Secondary responsein vitro to antigen ΦX 174 phage

In a model secondary reactionin vitro, a correlation was demonstrated between the size of the antigen dose used for the prestimulation of spleen tissue donors and the type of antibodies formed in the anamnestic reaction. After a small dose of antigen ΦX 174, the antibody response three days after prestimulation was of the 19 S (IgM) type, but later secondary contactin vitro (after four months) did not produce a 19 S anamnestic reaction. After large primary doses of antigen, a short interval between primary and secondary contact led to the formation of 19 and 7 S type antibodies, while after a long interval only 7 S (IgG) type antibodies were formed. The results are discussed in relation to differences in the size of the antigen dose needed to induce short-term 19 S and long-term 7 S immunological memory.     

Anti-IgD enhancement of primary antibody responses in rats

The role of membrane IgD in immune responses was examined by treating adult rats with anti-IgD. Anti-IgD when administered to rats in conjunction with optimal or suboptimal doses of either SRBC, a T-dependent antigen, or DNP-Ficoll, a T-independent antigen, enhanced the antibody responses. The greatest enhancement was obtained when anti-IgD was administered before the antigen. The effects of anti-IgD on antibody responses to SRBC were: (i) significant antibody responses to suboptimal antigen concentrations; (ii) greater antibody responses to optimal antigen concentrations; (iii) accelerated antibody responses; (iv) an early shift from IgM to IgG antibodies; (v) prolonged antibody responses. Similar effects on the immune response to DNP-Ficoll were observed with the exception that all antibodies were 2ME sensitive (IgM). These results suggest that an anamnestic type of immune response can be induced in anti-IgD-treated rats when given a primary antigen exposure. Injection of anti-IgD without SRBC or DNP-Ficoll induces B-cell proliferation without detectable antibody production to these antigens, indicating at least two signals are required for the enhanced antibody responses.     

Immunological memory to tetanus toxoid is established and maintained in the vitamin A-depleted rat

We have previously shown that vitamin A deficiency severely impairs the young rat's ability to produce specific antibodies after primary immunization with tetanus toxoid (TT). In the present studies, we asked whether immunologic memory to TT is established even in the vitamin A-depleted animal, and if so, whether such memory can be elicited after subsequent repletion with retinol. Vitamin A-depleted rats produced very low concentrations of TT-specific IgM and IgG antibodies in both the primary and secondary responses; however, the ratios of secondary to primary IgM anti-TT and of IgG anti-TT were normal. When rats were repleted with retinol 1 day after immunization, IgM and IgG anti-TT concentrations in both the primary and secondary responses were at least as great as those of control rats. For rats repleted with retinol 2 days before the booster immunization, secondary IgM and IgG anti-TT concentrations were equal in magnitude to those of vitamin A-sufficient controls. For all groups, the kinetics of the antibody response were similar. We conclude that immunological memory is intact in the vitamin A-depleted animal, as shown by 1) the normal ratio of its secondary to primary antibody responses, 2) the restoration of a quantitatively normal secondary antibody response in previously vitamin A-depleted animals repleted with retinol just before boosting with TT, and 3) a normal class switch from IgM to IgG. Retinol deficiency is also characterized by an abnormal elevation of total plasma IgG, despite the inability of the vitamin A-depleted animal to produce normal quantities of specific antibodies after challenge with antigen.     

Influence of age and physical activity on the primary in vivo antibody and T cell-mediated responses in men.

