Differentiation of human CD8(+) T cells from a memory to memory/effector phenotype

Previous studies of perforin expression and cytokine production in subsets of peripheral human CD45RA(-)CD8(+) T cells with different CD28/CD27 phenotypes showed that CD28(+)CD45RA(-)CD8(+) and CD27(+)CD45RA(-)CD8(+) T cells have characteristics of memory T cells, whereas CD28(-)CD45RA(-)CD8(+) and CD27(-)CD45RA(-)CD8(+) T cells have characteristics of both memory and effector T cells. However, the differentiation pathway from memory CD8(+) T cells into memory/effector CD8(+) T cells has not been completely clarified. We investigated this differentiation pathway using EBV- and human CMV (HCMV)-specific CD8(+) T cells. Three subsets of CD45RA(-)CD8(+) T cells were observed in both total CD8(+) T cells and EBV- or HCMV-specific CD8(+) T cells: CD27(+)CD28(+), CD27(+)CD28(-), and CD27(-)CD28(-). A significant number of the CD27(-)CD28(+) subset was observed in total CD8 T cells. However, this subset was barely detectable in EBV- or HCMV-specific CD8(+) T cells. Analysis of perforin expression and cytotoxic activity in the first three subsets suggested the following differentiation pathway: CD27(+)CD28(+)CD45RA(-)-->CD27(+)CD28(-)CD45RA(-)-->CD27(-)CD28(-)CD45RA(-). This was supported by the observation that the frequency of CCR5(+) cells and CCR7(+) cells decreased during this sequence. Analysis of CCR5 and CCR7 expression in the CD27(+)CD28(+) memory cell subset demonstrated the presence of three CCR5/CCR7 populations: CCR5(-)CCR7(+), CCR5(+)CCR7(+), and CCR5(+)CCR7(-). These findings suggested the following differentiation pathway: CD27(+)CD28(+)CD45RA(-) (CCR5(-)CCR7(+)-->CCR5(+)CCR7(+)-->CCR5(+)CCR7(-))-->CD27(+)CD28(-)CD45RA(-)-->CD27(-)CD28(-)CD45RA(-). The presence of a CD27(-)CD28(+) subset with a CCR5(+)CCR7(-) phenotype implies a specialized role for this subset in the differentiation of CD8(+) T cells.     

Activation of 5-HT(1A) but not 5-HT(1B) receptors attenuates an increase in extracellular dopamine derived from exogenously administered L-DOPA in the striatum with nigrostriatal denervation

In order to determine whether L-DOPA-derived extracellular dopamine (DA) in the striatum with dopaminergic denervation is affected by activation of serotonin autoreceptors (5-HT(1A) and 5-HT(1B) receptors), we applied in vivo brain microdialysis technique to 6-hydroxydopamine-lesioned rats and examined the effects of the selective 5-HT(1A) receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) and the selective 5-HT(1B) receptor agonist CGS-12066 A on L-DOPA-derived extracellular DA levels. Single L-DOPA injection (50 mg/kg i.p.) caused a rapid increase and a following decrease of extracellular DA, with a peak value at 100 min after L-DOPA injection. Pretreatment with both 0.3 mg/kg and 1 mg/kg 8-OH-DPAT (i.p.) significantly attenuated an increase in L-DOPA-derived extracellular DA and the times of peak DA levels were prolonged to 150 min and 225 min after L-DOPA injection, respectively. These 8-OH-DPAT-induced changes in L-DOPA-derived extracellular DA were antagonized by further pretreatment with WAY-100635, a selective 5-HT(1A) antagonist. In contrast, intrastriatal perfusion with the 5-HT(1B) agonist CGS-12066 A (10 nM and 100 nM) did not induce any changes in L-DOPA-derived extracellular DA. Thus, stimulation of 5-HT(1A) but not 5-HT(1B) receptors attenuated an increase in extracellular DA derived from exogenous L-DOPA. These results support the hypothesis that serotonergic neurons are primarily responsible for the storage and release of DA derived from exogenous L-DOPA in the absence of dopaminergic neurons.     

