Range Of Antigen Concentrations Research Articles

Altered peptide ligands and wild-type peptide induce indistinguishable responses of a human Th0 clone.

The response of the human influenza hemagglutinin (HA)-specific T helper clone HA1.7 to its wild-type (wt) HA307-319 peptide ligand and related altered peptide ligands (APL) was examined over a wide range of antigen concentrations. The time course of cytokine production and surface expression of CD40 ligand and CD3 was followed at the single-cell level by flow cytometry and compared with the induction of proliferation. We observed that the APL induced responses that were indistinguishable from those induced by the wt HA ligand, albeit at higher antigen densities. Moreover, the activation parameters were induced with identical kinetics. Blocking of CD4 co-ligation inhibited the recognition of the weakly stimulatory APL, but not of the wt HA307-319 peptide. Finally, in all cases the response of the T cell clone correlated with down-regulation of surface TCR/CD3 complexes. Together, these observations support a quantitative model of T cell activation.

A Descriptive Model for the Kinetics of a Homogeneous Fluorometric Immunoassay

A descriptive mathematical model was chosen to fit the antigen-antibody association kinetics of a new homogeneous immunometric assay for prolactin, involving time-resolved fluorescence detection (TRACE® technology, Time Resolved Amplified Cryptate Emission). We paid special attention to the methodology and criteria applied, to yield a convenient and statistically valid model, designed to allow potential exploitation of kinetic information in the data processing of the assay. We compared specific parameterizations of an hyperbolic model, the Gompertz, and the monomolecular models on the basis of morphological considerations, a statistical analysis of fit, and an assessment of the parameters estimation quality, over a wide range of antigen concentrations. The monomolecular model gave the best fit, and the most precise and stable estimation of its parameters. The study of parameter properties confirmed this choice.

Quantification of CYP1A1 and 2B1/2 in rat hepatocytes cultured in microwells by immunological methods

The quantification of P-450 enzymes (CYP proteins) is of great relevance for drug metabolism and toxicity studies. Their measurement in cultured cells requires a sufficient amount of cellular protein to isolate a minimal amount of functional microsomes. The difficulties in obtaining microsomes from a small number of cultured hepatocytes make it necessary to use more sensitive methods. Immunological methods can overcome such drawbacks. However, their use is prevented by the fact that P-450 enzymes are membrane proteins that require the use of detergents for adequate solubilization which, in turn, can interfere with the antigen-antibody recognition. A sensitive immunoassay (competitive indirect ELISA) was developed which allowed quantification of CYP1A1 and CYP2B1/2 in small culture plates with a reduced number of cells. The main advantages of this ELISA were: (1) precise measurement of both proteins over a wide range of antigen concentration (10–10,000 fmol CYP proteins); (2) minimal sample handling allowing reproducible titrations in small number of cells; (3) minimization of interference by detergents; (4) an excellent correlation (r = 0.96–0.98) between the immunologically determined CYP1A1 and CYP2B1/2, and the activities of ethoxyresorufin- O-deethylase (EROD) and pentoxyresorufin- O-depentylase (PROD), respectively. This assay allowed a precise monitoring of the expression of CYP proteins in cultured cells. Upon incubation of hepatocytes with 3-methylcholanthrene the levels of immunoreactive CYP1A1 and CYP2B1/2 were paralleled by an increase of the enzymatic activities of EROD and PROD, respectively. In cultured rat hepatocytes treated with phenobarbital, significant EROD activity was detected in the absence of immunoreactive CYP1A1 induction. Analogously, there was a dissimilarity between enzymatic activity and protein levels in ageing cultures: the decrease of PROD activity preceded by several hours that of the immunoreactive CYP2B1/2, which emphasizes the need to determine individual CYPs, in addition to their enzymatic activities.