The aging immune system is characterized by the progressive decline in the antibody and T cell-mediated responses to antigen. Little is known, however, about the benefits of exercise in aging on the generation of a primary immune response to antigen and the subsequent antibody and memory T cell-mediated response. Most in vivo immune research to date has utilized vaccines or recall antigens to elicit an immune response. Therefore, the purpose of this experiment was to examine the association of aging and physical activity on the primary antibody and T cell response to the novel protein antigen keyhole-limpet hemocyanin (KLH). Forty-six physically active and sedentary, young (20-35 yr) and older (60-79 yr) men were recruited. Subjects were intramuscularly immunized with 100 microg of KLH, and blood samples were collected at days 0, 7, 14, 21, and 28. Samples were measured for anti-KLH IgM, IgG, IgG1, and IgG2 by ELISA. On day 21 after intramuscular KLH administration, subjects received an intradermal injection with 1 microg of KLH of inflammation recorded at 24, 48, 72, 96, and 120 h to assess anti-KLH delayed-type hypersensitivity response. There was a significant reduction in all anti-KLH measures with aging except for anti-KLH IgG2. The physically active older group had significantly higher anti-KLH IgM, IgG, IgG1, and delayed-type hypersensitivity responses, but not IgG2 compared with the sedentary older group. In conclusion, regular physical activity in older men is associated with a more robust immune response to novel antigenic challenge.     

Immunological memory is compromised by food restriction in deer mice Peromyscus maniculatus

The immune system protects organisms against infection, but this protection presumably comes at a cost. Here, we asked whether food restriction would compromise the ability of an organism to generate an immune response on reexposure to an antigen, which would represent a functional cost of immunological memory. Immunological memory is generated when B and T lymphocytes sensitive to components of pathogens (i.e., antigens) proliferate after exposure and persist in circulation to hinder reinfection. To test the possibility that B cell memory, the component of the immune system responsible for antibody production, is expensive to maintain, secondary antibody production against a novel protein [keyhole limpet hemocyanin (KLH)] was compared in food-restricted and ad libitum-fed male deer mice (Peromyscus maniculatus). To determine whether compromised secondary antibody production was solely due to elevated corticosterone independent of resource availability, some food-restricted and ad libitum-fed mice were subjected to unpredictable, chronic (2 h/day) restraint. Mice fed 70% of their ad libitum diet 2 wk after primary antigen challenge produced approximately 95% less IgG against KLH after a second antigen challenge than mice fed ad libitum, even though all mice were fed ad libitum during the secondary antibody response period. Restraint had no effect on secondary IgG production in response to KLH, and corticosterone concentrations 1 day after food restriction did not differ between food-restricted and ad libitum-fed mice. Together, these data imply that secondary antibody responses and the benefits of immunological memory are energetically costly in this species.     

In vivo immune response to TNP hapten coupled to thymus-independent carrier lipopolysaccharide

Lipopolysaccharide has been utilized as a carrier for the TNP hapten, producing an antigen which induces an in vivo thymus-independent antibody response to TNP as determined using athymic nude mice and their normal littermates. The immune response to TNP-LPS was investigated at both the antibody-forming cell and the serum antibody levels.The primary response to an optimal dose of TNP-LPS (1.0 μg) exhibited unusual kinetics reaching a sharp peak on day 3 of 58,000 anti-TNP PFC/spleen. Serum antibody to TNP was first detected on day 3 and reached a maximum log₂ titer of 17.5 on day 5, an uncommonly high level for hapten-carrier conjugates and most carriers. Both the anti-TNP serum antibody and PFCs were exclusively IgM. No IgG antibody was detected in the primary response through 28 days postimmunization, nor was any detected in any experiment described in this paper. The primary PFC response to 1.0 μg of TNP-LPS was specific for TNP, producing no evidence of polyclonal antibody synthesis. The relative affinities of PFC-secreted antibody were investigated using hapten inhibition. The hapten inhibition curves for TNP-LPS and TNP-SRBC were very similar, indicating that relatively high affinity antibody was elicited by TNP-LPS. The secondary response to this dose following priming with TNP-SRBC or TNP-LPS was similar to the primary response, though the peak was less sharp in both cases. The response to the homologous secondary challenge shifted somewhat, reaching a peak on days 3–4. The effect of various doses in priming or challenging for the secondary response to TNP-LPS was investigated. Using an increased PFC response as a criterion, no dose was optimal for priming or immunological memory to TNP-LPS. While the adoptive primary response to TNP-LPS reached a low level peak on day 7, the adoptive secondary attained a maximum on day 6. This shift in kinetics in intact mice and in adoptive hosts in comparing primary to secondary responses indicated that a state of B cell priming may be induced. However, its full expression may be suppressed by endogenous factors at the time of priming, such as the high level of circulating anti-TNP antibody or residual antigen. Adoptive transfer would remove the cells from these influences, allowing such B cell priming to manifest itself fully.     

