Resolved-Sideband Laser Cooling in a Penning Trap.

We report the laser cooling of a single ^{40}Ca^{+} ion in a Penning trap to the motional ground state in one dimension. Cooling is performed in the strong binding limit on the 729-nm electric quadrupole S_{1/2}↔D_{5/2} transition, broadened by a quench laser coupling the D_{5/2} and P_{3/2} levels. We find the final ground-state occupation to be 98(1)%. We measure the heating rate of the trap to be very low with n[over ¯][over ˙]≈0.3(2) s^{-1} for trap frequencies from 150-400 kHz, consistent with the large ion-electrode distance.

Enhanced antigen immunogenicity induced by bispecific antibodies.

The binding of protein antigens to APC with heterocrosslinked bispecific antibodies (HBAs) enhances their processing and presentation to Th cells in vitro. Here we have asked whether HBAs could also increase immune responses in vivo. We immunized mice with hen egg lysozyme (HEL) in the presence or absence of HBA, and followed antibody production after the primary challenge and after a secondary boost. We found that HBAs that bind antigen to MHC class I or II molecules, to Fc gamma R, but not to surface IgD, enhance the immunogenicity of HEL. HBAs that bound HEL to MHC class II molecules, for examples, decreased the amount of antigen required to elicit a primary anti-HEL antibody response in mice by 300-fold, and the amount required to prime for a secondary response by 10(3)- to 10(4)-fold. In fact, HBAs were as effective as IFA in generating antibody responses. Since adjuvants cannot be used in humans, HBAs could prove useful for immunizing people, especially in cases where, due to scarcity or toxicity, minute doses of antigen must be used.

Specific targeting of human peripheral blood T cells by heteroaggregates containing anti-T3 crosslinked to anti-target cell antibodies.

Antibody heteroaggregates have been used to render human peripheral blood T cells lytic for specified targets. The heteroaggregates contain anti-T3 covalently linked to antibodies against nominal target cell antigens. Such heteroaggregates bind target cells directly to T3 molecules on effector cells and trigger target cell lysis. Freshly prepared human PBL, when coated with anti-T3-containing heteroaggregates, are lytic without further stimulation, although brief exposure to crude lymphokine-containing supernatants or recombinant IL-2, but not recombinant IFN-gamma, enhances the activity. The effector cells are T8+, and when fully stimulated, their lytic activity approaches that of some cloned CTL. When T cells are treated with heteroaggregate, washed, and incubated at 37 degrees C in medium not containing heteroaggregate, they retain activity for at least 24 h. The results of this study suggest a strategy in which heteroaggregate-coated T cells could be used in vivo to mount a lytic response against pathogenic cells such as tumor cells or virus-infected cells.

Production of target-specific effector cells using hetero-cross-linked aggregates containing anti-target cell and anti-Fc gamma receptor antibodies.

Rabbit anti-2,4-dintrophenyl (DNP) antibodies or their F(ab')2 fragments were chemically cross-linked to the anti-mouse Fc gamma R monoclonal antibody 2.4G2 or to its Fab fragment. P388D1 cells were incubated with heteroaggregates between 2.4G2 and anti-DNP (anti-Fc gamma R X anti-DNP) and washed. The resulting cells lysed 2,4,6-trinitrophenyl chicken erythrocytes (TNP CRBC) in a hapten-specific manner. The lysis was inhibited by free hapten but was resistant to inhibition by immune complexes. Other cells coated with antibody heteroaggregates also mediated lysis of TNP-modified target cells. For example, mouse resident peritoneal exudate cells (PEC) lysed TNP CRBC and bacillus Calmette-Guérin-activated PEC lysed both TNP CRBC and TNP tumor targets. Human neutrophils, when incubated with heteroaggregates containing the anti-human neutrophil Fc gamma R antibody 3G8 and anti-DNP also lysed TNP CRBC and TNP-modified tumor cells. To test whether linkage to Fc gamma R was required for lysis, F(ab')2 fragments from the anti-KdDd monoclonal antibody 34-1-2 were cross-linked to anti-DNP F(ab')2 fragments. P388D1 cells (which express Kd and Dd) were then incubated with these heteroaggregates and washed, and their abilities to form conjugates and lyse TNP CRBC were compared with those of P388D1 cells treated with anti-Fc gamma R X anti-DNP. In both cases, P388D1 cells formed conjugates. However, only the cells treated with anti-Fc gamma R X anti-DNP mediated lysis to a significant extent. We conclude that heteroaggregates containing anti-Fc gamma R and anti-target cell antibodies can be used to create potent effector cells against red cell and tumor targets and that bridging of effectors with target cells directly to Fc gamma R on effector cells is required for lysis.