Two Signal Activation as an Explanation of High Zone Tolerance: A Mathematical Exploration of the Nature of the Second Signal

Much work on the induction of non-responsiveness in lymphocytes suggests that it takes two signals to activate a cell correctly, and that receipt of the first signal alone not only causes suboptimal response, but also subsequent non-responsiveness or anergy. This paper discusses not anergy, but the original suboptimal response in the context of the well known but poorly understood phenomenon of high zone tolerance in experiments studying immunological responses to a range of antigen concentrations (low to high "zones"). We proceed to ask specific questions about the nature and timing of the signals involved. We construct three different models describing how the second signal might be received and use them to predict the shapes of dose-response curves. Comparison with data enables us to select, from a dynamical perspective, the most likely mechanism and to tentatively exclude a number of proposed mechanisms for high zone tolerance. We suggest that the stage at which a cell becomes susceptible to anergy is crucial and that it is over-contact, rather than lack of contact, which is important in the induction of tolerance.

An artificial glycoconjugate containing the bisphosphorylated glucosamine disaccharide backbone of lipid A binds lipid A monoclonal antibodies

Monoclonal antibodies (MAbs) against lipid A, the endotoxic component of lipopolysaccharide (LPS) of gram-negative bacteria, are presently discussed as therapeutic agents against lethal gram-negative infections; however, their binding specificities are controversial. We have isolated from the LPS of Escherichia coli J-5 the 1,4'-bisphosphorylated beta 1-->6-linked glucosamine disaccharide backbone of its lipid A moiety, which was covalently linked to bovine serum albumin. It was shown by solid-phase enzyme immunoassay that one antibody (MAb A6) bound equally well to the glycoconjugate and synthetic E. coli-type lipid A over a broad range of antigen concentrations whereas two other MAbs (IC3 and S1-15) bound better to the conjugate at low antigen concentrations and better to the lipid A when high concentrations of antigen were used. This proves in a direct way that there exist lipid A MAbs with different specificities which bind to epitopes in the hydrophilic backbone of lipid A and which do not require the presence of fatty acids.

Neonatal intubation with the Cole tube

<h3>Summary</h3> 1.Specific white cell lysis is produced by the addition of homologous antigen to fresh heparinized blood of certain rabbits immunized with crystalline hen ovalbumin or with bovine gamma globulin (Armour9s Fraction II). 2.Specific lysis may be produced in white cells of fresh normal rabbit blood by the addition of antigen and the serum of a sensitized animal. 3.There is an optimum concentration of antigen or range of antigen concentrations giving maximal lysis when the amount of antibody is constant. 4.Dilution of antiserum results in a corresponding reduction in the amount of antigen producing maximal lysis and in a reduction of its lytic potency. 5.Heating at 55–58 C for 4 hours failed to destroy the lytic capacity in 3 of 4 antisera tested. 6.The cells affected by specific lysis were polymorphonuclear leukocytes in tests of 5 different antisera (platelets were not studied). 7.The specific lytic capacity of an antiserum is not related to its content of precipitable antibody N or to its equivalence ratio R. No loss of lytic power results from removal of the precipitable antibody by small successive additions of antigen. Removal of all precipitable antibody by a single addition of antigen removed all lytic power from one of 2 sera so studied. Certain specifically lytic anti-ovalbumin sera showed lysis with an immunochemically uniform ovalbumin preparation and not with the common ovalbumin contaminants. These also showed a single band of precipitate by the Oudin agar diffusion technic, whereas certain nonlytic sera showed more than one band. 8.White cell lytic power was moderately well correlated with the ability to give passively transferred urticarial reactions in human or guinea pig skin, but not with the ability to cause passive anaphylaxis in the guinea pig. 9.Development of specifically lytic antibody was more evident in rabbits immunized by single intraperitoneal or intravenous doses of antigen in solution or repeated subcutaneous doses of alum-precipitated antigen than in rabbits receiving repeated subcutaneous or intravenous doses of antigen in solution.