The influence of epitope density on the immunological properties of hapten-protein conjugates. I. Characteristics of the immune response to hapten-coupled albumen with varying epitope density

The relationship between the epitope density of hapten-protein conjugates (DNP· BSA), and their immunogenicity in mice has been investigated. As others have found, lightly substituted protein (DNP₅BSA) elicited primary and secondary antibody responses which were mainly IgG. In contrast, DNP₅₀BSA induced a primary IgM response with relatively little IgG, and little or no immunological memory. The transition in immunogenic behaviour from “low” to “high” occurred with a hapten: protein molar ratio around 30. DNP₅₀BSA does not contain any serologically detectable native BSA determinants or neodeterminants resulting from dinitrophenylation. Although this antigen elicits a mainly IgM response as do thymus-independent antigens, antibody production to both DNP₅BSA and DNP₅₀BSA is highly thymus dependent. The possible reasons for the thymus dependence of immune responsiveness to highepitope-density hapten-protein conjugates are discussed.     

Studies on B-cell memory. IV. Effects of lipopolysaccharide on primary and secondary antibody responses to T-independent type-2 (TI-2) antigen in mice

Effects of LPS on primary and secondary antibody responses to typical TI-2 antigens were investigated in mice. Simultaneous injection of LPS with a TI-2 antigen showed only little adjuvant effect on the following primary antibody response to the antigen. In contrast, either a single or multiple injections of LPS, prior to the immunization with a TI-2 antigen, significantly augmented the following primary antibody response to the antigen. LPS, however, inhibited the development of B-cell memory to a TI-2 antigen when administered together with the antigen. Moreover, an injection of LPS in mice, which had strong IgM and IgG B-cell memories to a TI-2 antigen, caused disappearance or profound reduction of the memories. The results suggest that LPS produced by gram-negative bacteria exerts inhibitory effects on the development and continuation of B-cell memory to bacterial infections.     

Adjuvant Action of Capsular Polysaccharide of Klebsiella pneumoniae on Antibody Response

A study was performed to clarify the roles of primary and secondary injections of antigen and adjuvant (capsular polysaccharide of Klebsiella pneumoniae, CPS-K) in induction of antibody responses and in development of immunological memory in mice to bovine serum albumin (BSA). A primary injecion of BSA alone neither induced significant primary antibody response nor increased immunological memory for a secondary antibody response but, if primary injections of BSA and CPS-K were performed simultaneously, high antibody responses were induced. Moreover, a prior injection of BSA alone or CPS-K alone decreased the level of primary antibody response and the degree of increase in memory following the subsequent injection of BSA mixed with CPS-K. In contrast, a secondary injection of BSA alone into mice once primed with a mixture of BSA and CPS-K elicited very high secondary type antibody response and increased secondarily the memory for a tertiary antibody response. Injection of CPS-K simultaneously with or shortly before or after the secondary injection of BSA did not increase the level of the secondary antibody response and the degree of the secondary increase in memory. Augmentation of the secondary antibody response was elicited by simultaneous injection of CPS-K only when the secondary response was induced inadequately by a suboptimum or supraoptimum dose of antigen.     

Primary and secondary immune response of mice to polysaccharide antigens of meningococcal serogroups A and C

The peculiarities of the primary immune response, the formation of immunological memory and the secondary immune response to serogroup A and C meningococcal polysaccharides were studied in 7 strains of inbred mice, hybrids F1 and in noninbred animals. The passive local hemolysis test and the passive hemagglutination test indicated that the intensity of immune response to A and C polysacchardies depended on the genotype of the animals: both antigens induced the most intense response in CBA and BALB/c mice. The primary immune response to the both antigens was characterized by a short latent period, a rapid (by days 4-5) increase in the amount of antibody-producing cells in the spleen and in antibody titer in the blood serum to the maximum level, and a pronounced decrease inantibody formation by days 6-7 followed by a gradual extinction of the response. A single injection of A and C polysaccharides in a dose of 0.5 microgram induced the formation of immunological memory in mice, persisting for at least 4 weeks and manifesting after reimmunization as the increased or more prolonged synthesis of IgM and IgG.     

