Nigel Killeen

The TNF and TNF Receptor Superfamilies: Integrating Mammalian Biology

The authors regret the inability to cite all of the primary literature contributing to this review due to length considerations. The authors thank F. Chan, T. Migone, M. Peter, J. Puck, R. Siegel, H. Walczak, and J. Wang for insightful comments on the manuscript. N. K. is a Scholar of the Leukemia Society. Supported in part by grants from the National Institutes of Health (R. M. L., N. K.).

Asymmetric T Lymphocyte Division in the Initiation of Adaptive Immune Responses

A hallmark of mammalian immunity is the heterogeneity of cell fate that exists among pathogen-experienced lymphocytes. We show that a dividing T lymphocyte initially responding to a microbe exhibits unequal partitioning of proteins that mediate signaling, cell fate specification, and asymmetric cell division. Asymmetric segregation of determinants appears to be coordinated by prolonged interaction between the T cell and its antigen-presenting cell before division. Additionally, the first two daughter T cells displayed phenotypic and functional indicators of being differentially fated toward effector and memory lineages. These results suggest a mechanism by which a single lymphocyte can apportion diverse cell fates necessary for adaptive immunity.

OX40 Promotes Bcl-xL and Bcl-2 Expression and Is Essential for Long-Term Survival of CD4 T Cells

It is important to understand which molecules are essential for long-lived immunity. We show that OX40 (CD134) is required with CD28 for the survival of CD4 T cells following antigen-driven expansion. In contrast to CD28-/- T cells, which show defects early, OX40-/- T cells are relatively unimpaired in IL-2 production, cell division, and expansion. However, OX40-/- T cells fail to maintain high levels of Bcl-xL and Bcl-2 4-8 days after activation, and undergo apoptosis. Conversely, OX40 stimulation promotes Bcl-xL and Bcl-2 and suppresses apoptosis. Moreover, retroviral transduction of OX40-/- T cells with Bcl-xL or Bcl-2 reverses their survival defect. Thus, a temporal relationship exists between CD28 and OX40, with OX40 being a critical regulator of antigen-driven T cell survival.

Conditional deletion of Gata3 shows its essential function in TH1-TH2 responses

Expression of the transcription factor GATA-3 is strongly associated with T helper type 2 (TH2) differentiation, but genetic evidence for its involvement in this process has been lacking. Here, we generated a conditional GATA-3-deficient mouse line. In vitro deletion of Gata3 diminished both interleukin 4 (IL-4)–dependent and IL-4-independent TH2 cell differentiation; without GATA-3, TH1 differentiation occurred in the absence of IL-12 and interferon-γ. Gata3 deletion limited the growth of TH2 cells but not TH1 cells. Deletion of Gata3 from established TH2 cells abolished IL-5 and IL-13 but not IL-4 production. In vivo deletion of Gata3 using OX40-Cre eliminated TH2 responses and allowed the development of interferon-γ-producing cells in mice infected with Nippostrongylus brasiliensis. Thus, GATA-3 serves as a principal switch in determining TH1-TH2 responses.

Role of CXCR5 and CCR7 in Follicular Th Cell Positioning and Appearance of a Programmed Cell Death Gene-1High Germinal Center-Associated Subpopulation

Th cell access to primary B cell follicles is dependent on CXCR5. However, whether CXCR5 induction on T cells is sufficient in determining their follicular positioning has been unclear. In this study, we find that transgenic CXCR5 overexpression is not sufficient to promote follicular entry of naive T cells unless the counterbalancing influence of CCR7 ligands is removed. In contrast, the positioning of Ag-engaged T cells at the B/T boundary could occur in the absence of CXCR5. The germinal center (GC) response was 2-fold reduced when T cells lacked CXCR5, although these T cells were able to access the GC. Finally, CXCR5highCCR7low T cells were found to have elevated IL-4 transcript and programmed cell death gene-1 (PD-1) expression, and PD-1high cells were reduced in the absence of T cell CXCR5 or in mice compromised in GC formation. Overall, these findings provide further understanding of how the changes in CXCR5 and CCR7 expression regulate Th cell positioning during Ab responses, and they suggest that development and/or maintenance of a PD-1high follicular Th cell subset is dependent on appropriate interaction with GC B cells.

A lineage-specific transcriptional silencer regulates CD4 gene expression during T lymphocyte development

During development of T lymphocytes, differential regulation of expression of the CD4 and CD8 glycoproteins is coupled to the choice of one of two pathways of differentiation. Thymocytes that express both of these coreceptors commit to either the helper lineage, shutting off CD8, or the cytotoxic lineage, shutting off CD4. We have used transgenic mice to identify an intronic regulatory region that controls CD4 gene expression during development. This region selectively extinguishes transgene expression in CD4-CD8+, but not CD4+CD8- nor CD4+CD8+ T cells. It also represses gene expression in CD4-CD8- immature thymocytes, indicating that the CD4 gene is derepressed during differentiation from the CD4-CD8- to the CD4+CD8+ stage. The negative element(s) is both orientation and position independent and acts also on heterologous regulatory sequences. Its properties are functionally similar to those of silencers described in yeast and in Drosophila, suggesting that we have identified a developmentally regulated vertebrate transcriptional silencer.

