Novel approaches for identifying target antigens o

Authors: Klaus Dornmair Edgar Meinl Reinhard Hohlfeld

Publish Date: 2009/09/11

Volume: 31, Issue: 4, Pages: 467-477

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Abstract

Antigenspecific immune responses in multiple sclerosis have been studied for decades but the target antigens of the putatively autoaggressive B and T cells still remain elusive Here we summarize recent strategies which are based on the direct analysis of biopsy or autopsy specimens from patients Since this material is extremely scarce the experimental methods need to be exceptionally sensitive We describe technologies to distinguish auto aggressive T cells from irrelevant bystander lymphocytes by analyzing clonal expansions in relation to the morphological location of the cells in the tissue lesions We then discuss approaches to clone matching α and βchains of the antigenspecific T cell receptor TCR molecules from single T cells This is necessary because usually several clones are expanded and are diluted by many irrelevant cells The matching TCR chains from individual T cells can be resurrected in hybridoma cells which may then be used for antigen searches We discuss strategies to identify antigens of γδ and αβTCR molecules such as biochemical methods candidate antigens human leukocyte antigen requirements synthetic peptide and cDNA libraries These strategies are tailored to characterize the antigens of the membraneanchored lowaffinity TCR molecules The strategies to identify auto reactive B cells or immunoglobulin Ig molecules are fundamentally different because Ig molecules are watersoluble and have high affinities We further discuss proteomebased approaches techniques that analyze Igchains from single B cells and a repertoirebased method that compares Igproteomes and Igtranscriptomes The first method detects Ig antigens directly whereas the latter two methods allow reconstruction of Ig molecules which can be used for antigen searchesThe current concepts of multiple sclerosis MS as an autoimmune disease are mainly based on the animal model “experimental autoimmune encephalomyelitis” EAE 1 2 In different EAE models several distinct molecularly defined antigens could be demonstrated to induce pathogenic T and/or B cell responses However the target antigens recognized by immune cells in the different forms of human MS still remain speculative In the past most investigations of putatively pathogenic human B and T cells relied mainly on blood as the most easily accessible source Obviously this is far from ideal because any relevant cells are a remote from the target tissue b heavily diluted with irrelevant cells and c likely to have phenotypical and functional properties completely different from the tissueinfiltrating cells Most importantly all investigations of isolated circulating or migrating cells necessarily lack crucial morphological information on how the cells interact with the surrounding tissue The most direct approach would of course be to focus on the autoimmune target tissue as the most valuable source of information This approach is far from trivial because it demands a set of new techniques for obtaining maximum information from scarce human autopsy and biopsy materialThe central problem of such a direct strategy is the availability of tissue samples For most diseases wellpreserved biopsy specimens are extremely rare For example brain or spinal cord lesions are usually not biopsied in MS patients The same holds true for pancreas samples of type I diabetes patients etc There are only few autoimmune diseases where diagnostic biopsies are taken on a regular basis One such example is provided by the inflammatory myopathies IM This situation contrasts with animal experiments where diseases may be induced and manipulated experimentally and where tissue samples may be collected at any time point of choice even at preclinical stagesFor many years we have investigated antigendirected immune responses in MS and IM target tissues that is brain and muscle Both in MS and IM T cells—especially CD8+ cells—infiltrate the organs It is not known how these cells are attracted or how the local inflammatory response is sustained The target antigens are also unknown The B cell responses ie the auto antibody responses are somewhat better understood This is mainly due to the fact that soluble immunoglobulin Ig proteins are easier to investigate than autoaggressive T cells where the autoaggressive T cell receptor TCR is a member of a multimeric complex of membrane proteins During the course of our studies we have developed a series of technologies that allow analysis of T and B cell responses in IM and MSAutoaggressive T cells attack muscle fibers In the schematic representation left the autoaggressive T cells are shown in red Many other bystander T cells and T cells in blood vessels are present in the same tissue blue and green On the right panel we show an immunohistochemical stained cryosection from the muscle of a PM patient CD8+ T cells are stained in green In the scheme and in the cryosection muscleattached single T cells and T cell clusters that focally invade the muscle fibers are seen modified from 23 with permissionThe first step of our analysis is the identification of cell clones or Ig molecules that are expanded in the target tissue driven by antigen recognition Such “repertoire studies” help us to distinguish between pathogenic and irrelevant cells or molecules In the next step we focus on individual T cells and their antigenspecific receptor or on clonally expanded antibodies We then amplify the TCR or Ig chains by PCR and express them in vitro The transfectants are then used for antigen searches In a first series of experiments “best guess” candidate antigens may be screened Such candidates usually come from animal experiments A more unbiased approach is to screen cDNA expression libraries The cDNA libraries may be generated from the affected organs or—preferred—from the biopsy specimen of the patient Depending on whether B or T cell antigens are investigated the libraries are either expressed and screened directly or must be introduced into the classI or classII major histocompatibility complex MHC presentation pathway before they are screenedHere we will review the current state of antigen detection efforts in MS research Both for B cell and for T cell antigens several technical challenges have to be overcome Since the experimental strategies for identifying B and T cell antigens are quite different we will discuss the respective approaches separately Needless to say these new techniques may also be applied to tissues from patients with other autoimmune neoplastic or inflammatory diseases where adaptive immune responses occurTissueinfiltrating T cells are observed in all patients with MS or IM In most cases the T cell infiltrates are composed of αβT cells whereas γδT cells are rather an exception 8 9 10 In MS CD8+ T cells usually outnumber the CD4+ population 11 In IM it depends on the subtype of the disease While in inclusion body myositis and polymyositis CD8+ T cells clearly dominate while CD4+ T cells are more prominent in dermatomyositis 12 13 We have intensively studied the αβTCR repertoire of infiltrating CD8+ T cells in MS brain specimens 14 15 16 and in myositis muscle tissue of polymyositis and inclusion body myositis patients 17 18 19 Using CDR3spectratyping we found that in these diseases CD8+ T cells are expanded in the target tissues and blood and that these expanded clones may persist for many years in some patients We investigated the TCR repertoire in muscle samples and blood of several patients with IM 19 and in brain tissue cerebrospinal fluid CSF and blood of MS patients 15 In the myositis study we identified expanded T cell clones in muscle biopsy tissue of ten patients From four patients we isolated single morphologically characterized T cells by laser microdissection and analyzed the TCR βchains by single cell PCR These T cells were most probably autoaggressive because they belonged to expanded clones were in direct contact with their target cells and carried silent nucleotide exchanges in the TCR CDR3Nregions This indicates that the clonal expansions were driven by particular antigens Expanded clones were not only detectable in muscle tissue but also in the CD8+ T cell compartment of peripheral blood where they persisted for many years Persistent CD8+ T cell clones were also detected in MS patients 15 We found that these CD8+ clones were also present in brain tissue CSF and peripheral blood By contrast the CD4+ population was polyclonal Strikingly some of the CD8+ T cells that had been identified in brain tissue persisted in peripheral blood and CSF for more than 7 years These repertoire studies were the basis of a general strategy that leads to the molecular characterization of αβTCR molecules from autoaggressive T cell 3

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