A monoclonal antibody raised to lipomodulin recognizes T suppressor factors in two independent hapten-specific suppressor networks

Five different Ag-binding suppressor factors from two types of hapten-specific Ts cell hybridomas (TsF1 inducer and TsF3 effector factors) were bound by an anti-lipomodulin mAb (141-B9), that crossreacts with rodent glycosylation inhibition factor (GIF). The Ag-specific suppressor activity in these hybridoma supernatants was bound by anti-lipomodulin columns and could be recovered by elution at acid pH. Additional evidence for the expression of lipomodulin/GIF activity on these TsF molecules was demonstrated by the ability of the eluted fractions to inhibit the glycosylation of IgE-binding peptides during their biosynthesis. The same biologic activity is associated with GIF and lipomodulin. The relationship between TsF and lipomodulin/GIF was confirmed in a serologic assay, which showed that TsF1 and TsF3 molecules, whether purified over Ag, anti-IJ or anti-TsF columns, are recognized by the mAb. 141-B9. The combined results indicate that Ag-binding Ts factors share a common antigenic determinant with phospholipase inhibitory proteins such as lipomodulin and GIF. In addition, the demonstration of glycosylation regulatory activity carried on these TsF molecules suggests a possible mode for their bioactivity.     

Serologic, biologic, and western blot analysis of a T suppressor factor with specificity for the hapten 4-hydroxy-3-nitrophenyl acetyl derived from serum-free medium

A T cell hybridoma producing a T suppressor factor (TsF) with specificity for the hapten nitrophenyl was converted to long term growth in serum-free medium and its product tested by serology, bioactivity, and Western blot analysis. Results indicated that Ag-specific suppressive activity was present in serum-free medium and this TsF could exhibit the characteristics ascribed to it by various groups: it could bind nominal Ag with specificity, it was bound by anti-TsF mAb, and it could mediate Ag-specific suppression both in vivo and in vitro. Western blot and SDS-PAGE analysis of this purified TsF revealed a 43-kDa single chain protein.     

Genetic basis for altered idiotype expression in the hyperimmune response to (4-hydroxy-3-nitrophenyl)acetyl hapten

To assess the significance of somatic point mutation in the hyperimmune response to the hapten NP, an in vivo enrichment procedure was followed. Mice that expressed high titers of B1-8 idiotopic determinants were selected as donors for serial transfer of small numbers of immune spleen cells into syngeneic irradiated recipient mice. Cells expressing B1-8 idiotopic determinants were chosen for enrichment because B1-8 cross-reactive determinants constitute a significant portion of the primary response. Furthermore, B1-8 is a monoclonal antibody derived from a primary response to NP, and its heavy and light chains are unmutated products of the germ-line genes VH186.2 and VL lambda 1, respectively. The germ-line sequence is thus available for comparison with the somatic mutants that arise during enrichment and hyperimmunization. The data show that serial transfer of spleen cells from mice with a high titer of idiotypic determinants results in a dramatic decrease in the titers of antibodies that bind antigen. Three lines of evidence indicate that progeny cells from the initial lambda-positive, idiotype-bearing, antigen-binding cells are successfully transferred and expanded during successive adoptive transfers. First, the proportion of lambda-bearing antibodies relative to NP-specific lambda-bearing antibodies increases with transfer, which is consistent with mutation away from antigen binding. Second, analysis of serum antibodies and hybridoma proteins derived from transfer-recipient mice confirm the presence of idiotype-positive antibodies that do not bind antigen. Third, RNA dot blot analysis of hybridomas constructed from a recipient mouse in the fourth transfer indicates a high frequency of expression of the VH gene predominantly used in the NP response. Many of the antibodies expressed by these hybridomas not only do not bind antigen, but have also lost the determinants recognized by the anti-idiotypic reagents. Most of these VH-positive hybridomas express lambda L chain. The most likely interpretation of the data is that somatic mutation is occurring during the hyperimmune response. Because we selected donor mice that expressed a high titer of idiotype-positive, antigen-specific antibody and immunized the recipient mice, we expected to observe a selective expansion of somatic variants that bound antigen. This was not the case. The observed loss of antigen binding suggests that the majority of mutations arising result in antibodies with lower affinity for the immunizing antigen.     