Impaired processing and presentation of cytotoxic-T-lymphocyte (CTL) epitopes are major escape mechanisms from CTL immune pressure in human immunodeficiency virus type 1 infection

Investigating escape mechanisms of human immunodeficiency virus type 1 (HIV-1) from cytotoxic T lymphocytes (CTLs) is essential for understanding the pathogenesis of HIV-1 infection and developing effective vaccines. To study the processing and presentation of known CTL epitopes, we prepared Epstein-Barr virus-transformed B cells that endogenously express the gag gene of six field isolates by adopting an env/nef-deletion HIV-1 vector pseudotyped with vesicular stomatitis virus G protein and then tested them for the recognition by Gag epitope-specific CTL lines or clones. We observed that two field variants, SLFNTVAVL and SVYNTVATL, of an A*0201-restricted Gag CTL epitope SLYNTVATL, and three field variants, KYRLKHLVW, QYRLKHIVW, and RYRLKHLVW, of an A24-restricted Gag CTL epitope KYKLKHIVW escaped from being killed by the CTL lines, despite the fact that they were recognized when the synthetic peptides corresponding to these variant sequences were exogenously loaded onto the target cells. Thus, their escape is likely due to the changes that occur during the processing and presentation of epitopes in the infected cells. Mutations responsible for this mode of escape were located within the epitope regions rather than the flanking regions, and such mutations did not influence the virus replication. The results suggest that the impaired antigen processing and presentation often occur in HIV-1 field isolates and thus are one of the major mechanisms that enable HIV-1 to escape from CTL recognition. We emphasize the importance of testing HIV-1 variants in an endogenous expression system.     

Early-phase redistribution of the cation-independent mannose 6-phosphate receptor by U18666A treatment in HeLa cells

It has been shown that the treatment with 3-[2-(diethylamino)ethoxy] androst-5-en-17-one (U18666A) causes the accumulation of cholesterol and the cation-independent mannose 6-phosphate receptor (CIMPR) in late endosomal/lysosomal compartments in BHK cells. The present study reports on a study of the effect of U18666A on CIMPR distribution in more detail in HeLa cells. When cells were treated with U18666A for 20 h, the intense perinuclear signal for CIMPR corresponding to the trans-Golgi network (TGN) disappeared and lamp1-negative punctate signals, scattered in the perinuclear region were detected. CIMPR then began to accumulate in lamp1-positive compartments 48 h after addition of the drug. Double immunofluorescence microscopy showed that U18666A-induced mannose 6-phosphate receptor-containing compartments (U-MPRCs), which were formed in the early phase of the redistribution, contained no marker for the TGN, late endosomes or lysosomes. Approximately half of the structures contained transferrin that had been internalized for 20 min, and cathepsin D, the majority of which appeared to be its precursor form. Immunoelectron-microscopic analysis revealed that U-MPRCs are composed of multivesicular bodies, irregularly shaped structures, and vesicular structures adjacent to the multivesicular bodies. These results suggest that U18666A treatment primarily suppresses the CIMPR transport pathways to late endosomes and from transferrin-containing endosomes, both of which may be dependent on cholesterol function.This work was supported by grants from Japan Ministry of Education, Culture, Sports, Science, and Technology.     

D1-like dopamine receptor-mediated function in congenic mutants with D1 vs. D5 receptor "knockout"

Current understanding of the functional roles of individual dopamine D1-like [D1, D5] and D2-like [D2L/s, D3, D4] receptor subtypes remains incomplete. In particular, the lack of pharmacological agonists and antagonists able to distinguish between D1 and D5 receptors means that any differential roles in the regulation of behavior are poorly understood. Mutant mice with targeted gene deletion ("knockout") of individual dopamine receptor subtypes offer an important alternative approach to resolving these functional roles. In congenic D1 mutants examined ethologically, progressive increases in specific topographies of behavior over wildtypes were considerably greater than those in D1 mutants on a mixed genetic background; D1 knockout appears to influence the neuronal substrate(s) of habituation to disrupt sculpture of the changing topography of behavior from initial exploration through to quiescence. Similarly, the D1 receptor appears to regulate specific topographies of orofacial movement in the mouse as these are "sculpted" in a time-dependent manner. Although the well-recognized role of the D1-like family in regulating several aspects of behavioral topography has been assumed to involve primarily D1 receptors, this presumption may require modification to accommodate a subtle but not negligible role for their D5 counterparts as evidenced in the phenotype of congenic D5 mutants.     

