Increase in the number of splenic plaque forming cells with length of gestation in untreated pregnant mice

The number of plaque forming cells (PFC) in spleens from untreated virgin and syngeneically pregnant CBA mice has been evaluated by use of two different hemolytic plaque assays. The total number of splenic IgM- and IgG-PFC in virgin and 16 day pregnant mice was determined by protein A-sheep red blood cell (SRBC) plaque assay. The number of both IgM- and IgG-PFC was significantly increased in spleens from pregnant mice, but the total number of IgM-PFC was several times higher than that of the IgG-PFC. In the same experiment the number of splenic anti-trinitrophenyl (TNP) PFC was determined simultaneously. The values obtained for anti-TNP PFC in the different animals were found to correlate with the values for IgM-PFC in the protein A-SRBC plaque assay. Finally, the number of splenic anti-TNP PFC was determined at different occasions during pregnancy (day 4, 8, 12, and 16). It was found that the number of anti-TNP PFC was elevated as early as the 4th day of pregnancy, and that the number of PFC per spleen increased with the length of gestation.     

Antibody formation in mouse bone marrow: III. Effects of route of priming and antigen dose

The influence of the route of priming and the dose of sheep red blood cells (SRBC) on the IgM-, IgG- and IgA-plaque-forming cell (PFC) activity in mouse bone marrow during the secondary response to SRBC was studied. After intraperitoneal and subcutaneous priming the number of IgM-, IgG- and IgA-PFC during the secondary response increased with the number of SRBC used for priming. After intravenous (i.v.) priming the priming dose—secondary response relationship was found to be an optimum curve for IgM-PFC as well as for IgG- and IgA-PFC. A peak secondary response in the bone marrow was found after priming with 10⁷ and with 10⁸ SRBC i.v.

The appearance of IgG- and IgA-memory cells in the bone marrow after i.v. immunization with SRBC was shown to be dependent on the priming dose: the appearance of IgG-memory cells required a higher dose of SRBC than the appearance of IgM-memory cells, and the appearance of IgA-memory cells required even more antigen.

The effect of the booster dose upon the PFC activity was studied in mice primed with 10⁷ SRBC i.v. and boosted with 10⁴, 10⁶, or 10⁹ SRBC i.v. 2 months later. IgM-PFC as well as IgG- and IgA-PFC were present in spleen and bone marrow at each booster dose tested. In each group of mice the PFC activity in the bone marrow rose to a level which surpassed the level in the other lymphoid organs between the 7th and 10th day after the booster injection. Thus, independently of the booster dose, mouse bone marrow is the major source of PFC during the late phase of the secondary response to SRBC. Mice boosted with 10⁹ SRBC i.v. showed a prolonged presence of IgM-, IgG- and IgA-PFC in the thymus. Light and electron microscopic studies revealed the presence of plasma cells and macrophages studded with phagocytosed material in the thymic medulla of these mice, thus providing evidence for a real PFC response within the thymus. A thymic PFC response was absent in mice boosted with either 10⁴ or 10⁶ SRBC i.v. Therefore the thymic PFC activity during the secondary response to SRBC does depend on the booster dose.

     

Differential induction of help and suppression in mice by bifunctional antigens administered via different routes

Bifunctional antigens composed of one L-tyrosine-p-azobenzenearsonate (Tyr-ABA) carrier epitope and one dinitrophenyl (DNP) haptenic epitope separated by 6-aminocaproyl or polyprolyl spacers induced weak IgM anti-DNP plaque-forming cell (PFC) responses in the spleens of mice immunized intraperitoneally, without detectable IgG PFC. However, the same antigens introduced into the footpads induced IgG PFC responses in the draining lymph nodes which rose to levels greater than 100/10(6) viable lymphocytes. Moreover, the response in the lymph nodes to booster injections of antigen was characteristic of secondary T-dependent antibody responses, whereas the splenic secondary response simply mirrored the primary. The magnitude of the IgG PFC response was influenced by the size of the spacer and by the strain of mice, although genetic control did not map to the major histocompatibility complex. Prior i.p. immunization suppressed the IgG response to subsequent immunization in the footpads. This suppression could be transferred to normal syngeneic recipient mice with spleen cells from suppressed donors. Suppressor activity was eliminated by treating the spleen cells with anti-Thy-1 antibody prior to transfer, establishing the T-cell dependency of suppression. Suppression was also induced by Tyr-ABA itself, but not by DNP-lysine, indicating the epitope specificity of the suppressor cells. Thus, bifunctional antigens induce dominant suppression in the spleen but significant help in lymph nodes.     

