The electrophoretic hemolytic plaque assay - theory

We use the mathematical theory of plaque growth to determine if there is merit in performing a hemolytic plaque assay in the presence of an external electric field. In particular, we study the effects of an electric field on the transport of anti-bodies secreted by a single lymphocyte and on the size and shape of the plaques they produce. Our results indicate that in the presence of an applied electric field: (1) The mobility of the antibodies produced by the antibody forming cell can be determined from the plaque shape. (In the electric field the plaques are no longer circular, but cigar shaped.) (2) By changing the magnitude or direction of the applied electric field more than one plaque can be generated by a single AFC. Thus changes in mobility or the rate of antibody secretion can be assayed. (3) Plaques will reach a steady state size; for good emitters (cells that secrete antibodies at a high rate or that secrete high affinity antibodies) this steady state will be achieved rapidly.Equations are given which describe both the temporal development and steady state plaque size and shape. From the equations, computer generated plots of plaques produced by typical antibody farming cells are presented. These plots are then used to show how pictures of plaques formed in an electric field can be analyzed to determine the antibody mobility.     

Oscillations of IgM antibody affinity at the level of single immunocytes

IgM antibody affinity was measured by hemolytic plaque inhibition assays on spleen cells from mice immunized with a single injection of DNP-dextran. Maturation of affinity was found to occur with time after 1 to 1000 microgram of immunogen and to be characterized by rapid oscillations independent from changes inFC number/spleen and in antibody secretion rate. Analysis of affinity heterogeneity showed that such oscillations occur in higher affinity PFC subpopulations. The origin of affinity oscillations was discussed in terms of interactions among antigen and the elements of the immune network.     

Structural requirements for in vivo and in vitro immunogenicity in hapten-specific delayed hypersensitivity

Affinity changes of hapten-specific IgM antibodies were analysed by plaque inhibition assays and by inhibition with varying quantities of monovalent inhibitor. The resulting data were used for cell population studies, based on the affinity of plaque-forming antibody. Maturation of IgM plaque-forming cell populations was time- and antigen dose-dependent. Maturation was characterized by a consistent increase in the proportions of high affinity and medium affinity cells and a decrease in the proportion of low affinity cell populations. Thus progressive selection of high affinity cells occurred over the entire dose range of immunization. The hapten concentration at which 20% of cells was inhibited decreased with time at all administered antigen concentrations. The inhibitory hapten concentration at which 50 or 70% of cells was inhibited did not change after administration of very large immunizing doses. Thus recruitment of this type of plaque-forming cell depended on dose. In the secondary response, high affinity cells appeared initially but very transiently, and the subsequent rate of population changes was faster than that observed in the primary response. The short productive life of IgM plaque-forming populations may be responsible for the time restriction in IgM as compared to IgG maturation.     

Secretion rate independent evaluation of IgM antibody avidity at the level of single immunocytes.

The hemolytic plaque inhibition assay has been performed on spleen cells from mice immunized with TNP-HRBC to evaluate avidity of anti-TNP IgM antibodies. At different times after immunization direct plaques were inhibited by soluble TNP-EACA, TNP61-BGG, or anti-mu antiserum. Analysis of the inhibition data provided independent estimates of antibody avidity and secretion rate. Avidity was found to increase with time, to reach a maximum when the antibody response attained the peak value, and then to decline as the response was waning. There was a decrease followed by increase of the secretion rate concomitant with the rise and fall of the antibody response and avidity.     

