Coronin-1A Links Cytoskeleton Dynamics to TCRαβ-Induced Cell Signaling

Actin polymerization plays a critical role in activated T lymphocytes both in regulating T cell receptor (TCR)-induced immunological synapse (IS) formation and signaling. Using gene targeting, we demonstrate that the hematopoietic specific, actin- and Arp2/3 complex-binding protein coronin-1A contributes to both processes. Coronin-1A-deficient mice specifically showed alterations in terminal development and the survival of αβT cells, together with defects in cell activation and cytokine production following TCR triggering. The mutant T cells further displayed excessive accumulation yet reduced dynamics of F-actin and the WASP-Arp2/3 machinery at the IS, correlating with extended cell-cell contact. Cell signaling was also affected with the basal activation of the stress kinases sAPK/JNK1/2; and deficits in TCR-induced Ca²⁺ influx and phosphorylation and degradation of the inhibitor of NF-κB (IκB). Coronin-1A therefore links cytoskeleton plasticity with the functioning of discrete TCR signaling components. This function may be required to adjust TCR responses to selecting ligands accounting in part for the homeostasis defect that impacts αβT cells in coronin-1A deficient mice, with the exclusion of other lympho/hematopoietic lineages.     

Synaptic transfer by human gamma delta T cells stimulated with soluble or cellular antigens

B, alpha beta T, and NK lymphocytes establish immunological synapses (IS) with their targets to enable recognition. Transfer of target cell-derived Ags together with proximal molecules onto the effector cell appears also to occur through synapses. Little is known about the molecular basis of this transfer, but it is assumed to result from Ag receptor internalization. Because human gamma delta T cells recognize soluble nonpeptidic phosphoantigens as well as tumor cells such as Daudi, it is unknown whether they establish IS with, and extract molecules from, target cells. Using flow cytometry and confocal microscopy, we show in this work that Ag-stimulated human V gamma 9/V delta 2 T cells conjugate to, and perform molecular transfer from, various tumor cell targets. The molecular transfer appears to be linked to IS establishment, evolves in a dose-dependent manner in the presence of either soluble or cellular Ag, and requires gamma delta TCR ligation, Src family kinase signaling, and participation of the actin cytoskeleton. Although CD45 exclusion characterized the IS performed by gamma delta T cells, no obvious capping of the gamma delta TCR was detected. The synaptic transfer mediated by gamma delta T cells involved target molecules unrelated to the cognate Ag and occurred independently of MHC class I expression by target cells. From these observations, we conclude that despite the particular features of gamma delta T cell activation, both synapse formation and molecular transfer of determinants belonging to target cell characterize gamma delta T cell recognition of Ags.     

The crystal structure of murine coronin-1: a regulator of actin cytoskeletal dynamics in lymphocytes

Mammalian coronin-1 is preferentially expressed in hematopoietic cells and plays a poorly understood role in the dynamic reorganization of the actin cytoskeleton. Sequence analysis of coronin-1 revealed five WD40 repeats that were predicted to form a beta propeller. They are followed by a 130 residue extension and a 30 residue leucine zipper domain that is responsible for multimerization of the protein. Here, we present the crystal structure of murine coronin-1 without the leucine zipper at 1.75 A resolution. Coronin-1 forms a seven-bladed beta propeller composed of the five predicted WD40 repeats and two additional blades that lack any homology to the canonical WD40 motif. The C-terminal extension adopts an extended conformation, packs tightly against the bottom surface of the propeller, and is likely to be required for the structural stability of the propeller. Analysis of charged and conserved surface residues delineate possible binding sites for F-actin on the beta propeller.     

CD3 delta establishes a functional link between the T cell receptor and CD8

T cells expressing T cell receptor (TCR) complexes that lack CD3 delta, either due to deletion of the CD3 delta gene, or by replacement of the connecting peptide of the TCR alpha chain, exhibit severely impaired positive selection and TCR-mediated activation of CD8 single-positive T cells. Because the same defects have been observed in mice expressing no CD8 beta or tailless CD8 beta, we examined whether CD3 delta serves to couple TCR.CD3 with CD8. To this end we used T cell hybridomas and transgenic mice expressing the T1 TCR, which recognizes a photoreactive derivative of the PbCS 252-260 peptide in the context of H-2K(d). We report that, in thymocytes and hybridomas expressing the T1 TCR.CD3 complex, CD8 alpha beta associates with the TCR. This association was not observed on T1 hybridomas expressing only CD8 alpha alpha or a CD3 delta(-) variant of the T1 TCR. CD3 delta was selectively co-immunoprecipitated with anti-CD8 antibodies, indicating an avid association of CD8 with CD3 delta. Because CD8 alpha beta is a raft constituent, due to this association a fraction of TCR.CD3 is raft-associated. Cross-linking of these TCR-CD8 adducts results in extensive TCR aggregate formation and intracellular calcium mobilization. Thus, CD3 delta couples TCR.CD3 with raft-associated CD8, which is required for effective activation and positive selection of CD8(+) T cells.     