A fraction (FAd) from Trypanosoma cruzi epimastigotes depresses the immune response in mice.

The primary immune response to SRBC in BALB/c mice was depressed when they were injected with a fraction (FAd) obtained from Trypanosoma cruzi epimastigotes grown in LIT medium. Plaque-forming cell (PFC) number was 50% less than controls when FAd was injected i.v. 15 min before antigen in doses ranging from 70 microgram up to 400 microgram of protein. Similar depression was observed when 100 microgram FAd was injected up to 6 h before antigen. There was no shift in the peak response to SRBC, neither was depression detected, when a total of 100 microgram FAd protein was given in 20 microgram amounts twice a day before immunization. Mice injected with FAd fraction only showed no increase in background PFC. Both secondary IgM and secondary IgG PFC were depressed when FAd was given before the boosting injection. However, only IgG PFC were depressed when FAd was injected before the priming dose. The delayed-type hypersensitivity reaction to DNFB was depressed when animals were injected either during the 3 days after sensitization or with a single dose of 100 microgram of protein of FAd on day of challenge. Bone marrow colony-forming units in spleens of mice injected with FAd were depressed and nodules in the treated animals were smaller than in controls. We conclude that FAd affects humoral and cell-mediated immune responses by interfering with cell division at some stage of the cell cycle.     

Flagella-induced immunity against experimental cholera in adult rabbits.

The adult rabbit ligated ileal loop model was used to evaluate the prophylactic potential of a crude flagellar (CF) vaccine produced from the classical. Inaba strain CA401. A greater than 1,000-fold increase in the challenge inoculum was required to induce an intestinal fluid response in actively immunized adult rabbits equivalent to that produced in unimmunized animals. Similar protection was afforded against challenge with classical and El Tor biotypes of both Inaba and Ogawa serotypes. Highly virulent 35S-labeled vibrios were inhibited in their ability to associated with the intestinal mucosa of CF-immunized rabbits. The protection conferred by CF immunization was found to be superior to that of a commercial bivalent vaccine and also to that of glutaraldehyde-treated cholera toxoid. The critical immunogenic component of CF appears to be a flagella-derived protein. The immunogenicity of CF was destroyed by heat treatment, and absorption of CF-immune serum with aflagellated mutant vibrios did not diminish its ability to confer a high level of passive protection. The intestinal protection of CF-immunized rabbits was completely reversed by the introduction of both goat anti-rabbit immunoglobulins A and G, but by neither alone.     

Deficient antibody formation in the bone marrow of nude mice

The bone marrow of young adult nude mice was investigated as a site of antibody formation after intravenous immunization with the thymus-independent antigen Escherichia coli lipopolysaccharide (LPS). Mice heterozygous for the nu-gene were found to be capable of a plaque-forming cell (PFC) response in both spleen and bone marrow after primary and secondary immunization with LPS. Primary immunization of nude mice with LPS induced a normal PFC response in the spleen, but did not evoke the appearance of PFC in the bone marrow. During the secondary response the nude mice did show PFC activity in the bone marrow, but at a much lower level than their heterozygous littermates. At all time points after secondary immunization the number of splenic PFC was higher in nude mice than in the control mice.

