The induction of virus-specific cytotoxic T lymphocytes with solubilized viral and membrane proteins.

Reconstituted membranes were prepared from detergent solubilized P815 (H-2d) tumor cell membranes and solubilized Sendai virus protein. These reconstituted membranes stimulated a virus-specific H-2-restricted secondary CTL response. Stimulating activity was dependent upon the presence of both viral and P815 protein in the same membrane and was restricted to the H-2 specificity present in the reconstituted membrane. Liposomes prepared from solubilized Sendai virus proteins and partially purified H-2 alloantigen also had activity for CTL induction. The results demonstrate the feasibility of using detergent solubilized membrane proteins to study antigen recognition by virus-specific, H-2 restricted cytolytic T lymphocytes.

The induction of secondary cytolytic T lymphocytes by solubilized membrane proteins.

Membrane-bound antigens responsible for induction of a secondary allogeneic murine cytolytic T-cell (CTL) response have been obtained in a soluble, biologically active form by deoxycholate solubilization of tumor cell plasma membranes. The active proteins are soluble by the criteria of both ultracentrifugation and gel filtration. The immunological specificity of the induced CTL and removal of the activity from solution by treatment with B6 anti-P815 (anti-H-2d) antiserum and Protein A-Sepharose demonstrate that the CTL-inducing activity is dependent upon solubilized major histocompatibility complex antigens.

Carbohydrate moieties of major histocompatibility complex class I alloantigens are not required for their recognition by T lymphocytes.

The ability to generate specific cytotoxic responses using purified major histocompatibility complex (MHC) antigen in liposomes has made it possible to directly assess the importance of class I carbohydrate moieties in T cell recognition of alloantigen. Deglycosylation of affinity-purified H-2Kk to yield a single glycan-free product did not alter the specificity, the magnitude, nor the dose range of the cytotoxic T lymphocyte (CTL) response to the class I antigen. It can be concluded that carbohydrate moieties are not required to maintain the necessary conformation of the MHC protein, nor to interact with either the antigen-specific receptor or accessory proteins on precursor CTL.

The thymus leukemia antigen binds human and mouse CD8.

The thymus leukemia antigen (TLA) is a class Ib, or 'nonclassical' class I molecule, one of several encoded within the Tla locus of the mouse major histocompatibility complex (MHC). It structurally resembles the H-2K, D, and L class I transplantation antigens, which present processed peptides to cytotoxic T lymphocytes (CTLs). Although their function(s) are unknown, there has been recent speculation concerning the possibility that class Ib molecules may present antigens to T cells that express gamma delta T cell antigen receptors (TCRs). In this report, using both a cell-cell adhesion assay and adhesion of T lymphocyte clones to purified plate-bound TLA, we provide evidence that TLA can bind to both human and mouse CD8. We also show that a chimeric class I molecule containing the peptide antigen binding site of Ld and the alpha 3 domain, transmembrane, and cytoplasmic segments of TLA, can support a CD8-dependent immune response by CTLs. These results demonstrate for the first time binding of a class Ib molecule to CD8 with a functional outcome, as is observed for the class I transplantation antigens. The capacity to interact with CD8 has been conserved despite the extensive sequence divergence of TLA in the peptide antigen binding site, suggesting this interaction is highly significant. TLA is expressed by epithelial cells in the mouse small intestine. As these epithelial cells are in close contact with intestinal intraepithelial lymphocytes that are nearly all CD8+, and many of which express the gamma delta TCR, the data are consistent with the hypothesis that TLA is involved in antigen presentation, perhaps to gamma delta-positive lymphocytes in this site.

Cytoskeletal function in CD8- and T cell receptor-mediated interaction of cytotoxic T lymphocytes with class I protein.

Cloned allospecific cytolytic T lymphocytes (CTL) adhere to purified class I alloantigen immobilized on plastic and degranulate in response to it. Binding and degranulation are inhibited by drugs that impair cytoskeletal function. Cytochalasins D and E, which interfere with microfilament function, and colchicine, which disrupts microtubules, were used and gave qualitatively similar results. Concentrations of these drugs that inhibited degranulation in response to alloantigen did not inhibit response to immobilized anti-T cell receptor (TCR) antibody. Neither did they inhibit response when alloantigen was co-immobilized with an antibody against class I on the CTL to promote adhesion between the CTL and antigen-bearing surface. Thus, neither transmembrane signal generation via the TCR nor degranulation per se were prevented. Instead, the drugs act to prevent the initial adhesion to alloantigen. CTL binding to alloantigen depends in part on CD8-class I interaction, and adhesion via CD8 is "activated" by crosslinking the TCR with soluble anti-TCR antibody. This adhesion, too, is shown to be cytoskeleton dependent.

