Inhibition of lymphocyte trapping by a passenger virus in murine ascitic tumors: characterization of lactic dehydrogenase virus (LDV) as the inhibitory component and analysis of the mechanism of inhibition.

Cell-free fluid from several ascites promoting tumors inhibits lymphocyte trapping. Lactic dehydrogenase virus (LDV), a common passenger virus in many mouse tumors, was found to be a trapping inhibitor component in these fluids. Procedures used to eliminate infective LDV, such as dilution, passage of the tumor through irradiated rats, ether fractionation, and ultraviolet (UV) irradiation abolished the trapping inhibitory capacity of the fluids. LDV, dissociated from tumors, was inhibitory. Lymph nodes in mice with acute, but not chronic, LDV infections were inhibited from trapping. LDV does not appear to inhibit the capacity of circulating cells to be trapped, and as measured by mitogen responsiveness, the virus does not directly interfere with T-cell function. LDV may inhibit trapping by indirectly affecting the T cell or directly affecting the macrophage in which it replicates. The known characteristics of LDV infection may explain a number of reported immunosuppressive attributes of tumor-associated ascitic fluids.

Human B cell activation. Evidence for diverse signals provided by various monoclonal anti-IgM antibodies.

Seven murine monoclonal antibodies (mAb) with different binding characteristics for human IgM varied markedly in their ability to induce proliferation of T cell-depleted human splenocytes. Two mAb (HB57 and 5D7) that bound to distinct epitopes on IgM were highly effective initiators of B cell proliferation at very low concentrations, in the presence of a T cell factor source. In the absence of T cell supernatant, both HB57 and 5D7 mAbs produced a markedly reduced degree of stimulation at all concentrations. Two additional anti-IgM mAb (VIIIE11 and Mu53) were distinctive in that, even at high concentrations, only limited proliferation was observed compared with the first group of mAb. This proliferation depended on the presence of T cell supernatant. Competitive-binding studies revealed that the epitope recognized by mAb Mu53 may be identical or very proximate to that recognized by HB57. Three other mAb (1G6, XG9, and P24) induced little or no proliferation. 1G6 bound to a unique epitope on the IgM molecule, whereas XG9 shared a determinant with VIIIE11 mAb. Regulatory influences of Fc receptor binding cannot account for all the diversity in proliferation observed with the individual anti-IgM mAb. Markedly augmented proliferation was obtained when B cells were cultured with certain combinations of anti-IgM mAb in the presence of exogenous T cell supernatant. The proliferation induced in the absence of T cell supernatant by high concentrations of mAb mixtures that included 1G6 approached that observed for the same mixtures in the presence of T cell supernatant. The data suggest that certain signals delivered through membrane IgM can bypass the need for T cell supernatant in the activation of human B lymphocytes.

T cell regulation of IgG subclass antibody production in response to T-independent antigens.

The effect of T lymphocytes on the IgM, IgG3, IgG1, IgG2b, and IgG2a responses of B lymphocytes to the type-2 T-independent antigens, trinitrophenylated (TNP)-Ficoll, and TNP-Levan, was investigated. T cell-bearing nu/+ mice were found to produce substantially higher IgG2 serum anti-TNP antibody than their athymic counterparts, and nu/nu and nu/+ IgG2a titers exhibiting more disparity than nu/nu and nu/+ IgG2b titers. The Igm, IgG3, and IgG1 anti-TNP levels in nu/nu and nu/+ mice were indistinguishable. By cell transfer experiments, it was determined that this variance in nude and heterozygote IgG2 responses could not be explained by B cell differences between the two strains or by suppressive effects on IgG2 production within nu/nu mice. Rather, the difference was shown to be the result of the absence of T cells at the time B cells were responding to antigen. In the absence of T cells, the strength of the nu/nu anti-TNP antibody response was found to be in the following order: IgM > IgG3 > IgG1 > IgG2b > IgG2a, a heirarchy identical with the recently proposed heavy chain gene order. The possibilities that T cells influence IgG2 production via their specific recognition of IgG2-bearing B cells or via signals to increase heavy chain switching of responding B cell clones are discussed.