Trinitrophenyl modification of H-2k and H-2b spleen cells results in enhanced serological detection of Kk-like determinants.

Several anti-H-2Kk but not anti-H-2Dd monoclonal antibodies (mAb) exhibited enhanced binding to B10.A murine spleen cells after modification of the cells with trinitrobenzene sulfonate (TNBS). The number of antibody molecules bound to TNP-modified B10.A spleen cells increased by a factor of two or more. The same anti-2Kk mAb that exhibited enhanced binding to modified B10.A cells did not bind to unmodified C57BL/10 spleen cells, as expected, but did bind to TNP-modified C57BL/10 spleen cells. This TNP-dependent binding was not a result of cross-reactions with cell surface TNP groups nor with Fc receptors. TNP modification of a variant cell line that does not express class I H-2 products did not result in enhanced binding by these mAb. These findings can account for preferential recognition of TNP-Kk by B10.A and B10.BR CTL, and also for cross-reactive lysis by C57BL/10 CTL stimulated by C57BL/10-TNP against unmodified H-2Kk targets.

Mechanism of target cell recognition by natural killer cells: characterization of a novel triggering molecule restricted to CD3- large granular lymphocytes.

In an attempt to identify a molecule in target recognition by CD3- large granular lymphocytes (LGL), we have generated a rabbit antiidiotypic (anti-ID) serum against a monoclonal antibody (mAb 36) that reacted with the cell membrane of K562. Flow cytometry analysis demonstrated that the anti-ID serum bound selectively to CD3- LGL and that F(ab')2 fragments of the anti-ID serum blocked both target cell binding and lysis by NK cells. Stimulation of CD3- LGL with F(ab')2 fragments resulted in the release of serine esterases and the secretion of interferon gamma. Furthermore, anti-ID F(ab')2 antibodies crosslinked to anti-DNP F(ab')2 mediated directed cytotoxicity of a non-natural killer (NK)-susceptible mouse target (YAC-1) via this surface ligand. These functional reactivities were only removed by adsorption with the specific idiotype. Protein analysis showed that the anti-ID serum immunoprecipitated 80-, 110-, and 150-kD proteins. Using this anti-ID, a partial cDNA was cloned and an antipeptide antiserum was made against the portion of the predicted amino acid sequence that corresponded to a portion of the ID binding region. This antipeptide serum exhibited similar functional and biochemical reactivities to those observed with the anti-ID serum. These data suggest that the cell surface moiety recognized by the anti-ID and anti-p104 is novel and is selectively involved in both recognition and triggering of NK-mediated lytic function.

Two-ion Coulomb crystals of Ca + in a Penning trap.

Results demonstrating laser cooling and observation of individual calcium ions in a Penning trap are presented. We show that we are able to trap, cool, image and manipulate the shape of very small ensembles of ions sufficiently well to produce two-ion 'Coulomb crystals' aligned along the magnetic field of a Penning trap. Images are presented which show the individual ions to be resolved in a two-ion crystal. A distinct change in the configuration of such a crystal is observed as the experimental parameters are changed. These structures could eventually be used as building blocks in a Penning trap based quantum computer.

Histamine regulates cytokine production in maturing dendritic cells, resulting in altered T cell polarization.