Contribution of lymphocyte function-associated-1/intercellular adhesion molecule-1 binding to the adhesion/signaling cascade of cytotoxic T lymphocyte activation.

A rapid induction of adhesion to immobilized intercellular adhesion molecule (ICAM)-1 occurs when cytotoxic T lymphocytes (CTL) are stimulated with either soluble anti-T cell receptor (TCR) monoclonal antibodies (mAb) or with immobilized alloantigen, and this binding is blocked by the addition of anti-lymphocyte function-associated (LFA)-1 mAbs. Requirements for activating LFA-1 adhesion to ICAM-1 are similar to those found for induction of binding to immobilized fibronectin (FN), but distinct from those for activating CD8-mediated adhesion to class I major histocompatibility complex. A distinct role for LFA-1 in co-signaling for TCR-dependent degranulation could not be demonstrated. In contrast, both CD8 and the FN-binding integrin provide costimulatory signals for this response. Thus, if co-signaling via LFA-1 occurs, it clearly differs from that provided by CD8 or the FN-binding integrin. On the basis of antibody blocking effects, alloantigen-dependent activation of adhesion to ICAM-1 involves both the TCR and CD8. These results support a view of CTL activation as a cascade of adhesion and signaling events, with different coreceptors making distinct contributions.

Growth-inhibitory activity of lymphoid cell plasma membranes. I. Inhibition of lymphocyte and lymphoid tumor cell growth.

Membranes isolated from normal spleen cells or lymphoid tumor cells were found to inhibit in vitro growth of several murine tumor cell lines including a B cell hybridoma, a thymoma, and a mastocytoma. 50% inhibition occurred at membrane protein concentrations of 60-100 micrograms/ml. A similar concentration dependence was found for inhibition of [3H]-thymidine incorporation by tumor cells and for the lipopolysaccharide-induced mitogenic response of normal spleen cells. The inhibitory activity co-purified with the plasma membrane upon fractionation of crude membranes. Membrane solubilization with deoxycholate followed by dialysis to remove the detergent gave good recovery of inhibitory activity in the resulting reconstituted membranes. Membrane-mediated growth inhibition resulted from a decreased rate of proliferation and not from increased cell death. A toxic effect of the membranes was further ruled out by the finding that increasing the fetal calf serum content of the medium could substantially reverse the growth inhibition. Thus, the plasma membrane of lymphoid cells contains a component that can slow or stop the growth of cells in culture. This membrane component may have a role in cell contact-mediated regulation of growth.

Agorins: major structural proteins of the plasma membrane skeleton of P815 tumor cells.

Plasma membranes of P815 mastocytoma cells contain a set of proteins that remain selectively insoluble upon extraction of the membranes with Triton X-100, and appear to form a membrane skeletal matrix independent of the filamentous cytoskeletal systems. EGTA treatment of the matrix was found to release approximately 25% of the protein as polypeptides of 70, 69, 38, and 36 kD, all of which appear to be peripheral components associated with the cytoplasmic face of the plasma membrane via divalent cation-dependent interactions. About 75% of the total matrix protein was recovered in the EGTA-insoluble fraction. Actin accounted for approximately 5% of the total protein in the EGTA-insoluble fraction. The rest was accounted for by two novel proteins of 20 and 40 kD which, despite their relatively low molecular weights, do not enter SDS PAGE gels. Together these proteins account for approximately 15% of the total plasma membrane protein, and are thus present in much higher amounts than any other characterized protein of nucleated cell plasma membranes. Based on the extensive associations of these proteins to form very large detergent-insoluble structures, we propose that they may be named agorin I, the 20-kD protein, and agorin II, the 40-kD protein, from the Greek agora meaning assembly. The amount and properties of these proteins and the appearance of the EGTA-insoluble material in thin-section electron micrographs indicate that the agorins are the major structural elements of the membrane matrix, and thus of the putative membrane skeleton.

Growth-inhibitory activity of lymphoid cell plasma membranes. II. Partial characterization of the inhibitor.

