CD4<Superscript>+</Superscript> T-cell recognition of human 5T4 oncofoetal antigen: implications for initial depletion of CD25<Superscript>+</Superscript> T cells

Background The human 5T4 (h5T4) oncofoetal antigen is expressed by a wide variety of human carcinomas including colorectal, ovarian, gastric and renal, but rarely on normal tissues. Its restricted expression on tumour tissues as well as its association with tumour progression and bad prognosis has driven the development of a MVA-based vaccine (TroVax) which has been tested in several early phase clinical trials and these studies have led to the start of a phase III trial in renal cell carcinoma patients. We have recently shown that CD8⁺ T cells recognizing h5T4 can be generated in the absence of CD4⁺ T cells from peripheral blood lymphocytes of human healthy individuals. Results We report the existence and expansion of human CD4⁺ T cells against h5T4 by stimulation with autologous monocyte-derived dendritic cells infected with a replication defective adenovirus encoding the h5T4 cDNA (Ad-h5T4). The h5T4-specific T-cell responses in normal individuals are enhanced by initial depletion of CD25⁺ cells (putative T regulatory cells) prior to the in vitro stimulation. We have identified a novel h5T4-derived 15-mer peptide recognized by CD4⁺ T cells in HLA-DR4 positive healthy individuals. Interestingly, CD4⁺ T cells spontaneously recognizing a different 5T4 epitope restricted by HLA-DR were identified in tumour-infiltrating lymphocytes isolated from a regressing renal cell carcinoma lung metastasis. Conclusion Our data show that CD4⁺ T cells recognizing h5T4 can be expanded and detected in healthy individuals and a renal cell carcinoma patient. Such h5T4-specific CD4⁺ T cells boosted or induced by vaccination could act to modulate both cell or antibody mediated anti-tumour responses. This work was supported by Cancer Research UK.     

Antitumor activity of a self-adjuvanting glyco-lipopeptide vaccine bearing B cell,CD4<Superscript>+</Superscript> and CD8<Superscript>+</Superscript> T cell epitopes

Molecularly defined synthetic vaccines capable of inducing both antibodies and cellular anti-tumor immune responses, in a manner compatible with human delivery, are limited. Few molecules achieve this target without utilizing external immuno-adjuvants. In this study, we explored a self-adjuvanting glyco-lipopeptide (GLP) as a platform for cancer vaccines using as a model MO5, an OVA-expressing mouse B16 melanoma. A prototype B and T cell epitope-based GLP molecule was constructed by synthesizing a chimeric peptide made of a CD8⁺ T cell epitope, from ovalbumin (OVA_257–264) and an universal CD4⁺ T helper (Th) epitope (PADRE). The resulting CTL–Th peptide backbones was coupled to a carbohydrate B cell epitope based on a regioselectively addressable functionalized templates (RAFT), made of four α-GalNAc molecules at C-terminal. The N terminus of the resulting glycopeptides (GP) was then linked to a palmitic acid moiety (PAM), obviating the need for potentially toxic external immuno-adjuvants. The final prototype OVA-GLP molecule, delivered in adjuvant-free PBS, in mice induced: (1) robust RAFT-specific IgG/IgM that recognized tumor cell lines; (2) local and systemic OVA_257–264-specific IFN-γ producing CD8⁺ T cells; (3) PADRE-specific CD4⁺ T cells; (4) OVA-GLP vaccination elicited a reduction of tumor size in mice inoculated with syngeneic murine MO5 carcinoma cells and a protection from lethal carcinoma cell challenge; (5) finally, OVA-GLP immunization significantly inhibited the growth of pre-established MO5 tumors. Our results suggest self-adjuvanting glyco-lipopeptide molecules as a platform for B Cell, CD4⁺, and CD8⁺ T cell epitopes-based immunotherapeutic cancer vaccines. Both I. Bettahi and G. Dasgupta have contributed equally to this work.     

