Anti-CD3 antibody-induced activated killer cells: cytokines as the additional signals for activation of killer cells in effector phase to mediate slow lysis.

This study examines the role of cytokines in activating the effector cells to mediate slow lysis. After activation of splenocytes by alpha CD3, further culturing the cells in the absence of alpha CD3 resulted in the generation of activated killer cells (CD3-AK-) to mediate slow lysis. In contrast to fast lysis which was not affected by a PKC inhibitor H-7, slow lysis was inhibited. These findings suggested that a PKC-dependent activation phase preceded the lytic phase in slow lysis. To explore the mechanism for activating the lytic machinery in slow lysis, we examined the roles of cytokines in these reactions. First, it was found that alpha IL-2 or an alpha IL-2/alpha IL-4 combination inhibited slow lysis but had no effect on fast lysis. Secondly, IL-2, IL-4, or TNF alpha converted a noncytolytic CD3-AK- cells to mediate slow lysis, but they did not augment fast lysis. IL-2 and IL-4 had additive effect, and TNF alpha synergized with IL-2 to further augment the CD3-AK- cytolytic activity. Exogenous IL-6 and INF did not have any appreciable effect on the cytolytic activity of the killer cells. Besides TNF alpha, these cytokines were not directly cytotoxic to the target cells, indicating that they were not cytotoxic factors per se. Treatment with cycloheximide for 24 hr abrogated the cytolytic activities of CD3-AK cells, suggesting that a cytotoxic factor(s) was continuously synthesized to be stored in activated killer cells and was catabolized within 24 hr. Our results indicated that in the effector phase of slow lysis, after activating the CD3-AK- cells by the first signal (appropriate target cells), IL-2 and/or IL-4 appeared to be the second signal to initiate a cascade of events which triggered the release of other cytokines (e.g., TNF). This process resembles the secondary (memory) type of immune response. These events lead to full activation of the killer cells and converted the preformed cytotoxic factors into active form to initiate the lytic reaction and completed the lytic process.     

Revaluation of the role of cholesterol in stabilizing rafts implicated in T cell receptor signaling

T lymphocytes contain two kinetic pools of cholesterol extractable with methyl-beta-cyclodextrin (m-beta-CD): a fast pool (31.5%, t1/2=17 s) and a slow pool (68.5%, t1/2=15 min). Purification of detergent-resistant membranes (DRMs) shows that the fast pool corresponds to buoyant cholesterol. Cholesterol extraction of the fast pool (i.e. cholesterol from rafts) still allows the buoyancy of signaling proteins and their phosphorylation under CD3 stimulation. Cholesterol depletion of the slow pool (i.e. cholesterol from membranes other than rafts) is accompanied by the extraction of the whole raft followed by the inhibition of CD3-induced tyrosine-phosphorylations. Cholesterol oxidase (COase) allows a specific oxidation of raft cholesterol into cholestenone. Cholestenone leaves the DRMs and accumulates as Triton X-100-soluble material. Specific cholesterol-rich raft disruption by COase does not inhibit the activation of either Jurkat cells or T CD4+ lymphocytes. Our study challenges the real role of cholesterol-rich rafts in CD3/TCR signaling and suggests that a cholesterol-poor subtype of rafts is involved in signal transmission via the TCR.     

Quantitative analysis of the antiviral activity of CD8(+) T cells from human immunodeficiency virus-positive asymptomatic patients with different rates of CD4(+) T-cell decrease

We have measured in 22 asymptomatic human immunodeficiency virus type 1-infected patients (10 rapid progressors and 12 slow progressors) the proviral load of CD4(+) T cells homogeneously superinfected by the same dose of a non-syncytium-inducing virus in the presence or in the absence of autologous CD8(+) T cells. We demonstrated that the antiviral activity of CD8(+) T cells was highly predictive of the rate of peripheral CD4(+) T-cell decline.     

