Immunoregulation in the rat: requirements for in vitro B cell responses to classical TI-1 and TI-2 antigens

The presence of suppressor cells and their mediators has made it difficult to induce B cell mitogenic or immune responses in rat spleen cell cultures. In the present study, we have defined culture conditions required for induction of in vitro thymic independent (TI) immune responses in the rat. Rat spleen cell cultures support low responses to various trinitrophenyl (TNP) haptenated antigens including TNP-Brucella abortus (TNP-BA), TNP-lipopolysaccharide [LPS; either phenol (Ph)- or butanol (Bu)-water extracted], TNP-Ficoll, and TNP-dextran. However, all of these antigens induced good splenic anti-TNP PFC responses when given at appropriate doses in vivo. When spleen cells were depleted of adherent cells and cultured with TI antigens in vitro, good anti-TNP PFC responses were seen with TNP-BA, whereas, lower responses were induced by TNP-LPS (Ph or Bu). No responses were observed in cultures incubated with either TNP-Ficoll or TNP-dextran. Purified splenic B cell cultures [prepared by panning on plates coated with anti-rat F (ab')2] supported good responses to TNP-LPS (Ph or Bu) and TNP-BA. The addition of irradiated splenic adherent cells (macrophages, M phi) to either M phi-depleted or purified B cell cultures completely abrogated in vitro responses to TNP-BA or TNP-LPS (Ph or Bu). Purified splenic B cell cultures generally responded poorly to TNP-Ficoll or TNP-dextran. Addition of indomethacin (IM) to spleen cell cultures abrogated suppression and allowed anti-TNP PFC responses to TNP-BA, TNP-LPS (Ph or Bu), TNP-Ficoll, and TNP-dextran. Furthermore, nude spleen cell cultures treated with IM, also allowed significant TNP-Ficoll and TNP-dextran immune responses; however, untreated cultures did not respond to these antigens. Our studies indicate that rat splenic B cell cultures are responsive to TI antigens, and highest responses occur with the murine TI-1 class, e.g., TNP-BA and TNP-LPS. Inhibition of suppression with IM restored splenic B cell responses to the murine TI-2 class, i.e., TNP-Ficoll and TNP-dextran.     

Delayed maturation of the antibody response to type 2 thymus-independent antigens in a partially inbred strain of chicken

The ontogeny of antibody responses to trinitrophenylated (TNP) thymus-independent (TI) antigens was compared in two partially inbred strains of chicken: the SC strain (B2/B2 genotype) and the FP strain (B15/B22 genotype). In the SC chicken, maturation of both the splenic anti-TNP plaque-forming cell (PFC) response and the 19S hemagglutinating antibody response to TI type 2 (TI-2) antigens, TNP-Ficoll and TNP-dextran, were delayed to a significantly later time in ontogeny (20 wk of age) than in the FP chickens (9 wk of age). Four- to 6-wk-old SC chickens were virtually immunologically unresponsive to stimulation with TI-2 antigens. The TI-1 antigen TNP-Brucella abortus was equally immunogenic in both FP and SC chickens of different age groups tested. Kinetic studies of the primary PFC response to TNP-Ficoll in immunologically mature chickens of the SC and FP strains demonstrated a peak PFC response 4 days after antigen injection, followed by a rapid decline in numbers of splenic PFC/spleen on day 6. The results of these studies are discussed in relation to earlier observations that suggested there may be a delay or a defect in the ontogeny of the thymus in the SC chicken.     

Immunoregulation in the rat: cellular and molecular requirements for B cell responses to types 1, 2, and T-dependent antigens

