Target cells for the activity of a synthetic adjuvant: muramyl dipeptide.

Muramyl dipeptide (MDP), a synthetic adjuvant, increased the primary response of CBA mice to sheep red blood cells (SRBC). In reconstituted irradiated recipients, cooperation between T and B lymphocytes was required for the expression of adjuvant activity and MDP increased the efficiency of SRBC-educated T cells. The role of T-derived lymphocytes in mediating the MDP adjuvant activity was also demonstrated in irradiated mice and in mice reconstituted with various splenic cellular types of donors which had received SRBC and MDP 24 hr earlier. In our experiments, the macrophage did not seem to be involved, since MDP did not increase the phagocytic capacity of peritoneal exudate cells and MDP- and SRBC-pretreated macrophages had no increased ability to induce an anti-SRBC immune response. These results demonstrate the importance of T lymphocytes as mediators of the adjuvant activity of MDP.     

The effect of Bordetella pertussis lymphocytosis-promoting factor (LPF) on antibody response in mice: its enhancing and suppressive effects

The effect of lymphocytosis-promoting factor (LPF) on antibody response in mice was estimated under different sets of experimental conditions. Four- and 6-week-old mice were intravenously inoculated with LPF. Three days later these mice were inoculated either intraperitoneally or intravenously with sheep red blood cell (SRBC) or human serum albumin (HSA) as an antigen. The adjuvant effect of LPF was demonstrated on antibody response in 6-week old mice to intraperitoneally inoculated SRBC but not to intravenously-inoculated one. When 4-week-old mice were immunized, hemagglutinin production in response to intraperitoneally inoculated SRBC was not enhanced by LPF. In addition, a rather suppressive effect of LPF at a comparatively high dose was demonstrated on hemagglutinin production in response to intravenously inoculated SRBC. Anti-HSA production was enhanced by inoculation of LPF in any combination of the mouse age and the route of antigen administration. These findings indicate that the adjuvant effect of LPF on antibody response in mice depends upon experimental conditions: the age of mice, the quality of antigen and the route of antigen administration used for immunization.     

Synergy between T cell-replacing factor and bacterial lipopolysaccharides (LPS) in the primary antibody response in vitro: a model for lipopolysaccharide adjuvant action.

Unfractionated spleen cells, B cells from normal mice, and nu/nu spleen cells respond to the addition of bacterial lipopolysaccharide (LPS) and T-cell-replacing factor (TRF) by production of plaque-forming cells (PFC) in excess of the number expected from the addition of LPS and TRF separately. This synergistic activity is dependent on the presence of the antigen, SRBC. Supernatants of both allogeneic spleen cell mixtures and spleen cells cultured with Con A are effective and synergize best at concentrations suboptimal for their ability to act as TRF alone. Culture supernatants of unstimulated normal or fractionated cell populations are ineffective. Synergy is not dependent on the presence of macrophages in the cultures. Purified LPS free from active contaminants, as well as commercially available LPS, show synergy with TRF. Synergy was seen when TRF was added at initiation of culture or 24 hr later. It is suggested that synergy is the equivalent of LPS adjuvant activity, that the role of T cells in LPS adjuvanticity is that of a conventional cooperating cell, and the LPS acts as an adjuvant by inducing B cells to become more sensitive to T cell helper factors.     

A study of the mechanism of Con A-induced immunosuppression in vivo

The plaque-forming cell (PFC) response to sheep erythrocytes (SRBC) is suppressed in a dose-related manner when concanavalin A (Con A) is administered intravenously to mice prior to or after immunization with antigen. The magnitude of suppression as well as the duration of the Con A effect greatly depends on the concentration of antigen used for immunization. Although profound suppression of the anti-SRBC PFC response is observed in intact mice pretreated with Con A for 4-24 hr, spleen cells from these mice do not exhibit suppressive activity when transferred into normal recipients or when cotransferred with normal spleen cells into irradiated recipients. Moreover, the cells from Con A-treated mice respond as normal spleen cells to SRBC when transferred alone into irradiated hosts. Suppression of the anti-SRBC PFC is only observed when adoptive hosts of cells from Con A-treated mice are also injected with Con A within 48 hr (but not 72 hr) of cell transfer and immunization. This time course of responsiveness to the suppressive effects of Con A is similar to that observed in normal mice and in irradiated recipients of normal spleen cells. The immune response to SRBC is also suppressed in adoptive hosts of normal spleen cells that are pretreated with Con A 4-24 hr prior to irradiation and cell transfer. Although functionally inactive when transferred into adoptive hosts, spleen cells from mice pretreated with Con A for 4-24 hr can suppress a primary antibody response to SRBC in vitro. The suppressive activity, which cannot be detected in the spleens of mice when the interval between pretreatment and assay is longer than 24 hr, is present in a subpopulation that bears the Thy 1.2 and Lyt 2 phenotype. Taken together the results obtained in in vivo and in vitro functional assays suggest that a suppressor cell population is activated following in vivo treatment with Con A, but that the cells rapidly lose their state of activation when removed from a Con A environment. This phenomenon is in all probability responsible for the failure to demonstrate suppressive activity in the spleens of Con A-treated mice using in vivo functional assays.     

