The Future of Blood Testing Is the Immunome

It is increasingly clear that an extraordinarily diverse range of clinically important conditions-including infections, vaccinations, autoimmune diseases, transplants, transfusion reactions, aging, and cancers-leave telltale signatures in the millions of V(D)J-rearranged antibody and T cell receptor [TR per the Human Genome Organization (HUGO) nomenclature but more commonly known as TCR] genes collectively expressed by a person's B cells (antibodies) and T cells. We refer to these as the immunome. Because of its diversity and complexity, the immunome provides singular opportunities for advancing personalized medicine by serving as the substrate for a highly multiplexed, near-universal blood test. Here we discuss some of these opportunities, the current state of immunome-based diagnostics, and highlight some of the challenges involved. We conclude with a call to clinicians, researchers, and others to join efforts with the Adaptive Immune Receptor Repertoire Community (AIRR-C) to realize the diagnostic potential of the immunome

Erforschung des humanen Immunglobulinrepertoires mittels Hochdurchsatz- Sequenzierung

The main role of the adaptive immune system is to protect the body against pathogens. Immunoglobulins are an essential part of this system. The great diversity of immunoglobulins is obtained by a complex mechanism of genetic reorganization from a predetermined set of gene segments on chromosomal level and subsequent affinity maturation. Thereby a specific affinity for a particular pathogen is finally achieved. For a successful immune response to occur, the effector functions of the constant region of the immunoglobulin are also essential - which are different in all immunoglobulin classes. The latest innovations in DNA sequencing technology allow nowadays to analyze and evaluate immunological questions in respect to cell-bound (i.e. B-cell receptors) and secreted immunoglobulins from human donors on cDNA level. The technological innovation, which is generally referred to as "next generation sequencing" and especially the so-called pyro-sequencing laid the basis to the present study. The aim was to develop a method for sequencing antibody cDNA by pyro-sequencing, which allows all genes and all immunoglobulin classes to be quantitatively and qualitatively measurable. With this novel approach the immunoglobulin repertoire from peripheral blood samples of healthy donors of different ages and gender ought to be analyzed. The newly developed immunoglobulin specific cDNA amplification method established during this thesis is independent of the use of V-gene specific primers. It further allows the verification of the corresponding immunoglobulin classes and sub-groups in the obtained sequences. Through the introduction of a newly developed emulsion PCR method, the influences of the sample preparation and the cDNA amplification procedure on the representative quality of the obtained sequences were minimized. Furthermore, it was necessary to create a new amplicon processing protocol for the 454 Roche GS FLX sequencer to achieve compatibility with the amplicons generated in the previous antibody amplification process. Using the novel immunoglobulin-amplification and sequencing method, more than 3.5 million sequences were obtained from a representative group of 14 healthy individuals of different age and gender. At first, a comprehensive analysis of the distribution of V(D)J genes in the donor was performed. Some genes, which were not covered in previous studies, could be included in the study and their relative occurrence evaluated. Moreover, for the first time, the VDJ gene distribution in conjunction with their immunoglobulin class has been analyzed. The results of the analyses show that there are differences in the immunoglobulin repertoire of young (19-30 years) and elderly people (> 50 years). A reduction in the ability to change the classes of antibodies is observed and this correlates with age. This is in line with current studies, where the reduction in class switch is discussed as a cause for decreased vaccine efficacy in the elderly population. In this work, it is shown that the influence of age on the ability to change the immunoglobulin classes can be analyzed by cDNA sequencing. The results also strongly suggest that the senescence of the immune system begins in an age range between 50 and 60 years. A correlation between gender and the ability to change the classes of antibodies could, however, not be detected in this study. The developed methods for sequencing immunoglobulin repertoire allow entirely new insights into the immune system. Due to the improved experimental set-up, the complexity