Immunohistochemical localization of xenogeneic antibodies against Iak lymphocytes on B cells and reticular cells

Rabbit antiserum against B10.AQR mouse spleen and lymph node cells (RAQR), after appropriate absorption, reacted with Ia^k-positive spleen and lymph node cells in cytotoxic and complement-fixing indicator systems. It reacted neither with Ia^k-positive thymocytes nor Ia^k-negative thymocytes, spleen, and lymph node cells. Cryostat sections of tissue from Ia^k-positive and Ia^k-negative mice were incubated with RAQR and either rabbit anti-mouse Ig or rabbit anti-T cell globulin. With the unlabeled antibody enzyme method, RAQR-stained lymphocytes were concentrated in the B-cell regions of spleen and lymph nodes of Ia^k-positive CBA mice. The tissues of mice bearingI-region haplotypes different fromk were negative. Reticular cells of the T cell-supporting network were also positive in Ia^k mice, but liver, gall bladder, and testicular cells were not. Macrophages of both Ia^k-positive and -negative mice were stained by RAQR and also by heat-aggregated, peroxidase-labeled Ig. Ia^k-positive reticular cells survived 900 R total body irradiation and persisted after grafting with Ia^k-negative bone marrow. The reticular cells were also seen in a thymus which was depleted of cortisone-sensitive lymphocytes.Abbreviations used in this paper are as follows RAQR rabbit anti-mouse-B10.AQR globulin - RAMTG rabbit anti-mouse T-cell globulin - RAMIG rabbit anti-mouse Ig - SARIG sheep anti-rabbit Ig - agg-HIg aggregated human Ig - PAP anti-peroxidase-peroxidase-complex     

Studies on Fc receptor function. I. IgG-mediated inhibition of B lymphocyte activation by T-dependent and T-independent antigens.

Mouse aggregated IgG, when continuously present in cultures of mouse spleen cells immunized with sheep erythrocytes, causes a dose dependent inhibition of the generation of plaque forming cells with a maximum of about 90% at 400 μg IgG/culture. Unaggregated IgG induces a similar inhibition, whereas treatment with mouse albumin or F (ab¹)₂, under the same conditions, does not affect the generation of plaque forming cells.It has been reported that unaggregated IgG binds poorly to Fc receptors of B lymphocytes and thus should not be expected to inhibit PFC generation if the effect is at the level of B lymphocyte Fc receptors. Competitive binding experiments were carried out and showed that aggregated and unaggregated IgG compete similarly with ¹²⁵I-labeled aggregated IgG for binding to Fc receptors of mouse spleen cells.The same inhibition of PFC can be induced by aggregated IgG in cultures of B lymphocytes immunized with the T-independent antigen DNP-Ficoll. When IgG is absorbed extensively with sheep erythrocytes and added to cultures immunized with sheep erythrocytes, PFC generation is inhibited to a level comparable to that of nonabsorbed IgG.These results suggest that IgG binding to Fc receptors leads to a severe inhibition of the induction of PFC by both T-dependent and T-independent antigens. This and other work from our laboratory indicate that this effect may be at the level of B lymphocyte Fc receptors.Taken together with reports from several laboratories, the data presented here suggest that Fc receptors may have a regulatory role on the activation of B lymphocytes by antigens or mitogens.     

T-independent but not T-dependent antigens maintain surface Ig of lymphocytes.

The effect of T-independent (TIA) and T-dependent (IDA) antigens on the surface Ig of 24-hr cultured rabbit spleen cells was investigated by two techniques: the proportion of cells bearing surface Ig was determined by direct rosette formation with anti-light chain allotype-coated erythrocytes; the total amount of surface Ig was estimated by labeling the cells with anti-allotype ¹²⁵I-labeled Fab fragments. The addition of TIA resulted in the maintenance of the proportion of Ig-bearing cells almost to the initial level, an effect which could not be obtained with any of the TDA tested. The same type of effect was observed when the total amount of surface Ig was measured, i.e., there was a slight reduction (about 24%) in the amount of surface Ig in cultures to which TIAs were added and an almost sixfold reduction (about 70%) in cultures to which TDA, Con A, or no antigen was added. Some but not all of the TIA were able to induce [³H]TdR incorporation in 3-day spleen-cell cultures. We concluded that the common feature of TIA is the ability to stimulate the turnover of B-cell surface Ig, a feature that can be used for an easy screening of TIA.     