Elicitation of antigen-induced primary and secondary murine IgE antibody responses in vitro

Described herein are methods for eliciting and quantitating primary and secondary murine IgE antibody responses in vitro, and the important role of antigen concentration in determining the level of IgE produced during an immune response. The methods for quantitating IgE antibody levels in culture supernatant fluids and in serum by ELISA are presented in detail. The specificity of such methods was confirmed in that (1) no other isotype of antibody registered in the IgE-ELISA, and (2) parallel determinations of IgE antibody concentrations could be obtained by independent analysis using Fc epsilon RI-dependent basophil degranulation. We examined various parameters of cell donor immunization and lymphoid cell culture which allow for optimal in vitro primary and secondary IgE responses. High relative antigen doses result in diminished IgE antibody responses in experimental animals, a finding confirmed herein. High antigen concentrations in vitro also result in relative suppression of IgE antibody synthesis. This was also true for in vitro production of IgG1 and IgA antibodies. Conversely, IgM and IgG2a responses were elicited at both low and high antigen concentrations; IgG2b and IgG3 were not produced under the conditions of priming and culture used herein. Finally, production of IgE in vitro depended on the presence of carrier-primed CD4+ T cells and hapten-specific B cells. Generation of maximal IgE antibody secretion, and hence elicitation of an allergic reaction, is thus dependent on the amount of antigen acting as stimulant for the immune response.     

A soluble inhibitor of B and T cell proliferation and antibody synthesis produced by dividing human T cells.

The increase in affinity and heterogeneity of antibody with respect to time after immunization to the 2,4,6-trinitrophenyl (TNP) determinant was studied using TNP-brucella (BA) and TNP-type III pneumococcal polysaccharide (SIII). Experimental evidence is presented in support of the maturation of 19S antibody affinity. The difficulties which have been encountered in some previous investigations in detecting such a maturation appear to be the tendency of the cells to switch from IgM to IgG synthesis early after the peak of the primary response. Data are presented indicating that this switch occurs in a non-antibody-secreting memory cell population prior to, or more likely very shortly after, boosting. We also present evidence that the use of an antigen that does not induce a massive switch from IgM to IgG antibody synthesis offers a way of unmasking maturation of the 19S response. Thus, with the T-independent antigen TNP-SIII, a definite increase in heterogeneity could be detected in the 19S response upon secondary boosting. A greater increase in heterogeneity was noted in nude mice and was possibly due to the absence of suppressor T cells.     

Immune Response to Listeria monocytogenes in Rabbits and Humans

Rabbits were immunized with listeria antigens, staphylococcus antigen, or with both, and the course of their immune response was monitored. Antibodies to Listeria and Staphylococcus were produced in both immunoglobulin M (IgM) and immunoglobulin G (IgG) classes in response to inoculation with the specific antigen. Cross-responses occurred in rabbits injected only with Listeria or only with Staphylococcus, as well as in rabbits injected with both antigens. L. monocytogenes serotype 4d appeared to be immunologically distinct from L. monocytogenes serotype 2 and its cross-reaction with S. aureus. Human sera from bacteriologically confirmed cases of listeriosis were examined to determine the nature of the immunological response of man to Listeria. In the sera studied, IgM was the predominant antibody produced to Listeria, whereas cross-reactions with Staphylococcus were observed in both the IgM and the IgG antibody classes.     

