Immunological responsiveness of neonatal A/J mice to isotypic determinants of syngeneic IgE.

We have previously shown that adult A/J mice produce high titers of anti-IgE with isotypic or idiotypic specificities in response to challenge with a conjugate of KLH with syngeneic monoclonal IgE. Thus, B cells that can synthesize anti-IgE are present in the mice. Adult mice are unresponsive to unconjugated IgE in CFA, suggesting that tolerance exists at the level of T cells. The present study shows that neonatal mice produce anti-IgE antibodies in response to unconjugated IgE in CFA, but that this capacity is lost after the age of 2-3 wk. The loss of responsiveness corresponds closely with the appearance of detectable IgE in serum, suggesting that the IgE may induce tolerance. The affinities of anti-IgE antibodies produced by neonatal mice fall in the range of values obtained with KLH-IgE in adult mice. Tolerance to unconjugated IgE in CFA can be induced in neonatal mice by administration of IgE in saline. In addition, the tolerant state can be induced by adoptive transfer of spleen cells from adult mice. The time-dependent acquisition of tolerance provides a useful model for studying mechanisms of tolerance and autoimmunity.

Antigen- and receptor-driven regulatory mechanisms. I. Induction of suppressor T cells with anti-idiotypic antibodies.

Delayed-type hypersensitivity (DTH) to the azobenzenearsonate (ABA) hapten can be readily induced in A/J mice injecting ABA-coupled syngeneic spleen cells subcutaneously. To further characterize this T-cell-dependent immunological phenomenon, the effect of passively administered anti-cross-reactive idiotype common to anti-ABA antibodies of A/J mice (CRI) antibodies on the development of ABA-specific DTH was investigated. Animals given daily injections (of minute amounts) of anti-CRI antibodies subsequent to immunization with ABA-coupled cells show significant reduction of ABA specific responses. This inhibition is antigen specific and requires the intact immunoglobulin molecule, as F(ab')2 treatments were ineffective in suppressing the reaction. Investigations of the mechanism of the anti-CRI-induced suppression of ABA DTH revealed that the observed suppression is a result of the activation of suppressor cells. Spleen cells taken from animals which received anti-CRI antibodies were able to adoptively transfer suppression to naive recipients. This suppression was shown to be mediated by T cells, as anti-Thy1.2 plus complement completely abrogated the transfer of suppression. In addition, animals pretreated with low doses of cyclophosphamide were not suppressed by the administration of anti-CRI antibodies. The genetic restriction of anti-CRI-induced suppression was demonstrated. Antibodies to the major cross-reactive idiotype, (CRI) associated with anti-ABA antibodies in A/J mice were unable to suppress the development of DTH to ABA in BALB/c mice (H-2d, Igh-1a). Such antibodies were, however, fully active in suppressing ABA DTH in the allotype-congenic C.AL-20 strain which has an allotype (Igh-1d) similar to that of A/J (Igh-1e) on a BALB/c background, and which produces humoral antibodies with the CRI.

Antigen- and receptor-driven regulatory mechanisms. II. Induction of suppressor T cells with idiotype-coupled syngeneic spleen cells.

Anti-p-azobenzenearsonate (ABA) antibodies, coupled covalently to normal syngeneic spleen cells and then given intravenously to normal animals, were found to be potent tolerogens for delayed-type hypersensitivity (DTH) to ABA. The ability of the antibody-coupled cells to induce tolerance was determined to be a result of the cross-reactive idiotype (CRI+) fraction of the antibodies, because anti-ABA antibodies lacking the CRI+ components when coupled to spleen cells were unable to cause any significant inhibition. Furthermore, genetic analysis revealed that the ability of CRI-coupled cells to inhibit ABA-specific DTH is linked to Igh-1 heavy chain allotype, in as much animals which possess heavy chain allotypes similar to that of A/J were sensitive to this inhibition. Adoptive transfer experiments provided evidence that CRI-coupled cells induce suppressor cells, and spleen cells or thymocytes from animals received CRI-coupled cells were able to transfer suppression to naive recipients. In addition, treatment with anti-Thy1.2 serum plus complement completely abrogated their ability to transfer suppression. Thus, this active suppression is a T-cell-dependent phenomenon. In investigating the specificity of these suppressor T cells, it was found that they functioned in an antigen-specific manner and were unable to suppress the development of DTH to an unrelated hapten 2,4-dinitro-1-fluorobenzene.