We have shown that plasma membranes from lymphoid cells have inhibitory activity for the growth of normal lymphocytes and lymphoid tumor cells (Stallcup, K. C., A. Dawson, and M. F. Mescher, J. Cell Biol. 99:1221-1226). This growth-inhibitory activity has been found to co-purify with major histocompatibility complex class I antigens (H-2K and D) when these cell surface glycoproteins are isolated from detergent lysates of cells by affinity chromatography on monoclonal antibody columns. When incorporated into liposomes, the affinity-purified H-2 antigens inhibited the growth of both normal lymphocytes and tumor cells at concentrations of 1-3 micrograms/ml. Inhibition was readily reversed upon removal of the liposomes from the cell cultures, even after several days of exposure of cells to the inhibitor. Inhibitory activity was insensitive to protease digestion or heat treatment, indicating that it was not due to the H-2 glycoproteins. This was confirmed by the demonstration that inhibitory activity could be separated from the H-2 protein by gel filtration in the presence of deoxycholate and could be extracted from membranes or H-2 antigen preparations with organic solvents. The results demonstrate that the growth-inhibitory component(s) of the plasma membrane is a minor lipid or lipid-like molecule which retains activity in the absence of other membrane components. The findings reported here and in the preceding article suggest that this novel membrane component may have a role in control of lymphoid cell growth, possibly mediated by cell contacts.

Triton X-100 extraction of P815 tumor cells: evidence for a plasma membrane skeleton structure.

It has been shown that a Triton X-100-insoluble protein matrix can be isolated from the plasma membranes of P815 tumor cells and murine lymphoid cells (Mescher, M. F., M. J. L. Jose and S. P. Balk, 1981, Nature (Lond.), 289:139-144). The properties of the matrix suggested that this set of proteins might form a membrane skeletal structure, stable in the absence of the lipid bilayer. Since purification of plasma membrane results in yields of only 20 to 40%, it was not clear whether the matrix was associated with the entire plasma membrane. To determine if a detergent-insoluble structure was present over the entire cell periphery and stable in the absence of the membrane bilayer or cytoskeletal components, we have examined extraction of whole cells with Triton X-100. Using the same conditions as those used for isolation of the matrix from membranes, we found that extraction of intact cells resulted in structures consisting of a continuous layer of protein at the periphery, a largely empty cytoplasmic space, and a nuclear remnant. Little or no lipid bilayer structure was evident in association with the peripheral layer, and no filamentous cytoskeletal structures could be seen in the cytoplasmic space by thin-section electron microscopy. Analysis of these Triton shells showed them to retain approximately 15% of the total cell protein, most of which was accounted for by low molecular weight nuclear proteins. 5'-Nucleotidase, a cell surface enzyme that remains associated with the plasma membrane matrix, was quantitatively recovered with the shells. Included among the polypeptides present in the shells was a set with mobilities identical to those of the set that makes up the plasma membrane matrix. The polypeptide composition of the shells further confirmed that cytoskeletal proteins were present to a very low extent, if at all, after the extraction. The results demonstrate that a detergent-insoluble protein matrix associated with the periphery of these cells forms a continuous, intact macrostructure whose stability is independent of the membrane bilayer or filamentous cytoskeletal elements, and thus has the properties of a membrane skeletal structure. Although not yet directly demonstrated, the results also strongly suggest that this peripheral layer is composed of the previously described set of plasma membrane matrix proteins. This article discusses possible roles for this proposed membrane skeletal structure in stabilizing the membrane bilayer and affecting the dynamics of other membrane proteins.

Local drug delivery with a self-contained, programmable, microfluidic system.

The development and optimization of many new drug therapies requires long-term local delivery with controlled, but variable dosage. Current methods for chronic drug delivery have limited utility because they either cannot deliver drugs locally to a specific organ or tissue, do not permit changes in delivery rate in situ, or cannot be used in clinical trials in an untethered, wearable configuration. Here, we describe a small, self-contained system for liquid-phase drug delivery. This system enables studies lasting several months and infusion rates can be programmed and modified remotely. A commercial miniature pump is integrated with microfabricated components to generate ultralow flow rates and stroke volumes. Solutions are delivered in pulses as small as 370 nL, with pulses delivered at any interval of 1 min or longer. A unique feature of the system is the ability to infuse and immediately withdraw liquid, resulting in zero net volume transfer while compounds are exchanged by mixing and diffusion with endogenous fluid. We present in vitro results demonstrating repeatability of the delivered pulse volume for nearly 3 months. Furthermore, we present in vivo results in an otology application, infusing into the cochlea of a guinea pig a glutamate receptor antagonist, which causes localized and reversible changes in auditory sensitivity.