The requirements for primary in vitro plaque-forming cell (PFC) development in cultures of purified rat splenic B cells have been examined. Rat B cells were directly responsive to the type 1 antigen trinitrophenyl-Brucella abortus (TNP-BA), but both T cells and adherent accessory cells were required for B cell responses to the type 2 antigen TNP-Ficoll and the T cell-dependent (TD) antigen sheep erythrocytes (SRBC). However, the cellfree supernatants from concanavalin A-induced spleen cells of rat or mouse origin replaced the requirement for T cells and macrophages, and resulted in PFC development in response to TNP-Ficoll and SRBC and augmented PFC numbers in response to TNP-BA. Culture supernatants from induced murine T cell and macrophage cell lines were used to partially deduce the molecular requirements for the support of PFC development by rat B cells to these three antigens. Supernatants from the EL-4 (EL-4 sup) and B151 K12 (B15 sup) T cell lines augmented TNP-BA responses, suggesting that B cell growth factor II (BCGF-II) mediated this effect. An admixture of purified interleukin 2 (IL 2) and B15 sup supported PFC development to SRBC; indicating that IL 2, BCGF-II, and the T cell-replacing factor in B15 sup (B15-TRF) were sufficient to support this response. In addition, the IL 2 plus B15 sup-supported anti-SRBC PFC response was increased by the addition of an interleukin 1-containing fraction from the supernatant of the macrophage line P388D1. PFC development in response to TNP-Ficoll had the most stringent requirements and only occurred in the presence of EL-4 sup and B15 sup (IL 2, BCGF-I, BCGF-II, EL-TRF, B15-TRF). These data indicate that different cellular and molecular requirements exist for PFC development in response to types 1, 2, and TD antigens by rat B cells.     

Immunocompetent cells in the chicken. II. Synergism between thymus cells and either bursa or bone marrow cells in the humoral immune response to sheep erythrocytes

Newly hatched F₁ hybrid chicks isogenic for the strong B histocompatibility locus were rendered immunologically incompetent by cyclophosphamide treatment and x-irradiation. They were then injected intravenously with thymus, bone marrow, or bursa cells together with sheep erythrocytes (SE) and received another iv injection of SE 3 days later. Splenic plaque-forming cells (PFC) and serum hemagglutinins were assayed 7 days after transfer. At donor ages of 14–26 days, cells from thymus (T) and bone marrow (BM) showed synergism when injected together, as indicated by a significantly higher geometric mean of PFC per recipient spleen in the BM + T group than in the BM group. The response of the T group was extremely low. With thymus and bursa cells from 6- to 28-day-old donors, significant synergism was demonstrated in 3 of 9 individual experiments. However, almost all the other 6 experiments showed marked differences in the same direction, and the combined probability for all experiments was < 0.001. The most striking demonstration of thymus + bursa synergism was made in 2 experiments using 1-week-old donors. Bone marrow cells from 1-week-old donors failed to cooperate with thymus, as did BM cells from older bursectomized agammaglobulinemic donors. This suggests that B cells from bone marrow originate in the bursa. Thymus-bursa cooperation was somewhat difficult to demonstrate in individual experiments using donors over 1 week of age, owing to the occurrence of some responses with bursal cells alone and to variability of response within bursa or bursa + thymus recipient groups. Synergism between thymus and bursa cells was more consistently demonstrable when irradiated normal spleen or low doses of bone marrow cells were added. These additions led to an increased response and a lowered coefficient of variation in the thymus + bursa recipient groups. The ‘third’ cell type needed for optimal response by the thymus and bursa cells together was tentatively identified as a macrophage.     

Recovery of the in vitro reactivity of splenic cells of CBA/N mice toward type 2 thymus-independent antigens

CBA/N mice bearing a chromosome X linked immunological deficiency (Xid) cannot respond to type 2 thymus independent antigens (TI-2). However, when their spleen cells are in vitro simultaneously stimulated by both a TI-2 (Fluorescein conjugated polyacrylamide, Flu-PAA) antigen and a type 1 thymus independent (Trinitrophenyl conjugated Brucella abortus, TNP-BA) antigen, their capacity to respond to the TI-2 antigen is recovered. On the contrary, thymus dependent (TD) Sheep red blood cells (SRBC) antigen did not produce any significant increase of the anti-TI-2 response.     

LPS regulation of the immune response: suppression of immune responses to orally administered T-independent antigen

