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Antigen valence determines the binding of nominal antigen to cytolytic T cell clones.

We have shown that cytotoxic T cell clones specific for the nominal antigen FL will bind high molecular weight (600,000 to 2,000,000) polyacrylamide and Ficoll polymers conjugated with 200-600 FL groups per molecule. Low molecular weight polymers (40,000) with the same epitope density did not give stable binding. A high molecular weight polymer with a lower epitope density also failed to bind. Taken together, these results suggest that a substantial degree of multivalence is a necessary factor in the stable binding of nominal antigen to T cell clones.

Acrylic Resins↗

Human monoclonal IgG isotypes differ in complement activating function at the level of C4 as well as C1q.

Humanized antibodies are likely to have a major role in therapy and it is important to define their interaction with physiological effectors. By comparing a matched series of chimeric human mAbs we found that igG1 was most efficient in complement lysis, although IgG3 bound more C1q. To resolve this paradox we compared the ability of human IgG1, IgG2, IgG3, IgG4, and IgE and rat IgG2b to cause C1q binding, C1 binding and activation, C4 activation, C4b binding, and C3b binding. Rat IgG2b was included because this isotype has already successfully been used for therapy. Human IgG1 was less efficient than IgG3 and fixing C1q and C1 on the cell surface, but the number of C4 molecules bound per C1 was 10-fold greater for IgG1 than for IgG3. This difference, amplified through later stages of the complement cascade, can account for the superiority of IgG1 for cell lysis. The efficiency of IgG1 in fixing C4 was not due to a favored binding site on the antibody molecule, since virtually all of the bound C4b was attached to the cells. Rather, it appeared that the activation of C4 by C1s was greatly favored by IgG1 compared with IgG3. It should be possible to combine the optimal properties of IgG1 and IgG3 antibodies to produce an improved therapeutic reagent.

Antibodies, Monoclonal↗

T cell receptor beta chain lacking the large solvent-exposed Cbeta FG loop supports normal alpha/beta T cell development and function in transgenic mice.

The striking and unique structural feature of the T cell receptor (TCR) beta chain is the bulky solvent-exposed FG loop on the Cbeta domain, the size of almost half an immunoglobulin domain. The location and size of this loop suggested immediately that it could be a crucial structural link between the invariant CD3 subunits and antigen-recognizing alpha/beta chains during TCR signaling. However, functional analysis does not support the above notion, since transgene coding for TCR beta chain lacking the complete FG loop supports normal alpha/beta T cell development and function.

Animals↗

Acceleration of intracellular targeting of antigen by the B-cell antigen receptor: importance depends on the nature of the antigen-antibody interaction.

The B-cell antigen receptor (BCR) internalizes bound antigen such that antigen-derived peptides become associated with emigrating major histocompatibility complex (MHC) class II molecules for presentation to T cells. Experiments with B-cell transfectants reveal that BCR confers a specificity of intracellular targeting since chimeric antigen receptors which internalize antigen by virtue of a heterologous cytoplasmic domain do not necessarily give rise to presentation. In contrast, however, previous studies have shown that antigen binding to irrelevant cell surface molecules (e.g. transferrin receptor, MHC class I) can ultimately lead to presentation. The solution to this paradox appears to be that the intracellular targeting by BCR actually reflects an acceleration of antigen delivery. Depending on the nature of the BCR-antigen interaction, this accelerated targeting can be essential in determining whether or not internalization leads to significant presentation. Physiologically, the accelerated delivery of antigen by BCR could prove of particular importance early in the immune response when antigen-BCR interaction is likely to be poor.

Animals↗

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.

Animals↗

The effect of antibody isotype and antigenic epitope density on the complement-fixing activity of immune complexes: a systematic study using chimaeric anti-NIP antibodies with human Fc regions.

A systematic study has been carried out to investigate the role of immunoglobulin isotype, epitope density, and antigen/antibody ratio on the capacity of immune complexes to activate the classical and alternative pathways of human complement and for the complexes subsequently to bind to erythrocyte C3b-C4b receptors (CRI). For this purpose, a series of chimaeric monoclonal anti-NIP antibodies was used, which all shared the same combining site but had different human constant domains. Antigen epitope density was varied by coupling different numbers of NIP hapten molecules to bovine serum albumin. All three parameters affect complement fixation. In general, complement activation is better in antibody excess and at equivalence than it is in antigen excess, and better at high epitope density than at low epitope density, although the effects are variable for different immunoglobulin isotypes and for the two pathways. It has been confirmed that IgG1 and IgG3 are good activators of the classical pathway and are tolerant to variations in both epitope density and antigen/antibody ratio. IgG4 and IgA do not activate the classical pathway in any circumstances. IgG2 activates the classical pathway only at high epitope density and at equivalence or antibody excess. IgM activates the classical pathway well only at the higher epitope densities and at equivalence or antibody excess but, in addition, shows an interesting and unexpected prozone phenomenon where immune complex in antibody excess inhibits complement activation by the classical pathway. The results of the alternative pathway activation are strikingly different. IgA is by far the best activator of the alternative pathway and is relatively tolerant to epitope density and to antigen/antibody ratio. IgM, IgG1 and IgG3 do not significantly activate the alternative pathway in any circumstances. IgG2 is the best IgG subclass for alternative pathway activation but requires high epitope density and equivalence or antibody excess. Binding to CR1 in general parallels the amount of complement fixed independent to the pathway by which it is fixed. However, IgG1 and IgG3 complexes in antigen excess activate complement well but bind poorly to CR1. Nascently formed complexes seem to bind complement in a way that is similar to that bound by preformed complexes, but are then less able to bind to red cell CR1. These observations help to explain the pathogenesis of complement activation in various autoimmune and immune complex diseases such as systemic lupus erythematosus, autoimmune thyroiditis and others.

