CD20, CD3, and CD40 ligand microclusters segregate three-dimensionally in vivo at B-cell-T-cell immunological synapses after viral immunity in primate brain

The clearance of virally infected cells from the brain is mediated by T cells that engage antigen-presenting cells to form supramolecular activation clusters at the immunological synapse. However, after clearance, the T cells persist at the infection site and remain activated locally. In the present work the long-term interactions of immune cells in brains of monkeys were imaged in situ 9 months after the viral inoculation. After viral immunity, the persistent infiltration of T cells and B cells was observed at the infection sites. T cells showed evidence of T-cell receptor signaling as a result of contacts with B cells. Three-dimensional analysis of B-cell-T-cell synapses showed clusters of CD3 in T cells and the segregation of CD20 in B cells, involving the recruitment of CD40 ligand at the interface. These results demonstrate that immunological synapses between B cells and T cells forming three-dimensional microclusters occur in vivo in the central nervous system and suggest that these interactions may be involved in the lymphocyte activation after viral immunity at the original infection site.     

A role for Rab7 in the movement of secretory granules in cytotoxic T lymphocytes

Cytotoxic T lymphocytes (CTL) are potent killers of virally infected and tumorigenic cells. Upon recognition of target cells, CTL undergo polarized secretion of secretory lysosomes at the immunological synapse (IS) that forms between CTL and target. However, the molecular machinery involved in the polarization of secretory lysosomes is still largely uncharacterized. In this paper, we investigated the role of Rab7 in the polarization of secretory lysosomes. We show that silencing of Rab7 by RNA interference reduces the ability of CTL to kill targets. GTP-bound Rab7 and Rab interacting lysosomal protein, RILP, interact and both localize to secretory lysosomes in CTL. Over-expression of RILP recruits dynein to the membranes of secretory lysosomes and triggers their movement toward the centrosome. Together, these results suggest that Rab7 may play a role in secretory lysosome movement toward the centrosome by interacting with RILP to recruit the minus-end motor, dynein.     

Cutting edge: cytotoxic granule polarization and cytolysis can occur without central supramolecular activation cluster formation in CD8+ effector T cells

The functional implication of molecular segregation within the immunological synapse remains uncertain. We recently reported that effector but not naive TCR transgenic murine CD8+ T cells formed immunological synapses containing a central supramolecular activation cluster (cSMAC), suggesting that execution of effector functions such as cytolytic activity might be facilitated by the cSMAC structure. We have now explored this hypothesis using two approaches. First, by simultaneously imaging cSMAC formation and mobilization of cytotoxic granules to the synapse, we observed no correlation between the presence of a cSMAC and granule reorientation. Second, we took advantage of the observation that CD28 costimulation markedly enhances cSMAC formation. Granule polarization to the contact site was indistinguishable with B7-1+ and B7-1- target cells, and cytolytic activity against B7-1+ or B7-1- targets was similar and granule-dependent. Together, our results indicate that the formation of a cSMAC is not required for cytolytic activity in CD8+ effector T cells.     

CD8(+) T cells secreting type 2 lymphokines are defective in protection against viral infection

Effector T cells secreting type 1 and/or type 2 lymphokines (Tc1, Tc0, Tc2) were generated in vitro from CD8(+) T cells of mice with a transgenic TCR recognizing lymphocytic choriomeningitis virus (LCMV) glycoprotein to compare their effector function in vitro and in vivo. Tc1, Tc2, and Tc0 showed similar Fas- and perforin-mediated cytotoxicity in vitro. Upon adoptive transfer, Tc2 and Tc0 effectors were less efficient than Tc1 at controlling LCMV or recombinant vaccinia virus expressing the LCMV glycoprotein in vivo. Tc2 and Tc0 had decreased surface VLA-4 density and deficient activation-induced LFA-1/ICAM-1-dependent homotypic adhesion in vitro. Therefore, the reduced antiviral activity in vivo of Tc2 and Tc0 compared with Tc1 is not due to reduced cytotoxic activity or IFN-gamma secretion but may be explained by defective homing to the target organ due to decreased expression and/or lower activity of adhesion molecules.     