Regulatory role of peritoneal NK1.1+ alpha beta T cells in IL-12 production during Salmonella infection.

NK1.1+ alpha beta T cells emerge in the peritoneal cavity after an i.p. infection with Salmonella choleraesuis in mice. To elucidate the role of the NK1.1+ alpha beta T cells during murine salmonellosis, mice lacking NK1.1+ alpha beta T cells by disruption of TCR beta (TCR beta-/-), beta 2m (beta 2m-/-), or J alpha 281 (J alpha 281-/-) gene were i.p. inoculated with S. choleraesuis. The peritoneal exudate T cells in wild type (wt) mice on day 3 after infection produced IL-4 upon TCR alpha beta stimulation, whereas those in TCR beta-/-, beta 2m-/-, or J alpha 281-/- mice showed no IL-4 production upon the stimulation, indicating that NK1.1+ alpha beta T cells are the main source of IL-4 production at the early phase of Salmonella infection. Neutralization of endogenous IL-4 by administration of anti-IL-4 mAb to wt mice reduced the number of Salmonella accompanied by increased IL-12 production by macrophages after Salmonella infection. The IL-12 production by the peritoneal macrophages was significantly augmented in mice lacking NK1.1+ alpha beta T cells after Salmonella infection accompanied by increased serum IFN-gamma level. The aberrantly increased IL-12 production in infected TCR beta-/- or J alpha 281-/- mice was suppressed by adoptive transfer of T cells containing NK1.1+ alpha beta T cells but not by the transfer of T cells depleted of NK1.1+ alpha beta T cells or T cells from J alpha 281-/- mice. Taken together, it is suggested that NK1. 1+ alpha beta T cells eliciting IL-4 have a regulatory function in the IL-12 production by macrophages at the early phase of Salmonella infection.     

Control of T cell development in vivo by subdomains within the IL-7 receptor alpha-chain cytoplasmic tail

IL-7/IL-7R signaling functions in both growth and differentiation during T cell development. In this study, we examined the extent these activities were controlled by signaling associated with distinct IL-7R alpha cytoplasmic domains by transgenic expression of wild-type or cytoplasmic deletion mutants of IL-7R alpha in the thymi of IL-7R alpha(-/-) mice. We show an essential requirement for the tyrosine-containing carboxyl-terminal T domain in restoring thymic cellularity, pro-/pre-T cell progression, and survival. In contrast, the functional differentiation of TCR alpha beta cells and the development of TCR gamma delta cells are partially independent of the T domain. Thus, separate cytoplasmic domains of the IL-7R alpha chain differentially control distinct functions during T cell development, whereas normal IL-7R-dependent thymic development requires the integrated activity of all these domains.     

Alpha beta T cell receptor and CD3 transduce different signals in immature T cells. Implications for selection and tolerance

Ligand binding to alpha beta TCR has different consequences in thymocytes at different developmental stages, causing alternatively positive selection, clonal deletion, or activation. These various functional consequences may be due to changes in the signaling properties of the receptor complex during development. In this report we show that alpha beta TCR engagement on immature thymocytes has different effects on intracellular free calcium concentrations than alpha beta TCR engagement on mature T cells. In contrast, CD3 engagement on immature thymocytes and mature T cells has the same effect on intracellular free calcium, suggesting that altered signal transduction in immature thymocytes may be due to inefficient alpha beta TCR-CD3 coupling. These studies also suggest that in certain T cell populations, activation events resulting from ligation of CD3 may not accurately reflect the activation events resulting from ligation of the physiologic receptor, alpha beta TCR.     

Human invariant V alpha 24-J alpha Q TCR supports the development of CD1d-dependent NK1.1+ and NK1.1- T cells in transgenic mice