The role of T cells in immunoglobulin class switching of specific antibody production system in vitro in humans

Only antibodies of the IgM class were produced in vitro by peripheral blood mononuclear cells stimulated with streptococcal carbohydrate. B cells of the peripheral blood mononuclear cells, however, synthesized both IgM and IgG class antibodies when combined with tonsillar T cells, suggesting that T cells inducing immunoglobulin class switching are present in the tonsils. Peripheral blood T cells also became capable of inducing B cells to produce IgG class antibodies when the T cells were incubated with antigen-pulsed macrophages. Surface IgM-positive, IgG-negative high-density B cells produced IgG antibodies for streptococcal carbohydrate in the presence of these T cells or tonsillar T cells. The culture supernatant solutions from these T cells or tonsillar T cells, however, failed to cause the B cells to produce IgG, indicating that class switching is not mediated by factors released from T cells. Lymphokines such as interleukin-2, human B cell growth factor, helper T cell factor, or interferon-gamma were also incapable of inducing IgG production. These results suggest that the cognate interaction between T cells and B cells is necessary for the immunoglobulin class switching.     

Growth inhibition of myeloma cells by anti-idiotype antibodies in the absence of membrane-bound immunoglobulin

Immunoglobulins are expressed as membrane-bound or secreted forms. Plasma cells produce little or no membrane immunoglobulin but secrete immunoglobulin molecules in large amounts. Immunoglobulin idiotypes of malignant B cells are tumor-specific antigens that may be targeted for immunotherapy. Thus, idiotype vaccination is being evaluated in clinical trials to control residual disease in multiple myeloma and non-Hodgkin's lymphoma. It is traditionally considered that anti-idiotype antibodies are not effective against plasma cell tumors, because the large amounts of immunoglobulin molecules secreted by the tumors block anti-idiotype antibodies, and because the absence of membrane immunoglobulin on the surface of these tumor cells renders them resistant to the effect of anti-idiotype antibodies. While the obstacle of abundant circulating idiotype may be obviated by reducing tumor burden to minimal residual disease, the absence of membrane immunoglobulin has been considered as a limiting factor that prevents tumor eradication by anti-idiotype antibodies. We demonstrate here that murine plasmacytoma cells can produce small amounts of membrane immunoglobulin M (IgM) heavy chains. However, the latter are precursor molecules that do not reach the cell surface. Although membrane-bound IgM is absent, the cells stain positively for surface IgM, reflecting molecules of the secreted form in the process of secretion. In spite of the relatively low levels of secreted immunoglobulin on the cell surface, anti-idiotype antibodies are effective in retardation of tumor growth in vivo. Thus, while there is no doubt that idiotype-specific cell-mediated responses are very important, myeloma patients in complete remission may additionally benefit from idiotype-specific humoral responses.     

Activity of a partially purified human BCGF on murine assays for B cell stimulatory factors. II. Synergy between two distinct BCGF II-like factors in promoting B cell differentiation

We have previously identified two BCGF II-like factors which can be distinguished by their differential reactivity in several murine BCGF II assays. Prototype sources of these two factors are a partially purified preparation derived from PHA-P-stimulated human peripheral blood T lymphocytes (designated human BCGF), and supernatant from antigen-stimulated D10.G4.1 murine T cells (designated D10 sup). Extending the characterization of these two factors, we show here that human BCGF and D10 sup both cause tritiated thymidine ([3H]TdR) incorporation and IgM secretion by T cell-depleted, in vivo-activated, large murine B cells. In contrast, only the human BCGF consistently induced proliferation and IgM secretion by T cell-depleted, small murine B cells. When simultaneously added to cultures, D10 sup and human BCGF synergized to produce optimal IgM secretion by large murine B cells and murine BCL1 B lymphoma cells. The same factors were tested in an IgM-specific plaque assay, and a similar synergistic response was observed for the large B cells, but not for the BCL1 cells. The combination of factors also produced maximal [3H]TdR incorporation by large murine B cells. In contrast, the addition of human BCGF totally abrogated D10 sup-induced BCL1 proliferation. Together, these data suggest that the synergies observed in IgM secretion result from an increased production of plaque-forming cells (PFC) in cultures of large B cells and an increase in IgM production per responding cell in BCL1 cells. Kinetic analysis of the time of action of the two BCGF II-like lymphokines in the induction of the PFC response by large B cells indicated that human BCGF was required within the first 24 hr of a 4-day culture period, while D10 sup could be added as late as the final 15 hr without significant diminution of the response. In summary, these data provide further support for the existence of two distinct B cell stimulatory factors which cause growth and differentiation of activated B cells, and indicate that these two factors synergize to produce optimal Ig secretion. For ease of discussion, the activity in the human BCGF preparation is referred to as BCGF IIA, and the activity in D10 sup is referred to as BCGF IIB.     