Cell adhesion molecules in the central nervous system

Cell-cell adhesion molecules play key roles at the intercellular junctions of a wide variety of cells, including interneuronal synapses and neuron-glia contacts. Functional studies suggest that adhesion molecules are implicated in many aspects of neural network formation, such as axon-guidance, synapse formation, regulation of synaptic structure and astrocyte-synapse contacts. Some basic cell biological aspects of the assembly of junctional complexes of neurons and glial cells resemble those of epithelial cells. However, the neuron specific junctional machineries are required to exert neuronal functions, such as synaptic transmission and plasticity. In this review, we describe the distribution and function of cell adhesion molecules at synapses and at contacts between synapses and astrocytes.Key words: synapses, cell adhesion molecules, cadherin superfamily, immunoglobulin superfamily, nerve tissue proteins, axons     

Ral GTPases regulate cell-mediated cytotoxicity in NK cells

NK cells are key components of the immune response to virally infected and tumor cells. Recognition of target cells initiates a series of events in NK cells that culminates in target destruction via directed secretion of lytic granules. Ral proteins are members of the Ras superfamily of small GTPases; they regulate vesicular trafficking and polarized granule secretion in several cell types. In this study, we address the role of Ral GTPases in cell-mediated cytotoxicity. Using a human NK cell line and human primary NK cells, we show that both Ral isoforms, RalA and RalB, are activated rapidly after target cell recognition. Furthermore, silencing of RalA and RalB impaired NK cell cytotoxicity. RalA regulated granule polarization toward the immunological synapse and the subsequent process of degranulation, whereas RalB regulated degranulation but not polarization of lytic granules. Analysis of the molecular mechanism indicated that Ral activation in NK cells leads to assembly of the exocyst, a protein complex involved in polarized secretion. This assembly is required for degranulation, as interference with expression of the exocyst component Sec5 led to reduced degranulation and impaired cytotoxicity in NK cells. Our results thus identify a role for Ral in cell-mediated cytotoxicity, implicating these GTPases in lymphocyte function.     

Strong TCR signaling, TLR ligands, and cytokine redundancies ensure robust development of type 1 effector T cells

T cell effector function is a central mechanism of adaptive immunity, and accordingly, protection of the host against pathogens. One of the primary effector molecules produced by T cells in response to such pathogens is the cytokine, IFN-gamma. Although the signaling pathways associated with the production of IFN-gamma are well established, disparate in vivo and in vitro results indicate that distinct pathways may become more prominent dependent upon the nature of the infection, inflammatory milieu and tissue localization. We have examined the roles and requirements of the major IFN-gamma-inducing pathways in vivo and in vitro, specifically: strength of TCR signal; paracrine release of IL-12, IL-23, and IL-18; and autocrine production of IFN-gamma. Our data show a dynamic interaction between these activation pathways, which allows the host a degree of flexibility and redundancy in the induction of IFN-gamma. Upon strong signaling through the TCR, IL-12, IL-18, and IL-23 play negligible roles in the induction of IFN-gamma, whereas autocrine IFN-gamma is an important component in sustaining its own secretion. However, the absence of any one of these factors during a weaker TCR signal, results in strikingly impaired T cell IFN-gamma production. Of note, TLR-activated dendritic cells (DCs) were capable of overcoming the absence of a strong TCR signal, IL-12, IL-23, or IL-18 revealing an important additional mechanism for ensuring a robust IFN-gamma response. Our findings clarify the hierarchical requirements of the major IFN-gamma inducing pathways and highlight the important role TLR ligand-activated DCs have to preserve them.     

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.     

TCR complex-activated CD8 adhesion function by human T cells

The CD8 receptor plays a central role in the recognition and elimination of virally infected and malignant cells by cytolytic CD8(+) T cells. In conjunction with the TCR, the CD8 coreceptor binds Ag-specific class I MHC (MHC-I) molecules expressed by target cells, initiating signaling events that result in T cell activation. Whether CD8 can further function as an adhesion molecule for non-Ag MHC-I is currently unclear in humans. In this study, we show that in human CD8(+) T cells, TCR complex signaling activates CD8 adhesion molecule function, resulting in a CD8 interaction with MHC-I that is sufficient to maintain firm T cell adhesion under shear conditions. Secondly, we found that while CD8 adhesive function was triggered by TCR complex activation in differentiated cells, including in vitro generated CTL and ex vivo effector/memory phenotype CD8(+) T cells, naive CD8(+) T cells were incapable of activated CD8 adhesion. Lastly, we examine the kinetics of, and signaling for, activated CD8 adhesion in humans and identify notable differences from the equivalent CD8 function in mouse. Activated CD8 adhesion induced by TCR signaling may contribute to the more rapid and robust elimination of pathogen-infected cells by differentiated CD8(+) T cells.     

