Amplification of B cell clones forming antibody to the 2,4-dinitrophenyl group

The selection of several single cell clones forming anti-2,4-dinitrophenyl (DNP) antibody in mice enabled us to study the life span of B memory cells, and to estimate the number of DNP-binding cells belonging to a single clone, as well as their amplification into anti-DNP plaque-forming cells. B memory cells belonging to clone E9 were found to survive for several weeks in the absence of antigen. Memory cells for this clone were transferred into recipient mice; antigen challenge after 2 or 4 weeks led to E9 anti-DNP formation at the same or half the level resulting from immediate antigen challenge. DNP-binding cells were labeled with ¹²⁵I-DNP on a heterologous carrier protein (Maia Squinudo hemocyanin); for 3 different DNP-clones the incidence of DNP-binding cells in spleen (2nd transfer generation, 8 weeks after cell transfer) was 1000 – 1900 per 10⁶ cells. This compares with 900/10⁶ spleen cells in a multiclonal mouse primed with DNP-ovalbumin; a proportion of these cells may represent T cells with specificity for DNP. Anti-DNP-forming cells in spleens of irradiated mice, receiving clonal memory cells and antigen, appeared after a lag of 4 – 5 days, reached a peak of 12000 – 18000 PFC between day 8 and 11 and then declined rapidly despite continued high levels of antibody production in recipient mice; this suggests antibody formation at sites other than spleen. The data are consistent with around 1 % effective homing of B precursor cells in spleen.     

Idiotypic profile of the response to phosphorylcholine induced in the absence of the homologous antigen

In the absence of the homologous antigen, an anti-phosphorylcholine (PC) plaque- forming cell (PFC) response has been induced in vitro which is restricted to the TEPC 15 idiotype (T15). Anti-T15 antibodies were used to focus either the keyhole limpet hemocyanin (KLH) or the fowl gamma-globulin (FGG) carrier molecules on the membrane of B cells carrying the T15 idiotype on their immunoglobulin receptors; thereafter, these cells were allowed to cooperate in vitro for 5 days with T cells primed to the appropriate carrier molecule. A response, virtually 100% T15⁺, could be induced both in normal BALB/c and in T15 neonatally suppressed mice which had lost the T15 ⁺ clonal dominance. The magnitude of this response is comparable to that obtained in the presence of the PC antigen. The role of the membrane-bound immunoglobulin receptor (sIg) in the expression of different anti-PC clones was also investigated. We have focused either the KLH or the FGG carrier molecules on the membrane of B cells via anti-H-2 antibodies and then cultured these cells for 5 days with the appropriate carrier-specific T cells. Under these conditions, B cells are activated in the absence of interaction at the sIg. The idiotypic profile of the anti-PC PFC obtained with the anti-H-2-mediated activation was then compared with the profile of the anti-PC response obtained in the presence of PC antigen. Since similar idiotypic profiles were obtained in both cases, it can be excluded that sIg plays a direct role in favoring the expression of T15⁺ over T15^? anti-PC clones.     

Distinct helper activities control growth or maturation of B lymphocytes

A clone (C-11) of C3H/HeJ Lyt-1+2-T cells with specificity for "minor" antigens of C3H/Tif has been isolated which, in contrast to other similarly derived clones, did not activate polyclonal plaque-forming cell (PFC) responses in T cell-depleted "target" spleen cells. This clone, however, showed unaltered proliferative responses to the naturally occurring antigen(s) on presenting cells, and strongly synergized with regular helper clones in the induction of PFC responses. Further analysis demonstrated that C-11 cells are competent to stimulate extensive "target" B cell proliferation, but lack the ability to produce (or participate in the production of) maturation factors for activated B cells. Thus, the defective PFC responses could be fully reconstituted with supernatants from regular clones stimulated with antigen, but not by supernatants prepared from the C-11 cells themselves. While it is not clear whether this clone represents a normal helper T cell subpopulation or a variant that has lost maturation-factor production, these results demonstrate that distinct factors control growth and maturation in T cell-dependent B lymphocyte responses.     