Distinct IL-3 activation profile induced by intravenous versus subcutaneous routes of immunization

Lymphoid cells from mice immunized i.v. or s.c. with Leishmania major antigens were analyzed for their capacity to produce lymphokines when stimulated with specific antigens in vitro. Spleen cells from BALB/c mice immunized by the s.c. route produced significantly higher levels of IL-3 and IL-3 mRNA than those from mice immunized by the i.v. route. The differential production of IL-3 was maintained at a wide range of antigen concentrations tested in vitro and for different culturing times. T cell enrichment procedures and treatment with CD4 + mAb in vitro confirmed the T cell nature of the IL-3 producer population. However, the IL-3 production in the two populations of spleen cells was equally high after Con A stimulation in vitro. The IL-2 production by the two populations of cells was also not significantly different after antigen or mitogen stimulation in vitro. To our knowledge, this is the first report of a differential synthesis of IL-3 mRNA and secretion of IL-3 induced by different routes of immunization followed by specific-antigen stimulation in vitro. These findings may also explain earlier observations that i.v. immunization with leishmanial antigen induces protection, whereas s.c. immunization leads to exacerbation of L. major infection, since IL-3 has been previously shown to promote leishmanial infection. The fact that the phenomenon also extends to other antigen systems suggests that this finding may have a broader implication in immune regulation and vaccine development.

A radioimmunoassay to screen for antibodies to native conformational antigens and analyse ligand-induced structural states of antigenic proteins

A radioimmunoassay is described in which antigenic protein was immobilized by incubating nitrocellulose filters of defined diameter with antigen-containing solutions. The amount of adsorbed antigen increased in a linear fashion over a wide range of antigen concentrations. The antigen-antibody reactions and the indicator reactions were performed by incubating the filters with appropriate solutions. During the test any contact of the antigen with air was avoided. Antigenic sites which are sensitive to protein denaturation by drying could be detected with the assay. The assay was also used to screen hybridoma supernatants for antibodies directed against Na + cotransport proteins from renal brush-border membranes. Monoclonal antibodies were selected which showed different binding characteristics depending on whether or not substrates of Na + cotransporters were present. Since binding of several antibodies was altered by two different substrates and not by non-transported control substances, these monoclonal antibodies were believed to interact with more than one transport system. One of the antibodies, which showed different antibody binding after addition of D-glucose or L-lactate, bound to a polypeptide component of the renal Na +- D-glucose cotransporter and was able to inhibit Na + gradient-dependent D-glucose uptake in brush-border membrane vesicles (Koepsell, Korn, Raszeja-Specht, Bernotat-Danielowski, Ollig, 1988, J. Biol. Chem., in press). To investigate the effects of D-glucose and L-lactate on the binding of this antibody concentration dependence was measured. High and low affinity binding sites for D-glucose and L-lactate were characterized thereby demonstrating that the radioimmunoassay permits investigations of the properties of high and low affinity substrate binding sites.

Comparison by leukocyte adherence inhibition of human immune response to cancer-associated immunogens, Thomsen-Friedenreich (T) and Tn, myelin basic protein, and organ-specific cancer neoantigens

Peripheral blood leukocytes from cancer patients exhibit nonadherence to glass as an index of antigen recognition when incubated individually with four distinct, soluble tumor-related substances. Crude cancer extracts, purified antigens, T and Tn, myelin basic protein (MBP), and organ-specific cancer neoantigens (OSN), all elicited narrow dose-dependent leukocyte adherence inhibition (LAI) response curves. The present study focused on the reasons for the narrow antigen dose-LAI response relationship. Between 9 and 20 pmol of antigens elicited the maximum number of nonadherent leukocytes; cleavage products of T antigen and the nonapeptide (T18) of MBP required about a 10-fold increase in molar concentration for the same LAI response. When crude cancer extracts were combined with pure antigen or the pure antigens were combined at concentrations shown to give maximum LAI responses, the positive LAI responses were negated. The chemoattractant LTB 4 at 10 −11 M triggered maximum LAI. But when MBP was added with the LTB 4 at progressively increasing concentrations, there was dose-dependent negation of LAI. The magnitude of LAI depended on the total amount of mediator released rather than the rate of release. When leukocytes from cancer patients were incubated with optimum to high concentrations of MBP, the supernatants contained a mediator that gave similar bell-shaped dose-LAI responses on control leukocytes indicating that leukocytes from cancer patients react to a much broader range of antigen concentration than indicated by the LAI assay alone. High antigen dose negated LAI because of excess mediator production. Antigen-generated mediators had a biphasic effect inducing nonadherence and then adherence of leukocytes.

