Showing papers in "European Journal of Immunology in 1985"

Isolation and characterization of an Fc receptor from neonatal rat small intestine

Abstract: Receptors for the Fc region of IgG from neonatal rat intestinal brush borders were solubilized using 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propane sulfonate and purified by affinity chromatography. Analysis of IgG-binding material by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions reveals two components with apparent Mr of 41 000-50 000 and 15 000. The larger component is glycosylated and may dimerize, giving a 100-110-kDa band on nonreduced gels. Both proteins are localized in the proximal small intestine, where IgG is specifically taken up during the first three weeks of neonatal life, and disappear when specific transport stops after weaning. Electron irradiation of brush borders shows that the functional unit for IgG binding has a molecular weight in situ of 110 kDa. These data suggest that a dimer of the 41-50-kDa protein together with the 15 kDa and other proteins may mediate intestinal transport of maternal IgG.

Molecular and antigenic heterogeneity of the rat leukocyte-common antigen from thymocytes and T and B lymphocytes.

Abstract: The molecular forms and antigenic heterogeneity of the leukocyte-common antigen (L-CA) of rat lymphocytes have been analyzed. Thymocytes show one main band at 180 kDa, T cells four bands at 180, 190, 200 and 220 kDa and B cells one broad band at about 240 kDa. T helper and T cytotoxic cell subsets show the same four bands with some differences in the proportion of each. Four mouse monoclonal antibodies (MRC OX-1, 28, 29 and 30) reacted with all molecular forms of L-CA and fell into two sets that were noncompetitive in binding to L-CA (MRC OX-1, 28, 29 vs. OX-30). The antigenic determinants seen by all these antibodies were lost when L-CA was reduced and alkylated. Three antibodies (MRC OX-22, 31 and 32) reacted selectively with B cells, T cytotoxic cells and about 2/3 of T helper cells. OX-22 and OX-31 competed for binding but were noncompetitive with OX-32. All these antibodies bound to a subfraction of the 190, 200 and 220-kDa forms of T cell L-CA but not at all to the 180-kDa form of T cells or thymocytes. One antibody bound to B cells only (MRC OX-33) and precipitated a subfraction of B cell L-CA. With all the antibodies that did not label thymocytes the antigenic determinants survived reduction and alkylation. Subsequent proteolysis with trypsin then destroyed all determinants except the one reacting with MRC OX-22 antibody. In this case tryptic peptides retained full antigenic activity which was, however, destroyed by further proteolysis with pronase.

Structure of primary anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibodies in normal and idiotypically suppressed C57BL/6 mice.

Abstract: Eight monoclonal antibodies from the primary response of C57BL/6 mice against the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP) were isolated. The antibodies carry lambda 1 light chains and have similar affinities for the immunizing hapten. Sequence analysis at the level of mRNA reveals that all antibodies express the VH gene 186.2 and all but one the DFl 16.1 gene segment. The J segment of the heavy chain is JH2 in six cases and JH4 in two. Somatic point mutations are scarcely detectable in the antibodies, but there is extensive sequence variability at the boundaries of the D gene segment, mainly at its 5' end. However, seven of eight antibodies express tyrosine in position 99 of the heavy chain, encoded either by the 5' codon of DFl 16.1 or by presumed N sequences. In the former case, the tyrosine is the first of a stretch of three (positions 99-101). In the latter, a similar stretch (positions 99, 101, 102) is interrupted by aspartic acid, asparagine or cysteine in position 100. These variations profoundly affect idiotypic specificity. Six of the eight monoclonal antibodies came from mice neonatally suppressed by an anti-idiotope antibody whose target idiotope is regularly expressed in primary anti-NP responses and depends upon a non-germ-line-encoded aspartic acid in position 100 of the heavy chain. The sequence data show that the mice circumvent suppression by expressing antibodies which lack this aspartic acid but are otherwise structurally very similar to anti-NP antibodies from normal animals. Since suppression in the animals is partly controlled by regulatory T cells, we conclude that these T cells are highly restricted in their specificity in that they preferentially see a determinant which also depends upon the aspartic acid in position 100. The data suggest that the VH to D boundary serves as a target of idiotypic selection.

Biochemical and functional characteristics of the human leukocyte membrane antigen family LFA-1, Mo-1 and p150,95.

Abstract: The human leukocyte function-associated (LFA-1) antigen, the monocyte differentiation antigen Mo-1 which is characterized as the C3bi receptor and the glycoprotein p150,95 are characterized biochemically. Immunoprecipitations carried out with 6 different monoclonal antibodies (mAb) against LFA-1 indicated that four mAb (SPV-L1, SPV-L5, SPV-L7 and SPV-L11) were directed against the alpha chain, whereas mAb CLB54 and MHM-23 were found to react with the common beta chain of LFA-1, Mo-1 and p150,95. LFA-1 and Mo-1 expressed on KG-1 cells or lymphocytes, monocytes and granulocytes from one donor were homogeneous. Interestingly the alpha chain of p150,95 showed heterogeneity. The molecular weight of the alpha chain expressed on monocytes was consistently higher than that of the alpha chain on granulocytes. The beta subunits of LFA-1 and Mo-1 (as detected by mAb Bear-1) are not only similar in molecular weight and isoelectric focusing patterns, but it is demonstrated here that they are also identically glycosylated and have similar protein backbones as judged by tryptic peptide mapping. In spite of their structural similarities. LFA-1 and Mo-1 differ completely in some of their biological functions. Anti-LFA-1 mAb strongly inhibited monocyte-dependent T cell proliferation induced by tetanus toxoid or Helix pomatia hemocyanin and pokeweed mitogen-driven specific antibody production in vitro, whereas the anti-Mo-1 antibody Bear-1 was ineffective. These results suggest that the differences in these biological functions of LFA-1 and Mo-1 may be related to their different alpha subunits, which may recognize specific counter structures.