Animals↗

The use of mouse/human chimaeric antibodies to investigate the roles of different antibody isotypes, including IgA2, in the killing of Schistosoma mansoni schistosomula by eosinophils.

We report the use of a matched set of mice/human chimaeric antibodies, directed against the 5-iodo-4-hydroxyl-3-nitrophenacetyl (NIP) hapten, to investigate the roles of different human isotypes in antibody-mediated eosinophil-dependent killing of schistosomula. The chimaeric antibodies consist of mouse VH, VL and CL regions with human gamma 1, gamma 2, gamma 3 (2 allotypes), gamma 4, alpha 2, mu or epsilon CH regions and were used in in vitro assays with human eosinophils and NIP-coated S. mansoni schistosomula. Some anti-NIP isotypes mediated high levels of killing, which was specific for NIP-coated larvae, and we suggest that these antibodies will be a valuable tool for studies on the role of antibody isotypes in anti-schistosome immune effector mechanisms. In particular, this method directly demonstrated, for the first time, that IgA is highly effective in mediating the killing of metazoan parasites by human eosinophils.

Animals↗

The role of B-cell memory in secondary IgG and IgM responses.

Mice were primed with the hapten 3-nitro-4-hydroxy-5 iodophenacetic acid (NIP) conjugated to chicken globulin (cg) and were boosted 2, 6, or 12 months later with CG conjugates of the related haptens 3,5-diiodo-4-hydroxyphenacetic acid (DIP) or 3-nitro-4-hydroxphenacetic acid (NP). Accelerated secondary responses were demonstrated both in the 7S and 19S class. Fine-specificities of secondary-response antibodies were studied by the hapten inhibition method of haptenated bacteriophage inactivation. 7S antibodies were found to have the fine-specificity of anti-NIP antibodies regardless of whether DIP or NP was the booster hapten ('original antigenic sin'). 19S antibodies had the fine-specificity of anti-DIP when DIP was the booster hapten. NP as the booster hapten resulted in 19S antibodies whose fine-specificity was intermediate between anti-NIP and anti-NP. A strong B-cell memory could thus be demonstrated in the 7S antibody response and a weak B-cell memory in the 19S antibody response.

Animals↗

IgA in secondary anti-hapten responses in vitro.

Production of IgA anti-hapten antibodies in vitro was studied in suspension and fragment cultures of spleens from mice immunized with NIP-CG (4-hydroxy-5-iodo-3-nitrophenylacetyl coupled to chicken globulin) a few weeks earlier. IgA anti-hapten production tended to be of much shorter duration than IgG anti-hapten production. IgA produced in fragment cultures had sedimentation coefficients of 9S, 11S, and 13S. 9S IgA anti-hapten was more prominent later during the in vitro response than earlier. In suspension cultures IgM anti-hapten production preceded IgA and IgG anti-hapten production. Very little IgM anti-hapten was seen in fragment cultures, although the responses on a cell-to-cell basis tended to be much higher in fragment than in suspension cultures.

Animals↗

T helper lymphocytes recognize the VL domain of the isologous mouse myeloma protein 315.

The localization of a major determinant on an isologous myeloma protein (M315) which stimulates BALB/c helper T cells was investigated. Augmentation of the adoptive secondary antibody response to NIP-M315 and the idiotype of M315 (Id315) was used as an indicator of helper effects. Spleen cells primed with the light chain of M315 (L315) and its V-domain (VL315) were highly efficient helpers; priming with the fragment containing the two V-domains of M315 (FV315) induced a somewhat weaker helper effect than L315 or VL315. The helper effect was abolished or markedly reduced by treating the primed cells with rabbit anti-brain theta + C. Cells primed with the heavy chain of M315 (H315) effected weak but significant help. The V-domain of H315 (VH315) was incapable of eliciting cells with detectable helper effect. The data indicate that the VL315 embodies a major determinant for T helper lymphocytes. This determinant is expressed on the free VL315 as well as on the complete M315. In contrast, previous studies have shown that BALB/c antibodies produced against Id315 recognize antigenic sites that are only displayed on associated (VL315 + VH315) domains.

Animals↗

Carrier effect of concanavalin A-reactive and -non-reactive material in tuberculin PPD.