Specificity change of antibody to (4-hydroxy-3-nitrophenyl)acetyl haptens by somatic hypermutation

Change in the specificity of anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibodies (Abs) with time after immunization was studied. The early anti-NP Abs was specific to the ionized (phenolate) form of NP. The specificity changed with time and the late Abs became able to bind to the protonated (phenolic) form as well as the phenolate form of NP. The nucleotide sequences of mRNA coding for variable regions of heavy and light chains suggested that somatic hypermutation contributed to this change of the specificity.     

Fine-specificity of the contact sensitivity to 4-hydroxy-3-nitrophenyl acetyl (NP)

Dorf and colleagues (1-4) found that the contact sensitivity (CS) primed with (4-hydroxy-3-nitrophenyl)acetyl (NP) could be elicited as easily with the iodoanalog (NIP) as with NP when studied in Igh-1b mice but could only be elicited with NP, not NIP, in Igh-1j mice. Since this fine-specificity was parallel to the fine-specificity of anti-NP antibodies in the two types of mice and since anti-NP antibodies of Igh-1b mice are controlled by gene Igh-NPb the authors concluded that CS also was controlled by the Igh-NPb gene. The aim of this study was to confirm their findings with a more quantitative method (5). We confirmed equality of NP and NIP as elicitors of NP-primed CS in Igh-1b mice when the priming antigen was given subcutaneously into non-cyclophosphamide-treated mice (their method). We also found that this priming induced an anti-NP antibody response detectable at the time of challenge. Most experiments were carried out with a method that does not induce a detectable antibody response (pretreatment of mice with 200 mg/kg of cyclophosphamide; application of the sensitizing compound on skin). Since the NP-primed (and NBrP-primed) CS reactions exhibited "expected specificities," the immunizing compound was clearly the most efficient elicitor (relative efficiencies of homologs varied from 2 to 4). The Igh-NPb gene appears not to have a role in "antibody-free" reactions.     

4-Hydroxy-3-nitrophenyl (NP) acetyl-hapten specific lymphocyte proliferation. I. Mice bearing Igh-1b allotype can cross-react with 4-hydroxy-5-iodo-3-nitrophenyl (NIP) acetyl hapten

Hapten specific T cell proliferation was induced in several strains of mice. When lymph node T cells from 4-hydroxy-3-nitrophenyl acetyl-keyhole lympet hemocyanin (NP-KLH)-primed mice were stimulated in vitro with NP-polymer glutamic acid-lysine-phenyl alanine (NP-GL phi) or NP-ovalbumin (NP-OVA), they displayed a good level of proliferative responses. It was observed that NP-GL phi could induce NP-hapten specific proliferation even with NP-KLH lymphocytes from GL phi nonresponder strains. NP-KLH primed lymphocytes from C57BL/6 (H-2b, Igh-1b), CKB (H-2k, Igh-1b), CWB (H-2b, Igh-1b), and B10.BR (H-2k, Igh-1b) mice showed good proliferative responses to both 4-hydroxy-5-iodo-3-nitrophenyl (NIP) acetyl-GL phi and NIP-OVA antigens. However, NP-KLH primed lymphocytes from C3H/He (H-2k, Igh-1j) and C3H. SW (H-2b, Igh-1j) mice displayed poor proliferative responses to NIP-GL phi and NIP-OVA antigen. These results suggested that the gene coding for the NIP-cross-reaction might be mapped in the Ig heavy-chain linked locus.     

Cross-induction of predominant NPb idiotypic antibodies with derivatives of (4-hydroxy-3-nitrophenyl) acetyl