Antigen-specific cytotoxic T cell and antigen-specific proliferating T cell clones can be induced to cytolytic activity by monoclonal antibodies against T3.

Abstract: T3 is a human differentiation antigen expressed exclusively on mature T cells. In this study it is shown that anti-T3 monoclonal antibodies, in addition to their capacity to induce T cells to proliferate, are able to induce antigen-specific cytotoxic T lymphocyte clones to mediate antigen nonspecific cytotoxic activity. It is furthermore shown that anti-T3 reagents are able to trigger lytic activity in T cell clones characterized as noncytotoxic antigen-specific proliferating T cells. The data presented indicate that perturbation of T3 can trigger the lytic machinery in cytolytic as well as noncytolytic T cell clones.

p150/95, Third member of the LFA-1/CR3 polypeptide family identified by anti-Leu M5 monoclonal antibody.

Abstract: Monoclonal antibody (mAb) anti-Leu M5 reacts with a two-chain molecule composed of a 150-kDa alpha subunit noncovalently associated with a 95-kDa beta subunit and probably is specific for an epitope on the 150-kDa alpha chain. This p150/95 antigen is the third member of a family of polypeptides sharing a common 95-kDa beta chain, which includes the lymphocyte function-associated antigen LFA-1 (p177/95) and complement receptor CR3 (Mo1/MAC-1/OKM1; p165/95) antigens. Sequential immunoprecipitation with anti-p95 beta chain mAb specifically removed the antigens detected by anti-LFA-1, anti-CR3 and anti-Leu M5 mAb. Certain patients with recurrent bacterial infections are genetically deficient in expression of the LFA-1 and Mo1 antigens, and have impaired granulocyte function. Granulocytes from a patient with this disease also failed to react with anti-Leu M5. Stimulation of normal granulocytes with f-Met-Leu-Phe, C5a-desArg, or calcium ionophore resulted in increased expression of Mo1 and Leu M5 antigens on the cell surface, but did not significantly increase expression of LFA-1 antigen. In functional assays, anti-Leu M5 did not inhibit T cell-mediated or natural killer cell-mediated cytotoxicity. In addition, anti-Leu M5 neither inhibited the binding of complement-coated particles to CR1 or CR3 nor did it affect the binding of EC3dg to neutrophils (CR4). These studies clearly indicate that the p150/95 antigen recognized by the anti-Leu M5 antibody is a structurally distinct member of the LFA-1/CR3 family.

VLA‐1: a T cell surface antigen which defines a novel late stage of human T cell activation

Abstract: The VLA-1 protein complex defines a previously undescribed very late stage of activated T cell differentiation, following either alloantigen or mitogen activation. This protein appears after 2-3 weeks of activation, considerably later than the early T cell activation antigens such as the interleukin 2 (IL 2) receptor, transferrin receptor, 4F2 antigen, T10 and HLA-DR, and has therefore been termed very late antigen-1 (VLA-1). Unlike the IL 2 receptor, VLA-1 expression does not require restimulation with antigen, and in fact, VLA-1 expression was high on T cells that had lost their IL 2 receptors. Expression of VLA-1 was found on all or nearly all long-term-activated T cells including T4+ and T8+ clones, bulk cultures, long-term T cells from adults and newborns and long-term T cells maintained in pure or crude IL 2 preparations. VLA-1 was also found on HTLV-1 infected T cell populations. Immunoprecipitation experiments confirmed that the VLA-1 protein complex (210 000/130 000 Mr) can be co-expressed with another protein complex called VLA-2 (165 000/130 000 Mr) on the same T cell clones. However, co-expression was not obligatory because in some long-term cultures little or no VLA-2 was present relative to VLA-1. Because VLA-1 defines a novel late stage of T cell activation, being present on all or most all types of long-term activated T cells, and not on any other cell types in peripheral blood, it has unique potential as a marker for activated T cells in vivo and may provide a clue towards elucidating novel long-term T cell functions or growth requirements of this late stage of T cell differentiation.

Splenic dependence of the antibody response to thymus-independent (TI-2) antigens

Abstract: The murine antibody response to T-independent (TI)-2 antigens [2,4-dinitrophenyl-Lys-Ficoll (DNP-FIC) and DNP-hydroxyethyl starch (HES)] was impaired long after splenectomy, while responses to TI-1 [trinitrophenylated lipopolysaccharide (TNP-LPS)] and thymus-dependent [TNP-keyhole limpet hemocyanin (KLH)] antigens were largely unaffected. The antibody response to these TI-2 antigens was exclusively against the conjugated epitopes [DNP-, fluorescein isothiocyanate (FITC)- or tetramethylrhodamine isothiocyanate (TRITC)-Ficoll or -HES]. Fluorescent conjugates of Ficoll and HES localize selectively to the splenic marginal zone macrophages. This localization was not affected by 750 cGy of X-irradiation, but the antibody response to the TI-2 antigens was abrogated for 14 days. Administration of spleen cells restored the antibody response to these TI-2 antigens in otherwise intact irradiated mice but not if they had been splenectomized. Our findings indicate that the antibody response to TI-2 antigens depends upon stimulation of B cells in a splenic environment. This probably involves antigen presentation by marginal zone macrophages.