T cell regulation of immunoglobulin class expression in the antibody response to trinitrophenyl-ficoll. Evidence for T cell enhancement of the immunoglobulin class switch.

In the absence of T cells, B cells were found to respond to the type 2 T-independent (TI-2) antigen, trinitrophenyl (TNP)-Ficoll, with a characteristic hierarchy of IgM and IgG subclass Ab production which directly correlated with 5' to 3' Igh-C gene order, i.e., IgM greater tha IgG3 greater than IgG1 greater than IgG2b greater than IgG2a. This was evident when immune serum Ab titers were analyzed, when in vitro secretion of antibody from immune cells was measured and when TNP-Ficoll-stimulated clones in a splenic focus assay were analyzed for isotype production. T cells were found to cause a preferential relative increase in the amount of IgG2a antibody produced to TNP-Ficoll. The T cell responsible was present in anti-IgM neonatally suppressed mice and was needed early in the response, i.e., on the day of immunization or earlier. T cells were found to increase the frequency of TNP-Ficoll-responsive B cell clones that produced IgG2a in the splenic focus assay. The great majority of these IgG2a-positive clones also produced IgM and all or nearly all of the IgG isotypes whose genes are encoded 5' to the Igh-gamma 2a gene. The data are discussed in terms to T cell enhancement of IgG2a Ab synthesis being mediated through T cell enhancement of the Igh-C gene switching mechanism within TNP-Ficoll-responsive B cell clones. Thus, isotypes encoded by genes on the 3' end of the Igh-gamma gene complex, which in the absence of T cells have a low probability of being switched to, are the most influenced by T cell help.

Anti-immunoglobulin antibodies. I. Expression of cross-reactive idiotypes and Ir gene control of the response to IgG2a of the b allotype.

The anti-allotype antibody response to the b allotypic form of IgG2a is regulated by major histocompatibility complex (MHC)-encoded immune response (Ir) genes. Mice of d, b, p, q, r, and s haplotypes make a strong anti-allotype response on immunization with the CBPC101 myeloma protein (IgG2ab), whereas mice of the k, m, a, a1, u, and z haplotypes made no, or a very poor, response. All responder strains produce anti-IgG2ab antibodies which share common idiotypes (Id) without relation to the allelic forms of the Ig heavy-chain-constant region genes that the responding mice possess. Isoelectric focusing analysis of the anti-allotype antibodies produced in various strains of mice showed that they are of limited heterogeneity and quite similar from strain to strain. Five out of six hybridoma products with specificity for CBPC101 allotype expressed cross-reactive idiotypes (IdX). Two of hybridoma products expressing IdX identify CH3-domain determinants, and one has been assigned a CH2-domain specificity.

IgG subclass, IgE, and IgA anti-trinitrophenyl antibody production within trinitrophenyl-Ficoll-responsive B cell clones. Evidence in support of three distinct switching pathways.

The IgM, IgG subclass, IgE, and IgA anti-trinitrophenyl (TNP) antibody (Ab) response of B cells to the type 2 antigen TNP-Ficoll was studied in athymic nude mice and in the in vitro splenic focus assay. Results from the splenic focus assay in which purified B lymphocyte preparations had been transferred to irradiated nu/nu recipients indicate that many TNP-Ficoll stimulated B cell clones secrete multiple isotypes and hence appear to be undergoing intraclonal isotype switching. Although the frequency of clones secreting each of the IgG subclasses was found to correlate with 5' to 3' Igh-gamma gene order, the frequency of IgE and IgA-secreting clones did not appear to be influenced by the respective position of Igh-epsilon and Igh-alpha on the chromosome. Unlike clones that secreted anti-TNP Ab of the IgG subclasses, IgE and IgA anti-TNP Ab-secreting clones did not have a high propensity for coexpression of isotypes encoded by 5' Igh-C genes. These data suggest that three distinct switching pathways may be employed by B cells responding to TNP-Ficoll: a common IgG pathway, an IgE pathway, and an IgA pathway. The presence of T cells resulted in a preferential enhancement of the production of anti-TNP Ab of those IgG subclasses which were least represented in the absence of T cells, i.e., IgG2b and IgG2a. No significant enhancement of IgE anti-TNP clonal frequency was found in the presence of T lymphocytes, but T cells were found to significantly enhance the clonal expression of IgA anti-TNP Ab. Although a relatively large number of B cell clones were found to synthesize IgE and IgA anti-TNP Ab in the splenic focus assay, relatively little or no secretion of these isotypes was detected in immune mice. Possible explanations for this apparent discrepancy are discussed.