Differences in the repertoire of the Lewis rat T cell response to self and non-self myelin basic proteins.

We have examined the fine specificity of a panel of cloned T cell hybridomas generated from Lewis rats immunized with guinea pig (GP) or Lewis rat myelin basic protein (MBP) to determine the autoimmune T cell repertoire that develops in experimental allergic encephalomyelitis (EAE). This analysis has demonstrated that GP MBP, which was approximately 10-fold more potent for EAE induction than the autologous rat MBP, produced a population in which almost one-fourth of the members responded to GP-unique determinants and displayed no crossreactivity on the self antigen. The remaining majority of GP MBP-induced clones were specific for the 68-88 encephalitogenic determinant and could be subdivided into three groups based on their varying responses to the 68-88 peptide and rat and rabbit MBPs. Surprisingly, one of these groups showed equal reactivity to GP and rat MBPs. In contrast, the clonotype composition of the T cell population induced by rat MBP was quite different. One-half of these clones comprised a single group responding to the 68-88 determinant, reacting equally with GP and rat MBP. All of these responded to the same range of antigen concentrations as their GP-induced counterparts. The remaining half of that population contained a collection of clones that was nearly as encephalitogenic as the 68-88 population after propagation as a short-term T cell line. These clones were specific for at least three distinct antigenic determinants, all displaying extensive cross-species reactivity, and required as little or less rat MBP for maximal stimulation as did the 68-88-reactive clones. We therefore conclude that the T cell repertoire for MBP does include clones with reactivity to both 68-88 and non-68-88 determinants of GP and rat MBPs, and that both MBPs appear to be equally capable of stimulating these clones in vitro. However, the differences in the clonotype composition of the populations induced by immunization with these two antigens suggest that rat and GP MBPs are subject to different immunoregulatory constraints in the animal and may account for the difference in the encephalitogenic potential of these two antigens.

Interleukin 4 enhances the ability of antigen-specific B cells to form conjugates with T cells.

T cell-B cell conjugates are formed when trinitrophenyl-specific B cells are exposed to trinitrophenyl-ovalbumin and ovalbumin-specific T hybridoma cells. The proportion of conjugates was increased two- to threefold when antigen-pulsed trinitrophenyl-specific B cells, but not T cells, were pre-exposed to interleukin 4. Antigen-specific B cells pretreated with antigen and interleukin 4 and cultured in the presence of specific T helper cells also produced a larger proportion of antibody-secreting cells as compared to cells pretreated with antigen alone. The interleukin 4-induced enhancement of T/B conjugate formation occurred over a wide range of antigen concentrations, was dependent on the concentration of interleukin 4, and was inhibited by the monoclonal anti-interleukin 4 antibody, 11B11. The importance of Ia antigens in the enhancement of conjugate formation and generation of antibody-secreting cells is suggested by a) the fact that the interleukin 4-mediated increase in the density of Ia antigens on the antigen-specific B cells correlated with their enhanced ability to form T/B conjugates, b) the kinetics of the interleukin 4-mediated increase in conjugate formation and surface Ia expression were similar, c) 10- to 20-fold higher concentrations of anti-I-A antibody were required to inhibit T/B conjugate formation by 50% with interleukin 4-treated antigen-specific B cells compared with untreated antigen-specific B cells, and d) interferon-gamma, which inhibits the interleukin 4-mediated increase in Ia antigens, inhibited the interleukin 4-induced enhancement of T/B conjugate formation. These results indicate that the interleukin 4-induced increase in the expression of Ia antigens on B cells plays an important role in the enhancement of T/B cell interactions and the subsequent differentiation of antigen-specific B cells into antibody-secreting cells.

