30 research outputs found
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 groĂźe 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. FĂĽr eine erfolgreiche Immunantwort sind auĂźerdem die Effektor-
Funktionen der konstanten Region des Immunoglobulins, die in allen
Immunglobulin-Klassen unterschiedlich sind, entscheidend. Die jĂĽngsten
Innovationen in der Sequenz-Analyse von DNA erlauben seit kurzem,
zellgebundene (B-Zell-Rezeptoren) und sezernierte Immunglobuline von
menschlichen Spendern im groĂźen 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. DafĂĽr 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 fĂĽr
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 durchgefĂĽhrt. Dabei konnten auch Gene, die in frĂĽheren
Studien nicht abgedeckt wurden, in die Untersuchung einbezogen werden und
deren relatives Vorkommen evaluiert werden. Ăśberdies 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
schlieĂźen, 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 fĂĽr
zukĂĽnftige 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<sub>AD</sub></i> for antibody type <i>A</i> and donor <i>D</i> was calculated from the number of occurring VDJs <i>n<sub>AD</sub></i> and the total number of occurring VDJs in the donor <i>D n<sub>D</sub></i> as <i>n<sub>AD</sub>/n<sub>D</sub></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<sub>both</sub>/</i>max(n<sub>A</sub>, n<sub>B</sub>), where <i>n<sub>both</sub></i> is the number of VDJs present in both sets of VDJs and n<sub>A</sub> and n<sub>B</sub> represent the sizes of the sets. Blue and pink colors represent male and female, respectively.</p