On the mechanism of human stefin B folding: I. Comparison to homologous stefin A. Influence of pH and trifluoroethanol on the fast and slow folding phases

The folding of human stefin B has been studied by several spectroscopic probes. Stopped-flow traces obtained by circular dichroism in the near and far UV, by tyrosine fluorescence, and by extrinsic probe ANS fluorescence are compared. Most (60 ± 5%) of the native signal in the far UV circular dichroism (CD) appeared within 10 ms in an unresolved “burst” phase, which was followed by a fast phase (t = 83 ms) and a slow phase (t = 25 s) with amplitudes of 30% and 10%, respectively. Similar fast and slow phases were also evident in the near UV CD, ANS fluorescence, and tyrosine fluorescence. By contrast, human stefin A, which has a very similar structure, exhibited only one kinetic phase of folding (t = 6 s) detected by all the spectroscopic probes, which occurred subsequent to an initial “burst” phase observed by far UV CD. It is interesting that despite close structural similarity of both homologues they fold differently, and that the less stable human stefin B folds faster by an order of magnitude (comparing the non-proline limited phase). To gain more information on the stefin B folding mechanism, effects of pH and trifluoroethanol (TFE) on the fast and slow phases were investigated by several spectroscopic probes. If folding was performed in the presence of 7% of TFE, rate acceleration and difference in the mechanism were observed. Protein 32:296–303, 1998. © 1998 Wiley-Liss, Inc.     

Decay kinetics of human immunodeficiency virus-specific CD8+ T cells in peripheral blood after initiation of highly active antiretroviral therapy.

We measured the longitudinal responses to 95 HLA class I-restricted human immunodeficiency virus (HIV) epitopes and an immunodominant HLA A2-restricted cytomegalovirus (CMV) epitope in eight treatment-naive HIV-infected individuals, using intracellular cytokine staining. Patients were treated with highly active antiretroviral therapy (HAART) for a median of 78 weeks (range, 34 to 121 weeks). Seven of eight patients maintained an undetectable viral load for the duration of therapy. A rapid decline in HIV-specific CD8(+) T-cell response was observed at initiation of therapy. After an undetectable viral load was achieved, a slower decrease in HIV-specific CD8(+) T-cell response was observed that was well described by first-order kinetics. The median half-life for the rate of decay was 38.8 (20.3 to 68.0) weeks when data were expressed as percentage of peripheral CD8(+) T cells. In most cases, data were similar when expressed as the number of responding CD8(+) T cells per microliter of blood. In subjects who responded to more than one HIV epitope, rates of decline in response to the different epitopes were similar and varied by a factor of 2.2 or less. Discontinuation of treatment resulted in a rapid increase in HIV-specific CD8(+) T cells. Responses to CMV increased 1.6- and 2.8-fold within 16 weeks of initiation of HAART in two of three patients with a measurable CMV response. These data suggest that HAART quickly starts to restore CD8(+) T-cell responses to other chronic viral infections and leads to a slow decrease in HIV-specific CD8(+) T-cell response in HIV-infected patients. The slow decrease in the rate of CD8(+) T-cell response and rapid increase in response to recurrent viral replication suggest that the decrease in CD8(+) T-cell response observed represents a normal memory response to withdrawal of antigen.     

The biological activity of human CD20 monoclonal antibodies is linked to unique epitopes on CD20

We have previously defined a panel of fully human CD20 mAb. Most of these were unexpectedly efficient in their ability to recruit C1q to the surface of CD20-positive cells and mediate tumor lysis via activation of the classical pathway of complement. This complement-dependent cytotoxicity (CDC) potency appeared to relate to the unusually slow off-rate of these human Abs. However, we now present epitope-mapping data, which indicates that all human mAb bind a novel region of CD20 that may influence CDC potency. Epitope mapping, using both mutagenesis studies and overlapping 15-mer peptides of the extracellular loops of CD20, defined the amino acids required for binding by an extensive panel of mouse and human mAb. Binding by rituximab and mouse CD20 mAb, had an absolute requirement for alanine and proline at positions 170 and 172, respectively, within the large extracellular loop of CD20. Surprisingly, however, all of the human CD20 mAb recognize a completely novel epitope located N-terminally of this motif, also including the small extracellular loop of CD20. Thus, although off-rate may influence biological activity of mAb, another critical factor for determining CDC potency by CD20 mAb appears to be the region of the target molecule they recognize. We conclude that recognition of the novel epitope cooperates with slow off-rate in determining the activity of CD20 Ab in activation of complement and induction of tumor cell lysis.     