Immunization of C57BL/6 mice with bovine gamma-globulin (BGG) conjugated with (4-hydroxy-3-nitrophenyl) acetyl (NP) induced a population of anti-NP antibodies that bear predominantly lambda light chain, exhibit heteroclitic affinity for heterologous NP derivatives, and share NPb idiotype. The present study analyzes the idiotypes of antibodies induced with BGG conjugated with the iodo-, bromo-, or nitro-NP derivatives (NIP, NBrP, and NNP). NIP-BGG, NBrP-BGG, and NNP-BGG, induce specific antibodies bearing NPb idiotype in C57BL/6 or C57BL/10 congenic mice, but not in many other inbred strains. Furthermore, the quantity of NPb idiotype in immune sera from various mouse strains immunized with NIP-BGG, NBrP-BGG, and NNP-BGG was similar to that in sera from mice immunized with NP-BGG. Anti-idiotypic antisera against C57BL anti-NP, anti-NIP, or anti-NBrP antibodies exhibit extensive idiotype binding of specifically purified B6 anti-NP, -NIP, -NBrP, and -NNP antibodies. These purified antibodies contain a high percentage (greater than 70%) of lambda-chain-bearing molecules. The data indicate that an extensively shared repertoire composed of predominantly lambda-bearing NPb-positive idiotypic antibodies is used in response to NP and its derivatives in C57BL mice.     

A monoclonal antibody raised against Alzheimer cortex that specifically recognizes a subpopulation of microglial cells

A monoclonal antibody (MAb), termed AMC30, was raised after in vitro immunization with sonicated neurofibrillary tangle (NFT)-enriched fractions prepared from Alzheimer's brain. The antigen to which AMC30 is directed was expressed by microglial cells in senile plaques of Alzheimer's disease (AD). Microglia in the parenchyma surrounding brain tumors or infarctions, multinuclear giant cells, perivascular and parenchymal macrophages throughout the brain of AIDS patients were also labeled. Different non-nervous system lesions in which macrophages participate were also stained. Microglial cells in normal areas of the cortex or white matter were not labeled with MAb AMC30. The antigen to which AMC30 is directed was not detected in normal bone marrow, lymph nodes, lung, or spleen monocytes or macrophages. The epitope recognized by MAb AMC30 was present after formalin fixation and paraffin embedding. Our findings suggest that this MAb is directed against an antigen that is specifically expressed in a subpopulation of microglial cells and macrophages reactive to various pathological conditions.     

Anti-tumor effects of an antiserum raised in syngeneic mice to a tumor-specific T suppressor factor

Summary DBA/2 mice inoculated with either cells from the syngeneic P815 tumor or tumor cell membrane extracts develop T suppressor cells which suppress the in vitro generation of cytotoxic T lymphocytes with specificity for the tumor. A soluble suppressor factor with similar properties can be isolated from suppressor cell-enriched populations. It can be highly purified by appropriate immunoadsorption. Antisera to this suppressor factor raised in either DBA/2 or C57BL/6 mice can specifically absorb out suppressor factor and eliminate suppressor cells in the presence of complement. The in vivo effects of these antisera were tested for their ability to modulate the growth of P815 tumors in DBA/2 mice. It was found that the antiserum raised in syngeneic (DBA/2) but not allogeneic (C57BL/6) mice was able to significantly slow the rate of tumor growth and to prolong survival in treated mice. The antiserum was effective in this way only if it was administered early in the course of tumor growth. It was shown that this effect was not attributable to the presence in the serum of antibodies directed to antigens present on P815 cells, and it therefore appears to be due to interference with the function of T suppressor cells arising early in the immune response to the tumor cells.     

Differences in antibody repertoires for (4-hydroxy-3-nitrophenyl)acetyl (NP) in splenic vs immature bone marrow precursor cells

To evaluate the contribution of environmental regulatory mechanisms in fashioning the primary B cell repertoire, we have compared the repertoire of (4-hydroxy-3-nitrophenyl)acetyl (NP)-specific primary splenic B cells with that of precursor cells present as surface immunoglobulin-negative (sIg-) cells in adult bone marrow of C.B20 (Ighb) mice. Previous analyses using a variety of antigens have led to the conclusion that the antibody repertoire expressed in the spleen is similar to that expressed in newly generated B cell precursors with respect to both repertoire diversity and the representation of various predominant clonotypes. However, in the response to NP of C.B20 precursor cells, two marked disparities have been identified between the repertoire of sIg- bone marrow cells vs splenic precursor cells. The first concerns precursor cells that give rise to lambda-bearing NP-specific antibodies with heteroclitic fine specificity. Such antibodies normally dominate the primary response of Ighb mice; however, the representation of precursor cells giving rise to lambda-bearing antibodies is disproportionately low in the sIg- bone marrow cell population of C.B20 mice. Thus, during the maturation of these cells, post-sIg receptor expression, there is an apparent increase in the proportionate representation of lambda-bearing NP-specific cells. The second disparity concerns precursor cells whose antibody products bear kappa-light chains and exhibit high affinity and homoclitic binding for the NP haptenic determinant. Such precursor cells are poorly represented in the spleen, but represent a sizeable proportion of the sIg- NP-specific precursor cell population. Thus, there seems to be a selective elimination of high affinity, kappa-homoclitic anti-NP antibody-bearing cells as they acquire their sIg receptors. The elimination of this cell population could partially account for the dominance of lambda-heteroclitic antibodies in the serum responses to NP of C.B20 mice.     