Activation of polyphosphoinositide hydrolysis in T cells by H-2 alloantigen but not MLS determinants.

Murine minor lymphocyte-stimulating (Mls) determinants are cell surface antigens that stimulate strong primary T cell responses; the responding T cells display restricted T cell receptor (TCR) V beta gene usage. Interaction of T cells with mitogens or major histocompatibility complex (MHC) antigens activated the polyphosphoinositide (PI) signaling pathway, but this pathway was not triggered by Mls recognition. However, interleukin-2 (IL-2) secretion and proliferation to all three stimuli were comparable. Thus, although recognition of both allo-H-2 and Mls determinants is thought to be mediated by the TCR, these antigens appear to elicit biochemically distinct signal transduction pathways.

MR imaging contrast enhancement based on intermolecular zero quantum coherences.

A new method for magnetic resonance imaging (MRI) based on the detection of relatively strong signal from intermolecular zero-quantum coherences (iZQCs) is reported. Such a signal would not be observable in the conventional framework of magnetic resonance; it originates in long-range dipolar couplings (10 micrometers to 1 millimeter) that are traditionally ignored. Unlike conventional MRI, where image contrast is based on variations in spin density and relaxation times (often with injected contrast agents), contrast with iZQC images comes from variations in the susceptibility over a distance dictated by gradient strength. Phantom and in vivo (rat brain) data confirm that iZQC images give contrast enhancement. This contrast might be useful in the detection of small tumors, in that susceptibility correlates with oxygen concentration and in functional MRI.

Lactate turnover in rat glioma measured by in vivo nuclear magnetic resonance spectroscopy.

Elevated tissue lactate concentrations typically found in tumors can be measured by in vivo nuclear magnetic resonance (NMR) spectroscopy. In this study, lactate turnover in rat C6 glioma was determined from in vivo 1H NMR measurements of [3-13C]lactate buildup during steady-state hyperglycemia with [1-13C]glucose. With this tumor model, a narrow range of values was observed for the first-order rate constant that describes lactate efflux, k2 = 0.043 +/- 0.007 (n = 12) SD min-1. For individual animals, the standard error in k2 was small (< 18%), which indicated that the NMR data fit the kinetic model well. Lactate measurements before and after infusing [1-13C]glucose showed that the majority of the tumor lactate pool was metabolically active. Signals from 13C-labeled glutamate in tumors were at least 10-fold smaller than the [3-13C]lactate signal, whereas spectra of the contralateral hemispheres revealed the expected labeling of [4-13C]glutamate, as well as [2-13C] and [3-13C]glutamate, which indicates that label cycled through the tricarboxylic acid cycle in the brain tissue. Lack of significant 13C labeling of glutamate was consistent with low respiratory metabolism in this glioma. It is concluded that lactate in rat C6 glioma is actively turning over and that the kinetics of lactate efflux can be quantified noninvasively by 1H NMR detection of 13C label. This noninvasive NMR approach may offer a valuable tool to help evaluate tumor growth and metabolic responsiveness to therapies.

A portable and reconfigurable multi-organ platform for drug development with onboard microfluidic flow control.

The drug development pipeline is severely limited by a lack of reliable tools for prediction of human clinical safety and efficacy profiles for compounds at the pre-clinical stage. Here we present the design and implementation of a platform technology comprising multiple human cell-based tissue models in a portable and reconfigurable format that supports individual organ function and crosstalk for periods of up to several weeks. Organ perfusion and crosstalk are enabled by a precision flow control technology based on electromagnetic actuators embedded in an arrayed format on a microfluidic platform. We demonstrate two parallel circuits of connected airway and liver modules on a platform containing 62 electromagnetic microactuators, with precise and controlled flow rates as well as functional biological metrics over a two week time course. Technical advancements enabled by this platform include the use of non-sorptive construction materials, enhanced scalability, portability, flow control, and usability relative to conventional flow control modes (such as capillary action, pressure heads, or pneumatic air lines), and a reconfigurable and modular organ model format with common fluidic port architecture. We demonstrate stable biological function for multiple pairs of airway-liver models for periods of 2 weeks in the platform, with precise control over fluid levels, temperature, flow rate and oxygenation in order to support relevant use cases involving drug toxicity, efficacy testing, and organ-organ interaction.

Structural heterogeneity of murine IgD during ontogeny.