Anti-IgM-mediated B cell signaling. Molecular analysis of ligand binding requisites for human B cell clonal expansion and tolerance.

The ligand binding requisites for membrane IgM-mediated signaling of human B lymphocyte clonal expansion and B cell tolerance were investigated with a well-characterized set of soluble murine anti-human IgM mAbs. Evaluation of the impact of mu chain domain specificity, affinity, and binding stoichiometry for membrane IgM on antibody-induced regulation of normal and leukemic B cell DNA synthesis revealed that the ligand binding requisites for inducing or, alternatively, suppressing B cell DNA synthesis are significantly different. First, while the induction of S phase entry required micrograms/ml concentrations of ligand, orders of magnitude lower concentrations of ligand sufficed for inhibitory signaling. Second, while an upper affinity threshold for achieving maximal stimulation of B cell DNA synthesis was never detected, inhibitory signaling by bivalent ligands appeared to become relatively affinity independent at Fab binding affinities greater than 7.0 x 10(6) M-1. Third, while a C mu 1-specific mAb with an enhanced incidence of monogamous binding to mIgM was ineffective at inducing B cell DNA synthesis, the antibody was not significantly compromised in ability to initiate inhibitory signals. These differences could be observed in a clonal B cell population which positively or negatively responded to mIgM ligation depending upon its state of activation. The accumulated observations indicate that the ligand binding requisites for inhibitory signal transduction in human B lymphocytes are much less rigorous than those for stimulatory signal transduction and suggest that many physiologically relevant anti-Ig antibodies are more likely to function in the negative feedback regulation of B cell responses than in the direct triggering of human B cell clonal expansion.

Role of the thymus in directing the development of a subset of B lymphocytes.

In an effort to evaluate the role of the thymus in influencing the development of Lyb-5- B lymphocytes, mice expressing both the xid and nu gene defects were studied. Mice expressing either of these defects respond to both trinitrophenylated Brucellus abortus and lipopolysaccharide; whereas mice with the combined defect show markedly suppressed responses. The other abnormalities included: (a) greater than 80 percent diminution of serum Ig levels; (b) significant increase in the number of sIgM+ sIgD- B lymphocytes; (c) reduced expression of IgD on sIgD+ cells; and (d) a strikingly abnormal histology of their lymphoid tissue. Because nu/nu mice that do not express the xid defect appear relatively normal, it would suggest that the development of Lyb-5- B lymphocytes require a thymic influence for normal maturation, whereas, Lyb-5+ B lymphocytes are relatively independent of such influences.

Immune response to a thymus-dependent form of B512 dextran requires the presence of Lyb-5+ lymphocytes.

Studies of the ontogeny of the immune response to B512 dextran (Dex) show that antibody responses equal to those of adult mice are not attained until 12 wk of age. We have examined the anti-Dex response after immunization with a thymus-dependent antigen isomaltohexaosyl-keyhole limpet hemocyanin (IM6-KLH) and have shown that the development of the cross-reacting anti-Dex response parallels the development of Lyb-5+ B cells. Adult levels of anti-Dex antibody after immunization with IM6-KLH are achieved in mice between 3 and 12 wk of age, a time when Lyb-5+ cells have reached adult levels. Neonatal mice, immunized at 1 d or 1 wk after birth, failed to produce a significant amount of anti-Dex antibodies, although they did produce IM6-specific antibodies after immunization with IM6-KLH. Data, which support the conclusion from these experiments that Lyb-5+ cells are required for an anti-polysaccharide response even when the immunizing antigen is thymus-dependent, include the failure of IM6-KLH to stimulate a normal anti-Dex response in mice with the xid defect and the direct demonstration in normal adult mice that elimination of Lyb-5+ cells from spleens of mice primed with IM6-KLH abolishes the ability of these cells to transfer an anti-Dex response. The data imply that the expressed B cell repertoire in adult animals is skewed such that the vast majority of B cells capable of responding to polysaccharide determinants are in the Lyb-5+ subset.

