Malaria, caused by the apicomplexan parasite Plasmodium, is a major cause of morbidity and mortality throughout the world. This study has focused on the role of the spleen in the control of the blood stage of infection. Three aspects have been examined specifically: the effect of infection on the architecture of the spleen, the role of the spleen in parasite clearance and the formation of B cell memory. Firstly, the effect of infection on the splenic microarchitecture was examined. An essential component of the splenic architecture is the marginal zone (MZ), an area of the spleen that separates the reticuloendothelial red pulp of the spleen from the lymphoid white pulp compartment. Two unique populations of macrophages are found in the marginal zone: marginal zone macrophages (MZM) and marginal metallophilic macrophages (MMM). In the current study, parasitised red blood cells (pRBC) as well as normal RBC located to the MZ thirty minutes after intravenous injection and formed close associations with both MMM and MZM. Eight days after infection, at the time of peak parasitemia, a complete loss of both MMM and MZM was observed. Assays to detect cell death revealed that the loss of both MMM and MZM appeared to occur as a result of apoptosis. The apoptosis was not induced by up regulation of the inflammatory cytokines tumour necrosis factor or interferon-γ and could not be blocked by over expression of the apoptosis inhibitor Bcl2. Significantly, MMM were retained in the absence of CD8+ T cells implicating CD8+ T cells in the loss of MMM. Finally, infection of CD95-/- mice demonstrated that CD95/CD95-ligand (Fas/Fas-ligand) interactions were responsible for some of the CD8+ T cell-mediated loss of MMM. These data provide evidence for a novel interaction between MMM and CD8+ T cellsfollowing infection with Plasmodium. Secondly, the role of the spleen in the control of parasitemia and disease was monitored with an emphasis on determining the role of splenic macrophage populations (MMM, MZM and red pulp macrophages [RPM]) in parasite clearance. A clodronate liposome-mediated macrophage depletion technique was used, and caused a complete loss of all three macrophage sub-populations, as well as 50% of splenic dendritic cells, within 24 hours of administration. Each of the macrophage populations, as well as splenic DC, demonstrated different repopulation kinetics following their depletion from the spleen and these kinetics were utilised to examine each cell population in isolation. RPM depleted mice had significantly higher peak parasitemias than the controls. This peak returned to the level observed in undepleted control animals only after the repopulation of RPM was complete, suggesting that RPM play a role in the control of peak parasitemia following infection. Neither MMM nor MZM played a role in the control of parasitemia. The role of non-splenic macrophages and splenic dendritic cells also was investigated and shown to be insignificant in the absence of splenic macrophages. Finally, the role of RPM in mice immune to infection was investigated and their role shown to be dispensable, with immune mice clearing parasitemia efficiently in the absence of RPM. RPM therefore are important for the innate control of infection with P. chabaudi but are dispensible once adaptive immunity is established. Finally, the role of the spleen in the development of parasite-specific B cell memory was examined. Initial studies demonstrated that germinal centre (GC) development was compromised following infection with P. chabaudi, with an involution of B cell follicles noted early in infection. Adoptive transfer of memory B cells from immunised to naïve mice demonstrated that some protection was conferred on recipient mice by parasite-specific memory B cells. But, the memory B cells could not protect the host from developing parasitemia and did not produce significant amounts of parasite-specific immunoglobulin within seven days of challenge infection. Memory B cells could not be detected ten weeks after infection, indicating that the development, or survival, of parasite-specific memory B cells was compromised. The development of bystander memory B cells was not affected by infection. Finally, long-lived plasma cells were shown to develop in response to infection, although re-exposure of the cells to parasites in the form of recrudescent parasitemia resulted in their loss. This study therefore has identified a defect in the development of long-term, B cell-mediated, protection against infection with P. chabaudi. Each of these factors has significant implications for the understanding of how the spleen contributes to the control of infection with Plasmodium and potential applications for the further development of malaria vaccines and treatment regimens.

Plasmazellen gehören zu den Effektorzellen des adaptiven Immunsystems. Langlebige Plasmazellen tragen durch kontinuierliche Sekretion protektiver Antikörper wesentlich zum humoralen Gedächtnis bei und überleben hauptsächlich in spezialisierten Nischen des Knochenmarks. Bislang ist jedoch kein Marker bekannt, mit dessen Hilfe langlebige Plasmazellen eindeutig identifiziert werden können. Die vorliegende Arbeit befasst sich mit der molekularbiologischen, phänotypischen und funktionellen Charakterisierung von reifen Plasmazellen im gesunden humanen Knochenmark, die sich durch die differentielle Expression von CD19 unterscheiden. Dabei konnte festgestellt werden, dass CD19negative Plasmazellen durch eine vergleichsweise geringere Expression von CD45 und HLADR einen höheren Reifegrad aufweisen als CD19positive Plasmazellen. Zudem lässt die vermehrte Expression von CD28, Mcl1, Bcl2 sowie die schwächere Expression u.a. von CD95 darauf schließen, dass CD19negative Plasmazellen im Knochenmark eine bessere Überlebenskapazität besitzen als CD19positive Plasmazellen. Da beide Plasmazellpopulationen ähnliche Antigen-Spezifitäten aufweisen, Plasmazellen im Knochenmark von Säuglingen ausschließlich CD19 exprimieren und nach sekundärer Vakzinierung im