We have studied the expression of the two membrane delta heavy chains (delta 1 and delta 2) and the two native IgD structures (IgDI and IgDII) in neonatal mice. Both delta-chains appear simultaneously during development and neonatal mice, like adults, express equal amounts of delta 1 and delta 2. IgDI and IgDII also appear simultaneously during ontogeny and in the same ratio as expressed by adult mice of the same strain. Thus, when IgD first appears during ontogeny it shows the same structural heterogeneity as observed in adult mice.

Solid-phase binding of class I and II MHC proteins: immunoassay and T cell recognition.

Detergent-solubilized, affinity-purified class I and II MHC antigens can be immobilized on plastic by a simple detergent dilution procedure. The bound antigens retain allogeneic serological determinants and can be precisely quantitated by ELISA. The ability to quantitate immobilized antigen greatly facilitates purification by affinity chromatography. It is shown that differential elution can be used to highly enrich H-2Kd and Dd antigens from a single monoclonal antibody column which binds both. Binding of membrane proteins (class I, class II and plasma membrane protein) to plastic could be distinguished from binding of non-membrane proteins (bovine serum albumin and immunoglobulin) in competition studies and by comparison of their susceptibilities to inhibition by detergent. These contrasting properties suggested that MHC proteins may bind via their exposed hydrophobic regions and thus be oriented on the plastic surface. This was supported by the demonstration that immobilized class I is effectively recognized by alloantigen-specific cloned CTL to trigger the antigen-dependent degranulation response. Direct immobilization of MHC antigens, and probably other membrane proteins, provides an effective approach to the study of T cell recognition and triggering by physiological ligands.

IgE receptor-mediated arachidonic acid release by rat basophilic leukemia (RBL-2H3) cells: possible role in activating degranulation.

Aggregation of the IgE receptor on rat basophilic leukemia (RBL-2H3) cells triggers increased hydrolysis of polyphosphoinositides (PI), secretion of arachidonic acid (AA) and its metabolites, and degranulation to release 5-hydroxytryptamine. Despite the documented involvement of second messengers produced by the PI pathway in RBL cell exocytosis, recent evidence has suggested that additional signalling events are also necessary. We have, therefore, examined PLA2 activation and AA metabolite production by these cells in response to Ag stimulation, and evaluated the potential role of these in activating degranulation. The time course and antigen dose dependence for release of AA and its metabolites were comparable to those for degranulation and production of inositol phosphates (InsPs) when examined in parallel. Stimulated fatty acid release was highly selective for AA (compared with oleic or linoleic acids) and appeared to result predominantly from PLA2 activation. AA released upon antigen stimulation is rapidly metabolized to produce prostaglandin and leukotrienes. These are not required for activating degranulation, since BW755c completely inhibited AA metabolite production without affecting AA release, degranulation or InsP production. In contrast, the PLA2 inhibitors quinacrine and quercetin inhibited both AA release and degranulation in parallel, without significantly affecting levels of InsP production, and this inhibition could be partially reversed by exogenous addition of AA and lysophospholipid. These results demonstrate that activation of IgE-receptor mediated exocytosis of RBL cells does not require AA metabolites, and strongly suggest that PLA2 activation and release of AA and lysophospholipid may be involved in triggering this response.

Large-scale purification of murine I-Ak and I-Ek antigens and characterization of the purified proteins.

Detailed analysis of the role of the structural characteristics of these molecules will require isolation of relatively large amounts of these antigens in serologically active form. We have purified murine Ia antigens on a large scale by affinity chromatography using monoclonal antibodies coupled to Sepharose 4B. Both I-Ak and I-Ek were isolated by sequential passage of cell lysate over columns prepared using specific monoclonal antibodies. Elution of the bound antigens required high pH (11-12) but, nonetheless, the purified material was 50-75% serologically active. Using LPS-stimulated spleen cells or B-lymphocyte tumor cells as starting material, 0.5 mg of each antigen can be readily purified. Based on antigen yields, it can be estimated that normal B-cells have about the same surface density of Class I and Class II MHC antigens. LPS blasts, in contrast, have normal levels of Class I antigen but 3-5 times higher levels of Class II antigens. We have now purified I-Ak and I-Ek from a number of different cell sources and have noted differences in both the mol. wts of the alpha- and beta-chains and in their apparent associations with cytoskeletal components. Proteins having the same apparent mol. wts as actin and myosin co-purify with both I-Ak and I-Ek antigens from various sources. These proteins do not co-purify with H-2K and D molecules obtained by similar methods, suggesting that Ia antigens may specifically interact with cytoskeletal elements.