ANTIGEN BINDING TO LYMPHOID CELLS OF UNIMMUNIZED MICE

In order to determine the cell type responsible for the antigen-binding reaction in the bone marrow and spleen of mice, cells derived from pure in vitro derived colonies of neutrophils, eosinophils, macrophage-megakaryocytes and B lymphocytes were tested for their ability to bind fluorescent protein antigens. Only B lymphocytes bound antigen. An unexpectedly high percentage of bone marrow B lymphocytes (20%) bound a given antigen. This frequency was considerably higher than that found for spleen cells. As might be expected from such high binding frequencies, some cells bound two fluorchromated antigens when these are added together. As a direct test of the clonality of antigen binding to bone marrow B lymphocytes, whole colonies of B cells were tested for antigen binding of two non-cross-reacting protein antigens. The frequency of antigen-binding clones, including double antigen-binding clones, reflects exactly the frequencies observed for dispersed colony B cells and for in vivo derived Ig-bearing bone marrow B cells. The frequency of double antigen-binding colonies was equal to the product of the frequencies of the colonies binding each of the two antigens alone. No ‘mixed’ colonies containing single binding cells for each antigen were found. Thus, the ability to bind any two given antigens is a clonally distributed property of the bone marrow B lymphocyte population. Heterogenous receptors for multiple antigen binding on each cell are either randomly distributed among the B cell population, or homogenous antigen-binding receptors on each cell have a random chance of cross-reaction with the two antigens tested.     

Clonal dominance and the preservation of clonal memory cells mediated by antigen-antibody.

Selected B-cell clones and their well characterized monoclonal antibody products were used to analyse the role of antibody in clonal dominance and the regulation of memory cell supplies. The experimental design was to permit contact between spleen cells and antigen in vitro, and to administer antibody to DNP prior to or following cell transfers into irradiated recipients. The anti-hapten response was strongly suppressed by the clone's own antibody or higher affinity antibody administered on day 0. Antigen-antibody inhibited memory cell generation. The suppressive effect was temporary, and reversible with time and further antigen and the same clone could be induced to produce antibody again, analysed by isoelectric focusing. We were therefore not dealing with clonal deletion. Change in the source of clonal anti-hapten excluded possible effects of antibody to carrier protein or idiotypic determinants in this system. The timing of antibody administration indicates that clones already triggered in the first 4 days after antigen contact could not be suppressed by antibody. Passive antibody to DNP only suppressed when both B and T cells had been permitted contact with hapten-carrier protein. Alteration of the carrier protein enabled us to study the effect of antigen-antibody on B and T cells separately. B cells binding antigen and antibody to hapten were triggered more efficiently by fresh T cells recognizing the carrier protein than after antigen uptake alone. Antibody to DNP suppressed only when both B and T cells had taken up hapten-protein, suggesting that antigen-antibody acts centrally at the level of both B memory cells and T helper cells. This reversible antigen-antibody blockade appears to favour the preservation of a pool of long-lived memory cells rather than the priming of new clones developing from short lived precursor cells; clonal dominance ensues.     

Patterns of isotype expression by B cell clones responding to thymus-dependent and thymus-independent antigens in vitro

It was found that the Type 2 thymus-independent (TI-2) antigens bacterial levan, trinitrophenyl-Ficoll, and pneumococcal carbohydrate vaccine (PnC) stimulate clonal expansion and antibody secretion in splenic fragments from either hemocyanin-primed or unprimed irradiated recipients bearing B cells from unprimed donors. The in vitro stimulation with TI-2 antigens leads to the expression of isotype switching and provides a more balanced variety of isotypes than is usually observed in vivo. Still, some characteristic patterns of isotypes expressed in vivo to either TI-2 or thymus-dependent (TD) antigens are preserved in vitro. Frequencies of phosphocholine (PC)-reactive B cells responding to either PnC or to PC-hemocyanin (PC-Hy) suggest an appreciable overlap in populations responding to these TI and TD forms of antigen. The existence of a population responsive to either form of PC determinant is supported by the observation that many clones arising in the presence of both forms of antigen express patterns of isotypes that appear as summations of those distinct patterns shown by clones responding to only one form or the other. These data suggest that PC-Hy- and PnC-responding cells may derive from a linear rather than a branched pathway of B cell development and that expression of isotype switching over the lifetime of a developing B cell clone may be regulated in a manner dependent on the form of the stimulating antigen.     