Blut detektierbare Plasmablasten und Plasmazellen ebenfalls CD19 auf ihrer Oberfläche exprimieren, weist die Gesamtheit der Daten darauf hin, dass sich CD19negative Plasmazellen im Kindesalter in situ aus reifen CD19positiven Plasmazellen im Knochenmark entwickeln. Die CD19negative Plasmazellpopulation leistet durch hohe Halbwertszeit und Stabilität einen konstanten Beitrag zur Aufrechterhaltung des humoralen Gedächtnisses. Die CD19positive Plasmazellpopulation stellt hingegen eine flexible Komponente dar, die eine Anpassung der humoralen Immunität und des humoralen Gedächtnisses an aktuelle Herausforderungen auch im Erwachsenenalter ermöglicht. / Plasma cells are effector cells of the adaptive immune system. Humoral memory is sustained by long-lived plasma cells that continuously secrete protective antibodies and mostly reside in specialized niches in the bone marrow. So far, no marker is known that could distinguish long-lived plasma cells from short-lived ones. The present work addresses the biomolecular, phenotypical and functional characterization of mature plasma cells in healthy human bone marrow that differ in their expression of the surface marker CD19. CD19negative plasma cells showed higher maturity than CD19positive plasma cells as they expressed lesser amounts of CD45 and HLADR. Moreover, higher expression of CD28, Mcl1 and Bcl2 and lesser expression of CD95 argues for a better survival capacity of CD19negative plasma cells. Both plasma cell populations showed similar antigen specificities. All plasmablasts and plasma cells detectable in blood after secondary vaccination expressed CD19, as well as all plasma cells isolated from infant bone marrow. These results indicate that CD19negative plasma cells mainly develop during childhood by further differentiation of mature CD19positive plasma cells in situ in the bone marrow. CD19negative plasma cells represent a long-lived and stable component of the adaptive immune system and humoral memory, whereas the CD19positive plasma cell population displays a flexible element allowing for adaption of humoral immunity to new challenges throughout a lifetime.

Das humorale Immungedächtnis wird von reifen Plasmazellen des Knochenmarks vermittelt, welche bei Immunreaktionen aus aktivierten B-Lymphozyten gebildet werden. Dabei sind im Blut Plasmablasten als unmittelbare Vorläufer der Plasmazellen nachweisbar, die von dort aus in das Knochenmark einwandern. Anhand der durchflusszytometrischen Detektion spezifischer Plasmablasten gelang es hier, das simultane Auftauchen von Wellen neu generierter, migratorischer Plasmablasten und reifer, nicht-migratorischer Plasmazellen im Blut eine Woche nach einer Tetanusimpfung nachzuweisen. Plasmablasten und Plasmazellen lagen stets im Gleichgewicht vor, wodurch auf die stöchiometrische Mobilisierung reifer Plasmazellen des Knochenmarks durch systemisch induzierte Plasmablasten geschlossen wurde. Ein solcher Verdrängungsmechanismus wird hier erstmalig als Anpassungsmechanismus des humoralen Immungedächtnisses dargestellt, der die Aufnahme neuer Spezifitäten in das Gedächtnis unter Wahrung der Stabilität präexistierender Spezifitäten erlaubt. Anders als systemisch induzierte Plasmablasten, weisen Plasmablasten, die im immunologischen Ruhephase zirkulieren, Kennzeichen mukosaler Immunreaktionen auf: sie exprimieren IgA sowie die mukosalen Zellmigrationsrezeptoren alpha4beta7-Integrin und CCR10. Wahrscheinlich wandern sie in mukosale Plasmazelldepots ein und interferieren nicht mit den Plasmazellen des Knochenmarks, sodass die Stabilität des humoralen Gedächtnisses in der Ruhephase gewahrt bleibt. Eine Anpassung des humoralen Gedächtnisses findet somit nur im Rahmen systemischer Immunreaktionen statt. Bei splenektomierten Patienten und unter der B-Zell-Depletionstherapie bei Rheumapatienten bleiben mukosale Plasmablasten im Blut nachweisbar. Dies belegt deren autonome Bildung aus mukosalen, therapie-refraktären B-Zellen. Insgesamt wird hier eine bisher unbeachtete Komplexität menschlicher peripherer Plasmablasten und Plasmazellen und ihren Beziehungen zum humoralen Immungedächtnis dargestellt. / Humoral memory, i.e. persistence of specific antibody titers, is provided by plasma cells in the bone marrow, which are generated from activated B cells during immune responses. At this, immediate plasma cell precursors, the plasmablasts, migrate via the blood to the bone marrow. Using cytometric detection of antigen-specific plasmablasts, synchronous circulation of waves of recently generated, migratory plasmablasts and non migratory plasma cells with a mature phenotype was demonstrated one week after tetanus vaccination. Circulating plasmablast and plasma cell numbers were always in homeostasis, so that the stoichiometric mobilization of old bone marrow plasma cells by recently generated plasmablasts was hypothesized. This plasma cell replacement mechanism is herein described for the first time as an adaption mechanism of the humoral memory that allows incorporation of new antibody specificities while maintaining pre-existing ones. In immunological steady state, very low numbers of plasmablasts are detectable in any donor. These express IgA and receptors for mucosal homing, alpha4beta7 integrin and CCR10, and therefore most likely migrate into mucosal plasma cell depots and do not interfere with plasma cells of the bone marrow, preserving the stability of humoral memory during steady state. Hence, adaption of humoral memory is only possible during systemic immune reactions. Circulating mucosal plasmablasts produced during steady state remain detectable in patients with rheumatoid arthritis during B cell depletion therapy as well as in asplenic patients. Hence, this type of plasmablasts is self-sufficiently generated from mucosal B cells that are refractory to B cell depletion therapy. This work demonstrates a hitherto disregarded complexity of peripheral plasmablast and plasma cell subsets in healthy humans, with implications for the regulation of induction and maintenance of humoral memory.