CD4<Superscript>+</Superscript> T cells kill Id<Superscript>+</Superscript> B-lymphoma cells: FasLigand-Fas interaction is dominant in vitro but is redundant in vivo

B-lymphoma cells express a highly tumor-specific antigen, monoclonal Ig, which is a promising target for immunotherapy. Previous work has demonstrated that B-lymphoma cells spontaneously process their endogenous monoclonal Ig and present variable (V) region peptides (Id-peptides) on their MHC class II molecules to CD4⁺ T cells. Id-specific CD4⁺ T cells protect mice against B-lymphoma cells in the absence of anti-idiotypic antibodies. The molecular mechanism by which Id-specific CD4⁺ T cells kill B-lymphoma cells is hitherto unknown. We here demonstrate in an Id-specific T-cell receptor (TCR)–transgenic mouse model that Id-specific CD4⁺ T cells induce apoptosis of Fas⁺ B-lymphoma cells in vitro by FasLigand (FasL)–Fas interaction. Moreover, the rare B lymphomas that had escaped rejection in TCR-transgenic mice had down-regulated their sensitivity to Fas-mediated apoptosis. Although these results suggest that FasL-Fas interaction is important, Id-specific CD4⁺ T cells could eliminate Id⁺ B-lymphoma cells in vivo by other mechanisms, since three independent ways of blocking FasL-Fas–mediated killing failed to abrogate tumor protection in TCR-transgenic mice. These results suggest that there are several redundant pathways by which Id-specific CD4⁺ T cells eliminate Id⁺ B-lymphoma cells in vivo, of which FasL-Fas interaction is only one.Supported by grants from the Norwegian Cancer Society, the Research Council of Norway, and the Multiple Myeloma Research Foundation.     

Calcium Homeostasis Change in CD4<Superscript>+</Superscript> T Lymphocytes from Human Peripheral Blood during Differentiation <Emphasis Type="Italic">in vivo</Emphasis>

Resting naive CD4⁺CD45R0^?CD45RA⁺ T cells are sensitive to ionomycin. In contrast, resting CD4⁺CD45RA^?CD45R0⁺ memory T cells show resistance to this Ca²⁺ ionophore. In the present study, the ability of activated T lymphocytes to respond to ionomycin during the transition from naive precursors into memory T cells has been analyzed. Activated CD4⁺CD45RA⁺CD45R0⁺ T cells are always present both in human peripheral blood (HPB) and in the ionomycin-resistant (IR) fraction. Therefore, some activated T cells are resistant toward the Ca²⁺ ionophore. CD69 molecules are markers of the very early stage of T cell activation. However, CD4⁺CD69⁺ T cells have never been found in the IR fraction. Thus, the majority of CD4⁺ T lymphocytes at the early stage of activation are ionomycin-sensitive cells. The proportion of CD4⁺CD25⁺ T cells did not differ significantly in HPB and in the IR fraction. The presence of CD4⁺CD25⁺ T lymphocytes in the IR fraction reflects changes in the Ca²⁺-signaling pathway at this differentiation step of activated cells. Depending on the expression level of CD25 molecules, the population of CD4⁺CD25⁺ cells is divided in T-regulatory (CD25^high) and proliferating (CD25^low) subpopulations. The action of ionomycin results in a decrease in the portion of the CD4⁺CD25^low T-cells, but it leads to an increase in the proportion of the CD4⁺CD25^high T lymphocytes. Consequently, greater portion of CD4⁺CD25^high T lymphocytes and smaller portion of CD4⁺CD25^low T cells are IR cells. Expression of HLA-DR molecules can be used as the marker for the late activation step. The IR fraction is significantly rich in CD4⁺HLA-DR⁺ T lymphocytes in comparison to the blood of the same donor. The link between different differentiation steps of CD4⁺ T-lymphocytes and alterations in calcium ion homeostasis is discussed.     

Apoptosis of CD4<Superscript>+</Superscript>CD25<Superscript>high</Superscript> T cells in response to Sirolimus requires activation of T cell receptor and is modulated by IL-2

Targeted molecular therapies inhibit proliferation and survival of cancer cells but may also affect immune cells. We have evaluated the effects of Sirolimus and Sorafenib on proliferation and survival of lymphoid cell subsets. Both drugs were cytotoxic to CD4⁺CD25^high T cells, and were growth inhibitory for CD4⁺ and CD8⁺ T cells. Cytotoxicity depended on CD3/CD28 stimulation and was detectable within 12 h, with 80–90% of CD4⁺CD25^high cells killed by 72 h. Cell death was due to apoptosis, based on Annexin V and 7AAD staining. Addition of IL-2 prevented the apoptotic response to Sirolimus, potentially accounting for reports that Sirolimus can enhance proliferation of CD4⁺CD25^high cells. These results predict that Sirolimus or Sorafenib would reduce CD4⁺CD25^high cells if administered prior to antigenic stimulation in an immunotherapy protocol. However, administration of IL-2 protects CD4⁺CD25^high T cells from cytotoxic effects of Sirolimus, a response that may be considered in design of therapeutic protocols.     

