B cells are capable of producing antibodies of diverse antigen specificities and effector functions to counter infection by a wide range of pathogens. The diversification of immunoglobulin (Ig) is achieved through a series of programmed DNA recombination and mutagenic events during B cell maturation. A key factor involved in the Ig diversification process is Activation Induced Cytidine Deaminase (AID). AID is a B cell specific enzyme that is critical for three distinct pathways of Ig diversification: class switch recombination, somatic hypermutation and Ig gene conversion. AID functions by deaminating cytosine to uracil in target DNA at the Ig loci. Although essential for effective immunity, the mutagenic activity of AID needs to be confined to the Ig loci in order to protect genomic integrity, but the underlying mechanism is not fully understood. In this study, I show that two lymphoid specific transcription factors, PU.1 and IRF4, play important roles in regulating AID function in chicken B cells. PU.1 and IRF4 have been implicated in many aspects of B cell development and function. The two factors could form a heterodimer and regulate target gene expression cooperatively. However, we found that PU.1 and IRF4 appear to have different impacts on AID function. We show that PU.1 is important for the expression of AID gene in chicken B cells, and the regulation appears to involve direct interaction of PU.1 with the AID gene. By comparison, IRF4 plays a minor role in AID expression. On the other hand, both PU.1 and IRF4 are required for efficient gene conversion that is mediated by AID at the Igλ locus. Moreover, the gene dosage of PU.1 is critical for AID function, since a severe gene conversion defect is observed in PU.1+/- cells. The function of PU.1 and IRF4 in AID-mediated gene conversion involves binding sites for the PU.1/IRF4 complex within a regulatory element at the Igλ locus. Future studies will be directed at understanding how PU.1 and IRF4 regulate AID-mediated gene conversion. / text

La maladie de Whipple (MW) est une maladie infectieuse rare, sévère et chronique qui ne touche qu’une minorité d’individus infectés par Tropheryma whipplei (T. whipplei). Le portage chronique et asymptomatique de T. whipplei est moins rare. La pathogénie de la MW reste largement inconnue. Par une approche génétique combinant une analyse de liaison du génome entier et le séquençage de l’ensemble des exons, nous avons testé l’hypothèse d’une prédisposition génétique à la MW. Nous avons étudié une famille multiplexe comprenant quatre patients atteints de la MW mais n’ayant pas d’autres pathologies associées et cinq porteurs asymptomatiques de T. whipplei, pour laquelle nous avons envisagé un modèle autosomique dominant (AD) avec une pénétrance incomplète liée à l’âge. Nous avons montré que la mutation c.292 C>T (p.R98W) dans le gène IRF4 était le seul variant hétérozygote très rare et non-synonyme commun aux quatre patients. Chez la souris, le gène Irf4 est un facteur de transcription qui joue un rôle pléiotrope dans l’immunité. La caractérisation moléculaire de l’allèle muté a montré un effet délétère par un mécanisme d’haplo-insuffisance pour la fonction de facteur de transcription de la protéine. Une augmentation de la localisation cytoplasmique de la protéine a également été observée. De plus, le défaut IRF4 étudié confère une réponse transcriptomique distincte dans les leucocytes stimulés par le BCG ou T. whipplei. En conclusion, nous avons identifié la première étiologie génétique associée à la MW. Le mode d’hérédité est AD avec une pénétrance incomplète, le portage chronique précédant probablement la MW de plusieurs décennies chez les individus hétérozygotes infectés par T. whipplei. Ces travaux permettront de mieux comprendre la pathogénie de la MW, de mieux définir les mécanismes de l’immunité contre T. whipplei et de pouvoir offrir un diagnostic moléculaire et génétique adapté aux familles. / Whipple's disease (WD) is a rare, severe and chronic infectious disease that affects only a small minority of individuals infected with Tropheryma whipplei (T. whipplei). The chronic and asymptomatic carriage of T. whipplei is less rare. The pathogenesis of WD remains largely unknown. Using a genetic approach combining genome-wide linkage and whole exon sequencing, we tested the hypothesis of a genetic predisposition to WD. We studied a multiplex family containing four otherwise healthy WD patients and five asymptomatic T. whipplei carriers. We tested the hypothesis that WD follows autosomal dominant (AD) inheritance with age-dependent incomplete penetrance. We showed that the c.292 C> T mutation (p.R98W) of IRF4 gene was the only heterozygote variant that was very rare and non-synonymous for all four patients. In mice, the Irf4 gene is a transcription factor that plays pleiotropic roles in immunity. Molecular characterization of the mutated allele showed a deleterious effect by a haplo-insufficiency mechanism for the transcription factor function of the protein. Increase localization of the protein in the cytoplasmic has also been observed. In addition, the defect IRF4 studied confers a distinct transcriptomic response in leukocytes stimulated by BCG or T. whipplei. In conclusion, we identified the first genetic etiology associated with WD. The mode of inheritance is AD with incomplete penetrance, chronic carriage probably preceding WD for several decades in heterozygous individuals infected with T. whipplei. This work will help to better understand the pathogenesis of WD, to better define the mechanisms of immunity against T. whipplei and to be able to offer a molecular and genetic adapted diagnosis to families.