Hapten-specific T cell responses to 4-hydroxy-3-nitrophenyl acetyl. XIII. Characterization of a third T cell population involved in suppression of in vitro PFC responses

The involvement of a third-order suppressor T cell population (Ts3) in the suppression of in vitro PFC responses was analyzed. It was shown that Ts2 effector-phase suppressor cells, induced by the i.v. injection of NP-coupled syngeneic spleen cells, require a third suppressor T cell population to effect NPb idiotype-specific suppression of an in vitro B cell response. This Ts3 population was shown to be present in NP-primed but not unprimed donors. The Ts3 population specifically binds NP and is Lyt-1-, Lyt-2+, I-J+ and bears NPb idiotypic determinants. The involvement of the Ts3 population in a suppressor pathway that requires recognition of idiotypic determinants is discussed.     

The frequency and fine specificity of B cells responsive to (4-hydroxy-3-nitrophenyl)acetyl in aged mice

The B-cell response to NP-Hy of two murine strains has been analyzed in order to evaluate the affects of aging on B-cell repertoire expression. The results indicate that for both BALB/c mice (Igha) and B10.D2 mice (Ighb) the frequency of (4-hydroxy-3-nitrophenyl)acetyl (NP)-responsive splenic B cells is approximately twofold lower, on a per B cell basis, in aged mice as compared to young adults. However, as with previous assessments of the response to DNP-Hy in aged mice, the frequency of newly generated surface immunoglobulin negative bone marrow precursor cells specific for NP in aged BALB/c mice is the same as in young mice. The decrease in frequency of responsive splenic B cells is not accompanied by a measurable decrease in repertoire diversity or changes in clonotype distribution as assessed by representation of kappa vs lambda light chain-bearing antibodies, binding of monoclonal antibodies to a panel of analogues of NP, and the proportionate representation of B10.D2 monoclonal antibodies that bear NPb idiotypic determinants. By these criteria it appears that down-regulation of B cells as they mature and emerge from the bone marrow of aged mice is pan-specific and does not disproportionately affect B cells of a predominant clonotype family. Consistent with other investigations which have demonstrated differences in secreted antibodies of immunized aged vs young mice, we have observed that 4 weeks after immunization of B10.D2 mice with NP-BSA, the frequency of newly generated secondary B cells is lower in aged than in young mice and the generation of lambda-bearing secondary precursor cells is particularly low. Thus, clonotype-specific down-regulation may play a role in shaping the B-cell repertoire subsequent to immunization of aged mice.     

Heterogeneity of human T4+ inducer T cells defined by a monoclonal antibody that delineates two functional subpopulations