Preservation of functional virus-specific memory CD8+ T lymphocytes in vaccinated, simian human immunodeficiency virus-infected rhesus monkeys

Functional impairment of virus-specific memory CD8(+) T lymphocytes has been associated with clinical disease progression following HIV, SIV, and simian human immunodeficiency virus infection. These lymphocytes have a reduced capacity to produce antiviral cytokines and mediators involved in the lysis of virally infected cells. In the present study, we used polychromatic flow cytometry to assess the frequency and functional capacity of central memory (CD28(+)CD95(+)) and effector memory (CD28(-)CD95(+)) subpopulations of Gag-specific CD8(+) T cells in SIV/simian human immunodeficiency virus-infected rhesus monkeys. The aim of this study was to determine whether Ag-specific, memory CD8(+) T cell function could be preserved in infected monkeys that had been immunized before infection with a vaccine regimen consisting of a plasmid DNA prime followed by a recombinant viral vector boost. We observed that vaccination was associated with the preservation of Gag-specific central memory CD8(+) T cells that were functionally capable of producing IFN-gamma, and effector memory CD8(+) T cells that were capable of producing granzyme B following viral Ag exposure.     

Generation of activated and antigen-specific T cells with cytotoxic activity after co-culture with dendritic cells

Co-culturing of immunological effector cells with antigen-pulsed DC leads to an increase of cytotoxic activity against antigen-expressing tumour cells. Using this approach, we could detect up to 2.8% antigen-specific CTLs after co-culture with antigen-pulsed DC. However, the required high effector cell numbers remain a major obstacle in immunotherapy. In this study, we show an approach for generating activated and antigen-specific effector cells that enables us to decrease effector to target cell ratios. We used an interferon-gamma secretion assay to enrich activated effector cells after co-culture with antigen-pulsed dendritic cells (DC). Purified immunological effector cells lysed 58.3% of antigen-expressing tumour cells at an effector to target ratio of 1:1. Furthermore, using MHC-IgG complexes, we enriched effector cells expressing antigen-specific T-cell receptor after co-culture with DC. Performing ELISpot, flow cytometry and TCR analysis, we could show a significant increase of activated and specific TCR-expressing effector cells after co-culture with DC.     

The level of viral antigen presented by hepatocytes influences CD8 T-cell function

CD8 T cells exert their antiviral function through cytokines and lysis of infected cells. Because hepatocytes are susceptible to noncytolytic mechanisms of viral clearance, CD8 T-cell antiviral efficiency against hepatotropic viruses has been linked to their capacity to produce gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha). On the other hand, intrahepatic cytokine production triggers the recruitment of mononuclear cells, which sustain acute and chronic liver damage. Using virus-specific CD8 T cells and human hepatocytes, we analyzed the modulation of virus-specific CD8 T-cell function after recognition peptide-pulsed or virally infected hepatocytes. We observed that hepatocyte antigen presentation was generally inefficient, and the quantity of viral antigen strongly influenced CD8 T-cell antiviral function. High levels of hepatitis B virus production induced robust IFN-gamma and TNF-alpha production in virus-specific CD8 T cells, while limiting amounts of viral antigen, both in hepatocyte-like cells and naturally infected human hepatocytes, preferentially stimulated CD8 T-cell degranulation. Our data document a mechanism where virus-specific CD8 T-cell function is influenced by the quantity of virus produced within hepatocytes.     

Antiviral cytokines induce hepatic expression of the granzyme B inhibitors, proteinase inhibitor 9 and serine proteinase inhibitor 6

Expression of the granzyme B inhibitors, human proteinase inhibitor 9 (PI-9), or the murine orthologue, serine proteinase inhibitor 6 (SPI-6), confers resistance to CTL or NK killing by perforin- and granzyme-dependent effector mechanisms. In light of prior studies indicating that virally infected hepatocytes are selectively resistant to this CTL effector mechanism, the present studies investigated PI-9 and SPI-6 expression in hepatocytes and hepatoma cells in response to adenoviral infection and to cytokines produced during antiviral immune responses. Neither PI-9 nor SPI-6 expression was detected by immunoblotting in uninfected murine or human hepatocytes. Similarly, human Huh-7 hepatoma cells were found to express only very low levels of PI-9 relative to levels detected in perforin- and granzyme-resistant CTL or lymphokine-activated killer cells. Following in vivo adenoviral infection or in vitro culture with IFN-alphabeta or IFN-gamma, SPI-6 expression was induced in murine hepatocytes. Similarly, after culture with IFN-alpha, induction of PI-9 mRNA and protein expression was observed in human hepatocytes and Huh-7 cells. IFN-gamma and TNF-alpha also induced 4- to 10-fold higher levels of PI-9 mRNA expression in Huh-7 cells, whereas levels of mRNA encoding a related serine proteinase inhibitor, proteinase inhibitor 8, were unaffected by culture of Huh-7 cells with IFN-alpha, IFN-gamma, or TNF-alpha. These findings indicate that cytokines that promote antiviral cytopathic responses also regulate expression of the cytoprotective molecules, PI-9 and SPI-6, in hepatocytes that are potential targets of CTL and NK effector mechanisms.     

