IL-2 has multiple functions in T cell-mediated adaptive immunity. The stringent control of its expression is important for T cell activation, proliferation and the subsequent T cell clone contraction. Our lab has recently shown that the transcriptional repressor Blimp-1 is part of a negative feedback loop which controls IL-2 gene expression in mice. Understanding the molecular mechanisms of this signaling loop in T cells might help us to better understand the regulation as well as the role of IL-2 in T cell immunity. The human ortholog to murine Blimp-1 is termed PRDI-BF1 (each encoded by the respective Prdm1 gene). Both genes contain five zinc finger regions, whereby the first two zinc fingers are dispensable for DNA binding. In case of the human protein they are instead required to recruit the G9?Ñ methyltransferase to the gene promotor. We found that the human wild-type PRDI-BF1 protein suppressed IL-2 production in murine T cells, while deletion of the first two zinc fingers abolished this ability. Thus, a similar Blimp-1-mediated methylation mechanism might exist in IL-2 gene silencing. IL-2/IL-2R signaling is indispensable for Blimp-1 induction. PI-3Kinase and Stat5 are downstream of the IL-2 receptor complex and are known to contribute to IL-2 inhibition in T cells from C57BL/6 mice. However, activating only these two pathways are still not sufficient to induce Blimp-1 or suppress IL-2 expression in in IL-2R beta-/- mice. The Blimp-1-dependent IL-2 self regulatory loop is not functional in IL-2R beta-/-mice. In order to conveniently study this dysregulation we crossed these mice with a GFP transgenic strain in which the GFP transgene is under the control of IL-2 promoter sequence. In IL-2R beta-/-IL-2p-GFP mice about five times as many spleenic CD4+ T cells transcribe IL-2pGFP, compared to the littermate IL-2R beta+/-IL-2p-GFP control animals. And most of the GFP cells demonstrate activated phenotype (CD44HighCD62Llow). Blimp-1 is known as a master regulator of B cell terminal differentiation. Since a recent report indicated that IL-2 signaling via STAT5 constrains Th17 Cell differentiation, we speculated that Blimp-1 might play a similar role in effector T cell differentiation. In order to evaluate this possibility, activated CD4+ T cells from C57BL/6 mice were transduced with Blimp-1 and cultured under Th17 polarizing conditions. Blimp-1 overexpression in did not change the profile of IL-17 production.

In order to eradicate HIV-1 infection the virus needs to be specifically eliminated from latently infected memory CD4+ T cells. There does not seem to be a single mechanism that promotes HIV-1 latency. RNA Polymerase II (RNAP II) pausing, chromatin structure, tissue specific transcriptional repressors and transcriptional interference have been implicated in regulating HIV-1 transcription. The transcription factor B Lymphocyte-Induced Maturation Protein 1 (Blimp-1) is expressed in B and T cells and upregulated in patients chronically infected with HIV-1. I hypothesized that Blimp-1 is a T cell intrinsic factor that binds to HIV-1 LTR, inhibits HIV-1 transcription and contributes to HIV-1 latency. Blimp-1 is expressed in primary peripheral blood CD4+ T cells and is further induced by T cell activation. Importantly, Blimp-1 is highly expressed in memory CD4+ T cells compared to naïve CD4+ T cells. Ectopic expression of Blimp-1 in CD4+ T cells represses HIV-1 transcription, whereas decreasing Blimp-1 in memory CD4+ populations activates HIV-1 transcription. Reduction of Blimp-1 in infected primary T cells increases RNAP II processivity and histone H3 acetylation. Blimp-1 binds downstream of the HIV-1 5'-LTR to the interferon-stimulated response element (ISRE) in resting primary CD4+ T cells and strongly represses Tat-dependent HIV-1 transcription. Upon T cell activation, Blimp-1 is released from the HIV-1 ISRE and this correlates with significant increase in HIV-1 transcription. These results demonstrate that Blimp-1 acts to limit HIV-1 transcription in memory CD4+ T cells and promotes the establishment and maintenance of latency. I also examined whether neighboring host promoters could impact HIV-1 transcription. Using a set of inducible cell lines I observed that neighboring promoters have minimal impact on HIV-1 transcription and that enabling release of paused RNAP II by diminishing negative elongation factor (NELF) is sufficient to reactivate transcriptionally repressed HIV-1 provirus. The implications of my results in the different mechanisms regulating HIV-1 latency are discussed.

The studies presented within this dissertation provide the first description of PRDM1 (also known as Blimp-1 or PRDI-BF1) function in natural killer cells. NK cells are major effectors of the innate immune response via antigen-independent cytotoxicity and link to the adaptive immune response through cytokine release. Molecular mechanisms mediating NK activation are relatively well-studied; however, much less is known about the mechanisms that restrain activation. In the first study, the transcriptional repressor PRDM1 is shown to be a critical negative regulator of NK function. Microarray analysis was used to characterize transcriptional changes associated with cytokine-mediated activation. PRDM1 is expressed at low levels in resting NK cells and three distinct PRDM1 isoforms are selectively induced in the CD56dim NK population in response to activation. PRDM1 coordinately suppresses the production of IFNγ, TNFα and TNFβ through direct binding to multiple conserved regulatory regions. Ablation of PRDM1 expression leads to enhanced production of IFNγ and TNFα but does not alter cytotoxicity, whereas over-expression blocks cytokine production. PRDM1 response elements are defined at the IFNG and TNF loci. To further delineate the targets of PRDM1-mediated regulation in NK cells, global approaches were utilized. Experiments utilizing chromatin immunoprecipitation coupled to promoter tiling arrays identified 292 novel direct targets of PRDM1 binding. These studies revealed widespread binding of PRDM1 to the genome, which was not limited to proximal promoter regions. Furthermore, microarray analysis of stimulated NK cells combined with PRDM1 knockdown has enabled the identification of genes responsive to PRDM1 knockdown using primary cells. Collectively, these experiments identify both direct and indirect targets of PRDM1 regulation and help define a PRDM1-centered gene regulatory network in NK cells. Data presented in the final chapter pertains to an independent project aimed at identifying small molecule inhibitors of the methyltransferase G9a, which is recruited by PRDM1 and is required for silencing of target genes. A mass spectrometry-based assay was developed and used to screen a small molecule library. Several hits were identified and combinatorial chemistry yielded several compounds with < 20µM IC50 values. In cell-based assays, however, treatment with the small molecules had limited efficacy, indicating additional chemical modifications are necessary to yield bioactive compounds. The data presented here demonstrate a key role for PRDM1 in the negative regulation of NK activation and position PRDM1 as a common regulator of the adaptive and innate immune response.