Determination by immunofluorescence of cells containing cytoplasmic immunoglobulin (C-Ig cells) in the bone marrow of young adult nonimmune nude and heterozygous mice, revealed a three times higher number of C-IgM cells in the bone marrow of the heterozygous thymus-bearing mice. On the other hand, the number of splenic C-IgM cells was higher in the nude mice than in their heterozygous littermates. These results suggest that the presence of the thymus facilitates the appearance of mature antibody-forming cells in the bone marrow of young adult mice, irrespective of whether the generation of these cells is initiated by so called thymus-dependent or thymus-independent antigens.

     

Antibody formation in mouse bone marrow: I. Evidence for the development of plaque-forming cells in situ

Mouse bone marrow as a source of plaque-forming cells (PFC) was studied by single and multiple intravenous injections of sheep erythrocytes (SRBC). In the primary response a great number of PFC appeared in the spleen while only a few showed up in the marrow. In the secondary response there is a clear PFC-response in both the spleen and the bone marrow. The spleen contains the majority of PFC until about 9 days after the second injection. In the course of the reaction, however, the number of PFC in the bone marrow rises to a level which surpasses the level in the spleen. IgM-PFC as well as IgG-PFC and IgA-PFC could be demonstrated in the marrow. In the lymph nodes and Peyer's patches the number of PFC did not increase above the normal background level at any time after the primary or secondary immunization. In order to test whether the bone marrow PFC-response is caused by a migration of PFC from the spleen or by development in situ, mice were splenectomized shortly before the second injection of SRBC. It could be shown that splenectomy does not prevent the bone marrow PFC-response. Because no activity in the other lymphoid organs was observed, it is concluded that the PFC activity of the marrow is caused by development in situ.     

Antibody formation in mouse bone marrow: VI. The regulating influence of the spleen on the bone marrow plaque-forming cell response to Escherichia coli lipopolysaccharide

Mouse bone marrow is capable of a distinct plaque-forming cell (PFC) response after i.v. immunization with the thymus-independent antigen E. coli lipopolysaccharide (LPS). Both during the primary and secondary response to i.v. administered LPS the spleen contained the majority of PFC until about 5 days after immunization. In the course of the reaction the number of PFC in the bone marrow rose to a level which surpassed the level in the spleen. This paper deals with the regulating influence of the spleen on the primary and secondary anti-LPS PFC response in the bone marrow.

Splenectomy prior to the first injection of 5 μg LPS i.v. initially did not affect the bone marrow PFC response. However, at the 7th day after immunization the PFC response in the bone marrow fell to only 10 per cent of the bone marrow PFC activity in sham-splenectomized mice. In contrast to the primary response no regulating influence of the spleen on the bone marrow PFC activity could be demonstrated during the secondary response. The influence of splenectomy on the appearance of B-memory cells in the bone marrow depended on the priming dose. The appearance of LPS-specific B-memory cells in the bone marrow was not affected by splenectomy at priming doses of LPS as high as 1 and 0.1 μg. On the other hand splenectomy before 0.01 μg LPS i.v. reduced, and splenectomy prior to 0.001 μg LPS i.v. completely prevented the appearance of B-memory cells in the bone marrow.

     

Mucosal and Systemic Immune Responses in Humans after Primary and Booster Immunizations with Orally Administered Invasive and Noninvasive Live Attenuated Bacteria

