Enhancement of LFA-1-mediated cell adhesion by triggering through CD2 or CD3 on T lymphocytes

The lymphocyte function-associated molecule LFA-1 (CD11a/CD18) plays a key part in lymphocyte adhesion. Lymphocytes do not adhere spontaneously; activation of protein kinase C (PKC) by phorbol esters, however, gives rise to strong LFA-1-dependent adhesion, indicating that activation of LFA-1 is required to induce cell adhesion. We have now investigated whether the functionally important CD2 and CD3 surface structures on T lymphocytes are involved in the activation of LFA-1. The stimulation of these molecules, which causes activation of PKC, strongly promoted LFA-1-dependent adhesion. Furthermore, we demonstrate by using cells from an LFA-1-deficient patient that this enhanced lymphocyte adhesion is caused by activation of the LFA-1 molecule and not by activation of its ligands. LFA-1 was persistently activated by triggering through CD2 but only transiently by triggering through CD3. We postulate that CD2 and CD3 can differentially regulate the affinity of LFA-1 for its ligands by modulating its molecular conformation through PKC-dependent mechanisms.     

Recognition of H-2 domains by cytotoxic T lymphocytes

The polymorphic major histocompatibility antigens (H-2) have a crucial role in the activation of antigen-specific T lymphocytes. Thus, H-2 antigens are not only recognized by allogenic lymphocytes leading to generation of cytotoxic T lymphocytes (CTLs), but it has also been demonstrated that in syngeneic systems most T cells are only able to recognize foreign antigens in conjunction with their own MHC (major histocompatibility complex) antigens. This phenomenon, termed H-2 restriction, may be the key to our understanding to the biological function of MHC antigens. It is not clear whether recognition by T cells of H-2 on a molecular level is confined to particular domains on the H-2 molecule, nor whether the same polymorphic H-2 sites, which are characterized by antibodies, are recognized by allogeneic as well as by H-2 restricted syngeneic CTLs. Previous findings indicate the existence of at least two major polymorphic domains on the H-2Kk molecule as defined by antibodies. Here we show the existence of CTLs with specifity for these polymorphic domains, and the preferential recognition of a particular domain by both alloreactive as well as H-2 restricted CTLs.     

Sae2, Exo1 and Sgs1 collaborate in DNA double-strand break processing

DNA ends exposed after introduction of double-strand breaks (DSBs) undergo 5'-3' nucleolytic degradation to generate single-stranded DNA, the substrate for binding by the Rad51 protein to initiate homologous recombination. This process is poorly understood in eukaryotes, but several factors have been implicated, including the Mre11 complex (Mre11-Rad50-Xrs2/NBS1), Sae2/CtIP/Ctp1 and Exo1. Here we demonstrate that yeast Exo1 nuclease and Sgs1 helicase function in alternative pathways for DSB processing. Novel, partially resected intermediates accumulate in a double mutant lacking Exo1 and Sgs1, which are poor substrates for homologous recombination. The early processing step that generates partly resected intermediates is dependent on Sae2. When Sae2 is absent, in addition to Exo1 and Sgs1, unprocessed DSBs accumulate and homology-dependent repair fails. These results suggest a two-step mechanism for DSB processing during homologous recombination. First, the Mre11 complex and Sae2 remove a small oligonucleotide(s) from the DNA ends to form an early intermediate. Second, Exo1 and/or Sgs1 rapidly process this intermediate to generate extensive tracts of single-stranded DNA that serve as substrate for Rad51.     

Inhibition of nociceptors by TRPV1-mediated entry of impermeant sodium channel blockers

Most local anaesthetics used clinically are relatively hydrophobic molecules that gain access to their blocking site on the sodium channel by diffusing into or through the cell membrane. These anaesthetics block sodium channels and thereby the excitability of all neurons, not just sensory neurons. We tested the possibility of selectively blocking the excitability of primary sensory nociceptor (pain-sensing) neurons by introducing the charged, membrane-impermeant lidocaine derivative QX-314 through the pore of the noxious-heat-sensitive TRPV1 channel. Here we show that charged sodium-channel blockers can be targeted into nociceptors by the application of TRPV1 agonists to produce a pain-specific local anaesthesia. QX-314 applied externally had no effect on the activity of sodium channels in small sensory neurons when applied alone, but when applied in the presence of the TRPV1 agonist capsaicin, QX-314 blocked sodium channels and inhibited excitability. Inhibition by co-applied QX-314 and capsaicin was restricted to neurons expressing TRPV1. Injection of QX-314 together with capsaicin into rat hindpaws produced a long-lasting (more than 2 h) increase in mechanical and thermal nociceptive thresholds. Long-lasting decreases in pain sensitivity were also seen with regional injection of QX-314 and capsaicin near the sciatic nerve; however, in contrast to the effect of lidocaine, the application of QX-314 and capsaicin together was not accompanied by motor or tactile deficits.     