X-gp70: a third molecular species of the envelope protein gp70 of murine leukemia virus, expressed on mouse lymphoid cells.

Three variants of the gp70 envelope component of MuLV are now recognizable serologically: GIX-gp70, 0-gp70, and X-gp70. The last of these, X-gp70, has so far been found only in mice or cells producing abundant C-type virus. This distinguishes X-gp70, provisionally, from the GIX-gp70 and 0-gp70 variants, each of which can be expressed on normal thymocytes without accompanying virus production, as exemplified by mouse strains 129 and B6, respectively. The X-gp70 genotype, however, is not limited to strains of mice-producing abundant virus, because X-gp70+ leukemias occur in strains of mice which do not produce a great deal of virus and whose thymocytes and other tissues are X-gp70-; this is analogous to the appearance of GIX+ leukemias in GIX- mouse strains.

Properties of the antigen-specific suppressive T-cell factor in the regulation of antibody response of the mouse. III. Dual gene control of the T-cell-mediated suppression of the antibody response.

The antigen-specific suppressive T-cell factor of mice, which had previously been shown to be an I region gene product, could effectively suppress the in vitro secondary antibody response of spleen cells from syngeneic or H-2 compatible mouse strains but not that of H-2 incompatible strains. The identities among genes in the left side half (K, I-A, and I-B) of the H-2 complex between the donor and recipient strains were found to be both necessary and sufficient for the induction of suppression. This suggests that the acceptor site for the suppressive T-cell factor is also determined by the gene present in the left side half of the H-2 complex. The cell type which expresses the acceptor site was found to be a subset of T cell. In general, the suppressive T-cell factor obtained from F1 mice could suppress the responses of both parental strains, and the parental factors could suppress the response of F1 mice. The results indicate that both suppressor and acceptor molecules are codominantly expressed on F1 T cells. There were found two types of defects in the expression of suppressor and acceptor molecules among mouse strains: A/J mice could not produce the suppressive T-cell factor despite that they could accept the factor produced by other H-2 compatible mouse strains. In contrast, all the B10 congeneic lines could produce the T-cell factor, but could not accept the factor produced by syngeneic and H-2 compatible non-B10 congeneic lines. The F1 hybrid of A/J and B10. A could both produce and accept the T-cell factor, and thus the expressions of suppressor and acceptor molecules were found to be dominant traits. These results indicate that the antigen-specific T-cell-mediated suppression is regulated by at least two genes both present in the H-2 complex, and that the complementation of these two genes is required for the induction of suppression.

Regulation of azophenylarsonate-specific repertoire expression. 1. Frequency of cross-reactive idiotype-positive B cells in A/J and BALB/c mice.

A large proportion of p-azophenylarsonate (ARS)-specific antibodies from A/J mice share a cross-reactive idiotype (CRIA) that comprises a family of closely related but nonidentical clonotypes. I determined that only 2.6 % (7 out of 267) A/J ARS-specific monoclonal antibodies generated in the splenic focus system possess the predominant CRIA. Because ARS-specific B cells are present at a frequency of 1/68,000 B cells, the frequency of the entire idiotype family is 1 per 2.8 X 10(6) splenic B cells. Thus, there is a striking discrepancy between the representation of this idiotype at the clonal precursor cell level and the serum antibody response. In addition, BALB/c mice have the potential to generate CRIA-positive precursor cells within their nonimmune repertoire. When A/J mice are immunized with ARS-protein conjugates, the serum antibody response and precursor cell population are both dominated by CRIA. The frequency of CRIA-positive B cells increases over 100-fold after immunization, whereas CRIA-negative precursor cells may initially decrease, followed by a later rise in frequency. Finally, although ARS-specific precursor cells are present in high frequency at birth, CRIA-positive monoclonal anti-ARS antibodies are not observed during the early neonatal period. These data provide evidence to suggest that complex regulatory networks influence precursor cell and serum antibody expression.

Idiotypic analysis of lymphocytes in vitro. I. Specificity and heterogeneity of B and T lymphocytes reactive with anti-idiotypic antibody.