The mucosal and systemic immune responses after primary and booster immunizations with two attenuated live oral vaccine strains derived from a noninvasive (Vibrio cholerae) and an invasive (Salmonella typhi) enteric pathogen were comparatively evaluated. Vaccination with S. typhi Ty21a elicited antibody-secreting cell (ASC) responses specific for S. typhi O9, 12 lipopolysaccharide (LPS), as well as significant increases in levels of immunoglobulin G (IgG) and IgA antibodies to the same antigen in serum. A strong systemic CD4(+) T-helper type 1 cell-mediated immune (CMI) response was also induced. In contrast to results with Ty21a, no evidence of a CMI response was obtained after primary immunization with V. cholerae CVD 103-HgR in spite of the good immunogenicity of the vaccine. Volunteers who received a single dose of CVD 103-HgR primarily developed an IgM ASC response against whole vaccine cells and purified V. cholerae Inaba LPS, and seroconversion of serum vibriocidal antibodies occurred in four of five subjects. Serum IgG anti-cholera toxin antibody titers were of lower magnitude. For both live vaccines, the volunteers still presented significant local immunity 14 months after primary immunization, as revealed by the elevated baseline antibody titers at the time of the booster immunization and the lower ASC, serum IgG, and vibriocidal antibody responses after the booster immunization. These results suggest that local immunity may interfere with colonization of the gut by both vaccine strains at least up to 14 months after basis immunization. Interestingly, despite a low secondary ASC response, Ty21a was able to boost both humoral (anti-LPS systemic IgG and IgA) and CMI responses. Evidence of a CMI response was also observed for one of three volunteers given a cholera vaccine booster dose. The direct comparison of results with two attenuated live oral vaccine strains in human volunteers clearly showed that the capacity of the vaccine strain to colonize specific body compartments conditions the pattern of vaccine-induced immune responses.     

The role of Peyer's patch cells in antibody formation

The role of Peyer's patches in primary and secondary immune response was studied with Vibrio cholerae as the antigen. V. cholerae E1 Tor ME 7 is a typical enteric antigen which was shown to be suitable for the demonstration of antibody forming cells by localized lysis in agar. No plaque-forming cells (PFC) against this antigen were found in the organs of non-immunized mice. Consequently a primary immune response could be studied. In vitro an immune response against this antigen could only be obtained with spleen cells when the donor animal had been primed.

After primary intraperitoneal or enteral immunization many PFC were found in the spleen but none in Peyer's patches. This cannot be explained by insufficient penetration of the antigen into Peyer's patches as secondary responses were obtained with both routes of immunization.

After local injection of antigen directly into Peyer's patches, PFC only appeared after 5 days. This is an indication that an essential cell type for the immune response is lacking in Peyer's patches. The cells from Peyer's patches were unable to restore the immune response in lethally irradiated mice, unless they were injected together with thymus cells or cells from peripheral lymph nodes. This suggests the absence of active T cells and indicates the presence of B cells in Peyer's patches. The combination of thymus cells and bone marrow cells was inactive in restoring the immune response. This is an indication that the B cells in Peyer's patches are immunologically more mature than bone marrow cells.

     

The glucuronoxylomannan of Cryptococcus neoformans serotype A is a type 2 T-cell-independent antigen.

The humoral immune response of inbred mice to immunization with the glucuronoxylomannan (GXM) of Cryptococcus neoformans was investigated both serologically and in plaque-forming cells (PFCs). The T-helper-cell-independent quality of the GXM was demonstrated by using BALB/c nu/nu mice. Primary and secondary dose responses to three antigenic forms of GXM, (i) the native antigen, (ii) a GXM-bovine serum albumin protein conjugate, and (iii) a cryptococcal whole-cell vaccine, revealed a lack of isotype class switching and anamnestic responses. Both the levels of complement-fixing anti-GXM antibody in serum and the PFC responses in the athymic mice showed no significant differences from those in the wild-type controls. However, T cells are involved in the suppression of the primary response to GXM. When BALB/cBy mice were given rabbit anti-mouse thymocyte serum along with 0.5 microgram of GXM, both antibody levels in serum and PFC responses were significantly increased over those of control mice that received GXM and normal rabbit serum. In addition, T cells were also shown to enhance the primary immune response to GXM. BALB/cBy mice were given GXM and anti-mouse thymocyte serum on day 1. On day 2, the experimental group was given anti-mouse thymocyte serum and the control group was given saline. On day 5, comparison of the PFC responses and anti-GXM antibody titers of the two groups revealed a significant increase in the immune response of the control over the experimental group. The type 2 T-cell-independent quality of GXM was also demonstrated in CBA/cHN xid mice. These mice lack the Lyb+ subset of B cells and are unable to respond to type 2 T-independent antigens but respond normally to type 1 T-independent antigens. Type III pneumococcal polysaccharide, a type 2 T-independent antigen, was used as a negative control, and trinitrophenyl-lipopolysaccharide, a type 1 T-independent antigen, was used as a positive control. The CBA/cHN xid mice failed to respond to either type III pneumococcal polysaccharide or GXM but did not respond to immunization with trinitrophenyl-lipopolysaccharide. BALB/cBy mice responded normally to all three antigens.     