Actin integrity is indispensable for CD95/Fas-induced apoptosis of HIV-specific CD8<Superscript>+</Superscript> T cells

We have recently provided data suggesting a potential role for mitochondria and Bcl-2-family molecules in apoptosis sensitivity of HIV-specific CD8⁺ T cells. Here, we report on the role of filamentous (F) actin in this process. Disruption of actin by cytochalasin D (cytD) or lantrunculin A remarkably reduced CD95/Fas-induced apoptosis of HIV-specific CD8⁺ T cells while their spontaneous apoptosis was unaffected. This inhibition cannot be attributed to changes of CD95/Fas distribution or levels in these cells. Furthermore, cytD treatment reduced CD95/Fas-induced apoptosis of CD8⁺ T cells from HIV⁺ patients independently of their differentiation status. CD95/Fas-induced apoptosis of both CD38⁺ and CD38⁻ HIV-specific CD8⁺ T cells was inhibited by cytD treatment indicating that actin mediates this apoptotic process independently of the activation level of these cells. CytD was found to reduce the activation of caspase-8 induced by short treatment of purified CD8⁺ T cells from HIV⁺ patients with anti-CD95/Fas. Our data reveal actin as a critical mediator of HIV-specific CD8⁺ T cell apoptosis; further analysis of the molecular mechanisms governing this process may potentially contribute to design new therapies targeting the enhancement of the immune system in HIV infection.     

Signaling Pathways in the Biphasic Effect of ANG II on Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> Exchanger in T84 Cells

The effect of ANG II on pH_i, [Ca²⁺]_i and cell volume was investigated in T84 cells, a cell line originated from colon epithelium, using the probes BCECF-AM, Fluo 4-AM and acridine orange, respectively. The recovery rate of pH_i via the Na⁺/H⁺ exchanger was examined in the first 2 min following the acidification of pH_i with a NH₄Cl pulse. In the control situation, the pH_i recovery rate was 0.118 ± 0.001 (n = 52) pH units/min and ANG II (10⁻¹² M or 10⁻⁹ M) increased this value (by 106% or 32%, respectively) but ANG II (10⁻⁷ M) decreased it to 47%. The control [Ca²⁺]_i was 99 ± 4 (n = 45) nM and ANG II increased this value in a dose-dependent manner. The ANG II effects on cell volume were minor and late and should not interfere in the measurements of pH_i recovery and [Ca²⁺]_i. To document the signaling pathways in the hormonal effects we used: Staurosporine (a PKC inhibitor), W13 (a calcium-dependent calmodulin antagonist), H89 (a PKA inhibitor) or Econazole (an inhibitor of cytochrome P450 epoxygenase). Our results indicate that the biphasic effect of ANG II on Na⁺/H⁺ exchanger is a cAMP-independent mechanism and is the result of: 1) stimulation of the exchanger by PKC signaling pathway activation (at 10⁻¹² – 10⁻⁷ M ANG II) and by increases of [Ca²⁺]_i in the lower range (at 10⁻¹² M ANG II) and 2) inhibition of the exchanger at high [Ca²⁺]_i levels (at 10⁻⁹ – 10⁻⁷ M ANG II) through cytochrome P450 epoxygenase-dependent metabolites of the arachidonic acid signaling pathway.     

Activation and route of administration both determine the ability of bone marrow-derived dendritic cells to accumulate in secondary lymphoid organs and prime CD8<Superscript>+</Superscript> T cells against tumors

Aims

To examine the effects of route of administration and activation status on the ability of dendritic cells (DC) to accumulate in secondary lymphoid organs, and induce expansion of CD8⁺ T cells and anti-tumor activity.

Methods

DC from bone marrow (BM) cultures were labeled with fluorochromes and injected s.c. or i.v. into naïve mice to monitor their survival and accumulation in vivo. Percentages of specific CD8⁺ T cells in blood and delayed tumor growth were used as readouts of the immune response induced by DC immunization.