Studies on the regulation of igm immune response. VII. changes in the affinity of antibodies and cell receptors after immunization with the T-cell independent antigen DNP-Ficoll.

A/J-mice immunized by a single injection with DNP21-Ficoll respond on the humoral level exclusively with IgM antibodies. The intrinsic association constants (K0) of IgM anti-DNP to monovalent hapten E-DNP-L-lysine are within the range of 105-106 M-1 and do not change significantly during the immune response. On the other hand, the functional association constants (KF) of pentameric IgM to multivalent DNP-T4 bacteriophage increase from 1010 M-1 at 3rd day up to 1012 M-1 at 8th day. Subsequently, a decrease of KF to 1011 M-1 can be observed. This rise and fall of the affinity of IgM antibodies of multivalent DNP-conjugate can be detected at the cellular level also by inhibition of plaque formation. The concentration of DNP15-BSA needed for 50% inhibition of plaque formation (I50) decreases from second day to 8 th day by 4 orders, which represents a strong increase of functional affinity. In contrast, the I50 of E-DNP-L-lysine slightly decreases only until day 4 and does not change until day 21. the inhibition of rosette formation by mono- and multivalent ligands was used to study the affinity of lymphocyte receptors. In the course of immunization antigen-binding cells carrying receptors with increasingly higher affinity for multivalent DNP-conjugates occur. These results are discussed with regard to the importance of functional affinity of lymphocyte receptors for the antigen-driven selection of high affinity anti-DNP-cell clones producing IgM antibodies.     

Restricted maturation of antibody-binding characteristics for hapten-specific IgM-plaque-forming cells in mice

Inhibition of plaque formation was used as a method for studying changes in binding characteristics of IgM antibody specific for the hapten, 2,4,6-trinitrophenyl (TNP). By incorporating successive concentrations of TNP ligand into specific hemolytic plaque assays, we were able to obtain inhibition curves and to determine relative ligand binding of anti-TNP antibodies secreted by plaque-forming cell (PFC) populations. The concentration of free hapten required to obtain 50% reduction in IgM PFC decreased with time after immunization, indicating that an increase in average binding affinity of their secreted antibodes occurred. The slopes of the inhibition curves also increased with time after immunization. Maturation with regard to the above two properties was virtually complete for IgM PFC 5–7 days after immunization. The manner by which the slopes of the inhibition curves changed suggested that maturation occurred as the result of a simple selective process, and it appeared to involve primarily precursor cells existing prior to administration of antigen.     

Serum antibody and cellular immune response in mice to dextran B512.

Serum antibodies to dextran started to appear 3 days after immunization of C57BL/6 mice. Synthesis of IgM antibodies was followed by IgG3 and IgGA. Other immunoglobulin classes (IgG1, IgG2b, and IgG2a) were very low or absent. The immune response to dextran was also thymus independent with regard to IgG3 and IgA synthesis as demonstrated by the use of nu/nu mice. CBA and C57BL/6 mice were high responders to dextran with regard to IgM synthesis. C57BL/6 mice produced high levels of IgG3 and IgA antibodies, whereas CBA, A/J, and A.TL only synthesized IgM antibodies. A/J and A.TL strains were most frequently low responders with regard to IgM synthesis and CBA/N mice were completely nonresponders with regard to all immunoglobulin classes. The ability to produce anti-dextran antibodies increased with age in high responder strains. This was most pronounced for IgG3 and IgA antibodies, which reached adult levels 3 months after birth. The affinity of anti-dextran antibodies was high and homogeneous in antisera from C57BL/6 mice. Preimmune matural antibodies and antibodies from immunized low responder strains had a low and variable affinity for dextran.     

