5 research outputs found
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 âclass
switchedâ 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 ârecirculationâ 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 ârecirculationâ 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 fĂŒr die Entstehung des immunologischen GedĂ€chtnisses und
der Produktion hoch-affiner Antikörper von entscheidender Bedeutung. Hierbei
handelt es sich um transiente Strukturen die nach Immunisierung mit einem T
Zell-abhÀngigen Antigen in sekundÀr lymphatischen Geweben wie der Milz und den
Lymphknoten entstehen. Im Laufe einer Immunantwort, wandern aktivierte B
Zellen in die sekundÀr lymphatischen Organe ein und erreichen dann die T-Zell-
Bereiche. Dort befinden sich fĂŒr 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-evolutionÀren Prozessen, B-Zellen mit hoher AntigenaffinitÀt, diese
erhalten ĂŒberlebenswichtige Signale von T-Zellen und können die Keimzentren
verlassen um zu langlebigen Plasmazellen oder B-GedÀchtniszellen zu
differenzieren. Diese B-GedÀchtniszellen sezernieren auch Antikörper anderer
Immunglobulin-Klassen als IgM. Die Sezernierung solch âKlassengewechselterâ
Antikörper ist fĂŒr die Optimierung der Immunantwort von essentieller
Bedeutung, da die âImmunglobulin-klasseâ eines Antikörpers bestimmend fĂŒr
seine Effektor-Funktion ist. Beispiele solcher Funktionen wÀren die
Aktivierung des Komplementsystems, Opsonisierung, Neutralisation bakterieller
Toxine oder Mastzellen Aktivierung. Obgleich GCs unerlĂ€sslich fĂŒr die
Entstehung des immunologischen GedÀchtnisses sind, spielen ektopische
Keimzentren, also Keimzentren die sich auĂerhalb des sekundĂ€ren 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 groĂe 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
durchgefĂŒhrt: i) Es wurde eine Kinetik sĂ€mtlicher 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 primÀren und sekundÀren 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 trÀgt durch folgende Erkenntnisse zum VerstÀndnis der
GC Entwicklung und Dynamik bei: 1) Eines der wichtigsten Erkenntnisse dieser
Arbeit war die Detektion von B-Zellen mit einem GC PhÀnotyp im Blut. Die
durchflusszyometrischen Untersuchungen zeigten dass diese Zellen eindeutig
reife B-Zellen sind und follikulÀren Ursprung haben. Die Entdeckung von
GC-B-Zellen im Blut fĂŒhrte zu der Hypothese der âRezirkulationâ; die besagt
das eine Fraktion von GC-B-Zellen nach Verlassen der GC ins periphere Blut
wandern, ohne ihren PhÀnotyp zu verlieren. Das Zirkulieren dieser Zellen im
Blut ermöglicht es ihnen in jedes beliebige sekundÀre lymphatische Gewebe
einzuwandern um dort, in den lokalen GC Reaktionen teilzunehmen. Solch ein
Schema wĂŒrde zu einer höheren GC-DiversitĂ€t und einer schnelleren
AffinitĂ€tsreifung fĂŒhren. 2) Um der âRezirkulation-Hypotheseâ eine
experimentelle Basis zu geben, wurden die im Blut detektierten GC-B-Zellen
isoliert und in Rezipienten transferiert, die sich in der frĂŒhen Phase oder
kurz vor dem Höhepunkt der GC Reaktion befanden. Die Resultate zeigten dass
die transferierten GC-B-Zellen in das sekundÀre lymphatische Gewebe einwandern
und an laufenden GC Reaktionen teilnehmen können um dann weiter zu
Plasmazellen zu differenzieren. 3) Um zu prĂŒfen ob die FĂ€higkeit in sekundĂ€r
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 spÀteren Entwicklungsstadium der GC B-Zellontogenese waren, auf
dieselbe Weise transferiert. Hierbei handelte es sich um zwei potentielle
GedÀchtnis-B Zellpopulationen; CD38hiIgG1+ und CD38hiIgM+ B-Zellen. Die
Analysen ergaben dass diese zwei B-Zellpopulationen in der Tat ein
unterschiedliches Migrationsverhalten aufweisen. WĂ€hrend 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