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.     

Antigen-induced in vitro inhibition of immune responsiveness

Addition of the dinitrophenyl derivative of the copolymer of d-glutamic acid and d-lysine (DNP-d-GL) or dinitrophenyl bovine γ-globulin (DNP-BGG) to spleen cell cultures specifically inhibited their capacity to produce an anti-DNP plaque-forming cell (PFC) response to the T-independent antigen dinitrophenylated polyacrylamide beads (DNP-PAA) or to the T-dependent antigen TNP-burro erythrocytes. The degree of unresponsiveness was dependent upon the tolerogen concentration and the duration over which the tolerogen was present in the culture. Treatment with rabbit anti-mouse brain antiserum and complement did not alter the induction of unresponsiveness suggesting a state of B-cell tolerance. Culture of spleen cells for 4 days in the absence of antigen led to the appearance of nonspecific suppressor activity which was demonstrable by its effect on the response of fresh spleen cells to antigen. Preculture in the presence of the immunogen DNP-PAA induced both nonspecific and specific suppressor activities. Induction of specific suppressor activity was not prevented by the presence of the tolerogen DNP-D-GL in the culture. The suppressor activity resided in an adherent T-cell population and did not appear to require macrophages for its induction.     

Murine NK cell heterogeneity: subpopulations of C57BL/6 splenic NK cells detected by NK-1.1 and NK-2.1 antisera

NK-1.1 antiserum - (BALB/c X C3H)F1 anti-CE - and NK-2.1 antiserum - NZB anti-BALB/c - detect genetically distinct alloantigens on C57BL/6 natural killer (NK) cells. We have analyzed whether these two alloantigens are associated with functional subsets of NK cells. For this study, nylon wool nonadherent C57BL/6 spleen cells (SC) were treated with complement (C) and NK-1.1 or NK-2.1 antisera and then tested for NK activity against a panel of tumor targets in 6- and 19-hour 51Cr release assays. The NK activity against the prototype NK target YAC-1 was reduced equally by both antisera. Similar reductions by both antisera were also observed when SC were tested against another murine lymphoma target, L5178c127v, against the C57BL/6 melanoma B16, and against the human liver cell line Chang. In contrast, NK activity to the lymphoma FBL-3 and the human erythroleukemia K562 was significantly reduced in SC treated with NK-2.1 antiserum and C, whereas SC treated with NK-1.1 antiserum and C showed either less reduction or no reduction in activity against these two cell lines. With two other targets, E male G2 and RBL-5, neither serum produced significant depletion of activity, Analysis of SC indirectly labeled with either NK-1.1 or NK-2.1 antiserum and fluorescein-labeled goat anti-mouse Ig, however, did not detect significant differences between NK-1+ and NK-2+ cell populations.     

Age-related changes in in vitro immunoglobulin secretion: Comparison of responses to T-dependent and T-independent polyclonal activators

In vitro Ig secretion by peripheral blood mononuclear cells (MNCs) from old and young donors, in response to T-dependent (TD) [pokeweed mitogen (PWM)] and T-independent (TI) [Salmonella paratyphii B (SPB)] activation were compared. In older donors, the IgG and IgA responses to PWM were comparable to those of young donors; the IgM response was reduced in the elderly. With SPB activation, IgA response was again preserved, whereas IgG response was reduced and IgM secretion was markedly decreased. These data indicate class-specific changes in Ig responsiveness to both TD and TI cell activators with age. The reduction in TI-induced IgG and IgM responses in the elderly suggest that changes in B cells themselves have occurred. The preservation of the TD IgG response in concert with reduced TI response indicates that a decline in T-suppressor influences over B cells in the elderly coupled with reduced B-cell synthesizing capacity can result in apparent “preservation” of the final Ig response. In keeping with the above postulate, analysis of individual elderly donors' responses indicated that some of the old donors responded to PWM, but not SPB; none of the old donors responded to SPB and not PWM. In contrast, some young donors did respond to SPB, but not PWM. These results also suggest that nonresponse to PWM in young donors relates to an override of functionally intact B cells by T-regulator influences.     

