A minimal model of peptide binding predicts ensemble properties of serum antibodies

<p/> <p>Background</p> <p>The importance of peptide microarrays as a tool for serological diagnostics has strongly increased over the last decade. However, interpretation of the binding signals is still hampered by our limited understanding of the technology. This is in particular true for arrays probed with antibody mixtures of unknown complexity, such as sera. To gain insight into how signals depend on peptide amino acid sequences, we probed random-sequence peptide microarrays with sera of healthy and infected mice. We analyzed the resulting antibody binding profiles with regression methods and formulated a minimal model to explain our findings.</p> <p>Results</p> <p>Multivariate regression analysis relating peptide sequence to measured signals led to the definition of amino acid-associated weights. Although these weights do not contain information on amino acid position, they predict up to 40-50% of the binding profiles' variation. Mathematical modeling shows that this position-independent ansatz is only adequate for highly diverse random antibody mixtures which are not dominated by a few antibodies. Experimental results suggest that sera from healthy individuals correspond to that case, in contrast to sera of infected ones.</p> <p>Conclusions</p> <p>Our results indicate that position-independent amino acid-associated weights predict linear epitope binding of antibody mixtures only if the mixture is random, highly diverse, and contains no dominant antibodies. The discovered ensemble property is an important step towards an understanding of peptide-array serum-antibody binding profiles. It has implications for both serological diagnostics and B cell epitope mapping.</p

Migration und Differenzierung von aus Keimzentren abstammenden B-Zell-Subtypen im Laufe der NP-spezifischen Immunantwort im Mausmodell

1 INTRODUCTION 4 1.1 THE ADAPTIVE AND INNATE IMMUNITY 4 1.2 LYMPHOCYTES AND LYMPHATIC ORGANS 5 1.2.1 T CELLS AND THEIR TYPES AND FUNCTIONS 5 1.2.2 B CELLS; FUNCTIONS AND THEIR ANTIBODIES 6 1.2.3 PRIMARY LYMPHOID ORGANS 8 1.2.4 SECONDARY LYMPHOID ORGANS AND TISSUES 11 1.3 ACTIVATION OF B CELLS BY T-INDEPENDENT ANTIGENS 13 1.4 ACTIVATION OF B CELLS BY T-DEPENDENT ANTIGENS 14 1.5 SELECTION, SURVIVAL AND DIFFERENTIATION OF B CELLS IN THE T-DEPENDENT IMMUNE RESPONSE 15 1.5.1 THE EXTRAFOLLICULAR RESPONSE 16 1.5.2 THE GC REACTION 17 1.5.3 MEMORY B CELLS AND PLASMA CELLS 18 2 OBJECTIVES 22 3 MATERIALS AND METHODS 23 3.1 BUFFERS, REAGENTS AND SOLUTION 23 3.2 ANTIBODIES AND REAGENTS FOR FACS AND IMMUNOFLUORESCENCE 24 3.3 MICE, ANTIGEN AND IMMUNIZATIONS 25 3.4 IMMUNOFLUORESCENCE 25 3.5 ADOPTIVE TRANSFERS 27 3.6 FLOW CYTOMETRY 28 4 RESULTS 29 4.1 KINETIC OF NP-KLH SPECIFIC RESPONSE AFTER PRIMARY AND SECONDARY CHALLENGE 29 4.1.1 DIFFERENCES IN ABSOLUTE LYMPHOCYTE NUMBERS OF C57BL/6 MICE AFTER PRIMARY OR SECONDARY CHALLENGE DO NOT CORRELATE WITH THE STATE OF IMMUNE RESPONSE 29 4.1.2 KINETICS AND MATURATION OF NP-KLH INDUCED B CELL SUBSETS DURING PRIMARY AND SECONDARY IMMUNE RESPONSE 33 4.1.3 IDENTIFIED B CELLS WITH GC PHENOTYPE IN BLOOD ARE MATURE B CELLS WITH FOLLICULAR ORIGIN 38 4.1.4 DYNAMICS OF THE NP-KLH INDUCED PLASMABLAST AND PLASMA CELL RESPONSE 42 4.2 MIGRATORY BEHAVIOR OF BLOOD-DERIVED B CELL SUBSETS AFTER TRANSFER INTO AT AN EARLIER TIME POINT AFTER IMMUNIZATION 47 4.2.1 TRANSFERRED B CELLS NUMBERS INCREASE AS EARLY AS 2 DAYS AFTER TRANSFER AND DO NOT VARY IN FREQUENCIES UNTIL DAY EIGHT 48 4.2.2 TRANSFERRED GC B CELLS HOME TO SECONDARY LYMPHOID ORGANS, KEEP THEIR