Geographic classification of dengue-2 virus strains by antigen signature analysis

Dengue-2 virus strains from different locations were compared by T 1-RNAse-resistant oligonucleotide fingerprinting and antigen signature analysis. The latter technique involved construction of radioimmunoassays using monoclonal antibodies that recognize nine distinct dengue-2 type-specific and flavivirus cross-reactive epitopes over a range of antigen concentrations. A statistical method was used to align unknown dengue antigen concentrations in different strain preparations, allowing comparison of binding profiles. Twentysix dengue-2 virus strains were separated into five distinct groups (topotypes) on the basis of unique RNA fingerprints. Two of these were represented by New Guinea C, the prototype virus isolated in 1944, and a Philippine strain; others were segregated on the basis of ≥80% shared oligonucleotides into similarity groups representing Burma/Thailand (8 strains), Puerto Rico (12 strains), and Jamaica (4 strains). Signature analysis of the prototype and four geographic topotype strains revealed striking antigenic differences. In contrast, a high degree of antigenic similarity was found among strains from the same geographic region. Variation between antigenically distinct strains occurred at both type-specific and group-reactive epitopes, but the widest differences appeared at group-reactive determinants. Signature analysis provides a more rapid and simpler means than RNA fingerprinting of monitoring changes or new introductions of dengue virus populations in a geographic region.

Immune sensitization to syngeneic organ-specific intestinal antigens in the Lewis rat

In human chronic inflammatory bowel disease involving mucosal epithelium, sera and lamina propria mononuclear cells are reactive with cell surface components isolated from gut epithelial cells. To define a model system in which the disease-inducing potential of such immune factors could be rigorously evaluated, we sought (1) to immunologically sensitize inbred murine strains to syngeneic colonic epithelial cell-associated components (ECAC-C), (2) to define precise in vivo and in vitro conditions to optimize ECAC-C reactivity, and (3) to initially explore whether such cells could elicit tissue injury in epithelium after adoptive transfer to naive animals. Following footpad immunization, Day 42 lymph node cells but not splenocytes were reactive with syngeneic ECAC-C, as shown by a linear increase in [ 3H]thymidine incorporation over a wide range of antigen concentration (0.5 to 100 μg/ml). A subsequent 48-hr exposure to ECAC-C and/or interleukin 2 resulted in a more restricted responsiveness, proliferation occurring only in the presence of ECAC-C or mitogen and not to a coimmunogen (PPD). Further evidence that lymph node cells from ECAC-C/CFA immunized animals were indeed sensitized to syngeneic ECAC-C included (1) ability of donor animals to mount highly significant earlobe DTH responses to ECAC-C, indicating the presence of antigen-specific T-DTH cells, and (2) the failure of polymyxin B, in doses sufficient to inhibit LPS-induced mitogenesis, to reduce lymph node cell responsiveness to ECAC-C, known to be contaminated with LPS. ECAC-C-specific circulating antibody and T-cytotoxic cells were not detected. Adoptive transfer of Day 42 lymph node cells, sensitized in vivo and conditioned in vitro, was not associated with tissue injury in syngeneic recipients in preliminary experiments. This model system, with antigen-specific cells analogous to those present in diseased mucosa of human chronic inflammatory bowel disease, may be an important means to determine the pathophysiologic significance of anti-epithelial cell immune responses in these disorders.

Selective impairment of B cell function by Neisseria meningitidis

Spleen cells from CBA/J mice infected with Neisseria meningitidis displayed depressed in vitro plaque-forming cell (PFC) responses to T-dependent (sheep red blood cell; SRBC) and T-independent (TNP-LPS, TNP-Ficoll) antigens. The inhibition was observed over a wide range of antigen concentrations. The decreased responsiveness of splenocytes from infected mice was due to a selective impairment of B-cell function since (1) helper-T-cell activity was intact in infected mice as shown by the ability of T-enriched lymphocytes to cooperate with normal B-enriched lymphocytes in the generation of an anti-SRBC response, (2) accessory macrophage function was preserved since adherent spleen cells from bacteria-injected mice were shown to produce normal or increased levels of IL-1 and were able to cooperate with normal non-adherent spleen cells in the generation of PFC against SRBC. Addition of peritoneal cells from normal animals or extraneous IL-1 both failed to restore normal PFC responses in cultures of splenocytes from infected mice. Finally, (3) B-enriched lymphocytes from infected mice produced poor anti-SRBC responses when cultured with either Con A supernatant or T-enriched lymphocytes from normal or infected mice. Cell-mixing experiments failed to detect the presence of suppressor cells in cultures of unfractionated spleen cells or B-enriched lymphocytes from infected mice. Therefore, the immunological unresponsiveness associated with a Neisseria meningitidis infection was attributed to a meningococcus-induced defect(s) in B-cell function. In vivo polyclonal B-cell activation leading to clonal exhaustion did not play a major role in the depression of humoral responses since meningococcal infection induced little or no polyclonal Ig secretion.