Restricted recognition of beta 2-microglobulin by cytotoxic T lymphocytes

Recognition of foreign antigen by cytotoxic T lymphocytes (CTL) is restricted by class I major histocompatibility complex (MHC) products. Class I heavy chains (relative molecular mass (Mr) 45,000-48,000) are reversibly and noncovalently associated with beta 2-microglobulin (beta 2M, Mr = 12,000). Cells expressing human or murine class I heavy chains can exchange their native beta 2M for exogenously added free beta 2M, which is present in serum. Two allelic forms of beta 2M exist among the common laboratory mouse strains, beta 2M-A and beta 2M-B, which are represented in BALB and C57BL mice, respectively. The two forms differ at a single amino acid at position 85, the gene (beta 2m) is located on chromosome 2 linked to a minor histocompatibility (H) region, H-3. It has been proposed that one of the H-3 loci is identical with beta 2m, and that CTL raised across certain H-3 incompatibilities are actually specific for beta 2M. Here we describe CTL raised in such a combination which recognize endogenous as well as exogenous beta 2M-B in the context of H-2Kb. This represents a unique case of CTL recognition, as CTL usually recognize antigens inserted into the membrane, and it is the first molecular identification of the product of a minor H locus.     

Netrin-1-mediated axon outgrowth and cAMP production requires interaction with adenosine A2b receptor

The netrins, a family of laminin-related secreted proteins, are critical in controlling axon elongation and pathfinding. The DCC (for deleted in colorectal cancer) protein was proposed as a receptor for netrin-1 in the light of many observations including the inhibition of netrin-1-mediated axon outgrowth and attraction in the presence of an anti-DCC antiserum, the similitude of nervous system defects in DCC and netrin-1 knockout mice and the results of receptor swapping experiments. Previous studies have failed to show a direct interaction of DCC with netrin-1 (ref. 10), suggesting the possibility of an additional receptor or co-receptor. Here we show that DCC interacts with the membrane-associated adenosine A2b receptor, a G-protein-coupled receptor that induces cAMP accumulation on binding adenosine. We show that A2b is actually a netrin-1 receptor and induces cAMP accumulation on binding netrin-1. Finally, we show that netrin-1-dependent outgrowth of dorsal spinal cord axons directly involves A2b. Together our results indicate that the growth-promoting function of netrin-1 may require a receptor complex containing DCC and A2b.     

Peptide-dependent recognition of H-2Kb by alloreactive cytotoxic T lymphocytes

Antigen-specific T lymphocytes appear to recognize foreign antigens in the form of peptide fragments presented within the antigen-binding groove of class I or class II molecules encoded by the major histocompatibility complex (MHC). Alloreactive T cells also show specificity for MHC molecules, and various reports suggest that residues of the MHC molecules constitute at least part of the ligand to which alloreactive T-cell receptors bind. The X-ray crystal structure of the human MHC class I molecule, HLA-A2, has provided evidence to strengthen the argument that MHC-bound self-peptide might also contribute to such recognition. We now provide direct evidence for this, showing that at least some alloreactive cytotoxic T lymphocyte clones recognize peptide fragments derived from cytoplasmic proteins. We reasoned that if self-peptides were involved in allorecognition, then the sequence of some of these peptides could vary between species, resulting in species-restricted distribution of the relevant ligand(s). Several alloreactive cytotoxic T lymphocyte clones specific for H-2Kb, expressed by the murine cell line EL4, did not lyse a human-cell transfectant expressing the H-2Kb molecule (Jurkat-Kb cells). However, these clones were able to lyse Jurkat-Kb cells sensitized by preincubation with an EL4 cytoplasmic extract cleaved by cyanogen bromide. The sensitizing activity from this extract was destroyed by protease and appeared to be due to a peptide consisting of 10 to 15 amino acids.     

Generation of F1 hybrid cytotoxic T lymphocytes specific for self H-2

Cytotoxic T lymphocytes specific for self H-2 antigens are generated by murine F1 hybrid (H-2 heterozygous) spleen cells cultured with irradiated parental (H-2 homozygous) splenocytes. The effectors bind to heterozygous and homozygous cells bearing the appropriate H-2 alleles but only lyse homozygous targets. Autoreactivity for membrane-bound molecules of normal cells may be a mechanism for regulating cellular interactions.     