In vivo immunostimulating activity of the 163-171 peptide of human IL-1 beta

The stimulating effect of a synthetic nonapeptide (fragment 163-171) of human interleukin 1 beta (IL-1 beta) on antibody responses to both T helper-dependent and T helper-independent antigens was investigated. It was shown that the nonapeptide enhanced the antibody response, as evaluated in the hemolytic plaque assay, of spleen cells from mice immunized with sheep red blood cells (SRBC). The activity of the 163-171 peptide on the primary response to SRBC was dose-dependent, being maximal when the peptide was inoculated at 100 mg/kg together with the antigen. Moreover, the 163-171 peptide was also effective in enhancing the secondary response to SRBC. The effect of the 163-171 peptide was to augment the frequency of cells specific for the antigen, inasmuch as no increase was ever observed in spleen cell numbers after treatment. In all these studies, human recombinant IL-1 beta gave effects qualitatively comparable to those of the 163-171 peptide, with a maximal activity at 20 ng/kg. Both the 163-171 peptide and human recombinant IL-1 beta were also able to enhance the in vivo immune response to a T helper-independent antigen such as SIII, a poorly immunogenic polysaccharidic antigen from Streptococcus pneumoniae type III. It can therefore be proposed that this synthetic nonapeptide of human IL-1 beta may represent a good candidate for use as adjuvant in vaccines.     

Antibody formation in mouse bone marrow. IX. Peripheral lymphoid organs are involved in the initiation of bone marrow antibody formation.

Mouse bone marrow is barely capable of plaque-forming cell (PFC) activity during the primary response to sheep red blood cells (SRBC). However, during secondary-type responses, it becomes the major organ, containing IgM, IgG, and IgA PFC. In the present paper, the influence of splenectomy (Sx) upon the secondary bone marrow PFC response to SRBC was investigated. When previously primed mice were splenectomized just before the second intravenous (iv) injection of SRBC, the effect of Sx upon the height of the bone marrow PFC response was dependent on the booster dose. Sx just before a booster of 10⁶ SRBC iv almost completely prevented bone marrow PFC activity, whereas an iv booster dose of 4 × 10⁸ SRBC evoked a normal IgM, IgG, and IgA PFC response in Sx mice. Apparently low doses of iv administered antigen require the spleen in order to evoke antibody formation in the bone marrow. Experiments with parabiotic mice, consisting of Sx and sham-Sx mice, showed that this facilitating influence of the spleen upon bone marrow antibody formation occurs via the blood stream. In a subsequent study, it was investigated whether the spleen is required throughout the bone marrow PFC response or only during the few days of the initiation phase. Therefore, mice were splenectomized at different intervals after a booster injection of 10⁶ SRBC iv. It appeared that Sx 2 days after the booster injection could still prevent the normal bone marrow PFC activity, whereas Sx at Day 4 could no longer do so. Apparently, after an iv booster injection, the spleen is only required for initiation of the bone marrow PFC response and not for the maintenance of this PFC activity thereafter.     

Expression of a sheep red blood cell receptor by a murine lymphoma.

Murine 6C3HED lymphoma cells were found to rosette with sheep red blood cells (SRBC). Normal C3H lymphocytes did not exhibit this property. This rosetting capacity of 6C3HED cells was found to be an accurate and reproducible means for discriminating between normal and tumor cells. The SRBC receptor on these lymphoma cells appeared to be serologically distinct from that expressed by normal human T lymphocytes since reciprocal blocking experiments demonstrated that inhibitory antisera were not cross-reactive. The expression of the SRBC receptor by the 6C3HED cells appeared to correlate with the expression of a tumor-associated antigen and was spatially related to an antigen expressed by 6C3HED and normal neonatal but not adult mouse cells.     

