The Paf oncogene is essential for hematopoietic stem cell function and development.

Hematopoietic stem cells (HSCs) self-renew to maintain the lifelong production of all blood populations. Here, we show that the proliferating cell nuclear antigen-associated factor (Paf) is highly expressed in cycling bone marrow HSCs and plays a critical role in hematopoiesis. Mice lacking Paf exhibited reduced bone marrow cellularity; reduced numbers of HSCs and committed progenitors; and leukopenia. These phenotypes are caused by a cell-intrinsic blockage in the development of long-term (LT)-HSCs into multipotent progenitors and preferential loss of lymphoid progenitors caused by markedly increased p53-mediated apoptosis. In addition, LT-HSCs from Paf(-/-) mice had increased levels of reactive oxygen species (ROS), failed to maintain quiescence, and were unable to support LT hematopoiesis. The loss of lymphoid progenitors was likely due the increased levels of ROS in LT-HSCs caused by treatment of Paf(-/-) mice with the anti-oxidant N-acetylcysteine restored lymphoid progenitor numbers to that of Paf(+/+) mice. Collectively, our studies identify Paf as a novel and essential regulator of early hematopoiesis.

Requirements for eomesodermin and promyelocytic leukemia zinc finger in the development of innate-like CD8+ T cells.

Conventional and nonconventional T cell development occur in the thymus. Nonconventional thymocytes that bear characteristics typically associated with innate immune cells are termed innate-like lymphocytes (ILLs). Mice harboring a tyrosine to phenylalanine mutation in the adaptor protein Src homology 2 domain-containing leukocyte protein of 76 kDa at residue 145 (Y145F mice) develop an expanded population of CD8(+)CD122(+)CD44(+) ILLs, typified by expression of the T-box transcription factor eomesodermin. Y145F mice also have an expanded population of γδ T cells that produce copious amounts of IL-4 via a mechanism that is dependent on the BTB-ZF transcription factor promyelocytic leukemia zinc finger. Using mice with T cell-specific deletion of Eomes, we demonstrate that this transcription factor is required for CD8(+) ILL development in Y145F as well as wild-type mice. Moreover, we show that promyelocytic leukemia zinc finger and IL-4 are also required for the generation of this ILL population. Taken together, these data shed light on the cell-intrinsic and cell-extrinsic factors that drive CD8(+) ILL differentiation.

Development of PLZF-expressing innate T cells.

Recent studies have shown that the transcriptional regulator promyelocytic leukemia zinc finger (PLZF) controls the development of essentially all of the innate-like features of invariant Natural Killer T (NKT) cells. For example, PLZF-deficient NKT cells do not acquire an 'activated' phenotype nor do they acquire the capacity to secrete multiple cytokines upon primary stimulation. The function of a subset of γδ T cells has now also been shown to be dependent upon expression of PLZF. Furthermore, IL-4 produced by PLZF-expressing cells causes some CD8 T cells to acquire innate-like features. Therefore, it is becoming clear that PLZF has a broad impact on the immune response. Here we discuss the current understanding of how expression of PLZF, the innate T cell determinant, is initiated during T cell development.

Selective depletion of eosinophils or neutrophils in mice impacts the efficiency of apoptotic cell clearance in the thymus.

Developing thymocytes undergo a rigorous selection process to ensure that the mature T cell population expresses a T cell receptor (TCR) repertoire that can functionally interact with major histocompatibility complexes (MHC). Over 90% of thymocytes fail this selection process and die. A small number of macrophages within the thymus are responsible for clearing the large number of dying thymocytes that must be continuously cleared. We studied the capacity of thymic macrophages to clear apoptotic cells under acute circumstances. This was done by synchronously inducing cell death in the thymus and then monitoring the clearance of apoptotic thymocytes. Interestingly, acute cell death was shown to recruit large numbers of CD11b(+) cells into the thymus. In the absence of a minor CSF-1 dependent population of macrophages, the recruitment of these CD11b(+) cells into the thymus was greatly reduced and the clearance of apoptotic cells was disrupted. To assess a possible role for the CD11b(+) cells in the clearance of apoptotic cells, we analyzed mice deficient for eosinophils and mice with defective trafficking of neutrophils. Failure to attract either eosinophils or neutrophils to the thymus resulted in the impaired clearance of apoptotic cells. These results suggested that there is crosstalk between cells of the innate immune system that is necessary for maximizing the efficiency of apoptotic cell removal.

PLZF induces the spontaneous acquisition of memory/effector functions in T cells independently of NKT cell-related signals.