of the variables that can be analyzed from a single sample increases considerably. In future, changes of the immune system in healthy and diseased individuals can be measured and analyzed on a new, unrivaled level of complexity. The methods developed during my PhD and the obtained results provide a solid basis for future research addressing the analysis and verification of disease-specific changes in the immune system.Die wichtigste Rolle des adaptiven Immunsystems ist der Schutz des Körpers vor Pathogenen. Immunglobuline sind ein wesentlicher Bestandteil dieses Systems. Die große Vielfalt von Immunglobulinen wird durch einen komplexen Mechanismus der genetischen Reorganisation aus einem vorgegebenen Satz von Gensegmenten auf chromosomaler Ebene und anschließender Affinitätsreifung erhalten. Dadurch wird letztlich eine spezifische Affinität für ein bestimmtes Pathogen erreicht. Für eine erfolgreiche Immunantwort sind außerdem die Effektor- Funktionen der konstanten Region des Immunoglobulins, die in allen Immunglobulin-Klassen unterschiedlich sind, entscheidend. Die jüngsten Innovationen in der Sequenz-Analyse von DNA erlauben seit kurzem, zellgebundene (B-Zell-Rezeptoren) und sezernierte Immunglobuline von menschlichen Spendern im großen Umfang auf cDNA-Ebene zu sequenzieren und immunologisch zu bewerten. Diese technologischen Innovationen, die generell als „Next-Generation-Sequencing“ bezeichnet werden und speziell die der sogenannten Pyro-Sequenzierung bildeten die Basis der vorliegenden Untersuchung. Ziel war die Entwicklung eines Verfahrens zur Sequenzierung von Antikörpern durch Pyro-Sequenzierung von cDNA, das alle Gene sowie alle Immunglobulin-Klassen quantitativ und qualitativ messbar macht. Dafür sollte das Immunglobulin-Repertoire aus peripheren Blutproben von gesunden Spendern unterschiedlichen Alters und Geschlechts analysiert werden. Die in dieser Arbeit neu entwickelte Immunglobulin-spezifische cDNA-Amplifikationsmethode ist unabhängig von der Verwendung von Primern, die für V-Gene spezifisch sind. Sie ermöglicht zudem die Verifizierung der zugehörigen Immunglobulin-Klassen und ihrer Untergruppen in den erhaltenen Sequenzen. Der Einfluss der Vorbereitung auf die repräsentative Qualität der Proben und deren cDNA- Vervielfältigung wird durch eine entwickelte Emulsions-PCR-Methode minimiert. Des Weiteren war es notwendig, ein neues Amplicon-Aufarbeitungsverfahren für den 454 Roche GS FLX Sequenzer zu erstellen, um Kompatibilität mit den vorangegangenen Schritten der Amplicon-Generierung zu erreichen. Mittels der neuartigen Immunglobulin-Amplifikations- und Sequenzier-Methode konnten aus einer repräsentativen Gruppe von 14 gesunden Individuen unterschiedlichen Alters und Geschlechts mehr als 3,5 Millionen Sequenzen gewonnen werden. Im ersten Schritt wurde eine umfassende Analyse der Verteilung der V(D)J-Genen innerhalb der Spender durchgeführt. Dabei konnten auch Gene, die in früheren Studien nicht abgedeckt wurden, in die Untersuchung einbezogen werden und deren relatives Vorkommen evaluiert werden. Überdies wurde erstmalig die VDJ- Gen-Verteilung im Zusammenhang mit deren Immunglobulin-Klasse analysiert. Das Ergebnis der Analyse zeigt, dass junge (19-30 Jahre) und ältere Menschen (>50 Jahre) sich in Bezug auf ihr jeweiliges Immunglobulin-Repertoir unterschiedlich verhalten. Die Reduktion der Fähigkeit zum Wechsel der Antikörperklassen korreliert mit dem Alter – in aktuellen Untersuchungen wird dies als Ursache für verringerte Impfstoffwirksamkeit bei älteren Menschen diskutiert. In dieser Arbeit wird deutlich, dass der Einfluss des Alters auf die Fähigkeit zum Wechsel der Antikörperklassen durch Immunglobulin- Sequenzierung analysiert werden kann. Die Ergebnisse lassen zudem darauf schließen, dass in einem Alter zwischen 50 und 60 Jahren die Seneszenz des Immunsystems beginnt. Eine Korrelation zwischen Geschlecht und Fähigkeit zum Wechsel der Antikörperklassen konnte in dieser Untersuchung dagegen nicht nachgewiesen werden. Durch die hier entwickelten Verfahren zur Immunglobulin- Sequenzierung ergeben sich ganz neue Erkenntnisse in Bezug auf das Immunsystem. Aufgrund des verbesserten Versuchsaufbaus hat sich die Komplexität an Messgrößen, die aus einer einzigen Probe analysiert werden können, deutlich erhöht. In Zukunft können die Veränderungen des Immunsystems in Gesunden und Erkrankten in einer neuen, vorher unerreichten Komplexität gemessen und analysiert werden. Die im Rahmen meiner Promotion entwickelten Verfahren und die damit erhaltenen Ergebnisse bieten eine solide Grundlage für zukünftige wissenschaftliche Fragestellungen, die der Entdeckung und Verifizierung krankheitsspezifischer Veränderungen des Immunsystems dienen