The mechanism of OTUB1-mediated inhibition of ubiquitination

Histones are ubiquitinated in response to DNA double-strand breaks (DSB), promoting recruitment of repair proteins to chromatin. UBC13 (also known as UBE2N) is a ubiquitin-conjugating enzyme (E2) that heterodimerizes with UEV1A (also known as UBE2V1) and synthesizes K63-linked polyubiquitin (K63Ub) chains at DSB sites in concert with the ubiquitin ligase (E3), RNF168 (ref. 3). K63Ub synthesis is regulated in a non-canonical manner by the deubiquitinating enzyme, OTUB1 (OTU domain-containing ubiquitin aldehyde-binding protein 1), which binds preferentially to the UBC13～Ub thiolester. Residues amino-terminal to the OTU domain, which had been implicated in ubiquitin binding, are required for binding to UBC13～Ub and inhibition of K63Ub synthesis. Here we describe structural and biochemical studies elucidating how OTUB1 inhibits UBC13 and other E2 enzymes. We unexpectedly find that OTUB1 binding to UBC13～Ub is allosterically regulated by free ubiquitin, which binds to a second site in OTUB1 and increases its affinity for UBC13～Ub, while at the same time disrupting interactions with UEV1A in a manner that depends on the OTUB1 N terminus. Crystal structures of an OTUB1-UBC13 complex and of OTUB1 bound to ubiquitin aldehyde and a chemical UBC13～Ub conjugate show that binding of free ubiquitin to OTUB1 triggers conformational changes in the OTU domain and formation of a ubiquitin-binding helix in the N terminus, thus promoting binding of the conjugated donor ubiquitin in UBC13～Ub to OTUB1. The donor ubiquitin thus cannot interact with the E2 enzyme, which has been shown to be important for ubiquitin transfer. The N-terminal helix of OTUB1 is positioned to interfere with UEV1A binding to UBC13, as well as with attack on the thiolester by an acceptor ubiquitin, thereby inhibiting K63Ub synthesis. OTUB1 binding also occludes the RING E3 binding site on UBC13, thus providing a further component of inhibition. The general features of the inhibition mechanism explain how OTUB1 inhibits other E2 enzymes in a non-catalytic manner.     

Anti-HLA-A2 antibody-enhancement of peptide association with HLA-A2 as detected by cytotoxic T lymphocytes

Most cytotoxic T lymphocytes (CTL) not only recognize epitopes of viral or other foreign proteins in association with class I major histocompatibility complex (MHC) molecules, but also recognize target cells sensitized with short synthetic peptides representing the epitopes. There is increasing evidence that these synthetic peptides associate with the class I molecule both at the cell surface and intracellularly. We have now investigated the effect of a monoclonal antibody specific for HLA-A2 and HLA-B17 (B57/58) molecules (antibody MA2.1)3 on the sensitization of target cells with peptide for lysis by HLA-A2-restricted CTL. Previously, anti-HLA class I monoclonal antibodies have been shown to inhibit the recognition of target cells, infected with influenza A virus, by virus-specific CTL. We find, however, that target cells treated with MA2.1 antibody can be sensitized with peptide for CTL lysis much more rapidly than untreated cells, or at greater than 100-fold lower peptide concentration than that required for sensitization of untreated cells. This implies that the antibody, which is believed to bind to one side of the peptide-binding groove, directly affects the binding of peptide to the HLA-A2 molecule at the cell surface.     

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Inhibition of alloreactive cytotoxic T lymphocytes by peptides from the alpha 2 domain of HLA-A2

Class I major histocompatibility complex (MHC) molecules function in the recognition of antigens by cytotoxic T lymphocytes (CTL). Although this biological role is firmly established and much has been learnt about their structure and polymorphic variation, little is known of the regions of class I molecules that are involved in functional interactions with components of the T-cell surface. Here we show that peptides derived from residues 98-113 of the alpha 2 domain of HLA-A2 specifically inhibit the recognition of target cells by many HLA-A2-specific CTL. In addition to identifying a region that is probably involved in binding the T-cell receptor these results raise the possibility that alloreactive CTL may recognize degraded fragments of class I histocompatibility antigens.     