The mitogenic lectin from Phaseolus vulgaris does not recognize the T3 antigen of human T lymphocytes

Abstract: Human peripheral blood T lymphocytes are stimulated to grow and divide by lectins such as concanavalin A (Con A) and Phaseolus vulgaris phytohemagglutinin (PHA), as well as a few anti-T cell monoclonal antibodies. The latter antibodies recognize the T3 antigen. It has been suggested previously that PHA and Con A mediate T cell growth by interacting with T3. However, as reported in this study, affinity chromatography on immobilized lectins, and immunoprecipitation by lectin plus anti-lectin antibodies showed that T3 binds Con A but not PHA. Fab fragments of a monoclonal antibody against T3 (namely Leu-4) inhibited T lymphocyte proliferation induced by T3 antibodies and Con A, but not by PHA. Nevertheless, co-capping experiments performed with fluorescein-labeled lectins and rhodamine-labeled T3 antibodies showed that T3 co-caps with Con A and PHA receptors, although the co-capping with PHA was incomplete. Since the T cell receptor for antigen (Ti) has been shown to co-cap with T3 on the cell surface, we reasoned that PHA induced capping of the T3 antigen by interacting with Ti. A disulfide-linked heterodimer comprising subunits of about 49 000 and 41 000 mol. wt. that resembled the Ti molecule was detected in PHA-anti-PHA immunoprecipitates of various surface- and biosynthetically-labeled T cells, by two-dimensional (nonreduced vs. reduced) sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis. The results suggest that PHA triggers T lymphocytes by interacting with the carbohydrate moieties of Ti and imply that T lymphocytes can be stimulated by mitogens via at least two different cell surface molecules (Ti and T3).

Enhancement of human B cell proliferation by an antibody to the C3d receptor, the gp 140 molecule

Abstract: The C3d receptor is a specific marker of B lymphocytes. Recently we have shown that C3d receptor activity is carried by a gp 140 membrane antigen. A polyclonal antibody has been prepared by immunizing a rabbit with highly purified gp 140 molecule isolated from membranes of the human B lymphoblastoid cell line Raji and its high specificity was previously demonstrated. We tested the effect of this antibody to the C3d receptor on the B cell proliferative response. Purified B cells from human blood were induced to proliferate by a B cell growth factor (BCGF)-containing partially purified supernatant from activated T cells. The anti-C3d receptor F(ab')2 enhanced the BCGF-dependent B cell proliferation. This effect was dose dependent, was observed in the presence of different concentrations of BCGF and did not correspond to a change in the time course of the response. The anti-C3d receptor F(ab')2 had no mitogenic effect in the absence of T cell supernatant. In contrast the undigested anti-C3d receptor IgG suppressed the BCGF-dependent B cell proliferation. These results emphasize the potentialities of anti-gp 140 F(ab')2 to explore the involvement of the C3d receptor in the regulation of B cell response to T cell products.

Mechanisms by which accessory cells contribute in growth of resting T lymphocytes initiated by OKT3 antibody

Abstract: This study was undertaken to determine how accessory cells (AC) participate in growth of normal resting T cells initiated by anti-T3 monoclonal antibodies. Highly purified peripheral blood resting T cells were obtained by sequentially using three procedures (adherence to plastic surface, adherence to nylon wool columns and treatment with four monoclonal antibodies against antigens on AC and activated T cells plus complement). The assays for T cell growth were carried out at low cell density (104 cells/well) and with T cell populations where we could not detect cells bearing OKM1 and Ia antigens. Soluble OKT3 antibody, concanavalin A, recombinant interleukin 2 (IL2) or purified interleukin 1 (IL1) alone did not induce proliferation of purified resting T cells. Recombinant IL2 together with soluble OKT3 antibody stimulated significant growth whereas purified IL1 and two distinct preparations derived from AC containing IL1 activity did not. Nevertheless, purified IL1 amplified the proliferation of T cells induced by soluble OKT3 antibody in the presence of a small number of irradiated AC (3%). Phorbol myristate acetate (PMA) together with soluble OKT3 antibody activated purified resting T cells to proliferate, but PMA alone had little growth-promoting activity only. Soluble OKT3 antibody did not by itself induce a detectable number of resting T cells to express receptors for IL 2 as determined by direct immunofluorescence staining and FACS analysis with monoclonal anti-IL2 receptor antibody. Cloned IL2 or purified IL1 alone did not induce resting normal T cells to express receptors for IL2 either. In contrast, T cells exposed to both soluble OKT3 antibody and IL2 exhibited IL2 receptors. PMA alone stimulated some resting T cells to express IL2 receptors and this response was significantly increased when the drug was used together with soluble OKT3 antibody. Studies were performed with unfractionated mononuclear cells from a donor whose cells respond to OKT3 (IgG2) but not to Leu 4 (IgG1) anti-T3 antibodies. Recombinant IL2 but not purified IL 1 corrected the defective response to Leu 4 antibody. Finally, OKT3 antibody linked to beads, but not in soluble form, and purified IL1 replaced AC in growth of purified resting T cells. Based on these data I conclude the following: (a) AC participate in growth of resting normal T cells initiated by anti-T3 antibodies through their Fc receptors in two ways, namely, by providing a matrix to favour cross-linking of the T3 complex and simultaneously by secreting IL 1. (b) The contribution of IL 1 is that of amplifying rather than inducing the production of IL 2. This cytokine has no detectable effect on the expression of IL2 receptors by normal resting T cells, but it may improve the action of IL2, and (c) PMA bypasses the mechanisms by which AC contribute to T cell growth in this system. The efficacy of this drug is likely to rest on its capacity to both induce resting T cells to express IL2 receptors and increase the transcription of the gene encoding IL2. Both activities are most likely due to its property of binding to and activating protein kinase C. Finally, I propose that AC and PMA may ultimately act via the same mechanisms. Thus, both cross- linking of the T3 complex and IL 1 (the functions of AC) would stimulate the synthesis of diacylglycerol and a raise in T cell intracellular Ca2+. Diacylglycerol and Ca2+acting in concert efficiently activate protein kinase C and thereby, like PMA, trigger a sequence of events culminating in DNA synthesis.