PHENOTYPE BUT MAINLY DEVELOP FURTHER INTO POST GC B CELLS AND PLASMA CELLS 50 4.2.3 TRANSFERRED IGM+ CD38+PNALO B CELLS HOME TO SECONDARY LYMPHOID ORGANS, SWITCH THEIR ISOTYPE AND INDUCE A STRONG BUT RATHER SHORT LIVED PLASMA CELL RESPONSE 53 4.2.4 TRANSFERRED IGG1 B CELLS HOME TO SPLEEN AND BONE MARROW AND DOWN-REGULATE SURFACE B220 AT LATER TIME POINTS 56 4.2.5 TRANSFERRED GC B CELLS ARE INITIALLY LOCATED ALONG THE T CELL BORDER AND PARTIALLY PROLIFERATE WITHIN THE B CELL ZONE AND INTERFOLLICULAR ZONE WHEREAS ANOTHER FRACTION ENTERS THE DARK ZONE AND SUBSEQUENTLY ACCUMULATES IN THE GC LIGHT ZONE 58 4.2.6 TRANSFERRED B220+PNALOCD38HIIGM+ CELLS ACCUMULATE ALONG THE B/T BORDER AND WITHIN THE IF ZONE PRIOR TO ENTERING THE GC DARK AND LIGHT ZONES 63 4.3 MIGRATORY BEHAVIOR OF BLOOD-DERIVED B CELL SUBSETS AFTER TRANSFER INTO RECIPIENTS AT THE TIME POINT AFTER IMMUNIZATION 68 4.3.1 TRANSFERRED GC B CELLS AND IGM+ B CELLS PROLIFERATE 2 DAYS AFTER TRANSFER IN SPLEEN AND BONE MARROW BUT DECREASE IN NUMBERS AFTERWARDS, WHEREAS IGG1+ B CELLS ARE ONLY DETECTED IN BONE MARROW AND REMAIN THERE AT STABLE FREQUENCIES 69 4.3.2 TRANSFERRED BLOOD GC B CELLS HOME TO SECONDARY LYMPHOID ORGANS, PARTIALLY KEEP THEIR PHENOTYPE BUT MAINLY DIFFERENTIATE FURTHER AND INDUCE A SHORT LIVED PLASMA CELL RESPONSE 73 4.3.3 TRANSFERRED BLOOD RESIDING IGM+CD38+ B CELLS CAN REGAIN A GC PHENOTYPE FOR A FEW DAYS AND INDUCE A STRONG SHORT LIVED PLASMA CELL RESPONSE 76 4.3.4 BLOOD-DERIVED IGG1+ CD38+ B CELLS ACCUMULATE IN THE BONE MARROW AND CHANGE THEIR SURFACE EXPRESSION OF B220 AT EARLY TIME POINTS OF IMMUNE RESPONSE 78 4.3.5 T CELL ASSOCIATED PROLIFERATION OF BLOOD-DERIVED GC B CELLS WITHIN THE FOLLICLE PRECEDES THEIR MIGRATION TO THE GC DARK ZONE FOLLOWED BY THEIR RECRUITMENT TO GC LIGHT ZONE 79 4.3.6 THE B CELL FOLLICLE IS THE MAIN ASSEMBLY SIDE OF TRANSFERRED BLOOD-DERIVED PNALOCD38HIIGM+ B CELLS 84 5 DISCUSSION 90 5.1 KINETIC OF NP-KLH INDUCED B CELL SUBSETS BY ANALYSIS OF THEIR EXPRESSION PROFILE OF CELL SURFACE MARKERS AFTER PRIMARY AND SECONDARY CHALLENGE 90 5.1.1 IDENTIFICATION OF THE GC B CELL SUBSET 90 5.1.2 KINETIC OF THE GC B CELL SUBSETS 91 5.1.3 IDENTIFICATION OF THE MEMORY B CELL SUBSET 92 5.1.4 KINETIC OF THE MEMORY B CELL SUBSETS 92 5.1.5 IDENTIFICATION OF THE PLASMABLAST AND PLASMA CELL SUBSETS 93 5.1.6 KINETIC OF THE PLASMABLAST AND PLASMA CELL SUBSETS 93 5.2 DETECTION OF GC B CELLS IN BLOOD OF NP-KLH IMMUNIZED C57BL/6 MICE 95 5.3 TRANSFERRED BLOOD GC B CELLS REPOPULATE PERSISTING GCS AND DIFFERENTIATE INTO PLASMA CELLS 96 5.4 TRANSFERRED IGM AND IGG1 POTENTIAL MEMORY B CELLS DISPLAY DIFFERENT MIGRATION PATTERNS 99 6 SUMMARY AND PERSPECTIVES 104 7 ZUSAMMENFASSUNG 107 8 ABSTRACT 109 9 REFERENCES 111 10 APPENDIX 117Development of B cell memory and generation of high affinity antibodies are crucially dependent on germinal centers (GC). GCs are transient structures which arise after challenge with a T cell-dependent antigen within secondary lymphoid organs, such as the spleen and lymph nodes. During the immune response activated B cells migrate to the T cell zones within the secondary lymphoid organs and acquire help from the residing antigen-specific, activated T cells. Some of these activated B cells migrate toward the B cell zones, expand rapidly and found the GCs. A number of micro-evolutionary processes occur within the GCs, leading to the production of high-affinity B cells which acquire the necessary survival signals from T cells and leave the GCs in order to differentiate into plasma cells and memory B cells. These memory B cells are able to produce