The kinectics of proliferation and maturation of mitogen-activated bone marrow-derived lymphocytes

The B cell mitogens lipopolysaccharide (LPS), purified protein derivative of tuberculin (PPD) and fetal calf serum (FCS) all stimulate spleen cells from nude mice to increased DNA-synthesis 14 to 16 h after the initiation of mitogenic stimulation. The three mitogens also induce the maturation of B cells to plaque-forming cells (PFC). All three mitogens stimulate largely identical B cell populations. The extent to which B cells are stimulated to DNA synthesis and to the development of PFC varies from mitogen to mitogen. PFC arise by clonal growth through proliferation and maturation. A “hot pulse” of radioactive thymidine given between 0 and 20 h of stimulation has no effect on the development of PFC during subsequent mitogenic stimulation. Pulses given between 24 and 36 h after stimulation abolish the development of over 90 % of the PFC. The minimum pulse time required for maximum inhibition of the development of PFC during that time is 6 h. Beyond 36 h of stimulation by either of the three mitogens, PFC emerge rapidly. Between 60 and 72 h less than 10 % of all PFC emerging at 72 h are abolished by a “hot pulse”. The kinetics of the clonal development of PFC after mitogenic stimulation are compared to that of antigenic stimulation. The comparison suggests that B cells stimulated by antigen in the in vivo primary immune response culture system go through a longer period of proliferation than when they are stimulated by mitogen before maturation to PFC.     

Con A-propagated, auto-reactive T cell clones that secrete factors promoting high IgA responses

Four BALB/c T cell clones from among a set propagated in the presence of concanavalin A (Con A) were selected on the basis of their ability to produce supernatant factors promoting high IgA plaque-forming cell (PFC) responses by 2,4,6-trinitrophenyl-conjugated keyhole limpet hemocyanin (TNP-KLH)-primed splenic B cells in the presence of TNP-SRBC. Such clones could be derived from cultures containing T cells not only from gut-associated lymphoid tissue, but also from the spleen. The selected clones all proliferated well in the presence of syngeneic, irradiated APC without either Con A or exogenous IL-2, but required both APC and Con A to produce helper factors. Factors from three of the clones helped B cells both to proliferate and to differentiate into IgM, IgG and IgA PFC. Factors from the fourth clone helped B cells differentiate into IgA and IgG PFC and may have promoted switching to these isotypes but did not support either B cell proliferation or generation of IgM PFC. Cross-linking of B cell receptors for antigen was not required for the response to the helper factors since TNP-SRBC were unnecessary and high concentrations of them were actually inhibitory.     

Factors affecting the propagation of a B cell clone forming antibody to the 2,4-dinitrophenyl group

We have studied the factors controlling the propagation and sequential transfer of B cell clone, E9, forming IgGI antibody to DNP in syngeneic CBA/H mice. The history of this clone is documented here in detail. E9 antibody was recognized by its isoelectric spectrum, detected by binding of ¹³¹-DNP hapten after isoelectric focusing. During the life of this clone, induction of anti-DNP depended on the presence of antigen (1 – 10 μg provided an optimum dose) and on cooperation with T-derived cells recognizing carrier molecules. T cells contribute no information towards antibody formed by the B cells. During the first three transfer generations the clone produced high levels of E9 anti-DNP (2 – 3 mg/ml serum) and the reliability of clonal transfer was high; 53/54 recipient mice formed large amounts of E9 antibody. As the proliferative capacity of the clone decreased, transfer became less reliable. The optimum interval between consecutive spleen cell transfers was 3 – 5 weeks, although successful transfer of memory cells could still be accomplished 65 days after last contact with antigen. The pI spectrum of E9 anti-DNP remained constant throughout the life span of the clone, covering a period of one year and eight transfer generations and passage into 300 mice. We have therefore no evidence for any change or improvement in antibody produced by a single clone due to selective pressure. In other experimental series, limiting numbers (0.5 × 10⁶ - 2 × 10⁶) of spleen cells primed with DNP-BGG were distributed into irradiated syngeneic CBA/H mice. The incidence of single anti-DNP-forming clones sufficiently expanded for reliable sequential transfer is low; in 300 irradiated recipients we found 9 clones which produced high levels (0.3 - 3 mg/ml) of anti-DNP; some of these have been propagated by serial transfer. The high protein production appears to reflect overall expansion of the clone and to be a useful indicator of large numbers of memory cells. In general, low producing clones are not sufficiently expanded for effective propagation of the clone by serial spleen cell transfer.     

T cell-dependent mediator and B-cell clones.

Supernatants were collected from keyhole limpet haemocyanin (KLH) primed spleen cells which had been incubated in tissue culture medium with their priming antigen KLH. These non-specific factor (NSF) containing supernatants were then tested in a microculture system for their ability to facilitate an anti-SRBC response of nu/nu or AT times BM spleen cells. It was concluded that:(a) NSF was able to engage a large number of the available pool of sheep erythrocyte (SRBC) specific B cells into an antibody response (b) this response was characterized by the development of clones expressing plaque-forming cells (PFC), the number of PFC produced within a clone being dependent upon the amount of NSF available in that culture; (c) NSF probably acted directly on B cells, and not via other accessory cell types.     