Simian immunodeficiency virus infection induces severe loss of intestinal central memory T cells which impairs CD4+ T-cell restoration during antiretroviral therapy

BACKGROUND: Simian immunodeficiency virus (SIV) infection leads to severe loss of intestinal CD4(+) T cells and, as compared to peripheral blood, restoration of these cells is slow during antiretroviral therapy (ART). Mechanisms for this delay have not been examined in context of which specific CD4(+) memory subsets or lost and fail to regenerate during ART. METHODS: Fifteen rhesus macaques were infected with SIV, five of which received ART (FTC/PMPA) for 30 weeks. Viral loads were measured by real-time PCR. Flow cytometric analysis determined changes in T-cell subsets and their proliferative state. RESULTS: Changes in proliferative CD4(+) memory subsets during infection accelerated their depletion. This reduced the central memory CD4(+) T-cell pool and contributed to slow CD4(+) T-cell restoration during ART. CONCLUSION: There was a lack of restoration of the CD4(+) central memory and effector memory T-cell subsets in gut-associated lymphoid tissue during ART, which may contribute to the altered intestinal T-cell homeostasis in SIV infection.     

Fast progression of recombinant human myelin/oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis in marmosets is associated with the activation of MOG34-56-specific cytotoxic T cells

The recombinant human (rh) myelin/oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) model in the common marmoset is characterized by 100% disease incidence, a chronic disease course, and a variable time interval between immunization and neurological impairment. We investigated whether monkeys with fast and slow disease progression display different anti-MOG T or B cell responses and analyzed the underlying pathogenic mechanism(s). The results show that fast progressor monkeys display a significantly wider specificity diversification of anti-MOG T cells at necropsy than slow progressors, especially against MOG(34-56) and MOG(74-96). MOG(34-56) emerged as a critical encephalitogenic peptide, inducing severe neurological disease and multiple lesions with inflammation, demyelination, and axonal injury in the CNS. Although EAE was not observed in MOG(74-96)-immunized monkeys, weak T cell responses against MOG(34-56) and low grade CNS pathology were detected. When these cases received a booster immunization with MOG(34-56) in IFA, full-blown EAE developed. MOG(34-56)-reactive T cells expressed CD3, CD4, or CD8 and CD56, but not CD16. Moreover, MOG(34-56)-specific T cell lines displayed specific cytotoxic activity against peptide-pulsed B cell lines. The phenotype and cytotoxic activity suggest that these cells are NK-CTL. These results support the concept that cytotoxic cells may play a role in the pathogenesis of multiple sclerosis.     

Transitions from high to low molecular weight isoforms of CD45 (T200) involve rapid activation of alternate mRNA splicing and slow turnover of surface CD45R

The expression of CD45 isoforms and of CDw29 has been analyzed as a function of time by correlating cell surface phenotype with mRNA synthesis. After activation, T cells lose CD45R and acquire a high density of CD45 p180 and of CDw29. Throughout this transition the density of CD45 common determinants steadily increases, resulting in a net gain of surface CD45. The gradual loss of CD45R could reflect a rapid switch in CD45 mRNA splicing patterns followed by a slow loss of surface CD45R. Alternatively it could reflect a delayed activation of splicing to produce the 4.8-kb CD45 mRNA, or long lived 5.4-kb CD45 mRNA. Analysis of CD45 mRNA indicated that at 24 h postactivation, 5.4-kb CD45 mRNA is lost and only 4.8-kb mRNA is detectable. The amount of 4.8-kb mRNA increases until day 3 and then decreases somewhat. Thus our results support the interpretation that transitions in CD45 isoform mRNA expression occur within the first 24 h after activation and that the persistence of CD45R+ T cells until day 3 to 4 of culture results from slow turnover of surface CD45R glycoprotein.     