Thesis (Ph.D.)--Indiana University, 2009. / Title from screen (viewed on August 26, 2009). Department of Microbiology and Immunology, Indiana University-Purdue University Indianapolis (IUPUI). Advisor(s): Mark Kaplan. Includes vita. Includes bibliographical references (leaves 107-125).

Indiana University-Purdue University Indianapolis (IUPUI) / Adaptive and innate immune responses play a critical role in the protection against extracellular or intracellular pathogens. The function of these two types of immune responses is coordinated by CD4+ T-helper (Th) cells. Depending on the cytokine environment, Th progenitor (Thp) cells differentiate into three functionally different effector subsets. T-helper-1 (Th1) cells which mediate cell-mediated immunity, T-helper-2 (Th2) which orchestrates humoral immunity and T-helper-17 (Th17) cells key players in autoimmunity response. Cytokine induced transcription factors that are differentially expressed in Th cells are required for the development and commitment to a specific Th lineage. The population of Th2 cells can be subdivided in subpopulations depending on the level of a cytokine and the subsets of cytokines they produce. Very limited information is available about the regulation of cytokine production in this array of Th2 cells. We have recently identified the ETS family transcription factor PU.1 as regulating heterogeneity in Th2 populations. To define additional factors that might contribute to Th2 heterogeneity, we examined the PU.1 interacting protein IFN-regulatory factor (IRF)-4, a transcription factor expressed in lymphocytes and macrophages. When Th2 cells are separated based on levels of IL-10 secretion, IRF4 expression segregates into the subset of Th2 cells expressing high levels of IL-10. To investigate the role of IRF4 in cytokine heterogeneity, Th2 cells were infected with retrovirus expressing IRF4. The cells overexpressing IRF4 secreted significantly higher levels of IL-10 and IL-4 compared to cells infected with a control vector at the same time the level of IL-9 decreases. To understand the mechanism by which IRF4 regulates IL-10 expression in various Th2 cell subpopulations we used co-immunoprecipitation assays to determine transcription factors that interact with IRF4. Our data shows that PU.1, IRF4 and NFATc2 form a complex in Th2 nuclear extract. We also demonstrated by ChIP assay that IRF4 directly binds the Il10 and Il4 loci in a time dependent manner. The role of these protein-protein and protein-DNA complexes and their contribution towards Th2 heterogeneity will be further defined. Understanding the regulation of the anti-inflammatory cytokine IL-10 in Th2 cells may give us a tool to control inflammation.