antibodies other than IgM. The production of such “class switched” antibodies is important for optimizing the immune response to particular antigens, since the antibody class defines its effector functions, such as complement activation, opsonization, neutralization of bacterial toxins and mast cell activation. GCs play a major role in the development of protective immunological memory; however, they are responsible for the pathogenesis of several autoimmune and infectious diseases, such as rheumatic arthritis, hashimoto thyroiditis, sjogren syndrome, multiple sclerosis, HIV and chronic hepatitis C. It is, therefore, of significant importance to understand the dynamic of GCs and the regulating mechanisms which underlie their progress and termination. This work delivers a deeper insight into the mentioned topics by performing the following analyses: i) A kinetic of the GC B cell subsets was conducted by means of flow cytometric analyses and immunofluorescence microscopy methods. This kinetic included the spleen, blood and isolated bone marrow from femur and tibia and comprised several time points after primary and secondary challenge with NP-KLH, a model antigen often used to analyze the T-dependent immune response. ii) To monitor the migratory behavior of GC B cell emigrants, different B cell subsets, corresponding to distinct stages of GC B cell ontogeny were isolated and enriched from blood. Subsequently, these subsets were transferred into recipients at an early time point and shortly before the peak of their GC response. The localization and differentiation status of the donor B cells within spleen, bone marrow and mesenteric lymph nodes of recipients were determined during one week after transfer via flow cytometry and immunofluorescence microscopy. This thesis contributes the following insight into the development and dynamics of the GC response: 1\. The major finding of this work was the detection of B cells bearing a GC phenotype in blood. The flow cytometric analysis revealed that these cells consist of mature B cells of a follicular GC origin. The detection of GC B cells within blood led to the postulation of the “recirculation” hypothesis, which states that a fraction of GC B cells exits the GCs and enters the peripheral blood without losing the GC B cell phenotype. Consequently, the circulation through blood enables such emigrants to enter any secondary lymphoid organ, enabling them to be re- admitted to the local GC reactions. Supposedly, such a scheme could lead to faster affinity maturation and a higher diversity of GC B cells. 2\. To test the „recirculation“ hypothesis, a blood derived GC B cell fraction was isolated and transferred into recipients which were at an early time point, just before the peak of their GC response. The obtained results showed that the blood derived GC B cells immigrate into secondary lymphoid organs and are recruited to the already existing GC reaction. Furthermore, they differentiated into plasmablasts and subsequently plasma cells. 3\. To address the question whether the capability to immigrate into secondary lymphoid organs is a GC B cell specific feature, additional transfer experiments were conducted with blood derived CD38hiIgG1+ and CD38hiIgM+ potential memory B cells, which correspond to later stages of GC B cell ontogeny. Interestingly, these two subsets displayed a different migratory behavior. Whereas CD38hiIgG1+ B cells preferentially migrated into the bone marrow and differentiated into plasma cells, CD38hiIgM+ B cells migrated into the spleen and the lymph nodes and participated in the ongoing