CD4+CD25+Foxp3+ T regulatory (Treg) cells are critical in maintaining self tolerance and promoting the acceptance of allogeneic tissue/organ grafts. To be widely applied in clinical settings, there needs to be a readily available source of Treg cells, a requirement that is better met if non-histocompatible donor cells could be utilized in adoptive therapy. Therefore, to investigate the therapeutic potential of fully allogeneic Treg cells to control autoimmune disease or allograft rejection, we utilized IL-2R beta-deficient mice that exhibit rapid lethal autoimmunity due to low production of an ineffective population of Treg cells. We show that adoptive transfer of MHC-mismatched Treg cells into IL-2R beta-/- mice resulted in life-long engraftment of the donor cells, which exhibited skewed reactivity toward host alloantigens, and prevented autoimmunity. When such animals received skin grafts, they exhibited tolerance to those grafts that expressed MHC molecules from which the donor Treg cells were derived. Collectively, these data provide proof-of-principle that effective engraftment by allogeneic Treg cells controls autoimmunity and leads to favorable conditions for long-term acceptance of allografts. Current data indicates that CD4+CD25+Foxp3+ Treg cells exhibit a broad TCR repertoire. However, the relationship between this diversity and capacity to control a similarly diverse population of potentially autoreactive T cells remains to be defined. To investigate this issue, we assessed the TCR repertoire of chimeric donor Treg cells in IL-2R beta-/- mice that were adoptively treated with a diverse polyclonal Treg inoculums. We demonstrate that autoimmune disease was fully prevented by engrafted donor Treg cells in spite of a TCR repertoire that is less diverse than the input cells. However, in settings where the input TCR repertoire is limited by utilizing donor Treg cells that express a single TCR beta chain, control of disease was hampered, correlating with a limited TCR alpha repertoire within the engrafting donor Treg cells. Collectively, these findings suggest that for adoptive therapy, a diverse TCR repertoire of input Treg cell inoculums is an essential requirement for effective control of polyclonal autoreactive T cells but perturbations in the repertoire that results in significant limitation to this diversity may compromise Treg cell efficacy at fully keeping autoaggressive cells in check.

B lymphopoiesis in aged mice is characterized by reduced B cell precursors and an altered antibody repertoire. Aged mice maintain an ordinarily minor pool of early c-kit+ pre-B cells, indicative of poor preBCR expression, even as preBCR competent early pre-B cells are significantly reduced. Therefore, in aged mice, preBCR-mediated B2 B lymphopoiesis is significantly diminished; likely as a consequence of poor surrogate light chain expression. Notably, the remnant B1 B cell lineage present in adult bone marrow is retained in aged mice as evidenced by normal numbers (~0.3%) of Lin-CD19+B220low/- B1 B cell precursors. Of interest, B1 progenitors express substantially less lambda 5 surrogate light chain protein than do B2 pro-B cells and the surrogate light chain levels are further reduced in aged mice. B cells derived from putatively preBCR-deficient precursors, either B2 c-kit+ B cell precursors or B1 B cell progenitors, from either young or aged mice, generate new B cells in vitro that are biased to larger size, higher levels of CD43/S7, and decreased kappa light chain expression. Notably, immature B cells in aged bone marrow exhibit a similar phenotype in vivo, consistent with the changes seen in B cell precursor subpopulations. In aged mice, the B2 pathway is partially blocked with limited preBCR expression and signaling; however, continued B cell development via preBCR-deficient pathways, including B1 pathways, is observed. Increased generation of new B cells by these alternative pathways may contribute to altered phenotype, repertoire, and function in senescence.

Broadly neutralizing antibodies against HIV are rare; most patients never develop them at detectable levels. The discovery of four such antibodies therefore warrants research into their origins and their presumed unique characteristics. Such studies, however, require baseline knowledge about commonalities and biases affecting human immunoglobulin development. Obtaining that knowledge requires large sets of gene sequence data and the appropriate statistical techniques and tools. The Genbank repository provides a free and easily accessible source for such data. Several large datasets cumulatively comprising over 10,000 human Ig heavy chain genes were identified, downloaded, and carefully filtered. We then developed a special software tool called SoDA, which employs a unique dynamic programming algorithm to provide a statistical reconstruction of the events that led to a given antigen receptor gene. Once developed, tested, and peer-reviewed, we used SoDA to provide initial data about each downloaded gene with respect to gene segment usage, n-nucleotide addition, CDR3 length, and mutation frequency, thereby establishing the most precise estimates currently available for human Ig heavy chain gene segment usage frequencies. We compared data from productive non-autoreactive Ig to non-productive Ig and found evidence for gene segment usage biases, D/J segment pairing preferences resulting from multiple sequential D-to-J recombination events, and biases in TdT action between the V-D and D-J. Further analysis of autoreactive Ig genes yielded evidence that n-nucleotide addition comes at a cost: the higher the ratio of n-nucleotides to germline-encoded nucleotides for a given CDR3 length, the greater the probability of autoreactivity. These results suggest that the germline gene segments have been selected for lack of autoreactivity. It has previously been shown that human Ig gene segments have evolved efficient evolvability under somatic hypermutation. We have now extended these results, showing that Ig gene sequences are "tuned" to preferentially produce consequential mutations in the antigen-binding domains, and synonymous mutations in the framework regions. Together, these analyses provide new insights into the genetic and mechanistic biases shaping the human Ig repertoire. / Dissertation