The regulation of immune responses to gastrically administered TI antigens has been investigated, and the characterization of a regulatory cell population has been performed. Intragastric administration of TNP-haptenated homologous erythrocytes (TNP-MRBC) induced splenic IgM anti-TNP PFC responses in LPS nonresponsive C3H/HeJ mice that were higher than those in LPS-responsive C3H/HeN mice and similar to those noted in athymic (nu/nu) C3H/HeN animals. The simultaneous intragastric administration of LPS with TNP-MRBC augmented immune responses in a manner similar to that previously reported for parenterally administered LPS and antigen. Further, LPS-induced augmentation of TNP-MRBC responses was greater in athymic mice. These findings were substantiated using in vitro spleen cultures. Intragastric challenge with a 2nd TI antigen, TNP-LPS, induced approximately 8-fold higher splenic anti-TNP PFC responses in athymic C3H/HeN mice compared with those in euthymic littermates. By admixture of B and T cell populations, it was demonstrated that the host responsiveness to TNP-LPS was negatively regulated by suppressor cells. Suppressive activity resided in a Thy 1.2-bearing, irradiation-resistant, nylon wool-nonadherent cell population. These cells could be demonstrated in spleen and Peyer's patches from young or old LPS-responsive C3H/HeN mice, but not in tissues from LPS nonresponsive C3H/HeJ mice. The specificity of the regulator cells was not limited to TNP-LPS responses, since immune responsiveness to another TI antigen, TNP-dextran, was also under the control of this cell population. These studies confirm the TI nature of TNP-MRBC and indicate that immune responses to gastrically administered antigens such as TNP-LPS, TNP-dextran, and possibly TNP-MRBC are negatively regulated by a suppressor T cell population. A role for endogenous LPS in the generation of regulator cells and the effect of these cells on host responses to gut-derived antigens is discussed.     

Priming of peripheral lymph node B cells with TNP-Ficoll: role of lymphokines in B cell differentiation.

We have previously shown that peripheral lymph node (PLN) B lymphocytes of adult DBA/2J mice failed to make an antibody response to type 2 antigen TNP-Ficoll, but exhibited a good antibody response to type 1 antigen TNP-Brucella abortus. In the present study we wanted to find out whether the unresponsiveness of PLN B cells to TNP-Ficoll is due to defects in the early activation and proliferation stage or in the final differentiation stage of B cells. Therefore, we have used a two-step protocol of in vivo immunization of mice with TNP-Ficoll and the subsequent in vitro challenge with TNP-Brucella abortus and studied the anti-TNP plaque-forming cell (PFC) responses. The results indicate a three- to sixfold increase of PFC responses in PLN cell cultures derived from TNP-Ficoll-primed animals compared to saline control mice. This increased antibody response was TNP-specific as 93% of the PFC's were inhibited by TNP-lysine. Limiting dilution experiments confirm that the increase in anti-TNP PFC response from the TNP-Ficoll-primed animals was indeed due to an increase in TNP-specific precursor B cells. Further, the addition of rIL-5 or rIL-6 induced anti-TNP PFC in the TNP-Ficoll-primed and in control PLN cell cultures in the presence of antigen. However, in primed PLN cells lymphokines alone were sufficient to restore anti-TNP PFC response. In conclusion, our results show that in PLN, the TNP-Ficoll can induce proliferation of hapten-specific B cells but not final differentiation. These primed PLN B cells mature into antibody-secreting cells upon stimulation with TNP-BA or lymphokines.     

Production of auto-anti-idiotype antibody during the normal immune response. X. Response to TNP-Ficoll in the chicken

The spontaneous production of auto-anti-idiotype (Id) was demonstrated after injection of chickens with trinitrophenylated Ficoll (TNP-F) by: (a) the presence of hapten-augmentable plaque-forming cells (PFC), (b) the ability of serum and of hapten eluates from immune spleen cells to cause hapten-reversible inhibition of anti-TNP plaque formation, and (c) an enzyme-linked immunosorbent assay (ELISA). Tests for anti-Id using the ELISA and hapten-reversible inhibition of PFC correlated very well. As in the mouse, the incidence of hapten-augmentable PFC was reduced by thymectomy and increased by the transfer of TNP-F-immune spleen cells. Hapten-augmentable PFC were also observed during the immune response of chickens to p-azobenzene arsonate-conjugated Brucella abortus.     