Characterization of mouse thoracic duct B lymphocytes I. Evidence of functional heterogeneity

Thoracic duct lymphocytes (TDL) from C 3 H/Tif and BALB/c mice were studied for their in vitro reactivity to the B cell mitogens lipopolysaccharide (LPS) and lipoprotein (LP). Roughly 4% and 10% of the surface immunoglobulin (Ig)-positive cells in these populations could be stimulated by LPS and LP, respectively, to generate clones of IgM-secreting cells. Among LPS-reactive B cells, roughly 30% developed into clones which also produced IgG₃ or IgG₂, while only a very small fraction (1–2%) of all precursors could give rise to clones secreting IgG₁ and IgA. Freshly collected TDL from some batches of C 3 H/HeJ mice displayed a high proportion of Ig-containing B cell blasts (5–10%), which did not secrete enough Ig to be detected as plaque-forming cells (PFC). These cells, however, under appropriate culture conditions and stimulated by LP (but not by Nocardia mitogen), differentiated to PFC of the various Ig classes without dividing.     

Regulation of the specific plaque-forming cell response in man: restraint of B cell responsiveness.

Specific 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.     

A low antigen dose selectively promotes expansion of high-avidity autoreactive T cells with distinct phenotypic characteristics: A study of human autoreactive CD4+T cells specific for GAD65

T cells specific for pancreatic islet proteins can be detected in type 1 diabetes patients and at-risk individuals, suggesting a failure of the central tolerance and negative selection. We addressed the question, how antigen dose shapes the diversity of CD4+ autoreactive T cells specific for glutamate decarboxylase 65 (GAD65) in a healthy HLA-DR*0404+ individual, with a persistent GAD65-specific T-cell response. CD4+T cells from this subject were stimulated with decreasing concentrations of the GAD65 555-567 (557I) peptide, and T-cell clones were derived from the tetramer-binding cell population. Functional and structural avidity, TcR-Vβ usage, and cytokine profiles were investigated at a clonal level. T-cell clones established with a low antigen dose (0.1 and 1 μg/ml) displayed higher avidity in contrast to the clones established with the highest antigen dose (10 μg/ml; Mann–Whitney U test, p = 0.003 and 0.006, respectively). The T-cell clones stimulated with the lowest peptide dose also had a higher tetramer-binding affinity than clones stimulated with the highest dose (p = 0.026). The majority (60.0%) of the high-avidity clones expressed TcR-Vβ5.1 chain whereas only one (12.5%) low-avidity clone did. All clones displayed Th0/Th2 cytokine profiles, but intermediate and high-avidity clones produced more IL-10 than low-avidity clones (p = 0.032). The results demonstrate an important role of the antigen dose in the determination of characteristics of the responding T-cell repertoire. High IL-13 and IL-10 production by GAD65-reactive T cells suggests a more anti-inflammatory profile of this healthy individual underlying protection from T1D.     

Relative frequencies of secondary B cells activated by cognate vs. other mechanisms.

Three distinct mechanisms for the activation of secondary B cells to antibody-forming cells have been examined in splenic fragment cultures. The clonal response to a protein antigen [cytochrome c (cyt)] was quantified in terms of both the number of B cells activated and the amount of antibody produced by each clone. The vast majority of memory B cells required surface immunoglobulin (sIg) receptor-mediated uptake of antigen followed by cognate interactions with an antigen-specific T helper cell. Nevertheless, a significant number (as many as 12%) could be activated via soluble factor-mediated bystander activation, involving occupation of neither the sIg receptor nor class II major histocompatibility complex (MHC) molecules. This pathway, however, was relatively inefficient in that individual clones secreted less than half as much antibody as clones activated as a result of cognate collaboration with a T helper cell. A similar number of secondary B cells were activated following nonspecific uptake of high concentrations of antigen for which splenic fragment T cells had been primed [i.e., hemocyanin (Hy)], independent of sIg receptor occupancy. Antibody levels were similar to those in cultures where B cells were activated in a cognate manner with sIg receptor occupancy. When Hy-stimulated fragment cultures were supplemented with polymerized cyt, the frequency of activation via this latter pathway increased fourfold, despite the fact that no cyt-primed T cells were present. This observation supports the idea that receptor-mediated uptake of antigen serves not just to focus antigen but also provides an important signal in activating B cells. Bystander B cell activation, however, was not enhanced by providing a sIg cross-linking signal with polymerized cyt. The lower level of antibody production by B cells activated in a bystander fashion and the inability to enhance their frequency of activation with sIg receptor occupancy suggest that there is indeed a fundamental difference between soluble factor-mediated bystander activation and activation via T cell determinant-mediated cognate T helper-B cell interactions, perhaps involving signaling through class II MHC molecules.     

