Extensive replicative capacity of human central memory T cells

To characterize the replicative capacity of human central memory (T(CM)) CD4 T cells, we have developed a defined culture system optimized for the ex vivo expansion of Ag-specific CD4(+) T cells. Artificial APCs (aAPCs) consisting of magnetic beads coated with Abs to HLA class II and a costimulatory Ab to CD28 were prepared; peptide-charged HLA class II tetramers were then loaded on the beads to provide Ag specificity. Influenza-specific DR*0401 CD4 T(CM) were isolated from the peripheral blood of normal donors by flow cytometry. Peptide-loaded aAPC were not sufficient to induce resting CD4 T(CM) to proliferate. In contrast, we found that the beads efficiently promoted the growth of previously activated CD4 T(CM) cells, yielding cultures with >80% Ag-specific CD4 cells after two stimulations. Further stimulation with peptide-loaded aAPC increased purity to >99% Ag-specific T cells. After in vitro culture for 3-12 wk, the flu-specific CD4 T(CM) had surface markers that were generally consistent with an effector phenotype described for CD8 T cells, except for the maintenance of CD28 expression. The T(CM) were capable of 20-40 mean population doublings in vitro, and the expanded cells produced IFN-gamma, IL-2, and TNF-alpha in response to Ag, and a subset of cells also secreted IL-4 with PMA/ionomycin treatment. In conclusion, aAPCs expand T(CM) that have extensive replicative capacity, and have potential applications in adoptive immunotherapy as well as for studying the biology of human MHC class II-restricted T cells.     

SHP-1 and SHP-2 associate with immunoreceptor tyrosine-based switch motif of programmed death 1 upon primary human T cell stimulation, but only receptor ligation prevents T cell activation

To study the cis- and trans-acting factors that mediate programmed death 1 (PD-1) signaling in primary human CD4 T cells, we constructed a chimeric molecule consisting of the murine CD28 extracellular domain and human PD-1 cytoplasmic tail. When introduced into CD4 T cells, this construct mimics the activity of endogenous PD-1 in terms of its ability to suppress T cell expansion and cytokine production. The cytoplasmic tail of PD-1 contains two structural motifs, an ITIM and an immunoreceptor tyrosine-based switch motif (ITSM). Mutation of the ITIM had little effect on PD-1 signaling or functional activity. In contrast, mutation of the ITSM abrogated the ability of PD-1 to block cytokine synthesis and to limit T cell expansion. Further biochemical analyses revealed that the ability of PD-1 to block T cell activation correlated with recruitment of Src homology region 2 domain-containing phosphatase-1 (SHP-1) and SHP-2, and not the adaptor Src homology 2 domain-containing molecule 1A, to the ITSM domain. In TCR-stimulated T cells, SHP-2 associated with PD-1, even in the absence of PD-1 engagement. Despite this interaction, the ability of PD-1 to block T cell activation required receptor ligation, suggesting that colocalization of PD-1 with CD3 and/or CD28 may be necessary for inhibition of T cell activation.     

In vivo VL-targeted activation-induced apoptotic supraclonal deletion by a microbial B cell toxin

To interfere with host immune responses, some microbial pathogens produce proteins with the properties of superantigens, which can interact via conserved V region framework subdomains of the Ag receptors of lymphocytes rather than the complementarity-determining region involved in the binding of conventional Ags. In recent studies, we have elucidated how a model B cell superantigen affects the host immune system by targeting a conserved V(H) site on the Ag receptors of B lymphocytes. To determine whether these findings represent a general paradigm, we investigated the in vivo immunobiologic properties of protein L of Peptostreptococcus magnus (PpL), a microbial Ig-binding protein specific for a V region site on Ig L chains. Our studies confirmed that PpL binding is restricted to a subset of murine Vkappa-expressing B cells, and found that B cells with stronger PpL-binding activity are associated with certain B cell subsets: splenic marginal zone (CD21(high) CD23(low)), splenic CD1(+), peritoneal B-1a (IgD(low) CD5(+)), and CD21(high) CD24(high) B cells in peripheral lymph nodes, mesenteric lymph nodes, and Peyer's patches. Infusion of PpL triggered a sequence of events in B cell receptor (BCR)-targeted B cells, with rapid down-regulation of BCR, the induction of an activation phenotype, and limited rounds of proliferation. Apoptosis followed through a process heralded by the dissipation of mitochondrial membrane potential, the induction of the caspase pathway, DNA fragmentation, and the deposition of B cell apoptotic bodies. These studies define a common pathway by which microbial toxins that target V region-associated BCR sites induce programmed cell death.     