Guinea pig anti-idiotypic antibodies (anti-Id) of the IgG1 class, directed to an A/J antibody to Group A streptococcal carbohydrate (A-CHO), or directed to a BALB/c myeloma protein that binds the same antigen, stimulate B-precursor cells as well as T-helper cells when injected into mice of the appropriate strain. The strain-specific induction of both precursor and helper activity was detected by in vitro secondary responses of primed spleen cells to A-CHO or to 2,4,6-trinitrophenyl (TNP) upon challenge with Group A streptococcal vaccine (Strep.A) or with TNP-Strep.A, respectively. B- and T-cell populations primed with anti-Id were uniform with respect to the binding of antigen and of anti-Id. This was in contrast to cells primed with Strep.A, which were heterogenous. Taken together, B and T cells that possess the same antigen-binding specificity share idiotypic determinants, reveal the same idiotypic polymorphism, and may display similar degrees of heterogeneity with respect to the binding of antigen and anti-Id. Since the anti-Id used in this study detect Id determinants associated with the heavy chain of the variable region of mouse antibodies, the data suggest that this region of the immunoglobulin molecule is shared between T- and B-cell antigen receptors.

Evidence for the linkage of the IGC H locus to a gene controlling the idiotypic specificity of anti-p-azophenylarsonate antibodies in strain A mice.

Anti-p-azophenylarsonate (anti-Ar) antibodies elicited in all strain A/J mice tested share one or more idiotypic specificities. These specificities are also found in the anti-Ar antibodies of mice of the closely related strain, AL/N, but not in those of BALB/c mice. Anti-Ar antibodies were elicited in congenic mice in which the IgC(H) locus of AL/N mice, which controls allotypic markers in the constant regions of heavy chains, had been introgressively backcrossed for nine generations onto a BALB/c background; the mice were then rendered homozygous for the AL/N allotypic determinant. On the average, these antibodies were quantitatively equivalent, with respect to content of the cross-reactive idiotype, to those of AL/N mice. This indicates that the gene controlling the idiotype is closely linked to the IgC(H) locus. Since idiotype must be a function of V region sequences, the results suggest close linkage of V(H) and C(H) genes. The cross-reactive idiotype was found in nearly all F(1) mice (C57/BL x A/J or BALB/c x A/J) tested.

Antigen- and receptor-driven regulatory mechanisms. III. Induction of delayed type hypersensitivity to azobenzenearsonate with anti-cross-reactive idiotypic antibodies.

Delayed-type hypersensitivity (DTH) to p-azobenzenearsonate (ABA) can be induced in A/J mice with intravenous injection of minute amounts of anti-cross-reactive idiotypic (CRI) antibodies, providing that the animals have been pretreated 2 d earlier with low doses of cyclophosphamide (50 mg/kg). However intravenous injection of the F(ab')2 fragments of the anti-CRI antibodies or subcutaneous administration with anti-CRI antibodies induces comparable immunity in both cyclophosphamide-pretreated and normal nontreated animals. Furthermore adoptive transfer experiments indicate that lymph node cells taken from animals sensitized with anti-CRI 4 d earlier can adoptively transfer immunity to naive recipients. Transfer of immunity is mediated by a population of thymus-dependent (T) cells, which express idiotypic structures on their surface. Treatment of effector cells with either anti-theta serum or anti-idiotypic antibodies plus complement completely abrogated their ability to transfer immunity. In addition idiotype-bearing suppressor T cells induced with ABA-coupled spleen cells inhibit the development of ABA-specific DTH induced with anti-CRI antibodies. Genetic analysis revealed that the ability of anti-CRI antibodies to induce ABA-specific DTH was linked to Igh-1 heavy-chain allotype. Anti-idiotypic antibodies to the major CRI associated with anti-ABA antibodies in A/J mice failed to induce significant immunity in BALB/c mice (H-2d, Igh-1a). Nevertheless, they were able to induce significant immunity in C.AL20 mice (H-2d, Igh-1d) which possess a heavy-chain allotype similar to that of A/J mice.

Structural studies on induced antibodies with defined idiotypic specificities. IX. Framework differences in the heavy- and light-chain-variable regions of monoclonal anti-p-azophenylarsonate antibodies from A/J mice differing with respect to a cross-reactive idiotype.