Additional file 2: Figure S2. of The Repertoire Dissimilarity Index as a method to compare lymphocyte receptor repertoires

Changes in repertoire content correlate with diversity changes following clonal expansion. Individual naĂŻve and memory CD4+ and CD8+ V gene repertoires were tallied based on the full (molecular) dataset from Rubelt et al. Shannon entropy was calculated for each repertoire, and the fold change in entropy between the naĂŻve and memory repertoires of each patient/cell type. A) Absolute log2 fold change values of Shannon entropy are plotted against the estimated fold change in repertoire contents as calculated by RDI. B) Individual log-fold change values (tick marks) and a kernel density plot (curved line) are shown for each group. Significance was determined using a paired t-test. (PDF 16 kb

Onset of Immune Senescence Defined by Unbiased Pyrosequencing of Human Immunoglobulin mRNA Repertoires

<div><p>The immune system protects us from foreign substances or pathogens by generating specific antibodies. The variety of immunoglobulin (Ig) paratopes for antigen recognition is a result of the V(D)J rearrangement mechanism, while a fast and efficient immune response is mediated by specific immunoglobulin isotypes obtained through class switch recombination (CSR). To get a better understanding on how antibody-based immune protection works and how it changes with age, the interdependency between these two parameters need to be addressed. Here, we have performed an in depth analysis of antibody repertoires of 14 healthy donors representing different gender and age groups. For this task, we developed a unique pyrosequencing approach, which is able to monitor the expression levels of all immunoglobulin V(D)J recombinations of all isotypes including subtypes in an unbiased and quantitative manner. Our results show that donors have individual immunoglobulin repertoires and cannot be clustered according to V(D)J recombination patterns, neither by age nor gender. However, after incorporating isotype-specific analysis and considering CSR information into hierarchical clustering the situation changes. For the first time the donors cluster according to age and separate into young adults and elderly donors (>50). As a direct consequence, this clustering defines the onset of immune senescence at the age of fifty and beyond. The observed age-dependent reduction of CSR ability proposes a feasible explanation why reduced efficacy of vaccination is seen in the elderly and implies that novel vaccine strategies for the elderly should include the “Golden Agers”.</p> </div

In detail analysis of VDJ rearrangements.

<p>(<b>A</b>) Overall distribution of VDJ rearrangements in 14 donors. (<b>B</b>) IgA2-specific VDJ rearrangements in donor I200091-032. (<b>C</b>) IgA1-specific VDJ rearrangements in donor I200091-030. (<b>D</b>) IgG1-specific VDJ rearrangements in donor I200091-021. (<b>E</b>) Gauge; sphere volumes refer to their respective numerical proportion. Less than 10 ppm are represented by a fixed size sphere. Green color shading indicates number of reads constituting respective recombination. Other colors highlight selected V-genes; blue: IGHV1-2, yellow: IGHV2-5, red: IGHV4-34.</p

Analyses based on Ig-isotype distributions.

<p>(<b>A</b>) Relative frequency of isotype-specific sequences within donors sorted left to right according age. (<b>B</b>) Relative variability of isotypes on the basis of initial (IgM/D) and specific response (IgA/E/G). Variability: percentage of VDJs covered by a distinct isotype in each donor. Variability <i>V_AD</i> for antibody type <i>A</i> and donor <i>D</i> was calculated from the number of occurring VDJs <i>n_AD</i> and the total number of occurring VDJs in the donor <i>D n_D</i> as <i>n_AD/n_D</i>. (<b>C</b>) Clustering of donors (rows) according to coincident appearance of most frequent VDJ rearrangements in their isotypes (column) with age and gender. The hundred most frequently occurring VDJ rearrangements (or less if there were less than hundred) for each donor and isotype were selected. For each donor the overlap between each pair of isotypes was quantified (visualized from black to red) using the formula <i>n_both/</i>max(n_A, n_B), where <i>n_both</i> is the number of VDJs present in both sets of VDJs and n_A and n_B represent the sizes of the sets. Blue and pink colors represent male and female, respectively.</p