Functionally impaired HIV-specific CD8 T cells show high affinity TCR-ligand interactions

We eventually isolated two different clonotypic CD8 T cell subsets recognizing an HIV Pol-derived epitope peptide (IPLTEEAEL) in association with HLA-B35 from a chronic HIV-infected patient. By kinetic analysis experiments, the subsets showed a >3-fold difference in half-lives for the HLA tetramer in complex with the Pol peptide. In functional assays in vitro and ex vivo, both subsets showed substantial functional avidity toward peptide-loaded cells. However, the high affinity subset did not show cytolytic activity, cytokine production, or proliferation activity toward HIV-infected cells, whereas the moderate affinity one showed potent activities. Furthermore, using ectopic expression of each of the TCR genes into primary human CD8 T cells, the CD8 T cells transduced with the high affinity TCR showed greater binding activity toward the tetramer and impaired cytotoxic activity toward HIV-infected cells, corroborating the results obtained with parental CD8 T cells. Taken together, these data indicate that impaired responsiveness of T cells toward HIV-infected cells can occur at the level of TCR-ligand interactions, providing us further insight into the immune evasion mechanisms by HIV.     

Reserpine pretreatment prevents increases in extracellular striatal dopamine following L-DOPA administration in rats with nigrostriatal denervation

The influence of L-DOPA and reserpine on extracellular dopamine (DA) levels in the striatum of intact and dopaminergic denervated rats was studied using the brain microdialysis technique. In intact rats, reserpine (5 mg/kg s.c.) reduced extracellular DA levels to 4% of basal values. L-DOPA (50 mg/kg i.p.) had no effect on extracellular DA levels in reserpine-pretreated rats. In rats with 6-hydroxydopamine-induced lesion of the nigrostriatal dopaminergic system, basal levels of extracellular DA were low but markedly increased by L-DOPA (50 mg/kg i.p.). In 6-hydroxydopamine-lesioned rats, pretreatment with reserpine (5 mg/kg s.c.) diminished L-DOPA (50 mg/kg i.p.)-induced increases in extracellular DA levels to 16% of those obtained in denervated animals not pretreated with reserpine (p<0.01). These results suggest that in the intact striatum, extracellular DA stems mainly from vesicular storage sites and that in the striatum with dopaminergic denervation, a large part of the L-DOPA-derived extracellular DA is also derived from a vesicular pool that is released by an exocytosis mechanism.     

Nonstandard peptide binding revealed by crystal structures of HLA-B*5101 complexed with HIV immunodominant epitopes

The crystal structures of the human MHC class I allele HLA-B*5101 in complex with 8-mer, TAFTIPSI, and 9-mer, LPPVVAKEI, immunodominant peptide epitopes from HIV-1 have been determined by x-ray crystallography. In both complexes, the hydrogen-bonding network in the N-terminal anchor (P1) pocket is rearranged as a result of the replacement of the standard tyrosine with histidine at position 171. This results in a nonstandard positioning of the peptide N terminus, which is recognized by B*5101-restricted T cell clones. Unexpectedly, the P5 peptide residues appear to act as anchors, drawing the peptides unusually deeply into the peptide-binding groove of B51. The unique characteristics of P1 and P5 are likely to be responsible for the zig-zag conformation of the 9-mer peptide and the slow assembly of B*5101. A comparison of the surface characteristics in the alpha1-helix C-terminal region for B51 and other MHC class I alleles highlights mainly electrostatic differences that may be important in determining the specificity of human killer cell Ig-like receptor binding.