Martha S. Jordan

Thymic selection of CD4+CD25+ regulatory T cells induced by an agonist self-peptide.

Despite accumulating evidence that regulatory T cells play a crucial role in preventing autoimmunity, the processes underlying their generation during immune repertoire formation are unknown. We show here that interactions with a single self-peptide can induce thymocytes that bear an autoreactive T cell receptor (TCR) to undergo selection to become CD4+CD25+ regulatory T cells. Selection of CD4+CD25+ thymocytes appears to require a TCR with high affinity for a self peptide because thymocytes that bear TCRs with low affinity do not undergo selection into this pathway. Our findings indicate that specificity for self-peptides directs the selection of CD4+CD25+ regulatory thymocytes by a process that is distinct from positive selection and deletion.

T Cell Activation

This year marks the 25th anniversary of the first Annual Review of Immunology article to describe features of the T cell antigen receptor (TCR). In celebration of this anniversary, we begin with a brief introduction outlining the chronology of the earliest studies that established the basic paradigm for how the engaged TCR transduces its signals. This review continues with a description of the current state of our understanding of TCR signaling, as well as a summary of recent findings examining other key aspects of T cell activation, including cross talk between the TCR and integrins, the role of costimulatory molecules, and how signals may negatively regulate T cell function.Acronyms and DefinitionsAdapter protein: cellular protein that functions to bridge molecular interactions via characteristic domains able to mediate protein/protein or protein/lipid interactions Costimulation: signals delivered to T cells by cell surface receptors other than the TCR itself that potentiate T cell activation cSMAC: central supramolecular activation cluster Immunoreceptor tyrosine-based activation motif (ITAM): a short peptide sequence in the cytoplasmic tails of key surface receptors on hematopoietic cells that is characterized by tyrosine residues that are phosphorylated by Src family PTKs, enabling the ITAM to recruit activated Syk family kinases Inside-out signaling: signals initiated by engagement of immunoreceptors that lead to conformational changes and clustering of integrins, thereby increasing the affinity and avidity of the integrins for their ligands NFAT: nuclear factor of activated T cells PI3K: phosphoinositide 3-kinase PKC: protein kinase C PLC: phospholipase C pMHC: peptide major histocompatibility complex (MHC) complex pSMAC: peripheral supramolecular activation cluster PTK: protein tyrosine kinase Signal transduction: biochemical events linking surface receptor engagement to cellular responses TCR: T cell antigen receptor

Adaptors as central mediators of signal transduction in immune cells

Adaptors are molecular scaffolds that recruit effectors, which are critical for immune cell activation. Recent work has underscored the requirement for adaptors in propagating stimulatory signals as well as their ability to inhibit immune cell function. The mechanisms by which adaptors function rely not only on the intermolecular interactions they mediate, but also on where they are localized within the cell. The use of sophisticated genetic, biochemical, cellular and imaging approaches has provided important new insights into the biology of adaptor protein function. Here we focus on T lymphocytes as a model to illustrate the critical roles adaptors play as regulators of cellular activation.

Disruption of diacylglycerol metabolism impairs the induction of T cell anergy

Anergic T cells have altered diacylglycerol metabolism, but whether that altered metabolism has a causative function in the induction of T cell anergy is not apparent. To test the importance of diacylglycerol metabolism in T cell anergy, we manipulated diacylglycerol kinases (DGKs), which are enzymes that terminate diacylglycerol-dependent signaling. Overexpression of DGK-α resulted in a defect in T cell receptor signaling that is characteristic of anergy. We generated DGK-α-deficient mice and found that DGK-α-deficient T cells had more diacylglycerol-dependent T cell receptor signaling. In vivo anergy induction was impaired in DGK-α-deficient mice. When stimulated in anergy-producing conditions, T cells lacking DGK-α or DGK-ζ proliferated and produced interleukin 2. Pharmacological inhibition of DGK-α activity in DGK-ζ-deficient T cells that received an anergizing stimulus proliferated similarly to wild-type T cells that received CD28 costimulation and prevented anergy induction. Our findings suggest that regulation of diacylglycerol metabolism is critical in determining whether activation or anergy ensues after T cell receptor stimulation.

Enhanced T cell responses due to diacylglycerol kinase ζ deficiency

Much is known about how T cell receptor (TCR) engagement leads to T cell activation; however, the mechanisms terminating TCR signaling remain less clear. Diacylglycerol, generated after TCR ligation, is essential in T cells. Its function must be controlled tightly to maintain normal T cell homeostasis. Previous studies have shown that diacylglycerol kinase ζ (DGKζ), which converts diacylglycerol to phosphatidic acid, can inhibit TCR signaling. Here we show that DGKζ-deficient T cells are hyperresponsive to TCR stimulation both ex vivo and in vivo. Furthermore, DGKζ-deficient mice mounted a more robust immune response to lymphocytic choriomeningitis virus infection than did wild-type mice. These results demonstrate the importance of DGKζ as a physiological negative regulator of TCR signaling and T cell activation.