Studies of immune responses in mice prone to autoimmune disorders. II. Decreased down-regulation by auto-anti-idiotype antibody in autoimmune-prone mice

Three lines of evidence are presented which suggest that autoimmune-prone mice are deficient in the production of auto-anti-idiotype antibody during their immune response to trinitrophenylated Ficoll (TNP-F). NZB, MRL lpr/lpr and older BXSB male mice have no hapten-augmentable plaque-forming cells (PFC). Hapten-augmentable PFC have been previously shown to be cells whose secretion of antibody has been inhibited by the binding of auto-anti-idiotype antibody to cell surface idiotype. Sera from TNP-F immunized NZB mice lack PFC inhibiting activity (anti-idiotype antibody). Spleen cells from TNP-F immune NZB mice fail to transfer anti-idiotype antibody-mediated suppression to naive mice as do spleen cells from immune non-autoimmune-prone mice. Taken together these data suggest that autoimmune-prone mice are deficient in auto-anti-idiotype antibody-mediated downward regulation of their immune responses. It was further shown that the immune response of NZB mice to TNP-F shows a slower decline in splenic PFC and a greater heterogeneity of PFC affinity than do the responses of non-autoimmune-prone strains. Since athymic (nude) mice, which were previously shown to be defective in the production of auto-anti-idiotype antibody, also show a slower decline in splenic PFC and an increased heterogeneity of PFC affinity, it is suggested that these peculiarities of the immune responses of autoimmune-prone and athymic mice are also the consequences of the lack of auto-anti-idiotype antibody-mediated down-regulation.     

Newborn and Wiskott-Aldrich patient B cells can be activated by TNP-Brucella abortus: evidence that TNP-Brucella abortus behaves as a T-independent type 1 antigen in humans

TNP-Brucella abortus (TNP-Ba) has been classified as a T-independent type 1 (TI-1) antigen in the mouse on the basis that it activates neonatal and CBA/N (X-linked immunodeficient) murine B cells in contrast to T-independent type 2 (TI-2) antigens. Therefore, it was of interest to determine whether human newborn and X-linked Wiskott-Aldrich syndrome B cells could be triggered by TNP-Ba. Previous studies had shown that human B cells from both these latter sources were relatively insensitive to stimulation with T-dependent and polysaccharide antigens (TI-2 in mouse). In this study, we show that TNP-Ba can trigger human cord blood B cells to differentiate into anti-TNP plaque-forming cells (PFC) in a hapten-specific and T-independent manner. The dose response and kinetics were similar to those previously seen with adult cells. The newborn responses, however, were lower than adult PFC responses. Precursor frequency and clone size analyses revealed that this lower response was not due to newborn cells containing fewer precursors but was the result of a reduced ability of these anti-TNP clones to expand. The ability of TNP-Ba to activate immature newborn B cells implies that this antigen can be used to assess B cell function in very young children. It also implies that TNP-Ba behaves as a TI-1 antigen in humans as well as in mice. This was supported by the finding that B cells from Wiskott-Aldrich patients, which were unreactive to polysaccharide antigens, were generally responsive to TNP-Ba. Therefore, it would appear that human newborn and Wiskott-Aldrich patients do possess a functionally competent B cell subset possibly equivalent to Lyb-5- immature murine B cells.     

T cells and the anti-trinitrophenyl antibody response to fetal calf serum and 2-mercaptoethanol

Unprimed murine lymphocytes maintained in culture medium containing fetal calf serum (FCS) and 2-mercaptoethanol (2-ME) developed very high levels of anti-trinitrophenyl (TNP) plaque forming cells (PFC). Both FCS and 2-ME contributed to the response. The development of anti-TNP PFC during culture was accompanied by a 10-fold expansion in the number of immunoglobulin-secreting cells, indicating polyclonal stimulation. However, the number of anti-TNP PFC was disproportionately high and not accompanied by a similar increase in plaques specific for sheep red blood cells. The TNP-specific plaques were not artifacts of the plaque assay since they were 98% inhibited by specific antigen. The in vitro induction of anti-TNP PFC by FCS and 2-ME was common to a number of mouse strains, although some genetic variation occurred. Nylon-wool-separated B cells, nude mouse spleen cells, and bone marrow cells all produced high levels of anti-TNP after culture with medium containing FCS and 2-ME. The addition of T cells to B-cell cultures increased the numbers of anti-TNP PFC by 1.5- to 2.5-fold. The presence of a TNP-cross-reacting antigen in FCS probably contributed to the unexpectedly high anti-TNP response. The response to the antigen in FCS was potentiated by the enhancing activity of 2-ME.     

In vitro immune response to the 2,4,6-trinitrophenyl determinant in aged C57BL/6J mice:changes in the humoral immune response to, avidity for the TNP determinant and responsiveness to LPS effect with aging.