Gut SCP is an immune-relevant molecule involved in the primary immunological memory or pattern recognition in the amphioxus Branchiostoma belcheri

To understand the role of calcium-binding proteins of invertebrates in immunological response, amphioxus sarcoplasmic calcium-binding protein (SCP) was investigated in the present study. Following gene cloning, recombinant protein expression and purification and antibody preparation, the expression and alteration of SCP in the response to bacterial challenge were detected using Western blotting. SCP was not detected in the branchia, humoral fluid, gonad or in the gut of wounded animals, but it was abundant in muscle and appeared in the gut of healthy animals using Vibrio parahaemolyticus immunization and challenge. Furthermore, whether gut SCP possessed anamnestic response was investigated using cross-immune challenge between Gram-positive and -negative bacteria. Gut SCP showed stronger anamnestic activity or pattern-recognition in response to Gram-negative bacterium V. parahaemolyticus than Gram-positive bacterium Staphylococcus aureus. The response was faster and more species-specific to V. parahaemolyticus, whereas it was slower and longer to S. aureus. The reason why the response showed significant difference between Gram-positive and -negative bacteria awaits investigation. These results indicate that gut SCP is an immune-relevant molecule involved in the primary immunological memory or pattern recognition in the amphioxus Branchiostoma belcheri.     

Variations in the responses of C57BL and a mice to sheep red blood cells: II. Analysis of plaque-forming cells

The number of direct and indirect plaque-forming cells (PFC) and the serum hemolytic activity was determined for A/He, C57BL/6J, and B6AF1 mice responding to multiple injections of sheep red blood cells (SRBC). Although the kinetics of the primary response differed, all mice had high numbers of both direct and indirect PFC and low-titered 2-mercaptoethanol (2-ME) sensitive serum antibody. Following multiple SRBC injections, the A/He spleens contained predominantly IgG producing PFC. Their serum antibody activity was resistant to 2-ME signifying the presence of IgG. The serum activity of both the C57BL/6J and B6AF1 mice was sensitive to 2-ME (IgM antibody) over the course of immunization, and although there was a definite IgM PFC memory response, the presence of 7S memory PFC was questionable. The results are discussed in terms of the maturation of the antibody response to SRBC and of the question of the postulated IgM and IgG switch.     

IgM-mediated, T cell-independent suppression of humoral immunity.

The immunological unreactive state occurring in (T,G)-A-L nonresponder mice after secondary antigen challenge was investigated. Syngeneic IgM anti-(T,G)-A-L antibody-containing plasma, transferred at the time of the time of primary challenge, induced persistent suppression of autologous specific antibody production. Removal of plasma IgM with goat anti-mu antisera removed the ability of the plasma to supress. The induction and maintenance of the suppressed state were not different in thymectomized or sham-thymectomized animals. Primed animals subjected to graft-vs-host reaction (GVHR) at the time of secondary challenge switched over to IgG production. Animals suppressed by passive antibody transfer reacted to GVHR, at the time of secondary challenge, with specific IgM but not IgG antibody production. Transfused normal spleen cells partially abrogated suppression only when (suppressed) hosts had been lethally irradiated. Spleen cells from antigen-plus-antibody suppressed donors, upon transfer to previously normal, syngeneic hosts, were less immunocompetent than spleen cells from untreated donors. These data are consistent with a model of IgM mediated, T cell-independent persistent suppression of humoral immunity.     

Analysis of somatic mutation and class switching in naive and memory B cells generating adoptive primary and secondary responses

Clonal progeny of naive B cells (producing a primary antibody response) and of memory B cells (producing a secondary response) were identified in a cell transfer system. Primary response clones are typically derived from IgM precursors and express unmutated V regions. Multiple isotype switches occur in these clones. Secondary response clones derive from IgG1 precursors and express highly mutated V regions. Additional switches do not occur. With one exception, there was no evidence for somatic mutation during clonal expansion. The generation of mutated memory cells may thus represent a distinct differentiation pathway. Evidence is presented that, in this pathway, mutants that have lost antigen binding specificity but that remain available for stimulation by a different antigen arise upon antigenic stimulation.     