Establishment of an inbred line of mice that express a synergistic immune defect precluding in vitro responses to type 1 and type 2 antigens, B cell mitogens, and a number of T cell-derived helper factors.

Introduction of the CBA/N X-linked gene into C3H mice has resulted in the establishment of a new strain of mice that has profound immunologic defects. B cells from these mice show significantly impaired in vitro immune responses to the T cell-independent type 1 antigen trinitrophenyl-Brucella abortus (TNP-BA) as well as markedly reduced proliferative responses to a number of B cell mitogens when compared with the responses of the parental control mice. The in vivo response of such mice to TNP-BA is, however, comparable to that of CBA/N mice. Furthermore, B cells from C3.CBA/N mice are unresponsive to the plaque-forming cell enhancing effects induced by EL4-derived supernatant in the presence of TNP-BA, unlike B cells obtained from CBA/N or C3H/Hen mice whose responsiveness to TNP-BA can be significantly enhanced in the presence of EL4-derived supernatant. The model we have presented to best explain these results suggests that B cells from C3.CBA/N mice can be stimulated only under conditions in which they can interact with carrier-specific T cell help and not under conditions where factor-dependent responses are dominant.

Analysis of the domain specificity of various murine anti-human IgM monoclonal antibodies differing in human B lymphocyte signaling activity.

The domain binding specificity of 19 murine anti-human IgM monoclonal antibodies (MoAbs) that have shown considerable heterogeneity in the transduction of stimulatory and inhibitory signals to B lymphocytes was evaluated by competition radioimmunoassays. Through the use of: (i) enzymatic fragments of IgM which each encompass more than a single CH domain, i.e. Fc5 mu and F(ab')2 mu, (ii) isolated single domains, C mu 2, C mu 3, and C mu 4, and (iii) mu heavy chain disease proteins, nine anti-IgM MoAbs were found to have C mu 1 domain specificity, five to have C mu 2 specificity, and five others to have C mu 4 specificity. Ineffective binding to isolated mu chain demonstrated that C mu 1-specific MoAbs were directed to epitopes which require light chain for expression. The lack of binding of the C mu 4-specific MoAbs to CNBr cleavage fragments of Fc5 mu suggest that the determinants recognized by these MoAbs may also be conformational in nature. Cross-inhibition analyses were used to determine the number of unique epitopes recognized by the anti-IgM MoAbs. Results from these experiments showed that: (i) eight of the nine MoAbs specific for C mu 1 likely bind to a single epitope, or very proximate epitopes, (ii) the five C mu 2-specific MoAbs recognize at least three distinct epitopes, and (iii) the five C mu 4-specific MoAbs each recognize a separate determinant. A comparison of the known B cell activating properties of these MoAbs with their specificity for the various segments of the IgM molecule indicate that mitogenicity cannot be attributed to selective binding to any one domain.

Use of a solid-phase 3H-radioimmunoassay for the measurement of immunoglobulin produced in short-term cultures of antibody-secreting cells.

A slid-phase radioimmunoassay (RIA) for assaying immunoglobulin produced from antibody-secreting myeloma, hybridoma and immune spleen cells is described. Specific antibody is detected by culturing antibody-producing cells on antigen-coated flexible polyvinylchloride microtiter wells, washing away the cells, and measuring the bound specific antibody with tritiated affinity-purified anti-isotype reagents. Antibody produced from 10(3) myeloma cells can be detected with as little as 4 h of incubation. With 24-48 h of incubation, antibody from as few as approximately 3-15 myeloma, hybridoma or immune spleen plaque-forming cells (PFC) can be detected. This culture-well RIA has certain distinct advantages over the hemolytic PFC assay and RIA assays in which antibody in culture supernatants is measured.