GC reactions.Keimzentren (GC) sind für die Entstehung des immunologischen Gedächtnisses und der Produktion hoch-affiner Antikörper von entscheidender Bedeutung. Hierbei handelt es sich um transiente Strukturen die nach Immunisierung mit einem T Zell-abhängigen Antigen in sekundär lymphatischen Geweben wie der Milz und den Lymphknoten entstehen. Im Laufe einer Immunantwort, wandern aktivierte B Zellen in die sekundär lymphatischen Organe ein und erreichen dann die T-Zell- Bereiche. Dort befinden sich für dasselbe Antigen spezifische, bereits aktivierte T-Zellen. Diese, ermöglichen den B-Zellen zu proliferieren. Ein Teil jener B-Zellen wandert zusammen mit den T-Zellen, von denen sie aktiviert wurden, zu den B-Zell-Follikeln, wo sie an der Grenze zur T-Zell-Zone Keimzentren bilden. Innerhalb der Keimzentren entstehen durch einer Reihe von mikro-evolutionären Prozessen, B-Zellen mit hoher Antigenaffinität, diese erhalten überlebenswichtige Signale von T-Zellen und können die Keimzentren verlassen um zu langlebigen Plasmazellen oder B-Gedächtniszellen zu differenzieren. Diese B-Gedächtniszellen sezernieren auch Antikörper anderer Immunglobulin-Klassen als IgM. Die Sezernierung solch „Klassengewechselter“ Antikörper ist für die Optimierung der Immunantwort von essentieller Bedeutung, da die „Immunglobulin-klasse“ eines Antikörpers bestimmend für seine Effektor-Funktion ist. Beispiele solcher Funktionen wären die Aktivierung des Komplementsystems, Opsonisierung, Neutralisation bakterieller Toxine oder Mastzellen Aktivierung. Obgleich GCs unerlässlich für die Entstehung des immunologischen Gedächtnisses sind, spielen ektopische Keimzentren, also Keimzentren die sich außerhalb des sekundären lymphatischen Gewebes bilden, in der Entstehung und dem Verlauf diverser Erkrankungen wie z.B. Rheumatischer Arthritis, Hashimoto Thyreoiditis, Sjogren Syndrom, Multipler Sklerose, HIV und chronischer Hepatitis C eine große Rolle. Aus diesem Grund ist es unabdingbar, die Dynamik und die regulierenden Mechanismen, die dem Fortbestand und der Terminierung der Keimzentren zugrundeliegen zu verstehen. Deshalb wurden folgende Untersuchungen durchgeführt: i) Es wurde eine Kinetik sämtlicher wichtigen in der GC-Reaktion involvierten B Zellen mit Hilfe von durchflusszyometrischer Methoden erstellt. Diese Analyse umfasste die drei Organe, Milz, Blut und das isolierte Knochenmark aus Femur und Tibia, und wurde an mehreren aufeinanderfolgenden Zeitpunkten nach Induktion einer primären und sekundären Immunantwort mit dem Model Antigen NP-KLH vorgenommen. ii) Um das Migrationsverhalten von GC-B -Zell-Emigranten zu untersuchen, wurden verschiedene im Blut zirkulierende zu unterschiedlichen Zeitpunkten der GC-B-Zell-Entwicklung gehörige B-Zell- Populationen isoliert und in Rezipienten transferiert die zu unterschiedlichen Zeitpunkten zuvor immunisiert waren. Die Lokalisierung und weitere Differenzierung der transferierten B-Zellen wurden in der Milz und im Knochenmark mittels Durchflusszytometrie und in den mesenterischen Lymphknoten mit Immunofluoreszenz-Mikroskopie innerhalb einer Woche nach dem Transfer bestimmt. Diese Arbeit trägt durch folgende Erkenntnisse zum Verständnis der GC Entwicklung und Dynamik bei: 1) Eines der wichtigsten Erkenntnisse dieser Arbeit war die Detektion von B-Zellen mit einem GC Phänotyp im Blut. Die durchflusszyometrischen Untersuchungen zeigten dass diese Zellen eindeutig reife B-Zellen sind und follikulären Ursprung haben. Die Entdeckung von GC-B-Zellen im Blut führte zu der Hypothese der „Rezirkulation“; die besagt das eine Fraktion von GC-B-Zellen nach Verlassen der GC ins periphere Blut wandern, ohne ihren Phänotyp zu verlieren. Das Zirkulieren dieser Zellen im Blut ermöglicht es ihnen in jedes beliebige sekundäre lymphatische Gewebe einzuwandern um dort, in den lokalen GC Reaktionen teilzunehmen. Solch ein Schema würde zu einer höheren GC-Diversität und einer schnelleren Affinitätsreifung führen. 