Immune Response to Mycobacterium leprae: Plaque-Forming Cells in Mice

Intravenous immunization with a cell extract of Mycobacterium leprae produced a primary immune response of considerable magnitude, followed by an equally large response after secondary stimulation, as measured by assay of plaque-forming cells (PFC). Infection with M. leprae or immunization with cell extract by the footpad route produced a lower level of response than that seen in the intravenous group. Identical patterns of response, although not of the same magnitude, were observed after both primary and secondary challenges in the two footpad groups, one infected with viable M. leprae and the other immunized with M. leprae cell extract. The secondary response after a booster dose to all these groups appeared to be an enhanced immunoglobulin M response. Control studies confirmed that the immune response was a direct result of the host-parasite interaction and that the PFC observed resulted from stimulation of antibody-forming cells by antigens of M. leprae. The similarity in time of appearance of peak PFC levels in the two footpad groups may be attributed to the live challenge passing through a latent phase. Alternatively, the challenge is known to contain a large proportion on nonviable cells, and it may also contain soluble M. leprae antigens. Studies of the cross-reactivity of the antigens have extended previous observations on antigens shared between M. leprae and other mycobacterial species. Use of the two antigen-containing fractions of the M. leprae cell extract has suggested that one of the fractions contains some shared antigens, whereas the other has an antigen specific to M. leprae.     

Facb rosette-forming cells in mice: studies on their functional significance.

Lymphocytes bearing receptors for the Facb fragment of IgG have been shown previously to be elevated in the peripheral blood of patients with rheumatoid arthritis. The generation of these cells and their possible functional role in immune regulation have been investigated in mice. Facb rosette-forming (Facb-R+) lymphocytes were found to be elevated in the spleens of mice mounting a secondary plaque-forming cell (PFC) response to sheep erythrocytes but not during the primary response. Splenic Facb-R+ lymphocytes were also elevated when a cross-reacting antigen (goat erythrocytes) was used for the secondary immunization but not when a non-cross-reacting antigen (chicken erythrocytes) was used. Both primary and secondary immunization with bacterial lipopolysaccharide resulted in elevation of splenic Facb-R+ lymphocytes. Administration of antigen-specific Facb fragment in conjunction with antigen (calf erythrocytes) produced a suppression of the secondary PFC response. However, F(ab')2 fragments produced no such effect. This suppressive effect was shown to be antigen-specific since administration of Facb fragment of anti-calf erythrocyte IgG had no suppressive effect on the secondary PFC response to sheep erythrocytes. No change in splenic Facb-R+ lymphocytes was observed during delayed hypersensitivity responses to either sheep erythrocytes or the contact-sensitizing agent oxazolone. These results indicate that Facb-R+ lymphocytes are generated during secondary humoral responses but not cell-mediated immune responses, and suggest that these cells may exert a suppressive influence on antibody production. These findings are discussed in relation to the occurrence of these cells in patients with rheumatoid arthritis.     

Bispecific cells among IgM and IgG producers during the early phase of primary and secondary responses.