Lymphocyte function in experimental African trypanosomiasis. VIII. Loss of suppressor T cell function in lymph nodes

The immunosuppression that occurs in mice experimentally infected with African trypanosomiasis has been examined further. In the present study we have examined lymph node cells from Trypanosoma rhodesiense-infected C57Bl/6J mice for the ability to produce mitogen induced antigen-nonspecific suppressor T cells (Ts). Inguinal, mesenteric, and brachial lymph node cells were harvested from uninfected control mice and from mice at different periods of infection. These cells were cultured with or without concanavalin A (Con A) for 48 hr to induce Ts activity. After stimulation, the control and infected lymph node cells were passed over Sephadex G-10 columns to remove suppressor macrophages that arise during the infection from Con A-induced Ts. The column passed cells were then added to normal mouse responder spleen cells in a primary in vitro antibody response culture system with sheep erythrocytes (SRBC) as antigen. The resultant plaque-forming cell responses to SRBC indicated that Ts function was not induced in infected lymph node cell populations. However, early in the infection, a stimulatory signal was provided by both the untreated and Con A-treated infected lymph node cells, which was lost in the terminal stage. Determinations of T cell subpopulations revealed that the infected Lyt 2.2-bearing subpopulation was not significantly altered from normal controls. We conclude that T. rhodesense infected mice fail to mount normal lymph node cell antigen nonspecific Ts responses and that this loss of activity may be due to an intrinsic dysfunction in the suppressor T cell population.     

Relation between Enhanced Antibody Responses Elicited by Adjuvant Injection and Those Elicited by Secondary Antigenic Stimulation

An attempt was made to determine if there is any common mechanism in the enhanced antibody response caused either by injection of adjuvant, such as bacterial endotoxin (LPS) and complexed polynucleotides, or by secondary antigenic stimulation. LPS inoculated in mice 4 days before injection of sheep red blood cells (SRBC) and polyA:U invalidated the adjuvant effect of polyA:U injected together with SRBC, and the hemolysin plaque-forming cell (PFC) response of such mice was similar to that of the mice which received SRBC alone. When mice primed with SRBC 24 days in advance were injected with LPS and 4 days later re-stimulated with SRBC, their PFC response to the secondary stimulation was suppressed to less than one tenth of the normal secondary PFC response. The suppressive effect of LPS on the secondary antibody response was abolished if the serum collected from mice injected with LPS was given to the primed and LPS-injected mice at the time of the secondary antigenic stimulation. From these results we discussed the possibility that some common mediator might play a role in the enhanced antibody response elicited by either adjuvant injection or secondary injection of antigen.     

Functional characteristics of Peyer's patch lymphoid cells. IV. Effect of antigen feeding on the frequency of antigen-specific B cells.

The frequency of B cells in Peyer's patches from normal BDF(1) and sheep red blood cell (SRBC)-fed BDF(1) mice that could respond to antigenic determinants on SRBC and trinitrophenyl (TNP) was determined using an in vitro system of limiting dilution analysis. In normal mice, one B cell in 1.9 x 10(4) Peyer's patch cells could be induced to an anti-SRBC response and one B cell in 3.6 x 10(4) Peyer's patch cells could be induced to an anti-TNP response. The frequency of B cells capable of responding to SRBC in normal mice was similar in Peyer's patches and spleen. However, after feeding mice SRBC for 3 weeks, there was a 6-fold reduction in the frequency of B cells in Peyer's patches capable of responding to SRBC but no change in the frequency of B cells capable of responding to TNP. The average clone size of Peyer's patch B cells responding to SRBC was similar in normal and SRBC-fed mice. Although antigen-feeding does not stimulate Peyer's patch B cells in situ to humoral antibody synthesis, antigen-feeding can markedly alter the reactivity of the antigen-sensitive cell population in Peyer's patches. We previously demonstrated that T cells in Peyer's patches could be specifically carrier primed for helper function by SRBC feeding. We have now demonstrated that antigen-feeding reduced significantly the frequency of B cells in Peyer's patches capable of responding to the fed antigen. Peyer's patches appear to serve an important function as a sampling site for intestinal antigens.     