Regulation of clones responding to dextran B1355S. II. Response of T-dependent and T-independent precursors

The existence of precursors for TI and TD alpha 1 leads to 3 dextran antigens in BALB/c mice was demonstrated. A T-dependent dextran antigen was prepared by coupling dextran B1355S to hemocyanin and subsequent digestion with dextranase. The PFC response of BALB/c mice primed with hemocyanin to dextran-hemocyanin was found to be 8 times higher than in unprimed animals. The splenic focus assay was adapted for the analysis of precursors responding to T-dependent and T-independent dextran antigens. Pretreatment of recipients with anti-thymocyte serum abolished the response in fragment cultures to dextran-hemocyanin but did not affect the response to dextran B1355S. The frequencies of precursors in the adult BALB/c mouse responding to dextran and dextran-hemocyanin were determined by limiting dilution analysis. The frequency of T-dependent precursors was found to be almost 3 times greater than the frequency of T-independent precursors.     

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 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, 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, 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.     

Interferon-mediated inhibition of human polyclonal immunoglobulin synthesis

Human lymphoblastoid IFN suppressed human polyclonal Ig synthesis in a dose-related manner, with 71% mean inhibition of antibody production found when 1000 units of IFN were added to the cultures. This suppression was demonstrated to be mediated by an electrophoretically pure preparation of IFN. Furthermore, IFN inhibition of both the T-dependent and the T-independent polyclonal activators was not the result of direct cellular cytotoxicity. IFN-mediated suppression of human polyclonal Ig production does not result from induction of T suppressor cells, and in a T helper cell-independent system this inhibition appears to result from either a direct action on the B cells or an indirect effect via monocytes.     

Heterogeneity of B cell clones: immunoglobulin-secreting subsets

Staphylococcus aureus Cowan I bacteria (SpA CoI) is known to be a polyclonal B-cell activator of human lymphocytes. In this study, we investigated which of the B-cell subsets SpA CoI could stimulate and induce immunoglobulin (Ig) production. B-Cell subsets were separated from peripheral blood and tonsil lymphocytes by rosette formation with E, EA_IgG, EAC, anti-Ig-conjugated ox erythrocytes (OE-anti-Ig), and protein A-conjugated OE (OE-Pro A), or on a bovine serum albumin (BSA) discontinuous density gradient. The cells responding to SpA CoI included E^?, C3 receptor-positive (C3R⁺), Fc receptor-negative (FcR^?), and surface Ig-positive (SIg⁺) B-cell subsets. These B-cell populations responded well to SpA CoI and produced significant amounts of IgG, IgM, and a lesser amount of IgA. Among SIg⁺ B cells, IgG, IgA, and IgM⁺ B-cell subsets responded to SpA CoI and produced large amounts of Ig belonging to each corresponding Ig class. IgD⁺ B cells failed to produce Ig of any class, except for minimal amounts of IgG and IgM. While both the protein A receptor-positive (Pro A · R⁺) and negative (Pro A · R^?) cells responded well to SpA CoI, Pro A · R⁺ B cells produced IgG mainly and Pro A · R^? B cells produced IgM. Fractionation of B cells on a BSA gradient revealed that comparatively small-sized and denser B-cell subsets responded well to SpA CoI and produced every class of Ig.     

Induction of an immune response in athymic nude mice to thymus-dependent antigens by Pseudomonas aeruginosa exotoxin A

Exposure of spleen cells from athymic nude mice to Pseudomonas aeruginosa exotoxin A induces these cells to respond to the thymus-dependent (TD) antigen sheep erythrocytes (SRBC). The response induced by toxin is dose dependent, antigen specific, and not due to polyclonal B-cell activation. Enhancement of the anti-SRBC response can be observed when toxin addition precedes antigen stimulation by 24-48 hr, which decreases when toxin administration follows antigen stimulation. A significant response is also observed when toxin and antigen are added simultaneously. A significant anti-SRBC response can be observed out to Day 10 postantigen and toxin stimulation after attaining a peak response at Day 5. Cultures exposed to toxin in the presence or absence of antigen exhibited a higher cell number and relative number of B cells as compared to control cultures. Exposure of T-cell depleted B cells from euthymic +/nu mice to toxin plus antigen does not result in an anti-SRBC response indicating that exotoxin A alone is not sufficient to induce B-cell responsiveness to T-dependent antigens and that other cells and/or factors are involved in the toxin-induced responsiveness of nude mice to T-dependent antigens.     

A general method for studying the secretion of macromolecules by single cells. I. Detection of immunoglobulin-secreting cells.