Atopic diseases such as allergy and asthma are characterized by increases in Th2 cells and serum IgE antibodies. The binding of allergens to IgE on mast cells triggers the release of several mediators, of which histamine is the most prevalent. Here we show that histamine, together with a maturation signal, acts directly upon immature dendritic cells (iDCs), profoundly altering their T cell polarizing capacity. We demonstrate that iDCs express two active histamine receptors, H1 and H2. Histamine did not significantly affect the LPS-driven maturation of iDCs with regard to phenotypic changes or capacity to prime naive T cells, but it dramatically altered the repertoire of cytokines and chemokines secreted by mature DCs. In particular, histamine, acting upon the H2 receptor for a short period of time, increased IL-10 production and reduced IL-12 secretion. As a result, histamine-matured DCs polarized naive CD4(+) T cells toward a Th2 phenotype, as compared with DCs that had matured in the absence of histamine. We propose that the Th2 cells favor IgE production, leading to increased histamine secretion by mast cells, thus creating a positive feedback loop that could contribute to the severity of atopic diseases.

Bispecific antibodies in cancer therapy.

Based upon in vitro and animal studies, a number of Phase I and II clinical trials have been initiated to test whether bispecific antibodies could redirect immune effectors against tumor cells in cancer patients. Recently, results from those trials showed beneficial effects in some patients but it is clear many problems remain to be solved. In addition, molecular engineering approaches are providing new and improved sources of clinically relevant bispecific antibodies.

Enhanced in vitro lysis of human ovarian carcinomas with activated peripheral blood lymphocytes and bifunctional immune heteroaggregates.

Heteroaggregated monoclonal antibodies (HA) comprised of an anti-CD3 and an anti-tumor associated antigen (TAA) antibody were evaluated for their ability to lyse fresh human ovarian cancer cells. HAs were able to significantly enhance the in vitro lysis of fresh ovarian tumor cells by in vitro activated peripheral blood lymphocytes (PBLs). HAs did not increase tumor lysis using fresh PBLs or PBLs cultured overnight in vitro. HA efficacy required both anti-CD3 and anti-TAA antibodies to be present in the same molecule, implying that physical bridging between the effector and target cell by the HA may be a requirement for their activity in cell lysis. The presence of anti-CD3 antibody in the PBL cultures enhanced cell growth and did not block the efficacy of the anti-CD3 containing HAs. The frequent expression of multiple TAAs in human ovarian cancers and the ability to recruit cytotoxic T-cell activity with HAs in vitro give promise to the future of this form of immunotherapy in the clinical setting.

Human T-lymphocytes targeted against an established human ovarian carcinoma with a bispecific F(ab')2 antibody prolong host survival in a murine xenograft model.

A bispecific F(ab')2 fragment with anti-CD3 and antitumor specificity was used to target activated human peripheral blood lymphocytes (PBL) against OVCAR-3 human ovarian carcinoma cells growing i.p. in athymic mice. Mice were given injections of OVCAR-3 cells on day 0 and treated with i.p. injections of activated PBL coated with the [anti-CD3 (TR66) x antitumor (MOv18)] bispecific F(ab')2 on day 4, using an approximate effector:target ratio of 1:1. Treatment was evaluated for the ability either to block tumor growth at 15 days or to prolong survival of tumor-bearing mice. After 15 days, the incidence of mice with tumor growth was 20% among those given PBL coated with bispecific F(ab')2, whereas the incidence among mice untreated or treated with PBL alone or PBL with either parental antibody ranged from 80 to 94%. The mean survival time of tumor-bearing mice treated with PBL and bispecific F(ab')2 was 104 days, which was 3.5 times that of untreated mice and twice that of mice given PBL alone or PBL with either parental antibody. These results provide support for the concept that treatment of ovarian cancer patients with targeted T-cells could prove beneficial.

Targeting human T-lymphocytes with bispecific antibodies to react against human ovarian carcinoma cells growing in nu/nu mice.