Results

The route of DC administration was critical in determining the site of DC accumulation and time of DC persistence in vivo. DC injected s.c. accumulated in the draining lymph node, and DC injected i.v. in the spleen. DC appeared in the lymph node by 24 h after s.c. injection, their numbers peaked at 48 h and declined at 96 h. DC that had spontaneously matured in vitro were better able to migrate compared to immature DC. DC were found in the spleen at 3 h and 24 h after i.v. injection, but their numbers were low and declined by 48 h. Depending on the tumor cell line used, DC injected s.c. were as effective or more effective than DC injected i.v. at inducing anti-tumor responses. Pre-treatment with LPS increased DC accumulation in lymph nodes, but had no detectable effect on accumulation in the spleen. Pre-treatment with LPS also improved the ability of DC to induce CD8⁺ T cell expansion and anti-tumor responses, regardless of the route of DC administration.

Conclusions

Injection route and activation by LPS independently determine the ability of DC to activate tumor-specific CD8⁺ T cells in vivo.

     

The Na<Superscript>+</Superscript>-translocating ATPase in the plasma membrane of the marine microalga<Emphasis Type="Italic"> Tetraselmis viridis</Emphasis> catalyzes Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchange

Our previous investigations have established that Na⁺ translocation across the Tetraselmis viridis plasma membrane (PM) mediated by the primary ATP-driven Na⁺-pump, Na⁺-ATPase, is accompanied by H⁺ counter-transport [Y.V. Balnokin et al. (1999) FEBS Lett 462:402–406]. The hypothesis that the Na⁺-ATPase of T. viridis operates as an Na⁺/H⁺ exchanger is tested in the present work. The study of Na⁺ and H⁺ transport in PM vesicles isolated from T. viridis demonstrated that the membrane-permeant anion NO₃^– caused (i) an increase in ATP-driven Na⁺ uptake by the vesicles, (ii) an increase in (Na⁺+ATP)-dependent vesicle lumen alkalization resulting from H⁺ efflux out of the vesicles and (iii) dissipation of electrical potential, , generated across the vesicle membrane by the Na⁺-ATPase. The (Na⁺+ATP)-dependent lumen alkalization was not significantly affected by valinomycin, addition of which in the presence of K⁺ abolished at the vesicle membrane. The fact that the Na⁺-ATPase-mediated alkalization of the vesicle lumen is sustained in the absence of the transmembrane is consistent with a primary role of the Na⁺-ATPase in driving H⁺ outside the vesicles. The findings allowed us to conclude that the Na⁺-ATPase of T. viridis directly performs an exchange of Na⁺ for H⁺. Since the Na⁺-ATPase generates electric potential across the vesicle membrane, the transport stoichiometry is mNa⁺/nH⁺, where m>n.Abbreviations BTP Bis-Tris-Propane, 1,3-bis[tris(hydroxymethyl)methylamino]-propane - CCCP Carbonyl cyanide m-chlorophenylhydrazone - DTT Dithiothreitol - NCDC 2-Nitro-4-carboxyphenyl N,N-diphenylcarbamate - PMSF Phenylmethylsulfonyl fluoride - PM Plasma membrane     

Molecular mechanisms of extracellular adenine nucleotides-mediated inhibition of human Cd4<Superscript>+</Superscript> T lymphocytes activation

We have previously reported that ATPγS, a slowly hydrolyzed analog of ATP, inhibits the activation of human CD4⁺ T lymphocytes by anti-CD3 and anti-CD28 mAb. In this report we have partially characterized the signaling mechanisms involved in this immunosuppressive effect. ATPγS had no inhibitory effect on CD4⁺ T-cell activation induced by PMA and anti-CD28, indicating that it acts proximally to the TCR. It had no effect on the calcium rise induced by CD3/CD28 stimulation, but inhibited the phosphorylation of three kinases, ERK2, p38 MAPK and PKB, that play a key role in the activation of T cells. The receptor involved in these actions remains unidentified.     

