Studies on the control of antibody synthesis. XVI. Effect of immunodepression on antibody affinity

The relationship between depression in the magnitude of the immune response and a decrease in affinity of the antibody produced was examined in three different models of immuno-depression (B-cell clonal deletion tolerance, specific suppressor T-cell activity, and anti-genie competition). In B-cell clonal deletion tolerance and in antigen-specific, suppressor T-cell-mediated immunodepression, a small decrease in magnitude (50% or less) is associated with a marked decrease in high-affinity, plaque-forming cells. In contrast, with nonspecific immunodepression, due to antigenic competition, a depression in affinity is only seen when there is a marked (85%) reduction in the magnitude of the response. The results are consistent with the view that when the mechanism of immunodepression involves interaction of antigen with antigen-specific B cells there is a disproportionate loss of high-affinity, antibody-producing cells relative to the decrease in magnitude. In contrast, with nonspecific immuno-depression, where the decrease in affinity is presumably due to inefficient expansion of high-affinity clones, an effect on affinity is only observed in association with a marked depression in the magnitude of the response.     

Studies on the control of antibody synthesis. IX. Effect of boosting on antibody affinity.

The effect of boosting on antibody affinity was studied in a haptenic system. Generally, boosting results in the prompt synthesis of high affinity anti-hapten antibody. However, repeated boosting frequently leads to a decrease in the amount and affinity of the serum antibody. Repeated boosting with hapten on a carrier different from that used for priming selectively stimulates synthesis of the highest affinity anti-hapten antibody and does not result in a decrease in affinity. Priming with soluble antigen without adjuvants results in the synthesis of low affinity antibody. After such priming, boosting stimulates low affinity antibody synthesis and repeated boosting leads to a moderate increase in antibody affinity.     

Studies on the control of antibody synthesis. XIV. Role of T cells in regulating antibody affinity.

The effect of limiting the number of helper T cells on the affinity of the primary antibody response to a T-dependent antigen (DNP-BGG) was evaluated in a cell transfer system. Lethally irradiated, thymectomized mice were reconstituted with either bone marrow or anti-brain θ antiserum plus complement-treated spleen as the source of B cells. In addition, they received various numbers of thymus cells as a source of helper T cells. The animals were immunized with DNP-BGG 1 day after cell transfer and their splenic anti-DNP PFC response was assayed for magnitude and affinity 3 weeks later. A marked restriction in helper T-cell activity resulted in a primary response which was of low magnitude, which lacked indirect PFC, and which had a very low affinity and restricted heterogeneity. When sufficient thymus cells were given to permit a switch to indirect plaque formation, a highly heterogeneous, high-affinity primary response was elicited. Further increase in the number of thymic cells resulted in a progressive increase in the magnitude of the primary response but had no effect on affinity. Thus, a reduction of 50% in the magnitude of the response as a consequence of limiting the number of T-helper cells had no effect on the affinity of the PFC. The results are consistent with the interpretation that the effect of restriction in T-cell help on antibody affinity is not due to a direct effect on precursors of high-affinity PFC but is secondary to inefficient selection for high-affinity cells when the degree of cell proliferation is markedly reduced.     

Studies on the genetic control of antibody affinity: the independent control of antibody levels and affinity in Biozzi mice.

The levels (Abt) and relative affinity (KR) of antibody produced to protein antigens injected in saline have been measured in the 22nd generation of the Biozzi high (Ab/H) and low (Ab/L) antibody-producing mice. No significant differences in affinity were observed between the two lines of mice (p 0.10) but the levels of antibody (Abt) differed significantly (p 0.0025) when immunized with antigen in saline; however, both Ab/H and Ab/L mice were able to mount a high affinity response to protein antigens injected in Freund's complete adjuvant. These results substantiate earlier observations that in mice, antibody affinity (KR) and antibody level (Abt) are under independent genetic control.     

Studies on the control of ribosomal RNA synthesis in HeLa cells.

In many eucaryotic systems protein synthesis is coupled to ribosomal RNA synthesis such that shut-down of the former causes inhibition of the latter. We have investigated this stringency phenomenon in HeLa cells. The protein synthesis inhibitors cycloheximide and puromycin cause inactivation of both processes but valine starvation totally inhibits only the processing of 45-S RNA. DNA-dependent RNA polymerases from A, B and C (or I, II and III respectively) were extracted, separated partially by DEAE-cellulose chromatography and their activity levels determined. These do not decrease significantly during inhibition of protein synthesis. To find out whether or not form A is bound to its template under these conditions, proteins were removed from chromatin with the detergent sarkosyl. This does not affect bound RNA polymerase. Inhibition of protein synthesis caused up to 50% reduction in endogenous alpha-amanitin-insensitive chromatin-RNA-synthesising activity. This reduced level of activity was not affected by sarkosyl treatment. Levels in normal cells were stimulated. This result indicates that the form A RNA polymerase is not bound to its template when protein synthesis is inhibited.     

Studies on the synthesis of plasmid-coded proteins and their control in Bacillus subtilis minicells.

The minicell system of Bacillus subtilis has been used to study the expression of plasmid genes using several R plasmids derived from Staphylococcus aureus. pE194, pC194, and pUB110 as well as several mutant and in vitro recombinant derivatives of these plasmids segregate into minicells. A copy control mutant of pE194 was used to show that the extent of segregation is proportional to the copy number. The polypeptides specified by these plasmids were examined by SDS-polyacrylamide gel electrophoresis. Six proteins specified by pE194, an erythromycin resistance plasmid, were identified using cop mutants. These comprise about 90% of the potential coding capacity of the 2.4-Mdal pE194 plasmid. One of these proteins (29,000 daltons) is inducible by erythromycin in the wild type pE194 but is synthesized constitutively in a mutant derivative which also expresses antibiotic resistance constitutively. Several other proteins are detected only in copy control mutants. pUB110, a kanamycin resistance plasmid, expresses three major proteins which comprise 50% of the coding capacity of this 3.0-Mdal plasmid. Two additional minor proteins are occasionally observed. pC194 (2.0 Mdal), which confers chloramphenicol resistance, expresses two polypeptides comprising about 25% of its coding capacity. One of these polypeptides (22,000 daltons) is inducible by chloramphenicol. pBD9, an in vitro composite of pUB110 and pE194, probably expresses all of the major parental plasmid proteins with the exception of one from pUB110 and one from pE194.     

Studies on the synthesis of Lewis-y oligosaccharides

Lewis-y histo-blood group oligosaccharides are tumour-associated antigens prevalent in several different types of cancer, and they may also be secondary ligands for bacterial toxins from Escherichia coli and Vibrio cholerae. The key step in the synthesis of these sterically congested oligosaccharides involves difucosylation of partially protected lactosamine derivatives. Existing methods require either prolonged reaction times or elaborate glycosyl donors to ensure high stereoselectivity. Herein we report an optimised procedure for using a simple thioglycoside donor that leads to the desired products in high yield and excellent stereoselectivity. It is found that initial glycosylation of the 3′-hydroxy group of lactosamine derivatives in dichloromethane solution can inhibit subsequent glycosylation at the 2-position; however, reaction in toluene solution leads to Lewis-y oligosaccharides in high yield.