In the present study we tested whether human T-cells from normal donors can be targeted against human ovarian carcinoma cells and block i.p. growth of an established tumor in immunodeficient mice. For targeting we used chemically cross-linked bispecific monoclonal antibodies (mAbs) reacting with CD3 on the T-cells and with cell-surface antigens selectively expressed by tumor cells. The tumor model consisted of mice given i.p. injections of a human ovarian carcinoma cell line, OVCAR-3, whose growth includes development of massive ascites. Peripheral blood lymphocytes from normal human donors were cultured overnight with 50-100 units/ml recombinant interleukin 2, coated with bispecific antibodies, and injected i.p. into mice 4-6 days after tumor inoculation, at which time tumor cells were established and growing in about 85% of the hosts. Tumor growth was assessed by the number of tumor cells, and in some tests by cell-free tumor antigen, recovered in peritoneal lavage fluid collected 15 days after tumor priming. Treatment with lymphocytes retargeted with bispecific mAbs, prepared with anti-CD3 and three different antitumor mAbs, 113F1, OVB-3, and MOv19, gave highly significant increases in percentages of mice without detectable tumor. Controls showed that the antitumor activity of retargeted lymphocytes did not result simply from antibody-dependent cellular cytotoxicity or from heteroconjugates reacting only with CD3 or with lymphocyte major histocompatibility complex determinants and tumor cells. These results show that targeted T-lymphocytes can significantly decrease the growth of an established tumor in a fashion specific for antigens expressed by the neoplastic cells.

A single-chain bispecific Fv2 molecule produced in mammalian cells redirects lysis by activated CTL.

Single-chain Fv (sFv) molecules consist of the two variable domains of an antibody (Ab) connected by a polypeptide spacer and contain the binding activities of their parental antibodies (Abs). In this paper we have attached the C-terminus of 2C11-sFv (anti-mouse CD3 epsilon-chain) to the N-terminus of OKT9-sFv (anti-human transferrin receptor [TfR]) through a 23 amino acid inter-sFv linker consisting primarily of CH1 region residues from 2C11, to form a single-chain bispecific Fv2 [bs(sFv)2] molecule. The bs(sFv)2 was expressed in COS-7 cells, and was secreted at the same rate as the two parental sFvs. The secreted protein had both anti-CD3 and anti-TfR binding activities. Essentially all of the secreted bs(sFv)2 molecules bound TfR and the binding affinity of the bs(sFv)2 was comparable to that of OKT9 sFv and Fab. Thus, the attachment of the inter-sFv linker to the N-terminus of OKT9-sFv did not impair its binding function. The bs(sFv)2 retained both binding specificities after long-term storage at 4 degrees C or overnight incubation at 37 degrees C. It redirected activated mouse CTL to specifically lyse human TfR+ target cells at low (ng/ml) concentrations and was much more active than a chemically cross-linked heteroconjugate prepared from the same parental mAbs. Because bs(sFv)2 molecules secreted by mammalian cells are homogeneous proteins containing two binding sites in a single polypeptide chain, they hold great promise as an easily obtainable, economic source of a bispecific molecule suitable for in vivo use.

Correct disulfide pairing and efficient refolding of detergent-solubilized single-chain Fv proteins from bacterial inclusion bodies.

In vitro folding of denatured proteins has remained an inefficient and empirical process that has limited the use of bacterially expressed recombinant proteins. In this paper we show that in vitro folding of recombinant single-chain Fv (sFv) proteins is markedly facilitated when disulfide bonds are formed in detergent solution. sFv proteins from three different antibodies were expressed as bacterial cytoplasmic inclusion bodies and solubilized in the weak ionic detergent, sodium lauroylsarcosine (SLS). Upon oxidation in air in the presence of metal ion catalysts, all three sFvs quantitatively formed intrachain disulfide bonds which ran as a single band in SDS-polyacrylamide gel electrophoresis under non-reducing conditions. By contrast, oxidation from 6 M urea gave large amounts of disulfide linked aggregates, and three closely spaced bands of monomeric protein. Detergent was removed from the oxidized sFvs by addition of 6 M urea and absorption with an ion exchange resin. After dialysis and gel filtration in non-denaturing solution, moderate to high yields of monomeric sFv were obtained, depending upon the sFv. All three sFvs gave single bands on isoelectric focussing and SDS-PAGE gels and had similar or identical binding specificities and affinities as the parental Fabs, implying that the final products contained correctly paired disulfide bonds. The correct disulfide pairing suggests that the disulfide loops within the detergent-solubilized sFvs adopt a native-like structure.