Presentation of Antigen to T Lymphocytes by Non-Immune B-Cell Hybridoma Clones: Evidence for Specific and Non-Specific Presentation

Non-immune SJL (H-2^S) spleen cells were fused with (H-2^d) Balb/c 653-myeloma cells and the hybridomas were cloned by two limiting dilutions. The resulting hybrid B- cell clones were tested for their antigen presentation capability to SJL T-cell lines that were specific for either lysozyme or myoglobin. In proliferative assays, 53% of the antigen presenting B-cell clones were able to present both myoglobin and lysozyme (general presenters) while the other 47% presented specifically either myoglobin or lysozyme (specific presenters). The ability to selectively present either myoglobin or lysozyme indicates that antigen presentation at the clonal level can be specific or non-specific depending on the particular B-cell clone.     

B lymphocyte development in fetal liver. I. Development of reactivities to B cell mitogens "in vivo" and "in vitro".

Single cell suspensions of fetal liver cells, prepared from embryos at day 13 of gestation until birth, develop mitogen bacterial lipopolysaccharide (LPS)-reactive B cells “in vitro” within the same time as they do “in vivo”. The development of these mitogen LPS-reactive B cells in vitro is dependent on unknown components of fetal calf serum, found in some but not all batches. It does not require the presence of the corresponding B cell mitogens, i.e. LPS, in culture. Fetal liver cells, put in culture at different times of gestation, all acquire LPS reactivity at the equivalent of birth in vitro. When the cells have become LPS-reactive, they are then stimulated by LPS to growth and maturation into clones of IgM-secreting, IgA-secreting and IgG-secreting plaque-forming cells (PFC). The development of these PFC responses is mitogen-dependent: in the absence of LPS only 2-5 % of the LPS-stimulated PFC responses develop. Only the magnitude, but not the development in time, of LPS reactivity of fetal liver cells is different in vivo and in vitro. This may indicate either that an early precursor cell to LPS-reactive B cells can only divide in vivo but not in vitro, or that early precursors continue to migrate with time of gestation into fetal liver. After birth B cells in liver are immediately reactive to LPS; however, they rapidly leave the liver. Few, if any, dextrans sulfate or lipoprotein-reactive B cells, yielding a PFC response, develop in fetal liver cells either in vivo or in vitro. Neonatal spleen contains immediately mitogen-reactive B cells. Comparable numbers of LPS, dextran sulfate and lipoprotein-reactive cells are found. Development of fetal liver cells to Ig-secreting PFC, occurs, therefore, in two stages. The first (pre-B → B) is independent of externally added mitogen (such as LPS), the second (B → PFC) requires the addition of mitogen. The majority of spleen cells have already passed this first stage of development.     

T cell clones interacting with accessory and T helper cells for a proliferative response

The requirement for cell interactions in T cell activation has been studied with two continuously in vitro growing T cell clones. These clones are specific for minor histocompatibility antigens, are H-2K restricted, and one clone is functionally a cytolytic T lymphocyte. Both can proliferate when interleukin 2 is added to the cultures, but for continuous growth they require irradiated spleen cells carrying the specific minor histocompatibility antigen and the restricting H-2. In this study we show that for proliferation the clones require at least two cell populations in the stimulator spleen, one is a splenic-adherent cell (SAC), the other a T cell. The SAC are plastic adherent, Thy-1-, Ia+. The T cells are nylon wool nonadherent, Thy-1+, Lyt-1+2- and Ia-. Cell mixing experiments of stimulator cells (all were done with H-2-syngeneic cells), depleted of either SAC or T cells confirm the requirement for a specific interaction between these two cell types and the T clone. Neither SAC, syngeneic with the T clone when mixed with T cells of the stimulator type, nor T cells syngeneic with the clone added to stimulator SAC, can induce an optimal proliferative response. Such a response is obtained only if both cell types, SAC and T cells, are of the stimulating genotype. This suggests that, in addition to an interaction of clonal T cells with SAC, a specific recognition at the T cell level between T stimulator and T clone is necessary. The interaction of the T clones with stimulator SAC and T cells leads to an activation, mediated by antigen recognition, of all three cell populations. Since we also show that each of the stimulator cell types are impaired by ultraviolet light irradiation, we conclude that factor production by SAC and T helpers is the final prerequisite for clonal expansion.     