Perforin and gamma interferon-mediated control of coronavirus central nervous system infection by CD8 T cells in the absence of CD4 T cells

Infection of the central nervous system (CNS) with the neurotropic JHM strain of mouse hepatitis virus produces acute and chronic demyelination. The contributions of perforin-mediated cytolysis and gamma interferon (IFN-gamma) secretion by CD8(+) T cells to the control of infection and the induction of demyelination were examined by adoptive transfer into infected SCID recipients. Untreated SCID mice exhibited uncontrolled virus replication in all CNS cell types but had little or no demyelination. Memory CD8(+) T cells from syngeneic wild-type (wt), perforin-deficient, or IFN-gamma-deficient (GKO) donors all trafficked into the infected CNS in the absence of CD4(+) T cells and localized to similar areas. Although CD8(+) T cells from all three donors suppressed virus replication in the CNS, GKO CD8(+) T cells expressed the least antiviral activity. A distinct viral antigen distribution in specific CNS cell types revealed different mechanisms of viral control. While wt CD8(+) T cells inhibited virus replication in all CNS cell types, cytolytic activity in the absence of IFN-gamma suppressed the infection of astrocytes, but not oligodendroglia. In contrast, cells that secreted IFN-gamma but lacked cytolytic activity inhibited replication in oligodendroglia, but not astrocytes. Demyelination was most severe following viral control by wt CD8(+) T cells but was independent of macrophage infiltration. These data demonstrate the effective control of virus replication by CD8(+) T cells in the absence of CD4(+) T cells and support the necessity for the expression of distinct effector mechanisms in the control of viral replication in distinct CNS glial cell types.     

NK cells regulate CD8+ T cell effector function in response to an intracellular pathogen

We studied the role of NK cells in regulating human CD8+ T cell effector function against mononuclear phagocytes infected with the intracellular pathogen Mycobacterium tuberculosis. Depletion of NK cells from PBMC of healthy tuberculin reactors reduced the frequency of M. tuberculosis-responsive CD8+IFN-gamma+ cells and decreased their capacity to lyse M. tuberculosis-infected monocytes. The frequency of CD8+ IFN-gamma+ cells was restored by soluble factors produced by activated NK cells and was dependent on IFN-gamma, IL-15, and IL-18. M. tuberculosis-activated NK cells produced IFN-gamma, activated NK cells stimulated infected monocytes to produce IL-15 and IL-18, and production of IL-15 and IL-18 were inhibited by anti-IFN-gamma. These findings suggest that NK cells maintain the frequency of M. tuberculosis-responsive CD8+IFN-gamma+ T cells by producing IFN-gamma, which elicits secretion of IL-15 and IL-18 by monocytes. These monokines in turn favor expansion of Tc1 CD8+ T cells. The capacity of NK cells to prime CD8+ T cells to lyse M. tuberculosis-infected target cells required cell-cell contact between NK cells and infected monocytes and depended on interactions between the CD40 ligand on NK cells and CD40 on infected monocytes. NK cells link the innate and the adaptive immune responses by optimizing the capacity of CD8+ T cells to produce IFN-gamma and to lyse infected cells, functions that are critical for protective immunity against M. tuberculosis and other intracellular pathogens.     

Fine structural analysis of membrane changes during reaggregation culture of chick optic tectum

Thin section and freeze-fracture electron microscopy have been used to characterize the changes in membrane morphology of reaggregating cultures of chick optic tectum. The cells are rounded and freely dispersed at 0 hr after dissociation. Between 2 and 6 hr the cells become closely apposed on all sides by other cells and form small aggregates. At this time punta adhaerentia junctions and focal densities are seen along the membranes of neighboring cells. Between 1 and 5 days in vitro (DIV) neurites containing growth cone regions are present. At 5 DIV the first synaptic contacts are observed. Between 7 and 14 DIV, the number of synaptic contacts increase and fewer growth cone regions are observed. As early as 7 DIV profiles are observed which strongly resemble both astrocytic and oligodendroglial cell somata and processes. Freeze-fracture analysis of aggregates at 0–4 hr reveals a sparse particle distribution on the P and E faces of apposed cells. By 1 DIV small clusters of loosely packed, large sized particles are seen on the P face of apposed cell membranes which may represent junctional contacts. Apparent coated vesicle fusion sites are common on the P face at 1–2 DIV. By 7 DIV, E face particle arrays are seen on cell bodies and neurites which correspond to specializations characteristic of excitatory synaptic junctions. By 8–10 DIV particle arrays are seen on the P face of post-synaptic membrane which may represent inhibitory synaptic contacts. Other types of particle specializations seen in freeze-fracture replicas include: specializations characteristic of gap junctions between cells and orthogonal assemblies of particles thought to be characteristic of astrocytes.     