Antibody formation in mouse bone marrow. VII. Evidence against the migration of plaque-forming cells as the underlying cause for bone marrow plaque-forming cell activity: a study with parabiotic mice

After intravenous immunization of mice with Escherichia coli lipopolysaccharide (LPS) or sheep red blood cells (SRBC), the bone marrow can contain large numbers of plaque-forming cells (PFC). By means of parabiosis, it was studied whether or not this appearance of PFC in the bone marrow might be due to a migration of such cells from peripheral lymphoid organs into the marrow, as has been suggested in the literature. Using parabionts consisting of nonimmunized mice and mice immunized with LPS, only background numbers of PFC could be demonstrated in the bone marrow of the nonimmunized mice. In similar experiments, with SRBC as antigen, mice showing high anti-SRBC PFC activity in the bone marrow could only provide for minor numbers of anti-SRBC PFC in the bone marrow of affixed normal mice. These results suggest that migration of PFC can not be the main cause for bone marrow PFC activity in the mouse. This provides additional evidence for our view presented in previous papers of this series that the appearance of PFC activity in the bone marrow is dependent on local maturation of B cells into PFC rather than on immigration of PFC.     

Activation of T cells by glutaraldehyde-fixed erythrocyte antigens: radiosensitivity of cells mediating delayed-type hypersensitivity reactions in the mouse.

Mice immunized with glutaraldehyde-fixed sheep red blood cells (G-SRBC) show delayed-type hypersensitivity (DTH) reactions to G-SRBC or SRBC. The specificity of the DTH reaction of mice sensitized with glutaraldehyde-fixed antigens is similar to that found after sensitization with unfixed antigens. The dose-response curve for sensitization by glutaraldehyde-fixed SRBC was very different from the curve for normal SRBC. At low doses, both antigens were effective in sensitizing to show DTH but neither induced an antibody response. However, at high antigen doses, only the glutaraldehyde-fixed antigen was efficient in sensitizing to show DTH and it failed to raise an antibody titer. Spleen cells of mice sensitized with fixed RBC can transfer DTH locally but if the donor cells are irradiated (500 R), the transfer is abrogated. In contrast, the transfer of DTH by spleen cells of mice immunized with unfixed antigen is not affected by 500 R. The transfer of DTH by spleen cells of mice immunized with fixed antigen can be blocked by “in vitro desensitization” while the transfer of DTH by spleen cells from mice primed with normal antigen is resistant to “in vitro desensitization.” These results suggest that immunization of mice with different physical states of the same antigen can result in the activation of antigen-specific T cells which exhibit markedly different properties.     

Further Studies of the Polysaccharide of Klebsiella pneumoniae Possessing Strong Adjuvanticity: III. Augmentation of the Antibody Response to Subcutaneously Injected Sheep Red Blood Cells by the Adjuvant Polysaccharide

The adjuvant action of the O3 antigen of Klebsiella (KO3) on the antibody response to sheep red blood cells (SRBC) was elucidated by injecting both KO3 and SRBC subcutaneously at the right inguinal region of SMA mice. We demonstrated that KO3 exhibits a novel ability to augment anti-SRBC plaque-forming cell responses in both the local lymph node and the spleen at a relatively late stage of immunization. Escherichia coli lipopolysaccharide, dextran sulfate and concanavalin A showed such an action only minimally. In parallel with the development of the adjuvant action, KO3 definitely activated B cells in the local lymph node polyclonally for either IgM or IgG synthesis, suggesting that the mechanism of the adjuvant action includes direct stimulation of B cells by KO3 at the late stage. Neither increase in trapping of lymphocytes in the local lymph node nor change in tissue distribution of antigen was shown to be primarily involved in the mechanism of the adjuvant action.     

Recognition of xenogeneic erythrocytes: the GalNAc/Gal-particle receptor of rat liver macrophages mediates or participates in recognition

We studied mechanisms that mediate recognition of human erythrocytes (HRBC) and sheep erythrocytes (SRBC) by rat liver macrophages. We used an in vitro cell binding assay that allows spontaneous formation of cell contacts. Binding of HRBC to rat macrophages shows the following characteristics: inhibition studies with several monosaccharides and oligosaccharides yield complete inhibition of cell contacts with saccharides, which block the GalNAc/Gal-particle receptor on rat liver macrophages. We found the inhibition pattern: N-acetyl-D-galactosamine, lactose greater than D-galactose, D-fucose greater than L-fucose much greater than N-acetyl-D-glucosamine. Cell binding is dependent on the presence of calcium ions, but not influenced by heat-aggregated IgG or gangliosides. The inhibition pattern was the same after treatment of HRBC with neuraminidase. Therefore, binding of HRBC, as well as binding of neuraminidase-treated HRBC, is mediated by the GalNAc/Gal-particle receptor. Binding of SRBC is partly inhibited by galactose-related saccharides. Binding is also partly inhibited by heat-aggregated IgG, gangliosides, and L-fucose. Complete inhibition of cell contacts with SRBC is achieved by combination of all inhibitors. We therefore conclude that binding of SRBC is mediated by several different mechanisms, including the GalNAc/Gal-particle receptor. Binding of neuraminidase-treated SRBC, however, was found to be completely inhibited by saccharides, which block the GalNAc/Gal-particle receptor. We conclude that the GalNAc/Gal-particle receptor mediates or participates in recognition of non-self structures.     