Anti-immunoglobulin augments the B-cell antigen-presentation function independently of internalization of receptor-antigen complex.

All mouse splenic B cells, including small resting B cells, process and present the native globular protein antigens, pigeon and tobacco hornworm moth cytochromes c, to a cytochrome c-specific T-cell hybrid in a major histocompatibility complex-restricted fashion, in the micromolar to nanomolar antigen-concentration range. As is the case for macrophages, treatment with paraformaldehyde or the lysosomotropic agents chloroquine and ammonium chloride blocked processing of the native pigeon protein but did not affect the presentation of a carboxyl-terminal peptide fragment of pigeon cytochrome c (residues 81-104) which contained the T-cell antigenic determinant. However, in contrast to macrophages, whose antigen-processing and -presentation functions are insensitive to radiation, radiation blocked the processing of the native protein but not the presentation of the peptide fragment. The processing and presentation function of the B cells was augmented by F(ab')2 of rabbit anti-mouse Ig antibodies, in that 1/10th to 1/30th as many cells and 1/10th as much antigen were required to maximally activate the T-cell hybrid. This augmentation did not appear to be due to either crosslinking of the Ig receptors or to B-cell activation, as monovalent Fab fragments were nearly as effective as the bivalent reagent, and the concentrations of F(ab')2 anti-Ig used did not induce measurable proliferative responses. Furthermore, enhancement can occur in the absence of cytochrome c binding and internalization, since B cells that were fixed with paraformaldehyde after treatment with F(ab')2 anti-Ig were more effective in presenting the carboxyl-terminal peptide than were untreated fixed cells. The same phenomenon followed the binding of an irrelevant antigen (carboxydinitrophenylated bovine serum albumin) by antigen-binding B cells, resulting in enhanced processing and/or presentation of native pigeon cytochrome c. Thus, nonspecific enhancement of antigen processing and presentation can be obtained by either antigen or anti-Ig binding to the B-cell antigen receptor, both treatments presumably delivering the same signal without requiring internalization of the specifically bound antigen for subsequent processing.

An assay to measure antigen-specific immune complexes in food-allergy patients

An allergen- and isotype-specific assay to quantify the presence of serum immune complexes is described. This assay is based on a two-site recognition system so that only molecular complexes containing both antigen and antibody are counted. In this study affinity-purified antibodies to egg and milk whey were used on paper discs in conjunction with a soluble, labeled anti-immunoglobulin (IgE or IgG specific). To evaluate the assay, immune complexes were prepared in vitro by mixing antigen at different concentrations with serum from a patient with high levels of antibody. Complexes were detectable over a 5 log range of antigen concentration. The lower limit of sensitivity of the assay was estimated to be 5.8 ng of complexed IgE per milliliter of patient serum. To determine if the assay could detect natural in vivo-formed complexes, sera from patients with positive RAST scores to egg white or milk were selected for a preliminary study. Three of the nine egg white-positive sera and two of the nine milk-positive sera were found to contain significant levels of IgE immune complexes. The versatility and sensitivity of the assay should now make it feasible to design experiments to elucidate the role, if any, of circulating immune complexes in the etiology of food allergy.

Manifold amplification of in vivo immunity in normal and immunodeficient mice by ribonucleosides derivatized at C8 of guanine.