A sizable fraction of T cells expressing the NK cell marker NK1.1 (NKT cells) bear a very conserved TCR, characterized by homologous invariant (inv.) TCR V alpha 24-J alpha Q and V alpha 14-J alpha 18 rearrangements in humans and mice, respectively, and are thus defined as inv. NKT cells. Because human inv. NKT cells recognize mouse CD1d in vitro, we wondered whether a human inv. V alpha 24 TCR could be selected in vivo by mouse ligands presented by CD1d, thereby supporting the development of inv. NKT cells in mice. Therefore, we generated transgenic (Tg) mice expressing the human inv. V alpha 24-J alpha Q TCR chain in all T cells. The expression of the human inv. V alpha 24 TCR in TCR C alpha(-/-) mice indeed rescues the development of inv. NKT cells, which home preferentially to the liver and respond to the CD1d-restricted ligand alpha-galactosylceramide (alpha-GalCer). However, unlike inv. NKT cells from non-Tg mice, the majority of NKT cells in V alpha 24 Tg mice display a double-negative phenotype, as well as a significant increase in TCR V beta 7 and a corresponding decrease in TCR V beta 8.2 use. Despite the forced expression of the human CD1d-restricted TCR in C alpha(-/-) mice, staining with mCD1d-alpha-GalCer tetramers reveals that the absolute numbers of peripheral CD1d-dependent T lymphocytes increase at most by 2-fold. This increase is accounted for mainly by an increased fraction of NK1.1(-) T cells that bind CD1d-alpha-GalCer tetramers. These findings indicate that human inv. V alpha 24 TCR supports the development of CD1d-dependent lymphocytes in mice, and argue for a tight homeostatic control on the total number of inv. NKT cells. Thus, human inv. V alpha 24 TCR-expressing mice are a valuable model to study different aspects of the inv. NKT cell subset.     

CD3+4-8- alpha beta T cell population with biased T cell receptor V gene usage. Presence in bone marrow and possible involvement of IL-3 for their extrathymic development.

Analysis of TCR of a series of CD4-8- (double negative; DN) alpha beta T cell lines induced with IL-3 revealed that their V gene usage was biased for V alpha 4 and V beta 2. This has been confirmed in the primary short-term cultures. Thus, IL-3 induced the generation of DN alpha beta T cells with predominant V beta 2 gene expression from the CD4+/CD8+ T cell-depleted spleen or bone marrow (BM) cells of both normal and nude BALB/c mice within 10 days. It was further indicated that the V beta 2+ beta-chain genes contained few junctional N regions in both IL-3-induced primary DN alpha beta T cells and continuous lines. Search for the in vivo counterpart of in vitro IL-3-induced DN alpha beta T cells revealed that BM, but not spleens, of normal BALB/c and B6 mice did contain a significant proportion of DN alpha beta T cells, and that the majority of them expressed V beta 2+ beta-chain genes with few junctional N regions. The presence of V beta 2+ DN alpha beta T cells was similarly observed in the BM of BALB/c nude mice, but their proportion varied markedly among various strains of mice, which was not linked to H-2 haplotypes. The results indicated that V beta 2+ DN alpha beta T cells in the BM represented one of the thymus-independent T cell populations, whose development was under the major histocompatibility Ag complex-unlinked genetic control. TCR of these T cells were shown to be functional as judged by the proliferative response to anti-V beta 2 antibody. Taken together, present results suggested that IL-3 could induce differentiation and/or proliferation of DN alpha beta T cells with uniquely limited repertoire, which existed preferentially in BM in vivo, and implied the possible involvement of extrathymic endogenous ligands as a positive selection force.     

Antigen receptor regulation of phosphoinositide-dependent kinase 1 pathways during thymocyte development

Phosphoinositide-dependent kinase 1 (PDK1) is essential for T cell development but little is know about the stimuli that regulate PDK1 signaling in vivo. The thymus contains a heterogeneous mixture of cells at different stages of development making it difficult to use biochemical techniques to examine the activity of PDK1 pathways as thymocytes develop in situ. Herein, we use a single cell assay to quantify activation of the PDK1 target kinase ribosomal S6 kinase 1 (S6K1) in different murine thymocyte subsets immediately ex vivo. This technique allows an assessment of S6K1 activation as thymocytes respond to developmental stimuli in vivo. These studies reveal that only a small percentage of thymocytes show evidence for activation of PDK1 mediated signaling in situ. The thymic subpopulations that contain active PDK1/S6K1 are those known to be responding to signaling by the pre T cell receptor and the mature alpha/beta T cell antigen receptor (TCR). Moreover, loss of antigen receptor signaling in T cell progenitors that cannot rearrange their TCR beta locus prevents in vivo activation of S6K1. The present data identifying antigen receptor signaling as a key activator of PDK1 mediated signaling afford a molecular explanation for the important role of this molecule in T cells.     