Immunoglobulin M receptors on memory cells of immunoglobulin G antibody-forming cell clones

The memory cells of two antibody-forming cell clones had receptors of the IgM class, even though the clones had been producing IgG₁ or IgG_2a anti-2,4-dinitrophenyl antibodies for 9–15 months previously (on exposure to antigen). Thus a phenotypic switch in heavy chain constant region evidently occurred after re-exposure of these memory cells to antigen. To show that, we first removed the clonal cells' surface immunoglobulins by “capping” and “stripping”, with class- or subclass-specific antisera. Then, to assay their remaining receptor activity, the cells were incubated with antigen in vitro, washed and transferred (together with carrier-primed cells) to irradiated recipients, and their antibody responses to this in vitro boost were assayed by isoelectric focusing. Pretreatment with anti-μ serum, as well as with anti-Fab(k), prevented the responses of the IgG₁ and IgG_2a clones to an in vitro boost, while anti-γ₁, and anti-γ_2a antisera had no effect. An anti-serum to the putative mouse IgD also had no effect. The anti-μ serum failed to react with the IgG₁ and IgG_2a clonal serum antibodies in the test tube. Some other contaminating clones were suppressed completely only by the anti-Fab serum. This result strongly suggests that switching in class commitment may occur during the differentiation of memory cells to antibody producers, and may therefore be antigen-dependent. It also implies that some apparently naïve cells with surface IgM may., in reality, be B memory cells.     

Effect of Anti-δ Antibodies on Clonal Expansion and Maturation of B Lymphocytes

The effect of a monoclonal anti-δ antibody on the in vitro B cell response to the B cell mitogen lipopolysaccharide (LPS) has been studied.Titration of anti-δ antibody in mass cultures stimulated by LPS resulted in a dose-dependent reduction of thymidine incorporation. IgM and IgG plaque forming cell (PFC) responses were poorly affected at high concentration, and slightly increased at low concentrations of anti-δ antibody.By limiting dilution analysis it was shown that anti-δ antibody inhibit 30-50 % of LPSreactive B cells to grow as IgM secreting clones, while increasing the average size of clones that grew in the presence of anti-δ as compared to control cultures. Anti-δ also results in increased frequencies of IgG secreting clones.By immunofluorescence it was possible to show the presence of a higher relative number of cells containing immunoglobulin as an effect of anti-δ antibodies. Observations made at early times of culture indicate that the cells that do not proliferate in the presence of anti-δ undergo an early maturation to secretion.Experiments performed on LPS blasts suggest that the effects of anti-δ on cell proliferation require the presence of antibodies at early times in the response, while the effects on maturation can be manifested during clonal development.The relevance of membrane IgD and of the IgM-to-IgD ratio in the maintenance versus exhaustion of the clone is discussed.     

Antibody synthesis by chicken spleen cells in vitro. I. Requirements of B cells at various stages after immunization for T cells,macrophages and antigen

The requirements for T cells, macrophages and antigen during the induction of in vitro antibody responses were ascertained with chicken spleen cells obtained at various times after immunization with sheep red blood cells (SRBC). The IgM plaque-forming cell (PFC) response was T cell independent exclusively in cultures initiated 3 days after priming, but macrophage dependent at all time intervals tested. In cultures started 4 to 10 days after priming the IgG response was both T cell and macrophage independent and PFC numbers remained at a high plateau level throughout the culture period. In contrast, IgG responses initiated more than 15 days after priming showed a reversal to complete T cell and macrophage dependence and were characterized by a sharp increase in PFC numbers between days 2 and 4 of culture. Formaldehyde-fixed SRBC were immunogenic for IgG PFC 4 to 10 days after priming but failed to stimulate later IgG memory and all IgM responses. Contrasting antigen dose requirements for IgM and IgG responses were found in cultures initiated at various periods after priming. The results suggest that direct contact with fixed antigen was sufficient to maintain IgG antibody synthesizing PFC in vitro, while native antigen and cell co-operation were required for late secondary IgG and all IgM responses. These results are interpreted in terms of separate pathways of differentiation for IgM and IgG antibody-producing cells. A distinct 3rd day stage of T cell-independent but macrophage-dependent responsiveness for both classes of antibody was also defined.