An in vitro anti-TNP response of the spleen cells from aged C57BL/6J mice showed approximately 4-fold less PFC than did that from young adult mice. Anti-theta serum-treated young spleen cells gave an anti-TNP response that was definitely greater than the response of the anti-theta serum-treated aged spleen cells in the presence of the exogenous activated thymus cells as helper cells. These results suggest that the deficits in B cells may be partly responsible for the imparied anti-TNP response of the aged spleen cells. To examine further the capacity of stem cells in the bone marrow to generate B cells responsible for anti-TNP response in the spleen, we injected i.v. 1.5 to 2.0 times 10(7) bone marrow cells from young or aged mice into lethally irradiated syngeneic recipients that had previously been thymectomized. Four to 6 weeks later, 10(7) spleen cells from the two groups of these recipient mice were immunized with TNP-SRBC in the presence of the exogenous activated thymus cells and assayed for anti-TNP PFC. The response of the aged marrow-derived B cells was approximately one-half of that of the young marrow-derived B cells.The avidity for TNP determinant of the antibodies produced by the PFC was determined by the plaque-inhibition technique. The avidity of the antibodies produced by the aged mice was approximately 33 times lower than that by the young mice. Anti-TNP response of the young spleen cells were markedly enhanced by the addition of LPS to the cultures, whereas no or little enhancement of the response was induced in the aged spleen cells even in the presence of high concentration of LPS. In contrast, DNA synthesis of both the young and aged spleen cells was comparably stimulated by 1 mug/ml and 10 mug/ml of LPS, however, it was rather less in the aged spleen cells at a concentration of 100 mug/ml. Mechanisms responsible for the changes in avidity and responsiveness to LPS with aging are discussed.     

Size-dependent B lymphocyte subpopulations: relationship of cell volume to surface phenotype, cell cycle, proliferative response, and requirements for antibody production to TNP-Ficoll and TNP-BA

Murine splenic B lymphocytes were separated into size-dependent subpopulations by using counterflow centrifugation. Spleen cells were rigorously depleted of T lymphocytes to yield a population of cells that were greater than 90% surface immunoglobulin (Ig)-positive and that had a mean cell volume of 136.6 +/- 3.3 microns. From this population, five fractions of cells were obtained with mean cell volumes that ranged from 115.8 +/- 3.7 microns in fraction 1 to 168.0 +/- 6 microns in fraction 5. The cells in these five subpopulations were characterized by analysis on a fluorescence-activated cell sorter after staining with acridine orange to evaluate RNA and DNA content, and with fluorescein-conjugated anti-mu, anti-delta, and anti-Ia antibodies to evaluate their surface membrane phenotypes. DNA analysis revealed that virtually all of the cells in fractions 1 to 4 had 2 N DNA. Between 7 and 21% of fraction 5 cells were either in S-phase or contained 4 N DNA. In contrast, RNA content increased through the fractions, suggesting a transition from G0 to G1 in the subpopulations with increasing B cell size. As another measure of cell activation seen with increasing cell size, we observed a progressive increase in the expression of surface Ia and a decrease in the expression of surface IgD. In the absence of in vitro stimulation, the larger cells showed significantly higher levels of thymidine incorporation. When polyclonal B cell activators such as LPS or anti-Ig antibody were added, peak proliferative responses were similar in all of the fractions, but the time necessary to achieve a maximal response was shorter for the larger-sized cell subpopulations than it was for the smaller-sized cell subpopulations. Unprimed, size-dependent B lymphocyte subpopulations exhibited spontaneous or "background" antibody formation that occurred primarily in the subpopulations containing the largest cells. T cell factors present in EL4 supernatant enhanced the efficiency of in vitro differentiation of these same subpopulations. When cultured in the absence of T cell help, the thymus-independent type 1 (TI-1) antigen TNP-Brucella abortus (TNP-BA) or the thymus-independent type 2 (TI-2) antigen TNP-Ficoll induced the largest anti-TNP plaque-forming cell (PFC) responses in the fractions containing intermediate-sized cells, suggesting that in vitro, antigen-specific responses came primarily from B cells that have been influenced in vivo to leave their small resting state. The subpopulations containing the smallest size B cells required the presence of both a TI antigen and EL4 supernatant for efficient differentiation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Immunity to Plasmodium berghei yoelii in mice. II. Specific and nonspecific cellular and humoral responses during the course of infection.