A monoclonal antibody termed anti-T4 that detected approximately 60% of peripheral blood T lymphocytes was shown to define the human inducer population. In the present study, we characterized three additional monoclonal antibodies, anti-T4A, anti-T4B, and anti-TQ1, that were reactive with a similar percentage of T lymphocytes. Anti-T4A, anti-T4B, and anti-T4 delineated identical cell populations, while those defined by anti-TQ1 differed in several respects: 1) Anti-TQ1 stained a minority (less than 7%) of thymocytes, whereas the other antibodies stained a majority (80%); 2) Anti-TQ1 reacted with 70 to 85% of T4+ lymphocytes, but also stained 50% of T cells within the T4- (T8+) cytotoxic/suppressor subset; 3) The antigen defined by anti-TQ1 was not restricted in its expression to T cells; it defined a fraction of normal B and null lymphocytes as well as non-T cell lines. In vitro studies indicated that the subpopulations of T4+ T lymphocytes delineated by anti-TQ1 were functionally distinct. Although T4+TQ1+ and T4+TQ1- T cells proliferated in an equal fashion to soluble antigen and alloantigen, only the T4+TQ1+ subset was responsible for maximal proliferation in autologous MLR. This T4+TQ1+ subset contained a population of lymphocytes reactive with the previously defined JRA autoantibody. In contrast, the T4+TQ1-, but not the T4+TQ1+, subset provided the majority of T cell help for B cell immunoglobulin production in a pokeweed-driven system. We conclude that the subpopulation of T4+ inducer cells responsible for maximal helper activity in T-B interactions is restricted to a minor subpopulation of T4+ lymphocytes.     

A monoclonal antibody to animal centrosomes recognizes components of the basal-body root complex inChlamydomonas

Summary The mammalian centrosome monoclonal antibody MPM-13 recognized component(s) of the well defined MTOC basal-body root complex in the green plantChlamydomonas. The antibody reaction coincided in location with the basal-body root complex and the cruciate nature of the staining pattern corresponded to the configuration of the root microtubules. During mitosis the behaviour of MPM-13 stained material mirrored the duplication, separation and migration to the spindle poles of the basal body-root complex. It is suggested that conserved MTOC components were recognized and that these may have retained a similar, perhaps universal, function in microtubule organization.Abbreviations BSA bovine serum albumin - DAPI 4,6-diamidine-2-phenylindole dihydrochloride - mt mating type - MT microtubule - MTOC microtubule organizing centre - PFA paraformaldehyde - PBS phosphate buffered saline     

Amplification of suppressor inducer pathway with monoclonal antibody, anti-2H4, identifying a novel epitope of the common leukocyte antigen/T200 antigen

The 2H4 antigen, comprised of a 200/220-kDa glycoprotein of the leukocyte common antigen (LCA) family, is expressed on a suppressor inducer, but not a helper inducer subset of T4 cells. Earlier studies have demonstrated that the T4+2H4+ subset of cells maximally responded to the AMLR and this molecule has an important role in generated suppressor signals in AMLR/Con A-activated T cell systems. In the present study, we examined the effect of a series of monoclonal antibodies including anti-2H4 antibody on the initial activation of T4 cells in response to self-Ia antigens. We found that the addition of anti-2H4 antibody resulted in an augmentation of the proliferative response of T4 cells in AMLR, whereas other antibodies reactive with LCA/T200 antigens lacked this ability. Furthermore, anti-2H4 antibody enhanced both IL-2 production and IL-2R expression in this AMLR system. This enhancing effect was inhibited by anti-T3 antibody. Moreover, the suppressor inducer function of AMLR T4 cells was enhanced with anti-2H4 antibody by increasing the number of 2H4+ cells with high antigen density. Taken together, these results suggest that the 2H4 antigen may serve as an accessory structure for enhancing the activation of the T4+2H4+ suppressor inducer subset at initiation of cell triggering.     

Further characterization of the human inducer T cell subset defined by monoclonal antibody.

Evidence is presented that the OKTA+ T cell subset in man, defined by a monoclonal hybridoma antibody, provides help for B lymphocyte differentiation in a PWM driven system. Both B cell proliferation and intracytoplasmic immunoglobulin synthesis are facilitated by OKT4+ and not by OKT4- T cells. Given earlier studies demonstrating that OKT4+ T cells were necessary for generation of T cytotoxic cells and the present study that OKT+ T cells are necessary for the differentiation of B cells, it would appear that the OKT+ population is the major human T helper (inducer) subset.     

Comparison of T suppressor factors from tumour-bearing mice and mice immunized with a monoclonal anti-idiotypic antibody

Summary Certain dosage schedules of a monoclonal antiidiotypic antibody (related to a murine bladder carcinoma) were found to induce suppressor factor production by syngeneic mice. This suppressor factor resembled the factor from tumour-bearing mice with respect to idiotype specificity, possession of molecular markers (reactive with anti-IJ and B16G antibodies) and production by Lyt2⁺IJ⁺ T cells in spleen cell cultures. The two factors differed with respect to Igh restriction in an in vitro assay (leucocyte adherence inhibition) and ability to suppress the induction of delayed-type hypersensitivity to tumour antigen.     