6-hydroxydopamine lesions in anuran amphibians: a new model system for Parkinson's disease?

We investigated the effects of dopamine depletion on acoustically guided behavior of anurans by conducting phonotaxis experiments with female gray treefrogs (Hyla versicolor) before and 90 min after bilateral injections of 3, 6, or 12 microg 6-hydroxydopamine (6-OHDA) into the telencephalic ventricles. In experiments with one loudspeaker playing back a standard artificial mating call, we analyzed the effects of 6-OHDA on phonotactic response time. In choice tests we measured the degree of distraction from the standard call (20 pulses/s) by three different variants with altered pulse-rate (30/s, 40/s, 60/s). Five days after experiments, brains were immunostained for tyrosine hydroxylase. Labeled neurons were counted in the suprachiasmatic nucleus, posterior tuberculum, interpeduncular nucleus, and locus coeruleus, and correlation between neuronal numbers and behavioral scores was tested. Response times increased together with 6-OHDA concentrations, which was mainly due to longer immobile periods before the animals started movement. In choice tests the most irrelevant stimulus (60/s) distracted 6-OHDA injected females from the standard stimulus, while sham injected controls were undistracted. The number of catecholaminergic neurons decreased with increasing 6-OHDA concentration in the suprachiasmatic nucleus, posterior tuberculum, and interpeduncular nucleus. The normalized number of immunoreactive neurons in the posterior tuberculum was positively correlated with phonotaxis scores in the one-speaker test, demonstrating that motor deficits are a function of tubercular cell loss. We conclude that bilateral 6-OHDA lesions in anuran amphibians cause motor (difficulty to start movements) as well as cognitive symptoms (higher distraction by irrelevant stimuli) that have also been described for human Parkinson patients.     

Acid sphingomyelinase and inhibition by phosphate ion: role of inhibition by phosphatidyl-myo-inositol 3,4,5-triphosphate in oligodendrocyte cell signaling

There is ample evidence that both acid (ASMase) and neutral (NSMase) sphingomyelinases play a role in cell death so inhibitors of either enzyme could have significant value as protectors against neurodegeneration. We used a fluorogenic sphingomyelinase substrate, 6-hexadecanoylamino-4-methylumbelliferyl-phosphorylcholine, and a [(14)C]choline-labeled sphingomyelin substrate to screen large numbers of phosphocompounds for inhibition of ASMase in extracts of human oligodendroglioma cells (HOG) and neonatal rat oligodendrocytes. Non-competitive inhibition was observed with inorganic phosphate and AMP, which was a more potent inhibitor of ASMase than cyclic AMP, ADP or ATP. However, other nucleotide phosphates, sugar phosphates, nucleotide sugars and glycerol phosphate did not inhibit ASMase. Our key finding was that phosphatidyl-myo-inositol 3,4,5-triphosphate [PtdIns (3,4,5)P(3)] was a much more potent inhibitor of ASMase than lysophosphatidic acid or phosphatidyl-myo-inositol 4,5-diphosphate [PtdIns(4,5)P(2)]. When PtdIns(3,4,5)P(3) was added to cultured cells we observed 50% inhibition of ASMase but no inhibition of other lysosomal hydrolases. After transfection of HOG cells with the tumor supressor phosphatase and tensin homolog protein (PTEN), which hydrolyses PtdIns(3,4,5)P(3) to PtdIns(4,5)P(2), we observed a two-fold increase in ASMase activity. Furthermore, the phosphatidylinositol-3-kinase inhibitor wortmannin (which reduces PtdIns(3,4,5)P(3) levels) also resulted in activation of ASMase. We propose that the small amount of ASMase activity associated with detergent-resistant cell membranes (Rafts) is regulated by PtdIns(3,4,5)P(3) and is most likely involved in receptor clustering and capping.     