Antigen-specific lymphocyte activation is generally thought to be initiated by the binding of antigen to specific membrane receptors, followed by transduction of a signal across the cell membrane. In addition, at least two supplementary signals apparently are required for induction of specific antibody synthesis. Various exogenous agents have been shown to augment the magnitude of specific responses to antigen. Certain of these adjuvants are polyclonal activators as well. We recently described a new class of activator, the C8-substituted-guanine ribonucleosides, that traverses the cell membrane and bypasses classical triggering mechanisms to activate the lymphocyte at an intracellular site. The current studies demonstrate that this new class of activator exerts powerful in vivo adjuvant activity when administered to mice after antigen. This effect is highly dose- and time-dependent. When nucleoside administration is delayed until 3-4 days after immunization, enhancement of the response persists but the optimal dose of nucleoside decreases by 2 orders of magnitude, indicating that these compounds are highly effective modulators of the immune response well after the initiating events have occurred. Amplification of the antibody response occurs over a wide range of antigen concentrations but is maximal at optimal antigenic concentrations. Enhancement of antibody responses in vivo is absolutely dependent upon derivatization of guanosine at C8; native guanosine is ineffective. When administered after antigen (sheep erythrocytes) to male (CBA/N X CBA/CaJ)F1 mice (which bear the xid B-cell defect) in an effort to bolster their impaired response to this T cell-dependent antigen, the brominated nucleoside induced a degree of responsiveness equivalent to that generated in the normal (female) counterparts injected with antigen and 8-bromoguanosine.

A rapid photoelectric method for reading cell migration from agarose microdroplets

A rapid photoelectric method for reading cell migration from agarose microdroplets is described. Practically instantaneous, the method eliminates drawing and planimetry and makes feasible the use of a wide range of antigen concentrations. The results obtained are similar to those obtained by planimetry, but the photoelectric method is more sensitive. Enhancement of migration as well as inhibition were significantly demonstrated by this method. Migration inhibition of immune mouse spleen cells was found to be bizonal, with 2 peaks, one at very low antigen concentrations (10 −3 μg/ml ovalbumin) and one at 10 μg/ml.

Properties of a murine lymphokine that augments E-rosette formation.

Antigenic stimulation of spleen mononuclear cells from contact-sensitized mice produced a soluble factor that enhanced the formation of E-rosettes by human lymphocytes in vitro. Production of this murine E-rosette augmenting factor (E-RAF) occurred over a narrow range of antigen concentrations, was hapten-specific, and depended on T-lymphocytes. The factor was not dialysable, in contrast to other substances extracted from normal leucocytes. Murine E-RAF is thus a lymphokine whose action is not species restricted. It was readily detected 30 min after contact of cells with antigen. E-RAF formation by spleen cells and delayed-type hypersensitivity reactions were elicited over a similar period after contact sensitization of mice.

Regulation of the specific plaque‐forming cell responsein man: Restraint of B cell responsiveness

AbstractSpecific in vitro plaque‐forming cell (PFC) responses of normal human lymphocytesare antigen dose‐dependent, with bell‐shaped dose response patterns. Both the bell‐shaped antigen dose response characteristics as well as optimal PFC responseamplitudes are dictated by two distinct and separable T lymphocyte subpopulationswhich act on a nonlimiting set of PFC precursor B cells. Specific theophylline‐sensitive suppressor and theophylline‐resistant helper T cells are activated by antigen in adose‐dependent fashion. The initially small suppressor cell pool expands exponentially following antigen contact, whereas the larger helper cell population does not. This leaves a restricted range of antigen concentrations where net helper activity issufficient to promote a PFC response. Due to coinduced suppressor activity, PFCresponses showed an overall negative restraint at all antigen concentrations. Limitingdilution analysis suggested that it is the numerical balance between the two alternative T cell subsets which determines the extent of this negative restraint. These datadelineate a cellular mechanism for the translation of an antigenic stimulus into a B cellantibody response; theophylline‐sensitive suppressor cells are able to expand inresponse to this stimulus and represent the main regulative vector. Thus, in mixedlymphocyte populations, antigen concentration is a valuable probe for studies of thebalance of discrete T cell subpopulations but not for absolute B cell responsiveness.