Anaplastisch-großzellige Lymphome (ALCL) repräsentieren eine Untergruppe der peripheren T-Zell-Lymphome und können über das Vorhandensein einer Translokation, die das ALK-Gen betrifft, in ALK-positive und ALK-negative ALCL unterteilt werden. Präliminäre Daten implizieren eine starke Expression des Transkriptionsfaktors IRF4 bei ALCL-Patientenproben, unabhängig des ALCL-Subtyps. Allerdings war die Rolle von IRF4 bei der Pathogenese des ALCL bislang wenig verstanden. In unserer Studie konnten wir zeigen, dass eine shRNA-vermittelte Herunterregulation von IRF4 Zytotoxizität im ALCL-Zelllinienmodell induziert und in vivo das Tumorwachstum signifikant verlangsamt. Genexpressionsanalysen zeigten die globale Rolle von IRF4 im Genexpressionsnetzwerk des ALCL über die Deregulation essentieller zellulärer Prozesse, wie der Zellzykluskontrolle und der DNA-Reparatur, sowie bekannter onkogener Signalwege wie Notch oder MYC. Wir konnten zeigen, dass IRF4 direkt an der Expression des bekannten onkogenen Transkriptionsfaktors MYC beteiligt ist und bestätigten zusätzlich die Abhängigkeit der ALCL-Zelllinien von dessen Expression. Die pharmakologische Inhibierung von MYC führte zum Rückgang der Zellviabilität aller ALCL-Zelllinien. Zusammenfassend zeigen diese Resultate, dass die Expression von IRF4 essentiell für die Proliferation von ALCL-Zelllinien ist und dass die von IRF4 regulierte Expression von MYC ein vielversprechendes therapeutisches Ziel für die zukünftige Behandlung sowohl ALK-positiver als auch ALK-negativer ALCL-Patienten darstellt. / Anaplastic large cell lymphoma (ALCL) is a distinct entity of peripheral T-cell lymphomas. ALCLs can be divided into two subtypes with respect to the presence of translocations involving the ALK gene (ALK-positive and ALK-negative). It has been shown that the interferon regulatory factor 4 (IRF4) is highly expressed in both ALCL subtypes but its role in the pathogenesis of these lymphomas remained unclear. Our study reveals an addiction to the expression of IRF4 in cell lines of both ALCL subtypes. Furthermore, we were able to transfer this into an ALCL xenograft mouse model showing the significant impact of IRF4 deregulation on ALCL tumor growth in vivo. Gene expression profiling after IRF4 knockdown highlighted the function of IRF4 in anaplastic large cell lymphoma by significant downregulation of genes involved in essential cellular processes like cell cycle control and DNA repair as well as known targets of the oncogenic transcription factor MYC. We were able to identify MYC as a direct target of IRF4 in both ALCL subtypes and further studies revealed an addiction of ALCLs to MYC expression as well. Pharmacological inhibition of MYC was toxic to all tested ALCL cell lines, indicating that IRF4 and MYC signaling may represent promising targets for future therapies of patients diagnosed with anaplastic large cell lymphoma.

No description available.

TCR signal strength is critical for CD8+ T cell clonal expansion after antigen stimulation. Levels of the transcription factor IRF4 control the magnitude of this process through induction of genes involved in proliferation and glycolytic metabolism. The signaling mechanism connecting graded TCR signaling to the generation of varying amounts of IRF4 is not well understood. Here, using multiple methods to vary TCR signal strength and measure changes in transcriptional activation in single CD8+ T cells, we connect antigen potency to the kinetics of NFAT activation and Irf4 mRNA expression. T cells that transduce weaker TCR signals exhibit a marked delay in Irf4 mRNA induction resulting in decreased overall IRF4 expression in individual cells and increased heterogeneity within the clonal population. The activity of the tyrosine kinase ITK acts as a signaling catalyst that accelerates the rate of the cellular response to TCR stimulation, controlling the time to onset of Irf4 gene transcription. These findings provide insight into the signal transduction pathway accounting for the reduced clonal expansion of low affinity CD8+ T cells following infection. We also describe another context for ITK activity, autoreactive T cell migration. Here, we connect TCR signaling strength to modulation of selectin binding and autoreactive T cell-mediated pathology in an adoptive transfer model system of autoimmune disease. Understanding the signaling mechanisms linking changes in TCR signaling to CD8 T cell function is important in furthering the understanding of vaccine development and T cell adoptive immunotherapy.

