Dragana Cado

Fas-mediated apoptosis and activation-induced T-cell proliferation are defective in mice lacking FADD/Mort1

Programmed cell death, or apoptosis, is important in homeostasis of the immune system: for example, non-functional or auto-reactive lymphocytes are eliminated through apoptosis. One member of the tumour necrosis factor receptor (TNFR) family, Fas (also known as CD95 or Apo-1), can trigger cell death and is essential for lymphocyte homeostasis,. FADD/Mort1 (refs 3–6) is a Fas-associated protein that is thought to mediate apoptosis by recruiting the protease caspase-8 (refs 7, 8). A dominant-negative mutant of FADD inhibits apoptosis initiated by Fas and other TNFR family members,. Other proteins, notably Daxx, also bind Fas and presumably mediate a FADD-independent apoptotic pathway. Here we investigate the role of FADD in vivo by generating FADD-deficient mice. As homozygous mice die in utero, we generated FADD−/− embryonic stem cells and FADD−/− chimaeras in a background devoid of the recombination activating gene RAG-1, which activates rearrangement of the immunoglobulin and T-cell receptor genes. We found that thymocyte subpopulations were apparently normal in newborn chimaeras. Fas-induced apoptosis was completely blocked, indicating that there are no redundant Fas apoptotic pathways. As these mice age, their thymocytes decrease to an undetectable level, although peripheral T cells are present in all older FADD−/− chimaeras. Unexpectedly, activation-induced proliferation is impaired in these FADD−/− T cells, despite production of the cytokine interleukin (IL)-2. These results and the similarities between FADD−/− mice and mice lacking the β-subunit of the IL-2 receptor suggest that there is an unexpected connection between cell proliferation and apoptosis.

NKG2D-deficient mice are defective in tumor surveillance in models of spontaneous malignancy.

Ligands for the NKG2D stimulatory receptor are frequently upregulated on tumor lines, rendering them sensitive to natural killer (NK) cells, but the role of NKG2D in tumor surveillance has not been addressed in spontaneous cancer models. Here, we provided the first characterization of NKG2D-deficient mice, including evidence that NKG2D was not necessary for NK cell development but was critical for immunosurveillance of epithelial and lymphoid malignancies in two transgenic models of de novo tumorigenesis. In both models, we detected NKG2D ligands on the tumor cell surface ex vivo, providing needed evidence for ligand expression by primary tumors. In a prostate cancer model, aggressive tumors arising in NKG2D-deficient mice expressed higher amounts of NKG2D ligands than did similar tumors in wild-type mice, suggesting an NKG2D-dependent immunoediting of tumors in this model. These findings provide important genetic evidence for surveillance of primary tumors by an NK receptor.

An Activated Form of Notch Influences the Choice between CD4 and CD8 T Cell Lineages

Notch is a transmembrane receptor that controls cell fate decisions in Drosophila and whose role in mammalian cell fate decisions is beginning to be explored. We are investigating the role of Notch in a well-studied mammalian cell fate decision: the choice between the CD8 and CD4 T cell lineages. Here we report that expression of an activated form of Notch1 in developing T cells of the mouse leads to both an increase in CD8 lineage T cells and a decrease in CD4 lineage T cells. Expression of activated Notch permits the development of mature CD8 lineage thymocytes even in the absence of class I major histocompatability complex (MHC) proteins, ligands that are normally required for the development of these cells. However, activated Notch is not sufficient to promote CD8 cell development when both class I and class II MHC are absent. These results implicate Notch as a participant in the CD4 versus CD8 lineage decision.

Critical function for Naip5 in inflammasome activation by a conserved carboxy-terminal domain of flagellin

Inflammasomes are cytosolic multiprotein complexes that sense microbial infection and trigger cytokine production and cell death. However, the molecular components of inflammasomes and what they sense remain poorly defined. Here we demonstrate that 35 amino acids of the carboxyl terminus of flagellin triggered inflammasome activation in the absence of bacterial contaminants or secretion systems. To further elucidate the host flagellin-sensing pathway, we generated mice deficient in the intracellular sensor Naip5. These mice failed to activate the inflammasome in response to the 35 amino acids of flagellin or in response to Legionella pneumophila infection. Our data clarify the molecular basis for the cytosolic response to flagellin.

Notch Activity Influences the αβ versus γδ T Cell Lineage Decision

Abstract The choice between the αβ or γδ T cell fates is influenced by the production of functional, in-frame rearrangements of the TCR genes, but the mechanism that controls the lineage choice is not known. Here, we show that T cells that are heterozygous for a mutation of the Notch1 gene are more likely to develop as γδ T cells than as αβ T cells, implying that reduced Notch activity favors the γδ T cell fate over the αβ T cell fate. A constitutively activated form of Notch produces a reciprocal phenotype and induces thymocytes that have functional γδTCR gene rearrangements to adopt the αβ T cell fate. Our data indicate that Notch acts together with the newly formed T cell antigen receptor to direct the αβ versus γδ T cell lineage decision.