Recombinant interferon-γ can induce the expression of HLA-DR and-DC on DR-negative melanoma cells and enhance the expression of HLA-ABC and tumor-associated antigens

Abstract: Recombinant interferon-gamma (IFN-gamma) induced the expression of HLA-DR when added to the culture medium of HLA-DR- melanoma cell lines. In addition, IFN-gamma induced the expression of another class II antigen, HLA-DC, on a HLA-DR+ and -DC-melanoma cell line and to a lower level on a -DR- and -DC-melanoma line. IFN-gamma also enhanced the expression of HLA-ABC and beta 2-microglobulin, as well as HLA-DR on DR+ melanoma cells. In contrast, IFN-alpha gave no induction of expression of HLA-DR and DC on two DR- melanoma lines, while it did enhance the expression of HLA-ABC and of beta 2-microglobulin. The expression of 3 out of 6 melanoma-associated differentiation antigens was enhanced by IFN-gamma treatment. The modulation of antigens by IFN-gamma was both dose and time dependent. A minimum incubation time of 48 h was necessary for the appearance of HLA-DR on the two HLA-DR- melanoma lines, whereas HLA-ABC and beta 2-microglobulin were already increased after 24 h. A dose of 20 U/ml IFN-gamma started to induce the expression of HLA-DR and DC on melanoma cells GLL-19 and Me-43 and a plateau of maximum antigen expression was reached with 100 U/ml. Analyses of IFN-gamma-treated cells by flow microfluorometry showed a homogeneous distribution of increased staining intensity rather than the appearance of two cell populations. Immunoprecipitation experiments using detergent-solubilized 125I-labeled membrane proteins of IFN-gamma-treated melanoma cells and a monoclonal anti-HLA-DR antibody confirmed the presence of HLA-DR antigens. When IFN-gamma-treated cells were cultured without IFN the induced or enhanced expression of HLA antigens was reversible. Eight days after removal of IFN, the HLA-DR level was reduced by more than 90% and the level of HLA-ABC and beta 2-microglobulin by more than 50%. The demonstration of the ability of HLA-DR- melanoma cells to express HLA-DR after IFN-gamma treatment was extended to cells from other types of tumor such as gliomas, colon carcinomas and one cervical carcinoma cell line.

Enhancement of IgG anti-carrier responses by IgG2 anti-hapten antibodies in mice

Abstract: Isologous IgG2a and IgG2b anti-hapten antibodies injected along with trinitrophenylated or fluoresceinated keyhole limpet hemocyanin (KLH) were found to considerably stimulate primary IgG responses to the carrier protein in mice. No such stimulation was observed with IgG1, IgM and IgA anti-hapten antibodies. Depending on the antigen antibody combination, the amplification obtained after a single injection of IgG2-complexed haptenated KLH ranged from 20- to 1000-fold. By comparison, secondary responses were only marginally enhanced (approximately 3-fold) when IgG2-complexed rather than free antigen was used to boost irradiated recipients previously reconstituted with primed spleen cells. The IgG2-mediated enhancement was effective over a wide range of antigen/antibody ratios, did not require the use of high affinity antibodies and occurred whatever the route of injection. The stimulation was specific for the complexed antigen and developed to the same extent whether complexes were formed in vitro or in vivo. A comparable stimulation of the anti-carrier response was obtained with several other haptenated proteins but with formaldehyde-treated diphtheria and tetanus toxoids inhibition rather than stimulation was observed.

VH-gene expression in murine lipopolysaccharide blasts distributes over the nine known VH-gene groups and may be random.

Abstract: VH-gene expression in hybridomas derived from lipopolysaccharide-activated B cells was analyzed. Isolated cytoplasmic RNA was hybridized to probes representing the 9 known VH-gene groups or subjected to mRNA sequencing. In the collection of hybridomas VH genes of all 9 groups are expressed at frequencies which in most correlate reasonably well with the relative complexities of the groups. In 51 out of 54 RNA samples VH-gene transcripts could be identified and corresponded to one of the known VH-gene groups. It therefore appears that the latter essentially represent the VH-gene cluster of the mouse.