Effect of Neisseria meningitidis on the humoral response of various strains of mice immunized with ram erythrocytes

The capacity of heat-killed meningococci and the polysaccharide-protein-lipopolysaccharide fraction ( PPLF ) isolated from the microbial cell wall for changing nonspecific immunological reactivity was studied. In this investigation CBA mice with high response and C57BL/6 mice with low response to sheep red blood cells (SRBC) were used. Heat-killed N. meningitidis, serogroup A, and PPLF , serogroups A and B, were found to enhance and suppress humoral response to the heterologous antigen. The effect of modulation depended on the dose of the antigen, the serogroup of meningococci, the scheme of the experiment and the strain of mice. The immune response of the vaccinated animals to the heterologous antigen was characterized by the following stages: the state of the adjuvant effect was replaced by the state of temporary immunodeficiency and then by enhanced response to SRBC.     

Combinations of two synthetic adjuvants: synergistic effects of a surfactant and a polyanion on the humoral immune response

Synergistic effects of two synthetic adjuvants, dimethyldioctadecylammonium bromide (DDA) and dextran sulfate (DXS) on the humoral response to sheep red blood cells (SRBC) were investigated. Mice received intraperitoneal (ip) injections of adjuvant and antigen simultaneously. The number of plaque-forming cells (PFC) in the spleen were determined 5 days later and circulating anti-SRBC antibodies were measured till 16 weeks after immunization. Although combinations of DDA and DXS were very effective in enhancing the PFC response to both moderate (2 X 10(7] and low (2 X 10(6] doses of SRBC, synergy between the adjuvants was only observed at the low dose of SRBC. Optimal augmentation of the primary response to the low antigen dose was evoked by the combination of the highest dose tested of either adjuvant (1 mumol DDA and 1 nmol DXS) resulting in a 560-fold increase of the number of PFC in the spleen as compared to controls. Even combinations of relatively small amounts of both adjuvants were very effective in augmenting the response to SRBC. Mice receiving half the amounts of both adjuvants with 2 X 10(6) SRBC displayed increased numbers of PFC in the spleen at Day 5 as well as increased titers of total anti-SRBC antibodies at Week 1 and Week 2 and 2-mercaptoethanol-resistant antibodies from Week 4 till Week 16 as compared to the calculated sum of responses in mice which received either DDA (0.05 mumol per mouse) or DXS (0.05 nmol per mouse). The mechanism behind the synergy between these adjuvants is discussed and the possibility of discerning adjuvants on their modes of action is suggested.     

A monoclonal antibody that recognizes an Ly-6-linked antigen inhibits the generation of functionally active T cell subsets

A monoclonal antibody (mAb) generated against the chemically-induced BALB/c Meth A sarcoma, designated HD42, reacts in cytotoxic tests with Meth A as well as with BALB/c peripheral lymph node cells and mitogen-activated spleen cells. The antigen was detected by FACS analysis on BALB/c spleen and lymph node cells, and by absorption assays on all normal lymphoid cells of BALB/c but not B6 mice. The expression of the antigen was not found on normal adult lung fibroblasts, on brain, nor on an extensive panel of tumors of BALB/c and B6 origin. Because the strain distribution of the antigen is reciprocal to that of Ly-6.2 and is not expressed in congenic C3H.Ly-6b mice, we have tentatively defined it as Ly-6.1 and referred to the mAb as alpha-Ly-6.1. The presence of alpha-Ly-6.1 abrogates both the Con A-induced and the IL 2-dependent proliferative response of normal T cells, whereas the response of normal B cells to LPS remains unaffected. alpha-Ly-6.1 is a potent suppressor of the primary in vitro plaque-forming cell (PFC) response to SRBC. Pretreatment of normal splenic T cells with alpha-Ly-6.1 and complement had no effect on the ability of these cells to generate in vitro either T helper cells (TH) or T suppressor cells (TS) to SRBC. However, addition of antibody in the absence of complement during the generation of TH or TS, or posttreatment of these T cell subsets with antibody and complement after in vitro education, completely removed the functional activity of these cell types. Addition of alpha-Ly-6.1 to MLC suppressed the MLR as well as the generation of cytotoxic lymphocytes (CTL), whereas the presence of the antibody during a cell-mediated lympholysis (CML) had no effect. Therefore, it appears that alpha-Ly-6.1 recognizes an antigen that is important for the generation of TH and TS cell subsets.     