Amino terminal amino acid sequence analyses have been performed on the heavy and light chains of induced monoclonal antibodies with specificity for the hapten p-azophenylarsonate. Four of the eight antibodies react with conventional antisera to the previously described A/J anti-arsonate cross-reactive idiotype (CRI). Of the 16 chains analyzed, all but one contain sequence differences in their first framework segment (residues 1-30) that distinguish them from the heavy- and light-chain sequences found in anti-arsonate antibodies isolated from A/J serum or ascites fluid. The presence of such framework differences appears to be independent of whether or not the hybridoma antibodies bear the CRI. In spite of the framework substitutions, all four of the CRI-positive hybridoma antibodies have variable (V)-region frameworks that are very similar to each other and to the CRI-positive molecules found in A/J serum. Two of the four CRI-negative molecules are also structurally similar to the serum antibodies. Two others, however, are strikingly different from any serum anti-arsonate antibody thus far described and appear to reflect a completely separate repertoire of anti-arsonate antibodies in the A/J MOUSE. In addition, serological analyses with an anti-idiotypic antiserum generated against a CRI-positive hybridoma product suggest that each monoclonal antibody may possess individual antigenic specificities different from the determinant(s) detected with the conventional rabbit anti-CRI. The consistent appearance of framework substitutions in what has been thought to be a homogeneous antibody population has important implications for our understanding of the generation of antibody diversity and for the precise chemical definition of an idiotype.

Expression of equivalent clonotypes in BALB/c and A/J mice after immunization with phosphorylcholine.

Analysis of A/J antibody to phosphorylcholine (PC) revealed a striking degree of similarity to PC-binding myeloma proteins of BALB/c origin. By quantitative idiotypic analysis A/J anti-PC antibody was composed to antibodies bearing binding site idiotypic determinants indistinguishable from two different BALB/c myeloma proteins, T15 and M511. Idiotypic determinants of three other PC-binding proteins, W3207, M167, and M603 were not detected. Isoelectric focusing of the light chains verified the presence of antibodies similar to T15 and M511 and indicated the presence of a third antibody whose light chains had a pI identical to that of M603. When the sequence of A/J heavy chains were compared to the heavy chains of T15, M511, and M603, both the framework and first complementarity regions were identical in all cases. Sequences analysis of the light chains through part of the first complementarity region revealed three chains, one similar to each of the myeloma proteins T15, M603, and M167-M511. The latter two sequences differ by only a single amino acid (a single base substitution) in the first 23 residues, suggesting that the two light chains may be very similar if not identical. Thus, BALB/c and A/J mice which differ genetically at multiple loci including the heavy chain allotype complex locus show a remarkable preservation of their anti-PC antibodies. These results indicate that the genes encoding these antibodies are contained in the germ line.

Mechanism of unresponsiveness to the alpha 1-6 epitope of dextran B512 in a C57BL substrain.

C57BL/10ScCr mice are low responders to the alpha 1-6 epitope of dextran B512, although other C57BL mice are high responders. Both thymus-independent and thymus-dependent forms of dextran failed to induce an immune response in C57BL/10ScCr mice, but dextran functioned as a good carrier for antihapten responses in this strain. Dextran is a potent polyclonal B cell activator for cells from C57BL/10ScCr mice, although such cells are not activated by LPS. The C57BL/10ScCr mice possess the Igh-V gene coding for antibodies against dextran and the antidextran antibodies induced in (A X C57BL/10ScCr)F1 hybrids share an idiotype with antidextran antibodies produced in C57BL/10 mice. Bone marrow cells from C57BL/10ScCr mice do not respond to dextran when transferred into lethally irradiated C57BL/10 mice and C57BL/10 cells transferred into C57BL/10ScCr mice give a strong antidextran response. Thus, B cells having both the Igh-V gene coding for antibodies against dextran and activation receptors for dextran cannot be activated into antibody synthesis against any form of this immunogen. This determinant specific immunodeficiency suggests the existence of as yet unknown regulatory influences on Igh-V gene expression or B cell activation.

Elevated myc expression and c-myc amplification in spontaneously occurring B lymphoid cell lines.