A general method for detecting single cells secreting macromolecules has been developed and applied to the detection of mouse cells secreting antibodies. Secreted Ig molecules were precipitated in the immediate vicinity of an active cell with rabbit anti-mouse Ig antibody. Rapid removal of excess antibody not incorporated into immunoprecipitates was achieved with an electrophoresis technique. The immuno-precipitate surrounding the active cell was then stained with sheep anti-rabbit Ig antibody labeled with horseradish peroxidase. The use of enzyme markers has made the procedure much more convenient and rapid than previous precipitin assays for cell secretion that used radioiodine for detecting immunoprecipitates. Moreover, the availability of several enzyme markers makes possible the detection of cells secreting more than one molecular species. Experiments were also run in which cells producing antibodies specific for horseradish peroxidase (HRP) could be identified among the population of cells producing other immunoglobulins. Presumably, HRP-labeled antigens could be used to identify cells producing other specific antibodies. The generality of this procedure suggests that it may be useful for detecting single T-cells releasing regulatory molecules, since specific antisera are already available for several of these molecules.     

Rabbit antisera to human B cell alloantigens: effects on the mixed lymphocyte response.

Antisera were produced in rabbits to two glycoproteins (31,000 MW and 23,000 MW by SDS-gel electrophoresis) isolated from papain digests of membranes from a human B lymphoblastoid cell line (LCL). After minimal absorption with a T LCL the antisera reacted with two glycoproteins (35,000 MW and 27,000 MW) present in detergent-solubilized membranes from human B LCLs. The immunizing molecules are proposed to have arisen by proteolysis of the intact molecules in the detergent-solubilized membranes. The glycoproteins were detectable on human B LCLs and macrophages, but absent from T LCLs and fibroblasts. The molecules were identified as B cell alloantigens by their reactivity with alloantisera specific for human B cells. The rabbit antisera reacted with peripheral blood B lymphocytes, but not with T lymphocytes, platelets or erythrocytes.Pretreatment of human peripheral blood mononuclear cells with the rabbit antisera in a uni- or bidirectional mixed lymphocyte response (MLR) rendered them unable to stimulate, but they were able to respond. Addition of the antisera at various intervals during the MLR to block continuous stimulation indicated that cells were activated at different times. The presence of an F(ab′)₂ or Fab′ preparation of the antisera throughout the MLR did not inhibit the response at the concentrations tested. Further experiments suggest that, while a responding lymphocyte can replicate several times without restimulation, there is a delay between commitment to and commencement of division.     

Analysis of B-cell subpopulations. I. Relationships among splenic xid, Ly 1+, and Lyb 5+ B cells

The phenotypic and functional relationships among the various B-cell subsets is of importance for better understanding studies of the immune system. In this report, the realm of B cells encompassed by Lyb 5, Ly 1, and xid has been examined through the use of the alloantiserum anti-Lyb 5, the unique functional properties of Ly 1+ B cells, and the spontaneous autoantibody producing congenic xid mice, NZB.xid. By functional and phenotypic analysis, we have shown that (i) Lyb 5- B cells and B cells of xid mice are largely, but not completely, overlapping; (ii) xid spleen cells contain a population which is Lyb 5+; (iii) Ly 1+ B cells fall largely in the Lyb 5+ compartment; and (iv) the autoantibody-producing Ly 1+ B cells are predominantly Lyb 5+.     

Selective suppression by auto-anti-idiotypic antibody of B-cell idiotype repertoires generated after stimulation with the same hapten on T-dependent and T-independent carriers