Cytokine dependency of human B cell cycle progression elicited by ligands which coengage BCR and the CD21/CD19/CD81 costimulatory complex.

Coengagement of BCR and the C3dg binding CD21/CD19/CD81 costimulatory complex can profoundly reduce the BCR binding threshold for eliciting B cell S phase entry, provided cytokine is present. IL-4 is substantially better than IL-2, IL-13, and TNF-alpha at exhibiting synergy with BCR:CD21 coengaging ligand (anti-IgM:anti-CD21:dextran) in promoting B cell DNA synthesis. Synergy between IL-4 and anti-IgM:anti-CD21:dextran (a) is not explained by the viability-promoting function of IL-4, (b) occurs when the anti-CD21 moiety engages either C3dg binding or non-C3dg binding domains, (c) does not reflect reversal of FcgammaRII-mediated negative regulation, and (d) involves differing temporal requirements for BCR and IL-4R signal transduction during the activation process. The IL-4R signaling pathway appears to synergize directly with the BCR:CD21 signaling pathway(s) in promoting the progression of resting B cells past an early G1 checkpoint, as well as to promote independently the progression of activated B cells past a later G1 to S checkpoint.

Inhibition of lymphocyte trapping by cell-free ascitic fluids cultivated in syngeneic mice.

Several cellfree ascitic fluids were tested for their influence on the trapping of lymphocytes in peripheral lymph nodes after subcutaneous antigen administration. Fluid from three ascitic tumors cultivated in syngeneic mice diminished the trapping response significantly. Fluid from one of these ascitic tumors cultivated in allogeneic mice showed no inhibition of trapping. Syngeneic normal mouse serum did not significantly affect the trapping phenomenon. It was concluded that syngeneic ascitic fluids contain a substance capable of inhibiting lymphocyte trapping.

T cell regulation of the IgG2a response to TNP-Ficoll: evidence that allotype congenic mice contain both helper cells that preferentially enhance IgG2a synthesis and suppressor cells that specifically suppress IgG2 synthesis.

When unprimed C57BL T cells were transferred into C57BL nu/nu mice, a preferential enhancement of the IgG2a antibody response to TNP-Ficoll was observed. Unprimed splenic T cells from Igh allotype congenic (B.C8) mice were unable to enhance the IgG2a response. The failure of T cells from allotype congenic mice to augment IgG2a antibody production to TNP-Ficoll was due to the presence of a T cell that specifically suppressed IgG2a antibody synthesis. The suppressive activity could be demonstrated in nu/nu mice and nu/nu mice reconstituted with C57BL helper cells. The suppressive activity of the B.C8 T cell population could be abrogated by treatment with anti-Lyt-1 and anti-Lyt-2 antibodies and complement as well as by treatment of B.C8 T cells with anti-Lyt-2 alone. Removal of the T cells responsible for IgG2a suppression from B.C8 splenic T cell population unmasked a population of B.C8 T cells that could enhance IgG2a preferentially augment IgG2a antibody synthesis can be found in donor mice that differ in their Igh background from the responding B cells.

Lactic dehydrogenase virus alteration of lymphocyte circulation.

Acute infection with lactic dehydrogenase virus (LDV) causes a systemic alteration in lymphocyte circulatory patterns. Peripheral lymph nodes (LN) and spleens in acutely, but not chronically, infected mice retain a significantly greater proportion of injected 51Cr-labeled lymphocytes than the respective tissues in noninfected controls. This increase in lymphocyte localization in LN and spleen is dependent upon the dose of LDV injected and the timing of the infection. A relatively large dose of LDV (10(8) infectious units) causes an early but very transient increase in splenic lymphocyte localization accompanied by an early but prolonged increase in lymphocyte recovery in LN. Smaller doses of LDV cause more prolonged effects on splenic lymphocyte recovery and retarded effects on lymphocyte localization in LN. Increases in splenic recovery were always accompanied by decreases in hepatic recovery of lymphocytes. LDV-induced alteration in lymphocyte circulation may be responsible for many previously observed modifications of immune responses in LDV-infected mice.