Simultaneous immunization of mice with sheep (SRBC) and horse (HRBC) erythrocytes regularly resulted in the appearance of hemolytic plaque-forming cells (PFC) specific for each type of erythrocyte and also of PFC lysing both types of erythrocytes. After primary stimulation the highest number of bispecific cells (42/10(6) cells) was found among PFC as revealed by the direct procedure (IgM producers). Among PFC enhanced with anti-mouse Fab serum (IgG producers), bispecific cells were less numerous (8/10(6) cells). In preparations enhanced by anti-mouse-gammaFc serum which reveals IgG producers without inhibiting IgM antibody, the number of bispecific PFC equalled the sum of bispecific cells revealed by direct and anti-Fab enhanced procedures. The number of direct bispecific PFC during primary and secondary response was approximately the same, whereas the number of IgG-producing, bispecific PFC increased considerably during the secondary response. Another difference was the time limitation of the appearance of bispecific cells: after primary immunization direct bispecific PFC were detected only on days 3, 4 and 5, but enhanced bispecific PFC were present from day 4 up to day 12. However, during the secondary reaction, bispecific PFC were detected by all three procedures only between days 3 and 6. Studies on the cross-reactivity between SRC and HRBC gave negative results at the humoral level, even when the mice were primed with a minimal amount of both erythrocytes and then two months later, boosted with one of them. Studies at the cellular level showed that after immunization with one antigen, only 0.4 to 0.7 direct or enhanced PFC/10(6) cells could simultaneously lyse both erythrocyte types. Thus, a hundred times more bispecific PFC were constantly found after double immunization of the animals. Moreover, sudden disappearance of all bispecific PFC on the 7th day after secondary stimulation makes it unlikely that all bispecific PFC are simply cross-reacting cells.     

Water-soluble adjuvant obtained from Bacterionema matruchotii.

The adjuvant effect of a butanol-extracted water-soluble adjuvant (bu-WSA) obtained from Bacterionemia matruchotii, a gram-positive oral bacteria, was studied on the antibody response at the plaque-forming cell (PFC) level in murine spleens. Intraperitoneal injection of Bu-WSA caused significant increase in direct PFC numbers in spleens 1 to 3 days after the antigenic stimulation with sheep erythrocytes (SRBC). Injection of 100 to 800 microgram of Bu-WSA was effective, and 400 microgram of Bu-WSA seemed to be the optimum for induction of the adjuvant effect. The adjuvant effect was strongest when Bu-WSA was injected at the same time as the SRBC, but some effect was still observed when Bu-WSA was injected 7 days before or 1 day after the immunization. The adjuvant effect of Bu-WSA was greatest at high dose of antigen. The mice injected with Bu-WSA at the time of priming SRBC and then immunized with trinitrophenylated SRBC showed greater anti-trinitrophenyl PFC response than controls without the injection of Bu-WSA. These findings suggest that a part of the adjuvant effect of Bu-WSA depends on thymic cell function and another part does not.     

Safety, immunogenicity, and efficacy against cholera challenge in humans of a typhoid-cholera hybrid vaccine derived from Salmonella typhi Ty21a.

A live oral vaccine consisting of attenuated Salmonella typhi Ty21a expressing Vibrio cholerae O1 Inaba lipopolysaccharide (LPS) O antigen was constructed and tested in volunteers for safety, immunogenicity, and efficacy. Fourteen adults ingested three doses of 10(10) viable organisms with buffer. One month later, 8 vaccinees and 13 unimmunized controls were challenged with 10(6) pathogenic V. cholerae O1 E1 T or Inaba organisms. No significant adverse reactions to vaccination were observed. All volunteers had significant rises in serum immunoglobulin G (IgG) antibody to S. typhi LPS. Only 2 (14%) of 14 had significant rises in serum IgA or IgG antibody to Inaba LPS, and 5 (36%) of 14 had fourfold rises in vibriocidal antibody. In the challenge study, diarrhea occurred in 13 of 13 controls and 6 of 8 vaccinees (vaccine efficacy, 25%; P = 0.13). The vaccine significantly reduced the severity of the clinical illness (P less than 0.05) and caused decreased excretion of challenge vibrios (P less than 0.05). Although the typhoid-cholera hybrid vaccine did not provide significant protection overall against experimental cholera, this study demonstrates the importance of antibody to V. cholerae O antigen in ameliorating clinical illness and illustrates the use of an S. typhi carrier vaccine strain expressing a foreign antigen.     