Clones of B lymphocytes in individual follicles of the bursa of Fabricius.

Abstract: To discover whether individual bursal follicles can contain clones of B lymphocytes, we estimated the numbers of lymphoid cell precursors populating single follicles in two types of chicken chimera. The first type was produced by establishing parabiotic connections between blood vessels of embryo chorioallantoic membranes. Under these conditions, and most likely during normal development, most follicles are populated by more than one, but less than ten, precursor cells. However, in a second type of chimera, a cyclophosphamide-treated chick reconstituted with normal bursal cells, most follicles in the reconstituted bursa are clonal (their lymphocytes are derived from a single precursor cell). Individual follicles can readily be isolated from bursae of reconstituted birds and should be useful in studies of B cell development.

Regulation of Epstein-Barr virus infection by recombinant interferons: selected sensitivity to interferon-γ

Abstract: Interferons (IFN) are antiviral proteins that may be important in mediating cellular defenses against Epstein-Barr virus (EBV) infection. However, the means by which IFN-alpha, -beta and -gamma modify EBV infectivity are not clear. We have evaluated the effects of purified recombinant preparations of all three classes of IFN on EBV-induced B lymphocyte proliferation and Ig secretion. When added early after EBV infection, all three recombinant IFN reduced B cell outgrowth and Ig secretion. IFN-gamma exerted a 7-10-fold more potent antiviral effect than IFN-alpha or -beta. All three types of IFN act directly on B cells. Monocytes and natural killer cells are not necessary for the anti-EBV activity. Of the three recombinant IFN, only IFN-gamma reduced EBV-induced proliferation and Ig secretion when added 3-4 days after virus infection; IFN-alpha/beta were only effective up to 24 h. B lymphoblastoid lines already transformed by EBV are insensitive to the anti-proliferative actions of all three types of IFN. On the basis of these findings, we propose three phases of regulation during EBV infection. In the early phase, EBV-infected cells can be regulated by all IFN. Subsequently, there is an intermediate period where only IFN-gamma is capable of directly affecting EBV-induced B cell responses. In the third phase, B lymphocytes become insensitive to direct actions of all IFN and are now subject to regulation only by cytotoxic cells.

Stromal cell types in the developing thymus of the normal and nude mouse embryo

Abstract: The anatomical distribution of various nonlymphoid cell types in the embryonic mouse thymus in vivo and in vitro, as well as in the thymic rudiment of the nude mouse embryo, has been studied. For this purpose a panel of monoclonal antibodies, ER-TR3, 4, 5, 6 and 7, directed to various types of stromal cells of the mouse thymus, was used in combination with immunoperoxidase labeling on frozen sections. It was shown that as early as day 13 in thymic ontogeny distinction of TR4+ cortical epithelial cells and TR5+ medullary epithelial cells is possible. Thus, as far as stromal components are concerned, the thymus at day 13 in ontogeny is already subdivided into cortex and medulla. At day 13, Ia (TR3) was expressed in a focal pattern in the medulla subsequently appearing throughout both cortex and medulla by day 16. The thymic rudiment of the nude mouse embryo differs markedly from the normal embryonic thymus in its lack of demonstrable Ia antigen. Furthermore, TR4 and TR5 were only expressed on occasional epithelial cells lining the cysts of the nude thymus in a mutually exclusive fashion. The majority of stromal cells of the nude thymus, however, is negative for all ER-TR antibodies tested. In addition, we have shown that in organ cultures the organization of the stroma of thymic lobes remains intact, at least for a period of 11 days. Embryonic thymi cultured in the presence of deoxyguanosine, which causes depletion of lymphoid cells, also contain cortical and medullary areas as identified by the presence of TR3,4+ and TR5+ stromal cells. This indicates that the lack of organization in the nude thymus is not simply due to the absence of lymphoid cells.

Constraints in T-B cooperation related to epitope topology on E. coli β-galactosidase. I. The fine specificity of T cells dictates the fine specificity of antibodies directed to conformation- dependent determinants

Abstract: Experiments to test the relationship between the epitopes on a protein antigen recognized by T and B cells in their collaboration to produce antibody cannot rely solely on hapten-carrier models. In the present work we used E. coli beta-galactosidase, a molecule whose tertiary and quaternary epitopes have been well characterized, as the model antigen. T helper cells were raised by stimulating mice with the intact or the denatured molecule or with any of several beta-galactosidase cyanogen bromide peptides. In a series of in vitro helper T cell assays we confronted the various T populations with B cells preimmunized with the native antigen, and we tested their capacity to help production of (a) binding antibodies and (b) antibodies directed to single conformational epitopes, characterized by their capacity to protect the enzyme from heat denaturation or to activate defective beta-galactosidase. According to our results, (a) equivalent T cell help can be provided by T helper cells primed with native or denatured antigen, even for the production of "conformational" antibodies; (b) one of the peptides (CB-18) is most efficient in raising help for binding antibodies; and (c) two peptides (CB-20 and CB-21) rank highest in priming T helper cells for the eventual production of protecting and activating antibodies, respectively. Thus, not every beta-galactosidase-specific T helper cell is useful in providing help to B cells specific for any particular epitope on the molecule, but rather preferential pairings exist, possibly governed by a proximity rule.