Induction of immune tolerance by administration of monoclonal antibody to L3T4

Immune responses can be profoundly altered in mice by treatment with monoclonal antibodies (MAb) to L3T4, the mouse homologue for the CD4 antigen in humans. Treatment of mice with anti-L3T4 blocks both primary and secondary immune responses, delays allograft rejection, and retards autoimmunity. To determine whether anti-L3T4 could also be used to induce tolerance, we investigated the effect of treatment with rat MAb to L3T4 on the immune response to two other rat MAb: MAb to chicken egg ovalbumin (OVA) and MAb to T200, an antigen expressed on all mouse mononuclear blood cells. Treatment with anti-L3T4 prevented the primary humoral response to both of these MAb. Moreover, the anti-L3T4 MAb induced tolerance to itself, and it induced tolerance to the anti-OVA MAb when the two MAb were given concurrently. However, anti-L3T4 did not induce tolerance to the anti-T200 MAb when these MAb were given concurrently. These findings indicate that treatment with MAb to L3T4 may provide a new method for inducing tolerance to some, but not all, antigens. Because L3T4 in mice is homologous to CD4 in humans, our findings suggest that it may be possible to use anti-CD4 to induce tolerance to specific xenogeneic MAb, thereby facilitating their use as therapeutic agents in people.     

A monoclonal antibody to swine erythrocytes recognizes the B blood group on the major glycophorin

Recently monoclonal antibodies (mAbs) to swine red blood cells have been described. One of them (1AC11) was specific for the major swine glycoprotein with a molecular weight of 45 kDa and another mAb, 2G2, recognized the B ^a allele in the B system of swine blood groups. Immunoblotting experiments to characterize the mAb 2G2 indicated that it reacts with an antigen of 45 kDa, present on the aqueous phase, glycophorin fraction, of swine red blood cells with the B ^a allele and does not react with B ^bB^b homozygous cells. The antigen recognized by 2G2 has the same characteristics as the major glycophorin recognized by 1AC11, so we can conclude that the B system of the swine blood group is on the major glycophorin of swine erythrocyte membranes.     

A monoclonal antibody that recognizes an Ly-6-linked antigen inhibits the generation of functionally active T cell subsets

A monoclonal antibody (mAb) generated against the chemically-induced BALB/c Meth A sarcoma, designated HD42, reacts in cytotoxic tests with Meth A as well as with BALB/c peripheral lymph node cells and mitogen-activated spleen cells. The antigen was detected by FACS analysis on BALB/c spleen and lymph node cells, and by absorption assays on all normal lymphoid cells of BALB/c but not B6 mice. The expression of the antigen was not found on normal adult lung fibroblasts, on brain, nor on an extensive panel of tumors of BALB/c and B6 origin. Because the strain distribution of the antigen is reciprocal to that of Ly-6.2 and is not expressed in congenic C3H.Ly-6b mice, we have tentatively defined it as Ly-6.1 and referred to the mAb as alpha-Ly-6.1. The presence of alpha-Ly-6.1 abrogates both the Con A-induced and the IL 2-dependent proliferative response of normal T cells, whereas the response of normal B cells to LPS remains unaffected. alpha-Ly-6.1 is a potent suppressor of the primary in vitro plaque-forming cell (PFC) response to SRBC. Pretreatment of normal splenic T cells with alpha-Ly-6.1 and complement had no effect on the ability of these cells to generate in vitro either T helper cells (TH) or T suppressor cells (TS) to SRBC. However, addition of antibody in the absence of complement during the generation of TH or TS, or posttreatment of these T cell subsets with antibody and complement after in vitro education, completely removed the functional activity of these cell types. Addition of alpha-Ly-6.1 to MLC suppressed the MLR as well as the generation of cytotoxic lymphocytes (CTL), whereas the presence of the antibody during a cell-mediated lympholysis (CML) had no effect. Therefore, it appears that alpha-Ly-6.1 recognizes an antigen that is important for the generation of TH and TS cell subsets.