IgM induction in murine B hybridomas with Ia-restricted T cell clones: a monoclonal model for T and B cell interactions in antibody response

The mechanism of MHC-restricted T and B cell interactions in antibody response was studied with IgM-inducible B hybridomas and antigen-specific helper T cell clones. B hybridomas were prepared by fusion between splenic B cells from (CBA/N (H-2k) X BALB/c (H-2d)) F1 (NBF1) male mice and a B lymphoma cell line, M12.4.5. A B hybridoma clone, 1M70, which expressed I-Ad but not I-Ak determinants was chosen in the present study. IgM secretion was induced in 1M70 when it was cocultured with a "resting" KLH-specific and H-2d restricted helper T cell clone in the presence of KLH. A "resting" KLH-specific and H-2k restricted T cell clone did not induce IgM secretion in 1M70 even in the presence of KLH. However, when these KLH-specific T cell clones were activated by KLH and appropriate antigen presenting cells, both H-2d and H-2k restricted T cell clones induced IgM secretion in 1M70 even in the absence of KLH. A monoclonal anti-I-Ad antibody inhibited IgM secretion induced by a "resting" H-2d restricted T cell clone, but not by an "activated" T cell clone. These results indicated that T cell clones recognized antigens in the context of Ia molecules on B hybridomas in a MHC-restricted manner and were activated to produce B cell stimulatory factors which in turn acted on B hybridomas in a non-MHC-restricted manner and induced differentiation of B hybridomas into IgM secreting cells.     

Novel mechanisms of specific suppression of anti-hapten antibody response mediated by monoclonal anti-carrier antibody

The cellular mechanisms of the antibody-induced suppression of immune responses were analyzed in the keyhole limpet hemocyanin (KLH) system. Some of the monoclonal anti-KLH antibodies, like KLH-specific suppressor T cell factor (KLH-TsF), were demonstrated to suppress the anti-2,4-dinitrophenyl IgG but not IgM plaque-forming cell responses in a KLH-specific and H-2-restricted manner. The anti-KLH antibodies with suppressive activity reacted with, and in turn, stimulated the suppressor hybridoma (34S-281) with the anti-idiotypic receptor complementary to the idiotypic KLH-TsF of the inducer type. Moreover, because the suppressive activity of the anti-KLH antibody was completely abolished by the treatment of responding spleen cells with anti-Lyt-2 and complement, it was apparent that the suppressive antibody activated suppressor T cell pathways. The isotype or affinity of antibodies is not related to the suppressive activity, because suppressive and nonsuppressive antibodies possess a similar affinity belonging to the same Ig isotypes. It also has been demonstrated that the Fc portion is not the functional site, because the F(ab')2 fragment still has the activity. The antibody specificity is found to be important for determining whether the antibody is suppressive or not. In fact, anti-KLH 26, but not other antibodies without activity, recognizes the particular KLH epitope seen by KLH-TsF, and exclusively interacts with the anti-idiotypic suppressor T cells. Thus, the anti-idiotypic suppressor T cell receives signals both from the suppressive anti-KLH antibody and from KLH-TsF, and transmits the antibody-induced suppressor signals to the effector-suppressor pathway. The size of the repertoire of anti-idiotypic suppressor T cells involved in the suppression seems to be very limited, because only four out of 120 monoclonal anti-KLH antibodies were found to have suppressor activity. The possible mechanisms of the cell interaction mediated by the suppressive antibody are discussed.     

The kinetics of hemolytic plaque formation. IV. IgM plaque inhibition.