H2AX prevents CtIP-mediated DNA end resection and aberrant repair in G1-phase lymphocytes

DNA double-strand breaks (DSBs) are generated by the recombination activating gene (RAG) endonuclease in all developing lymphocytes as they assemble antigen receptor genes. DNA cleavage by RAG occurs only at the G1 phase of the cell cycle and generates two hairpin-sealed DNA (coding) ends that require nucleolytic opening before their repair by classical non-homologous end-joining (NHEJ). Although there are several cellular nucleases that could perform this function, only the Artemis nuclease is able to do so efficiently. Here, in vivo, we show that in murine cells the histone protein H2AX prevents nucleases other than Artemis from processing hairpin-sealed coding ends; in the absence of H2AX, CtIP can efficiently promote the hairpin opening and resection of DNA ends generated by RAG cleavage. This CtIP-mediated resection is inhibited by γ-H2AX and by MDC-1 (mediator of DNA damage checkpoint 1), which binds to γ-H2AX in chromatin flanking DNA DSBs. Moreover, the ataxia telangiectasia mutated (ATM) kinase activates antagonistic pathways that modulate this resection. CtIP DNA end resection activity is normally limited to cells at post-replicative stages of the cell cycle, in which it is essential for homology-mediated repair. In G1-phase lymphocytes, DNA ends that are processed by CtIP are not efficiently joined by classical NHEJ and the joints that do form frequently use micro-homologies and show significant chromosomal deletions. Thus, H2AX preserves the structural integrity of broken DNA ends in G1-phase lymphocytes, thereby preventing these DNA ends from accessing repair pathways that promote genomic instability.     

Productive dual infection of human CD4+ T lymphocytes by HIV-1 and HHV-6

Although infection by HIV-1 has been implicated as the primary cause of AIDS and related disorders, cofactorial mechanisms may be involved in the pathogenesis of the disease. For example, several viruses commonly detected in AIDS patients and capable of transactivating the long terminal repeat of HIV-1, such as herpesviruses, papovaviruses, adenoviruses and HTLV-I have been suggested as potential cofactors. Another candidate is human herpesvirus-6 (HHV-6, originally designated human B-lymphotropic virus), which has not only been identified in most patients with AIDS by virus isolation, DNA amplification techniques and serological analysis, but is also predominantly tropic and cytopathic in vitro for CD4+ T lymphocytes. Here we demonstrate that HHV-6 and HIV-1 can productively co-infect individual human CD4+ T lymphocytes, resulting in accelerated HIV-1 expression and cellular death. We also present evidence that HHV-6 transactivates the HIV-1 long terminal repeat (LTR). These observations indicate that HHV-6 might contribute directly or indirectly to the depletion of CD4+ T cells in AIDS.     

Inhibition by dopamine of (Na(+)+K+)ATPase activity in neostriatal neurons through D1 and D2 dopamine receptor synergism

The (Na(+)+K+)ATPase, an integral membrane protein located in virtually all animal cells, couples the hydrolysis of ATP to the countertransport of Na+ and K+ ions across the plasma membrane. In neurons, a large portion of cellular energy is expended by this enzyme to maintain the ionic gradients that underlie resting and action potentials. Although neurotransmitter regulation of the enzyme in brain has been reported, such regulation has been characterized either as a nonspecific phenomenon or as an indirect effect of neurotransmitter-induced changes in ionic gradients. We report here that the neurotransmitter dopamine, through a synergistic effect on D1 and D2 receptors, inhibits the (Na(+)+K+)ATPase activity of isolated striatal neurons. Our data provide unequivocal evidence for regulation by a neurotransmitter of a neuronal ion pump. They also demonstrate that synergism between D1 and D2 receptors, which underlies many of the electrophysical and behavioural effects of dopamine in the mammalian brain, can occur on the same neuron. In addition, the results support the possibility that dopamine and other neurotransmitters can regulate neuronal excitability through the novel mechanism of pump inhibition.     

Differential modulation of three separate K-conductances in hippocampal CA1 neurons by serotonin

The hippocampus receives a dense serotonin-containing innervation from the divisions of the raphe nucleus. Serotonin applied to hippocampal neurons to mimic the action of endogenous transmitter often produces complex and variable responses (see for example ref. 3). Using voltage-clamp methods and new ligands that are selective for subtypes of serotonin receptors, we have been able to clarify the mechanism of serotonin action on CA1 cells in rat hippocampal slices. We describe three distinct actions of serotonin (or 5-HT) on identified K-conductances in these cells. First, it activates a Ca-independent K-current which is responsible for neuronal hyperpolarization and is inhibitory. Second, it simultaneously suppresses the slow Ca-dependent K-conductance that is largely responsible for the accommodation of cell firing in CA1 neurons: this produces a paradoxical increase in neuronal discharge in response to a depolarizing input. Third, serotonin produces a more slowly developing and long-lasting suppression of an intrinsic voltage-dependent K-conductance, Im (ref. 9), leading to neuronal depolarization and excitation. The hyperpolarizing response is mediated by class 1A serotonin receptors, whereas the other responses are not. Modulation of these different conductances by endogenously released serotonin could therefore change the probability or the duration (or both) of neuronal firing in the mammalian brain in different ways to give inhibitory, excitatory or mixed effects.