Transcriptional regulation of CD8+ T cell differentiation during acute and chronic viral infections is an intricate web made up of many of transcription factors. While several transcription factors have been elucidated in this process, there are still many more that remain elusive. In this work, we look into the role of two transcription factors, IRF4 and Runx2, and their role in CD8+ T cell terminal effector cells and memory precursor cells during acute LCMV-Armstrong infection. We found that IRF4 expression was regulated by TCR signal strength during infection, and that IRF4 expression levels directly correlated with the magnitude of the effector cell response. IRF4 was also shown to regulate T-bet and Eomes, two transcription factors critical for CD8+ T cell differentiation into effector and memory cells. From these results, we were interested in the potential role of IRF4 during chronic LCMV-clone 13 infection, where ratios of T-bet and Eomes are critical for viral clearance. We found that haplodeficiency of IRF4 in the T cell compartment lead to an increase in the ratio of Eomes to T-bet in T cells, which in turn affected the proportion of Eomeshi versus T-bethi cells and resulted in a loss in ability to clear viral infection. Irf4+/-Eomes+/- compound heterozygous mice were generated to test if decreasing Eomes expression would rescue the Irf4+/- phenotype. Irf4+/-Eomes+/- mice were phenotypically similar to WT mice in terms of Eomes to T-bet ratios, and were able to clear viral infection, demonstrating a critical role of IRF4 in regulating T-bet and Eomes during chronic viral infection. Next we looked into the role of Runx2 during acute LCMV-Armstrong infection and found that Runx2-deficient pathogen-specific CD8+ T cells had a defect in the total number of memory precursor cells compared to WT controls. We further showed that Runx2 was inversely correlated with TCR signal strength, and that Runx2 expression was repressed by IRF4. From these work, we have introduced two more transcription factors that are critical for CD8+ T cells differentiation during acute and chronic viral infection. Given the sheer number of transcription factors known to regulate these processes, having a full understanding of the transcriptional network will allow us to find the best targets for therapeutic intervention for treatments ranging from vaccine development and autoimmunity to cancer immunotherapy and treatment of chronic viral infections.

Interferon (IFN) regulatory factors (IRFs) have crucial roles in immune regulation and oncogenesis. We have recently shown that IRF4 is activated through c-Src-mediated tyrosine phosphorylation in virus-transformed cells. However, the intracellular signaling pathway triggering Src activation of IRF4 remains unknown. In this study, we provide evidence that Epstein–Barr virus (EBV) latent membrane protein 1 (LMP1) promotes IRF4 phosphorylation and markedly stimulates IRF4 transcriptional activity, and that Src mediates LMP1 activation of IRF4. As to more precise mechanism, we show that LMP1 physically interacts with c-Src, and the phosphatidylinositol 3 kinase (PI3K) subunit P85 mediates their interaction. Depletion of P85 by P85-specific short hairpin RNAs disrupts their interaction and diminishes IRF4 phosphorylation in EBV-transformed cells. Furthermore, we show that Src is upstream of PI3K for activation of both IRF4 and Akt. In turn, inhibition of PI3K kinase activity by the PI3K-speicfic inhibitor LY294002 impairs Src activity. Our results show that LMP1 signaling is responsible for IRF4 activation, and further characterize the IRF4 regulatory network that is a promising therapeutic target for specific hematological malignancies.

The lymphocyte-specific transcription factor Interferon (IFN) Regulatory Factor 4 (IRF4) is implicated in certain types of lymphoid and myeloid malignancies. However, the molecular mechanisms underlying its interactions with these malignancies are largely unknown. In this study, we have first profiled molecular signatures associated with IRF4 expression in associated cancers, by analyzing existing gene expression profiling datasets. Our results show that IRF4 is overexpressed in melanoma, in addition to previously reported contexts including leukemia, myeloma, and lymphoma, and that IRF4 is associated with a unique gene expression pattern in each context. A pool of important genes involved in B-cell development, oncogenesis, cell cycle regulation, and cell death including BATF, LIMD1, CFLAR, PIM2, and CCND2 are common signatures associated with IRF4 in non-Hodgkin B cell lymphomas. We confirmed the correlation of IRF4 with LIMD1 and CFLAR in a panel of cell lines derived from lymphomas. Moreover, we profiled the IRF4 transcriptome in the context of EBV latent infection, and confirmed several genes including IFI27, IFI44, GBP1, and ARHGAP18, as well as CFLAR as novel targets for IRF4. These results provide valuable information for understanding the IRF4 regulatory network, and improve our knowledge of the unique roles of IRF4 in different hematological malignancies.