Secondary IgG responses to type III pneumococcal polysaccharide. I. Kinetics and antigen requirements.

Mice primed with a thymus- (T)3 dependent form of Type III pneumococcal polysaccharide (S3), i.e., S3 coupled to sheep or horse erythrocytes (S3-RBC), produce S3-specific IgG antibody after secondary challenge with either the T-dependent (S3-RBC) or T-independent (S3) form of the antigen. The potential to produce IgG antibody after challenge with S3-RBC appears earlier after priming than the potential to produce IgG after challenge with S3, suggesting that different "memory" cells may be involved in the two responses. The "memory" cells were shown to be S3-specific since S3 had to be present on the carrier in order for priming to occur and carrier specificity was not required for elicitation of the secondary response by S3-RBC.     

Selective suppression of primary IgM responses by induction of low dose paralysis to type III pneumococcal polysaccharide.

Pretreatment of mice with a low dose of Type III pneumococcal polysaccharide (S3)² decreases the response elicited by an optimal dose of S3 (low dose paralysis). The types of S3-specific responses which are affected by the induction of low dose paralysis were examined in the present study. The results indicate that pretreatment with low doses of S3 selectively suppresses responses of virgin IgM-producing B cells (Bμ). This was apparently due to a direct suppression at the level of the B cells, since S3-specific IgM responses were also suppressed when pretreated mice were challenged with S3 coupled to a thymus-dependent carrier, S3-HRBC. Pretreatment with low doses of S3 does not suppress primary IgG or secondary IgM or IgG responses to S3. Spleen cells from S3-pretreated mice did not suppress responses of normal cells after transfer to irradiated or intact recipients.     

Suppression of IgM responses to type III pneumococcal polysaccharide by lipopolysaccharide (LPS).

Lipopolysaccharide (LPS) suppressed the primary IgM response of mice to Type III pneumococcal polysaccharide (S3). LPS and S3 had to be given together in order for maximum suppression to occur and suppression was not due to a change in the time of the peak response in mice which received LPS. Suppression was not due to an effect of LPS on T cells since S3 responses of nude mice were suppressed by LPS. LPS did not suppress S3 responses of C₃H/HeJ mice and base hyrolysis of LPS destroyed the suppressive effect, i.e., suppression was dependent of B cell mitogenic activity of the LPS. The suppressive effect of LPS was presumably directed against virgin B cells since LPS did not suppress the S3 response of primed mice and did not suppress the development of IgG memory responses to S3.     

Circular dichroism and fluorescence studies of homogeneous antibodies to type III pneumococcal polysaccharide.

The near-ultraviolet circular dichroism (CD) of three homogeneous anti-type III pneumococcal antibodies in the absence and the presence of the specific hexasaccharide ligand was studied. In addition recombinations and hybridizations of H and L chains derived from two of these antibodies were carried out and the CD spectra of bound and free reconstituted IgG molecules were measured. The results indicate that the CD spectra of the native antibodies in the 260-310-nm range are very similar in shape and sign and exhibit a positive band at 285 nm. The homologous reconstituted antibody molecules exhibited CD spectra very similar in shape and sign to those of the native antibody molecules although recombinant molecules are no longer stabilized by interchain disulfide bonds. Upon addition of the hexasaccharide ligand, a significant decrease in amplitude of the CD spectra (18-21%) occurred in all three native antibodies and their Fab fragments as well as in the homologous recombinant molecules. No CD spectral changes could be detected upon interaction of the hapten ligand with the heterologous recombinants. All homogeneous antibodies studied exhibited fluorescence quenching upon oligosaccharide binding and a blue shift of the emission maximum. This property allowed the determination of the binding constant of one selected antibody to be made. Taken together, CD and fluorescence spectroscopic data suggest that oligosaccharide ligands induced detectable conformational changes in the Fab fragment of the antibody.     

