James J. Gibbons

Lower antibody response to tetanus toxoid associated with higher auto-anti-idiotypic antibody in old compared with young humans

The production of anti‐tetanus toxoid (TT) antibody and anti‐anti‐TT (auto‐anti‐Id) antibody has been measured in three young and three old persons. Both antibodies were measured using ELISA. We found that: (i) the anti‐TT response of old subjects was lower than that of young subjects, (ii) scrum auto‐anti‐Id antibody concentration was higher in old compared with young humans before boosting with TT, and (iii) anti‐TT antibodies from different humans shared Id cross‐reactivity when tested with a rabbit anti‐Id antibody. Thus, the serum anti‐TT antibody response to boosting was inversely correlated with the serum level of auto‐anti‐Id. This conclusion is consistent with the view that the higher level of auto‐anti‐antibody in older subjects contributes to their impaired antibody response to TT.

Isolation and purification of human C1q from plasma

C1q was purified to homogeneity from human plasma by a 3-step purification procedure. Plasma was euglobulin precipitated, and the redissolved precipitate chromatographed on a rabbit IgG-Sepharose column. The 1 M NaCl buffer eluate was passed directly through a rabbit anti-human IgG-Sepharose affinity column. C1q freed of IgG was present in the flow through. The rationale for this scheme to remove IgG free and that bound to C1q is discussed. Overall recovery of C1q was about 40% with IgG less than 4 microgram/mg C1q. In SDS-polyacrylamide electrophoresis with non-reducing conditions bands at 52,000 and 42,000 daltons were demonstrated while with reducing conditions bands at 26,000, 24,000 and 20,000 daltons were found as reported by others. C1q was found to be stable at 4 degrees C for 1 year in a 1 M NaCl, 0.4 M Tris, 10% sucrose, 0.005 M EDTA, 0.02% NaN3, pH 8.6 buffer.

Production of auto-anti-idiotypic antibody during the normal immune response. XII. An enzyme-linked immunosorbent assay for auto-anti-idiotype antibody☆

An enzyme-linked immunosorbent assay (ELISA) to detect anti-idiotype (Id) antibody is described. Using this assay auto-anti-Id was detected in the serum of aged mice immunized with 2,4,6-trinitrophenylated-Ficoll (TNP-F). Hapten eluates from anti-TNP-F immune spleen cells also contained readily detectable auto-anti-Id.

Regulation of the Anti-Trinitrophenyl Response by Anti-Idiotype Antibodiesa

It has been suggested by Jerne that the immune system is self regulated as a consequence of a network of interactions between idiotypes (ids) and anti-idiotypes (anti-id). One of the key predictions from such a hypothesis is that anti-id should be spontaneously produced during the immune response to traditional foreign antigens. Such spontaneous production of auto-anti-id has been observed by several groups2- including our own.61o We have employed the observation that in some cases the presence of a low concentration of hapten in the assay medium results in an increase in the number of plaque-forming cells (PFC) observed in a Jerne assay, as the basis for the detection of auto-anti-idiotype We suggested that auto-anti-id can bind to Ig on the surface of potential antibody secreting cells, thereby causing a reversible inhibition of antibody secretion. Hapten competes with anti-id for cell-surface antigen receptors (id). Therefore, in the presence of hapten, bound auto-anti-id is displaced, the inhibition of secretion is reversed, and an increase in the number of PFC is observed. Thus, hapten-augmentable PFC represent B cells whose secretion of antibody has been blocked as a consequence of the binding of auto-anti-id to cell surface idiotype. Evidence justifying the use of this assay has been previously published6* and includes (a) the ability to elute with hapten a factor from spleen cells that can cause a specific hapten-reversible inhibition of plaque formation and that has binding and antigenic properties consistent with its being an IgG auto-anti-id; (b) the fact that under certain experimental conditions the number of hapten-augmentable PFC detected in the spleen correlates with the results obtained using a conventional enzyme-linked immunosorbent assay (ELISA) for anti-id in the serum. With this approach we have demonstrated that auto-anti-id is produced spontaneously during the primary and secondary responses to both T-dependent and T-independent antigens;.* auto-anti-id is produced spontaneously by mice38*0 rabbits

Production of auto-anti-idiotypic antibody during the normal immune response. VIII. Effect of auto-anti-idiotypic antibody on contact sensitivity.