Immunogenicity of synthetic TF-KLH (keyhole limpet hemocyanin) and sTn-KLH conjugates in colorectal carcinoma patients

Mucins of colorectal carcinomas overexpress the cancer-associated disaccharides Thomsen-Friedenreich antigen (TF) and sialyl-Tn antigen (sTn), making these antigens suitable for active specific immunotherapy. Patients at high risk for recurrent colon cancer, but free from disease after surgical resection, were immunized with synthetic TF and sTn covalently attached by a two-carbon crotyl linker to keyhole limpet hemocyanin (KLH). Four groups of patients were treated with TF-KLH without adjuvant, TF-KLH plus the immunological adjuvant Detox, sTn-KLH plus Detox, or sTn-KLH plus the immunological adjuvant QS-21, and the serological response was monitored. Enzyme-linked immunosorbent assay (ELISA), do-blot immunostains, and inhibition assays were used to identify antibody responses against synthetic TF and sTn epitopes and against natural antigens, including asialoglycophorin expressing TF antigen, and ovine submaxillary mucin and the human colon cancer line LS-C expressing sTn antigen. Our results demonstrate that vaccines containing TF or sTn-KLH conjugates plus immunological adjuvants Detox and especially QS-21 induced high IgM and IgG antibody titers against the respective synthetic disaccharide epitopes. However, when tested against natural antigens expressing these disaccharide epitopes, IgM antibodies showed weak to moderate reactivity, while IgG antibodies were almost totally unreactive. On the basis of these results we are continuing to test modifications of synthetic TF and sTn epitopes to identify those that induce IgM and IgG antibodies that are more reactive with these antigens as they are expressed on tumor mucins.This work was supported by grants from the National Institutes of Health (CA33049, CA52491, CA08748) and the Chemotherapy Foundation     

The immune response in a cat-related outbreak of Q fever as measured by the indirect immunofluorescence test and the enzyme-linked immunosorbent assay

The isotypic immune response of 16 individuals who developed Q fever pneumonia following exposure to an infected parturient cat was studied. The enzyme-linked immunosorbent (ELISA) test was used to detect IgM, IgA, and IgG antibodies to phase I and phase II Coxiella burnetii whole-cell antigens and to the phase I lipopolysaccharide. The indirect immunofluorescent antibody (IFA) test was also used to detect antibodies to phase I and phase II whole cells. None of the 16 subjects developed antibodies to the phase I lipopolysaccharide. The ELISA was more sensitive than the IFA test. IgM antibodies to phase II antigen were detectable by ELISA in 80% of the subjects at the time of onset of symptoms and were still present in 7 of the 8 tested at 32 weeks following the onset of symptoms. In all instances (ELISA: IgG, IgM; IFA: IgG, IgM) phase II antibodies developed earlier and reached higher levels than did phase I antibodies. The absence of antibodies to phase I lipopolysaccharide in acute Q fever combined with our unpublished findings of antibodies to phase I lipopolysaccharide in chronic Q fever suggests that this test may be used to distinguish acute from chronic Q fever.     

Characterization of the primary IgM response to GAT and GT: conditions required for the detection of IgM antibodies.

Primary IgM antibody responses to synthetic linear copolymers of L-glutamic acid, L-tyrosine, and L-alanine were investigated. The appearance of primary IgM anti-GAT antibodies was detected in BALB/c mice by using a solid phase radioimmunoassay (SPRIA) procedure. The finding was verified for GAT in responder mice and GAT-MBSA and GT-MBSA in nonresponder mice in an indirect plaque forming cell (PFC) assay by using a rabbit antiserum directed against the mulambda myeloma protein, MOPC 104E. Facilitated IgM PFC could be inhibited by a purified muK myeloma protein, TEPC 183. Maximal facilitated IgM plaque response was found to precede the IgG response by several days. A direct plaque assay was developed for the detection of IgM anti-GAT plaques using poly-L-lysine (PLL) to couple GAT to sheep erythrocytes (SRBC). GAT-SRBC coupled by the PLL method optimally couple 4 to 5 times less antigen to the indicator cell surface than does the CrCl3-coupling method routinely employed in our laboratory. These findings were extended to a conventional antigen, chicken gamma globulin (CgammaG). We found that a less dense epitope coat on the indicator cell surface favors detection of direct IgM PFC, whereas a more densely coated indicator cell favors the detection of facilitated IgM and IgG PFC responses.