Effect of Mechlorethamine on SRBC-Induced Secondary Antibody Response in Mice

Abstract

The effect of low-dose mechlorethamine (5μg/kg) on secondary humoral response to sheep red blood cells (SRBC), depending on time of exposure to the drug in relation to priming and challenge was studied in Balb/c mice. It was found that mechlorethamine in a dose of 5 μg/kg stimulated primary humoral response to SRBC resulting in the increased number of the plaque forming cells (PFC) and hemagglutinin titre (19S + 7S). However, this effect waned 10 days after immunization. On the other hand, the same mechlorethamine dose potentiated secondary humoral response to SRBC and increased the number of PFC and anti-SRBC hemagglutinin titres (notably 7S), which was due to the challenging antigenic stimulus. In each immunization, mechlorethamine administration prolonged the potentiating effect of the drug on anti-SRBC hemagglutinin titre. When mechlorethamine was administered to the mice only after priming, the number of PFC increased, but anti-SRBC hemagglutinin titre (7S) remained unchanged. This was likely due to the fact that mechlorethamine administered after priming increases the number of long-lived lymphocytes B, which in turn affect secondary humoral response.     

Antibody Formation in Mouse Bone Marrow During Secondary Type Responses to Various Thymus-Independent Antigens

The data presented in this paper show that different thymus-independent (TI) antigens have a differential capacity of inducing antibody formation in mouse bone marrow, both after primary and secondary intravenous immunization. Primary immunization with certain TI antigens (e.g., lipopolysaccharide [LPS], TNP-LPS, DNP-Ficoll) induces the appearance of antibody-forming cells not only in the spleen, but also in the bone marrow. A single injection of certain other TI antigens (e.g., pneumococci [Pn], TNP-conjugated detoxified LPS [TNP-dLPS], TNP-conjugated Brucella abortus bacteria [TNP-BA]), on the other hand, induces antibody formation in the spleen only. After secondary immunization with these TI antigens only TNP-BA induces a PFC response in the bone marrow. Pn, TNP-dLPS and TNP-BA, but also DNP-Ficoll, are unable to induce bone marrow antibody formation after secondary injection of the antigen, in spite of the clear-cut secondary type character of the splenic response. Thus, the absence of a bone marrow PFC response after secondary immunization with these antigens is not due to a failure to induce memory B cells. This data implies that either two subpopulations of memory B cells exist, one giving rise to antibody formation in the spleen and the other accounting for the bone marrow response, or that antibody can selectively inhibit the secondary bone marrow antibody response to certain TI antigens.     

Stimulation of humoral antibody formation by polyanions. IV. The effects of dextran sulfate on the kinetics of secondary immune response in mice

Dextran sulfate enhances secondary immune response as well as primary immune response in mice primed and challenged with suboptimal doses of sheep red blood cells. Enhancement of primary response by dextran sulfate did not diminish secondary responses towards suboptimal antigen dose. However, in mice primed and challenged with an optimal antigen dose and injected at the time of priming with dextran sulfate, immunological memory seems to be increased. In primary as well as in secondary response the number of both 19 S and 7 S PFC were increased.     

Effect of Mechlorethamine on SRBC-Induced Secondary Antibody Response in Mice

The effect of low-dose mechlorethamine (5μg/kg) on secondary humoral response to sheep red blood cells (SRBC), depending on time of exposure to the drug in relation to priming and challenge was studied in Balb/c mice. It was found that mechlorethamine in a dose of 5 μg/kg stimulated primary humoral response to SRBC resulting in the increased number of the plaque forming cells (PFC) and hemagglutinin titre (19S + 7S). However, this effect waned 10 days after immunization. On the other hand, the same mechlorethamine dose potentiated secondary humoral response to SRBC and increased the number of PFC and anti-SRBC hemagglutinin titres (notably 7S), which was due to the challenging antigenic stimulus. In each immunization, mechlorethamine administration prolonged the potentiating effect of the drug on anti-SRBC hemagglutinin titre. When mechlorethamine was administered to the mice only after priming, the number of PFC increased, but anti-SRBC hemagglutinin titre (7S) remained unchanged. This was likely due to the fact that mechlorethamine administered after priming increases the number of long-lived lymphocytes B, which in turn affect secondary humoral response.