Structural polymorphism of human telomere G-quadruplex induced by a pyridyl carboxamide molecule

In this work, we described a kinetically slow (hour-scale) but thermodynamically favored G-quadruplex conversion induced by a pyridyl carboxamide molecule. This slow transition was observed through CD spectra and gels, and its final stable parallel conformation was identified by 2-aminopurine experiments. Kinetic experiments indicated that this slow process was a first-order reaction, implying it was a unimolecular conversion. Quite distinctly from other reported ligand-driven G-quadruplex conformation alteration, this slow conversion reveals a novel insight into G-quadruplex polymorphism, in accordance with the behavior of human telomere G-quadruplex in a molecular crowding environment in K(+) solution, further enriching the known structural polymorphism of human telomere DNA and providing new consideration for drug design based on G-quadruplexes.     

Folding kinetics of the protein pectate lyase C reveal fast-forming intermediates and slow proline isomerization

Pectate lyase C (pelC) is a member of the class of proteins that possess a parallel beta-helix folding motif. A study of the kinetic folding mechanism is presented in this report. Kinetic circular dichroism (CD) and fluorescence have been used to observe changes in the structure of pelC as a function of time upon folding and unfolding. Three folding phases are observed with far-UV CD and four phases are observed with near-UV CD. The two slowest phases have relaxation times on the order of 21 and 46 s in aqueous buffer. Double-jump refolding assays and the measured activation enthalpies (16.0 and 21.2 kcal/mol for the respective slow phases) suggest that these two phases are the result of the slow cis-trans isomerization of prolyl-peptide bonds. We have determined that the earliest observed folding phase involves the formation of most, if not all, of the secondary structure with a relaxation time of 0.25 s. We also observed a phase by near-UV CD on the order of 0.25 s. This suggests that along with the appearance of secondary structure, some tertiary contacts are made. There is one kinetic phase observed in the near-UV CD and fluorescence that has no corresponding far-UV CD phase. This occurs with a relaxation time of 1.1 s. The temperature dependence of the natural log of the folding rate constant suggests that folding occurs via a sequential mechanism in which an on-pathway intermediate in rapid equilibrium with the unfolded protein is present. Semiempirical CD calculations support the idea that the beta-helix region of pelC forms in the fast kinetic phase, yielding near-native secondary and tertiary structures in that region. This is followed by the slower formation of the loop regions connecting individual strands of the beta-helix.     

Human CD4+ CD25 high cells suppress proliferative memory lymphocyte responses to herpes simplex virus type 2

Lymphocytes with the regulatory CD4+ CD25+ phenotype frequently suppress memory T-cell responses. Murine herpes simplex virus type 1 (HSV-1) models have shown that CD4+ CD25+ cells can limit immunity-mediated corneal damage but slow viral clearance. We investigated the effect of CD4+ CD25+ cells from healthy HSV-2-infected humans on recall proliferative (CD4) responses to HSV-2. Depletion and reconstitution experiments were consistent with a suppressive effect of autologous blood-derived CD4+ CD25+ cells for whole HSV-2 antigen. Regulatory T cells may modulate human CD4 memory responses to HSV-2 and influence their antiviral and inflammatory functions.     

Hyaluronan Anchored to Activated CD44 on Central Nervous System Vascular Endothelial Cells Promotes Lymphocyte Extravasation in Experimental Autoimmune Encephalomyelitis

The extravasation of lymphocytes across central nervous system (CNS) vascular endothelium is a key step in inflammatory demyelinating diseases including multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). The glycosaminoglycan hyaluronan (HA) and its receptor, CD44, have been implicated in this process but their precise roles are unclear. We find that CD44^−/− mice have a delayed onset of EAE compared with wild type animals. Using an in vitro lymphocyte rolling assay, we find that fewer slow rolling (<1 μm/s) wild type (WT) activated lymphocytes interact with CD44^−/− brain vascular endothelial cells (ECs) than with WT ECs. We also find that CD44^−/− ECs fail to anchor HA to their surfaces, and that slow rolling lymphocyte interactions with WT ECs are inhibited when the ECs are treated with a pegylated form of the PH20 hyaluronidase (PEG-PH20). Subcutaneous injection of PEG-PH20 delays the onset of EAE symptoms by ∼1 day and transiently ameliorates symptoms for 2 days following disease onset. These improved symptoms correspond histologically to degradation of HA in the lumen of CNS blood vessels, decreased demyelination, and impaired CD4⁺ T-cell extravasation. Collectively these data suggest that HA tethered to CD44 on CNS ECs is critical for the extravasation of activated T cells into the CNS providing new insight into the mechanisms promoting inflammatory demyelinating disease.     