2) Um der „Rezirkulation-Hypothese“ eine experimentelle Basis zu geben, wurden die im Blut detektierten GC-B-Zellen isoliert und in Rezipienten transferiert, die sich in der frühen Phase oder kurz vor dem Höhepunkt der GC Reaktion befanden. Die Resultate zeigten dass die transferierten GC-B-Zellen in das sekundäre lymphatische Gewebe einwandern und an laufenden GC Reaktionen teilnehmen können um dann weiter zu Plasmazellen zu differenzieren. 3) Um zu prüfen ob die Fähigkeit in sekundär lymphatisches Gewebe zu immigrieren im Laufe der GC B-Zell Entwicklung beibehalten wird, wurden zwei weitere aus dem Blut gewonnene B-Zelltypen, die in einem späteren Entwicklungsstadium der GC B-Zellontogenese waren, auf dieselbe Weise transferiert. Hierbei handelte es sich um zwei potentielle Gedächtnis-B Zellpopulationen; CD38hiIgG1+ und CD38hiIgM+ B-Zellen. Die Analysen ergaben dass diese zwei B-Zellpopulationen in der Tat ein unterschiedliches Migrationsverhalten aufweisen. Während die IgG1+ B-Zellen bevorzugt in das Knochenmark einwanderten und zu Plasmazellen differenzierten, immigrierten die IgM+ B-Zellen auch in die Milz und die Lymphknoten wo sie teilweise an der laufenden GC Reaktion des Rezipienten teilnahmen

Innate Immune Recognition of Bacterial Viability Instructs Human T follicular Helper Cell Differentiation

Live attenuated vaccines are often superior to dead vaccines, yet the immunological mechanisms remain largely obscure. We have recently uncovered an inherent capacity of antigen-presenting cells (APC) to discriminate live from killed bacteria by virtue of vita-PAMPs. Here we found that innate recognition of bacterial viability strongly promotes the differentiation of fully functional T follicular helper (TFH) cells. We identify TLR8 and its signaling adaptor MyD88 as critical sensor for bacterial viability in human APC, activation of which is required and sufficient to induce selective transcriptional remodeling and the production of TFH promoting signals like IL-12. Activators of other TLRs including licensed vaccine adjuvants fail to do so. Consequently, vita-PAMP receptors such as TLR8 represent promising targets for adjuvants to improve the efficacy of modern inanimate subunit vaccines

Innate Immune Recognition of Bacterial Viability Instructs Human T follicular Helper Cell Differentiation

Live attenuated vaccines are often superior to dead vaccines, yet the immunological mechanisms remain largely obscure. We have recently uncovered an inherent capacity of antigen-presenting cells (APC) to discriminate live from killed bacteria by virtue of vita-PAMPs. Here we found that innate recognition of bacterial viability strongly promotes the differentiation of fully functional T follicular helper (TFH) cells. We identify TLR8 and its signaling adaptor MyD88 as critical sensor for bacterial viability in human APC, activation of which is required and sufficient to induce selective transcriptional remodeling and the production of TFH promoting signals like IL-12. Activators of other TLRs including licensed vaccine adjuvants fail to do so. Consequently, vita-PAMP receptors such as TLR8 represent promising targets for adjuvants to improve the efficacy of modern inanimate subunit vaccines