An analysis of the inhibition of hemolytic plaques formed against IgM antibodies is presented. The starting point is the equations of DeLisi &; Bell (1974) which describe the kinetics of plaque growth, and DeLisi &; Goldstein (1975) which describe inhibition of IgG plaques. However, the physical chemical models which were used previously to describe IgG inhibition data are shown to be inadequate for describing the characteristics of IgM inhibition curves. Moreover, it is shown that the experimental results place severe restrictions on the possible choices of physical chemical models for IgM upon which to base the calculations. It is argued that in order to account even qualitatively for all the data, one must assume (1) a very restricted motion of IgMs about the Fab hinge region and (2) a very narrow secretion rate distribution of IgM by antibody secreting cells.     

The role of B and T lymphocytes in forming cell clones producing antibodies

Previous experiments with cloning of immunocompetent cells indicate a switch from IgM to IgG during the multiplication of immunologically activated cells derived by proliferation from one precursor. An alternative explanation, cooperation of one T cell with two different B cells (IgM and IgG precursors) is experimentally studied in the present work. Control experiments indicate that all detected foci are dependent on the production of antibodies and the action of complement. Numbers of foci are linearly dependent on the quantity of cells transferred to isologous lethally irradiated mice. There is a time gap between the first detection of antibodies by the focus technique (on the 4th day after the transfer) and by the plaque technique with isolated cells (detectable only from the 6th day after the transfer); early foci contain antibodies but do not produce (secrete) them in sufficient amounts. Transfer of lymphocytes isolated from spleen, bone marrow and thymus showed that foci in the primary response are formed only by B lymphocytes. The transfer of a constant number of B lymphocytes with increased numbers of T lymphocytes did not change the quantity of Ab-forming foci; there is an increase, however, of the numbers of individual cells producing antibodies detected by the plaque technique,i.e. the number of Ab forming cells per individual clone (focus). Through the action of T lymphocytes the switch from IgM to IgG is made possible. The auxiliary role of T lymphocytes in the primary response is discussed. Dedicated to Prof. F. Patočka on his 70th birthday     

Development of a novel engineered antibody targeting human CD123

Antibody engineering involves a range of custom modifications of immunoglobulins to improve their affinity, valency, and pharmacokinetics, ensuring a better target therapy achievement. A number of therapeutic antibodies have been used for cell surface receptor blockage, interfering with the ligand binding and inhibiting receptor-driven activation of cells. Here we describe the construction and characterization of a recombinant bivalent single-chain Fv (biscFv) that targets CD123. On conversion of anti-CD123 scFv to biscFv format, the recognition of the cognate ligand is not altered. Moreover, the increased overall efficacy of the anti-CD123 biscFv in binding and inhibition of CD123/IL-3 (interleukin-3) interactions in TF-1 cells is demonstrated.     

Demonstration of an in vivo generated sub-picomolar affinity fully human monoclonal antibody to interleukin-8

The high specificity and affinity of monoclonal antibodies make them attractive as therapeutic agents. In general, the affinities of antibodies reported to be high affinity are in the high picomolar to low nanomolar range and have been affinity matured in vitro. It has been proposed that there is an in vivo affinity ceiling at 100 pM and that B cells producing antibodies with affinities for antigen above the estimated ceiling would have no selective advantage in antigen-induced affinity maturation during normal immune responses. Using a transgenic mouse producing fully human antibodies, we have routinely generated antibodies with sub-nanomolar affinities, have frequently rescued antibodies with less than 10 pM affinity, and now describe the existence of an in vivo generated anti-hIL-8 antibody with a sub-picomolar equilibrium dissociation constant. This confirms the prediction that antibodies with affinities beyond the proposed affinity ceiling can be generated in vivo. We also describe the technical challenges of determining such high affinities. To further understand the importance of affinity for therapy, we have constructed a mathematical model to predict the relationship between the affinity of an antibody and its in vivo potency using IL-8 as a model antigen.     

Primary anti-trinitrophenyl memory cells investigated by plaque-forming cells and ELISA.