Evidence that specific oligosaccharides block early events necessary for the expression of antigen-specific proliferation by human lymphocytes

We have previously shown that monosaccharides and disaccharides will block the expression of spontneous monocyte-mediated cytotoxicity. Our data were consistent with the hypothesis that human mononuclear cells express lectin-like receptors that are capable of binding to a variety sugar moieties found on target cell membranes. In this communication, we will present evidence that monosaccharides and disaccharides are also capable of blocking the expression of T cell reactivity as measured by an in vitro antigen-specific proliferative assay. The majority of sugars that blocked monocyte-mediated cytotoxicity had no effect on antigen-specific proliferation. Those sugars that did suppress antigen-induced proliferation had no effect on PHA-induced proliferation. Furthermore, some of these sugars only inhibited if they were added at the initiation of the assay; they failed to inhibit if added 24 hr after the initiation of the assay. Antigen-pulsing experiments suggested that these sugars did not block antigen uptake by human monocytes. These data suggest that a variety of cellular interactions may be mediated by receptors with specificity for simple sugars. The ability to block these naturally occurring lectins specifically both in vitro and in vivo may prove to be a powerful tool for dissecting out various forms of cellular recognition and collaboration.     

The influence of phase shift in the light-dark cycle on humoral immune responses of mice to sheep red blood cells and polyvinylpyrrolidone

The influence of a phase shift in the light-dark cycle on humoral immune responses against sheep red blood cells (SRBC), a thymus-dependent antigen, and against polyvinylpyrrolidone (PVP), a thymus-independent antigen, was studied by using 180 BALB/c mice and 150 C3H/HeN mice. Significant suppression of the immune response to SRBC and the number of splenic plaque-forming cells (PFC) and hemagglutination (HA) titers was observed on days 5 and 6 after inversion of the light-dark cycle. On the other hand, the number of splenic PFC and HA titers in the blood against PVP were minimally suppressed by the phase shift in C3H/HeN mice, except for distortion of the rhythmicity. Corticosterone levels in the blood on days 5 and 6 after inversion were higher than those under a normal lighting regimen. The appearance of the high corticosterone level in the blood after the inversion almost concurred with the suppression of the immune response to SRBC. A decrease of the proportion of splenic T cells was also observed on day 6 after the inversion. These results show that a phase shift in the light-dark cycle provokes suppression of the immune response to SRBC, possibly through an increase of secretion of corticosterone after light-dark inversion, which induces a decrease of the proportion of T lymphocytes in the spleen.     

Antibody formation in mouse bone marrow. II. Evidence for a memory-dependent phenomenon

Mouse bone marrow is barely capable of plaque-forming cell (PFC) activity in a primary response to sheep red blood cells (SRBC), while PFC activity in the secondary response to SRBC can be clearly demonstrated. This phenomenon was studied by means of cell transfer experiments.T cells, which are involved in an anti-SRBC PFC response, were shown to be very scarce in normal mouse bone marrow. This is considered to be the cause of the low PFC activity in the marrow during the primary response to SRBC.In normal mouse bone marrow precursors of IgM-PFC but not of IgG- and IgA-PFC could be found. Priming with SRBC induced the appearance of IgM-, IgG-, IgA- and T-memory cells in the marrow. These B- and T-memory cells were shown to be specific for the antigen which induced their appearance. It is thought that after a second injection of SRBC the IgM-, IgG- and IgA-memory cells can differentiate with the help of the T-memory cells within the bone marrow into IgM-, IgG- and IgA-PFC respectively.The sequence of appearance of the B-memory cells in the bone marrow was shown to be IgM-IgG-IgA.Six months after the intravenous injection of SRBC, the presence of B-memory cells could be demonstrated not only in spleen and bone marrow, but also in peripheral lymph nodes, mesenteric lymph node, Peyer's patches, thymus and blood. The increase in amount of B-memory cells was most prominent in the spleen.