Lectin-induced modulation of the antibody response to type III pneumococcal polysaccharide

Several lectins were tested for their capacity to alter the antibody response to type III pneumococcal polysaccharide (SSS-III). The antibody response was enhanced by concanavalin A (Con A), phytohemagglutinin (PHA), as well as lectins from Phytolacca americana (Pa-2), Pisum sativum (PSA), and Lens culinaris (LCH), when these lectins were given 2 days after immunization with SSS-III; however, suppression was obtained when Con A and Pa-2 were given at the time of immunization. By contrast the lectins from Vicia villosa (VVL) and Bauhinia purpurea (BPA) did not alter the antibody response. Since the lectins PSA and LCH bind to the same monosaccharide as Con A, whereas the other lectins bind to different monosaccharides, these findings indicate that there is no relationship between nominal monosaccharide specificity and the capacity to modulate the antibody response. Substantial increases in the magnitude of the IgG₁ antibody response was noted after the administration of Con A whereas profound enhancement of IgG_2a antibody response was noted after PHA was given.     

Requirements for activation of contrasuppressor T cells by type III pneumococcal polysaccharide

Either S3-coupled spleen cells (S3-SC) or soluble S3 activates two populations of regulatory T cells, T suppressor cells (Ts) and contrasuppressor T cells (Tcs). The latter cells function to mask the activity of Ts in unfractionated T cell populations, so that Ts can be detected only after removal of Tcs. Activation of Tcs by S3 may be required for induction of an antibody response to S3. This is suggested by the findings that Tcs are activated only by immunogenic doses of S3, that Tcs are not detectable in the spleens of mice tolerant to S3, and that (CBA/N X BALB/c)F1 male (xid) mice, which are genetically unresponsive to S3, do not develop Tcs after immunization with S3. Moreover, the kinetics of activation of Tcs by S3 closely parallels the kinetics of the antibody response to S3. Tcs have no detectable activity in the absence of Ts, indicating that these cells do not function as amplifier or helper T cells.     

Genetic control of the immune response of mice to type III pneumococcal polysaccharide

BALBc mice immunized with Type III pneumococcal polysaccharide (SIII) had higher numbers of IgM plaque-forming cells (PFC) in the spleen than similarly immunized C57BL/Ks mice. The F₁ hybrids of these two strains had intermediate numbers of SIII-specific PFC. Analysis of the responses of F₂ and backcross strains indicated that the observed responses were compatible with results expected for control of the immune response to SIII at a single autosomal locus.     

The effect of Bordetella pertussis on the antibody response in mice to type III pneumococcal polysaccharide.

The effect of an i.p. injection of Bordetella pertussis on the primary humoral immune response in mice to the thymus-independent antigen SIII has been studied. Suppression of the antibody response occurred when pertussis cells were injected at the same time as an optimal immunizing dose of SIII. In contrast, the antibody response to high doses of SIII was enhanced by B. pertussis. When SIII alone was injected, only 19S antibody was detected. However, when B. pertussis was administered with either optimal or high doses of SIII, 7S as well as 19S antibody against SIII was produced.     

Requirement for B cells for activation of contrasuppressor T cells by type III pneumococcal polysaccharide

Type III pneumococcal polysaccharide (S3) is unable to activate S3-specific contrasuppressor T cells (Tcs) in mice depleted of B cells by chronic anti-IgM treatment or in immune defective xid mice that lack the B cell subset required for anti-S3 antibody responses. The inability of S3 to activate Tcs in xid mice was shown to be due to a requirement of B cells for Tcs activation rather than to an absence of Tcs in xid mice. The B cells from normal mice that are required for Tcs activation apparently function to present the S3 Ag to Tcs. S3 physically coupled to spleen cells (S3-SC) prepared from normal BCF1 SC could activate Tcs in both xid and BCF1 mice whereas S3-SC prepared from xid SC or B cell-depleted BCF1 SC could not activate Tcs in either strain. B cell APC function was abrogated by 3000 R irradiation and by treatment of the B cells with either chloroquine or paraformaldehyde. Interestingly, B cells from mice previously immunized with S3 were unable to function in Tcs activation; preimmunization of B cell donors with an irrelevant Ag or with a T-dependent form of S3 had no effect on their ability to function as APC. These latter observations are discussed in terms of the in vivo persistence of polysaccharide Ag and their ability to induce B cell tolerance under the experimental conditions used for these experiments. The results of this study provide evidence that B cells play an important and apparently obligatory role in the activation of Tcs by S3; B cells apparently function to present Ag to Tcs, resulting in the activation of this regulatory T cell subset.     