Primary anti-trinitrophenyl antibody production was investigated from spleen cells of mice immunized with trinitrophenylated-keyhole limpet hemocyanin, using the plaque-forming cell method and ELISA. Cells taken 5 days after antigen injection do not produce IgE, but do produce IgM and IgG1 anti-trinitrophenyl antibodies as demonstrated by plaque-forming cells. Substantial increase of IgM, IgG1, and IgE antibody production was seen from cells taken 7 days after immunization, followed by a rapid decline. By ELISA it was seen that cells taken 3 days after immunization already produce small amounts of anti-trinitrophenyl antibodies. Presence of antigen from the start of the cultures did not increase antibody production from cells taken 3 days after immunization, but potentiated antibody secretions from cells taken 5 days or later after immunization. This potentiation was interpreted as recruitment of antibody-forming cells from early memory B cells. The presence of IL-4 from the start of the cultures had no appreciable effect. Cell sorting with specific antibody-coated magnetic beads showed that plaque-forming cells from nonsorted cells, membrane IgE+ or membrane IgE- cells secreted similar amounts of anti-trinitrophenyl IgG1 and IgE antibodies. No difference in anti-trinitrophenyl IgM, IgG1, or IgE production was found in controls; cells sorted negatively or positively for CD23. The data show that memory B cells can be demonstrated already on Day 5 after immunization, and their antigen-induced antibody secretion is IL-4 dependent.     

Heterogeneity among T helper cells. Interleukin 2 secretion and help for immunoglobulin secretion

T lymphocytes are thought to provide "help" for B cells by activating them from the resting state, by secretion of antigen-nonspecific lymphokines that promote B cell differentiation and maturation, and by providing signals that induce isotype switching. To clarify the extent to which these different forms of helper activity could be carried out by individual T cells, we set up cultures in which B cells activated, and were in turn themselves stimulated by, limiting numbers of T cells through differences at the H-2 or Mls loci. At T cell doses at which responses were likely to represent the activity of individual helper T cells (or their immediate clonal progeny), we found that some T cells were able both to produce interleukin 2 (IL-2) and to induce secretion of both IgM and IgG, whereas others induced immunoglobulin (Ig) secretion without detectable IL-2 production, and still others made IL-2 but did not promote antibody secretion. We could not detect B cell stimulatory factor 1 production by alloantigen-stimulated T cells, and the addition of antibodies to B cell stimulatory factor 1 did not prevent Ig production. Two results, however--higher Ig accumulation in those wells that received an IL-2-producing cell, and inhibition by anti-IL-2 receptor antibodies of B cell but not T cell function--are consistent with a direct stimulatory effect of IL-2 on B cells in this system. The pattern of helper functions exhibited by T cells freshly isolated from mice differs from that inferred from studies of cloned lines of T cells in long term cultures.     

Genetic control of B- and T-Lymphocyte abnormalities of NZB mice in crosses with B10.D2 mice

Parental NZB and B10.D2, F₁ and F₁ × B10.D2 mice were studied to determine the genetic control of (1) altered B-cell IgD expression, (2) plasma cell frequency, (3) IgM secretion per plasma cell, (4) primary in vitro cytotoxic T-cell responses to H-2-compatible cells, (5) production of thymocyte-binding antibodies, and (6) production of red-cell-specific antibodies. The results demonstrate that, in this cross, IgD abnormalities and production of red-cell-specific antibodies were recessive traits. There was a common genetic influence on plasma cell frequency, IgM secretion per plasma cell and production of thymocyte-binding antibodies which was distinct from the genes governing the ability to generate a cytotoxic T lymphocyte response to H-2-compatible cells.Abbreviations used in this paper CTL cytotoxic T lymphocyte - F1 anti-Fab fluorescein-labeled antimouse Fab - FMF flow microfluorometry - Ig immunoglobulin - IgM/PC IgM secretion per PC - PC plasma cell - sIg surface immunoglobulin - TBA thymocyte-binding antibody