Histology of vermiform appendix-like organ in slow loris.

The vermiform appendix-like organ (VALO) of the slow loris was investigated for its histology and immunohistochemical characteristics. The VALO has a much thinner wall with flat folded mucosa and shallower crypts than the cecal mucosa, while cellular components and population of the mucosa were similar to those of the cecum. No coalescent lymph nodules were seen in the submucosa. Immunohistochemically 5-HT-positive cells in the crypts and CD3- and CD8-positive lymphocytes in the lymph nodules were shown in the VALO as well as in the cecum. These findings suggest that the VALO is a low-differentiated vermiform appendix of the slow loris.     

Folding of dihydrofolate reductase from Escherichia coli

The urea-induced equilibrium unfolding transition of dihydrofolate reductase from Escherichia coli was monitored by UV difference, circular dichroism (CD), and fluorescence spectroscopy. Each of these data sets were well described by a two-state unfolding model involving only native and unfolded forms. The free energy of folding in the absence of urea at pH 7.8, 15 degrees C is 6.13 +/- 0.36 kcal mol-1 by difference UV, 5.32 +/- 0.67 kcal mol-1 by CD, and 5.42 +/- 1.04 kcal mol-1 by fluorescence spectroscopy. The midpoints for the difference UV, CD, and fluorescence transitions are 3.12, 3.08, and 3.18 M urea, respectively. The near-coincidence of the unfolding transitions monitored by these three techniques also supports the assignment of a two-state model for the equilibrium results. Kinetic studies of the unfolding and refolding reactions show that the process is complex and therefore that additional species must be present. Unfolding jumps in the absence of potassium chloride revealed two slow phases which account for all of the amplitude predicted by equilibrium experiments. Unfolding in the presence of 400 mM KCl results in the selective loss of the slower phase, implying that there are two native forms present in equilibrium prior to unfolding. Five reactions were observed in refolding: two slow phases designated tau 1 and tau 2 that correspond to the slow phases in unfolding and three faster reactions designated tau 3, tau 4, and tau 5 that were followed by stopped-flow techniques. The kinetics of the recovery of the native form was monitored by following the binding of methotrexate, a tight-binding inhibitor of dihydrofolate reductase, at 380 nm.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evolution of human immunodeficiency virus type 1 env sequence variation in patients with diverse rates of disease progression and T-cell function.

We examined the relationship between env sequence variation and disease progression in 10 human immunodeficiency virus type 1 (HIV-1)-seropositive subjects selected from a longitudinal cohort receiving zidovudine therapy. Five subjects were chosen for stable clinical status and CD4 counts (slow progressors), and five were selected for rapid clinical deterioration and CD4 count decline (rapid progressors). The slow progressors had significantly lower plasma viral RNA loads and greater lymphoproliferative responses to mitogens than the rapid progressors. DNA sequences representing the C1 through C3 regions of env were amplified from two peripheral blood mononuclear cell DNA samples from each subject separated by an average of 2.5 years. Molecular clones of these amplicons were then sequenced, and DNA sequence and deduced amino acid sequence distances were compared. Inter-time point sequence comparison showed a higher rate of sequence evolution for the rapid progressors in three of five matched pairs of rapid progressors and slow progressors and for the slow progressors in the remaining two subject pairs. However, intra-time point sequence comparisons showed that four of five slow progressors developed a more diverse quasispecies over time and one showed no change. In contrast, four of five rapid progressors showed no change in quasispecies diversity over time and one showed a significant decrease in diversity. The overall C1 through C3 region quasispecies diversity in the slow progressors at baseline was lower than that for the rapid progressors, but this difference was not significant at the follow-up time points. These diversity relationships were obscured if sequence analyses were limited to the 300-bp C2 to V3 region. Thus, HIV-1 quasispecies diversity increased over time in subjects with more functional immune systems.     

Slow-folding kinetics of ribonuclease-A by volume change and circular dichroism: evidence for two independent reactions.