Production and characterization of monoclonal anti-thy-1 antibodies that stimulate lymphokine production by cytolytic T cell clones

Abstract: In an effort to derive monoclonal antibodies (mAb) which can induce production of macrophage-activating factor (MAF) by cloned murine cytolytic T lymphocyte (CTL) lines, we have fused spleen cells from a rat immunized with a CTL clone with the nonsecreting mouse myeloma X63-Ag8.653. Three mAb (designated I-22, III-5 and V-8) were found to stimulate MAF production by the immunizing CTL clone and (with a single exception) two other unrelated CTL clones. However, none of these mAb inhibited the cytolytic activity of the clones. Immunoprecipitation studies indicated that the three mAb reacted primarily with a 25-30-kDa protein which could not be distinguished from that precipitated by either a reference anti-Thy-1.2 mAb or a polyclonal rabbit anti-Thy-1 antiserum. Moreover, competition binding experiments demonstrated that the three mAb competed with each other and with the reference anti-Thy-1.2 mAb. Flow cytofluorometric analysis of the strain distribution of the molecules defined by the mAb revealed that two of the antibodies (I-22 and III-5) were directed against nonpolymorphic determinants of Thy-1, whereas V-8 mAb reacted only with Thy-1.2+ lymphocytes. One of the mAb (III-5) was also able to stimulate proliferation and interleukin 2 secretion by normal splenic T cells. Since mAb directed against a number of other surface structures on CTL clones did not stimulate MAF production, it thus appears that Thy-1 (or molecules associated with Thy-1) may play a functional role in T lymphocyte triggering.

Murine intestinal intraepithelial lymphocytes I. Relationship of a novel Thy-1-,Lyt-1-,Lyt-2+, granulated subpopulation to natural killer cells and mast cells.

Abstract: Highly purified populations of intraepithelial lymphocytes (IEL) were obtained from the murine small intestine. We found that 84% of IEL expressed Lyt-2 and that 45-55% possessed the unique phenotype, Thy-1-,Lyt-1-,Lyt-2+. Sixty percent of IEL had granules in their cytoplasm and thus resembled the large granulated lymphocytes associated with natural killer (NK) activity. However, less than 15% of IEL had NK activity in a 6-h assay. This activity resided in a subpopulation of Lyt-2- lymphocytes, leaving a large population of Thy-1-,Lyt-1-,Lyt-2+ granulated cells (45-55% of IEL) with unknown function. Sensitive radioenzymic assays for histamine showed that IEL from healthy CBA mice do not contain this amine. IgE-binding assays revealed that IEL, unlike mast cells, do not carry high-affinity receptors for IgE. Thus, only a small percentage of granulated IEL (less than 15%) possess NK activity, whereas 45-55% of IEL have granules, lack typical characteristics of mast cells, express the unique antigenic phenotype, Thy-1-,Lyt-1-,Lyt-2+ and have unknown function.

Immune responses of thymus/lymphocyte embryonic chimeras: studies on tolerance and major histocompatibility complex restriction in Xenopus.

Abstract: A new model has been developed to address the question of whether T cells that traverse an allogeneic thymus during early and late life become restricted to interact, in vivo, with other leukocytes and target cells that display the major histocompatibility complex (MHC) antigens of the thymus haplotype. Chimeras were made microsur- gically with pairs of 24-h-old Xenopus embryos such that the anterior region of an embryonic chimera contained the thymus anlagen and was of one MHC genotype, whereas the posterior region contained the anlagen of all hemopoietic cells and was of another genotype. Assays to determine the MHC haplotype restriction specificity of T cells in chimeras that had been reared through metamorphosis involved: specific antibody responses (IgM and IgG) to dinitrophenylated keyhole limpet hemocyanin; rejection of minor H locus disparate skin grafts that expressed the MHC antigens of either the thymus donor or the lymphocyte donor; and mixed leukocyte culture. MHC-mismatched chimeras displayed split tolerance since they accepted skin grafts of the thymus haplotype but had lymphocytes that proliferated in response to MHC antigens of the thymus donor strain as well as to MHC antigens of third-party donors. IgM responses of MHC-matched and MHC-mismatched chimeras and of nonchimeric controls did not differ. However, the IgG responses of MHC-mismatched thymus/ lymphocyte chimeras peaked later than those of MHC-matched chimeras and normal controls. Data from skin grafting protocols were consistent with the proposition that there may be in vivo selection of T cells reactive to minor H antigens presented in association with the MHC antigens of the thymus rather than the MHC antigens of the lymphocytes themselves. These data suggest that although it is not absolute, there is thymic selection of the T cell repertoire in Xenopus.

Phytohemagglutinin binds to the 20-kDa molecule of the T3 complex.

Abstract: Current findings have suggested that the T3 molecular complex is an essential antigenic signal transducer during T cell activation. Lectins, such as phytohemagglutinin (PHA), activate T cells nonspecifically. Concesivably, lectins may mediate their stimulatory action by affecting the T3 complex. In the present investigation we have studied the involvement of the T3 molecular complex in the PHA-mediated activation of T cells. We selectively modulated the surface expression of T3 molecules by anti-T3 antibody and subsequently tested the ability of the modulated cells to respond to PHA. Reduction of T3 expression by 70% resulted in 80% inhibition of the PHA response. This effect was specific for T3 since modulation of other T cell surface molecules (T4, T8) did not affect the PHA-induced mitogenesis. To determine if PHA could interact directly with the T3 complex, immunoblotting (Western blot) analyses of anti-T3 immunoprecipitates were performed. A 20-kDa member of the T3 complex reacted not only with the anti-T3 antibody, but also with PHA itself. These results provide the first evidence for direct binding of PHA to one of the molecules of the T3 complex. The combined data suggest that a major pathway of PHA-induced T cell activation involves the T3 complex. Possible activation mechanisms are discussed.