Direct Assessment of MHC Class I Binding by Seven Ly49 Inhibitory NK Cell Receptors

Mouse NK cells express at least seven inhibitory Ly49 receptors. Here we employ a semiquantitative cell-cell adhesion assay as well as class I/peptide tetramers to provide a comprehensive analysis of specificities of Ly49 receptors for class I MHC molecules in eight MHC haplotypes. Different Ly49 receptors exhibited diverse binding properties. The degree of class I binding was related to the extent of functional inhibition. The tetramer studies demonstrated that neither glycosylation nor coreceptors were necessary for class I binding to Ly49 receptors and uncovered peptide-specific recognition by a Ly49 receptor. The results provide a foundation for interpreting and integrating many existing functional studies as well as for designing tests of NK cell development and self-tolerance.

FUNCTIONAL REDUNDANCY OF THE NUR77 AND NOR-1 ORPHAN STEROID RECEPTORS IN T-CELL APOPTOSIS

The transcription factor Nur77 (NGFI‐B), a member of the steroid nuclear receptor superfamily, is induced to a high level during T‐cell receptor (TCR)‐mediated apoptosis. A transgenic dominant‐negative Nur77 protein can inhibit the apoptotic process accompanying negative selection in thymocytes, while constitutive expression of Nur77 leads to massive cell death. Nur77‐deficient mice, however, have no phenotype, suggesting the possible existence of a protein with redundant function to Nur77. To explore this possibility, we have characterized the role of two Nur77 family members, Nurr1 and Nor‐1, in TCR‐induced apoptosis. We found that Nor‐1 and Nurr1 can transactivate through the same DNA element as Nur77, and that their transactivation activities can be blocked by a Nur77 dominant‐negative protein. In thymocytes, Nor‐1 protein is induced to a very high level upon TCR stimulation and has similar kinetics to Nur77. In contrast, Nurr1 is undetectable in stimulated thymocytes. Furthermore, constitutive expression of Nor‐1 in thymocytes leads to massive apoptosis and up‐regulation of CD25, suggesting a functional redundancy between Nur77 and Nor‐1 gene products. As in the case of our Nur77‐FL mice, FasL is not detectable in the thymocytes of Nor‐1 transgenic mice. Constitutive expression of Nur77 in gld/gld mice rescues the lymphoproliferative phenotype of the FasL mutant mice. Thus, Nor‐1 and Nur77 demonstrate functional redundancy in an apparently Fas‐independent apoptosis.

A role for the orphan steroid receptor Nur77 in apoptosis accompanying antigen-induced negative selection

The transcription factor Nur77, an orphan member of the nuclear hormone receptor superfamily, is highly expressed during T cell receptor-signaled apoptosis, suggesting a possible role for Nur77 in negative selection. We examined this by generating two sets of transgenic mice. In one set of mice, a dominant-negative Nur77 mutant is constitutively expressed and the other in which wild-type Nur77 protein is constitutively expressed in developing thymocytes. We report that inhibition of endogenous Nur77 by the dominant-negative mutant perturbed T cell development and inhibited antigen-induced negative selection in F5T cell receptor transgenic mice. Constitutive expression of wild-type Nur77 protein induced apoptosis in developing thymocytes, resulting in a decreased number of thymocytes and mature T cells. Together, these data support a role for Nur77 in the downstream signaling events in antigen-induced negative selection.

The aminopeptidase ERAAP shapes the peptide repertoire displayed by major histocompatibility complex class I molecules.

Major histocompatibility complex (MHC) class I molecules present thousands of peptides to allow CD8+ T cells to detect abnormal intracellular proteins. The antigen-processing pathway for generating peptides begins in the cytoplasm, and the MHC molecules are loaded in the endoplasmic reticulum. However, the nature of peptide pool in the endoplasmic reticulum and the proteolytic events that occur in this compartment are unclear. We addressed these issues by generating mice lacking the endoplasmic reticulum aminopeptidase associated with antigen processing (ERAAP). We found that loss of ERAAP disrupted the generation of naturally processed peptides in the endoplasmic reticulum, decreased the stability of peptide–MHC class I complexes and diminished CD8+ T cell responses. Thus, trimming of antigenic peptides by ERAAP in the endoplasmic reticulum is essential for the generation of the normal repertoire of processed peptides.

A conserved transcriptional enhancer regulates RAG gene expression in developing B cells.

Although expression of the RAG1 and RAG2 genes is essential for lymphocyte development, the mechanisms responsible for the lymphoid- and developmental stage-specific regulation of these genes are poorly understood. We have identified a novel, evolutionarily conserved transcriptional enhancer in the RAG locus, called Erag, which was essential for the expression of a chromosomal reporter gene driven by either RAG promoter. Targeted deletion of Erag in the mouse germline results in a partial block in B cell development associated with deficient V(D)J recombination, whereas T cell development appears unaffected. We found that E2A transcription factors bind to Erag in vivo and can transactivate Erag-dependent reporter constructs in cotransfected cell lines. These findings lead us to conclude that RAG transcription is regulated by distinct elements in developing B and T cells and that Erag is required for optimal levels of RAG expression in early B cell precursors but not in T cells.