The OX40 Costimulatory Receptor Determines the Development of CD4 Memory by Regulating Primary Clonal Expansion

The costimulatory receptor OX40 has recently been shown to be involved in primary CD4 responses to several defined Ags. However, to date there has been little information regarding the mechanism of action of OX40, such as whether it regulates T cell numbers, reactivity, or both, and whether it contributes to induction of long-term T cell responses. With an agonist Ab to OX40, and by tracking Ag-specific TCR transgenic T cells in vivo, we show that ligation of OX40 induces clonal expansion and survival of CD4 cells during primary responses, and results in the accumulation of greater numbers of memory cells with time. Significantly, OX40-deficient T cells, from mice generated by gene targeting, secrete IL-2 and proliferate normally during the initial period of activation, but cannot sustain this during the latter phases of the primary response, exhibiting decreased survival over time. Mice lacking OX40 develop only low frequencies of Ag-specific CD4 cells late in primary responses in vivo and generate dramatically lower frequencies of surviving memory cells. These results demonstrate that OX40-OX40L interactions control primary T cell expansion and the ability to retain high numbers of Ag-specific T cells. In this way, OX40 signals promote survival of greater numbers of T cells with time and control the size of the memory T cell pool.

Mammalian Target of Rapamycin Protein Complex 2 Regulates Differentiation of Th1 and Th2 Cell Subsets via Distinct Signaling Pathways

Many functions of the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) have been defined, but relatively little is known about the biology of an alternative mTOR complex, mTORC2. We showed that conditional deletion of rictor, an essential subunit of mTORC2, impaired differentiation into T helper 1 (Th1) and Th2 cells without diversion into FoxP3(+) status or substantial effect on Th17 cell differentiation. mTORC2 promoted phosphorylation of protein kinase B (PKB, or Akt) and PKC, Akt activity, and nuclear NF-kappaB transcription factors in response to T cell activation. Complementation with active Akt restored only T-bet transcription factor expression and Th1 cell differentiation, whereas activated PKC-theta reverted only GATA3 transcription factor and the Th2 cell defect of mTORC2 mutant cells. Collectively, the data uncover vital mTOR-PKC and mTOR-Akt connections in T cell differentiation and reveal distinct pathways by which mTORC2 regulates development of Th1 and Th2 cell subsets.

Integrin cytoplasmic tyrosine motif is required for outside-in αIIbβ3 signalling and platelet function

Integrins not only bind adhesive ligands, they also act as signalling receptors. Both functions allow the integrin αIIbβ3 to mediate platelet aggregation. Platelet agonists activate αIIbβ3 (inside-out signalling) to allow the binding of soluble fibrinogen. Subsequent platelet aggregation leads to outside-in αIIbβ3 signalling, which results in calcium mobilization, tyrosine phosphorylation of numerous proteins including β3 itself, increased cytoskeletal reorganisation and further activation of αIIbβ3 (ref. 2). Thus, outside-in signals enhance aggregation, although the mechanisms and functional consequences of specific signalling events remain unclear. Here we describe a mouse that expresses an αIIbβ3 in which the tyrosines in the integrin cytoplasmic tyrosine motif have been mutated to phenylalanines. These mice are selectively impaired in outside-in αIIbβ3 signalling, with defective aggregation and clot-retraction responses in vitro, and an in vivo bleeding defect which is characterized by a pronounced tendency to rebleed. These data provide evidence for an important role of outside-in signalling in platelet physiology. Furthermore, they identify the integrin cytoplasmic tyrosine motif as a key mediator of β-integrin signals and a potential target for new therapeutic agents.

Vesicular Transport Regulates Monoamine Storage and Release but Is Not Essential for Amphetamine Action

To assess the role of exocytotic release in signaling by monoamines, we have disrupted the neuronal vesicular monoamine transporter 2 (VMAT2) gene. VMAT2-/- mice move little, feed poorly, and die within a few days after birth. Monoamine cell groups and their projections are indistinguishable from those of wild-type littermates, but the brains of mutant mice show a drastic reduction in monoamines. Using midbrain cultures from the mutant animals, amphetamine but not depolarization induces dopamine release. In vivo, amphetamine increases movement, promotes feeding, and prolongs the survival of VMAT2-/- animals, indicating that precise, temporally regulated exocytotic release of monoamine is not required for certain complex behaviors. In addition, the brains of VMAT2 heterozygotes contain substantially lower monoamine levels than those of wild-type littermates, and depolarization induces less dopamine release from heterozygous than from wild-type cultures, suggesting that VMAT2 expression regulates monoamine storage and release.