In the present study, we investigated whether auto-anti-idiotypic antibody in the immune sera from old mice could recognize antitrinitrophenyl (TNP) plaque-forming cells (PFC) generated after stimulation with the T-dependent and T-independent forms of the hapten, TNP. Young and old C57BL/6J male mice were immunized with a variety of T-dependent (TNP-bovine gamma-globulin, TNP-BGG; TNP-keyhole Limpet hemocyanin, TNP-KLH; ovalbumin, OVA; bovine serum albumin, BSA; BGG) and T-independent (TNP-Brucella abortus, TNP-BA; TBP-Ficoll; TNP-polyacrylamide beads, TNP-PAA) antigens either in complete Freund's adjuvant (CFA) or in soluble form. Splenic anti-TNP or antiprotein PFC responses were assayed for anti-idiotype-blocked, hapten- or protein-augmentable IgM, IgG and IgA PFC, 1-2 weeks after immunization. It was found that 8-month-old mice produced significantly a higher percentage of hapten augmentable (26-42%) IgM PFC response to T-independent antigens as compared with the 2-month-old mice (3-6% augmentation). Similarly, old mice produced a significantly higher percentage of hapten or protein augmentable (25-129%) IgG PFC response to T-dependent antigens as compared with the 2-month-old group (2-6% augmentation). The data support the view that age-related regulation of auto-anti-idiotypic antibody is a general phenomenon for immune responses to T-dependent and T-independent antigens. Hapten-reversible inhibition of plaque formation was used to determine whether anti-idiotypic antibody containing antisera from old mice could inhibit B-cell idiotype repertoires generated after stimulation with the same hapten, TNP, on T-dependent and T-independent carriers. Pools of immune sera from 8-month-old mice primed with T-dependent TNP-BGG or TNP-KLH antigens but not with T-independent TNP-PAA or TNP-BA antigens, or with the proteins OVA, BSA, or BGG selectively inhibited IgM, IgG, and IgA anti-TNP PFC from 2-month-old mice that were previously primed with either TNP-BGG or TNP-KLH. In contrast, immune sera from old mice primed with TNP on either T-dependent or T-independent carriers inhibited anti-TNP PFC from mice primed with T-independent TNP-PAA or TNP-BA antigens. Immune sera from old mice primed with OVA or BSA only inhibited the respective antiprotein PFC. The immune sera from young mice did not show any appreciable inhibition of PFC generated after stimulation by any of the antigens studied.(ABSTRACT TRUNCATED AT 400 WORDS)     

Establishment of T-cell hybridoma constitutively producing macrophage-dependent T-cell replacing factor

A T-cell hybridoma was established by the fusion of concanavalin A-stimulated splenic T cells with BW 5147. The hybridoma cells secrete a factor constitutively to support antibody formation of spleen cells depleted of T cells against TNP-Ficoll but not against horse red blood cells. The activity was indicated not to be due to interleukin 2, B-cell growth factor I, B-cell growth factor II, or interferon. The factor-mediated antibody response to TNP-Ficoll required the presence of adherent cells. The adherent cell function could be replaced by the macrophage culture supernatant containing interleukin 1. B cells responding to TNP-Ficoll in the culture with hybridoma factor were indicated to be Lyb 5+ and to bear receptors for third component of complement.     

Cutting edge: gamma delta T cells provide help to B cells with altered clonotypes and are capable of inducing Ig gene hypermutation

It has not been resolved whether gammadelta T cells can collaborate with germinal center B cells and support Ig hypermutation during an Ab response to a truly defined T-dependent Ag. In this study, we show that in the absence of alphabeta T cells, immunization with the well-defined T-dependent Ag, (4-hydroxy-3-nitrophenyl) acetyl (NP) conjugate, was able to induce Ig hypermutation. However, the clonotypes of B cells responding to NP were dramatically altered in TCR beta(-/-) mice. Unlike B cells in wild-type mice that use canonical VDJ rearrangements, most NP-responding B cells in mutant mice use analog genes of the J558 gene family. In addition, the majority of anti-NP Abs produced in mutant mice use kappaL chain instead of lambda1L chain, which dominates in mice of Igh(b) background. Thus, the B cell population that collaborates with gammadelta T cells is distinct from B cells interacting with conventional alphabeta Th cells.     

The role of immunoglobulin receptors and T cell mediators in B lymphocyte activation. I. B cell activation by anti-immunoglobulin and anti-idiotype reagents

The possibility that the failure of anti-mouse immunoglobulin (Ig) antibody to induce antibody synthesis by B cells might be due to reversible receptor blockade was investigated. Murine spleen cells were cultured for 3 days in the presence of minute quantities of intact of (Fab') fragments of rabbit anti-mouse Ig antibody. Thereafter, the cells were washed and either trypsin treated or not before reculturing for 18 hr. Only cells that had been trypsinized after culturing with either intact or fragments of anti-Ig gave a vigorous polyclonal antibody response. This response was extremely T dependent, since T cells or culture supernatants from Con A-activated T cells were required for the B cell response. Moreover, anti-delta was much more effective than anti-mu in inducing antibody synthesis. Finally, the use of three different anti-idiotypic antisera rather than anti-Ig reagents selectively activated the specific idiotype in each instance. The findings demonstrate that anti-Ig reagents can potentiate the response of B cells to signals delivered by T cells.