Affinity requirements for induction of sequential phases of human B cell activation by membrane IgM-cross-linking ligands.

The affinity of Ag interaction with a B cell's membrane IgM (mIgM) receptors has long been considered to play a critical role in the in vivo clonal selection of B lymphocytes. This study has examined a possible basis for this affinity selection at the level of Ag induction of sequential B cell activation phenomena, i.e., elevated membrane class II MHC expression (G0* excitation), G1 entry, and S phase entry. Functional experiments with model bivalent Ag, i.e., a group of murine mAb of diverse intrinsic binding affinities for human IgM, revealed that the minimal affinity requisites for inducing the above phenomena vary significantly. At a ligand concentration of 100 micrograms/ml, the induction of increased class II MHC expression, G1 entry, and S phase had minimal affinity thresholds of Ka approximately 0.2 to 2 x 10(6) M-1; approximately 7 x 10(6) M-1; and approximately 1 x 10(8) M-1, respectively. Pulsing studies revealed that whereas high affinity ligand was essential at later periods in the prolonged (greater than 24 h) signaling period that leads to S phase entry, mAb with significantly lower affinity were competent at signaling during the first 24 h. Because all but the lowest affinity ligand (Ka = 2 x 10(5) M-1) could effectively modulate mIgM, and furthermore, because B cells show a substantial increase in surface area during activation, it appears likely that one factor contributing to the higher affinity requirements for induction of late activation phenomena is a progressive decrease in the density of mIgM on the responsive B cells. These studies suggest that whereas only a small proportion of B cells, i.e., those with relatively high affinity for an antigenic epitope, will be triggered to clonally expand on encountering a paucivalent Ag in the absence of T cell help, a much wider spectrum of the B cell repertoire will be triggered to a state of partial activation. How the presence of ancillary T cells and cytokines may facilitate the full clonal expansion of these latter cells is discussed.

Human B cell activation. Effect of T cell cytokines on the physicochemical binding requirements for achieving cell cycle progression via the membrane IgM signaling pathway.

Given the range of mIg-binding affinities expressed by Ag-specific B cells, the ligand:receptor affinity threshold for achieving full B cell activation via the mIgM-mediated signaling pathway is quite high. Several recombinant, or semi-purified, cytokines were found to reduce the very high mIgM:ligand affinity threshold for induction of human B cell S phase entry by bivalent, affinity-diverse anti-IgM mAbs without notably affecting the lower affinity threshold for G1-related RNA synthesis. Two-stage culture experiments suggested that one major means by which IL-4, IL-2, and low m.w. B cell growth factor lower the affinity threshold for S phase entry is an indirect one, i.e., rescue of B cells whose mIg engagements with Ag are of sufficient affinity for achieving G1 entry, but of insufficient affinity for initiating the late-phase mIgM-mediated signals needed for the G1-->S phase transition. IL-4 had additional effects in early G1. In contrast to the above cytokines, IFN-gamma, did not function as an independent cell cycle progression factor, but rather required the concomitant presence of mIgM-cross-linking ligand for enhancement. A greater potential of multivalent anti-IgM-dextran conjugates to trigger S phase entry in the absence of cytokines was found to reflect a greater potential for initiating mIgM signals during the late phase in B cell activation. The results indicate that progression of mIgM receptor-activated B cells past a G1-->S phase restriction point is dependent upon continued signal transduction via either the mIgM receptor and/or a cytokine receptor signaling pathway. When mIgM-engaging ligands are ineffective at initiating late-phase signals, due to limited size and binding site valency and/or affinity, ancillary signal transduction through cytokine receptors becomes most relevant.