The antiviral efficacy of the murine alpha-1 interferon transgene against ocular herpes simplex virus type 1 requires the presence of CD4(+), alpha/beta T-cell receptor-positive T lymphocytes with the capacity to produce gamma interferon

Alpha/beta interferons (IFN-alpha/betas) are known to antagonize herpes simplex virus type 1 (HSV-1) infection by directly blocking viral replication and promoting additional innate and adaptive, antiviral immune responses. To further define the relationship between the adaptive immune response and IFN-alpha/beta, the protective effect induced following the topical application of plasmid DNA containing the murine IFN-alpha 1 transgene onto the corneas of wild-type and T-cell-deficient mice was evaluated. Mice homozygous for both the T-cell receptor (TCR) beta- and delta-targeted mutations expressing no alpha beta or gamma delta TCR (alpha beta/gamma delta TCR double knockout [dKO]) treated with the IFN-alpha 1 transgene succumbed to ocular HSV-1 infection at a rate similar to that of alpha beta/gamma delta TCR dKO mice treated with the plasmid vector DNA. Conversely, mice with targeted disruption of the TCR delta chain and expressing no gamma delta TCR(+) cells treated with the IFN-alpha 1 transgene survived the infection to a greater extent than the plasmid vector-treated counterpart and at a level similar to that of wild-type controls treated with the IFN-alpha 1 transgene. By comparison, mice with targeted disruption of the TCR beta chain and expressing no alpha beta TCR(+) cells (alpha beta TCR knockout [KO]) showed no difference upon treatment with the IFN-alpha1 transgene or the plasmid vector control, with 0% survival following HSV-1 infection. Adoptively transferring CD4(+) but not CD8(+) T cells from wild-type but not IFN-gamma-deficient mice reestablished the antiviral efficacy of the IFN-alpha 1 transgene in alpha beta TCR KO mice. Collectively, the results indicate that the protective effect mediated by topical application of a plasmid construct containing the murine IFN-alpha 1 transgene requires the presence of CD4(+) T cells capable of IFN-gamma synthesis.     

Coronin-1 is phosphorylated at Thr-412 by protein kinase Cα in human phagocytic cells

Coronin-1, a hematopoietic cell-specific actin-binding protein, is thought to be involved in the phagocytic process through its interaction with actin filaments. The dissociation of coronin-1 from phagosomes after its transient accumulation on the phagosome surface is associated with lysosomal fusion. We previously reported that 1) coronin-1 is phosphorylated by protein kinase C (PKC), 2) coronin-1 has two phosphorylation sites, Ser-2 and Thr-412, and 3) Thr-412 of coronin-1 is phosphorylated during phagocytosis. In this study, we examined which PKC isoform is responsible for the phosphorylation of coronin-1 at Thr-412 by using isotype-specific PKC inhibitors and small interfering RNAs (siRNAs). Thr-412 phosphorylation of coronin-1 was suppressed by Gö6976, an inhibitor of PKCα and PKCβI. This phosphorylation was attenuated by siRNA for PKCα, but not by siRNA for PKCβ. Furthermore, Thr-412 of coronin-1 was phosphorylated by recombinant PKCα in vitro, but not by recombinant PKCβ. We next examined the effects of Gö6976 on the intracellular distribution of coronin-1 in HL60 cells during phagocytosis. The confocal fluorescence microscopic observation showed that coronin-1 was not dissociated from phagosomes in Gö6976-treated cells. These results indicate that phosphorylation of coronin-1 at Thr-412 by PKCα regulates intracellular distribution during phagocytosis.     

Human CD4- CD8- alpha beta+ T cells express a functional T cell receptor and can be activated by superantigens.

The CD4 and CD8 molecules play an important role in the stimulation of T cells and in the process of thymic education. Most mature T cells express the alpha beta TCR and either CD4 or CD8; however, there is a small population of alpha beta+ TCR T cells that lack both CD4 and CD8. Little is known of the biology of the CD4- CD8- (double-negative) alpha beta+ TCR T cells or the nature of the Ag to which they may respond. These cells not only represent a novel population of T cells but also provide useful biologic tools to study the roles that CD4 and CD8 play in T cell activation. In this study we have addressed two questions. Firstly, whether CD4- CD8- alpha beta+ TCR T cells have functionally active TCR and, secondly, whether CD4 or CD8 is required for the activation of T cells by bacterial enterotoxins. Six double-negative alpha beta+ TCR T cell clones, propagated from two healthy donors, were challenged with a panel of nine bacterial enterotoxins. The V alpha and V beta usage of their TCR was determined by polymerase chain reaction. All of the CD4-CD8- clones proliferated in response to at least one of the enterotoxins, in a V beta-specific manner. The proliferative response of the CD4-CD8- alpha beta+ TCR T cell clones was similar in magnitude to that exhibited by CD4+ T cell clones of known V beta expression. These data clearly show that the CD4 and CD8 molecules are not required for the activation of untransformed human T cells by bacterial enterotoxins. Furthermore, these results indicate that CD4-CD8- alpha beta+ TCR T cells, normally present in all individuals, are not functionally silent, because they can be stimulated via their TCR. Their physiologic role, like that of gamma delta T cells, remains to be elucidated.