The kinetics of various specific and nonspecific immunologic responses were examined in BALB/c mice infected with 17X nonlethal Plasmodium berghei yoelii (a self-limiting infection). The sequence of events after infection was characterized by rapid sensitization of splenic T cells to malaria antigen and polyclonal B cell activation, followed by a period of depressed splenic proliferative responses in vitro to mitogens (PHA and LPS) and malaria (specific) antigen. At the same time, suppressed primary in vitro splenic PFC responses to trinitrophenyl-aminoethylcarbamylmethyl-Ficoll (TNP-F) were seen. This suppression was an active process requiring adherent cells. During this period, levels of antimalarial antibody also increased exponentially. As the infection was cleared, splenic malaria antigen-specific proliferative responses were again observed and splenic PFC and in vitro mitogen responses returned to preinfection levels after variable periods of time. Both splenic proliferative responses to malaria antigen and antimalarial antibody responses remained persistently elevated. In addition, some responses were examined in mice infected with 17X lethal P.b. yoelii (a fatal infection); in comparison to the early responses of mice infected with the nonlethal substrain, there was a decrease and delay in the development of a splenic T cell response to malaria antigen and a blunted antimalarial antibody response.     

Ontogeny of B-lymphocyte function: XIII. Kinetics of maturation of neonatal B lymphocytes to produce a heterogeneous antibody response to a T-independent antigen

The ontogeny of the capacity of a B-cell population to produce a heterogeneous, relatively high-affinity plaque-forming cell (PFC) response to the T-independent antigen trinitrophenylated-Ficoll (TNP-F) was studied in a cell transfer system. Lethally irradiated mice were reconstituted with liver cells from neonatal donors and were immunized with TNP-F at various times thereafter. In contrast to the results of our previous studies on the ontogeny of the response to T-dependent antigens, it was found that, in the cell transfer recipient, the response of an immature B-cell population to a T-independent antigen matures slowly (21–28 days). Furthermore, this maturation does not appear to require the presence of adult thymus cells as does the maturation of the response of a B-cell population to T-dependent antigens. Thus, it appears that the acquisition of the capacity of a B-cell population to produce a high-affinity, heterogeneous, PFC response to T-dependent and T-independent antigens occurs under different regulatory influences.     

Transfer of agammaglobulinemia in the chicken. I. Generation of suppressor activity by injection of bursa cells

Spleen cells from adult agammaglobulinemic (bursectomized) chickens taken 1 to 3 weeks after an injection of histocompatible bursa cells can inhibit the adoptive antibody response to B. abortus of normal spleen or bursa cells in irradiated recipients. Spleen cells from Aγ chickens not injected with bursa cells generally do not. Moreover, bursectomized chickens which have been reconstituted with spleen cells within the first week after hatching do not respond with suppressor cell formation upon bursa cell injection. This apparent “autoimmunization” with bursa cells induces suppressor T cells which are only minimally sensitive to treatment with mitomycin C or to 5000 R γ irradiation. The suppressor activity is neither induced nor potentiated by concanavalin A in vivo. It is much stronger in spleen than in thymus cells and appears to be macrophage independent and to require intact cells. The cell component which stimulates the suppressor activity is more pronounced on bursa than on spleen cells, and is at most present to a very limited extent on bone marrow, thymus, or peritoneal exudate cells. It is better represented in comparable cell numbers of Day 17 than of Day 14 embryonic bursa. The inducing cell component is present in the membrane fraction of disrupted bursa cells. Immunization with bursa cells from B locus histoincompatible chickens leads to suppressor activity against histocompatible bursa cells. Although the removal of Ig-bearing cells from bursa greatly diminishes its immunizing capacity, injection of serum IgM and IgG does not induce suppressor cells. It is suggested that tolerance to a B-cell antigen is lacking in adult Aγ chickens, resulting in an autoimmune response upon exposure to B cells. The B-cell antigen may be a cell surface-specific form of Ig, a complex of Ig and a membrane component, or a differentiation antigen which appears simultaneously with Ig during ontogeny.     