The role of non-immune IgG in controlling IgG-mediated effector functions.

The majority of evidence supports the conclusion that IgG-dependent effectors respond to antibodies which have been polymerized artificially or by polyvalent antigens, but not to monomeric IgG antibodies. Effectors can distinguish polymerized IgG antibodies from monomeric IgG because they contain multiple receptor units and can interact multivalently with polymerized IgG. However, monomeric IgG is present at very high concns in plasma and interstitial fluids and will inhibit multivalent interactions in vivo between polymerized antibody and effectors. Such inhibition raises the question of how IgG-mediated effector responses could function in vivo. In this review we present a mathematical model which quantitatively predicts how polyvalent ligands interact multivalently with receptors in the presence of excess monovalent ligand. We then show that results from experiments in vitro using such diverse systems as the binding and endocytosis of immune complexes by macrophages, complement-mediated lysis of antibody-coated target cells, and ADCC can be explained qualitatively by the model. We conclude that monomeric IgG does not totally inhibit IgG-mediated effector functions but, rather, raises the threshold of antibody binding which is required to elicit a response. We then consider how non-immune IgG may serve as a homeostatic regulator of IgG-dependent responses, in vivo, perhaps for the purpose of inhibiting responses to low levels of cell-bound IgG autoantibodies.

Processing fate of protein antigen attached to IgD or MHC molecules on normal B lymphocytes using heterocrosslinked bispecific antibodies.

We have studied the internalization, processing and presentation of hen egg lysozyme (HEL) attached to surface IgD (sIgD) or MHC molecules on normal murine B cells, using heterocrosslinked bispecific antibodies (HBA). Nearly all HEL attached to sIgD was internalized within one hour, with at least a portion rapidly entering a chloroquine-sensitive, acidic environment. Degradation and presentation of HEL to hybridoma T cells began several hours after internalization. Degraded HEL was found in the medium after about 6 hr incubation, but at no time were significant amounts of HEL peptides found within the cells. When HEL was attached to class I or class II MHC molecules, its rate of internalization was low. The fraction of antigen bound to MHC molecules that was inside the cell was always low, even at later stages of culture, but the internalized antigen was located in an acidic environment. Degradation and presentation of HEL internalized via MHC molecules followed internalization. No difference was observed in the processing fate of HEL attached to class I or class II MHC molecules. These results suggest that the rate limiting step in antigen processing and presentation is antigen degradation, when the antigen is bound to sIgD, and internalization when bound to MHC molecules. The slow and steady processing of bound or internalized antigen could provide a sustained presence of antigen on the B cell surface and enhance the potential for its presentation to T cells.

Activation of human T cells obtained pre- and post-interleukin-2 (IL-2) therapy by anti-CD3 monoclonal antibody plus IL-2: implications for combined in vivo treatment.

The role of activated T cells in the mediation of antitumor responses has been documented in several experimental models. In some of these, interleukin-2 (IL-2) has been used as a means to induce and expand the antitumor effects of the T cells. IL-2 has been tested in clinical trials for cancer treatment. Surprisingly, T cells appear to be inactivated by IL-2 in these clinical trials. T cells obtained from peripheral blood after IL-2 therapy showed decreased responses to mitogens and alloantigens, did not proliferate in vitro in response to IL-2, and did not mediate non-major histocompatibility complex-restricted cytotoxicity or targeted lysis in the presence of bispecific monoclonal antibodies. In this study, we present evidence that these post-IL-2 therapy T cells are not irreversibly inactivated; they can be activated in vitro by anti-CD3 monoclonal antibody together with IL-2 to upregulate the p55 component of the IL-2 receptor and proliferate. Nevertheless, following activation by anti-CD3 and IL-2, the level of targeted T-cell cytotoxicity mediated by the post-IL-2 therapy T cells was significantly lower than that by pre-IL-2 therapy T cells. Although in vivo treatment with IL-2 alone induces natural killer (NK) cells to mediate lymphokine-activated killer activity, these data suggest that the T-cell lytic function is inhibited by this treatment and only partially reversible by subsequent T-cell receptor activation using anti-CD3 mAb. Exposure of T cells to anti-CD3 mAb prior to in vivo IL-2 treatment generates T-cell lytic activity in vitro. These results, together with preclinical murine studies, suggest that a combined in vivo protocol of anti-CD3 mAb and IL-2, starting first with the anti-CD3 mAb, may cause activation of the T cells in addition to the activation of NK cells and thus warrant clinical testing.