Involvement of the antiplatelet activity of magnesium sulfate in suppression of protein kinase C and the Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchanger

Magnesium sulfate is widely used to prevent seizures in pregnant women with hypertension. The aim of this study was to examine the inhibitory mechanisms of magnesium sulfate in platelet aggregation in vitro. In this study, magnesium sulfate concentration-dependently (0.6–3.0 mM) inhibited platelet aggregation in human platelets stimulated by agonists. Magnesium sulfate (1.5 and 3.0 mM) also concentration-dependently inhibited phosphoinositide breakdown and intracellular Ca⁺² mobilization in human platelets stimulated by thrombin. Rapid phosphorylation of a platelet protein of M_r 47,000 (P47), a marker of protein kinase C activation, was triggered by phorbol-12-13-dibutyrate (PDBu, 50 nM). This phosphorylation was markedly inhibited by magnesium sulfate (3.0 mM). Magnesium sulfate (1.5 and 3.0 mM) further inhibited PDBu-stimulated platelet aggregation in human platelets. The thrombin-evoked increase in pHi was markedly inhibited in the presence of magnesium sulfate (3.0 mM). In conclusion, these results indicate that the antiplatelet activity of magnesium sulfate may be involved in the following two pathways: (1) Magnesium sulfate may inhibit the activation of protein kinase C, followed by inhibition of phosphoinositide breakdown and intracellular Ca⁺² mobilization, thereby leading to inhibition of the phosphorylation of P47. (2) On the other hand, magnesium sulfate inhibits the Na⁺/H⁺ exchanger, leading to reduced intracellular Ca⁺² mobilization, and ultimately to inhibition of platelet aggregation and the ATP-release reaction.     

Functional Identification of H<Superscript>+</Superscript>-ATPase and Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> Antiporter in the Plasma Membrane Isolated from the Root Cells of Salt-Accumulating Halophyte <Emphasis Type="Italic">Suaeda altissima</Emphasis>

A membrane fraction enriched in plasma membrane (PM) vesicles was isolated from the root cells of a salt-accumulating halophyte Suaeda altissima (L.) Pall. by means of centrifugation in discontinuous sucrose density gradient. The PM vesicles were capable of generating ΔpH at their membrane and the transmembrane electric potential difference (Δψ). These quantities were measured with optical probes, acridine orange and oxonol VI, sensitive to ΔpH and Δψ, respectively. The ATP-dependent generation of ΔpH was sensitive to vanadate, an inhibitor of P-type ATPases. The results contain evidence for the functioning of H⁺-ATPase in the PM of the root cells of S. altissima. The addition of Na⁺ and Li⁺ ions to the outer medium resulted in dissipation of ΔpH preformed by the H⁺-ATPase, which indicates the presence in PM of the functionally active Na⁺/H⁺ antiporter. The results are discussed with regard to involvement of the Na⁺/H⁺ antiporter and the PM H⁺-ATPase in loading Na⁺ ions into the xylem of S. altissima roots.     

Pivotal Role of Reduced Glutathione in Oxygen-induced Regulation of the Na<Superscript><Emphasis Type="Bold">+</Emphasis></Superscript>/K<Superscript><Emphasis Type="Bold">+</Emphasis></Superscript> Pump in Mouse Erythrocyte Membranes

This study addresses the mechanisms of oxygen-induced regulation of ion transport pathways in mouse erythrocyte, specifically focusing on the role of cellular redox state and ATP levels. Mouse erythrocytes possess Na⁺/K⁺ pump, K⁺-Cl^– and Na⁺-K⁺-2Cl^– cotransporters that have been shown to be potential targets of oxygen. The activity of neither cotransporter changed in response to hypoxia-reoxygenation. In contrast, the Na⁺/K⁺ pump responded to hypoxic treatment with reversible inhibition. Hypoxia-induced inhibition was abolished in Na⁺-loaded cells, revealing no effect of O₂ on the maximal operation rate of the pump. Notably, the inhibitory effect of hypoxia was not followed by changes in cellular ATP levels. Hypoxic exposure did, however, lead to a rapid increase in cellular glutathione (GSH) levels. Decreasing GSH to normoxic levels under hypoxic conditions abolished hypoxia-induced inhibition of the pump. Furthermore, GSH added to the incubation medium was able to mimic hypoxia-induced inhibition. Taken together these data suggest a pivotal role of intracellular GSH in oxygen-induced modulation of the Na⁺/K⁺ pump activity.     