Amino acid 324 in the simian immunodeficiency virus SIVmac V3 loop can confer CD4 independence and modulate the interaction with CCR5 and alternative coreceptors

The V3 loop of the simian immunodeficiency virus (SIV) envelope protein (Env) largely determines interactions with viral coreceptors. To define amino acids in V3 that are critical for coreceptor engagement, we functionally characterized Env variants with amino acid substitutions at position 324 in V3, which has previously been shown to impact SIV cell tropism. These changes modulated CCR5 engagement and, in some cases, allowed the efficient usage of CCR5 in the absence of CD4. The tested amino acid substitutions had highly differential effects on viral infectivity. Eleven of sixteen substitutions disrupted entry via CCR5 or the alternative coreceptor GPR15. Nevertheless, most of these variants replicated in the macaque T-cell line 221-89 and some also replicated in rhesus macaque peripheral blood monocytes, suggesting that efficient usage of CCR5 and GPR15 on cell lines is not a prerequisite for SIV replication in primary cells. Four variants showed enhanced entry into the macaque sMagi reporter cell line. However, sMagi cells did not express appreciable amounts of CCR5 and GPR15 mRNA, and entry into these cells was not efficiently blocked by a small-molecule CCR5 antagonist, suggesting that sMagi cells express as-yet-unidentified entry cofactors. In summary, we found that a single amino acid at position 324 in the SIV Env V3 loop can modulate both the efficiency and the types of coreceptors engaged by Env and allow for CD4-independent fusion in some cases.     

The importance of seed reserves for seedling performance: an integrated approach using morphological,physiological, and stable isotope techniques

To investigate how seed reserves affect early seedling performance, we conducted a factorial greenhouse experiment using Lithocarpus densiflora (Tanoak). Seedlings were grown from large (5.8±0.7 g) and small (3.2±0.4 g) seeds and, following shoot emergence, seeds were either removed or left attached. Seedlings were harvested for quantification of biomass and ¹³C at seven time periods following seed removal (2, 4, 8, 16, 32, 64, 128 days) and seedling photosynthesis was measured three separate time periods (2–4, 49–82, 95–128 days after seed removal). Biomass increased for all seedlings, but the increase was significantly larger for seedlings with attached seeds than with removed seeds. Seed removal just after shoot emergence significantly decreased seedling biomass, but seed removal 64 days after shoot emergence had no effect on seedling biomass. Seedling photosynthesis per unit leaf area varied by time and seed presence, but not by seed size. At the first period, seedlings with attached seeds had significantly higher photosynthetic rates than seedlings with removed seeds, at the second period there was no effect of seed removal, and at the third time period seedlings with attached seeds had significantly lower photosynthetic rates than seedlings with removed seeds. Despite temporal variation in photosynthesis per unit leaf area, seedlings with attached seeds always had significantly greater leaf area than seedlings with removed seeds, resulting in significantly higher total plant photosynthesis at all three time periods. The ¹³C values of both the leaves and roots were more similar to that of the seed for seedlings with attached seeds than for seedlings with removed seeds, however, seed removal and seed size strongly affected root ¹³C. This study demonstrates that seed reserves have important effects on the early growth, physiology, and ¹³C of L. densiflora seedlings.     

Failure of reproductive assurance in the chasmogamous flowers of Polygala lewtonii (Polygalaceae), an endangered sandhill herb

Hypothetically, a species with both cleistogamous (CL) flowers and delayed selfing chasmogamous (CH) flowers should display high levels of reproductive assurance because, over time, obligate selfing by CL flowers should reduce inbreeding depression and delayed selfing in CH flowers should compensate for the absence of outcross pollen. We used pollinator-exclusion experiments to investigate reproductive assurance in the CH flowers of Polygala lewtonii, an herb with a mixed mating system. We followed CH flowers from bud-break to flower/fruit abscission to quantify fruit initiation and maturation and rates of floral development. We also evaluated the efficacy of the selfing mechanism, conducted pollinator watches to assess the likelihood of pollinator limitation, and performed regression analysis to determine the effect of flower position on fruit production. Pollinator exclusion significantly reduced fruit initiation and maturation. Investigation of floral development demonstrated that the selfing mechanism is largely dysfunctional in CH flowers, indicating the failure of reproductive assurance. Low observed rates of insect visitation appear to contradict high rates of CH fruit production in open-pollinated plants, particularly given the rarity of delayed selfing. In both treatments, flower position significantly affected fruit initiation, suggesting a role for resource limitation in both pollinator-excluded and open-pollinated flowers.