Il est connu que les lymphocytes B (LB) ont un rôle pathogène dans de nombreuses maladies auto-immunes. Afin de comprendre comment la perte de tolérance B survient dans le lupus érythémateux systémique, maladie auto-immune prototypique, nous avons tout d’abord développé, dans un modèle murin, une méthode de marquage des LB pathogènes en cytométrie en flux. D’autre part, nous avons trouvé qu’un défaut de réponse au TLR9 est une caractéristique unique des LB de patients lupiques et est associée à une diminution d’expression du complexe CD19/CD21 à la surface des LB. Ces anomalies pourraient favoriser la survie des LB autoréactifs, et permettre la survenue de troubles auto-immuns. Dans un second temps nous avons exploré les fonctions d’une population lymphocytaire B méconnue: les LB thymiques humains. Nos résultats appuient le rôle tolérogène de ces LB, avec notamment l’expression, par 5% d’entre eux, de la protéine AIRE (AutoImmune REgulator), qui joue un rôle majeur dans la sélection négative des lymphocytes T. L’ensemble de ces données montrent la dichotomie fonctionnelle des LB et nous rappelle la complexité des processus régissant la tolérance et son maintien. / It is well known that B cells play pathogenic roles in a variety of autoimmune diseases. In order to understand how B tolerance breakdown occurs during systemic lupus erythematosus (SLE), a prototypical autoimmune disease, we first developed a method to identify autoreactive B cells by flow cytometry in a mouse model. In addition we found that a defective in vitro response to TLR9 agonists is a specific feature of SLE B cells and is associated with a CD19/CD21 complex downregulation. These abnormal functions in SLE B cells may contribute to B cell tolerance breakdown in these patients. But B-cell involvement in autoimmune diseases should not lead us to forget the functional diversity of B cells. This prompted us, in a second project, to analyze the tolerogenic role of a B cell population considered as simple bystanders of thymopoiesis: the human thymic B cells. Our results support the tolerogenic role of thymic B cells, as 5% of them express AIRE (AutoImmune REgulator), which plays a major role in cells in T cells negative selection. All these data underline the functional dichotomy of B cells and remind us the complexity of the processes governing tolerance and its maintenance.

Obwohl B-Zellen in der Pathogenese der Rheumatoide Arthritis (RA) eine wichtige Rolle spielen, ist über ihre Aktivierung und Differenzierung im Synovialgewebe (SG) nicht viel bekannt. Ein Merkmal von RA ist das Auftreten von Autoantikörpern (auto-AK). Trotz dessen sind bisher kaum Daten über den Einfluss des SG auf die auto-AK-Produktion und die Frequenz autoreaktiver synovialer B-Zellen bekannt. Diese Arbeit beschäftigt sich mit der Charakterisierung der synovialen B-Zell-Antwort und der Spezifität synovialer B-Lymphozyten. Dazu wurden B- und Plasmazellen (PC) aus dem Synovialgewebe von RA-Patienten mittels Mikrodissektion und Durchflusszytometrie isoliert und ihr Immunglobulin(Ig)-Repertoire bestimmt. Die Analyse der VH-Gene zeigte, dass sowohl naive als auch Gedächtnis-B-Zellen in das SG einwandern können. Ein Vergleich der VDJ-Rearrangements aus B-Zellen und PC belegte, dass hauptsächlich Gedächtnis-B-Zellen Antigen-abhängig aktiviert werden, klonal expandieren und zu PC differenzieren. Dabei können aktivierte B-Zellen ihre Ig-Klasse wechseln. Im Gegensatz dazu wurden nur rudimentäre Anzeichen somatischer Hypermutation nachgewiesen. Um die Spezifität synovialer B-Lymphozyten zu ermitteln, wurden rekombinante AK aus synovialen B-Zellen und PC generiert. Der Polyreaktivitätstest zeigte, dass naive B-Zellen aus dem SG einen hohen Anteil polyreaktiver Zellen besitzen. Im Gegensatz dazu ist die Frequenz von autoreaktiven Gedächtnis-B-Zellen und PC gegenüber naiven B-Zellen erhöht. Daneben konnten auch spezifische AK gegen bakterielle Antigene (insbesondere gegen Parodontitis-auslösende Bakterien) und gegen das Auto-Ag MCV identifiziert werden. Eine Affinitätsmessung des MCV-spezifischen Auto-AK zeigte, dass im SG sezernierte Auto-AK eine sehr hohe Affinität erreichen können. Die hier gewonnenen Daten verdeutlichen, dass B-Lymphozyten entscheidend an der Aufrechterhaltung oder gar Entstehung von RA beteiligt sind / Although B cells have an important impact on the pathogenesis of Rheumatoid arthritis (RA) still surprisingly little is known about their activation and differentiation within the inflamed synovial tissue (ST). A hallmark of RA is the presence of auto-antibodies (auto-Ab). However, still little is known about the frequency of self reactive synovial lymphocytes and it is unclear to which extent the inflamed ST contributes to auto-Ab production. These thesis deals with the characterization of the synovial B cell response and the specificity of synovial B lymphocytes. B and plasma cells (PC) from RA patients were isolated either by Laser Capture Microdissection or by FACS and their immunoglobulin(Ig)-repertoire was determined. The analysis of the VH-genes revealed that both naïve and memory B cells can immigrate the ST. A comparison of VDJ-rearrangements of B cells and PC showed that in ST without ectopic germinal centres mainly memory B cells become activated, expand clonally and differentiate into PC. During this process B cells can switch their Ig-class but do only hypermutate slightly. To determine the specificity of synovial B lymphocytes, recombinant Ab from synovial B cells and PC were generated. The polyreactivity assay showed that particularly naïve B cells were polyreactive. In contrast, the frequency of autoreactive memory B cells and PC was much higher than that of naïve B cells. In addition, Ab specific for bacterial antigens (especially for periodontal bacterias) and for the autoantigen MCV were identified. The affinity measurement of the MCV-specific autoantibody revealed that auto-Ab secreted in the ST can exhibit very high affinities. The data presented here show that B cells seem to play an important role in the maintenance and possibly the development of RA.