A high incidence of cross‐reactive idiotypes among murine natural autoantibodies

Abstract: Anti-idiotypic (anti-Id) antibodies were produced in rabbits against two natural monoclonal IgM autoantibodies (NmAb), D23 and E7, which exhibited a broad reactivity and were derived from fusions of spleen cells from adult unprimed B ALB/c mice and nonsecreting myeloma cell lines. They were used to test the reactivities of 12 NmAb obtained from adult and newborn unprimed mice. Both anti-Id recognized cross-reactive idiotopes frequently shared by NmAb; 8 out of the 12 NmAb reacted with anti-IdD23, while 5 of them also reacted with anti-IdE7. All of the Id-bearing antibodies possessed widespread reactivity with structurally dissimilar self and non- self antigens. In most cases, their cross-reactive Id determinants seemed to be located outside of their antigen-binding sites. Furthermore, the presence in normal mouse sera of significant levels of D23 and E7 idiotopes correlated with the presence of natural antibody activity and was mainly associated with IgM and IgG2b fractions. Finally, D23 idiotope(s) were also found on induced murine anti-myosin antibodies. The high incidence of cross-reactive idiotopes found among NmAb produced by clones derived from different mice and their presence in normal BALB/c mouse serum Ig fractions suggest that families of germ-line genes may encode for at least a part of them.

Cytotoxic T cell clone-specific monoclonal antibodies used to select clonotypic antigen-specific cytotoxic T cells.

Abstract: Two rat monoclonal antibodies (mAb) have been produced which recognize a clone-specific determinant on the alloreactive cytotoxic T lymphocyte (CTL) clone 3 F9. CTL clone 3F9 of BALB/c origin is specific for H-2Db and can be grown by weekly restimulation with irradiated stimulator spleen cells expressing H-2Db in the presence of interleukin 2. Two mAb against T cell clone 3F9, 44-22-1(IgG2a) and 46-6 B5(IgM), have been proven to be clone specific: they inhibit cytotoxic activity of 3F9 only and bind specifically to 3 F9 when compared in a panel of different CTL clones, or cells from different mixed lymphocyte cultures (MLC), BALB/c thymus and spleen cells. The mAb 44-22-1 has been used to sort cells from a primary MLC BALB/c anti-H-2Db by fluorescence-activated cell sorter (FACS) to select CTL expressing 3 F9 clonotype-specific determinants. The lymphocytes reactive with 44-22-1 represent a minor subpopulation of the CTL of the primary MLC. The specific alloreactive cytotoxicity of unsorted lymphocytes of the bulk primary MLC could not be inhibited by the mAb 44-22-1 and 46-6 B5 whereas the sorted 3 F9 clonotype-positive cultures could be inhibited very effectively. All the CTL clones derived from the FACS-sorted clonotype-positive culture show all the same properties and are identical with clone 3 F9 with respect to antigen-specific cytotoxicity, inhibition of cytotoxicity by the mAb and surface markers.

Very large and isotypically atypical polyclonal plaque-forming cell responses in mice infected with Trypanosoma cruzi.

Abstract: Normal C3H/HeJ mice, acutely infected with T. cruzi, develop large numbers of splenic Ig-secreting plaque-forming cells (PFC). IgG2a, IgG2b and IgG1 PFC account for over 90% of all PFC, while the numbers of IgG3- and IgA-secreting PFC are lower than in normal animals. These effects appear to be due to both T helper-dependent regulation and to a mitogenic activity associated with the parasites themselves.

Hepatobiliary transport of plasma IgA in the mouse: contribution to clearance of intravascular IgA

Abstract: Labeled monomeric and polymeric (pIgA) mouse monoclonal IgA were injected intravenously into mice which were either sequentially bled for plasma turnover studies of IgA, or cannulated at their common bile duct, with excluded gallbladder, for quantitation of plasma-to-bile transport of pIgA. Our data show that mice do display a relatively high rate of biliary transport of plasma pIgA (22-28% of the injected 125I-labeled pIgA over 3 h), which accounts for approximately 90% of the total amount of pIgA (8.8 mg/kg/day) daily delivered by hepatic bile into the duodenal fluid of this species. However, in mice the absolute biliary output of pIgA does not exceed that of IgG (9.5 mg/kg/day) and the kinetics of the hepatobiliary transport of plasma pIgA appear to be slower than in the rat. Furthermore, as plasma survival studies of 125I-labeled pIgA yielded a plasma turnover of pIgA averaging 20.6 mg/kg/day, it can be approximated that the hepatobiliary pathway contributes for only 38% to the elimination of intravascular pIgA from mouse plasma, a figure to be compared to 89.8% in the rat and approximately 8.9% in man. We conclude that internal catabolism plays a dominant role in the clearance of intravascular pIgA in the mouse which appears as a model intermediate between rats and humans. Supporting this conclusion, serum pIgA two days after common bile duct ligation in 6 mice was increased by 2.5-fold vs. greater than 14-fold in ligated rats and 1.1-fold in humans with complete biliary obstruction.