Recently, a minor subpopulation of murine B lymphocytes, Ly-1+ B cells, has been distinguished by its unique ontogeny, tissue distribution, and prominence in certain autoimmune and neoplastic B cell diseases. We have previously described a simple murine spleen culture system that results in the spontaneous and exclusive outgrowth of long-term Ly-1+ B cell lines (B Ly-1 cells). Here, we report that the immortal growth property of B Ly-1 cells correlates with a 10-45-fold elevation of steady-state myc RNA and 2-10-fold amplification of the c-myc locus. While c-myc amplification has been observed in malignant cell lines derived from several tissues of origin, its occurrence in lymphoid cells has not been previously reported. The consistent c-myc amplification in B Ly-1 cells may reflect a unique state of this locus in the Ly-1+ B lymphocyte lineage, and contribute to the spontaneous immortalization of this B cell population in vitro, and its apparent predilection for malignant transformation in vivo.

Frequencies of mitogen-reactive B cells in the mouse. I. Distribution in different lymphoid organs from different inbred strains of mice at different ages.

Frequencies of mitogen-reactive B cells have been determined in vitro under culture conditions which allow every growth-inducible B cell to grow and mature into a clone of Ig-secreting PFC. The frequencies of LPS-reactive B cells in the spleen of 6- to 8-wk old mice were between 1 in 3 and 1 in 10 splenic B cells from the following inbred strains of mice: C3H/Tif; BALB/c; BALB/c nu/nu; C57BL/6J; DBA/2J; C57BL/6J x DBA/(2J)F(1); and CBA and A/J. Very similar frequencies are found for lipoprotein-reactive B cells in BALB/c, BALB/c nu/nu, C3H/Tif, and C3H/HeJ mice. No LPS-reactive cells but normal frequencies of lipoprotein-reactive cells were found in C3H/HeJ mice, genetically nonreactive to LPS. SJL mice had significantly lower frequencies of LPS- and of lipoprotein-reactive B cells (1 in approximately 30 B cells). The number of LPS- and of lipoprotein-reactive B cells in spleen was dependent upon the age of the mouse. Newborn spleen contained approximately 10 percent of the number of reactive cells found at 6- to 8-wk of age. From there the frequencies declined again to drop below 5 percent of the maximal number at ages beyond 11 mo. LPS-reactive B cells yielding IgM- and IgG-PFC responses could be found in mesenteric lymph nodes, bone marrow, thymus, thoracic duct, and peripheral blood of 6- to 8-wk old mice. Their frequencies were one in three to five lymph node cells, 1 in 50 to 100 bone marrow cells, one in 10(5) thymus cells, and 1 in 20 to 40 thoracic duct or peripheral blood cells.

The novel CD4+CD25+ regulatory T cell effector molecule fibrinogen-like protein 2 contributes to the outcome of murine fulminant viral hepatitis.

UNLABELLED: Fulminant viral hepatitis (FH) remains an important clinical problem in which the underlying pathogenesis is not well understood. Here, we present insight into the immunological mechanisms involved in FH caused by murine hepatitis virus strain 3 (MHV-3), indicating a critical role for CD4(+)CD25(+) regulatory T cells (Tregs) and production of the novel Treg effector molecule FGL2. Before infection with MHV-3, susceptible BALB/cJ mice had increased numbers of Tregs and expression of fgl2 messenger RNA (mRNA) and FGL2 protein compared with resistant A/J mice. After MHV-3 infection, plasma levels of FGL2 in BALB/cJ mice were significantly increased, correlating with increased percentage of Tregs. Treatment with anti-FGL2 antibody completely inhibited Treg activity and protected susceptible BALB/cJ mice against MHV-3-liver injury and mortality. Adoptive transfer of wild-type Tregs into resistant fgl2(-/-) mice increased their mortality caused by MHV-3 infection, whereas transfer of peritoneal exudate macrophages had no adverse effect. CONCLUSION: This study demonstrates that FGL2 is an important effector cytokine of Tregs that contributes to susceptibility to MHV-3-induced FH. The results further suggest that targeting FGL2 may lead to the development of novel treatment approaches for acute viral hepatitis infection.

Immunological analysis of A strain mice bearing the A-10 mammary adenocarcinoma.