Cloning and characterization of a Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter gene of the moderately halophilic bacterium <Emphasis Type="Italic">Halobacillus aidingensis</Emphasis> AD-6<Superscript>T</Superscript>

A gene encoding a Na(+)/H(+) antiporter was obtained from the genome of Halobacillus aidingensis AD-6(T), which was sequenced and designated as nhaH. The deduced amino acid sequence of the gene was 91% identical to the NhaH of H. dabanensis, and shared 54% identity with the NhaG of Bacillus subtilis. The cloned gene enable the Escherichia coli KNabc cell, which lack all of the major Na(+)/H(+) antiporters, to grow in medium containing 0.2 M NaCl or 10 mM LiCl. The nhaH gene was predicted to encode a 43.5 kDa protein (403 amino acid residues) with 11 putative transmembrane regions. Everted membrane vesicles prepared from E. coli KNabc cells carrying NhaH exhibited Na(+)/H(+) as well as Li(+)/H(+) antiporter activity, which was pH-dependent with the highest activity at pH 8.0, and no K(+)/H(+) antiporter activity was detected. The deletion of hydrophilic C-terminal amino acid residues showed that the short C-terminal tail was vital for Na(+)/H(+) antiporter activity.     

Adhesive interactions between CD34<Superscript>+</Superscript>-derived dendritic cell precursors and dermal microvascular endothelial cells studied by scanning electron microscopy

Dendritic cells are migratory cells. Before they extravasate from the circulation into the skin across capillary blood vessel walls, they have to interact with endothelial cells. Using a fluorimetric adhesion assay, we have recently shown that CD34⁺-derived dendritic cell precursors are able to bind to resting and stimulated dermal microvascular endothelial cells. In the present study, we attempted to visualize this process at an ultrastructural level. CD34⁺ progenitor cells were purified from human cord blood samples by means of immunomagnetic beads, and dendritic cells were generated by culture in the presence of GM-CSF, TNF- and hSCF for 5 days. Immature CD83^– CD86^low dendritic cells were added to human dermal microvascular endothelial cells grown to confluence on membrane chambers. After 2 h, unbound dendritic cell precursors were removed, and bound cells were prepared for routine scanning electron microscopy. We found that (1) dendritic cell precursors firmly adhere to microvascular endothelial cells, enveloping them with their surface processes; (2) dendritic cell precursors are extremely deformable as they squeeze through the dense network of microvascular endothelial cells; (3) microvascular endothelial cells form, in part, a multi-layered network rather than the typical cobblestone pattern as seen by phase-contrast microscopy. The morphology of dendritic cell precursors and of human dermal microvascular endothelial cells was examined here, for the first time, by scanning electron microscopy. These data further emphasize that CD34⁺-derived dendritic cells efficiently adhere to dermal microvascular endothelial cells.     

Rapid turnover of the CD8(+)CD28(-) T-cell subset of effector cells in the circulation of patients with head and neck cancer

CD8⁺ T cells in the circulation of patients with head and neck cancer (HNC) were previously shown to be significantly more sensitive to, and preferentially targeted for, apoptosis than CD4⁺ T cells (Hoffmann et al., Clin Cancer Res, 8:2553–2562, 2002). To distinguish global from CD8⁺ subset-specific apoptosis, we studied Annexin-binding to naïve, memory, and effector subsets of CD8⁺ cells by multicolor flow cytometry. Age-related changes in naïve and effector CD8⁺ cell subsets were observed in patients and normal controls (NC). The frequencies of naïve (CD28⁺CD45RO^-) CD8⁺ T cells were lower and those of memory (CD28⁺CD45RO⁺) and effector (CD28^-) CD8⁺ T cells significantly higher in the circulation of HNC patients relative to age-matched NC. Among CD8⁺ T cells, the CD28^- effector cell subset contained the highest proportion of Annexin-binding cells, while the naïve CD28⁺CD45RO^- subset contained the lowest. This suggested a high turnover rate of the CD8⁺CD28^- effector cell subset in patients with HNC, which was being compensated by a rapid transition of naïve CD8⁺ T cells to the effector cell pool. Following tumor resection, the frequency of CD8⁺CD28^- T cells normalized in the patients, an indication that the presence of tumor had an influence on the size of CD8⁺CD28^- T-cell pool. Ex vivo, in mixed lymphocyte-tumor cultures (MLTC) with semiallogeneic T cells as responders, CD8⁺CD28^- T cells could be generated from CD8⁺CD28⁺ cells by repeated stimulations with tumor cells. These CD8⁺CD28^- effector cells lysed the tumor, produced IFN- in response to the tumor, and strongly expressed granzyme B. Thus, the high rate of their apoptosis in the circulation of patients with HNC might be expected to contribute to tumor progression. However, the ex vivo generation of this cell subset was suppressed by strong CD28/B7 ligation or by overexpresson of MHC molecules on tumor cells, suggesting that adequate costimulation is necessary for protection from apoptosis. It appears that interactions of immune and tumor cells might determine the fate of this terminally differentiated effector cell subset.Supported in part by NIH grants: PO-1 DE 12321 and RO-1 CA 82016 to Theresa L. Whiteside.     