Characterization and activity of contrasuppressor T cells induced by type III pneumococcal polysaccharide

Optimally immunogenic amounts of type III pneumococcal polysaccharide (S3) activate a population of contrasuppressor T cells (Tcs), which have been shown to play an important role in the induction of anti-S3 antibody responses. These Tcs belong to a unique T cell subset that has the surface phenotype Lyt 1+2- L3T4- I-J+ I-A+. These Tcs are also cyclophosphamide (Cy)-sensitive and sensitive to antilymphocyte serum (ALS) and mitomycin C. Tcs have antigen-binding receptors, indicating that any interactions of Tcs with B cells or T suppressor cells (Ts) (both of which also have antigen-binding receptors) must be via an antigen bridge rather than an idiotype-anti-idiotype interaction. Tcs are also Igh restricted in their action. Contrasuppression is manifest only when the Tcs are Igh compatible with both the Ts and the responding B cells. Tcs apparently mediate their effects by releasing a soluble factor, since a soluble factor extracted from Tcs is able to abrogate the effects of S3-specific Ts.     

Sensitivity of amplifier T cells involved in the antibody response to type III pneumococcal polysaccharide to anti-lymphocyte serum.

Amplifier T cells responsible for enhancement of the antibody response to type III pneumococcal polysaccharide have been shown to be resistant to the effects of antilymphocyte serum (ALS) given at the time of immunization, a treatment that eliminates suppressor T cell activity. The resistance of amplifier T cells to ALS can be attributed to the fact that their activity develops after that of suppressor T cells. ALS given 1 or 2 days after immunization does abrogate amplifier T cell activity, independent of the mode by which that activity is elicited. The data emphasize the importance of kinetic considerations in understanding the effects produced by immunologically active agents such as ALS.     

Suppression of the antibody response to type III pneumococcal polysaccharide with antigen coupled to syngeneic lymphoid cells

Nine allophenic mice of the type C57BL/10Sn … A were analyzed quantitatively, at weekly intervals over a period of 6 weeks, for the relative parental contributions to their red blood cell and white blood cell populations. It was found that four of the mice showed a significant change (termed “chimeric drift”) in the parental composition of their peripheral white blood cells, as determined by cytotoxicity testing. Six of the mice analyzed showed chimeric drift in their red blood cell population, as determined by hemoglobin analysis on isoelectric focusing gels. The isoelectric points of the hemoglobins of six inbred strains of mice were determined as an outgrowth of this study. Chimeric drift was observed in the direction of either parental cell type, and was found to be independent of the coat color, age, or sex of the mice.     

Induction of primary and inhibition of secondary antibody response to hapten by hapten conjugates of type III pneumococcal polysaccharide.

Tri- or dinitrophenylated pneumococcal polysaccharide type III (TNP- or DNP SIII)) induced a primary 19S anti-TNP response without generating immunological memory to the hapten in LAF₁ mice. Hapten-hemocyanin (TNP-KLH) or hapten conjugates of B. abortus organisms (DNP-BA) induced both 19S and 7S primary responses and memory to the hapten. Spleen cells from mice immunized with TNP-KLH or DNP-BA did not give adoptive memory responses upon challenge with hapten-SIII and, in fact, were inhibited from responding to their homologous hapten conjugates by simultaneous injection of hapten-SIII. Incubation of TNP-KLH-primed spleen cells for as short as 5 min at 0 °C with 10 μg of TNP-SIII per milliliter virtually abolished their ability to give 19S and 7S memory responses to TNP-KLH upon transfer into irradiated recipients. It is suggested that a difference in avidity and/or number of anti-TNP receptors per cell between virgin and primed B cells may be an important factor in determining whether the cells will be stimulated or inhibited by exposure to hapten-SIII. Another factor may be a difference between virgin and memory cells in their requirement for T-cell help.     

Studies on immunological memory. I. Different requirements for induction and recall of memory in antibody-mediated cellular cytotoxicity.

A system of antibody-mediated cellular immunity against chemically defined haptens has been adopted in order to study the requirements for induction and recall of immunological memory. Hapten-protein conjugates with different molar ratios of hapten to protein were used for immunization. Primed spleen cells were incubated together with ⁵¹Cr-labeled CRBC on which the conjugate was coupled by ECDI. Release of radioactivity was assayed.The induction of memory to the hapten was possible regardless of the degree of substitution. Recall of memory, however, was only possible with low substituted conjugates on either homologous or heterologous carriers. The data demonstrate different requirements for induction and recall of memory which possibly are T cell-independent and T cell-dependent processes, respectively.