Modulation of Mouse Anti-Trinitrophenyl Plaque-Forming Cell Affinity by Adjuvants or Lectins

The effects of several kinds of adjuvants or lectins, such as Corynebacterium parvum, dextran, poly AU, poly IC, dibutyryl cAMP, concanavalin A (Con A), phytohemagglutinin (PHA) and pokeweed mitogen (PWM) on anti-trinitrophenyl (TNP) direct plaque-forming cells (PFC) in the spleen of mice and the affinity of antibodies produced by these PFC were examined. The numbers of anti-TNP PFC in the spleens of mice which had been injected with C. parvum 7 days in advance were greater than those in controls after immunization with TNP-coupled heterologous erythrocytes, while the affinity of antibodies released by these PFC was not affected. On the contrary, simultaneous injection of dextran with TNP-erythrocytes did not increase the numbers of splenic anti-TNP PFC, but heightened the affinity of antibodies released by these PFC. Copolymers of nucleotides, poly AU and poly IC, were capable of enhancing splenic anti-TNP PFC responses, but showed almost no altering of PFC affinity. Dibutyryl cAMP did not have any effect on this system. Con A had potencies to both augment the number of anti-TNP PFC and heighten the PFC affinity, while PHA seemed to lack these potencies. Injection of PWM in the presence of antigen increased the number of anti-TNP PFC and heightened slightly the PFC affinity. These results indicate that the heightening of the affinity at the cellular level is regulated in ways different from the augmenting effects on the number of anti-TNP PFC by adjuvants or lectins. These results are discussed in the light of the mode of action of the substances used.     

EA rosette forming lymphoid cells: distribution as to cell types and organs from chickens of different developmental stages.

The interaction of chicken spleen cells with sheep erythrocytes coated with chicken antibody (EA complexes) was studied using a rosette assay. The results reported in this paper indicate that subpopulations of chicken lymphocytes, monocytes, and heterophils have a receptor for EA. The formation of rosettes between chicken lymphoid cells and sheep erythrocytes (SRBC) was dependent upon the concentration of antibody used to sensitize the SRBC. In a developmental study of rosette-forming lymphocytes (RFL), the bursa was the first site of appearance of large numbers of RFL. The percentage of RFL in the bursa reached a peak at 17 days of embryonic life, and declined to a low by hatching. The percentage of RFL in the spleen, however, began to increase at the time of hatching and by 6 weeks of age the spleen far surpassed the bursa in percentage RFL. At no age were significant numbers of RFL detected in the thymus.     

Induction of interleukin-5 responsiveness in resting B cells by engagement of the antigen receptor and perception of a second polyclonal activation signal.

Experiments were performed to examine the nature of agents which could induce IL-5 responsiveness in small, resting splenic B lymphocytes. First, IL-5 increased plaque forming cell responses to the TI-1 antigen TNP-LPS. A second set of experiments using anti-IgM + LPS which allowed limiting dilution analysis showed induction of IL-5 responsiveness in about 20% of the resting B cell population. In the same system, IL-4 increased the percentage of proliferating cells by about 40%. A third system using the TI-2 analog conjugate anti-IgD-dextran (anti-delta-dextran) also rendered small, resting B cells responsive to IL-5. An additional system employing anti-IgM plus dextran sulfate, which also allowed limiting dilution analysis, induced IL-5 responsiveness in at least 10% of resting B cells. The features common to all four systems inducing B cell IL-5 responsiveness are at least twofold. Each system directly accesses the B cell antigen receptor and causes crosslinking. Second, each system also provides an additional polyclonal activating moiety, some of which may be similar to those in thymus independent antigens. These results suggest that some resting B cells may become IL-5 receptive after perception of at least two kinds of signals one of which perturbs sIg and the second being nonspecific and polyclonally activating.     

Functional relationships between B-cell subsets: evidence for an antigen-induced B1 leads to B2 switch

Priming of mice with the TI-2 antigen TNP-Ficoll results in an increased secondary anti-TNP response to the TD antigen TNP-KLH. Spleen cells obtained from mice primed with TNP-Ficoll or TNP-LPS, but not TNP-KLH, gave augmented responses to DNP-Dextran, TNP-LPS, or LPS. In vitro treatment of spleen cells with DNP-Dextran results in an increased anti-TNP response to TNP-LPS. This effect is also obtained if mitogenic doses of Dextran and LPS are used to induce cell proliferation. We conclude that the B_i2 subset (responding to TNP-Ficoll and DNP-Dextran) switches to the B2 subset (responding to TNP-KLH) after antigen activation. The B_i1 subset (responding to TNP-LPS) may represent an intermediate stage of B-cell differentiation.