Quantitation of Fc receptors and surface immunoglobulin is affected by cell isolation procedures using plasmagel and ficoll-hypaque.

When mononuclear leukocytes are isolated directly from whole human blood using Ficoll-Hypaque or Plasmagel, cytophilic immunoglobulin is detected on cell surfaces. Upon incubation at 37 degrees C, this cell-associated immunoglobulin is shed slowly into the medium. However, when cells are prewashed in phosphate-buffered saline prior to isolation, they appear to be free of cytophilic immunoglobulin. Compared to prewashed cells, populations retaining cytophilic immunoglobulin on their surfaces demonstrate a decreased binding of soluble immune complexes and radiolabelled trimeric rabbit IgG. The data suggest that Ficoll-Hypaque and Plasmagel cause serum IgG to bind with abnormally high affinity to human mononuclear leukocytes, probably via Fc receptors. This artifact of preparation can lead to erroneous estimates of the numbers of cells bearing Fc receptors or intrinsic membrane immunoglobulin within a given population of cells and to an inaccurate assessment of the average number of Fc receptors per cell.

Texas Red, a hydrophilic, red-emitting fluorophore for use with fluorescein in dual parameter flow microfluorometric and fluorescence microscopic studies.

The sulfonylchloride derivative of the red-emitting fluorophore, sulforhodamine 101, has been synthesized in order to provide a reagent for coupling to amino groups on proteins and other compounds, and it is now commercially available under the name 'Texas Red'. Texas Red conjugates of antibodies and other proteins have been prepared in high protein yields; these conjugates retained their biological activities and were strongly fluorescent. The excitation and emission spectra of Texas Red conjugates are widely separated from those of molecules labeled with fluorescein isothiocyanate. Texas Red is therefore an excellent reagent for use in single or dual label flow microfluorometric and fluorescent microscopic studies.

Comparison of the ability of red cells sensitized with a bispecific anti-D x anti-Fc gamma RIII Fab fragment to activate human K cells and peritoneal macrophages through Fc gamma RIII.

The functional activity of Fc gamma RIII on human K cells from peripheral blood was compared with that of Fc gamma RIII on peritoneal macrophages (PM) separated from the waste material of patients undergoing peritoneal dialysis. Fc gamma R function was assessed in vitro using human monoclonal IgG1 anti-D (AB5) or a bispecific antibody comprising Fab fragments of AB5 chemically linked to Fab fragments of monoclonal anti-Fc gamma RIII, 3G8 (AB5 x 3G8). In antibody-dependent cell-mediated cytotoxicity (ADCC) assays, K cells mediated the lysis of papainized red cells sensitized with the AB5 x 3G8 bispecific antibody but not with AB5. In contrast, red cell lysis by PM was not promoted by AB5 x 3G8 although AB5 was active. However, this lysis, being inhibited by monomeric IgG, was presumably mediated via Fc gamma RI. AB5 x 3G8 also failed to promote the binding and phagocytosis of both papainized and native red cells by PM although 99% of red cells and over 90% of peritoneal cells bound the bispecific antibody. In marked contrast to K cells therefore, Fc gamma RIII on PM was unable to mediate functional interactions with red cells sensitized with anti-D x anti-Fc gamma RIII bispecific antibody.