The broad complex, tramtrack, bric-a-brac-zinc finger (BTB-ZF) transcription factor promyelocytic leukemia zinc finger (PLZF) is required for development of the characteristic innate/effector functions of NKT cells. In this study, we report the characterization and functional analysis of transgenic mouse T cells with forced expression of PLZF. PLZF expression was sufficient to provide some memory/effector functions to T cells without the need for Ag stimulation or proliferation. The acquisition of this phenotype did not require the proliferation typically associated with T cell activation. Furthermore, PLZF transgenic cells maintained a diverse TCR repertoire, indicating that there was no preferential expansion of specific clones. Functionally, PLZF transgenic CD4 and CD8 lymphocytes were similar to wild type memory cells, in that they had similar requirements for costimulation and exhibited a similar pattern of cytokine secretion, with the notable exception that transgenic T cells produced significantly increased levels of IL-17. Whereas transgene-mediated PLZF expression was not sufficient to rescue NKT cell development in Fyn- or signaling lymphocytic activation-associated protein (SAP)-deficient mice, the acquisition of memory/effector functions induced by PLZF in conventional T cells was independent of Fyn and SAP. These data show that PLZF is sufficient to promote T cell effector functions and that PLZF acts independently of SAP- and Fyn-mediated signaling pathways.

Development of promyelocytic zinc finger and ThPOK-expressing innate gamma delta T cells is controlled by strength of TCR signaling and Id3.

The broad-complex tramtrack and bric a brac-zinc finger transcriptional regulator (BTB-ZF), promyelocytic leukemia zinc finger (PLZF), was recently shown to control the development of the characteristic innate T cell phenotype and effector functions of NK T cells. Interestingly, the ectopic expression of PLZF was shown to push conventional T cells into an activated state that seems to be proinflammatory. The factors that control the normal expression of PLZF in lymphocytes are unknown. In this study, we show that PLZF expression is not restricted to NK T cells but is also expressed by a subset of gammadelta T cells, functionally defining distinct subsets of this innate T cell population. A second BTB-ZF gene, ThPOK, is important for the phenotype of the PLZF-expressing gammadelta T cells. Most importantly, TCR signal strength and expression of inhibitor of differentiation gene 3 control the frequency of PLZF-expressing gammadelta T cells. This study defines the factors that control the propensity of the immune system to produce potentially disease-causing T cell subsets.

The BTB-zinc finger transcriptional regulator PLZF controls the development of invariant natural killer T cell effector functions.

Invariant natural killer T cells (iNKT cells) have an innate immunity-like rapidity of response and the ability to modulate the effector functions of other cells. We show here that iNKT cells specifically expressed the BTB-zinc finger transcriptional regulator PLZF. In the absence of PLZF, iNKT cells developed, but they lacked many features of innate T cells. PLZF-deficient iNKT cells accumulated in lymph nodes rather than in the liver, did not express NK markers and did not have the characteristic activated phenotype. PLZF-deficient iNKT cells failed to secrete large amounts of interleukin 4 and interferon-gamma after activation; however, some cells produced either interleukin 4 or interferon-gamma but not both. PLZF, therefore, is an iNKT cell-specific transcription factor that is necessary for full functionality.

Functional identification of distinct sets of antitumor activities mediated by the FKBP gene family.

Assigning biologic function to the many sequenced but still uncharacterized genes remains the greatest obstacle confronting the human genome project. Differential gene expression profiling routinely detects uncharacterized genes aberrantly expressed in conditions such as cancer but cannot determine which genes are functionally involved in such complex phenotypes. Integrating gene expression profiling with specific modulation of gene expression in relevant disease models can identify complex biologic functions controlled by currently uncharacterized genes. Here, we used systemic gene transfer in tumor-bearing mice to identify novel antiinvasive and antimetastatic functions for Fkbp8, and subsequently for Fkbp1a. Fkbp8 is a previously uncharacterized member of the FK-506-binding protein (FKBP) gene family down-regulated in aggressive tumors. Antitumor effects produced by Fkbp1a gene expression are mediated by cellular pathways entirely distinct from those responsible for antitumor effects produced by Fkbp1a binding to its bacterially derived ligand, rapamycin. We then used gene expression profiling to identify syndecan 1 (Sdc1) and matrix metalloproteinase 9 (MMP9) as genes directly regulated by Fkbp1a and Fkbp8. FKBP gene expression coordinately induces the expression of the antiinvasive Sdc1 gene and suppresses the proinvasive MMP9 gene. Conversely, short interfering RNA-mediated suppression of Fkbp1a increases tumor cell invasion and MMP9 levels, while down-regulating Sdc1. Thus, syndecan 1 and MMP9 appear to mediate the antiinvasive and antimetastatic effects produced by FKBP gene expression. These studies show that uncharacterized genes differentially expressed in metastatic cancers can play important functional roles in the metastatic phenotype. Furthermore, identifying gene regulatory networks that function to control tumor progression may permit more accurate modeling of the complex molecular mechanisms of this disease.