Tuberculin purified protein derivative (PPD) is a very potent T-cell reactive material in tuberculin-positive individuals, but the components responsible for this reactivity have not been adequately defined. Three purified mycobacterial proteins (MPB70, the BCG 65-kDa protein, and BCG antigen 85B) with different degrees of temperature sensitivity were iodine-labelled and added to BCG culture fluid, and the mixtures were autoclaved at 120 degrees C for 30 min to simulate the initial heating procedure used to prepare PPD. SDS-PAGE followed by protein staining and autoradiography showed that the banded pattern of unheated culture fluid was completely lost after heating, and only the labelled MPB70 preparation retained most of the radioactivity in a fraction with soluble protein of the same size. Most mycobacterial proteins are extensively denatured by these procedures, which explains the previous extensive difficulties in isolating defined constituents from PPD to characterize their behaviour in B- and T-cell reactions. In assays for the carrier effect of NIP-PPD for induction of anti-NIP production in BCG-vaccinated mice, the active fractions were heterogeneous in lectin reactivity as well as in SDS-PAGE. It appears most likely that a number of Mycobacterium tuberculosis proteins give rise to core peptides which resist proteolysis and heat denaturation to possess powerful T-cell-activating ability.

Animals↗

Opsonophagocytic activity induced by chimeric antibodies of the four human IgG subclasses with or without help from complement.

The opsonophagocytic activity of the four human IgG subclasses was studied using chimeric mouse-human antibodies with specificity for the hapten NIP. As target cells we used haptenized sheep red blood cells and N. meningitidis, labelled with different amounts of hapten. We used polymorphonuclear leucocytes (PMN) as effector cells to measure respiratory burst (RB), and U937 to measure phagocytosis/rosette formation. When the target cells were opsonized with antibody only, and PMN used as effector cells, IgG3 was highly efficient, while IgG1 revealed an intermediate activity and IgG2 and IgG4 were negative. The same pattern among the subclasses was obtained in the presence of complement source, when target cells with low hapten concentration were used. However, at high epitope concentration on the target cells, in the presence of complement source, IgG2 was highly active, while IgG4 was still negative or only slightly positive. When U937 were used as effector cells and complement was omitted, IgG1, IgG3 and IgG4 all revealed high phagocytic/rosette-forming activity, while IgG2 was negative. When the target cells were opsonized with antibody and complement, the phagocytic/rosette-forming activity was often suppressed. Our results reveal that all four human IgG subclasses possess opsonophagocytic capacity, but with different requirements concerning complement and Fc gamma Rs. They also enlighten us as to how IgG2 might perform its protective effect against harmful bacteria displaying high density of carbohydrate epitopes on their outside surface.

Animals↗

The specificity of T lymphocyte responses to chemically defined antigens.

A system is described that allows the definition of T cell receptor specificity with some precision. It involves immunization of guinea pigs with hapten coupled to mycobacteria. The T cells of such animals respond to many but not all carriers modified by that hapten. Such T cells recognize neither hapten nor carrier alone, but rather determinants involving both the hapten and the carrier. No evidence for hapten-specific T cells was found. A model of the antigen binding site of the T cell receptor emerged from these experiments. According to this model, the T cell receptor consists of a single site of relatively large extent involving multiple subsites which are of low and roughly equal affinity. Thus, the haptenic group is not immunodominant for T cells as it is for B cells and for anti-hapten antibody. This suggests that the antigen binding receptor on T cells differs in some fundamental way from that on B cells. It is proposed that antigen recognition by T cells is mediated by an immunoglobulin heavy chain variable region that is not paired with an immunoglobulin light chain variable region.

Animals↗

Immune response in mice to hapten conjugated sepharose.

Mice were injected intraperitoneally with Sepharose 4B beads coupled with hapten NIP, and their anti-NIP response was studied by counting antibody forming cells and determining serum titers. Mice responded well to doses of 0.7 ml of packed beads but 0.3 and 1.2 doses induced much weaker responses. Anti-NIP titers in recipients of 0.7 ml of the antigen lasted nearly constant for at least 7 weeks. Both T cell status of the recipient and use of adjuvant had an effect on the response. Antigen without adjuvant induced primarily IgM antibodies in normal mice, but IgM and IgG in nude mice. When Hemophilus pertussis or polyacrylic acid was used as adjuvant both normal and nude mice produced IgM and IgG antibodies, and normal mice produced in addittion IgA antibodies.

Adjuvants, Immunologic↗

Specific immunosuppression by immunotoxins containing daunomycin.

Daunomycin, when conjugated with a targeting antigen by an acid-sensitive spacer, remains inactive at the intravascular pH of 7 but becomes active after cleavage within the acidic lysosomal environment of the target cell. This observation made it possible to construct cytocidal compounds that caused antigen-specific suppression of murine lymphocyte function. When daunomycin was coupled to the hapten conjugate of ovalbumin by an acid-sensitive cis-aconityl group, it caused hapten-specific impairment of immunocompetence in murine B lymphocytes in vitro and in vivo. Furthermore, the response by T lymphocytes to concanavalin A in vitro was selectively eliminated by a conjugate between daunomycin plus the acid-sensitive spacer and a monoclonal antibody specific for T cells.

Animals↗