Effects of palytoxin on cation occlusion and phosphorylation of the (Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>)-ATPase

Palytoxin (PTX) inhibits the (Na(+) + K+)-driven pump and simultaneously opens channels that are equally permeable to Na+ and K+ in red cells and other cell membranes. In an effort to understand the mechanism by which PTX induces these fluxes, we have studied the effects of PTX on: 1) K+ and Na+ occlusion by the pump protein; 2) phosphorylation and dephosphorylation of the enzyme when a phosphoenzyme is formed from ATP and from P(i); and 3) p-nitro phenyl phosphatase (p-NPPase) activity associated with the (Na+, K+)-ATPase. We have found that palytoxin 1) increases the rate of deocclusion of K+(Rb+) in a time- and concentration-dependent manner, whereas Na+ occluded in the presence of oligomycin is unaffected by the toxin; 2) makes phosphorylation from P(i) insensitive to K+, and 3) stimulates the p-NPPase activity. The results are consistent with the notion that PTX produces a conformation of the Na+, K(+)-pump that resembles the one observed when ATP is bound to its low-affinity binding site. Further, they suggest that the channels that are formed by PTX might arise as a consequence of a perturbation in the ATPase structure, leading to the loss of control of the outside "gate" of the enzyme and hence to an uncoupling of the ion transport from the catalytic function of the ATPase.     

Sulfatide and Na<Superscript>+</Superscript>-K<Superscript>+</Superscript>-ATPase: A Salinity-sensitive Relationship in the Gill Basolateral Membrane of Rainbow Trout

We investigated the effect of salinity on the relationship between Na⁺-K⁺-ATPase and sulfogalactosyl ceramide (SGC) in the basolateral membrane of rainbow trout (Oncorhynchus mykiss) gill epithelium. SGC has been implicated as a cofactor in Na⁺-K⁺-ATPase activity, especially in Na⁺-K⁺-ATPase rich tissues. However, whole-tissue studies have questioned this role in the fish gill. We re-examined SGC cofactor function from a gill basolateral membrane perspective. Nine SGC fatty acid species were quantified by tandem mass spectrometry (MS/MS) and related to Na⁺-K⁺-ATPase activity in trout acclimated to freshwater or brackish water (20 ppt). While Na⁺-K⁺-ATPase activity increased, the total concentration and relative proportion of SGC isoforms remained constant between salinities. However, we noted a negative correlation between SGC concentration and Na⁺-K⁺-ATPase activity in fish exposed to brackish water, whereas no correlation existed in fish acclimated to freshwater. Differential Na⁺-K⁺-ATPase/SGC sensitivity is discussed in relation to enzyme isoform switching, the SGC cofactor site model and saltwater adaptation.This revised version was published online in June 2005 with a corrected cover date.     

Hematopoietic reconstitution of CD34<Superscript>+</Superscript> cells grown in static and stirred culture systems in NOD/SCID mice

The hematopoietic reconstitution of cord blood (CB) CD34⁺ cells grown in static and stirred system was studied. Static cultures were better than stirred cultures for cell expansion. Engraftment of stirred-culture hematopoietic stem cells (HSCs) was higher than static-culture HSCs. Stirred-culture HSCs had better multilineage reconstitution ability and colony-forming ability than static-culture HSCs. Static cultures thus favor the expansion of HSCs and stirred cultures are more effective in preserving functional HSCs.