptible to Listeria infection (13). The pronounced sensitivity of females to Listeria, which was revealed by significantly higher lethality rates, correlated also with increased bacterial numbers in organ tissues (spleen and liver), and several immunological changes in peripheral blood samples. Surprisingly, increased severity of infection in females was associated with elevated IL-10 plasma levels. Experiments using IL-10-knockout mice, in which no differences between the susceptibility of males and females to Listeria infection could be detected, confirmed the crucial role of this immunosuppressive cytokine for the outcome of disease. Our findings are likely to have clinical relevance, since similar sex differences in infection with Listeria monocytogenes and other intracellular pathogens have been reported in humans.
Outlook
The assays developed so far for the primary immunology screen at the GMC have proven to be sensitive and reproducible, and as already demonstrated by the first results, the methods were able to identify a substantial number of new immunological phenotypes in mouse mutant lines. We will analyze up to 26 different mouse mutant lines per year, discus the results and potential further in-depth analysis with the mouse provider, and provide a comprehensive summary of all immunological data.
The ELISA-based antibody measurements will be entirely exchanged by the relatively new “bead array” method, which allows determination of different parameters within the same (small) plasma sample and which is easy and fast to perform. As already mentioned, we have introduced a bead array reader (Bioplex) into our hardware setup, and we were able to establish a protocol for determination of all immunoglobulin subtypes of the primary screen in one single multiplex measurement. Especially when partially using self-generated mAbs, the bead array system is cost-efficient and much faster (results can be analyzed at the same day!) as compared to ELISA techniques.
Our group currently established new multi-parameter flow cytometry applications for high-speed cell analysis. These methods might be ideal to improve our flow cytometry based assays, not only by increasing the cell subpopulations detectable within a single sample, also by helping to reduce the blood volume required for the immunology screen. These techniques will be integrated into the GMC. Important to mention is also the collaboration with the microarray platform at the Technical University Munich; here we try to identify new marker candidates for our primary and secondary screening protocols.
Our group is specifically interested in the identification of immune-modulatory genes involved in the regulation of antigen-specific T cell responses and the development of protective immunity. We, therefore, want to establish further screening systems (e.g. based on infection with Listeria monocytogenes) to identify mouse mutant lines with phenotypes in this area. We mapped a variety of new Listeria-derived epitopes for different mouse strains and developed sophisticated tools (e.g. MHC multimer reagents) to precisely analyse the T cell responses after in vivo challenge.
After identification of mouse mutant lines with altered immunological parameters it will be necessary to perform additional in depth analyses in collaboration with the mouse providers to more clearly characterize the phenotype and potentially the mechanisms that lead to the phenotype. The establishment of such assay systems, which will differ from mouse line to mouse line, will represent a substantial part of our so-called “secondary screen”.
Lit.: 1 Mak, T. W., J. M. Penninger, and P. S. Ohashi. 2001. Knockout mice: a paradigm shift in modern immunology. Nat Rev Immunol 1:11-19. 2 Fischer, A. 2002. Natural mutants of the immune system: a lot to learn! Eur J Immunol 32:1519-23. 3 Bluethmann, H., and P. S. Ohashi (Eds.) 1994. Transgenesis and targeted mutagenesis in immunology. Academic Press, San Diego. 4 Rogner, U. C., and P. Avner. 2003. Congenic mice: cutting tools for complex immune disorders. Nat Rev Immunol 3:243-52. 5.Busch DH et al. (2000) Differing roles of inflammation and antigen in T cell proliferation and memory generation. J. Immunol. 164: 4063-4070. 6.Wolan DW et al. IA. Crystal structure of the murine NK cell-activating receptor NKG2D at 1.95 A (2001) Nature Immunol. 2; 248-254. 7.Benz C. et al. (2002). Activated virus-specific T cells are early indicators of anti-CMV immune reactions in liver transplant patients. Gastroenterology, 122; 1201-1215. 8.Knabel M et al.(2002). Reversible MHC multimer staining for functional isolation of T cell populations and effective adoptive transfer. Nature Medicine, 8; 631-637. 9.Krmpotic A et al. (2002). MCMV glycoprotein gp40 confers virus resistance to CD8+ T cells and NK cells in vivo. Nature Immunology, 3:529- 535. 10.Drexler I et al. (2003). Identification of vaccinia virus epitope-specific HLA-A*0201-restricted T cell responses and analysis of immunogenicity and protective capacity of smallpox vaccines. PNAS 100(1):217-22. 11.Heit A et al., (2003). Toll like receptor 9 (TLR9) expression is not required for CpG-DNA aided cross-presentation of DNA conjugated antigens, but essential for cross-priming of CD8 T-cells. J Immunol 15;170(6):2802-5. 12.Cosma A et al. (2003) Therapeutic vaccination with recombinant modified vaccinia virus Ankara-HIV-1 nef elicits a strong Nef specific T-helper cell response in chronic HIV infected individuals. Vaccine, 8;22(1): 21-29. 13.Pasche B et al. Sex dependent susceptibility to Listeria monocytogenes infection is mediated through differential IL10 production, Infect Immun 2005, 73 (9): 5952-60.