Anergy and suppression regulate CD4 + T cell responses to a self peptide

To examine the role of cognate peptide in establishing CD4+ T cell tolerance, we have mated transgenic mice that express the major I‐Ed‐restricted determinant (S1) from the influenza virus PR8 hemagglutinin (HA28 mice) with mice expressing a S1‐specific T cell receptor (TS1 mice). Surprisingly, S1‐specific CD4+ T cells were not substantially deleted in TS1xHA28 mice; indeed, lymph node cells expressing the S1‐specific TCR were as abundant in TS1xHA28 mice as in TS1 mice. The S1‐specific T cells in TS1xHA28 mice were, however, impaired in their ability to respond to S1 peptide both in vitro and in vivo, and contained two distinct populations. Approximately half expressed a unique cell surface phenotype (CD25hi / CD45RBint) and had been anergized by the neo‐self S1 peptide. The remainder responded normally to the S1 peptide if purified away from the anergic T cells, but their proliferation was suppressed when the anergic T cells were also present in unfractionated lymphnode cells or in mixed cultures. These findings establish that anergy and suppression are coordinated mechanisms by which autoreactive CD4+ T cells are regulated and that anergic / suppressor CD4+ T cells can develop in response to self peptides.

Asymmetric Proteasome Segregation as a Mechanism for Unequal Partitioning of the Transcription Factor T-bet during T Lymphocyte Division

Polarized segregation of proteins in T cells is thought to play a role in diverse cellular functions including signal transduction, migration, and directed secretion of cytokines. Persistence of this polarization can result in asymmetric segregation of fate-determining proteins during cell division, which may enable a T cell to generate diverse progeny. Here, we provide evidence that a lineage-determining transcription factor, T-bet, underwent asymmetric organization in activated T cells preparing to divide and that it was unequally partitioned into the two daughter cells. This unequal acquisition of T-bet appeared to result from its asymmetric destruction during mitosis by virtue of concomitant asymmetric segregation of the proteasome. These results suggest a mechanism by which a cell may unequally localize cellular activities during division, thereby imparting disparity in the abundance of cell fate regulators in the daughter cells.

Development of Promyelocytic Zinc Finger and ThPOK-Expressing Innate γδ 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 γδ 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 γδ T cells. Most importantly, TCR signal strength and expression of inhibitor of differentiation gene 3 control the frequency of PLZF-expressing γδ T cells. This study defines the factors that control the propensity of the immune system to produce potentially disease-causing T cell subsets.

Persistence of Cooperatively Stabilized Signaling Clusters Drives T-Cell Activation

ABSTRACT Antigen recognition triggers the recruitment of the critical adaptor protein SLP-76 to small macromolecular clusters nucleated by the T-cell receptor (TCR). These structures develop rapidly, in parallel with TCR-induced increases in tyrosine phosphorylation and cytosolic calcium, and are likely to contribute to TCR-proximal signaling. Previously, we demonstrated that these SLP-76-containing clusters segregate from the TCR and move towards the center of the contact interface. Neither the function of these clusters nor the structural requirements governing their persistence have been examined extensively. Here we demonstrate that defects in cluster assembly and persistence are associated with defects in T-cell activation in the absence of Lck, ZAP-70, or LAT. Clusters persist normally in the absence of phospholipase C-γ1, indicating that in the absence of a critical effector, these structures are insufficient to drive T-cell activation. Furthermore, we show that the critical adaptors LAT and Gads localize with SLP-76 in persistent clusters. Mutational analyses of LAT, Gads, and SLP-76 indicated that multiple domains within each of these proteins contribute to cluster persistence. These data indicate that multivalent cooperative interactions stabilize these persistent signaling clusters, which may correspond to the functional complexes predicted by kinetic proofreading models of T-cell activation.

Differential requirement for SLP-76 domains in T cell development and function.

The hematopoietic cell-specific adaptor protein, SLP-76, is critical for T cell development and mature T cell receptor (TCR) signaling; however, the structural requirements of SLP-76 for mediating thymopoiesis and mature T cell function remain largely unknown. In this study, transgenic mice were generated to examine the requirements for specific domains of SLP-76 in thymocytes and peripheral T cells in vivo. Examination of mice expressing various mutants of SLP-76 on the null background demonstrates a differential requirement for specific domains of SLP-76 in thymocytes and T cells and provides new insight into the molecular mechanisms underlying SLP-76 function.