The slow refolding of guanidine-HCl-denatured ribonuclease-A was studied by volume change and by kinetic CD at 222 and 276 nm. Dilatometric measurements revealed that on refolding there is a fast volume change of +232 mL/mol of protein. This is followed by a very slow nonexponential change that takes about 25 min to reach equilibrium. By adding varying amounts of (NH4)2SO4, the slow volume change curve was resolved into 2 concurrent reactions. The faster of the 2 slow events entails a negative volume change of -64 mL/mol of protein and appears to arise from proline isomerization. The slower process, attended by a positive change of +53 mL/mol of protein, has properties consistent with the "XY" reaction of Lin and Brands (1983, Biochemistry 22:563-573). This reaction is so named because the conformational nature of neither its initial (Y) nor its final state (X) is known; the transition is characterized solely by its absorbance and fluorescence kinetics. These are the first direct physical measures attributable to the "XY" process. The early formation of a compact structure in the event responsible for the rapid +232-mL/mol volume change, however, is consistent with the sequential model of folding (Cook KH, Schmid FX, Baldwin RL, 1979, Proc Natl Acad Sci USA 76:6157-6161; Kim PS, Baldwin RL, 1980, Biochemistry 19:6124-6129). The usefulness of volume change measurements as a method of detecting structural rearrangements was confirmed by finding agreement between time constants obtained from parallel volume change and kinetic CD experiments. The measured volume changes arise from both changes in hydration and changes in the packing of atoms in the interior of the protein.     

GPI-anchored receptor clusters transiently recruit Lyn and G alpha for temporary cluster immobilization and Lyn activation: single-molecule tracking study 1

The signaling mechanisms for glycosylphosphatidylinositol-anchored receptors (GPI-ARs) have been investigated by tracking single molecules in living cells. Upon the engagement or colloidal gold-induced cross-linking of CD59 (and other GPI-ARs) at physiological levels, CD59 clusters containing three to nine CD59 molecules were formed, and single molecules of Galphai2 or Lyn (GFP conjugates) exhibited the frequent but transient (133 and 200 ms, respectively) recruitment to CD59 clusters, via both protein-protein and lipid-lipid (raft) interactions. Each CD59 cluster undergoes alternating periods of actin-dependent temporary immobilization (0.57-s lifetime; stimulation-induced temporary arrest of lateral diffusion [STALL], inducing IP(3) production) and slow diffusion (1.2 s). STALL of a CD59 cluster was induced right after the recruitment of Galphai2. Because both Galphai2 and Lyn are required for the STALL, and because Lyn is constitutively recruited to CD59 clusters, the STALL of CD59 clusters is likely induced by the Galphai2 binding to, and its subsequent activation of, Lyn within the same CD59 cluster.     

Phosphoinositide hydrolysis in mitogen-stimulated human peripheral-blood T lymphocytes.

Both phytohaemagglutinin and antibodies to the CD3 molecule induced proliferation and phosphoinositide hydrolysis in human peripheral-blood T lymphocytes, but the magnitude of the inositol phosphate response was small and the rate of accumulation slow [significant increases in Ins(1,4,5)P3 were observed only after 10 min]. Hence this response differs from the well-characterized Ins(1,4,5)P3 responses of many other systems. This slow response, its abrogation in Ca2+-depleted medium, the slow and maintained increase in Ca2+ as measured by Quin-2, and the ability of the Ca2+ ionophore A23187 to stimulate Ins(1,4,5)P3 accumulation all suggest that the increase in Ins(1,4,5)P3 occurs, at least in part, as a result of receptor-mediated Ca2+ influx in mitogen-stimulated T lymphocytes.     

Kinetics of recombination of heavy and light chains of a human IgG1 myeloma protein.

The recombination of alkylated H and L chains of a human myeloma protein (Jo) was studied by means of circular dichroism (CD). Marked CD changes were observed at 295 and 235 nm when H and L chains recombined. The change in the CD maximum at 235 nm was followed with time after mixing preparations of H and L chains in the pH range between 4 and 6. The recombination reaction was slow and followed second order kinetics. The observed rate constants were markedly dependent on pH. The pH dependence of the rate constant was analyzed assuming that there are two forms of H chain which are in a pH-dependent equilibrium with each other.