Monoclonal antibody 9.3 and anti-CD11 antibodies define reciprocal subsets of lymphocytes.

Abstract: We have previously described antibody 9.3 which recognizes a 44-kDa polypeptide designated Tp44 expressed on 70-80% of peripheral blood T cells, including nearly all CD4+ cells and some CD8+ cells. Whereas the 9.3+ population contains helper cells, cytotoxic T cell precursors and cytotoxic T cell effectors, the 9.3− population has been reported in several models to contain precursors for suppressor cells. In this report, we demonstrate that 9.3− lymphocytes express CD11, an antigen which is also present on monocytes and granulocytes. Among lymphoid cells, Tp44 and CD 11 represent markers that identify reciprocal, nonoverlapping subsets, each of which contains both CD8+ cells and CD4+ cells. With Tp44 and CD11, and CD4 and CDS, the development and function of T cells can thus be examined within the framework of two distinct systems of reciprocally expressed antigens.

Investigation of Early T-Cell Activation - Analysis of the Effect of Specific Antigen, Interleukin-2 and Monoclonal-Antibodies on Intracellular Free Calcium-Concentration

Abstract: The three mitogenic anti-T3 antibodies, UCHT1, anti-Leu-4 and WT-32, all produce a rapid increase in T cell intracellular Ca2+ ( [Ca2+]i) in all individuals, as measured by quin 2 tetra-acetoxymethyl ester fluorescence. This indicates that the lack of responsiveness of approximately 30% of individuals to UCHT1 in proliferation assays is not due to failure of the antibody to elicit Ca2+ mobilization and that a rise in [Ca2+]i is per se not adequate to induce cell division. Another mitogenic antibody, WT-31, which is directed against the constant portion of the T cell receptor, did not, however, produce a rapid calcium rise in peripheral blood T cells. The clone HA1.7 gave a similar Ca2+ response to UCHT1. WT-31 did not induce a rise in [Ca2+]i, nor did the specific antigen to which the clone responded. Accessory cells may be required to induce Ca2+ mobilization with these ligands. There was no response to IL2, or an antibody (anti-Tac) to the IL2 receptor. In contrast to peripheral blood T cells treatment of HA1.7 with WT-31 led to an enhancement of the calcium response to subsequent UCHT1 addition. Furthermore, cross-linking of WT-31 on the surface of HA1.7 cells did produce a small rise in [Ca2+]i. The IL2-independent malignant T cell line, HUT78, exhibited a calcium response to both UCHT1 and WT-32. Both of these responses occurred without cross-linking. The T cell receptor is closely associated with cell-surface proteins, including the T3 antigen and these studies confirm the importance of the T3 antigen in T cell activation. They also suggest that the relationship between the T cell receptor and the T3 antigen may vary in T cells in different proliferative states.

Partial biochemical characterization of IgG1-inducing factor.

Abstract: IgG1, induction factor elevates the IgGl and suppresses the IgG3 and IgG2b responses in lipopolysaccharide-stimulated murine spleen cell cultures. By the use of a quantitative assay, it was found that the three activities, induction of IgG1 and reduction of IgG3 and IgG2b synthesis, were found in the same fractions after different chromato-graphic procedures, suggesting that the same molecule was responsible for the effects. The factor was precipitated by 60-90% saturation of ammonium sulfate and was sensitive to proteolytic cleavage and to treatment with a buffer of pH 10. It had an apparent molecular mass of 20 kDa as judged by gel filtration chromatography and was separated into two peaks after isoelectric focusing, PI 7.4-7.2 and 6.4-6.2, respectively. Finally it was weakly hydrophobic and negatively charged at pH 7.55. These characteristics indicate that the factor is different from many previously characterized lymphokines and similar or identical to the B cell stimulating factor-1 (BSF-pl). The relevance of these findings to the mechanism of the immunoglobulin class switch is discussed.

Various expressions of a unique anti‐human thyroglobulin antibody repertoire in normal state and autoimmune disease

Abstract: Polyclonal anti-human thyroglobulin (hTgb) antibodies (Ab) were purified from sera of rabbits immunized with human thyroglobulin, normal humans and patients suffering from Graves' disease, Hashimoto's thyroiditis and thyroid carcinoma. The avidity of the various Ab preparations for hTgb ranged from 0.3 X 10(10) -2.2 X 10(10) M-1. By using well characterized mouse monoclonal antibodies (mAb) directed against hTgb, it was shown that the fine specificities of induced anti-hTgb Ab in rabbits, natural Ab in normal subjects and autoantibodies in diseased patients were similar; however, they differed from that of rabbit anti-bovine and anti-porcine thyroglobulin Ab which were able to inhibit the hTgb binding of only a few of the mAb. Anti-hTgb in rabbits and in patients with thyroid carcinoma varied from those in normal subjects only by uniformly elevated serum titers. In contrast, patients with Graves' disease and Hashimoto's thyroiditis showed an increased concentration essentially restricted to Ab reacting with few of the antigenic determinants recognized by the mAb. Our data suggest that the repertoire of anti-hTgb Ab is similar in mouse, rabbit and human. Furthermore, the finding of identical fine specificities for anti-hTgb Ab in normal and pathological conditions implies that autoantibodies are produced in normal subjects and held to a low level by regulatory processes which fail with respect to selected epitopes in autoimmune diseases.