Delta 9-tetrahydrocannabinol decreases cytotoxic T lymphocyte activity to herpes simplex virus type 1-infected cells.

The purpose of this study was to examine the effect of delta 9-tetrahydrocannabinol (delta 9-THC), the major psychoactive component of marijuana, on T lymphocyte functional competence against herpes simplex virus Type 1 (HSV1) infection. Spleen cells from C3H/HeJ (H-2k) mice primed with HSV1 and exposed to delta 9-THC were examined for anti-HSV1 cytolytic T lymphocyte (CTL) activity. Flow cytometry was used to determine whether delta 9-THC altered T cytotoxic (Lyt-2+) and T helper (L3T4+) lymphocyte numbers or cell ratios. Nomarski optics microscopy was used to determine whether effector lymphocytes from drug-treated mice were able to bind to virally infected L929 (H-2k) target cells. Cytotoxicity assays demonstrated that CTL from mice exposed to delta 9-THC were deficient in anti-HSV1 cytolytic activity. delta 9-THC in vivo treatment had little effect on the number of T lymphocytes expressing the Lyt-2 or L3T4 antigens. Nomarski optics microscopy revealed that the CTL from the drug-treated mice were able to bind specifically to the HSV1-infected targets. However, delta 9-THC in vivo exposure affected CTL cytoplasmic polarization toward the virus-infected target cell. CTL granule reorientation toward the effector cell-target cell interface following cell conjugation occurred at a lower frequency in co-cultures containing CTL from drug-treated mice. These results suggest that delta 9-THC elicits dysfunction in CTL by altering effector cell-target cell postconjugation events.     

Types of neurons and synaptic connections at hypostome-tentacle junctions in Hydra

Using transmission electron microscopy of thin sections we have examined neuronal concentrations at hypostome-tentacle junctions in Hydra littoralis. A total of 194 ganglion cells were counted in 587 serial thin sections of a single hypostome-tentacle junction. We found two distinct types of ganglion cells: those with and those lacking stereocilia. The majority of the neurons observed lacked stereocilia; in a single hypostome-tentacle junction only 37% of the ganglion cells possessed a kinocilium surrounded by rodlike stereocilia. Most of the ganglion cells (55%) were clustered together in the oral or upper epidermis of the hypostome-tentacle junction: Nineteen percent were in the lateral and 26% in the aboral or lower epidermis. The two types of ganglion cells did not differ significantly in their distribution. Both types of ganglion cell had synaptic contacts with other neurons and with epitheliomuscular cells. More than 85% of the neuroneuronal and 61% of the neuroepitheliomuscular cell synapses were located in the oral epidermis of a hypostome-tentacle junction. In addition, two-way chemical synapses and a gap junction between neurons were observed at hypostome-tentacle junctions. Our morphological evidence of synaptic connectivity in neuronal clusters at hypostome-tentacle junctions suggests that primitive ganglia are present in Hydra.     

T cell-dendritic cell immunological synapses contain TCR-dependent CD28-CD80 clusters that recruit protein kinase C theta

Short-lived TCR microclusters and a longer-lived protein kinase Ctheta-focusing central supramolecular activation cluster (cSMAC) have been defined in model immunological synapses (IS). In different model systems, CD28-mediated costimulatory interactions have been detected in microclusters, the cSMAC, or segregated from the TCR forming multiple distinct foci. The relationship between TCR and costimulatory molecules in the physiological IS of T cell-dendritic cell (DC) is obscure. To study the dynamic relationship of CD28-CD80 and TCR interactions in the T cell-DC IS during Ag-specific T cell activation, we generated CD80-eCFP mice using bacterial artificial chromosome transgenic technology. In splenic DCs, endogenous CD80 and CD80-eCFP localized to plasma membrane and Golgi apparatus, and CD80-eCFP was functional in vivo. In the OT-II T cell-DC IS, multiple segregated TCR, CD80, and LFA-1 clusters were detected. In the T cell-DC synapse CD80 clusters were colocalized with CD28 and PKCtheta, a characteristic of the cSMAC. Acute blockade of TCR signaling with anti-MHC Ab resulted in a rapid reduction in Ca(2+) signaling and the number and size of the CD80 clusters, a characteristic of TCR microclusters. Thus, the T cell-DC interface contains dynamic costimulatory foci that share characteristics of microclusters and cSMACs.