The present investigation was undertaken to determine whether antitumor antibodies are produced by A strain mice during the growth of a transplantable mammary adenocarcinoma (A-10). The antibody response was monitored by a sensitive radioimmunoassay which can detect 1 ng of antibody. No evidence of a humoral antitumor response was observed in animals given i.p. or s.c. injections of A-10 ascites cells. Control experiments showed that a humoral response was detectable 1 week after the inoculation of an allogeneic tumor. Immunoglobulin binds nonspecifically to cells via an Fc portion of the immunoglobulin molecule, and this was seen with a tumor bearer serum pool and with immunoglobulin preparations eluted from A-10 ascites cells. No specific antitumor antibody was found in these sources. The A strain mice could not be immunized to reject a challenge of live A-10 cells with mitomycin C-treated A-10 cells, with neuraminidase-treated A-10 cells, or with A-10 membrane preparations. It was concluded that the A-10 tumor is not immunogenic in its host of origin.

A sensitive radioimmunoassay for the detection of monoclonal anti-idiotope antibodies.

A radioimmunoassay was developed in order to detect anti-idiotypic antibodies in the supernatants of hybrid cells. This assay is both sensitive and specific for anti-idiotypic (but not anti-allotypic) antibodies. Monoclonal antibodies present in test supernatants are bound by an anti-immunoglobulin coated solid phase. Subsequent incubation with a source of mouse immunoglobulin 'blocks' unreacted anti-immunoglobulin antibodies on the solid phase. Anti-idiotypic antibodies are then detected by their ability to bind 125I-labelled idiotype-bearing antibody. This paper describes the use of this assay to detect monoclonal anti-idiotypic antibodies in 2 systems: the cross-reactive idiotype of A/J anti-ABA antibodies, and the idiotype expressed by the myeloma protein HOPC 8. Similarly, 125I-labelled anti-idiotype antibodies may be used in this assay to detect monoclonal idiotype-bearing antibodies. Further modifications are described which would allow the detection of monoclonal anti-allotype antibodies.

The elution of antibodies from viable murine tumor cells.

Several low pH buffers were used in order to dissociate radioiodinated antibodies from sensitized living tumor cells. Three buffers were further tested for their dissociation abilities under different conditions of time and temperature, and for their influence on the eluted antibodies. The cytotoxicity mediated by these low pH buffers was also determined by viability assays. Optimal results were obtained with 0.1 M citrate buffer at pH 3.5.

Crossed immunoelectrophoresis of mouse serum.

Because crossed immunoelectrophoresis (X-IEP) is quantitative and offers more information than immunoelectrophoresis (IEP), we selected X-IEP to study various physiologic and pathologic effects on the antigen composition of mouse serum. We therefore needed and X-IEP map of normal mouse serum. This paper presents one that we have developed, along with brief descriptions of how the mouse serum antigens were identified. Cross-reactivity between several mouse and human serum antigens was especially helpful. Some data from using ratios of the areas of precipitation of detected antigens to that of an internal standard antigen, alpha-macroglobulin, are presented. They show that X-IEP readily detects quantitative abnormalities among any of the several serum antigens detected by this technique, and that 'normal' values for these antigens in one population of mice can be established in the same way of using ratios with just a few analyses.

Specific suppression of graft-versus-host responsiveness by incubation of donor lymphoid cells with a solubilized membrane fraction of host lymphoid cells.

BALB/c spleen cells were incubated with a solubilized membrane fraction (SMF) prepared from spleen and thymus cells of (BALB/c x C3H/He)F1 or (BALB/c x A/J)F1 hybrid mice. Cells incubated with (BALB/c x C3H/He)F1 SMF produced less graft-versus-host (GVH) splenomegaly in (BALB/c x C3H/He)F1 hosts than did untreated BALB/c cells. The reduction of GVH splenomegaly was specific, inasmuch as the GVH activity of (BALB/c x C3H/He)F1 SMF-treated and untreated cells was similar in (BALB/c x C57BL)F1 hosts, and BALB/c cells treated with (BALB/c x A/J)F1 SMF showed no alteration of GVH activity in either (BALB/c x C3H/He)F1 hosts or (BALB/c x C57BL) F1 hosts. The time course of splenomegaly did not differ for SMF-treated and untreated cells. Donor cells that were labeled with tritiated adenosine and treated with (BALB/c x C3H/He) F1 SMF produced a reduction in the amount of label appearing in (BALB/c x C3H/He)F1 host spleens but not in (BALB/c x C57BL)F1 host spleens. Mechanisms which could account for the ability of SMF to cause specific reductions in both GVH activity and host spleen labeling are discussed.