An eluate prepared by brief incubation of spleen cells from 2,4,6-trinitrophenyl (TNP) lysyl-Ficoll immunized mice with TNP-epsilon-amino-n-caproic acid causes a specific inhibition of the induction of contact sensitivity by 2,4,6-trinitrochlorobenzene skin painting. The active factor in the eluate binds to an anti-mouse immunoglobulin (Ig) immunoadsorbent column but not to a TNP immunoadsorbent column and is therefore Ig but not anti-TNP antibody. The active factor does bind to an immunoadsorbent prepared from anti-TNP antibody, suggesting that the factor has anti-idiotype specificity. Evidence based upon hapten-reversible inhibition of plaque formation and an enzyme-linked immunosorbent assay (ELISA) indicates that the eluates contain auto-anti-idiotype antibody specific for anti-TNP antibody. It is suggested that auto-anti-idiotype antibody spontaneously produced during the immune response to a T-independent antigen can specifically downregulate contact sensitization to the same epitope.

Studies on suppressor factors produced by T-cell hybridomas. I. Characterization of antigen-specific suppressor factors.

Antigen-specific and antigen-nonspecific suppressor T cells were generated when spleen cells prepared from C57BL/6J (H-2b) were incubated with trinitrophenylated polyacrylamide beads (TNP-PAA) in vitro. T hybridomas were prepared by fusion of spleen cells cultured with TNP-PAA for 4 days and the thymoma cell line BW5147. More than 100 hybridomas were generated, and 15 of them suppressed the anti-TNP PFC response of fresh spleen cells cultured with TNP-PAA. The suppression was antigen specific with three of these five hybridoma supernatants tested. Hybridomas that caused antigen-specific suppression secrete factors which bring about suppression of the anti-TNP PFC response by spleen cells cultured with TNP-PAA. These hybridoma supernatants which cause antigen-specific suppression typically depressed the anti-TNP PFC response by 60% while depressing anti-SRBC PFC response by only 10%. The antigen-specific suppressor factors were bound to a TNP-BGG column but not to a BGG column. The suppressor factors, purified by affinity chromatography on a TNP-BGG column, were bound to anti-I-Jb antibody.

Regulation of antibody secretion by hybridoma cells: I. Suppression of antibody secretion by coculture of hybridoma cells with idiotype-induced suppressor cells☆

In this study, we have demonstrated that antibody secretion by hybridoma cell lines can be down-regulated by idiotype-specific immune spleen cells or by nylon wool nonadherent spleen cells. This suppression of antibody secretion can be abolished by treating the idiotype-specific immune spleen cells with anti-Thy 1.2 plus complement. The hybridoma we used for most of our experiments secretes IgM specific for the cross-reacting haptens 2,4,6-trinitrophenyl (TNP) and 2,4-dinitrophenyl (DNP). Suppression was achieved by direct coculture of hybridoma cells with immune cells from animals which were injected with affinity-purified hybridoma antibody-coupled syngeneic spleen cells. The suppressed and control cultures contained similar numbers of viable hybridoma cells, suggesting that a simple cytotoxic effect is not responsible. Idiotype specificity was established in experiments showing that two idiotype immune animals immunized with antibody from two different IgM anti-TNP hybridomas could suppress the hybridoma to which they were immunized but could not affect the other hybridoma. Immune spleen cells required 3-4 days of coculture with hybridoma cells before maximum suppression was achieved. The kinetics of the response suggest that the final effector suppressor cell is generated during the coculture period and that a second signal, perhaps a product of the hybridoma cells, may be required.

Production of auto-anti-idiotype antibody during the normal immune response. XI. Ficoll-induced variations in auto-anti-idiotype production during the response to 2,4,6-trinitrophenyl-Ficoll.

The responses to 2,4,6-trinitrophenyl conjugates of different Ficoll preparations differ with respect to the magnitude of the accompanying auto-anti-idiotype (Id) response in both mice and chickens. Evidence is presented that reduced auto-anti-Id production in the chicken is due to the activation of suppressor activity by some preparations of Ficoll.