Antibody formation in mouse bone marrow. VIII. Dependence on potentially circulating memory cells: a study with parabiotic mice.

Mouse bone marrow barely contains antibody-producing plaque-forming cells (PFC) during the primary response to sheep red blood cells (SRBC). However, during the secondary response, the number of IgM, IgG, and IgA PFC in the bone marrow can rise to a level which surpasses the number of PFC in all the other lymphoid organs together. In the present paper we investigated whether the capacity of immune mice to react upon a booster injection of SRBC with a bone marrow PFC response can be transferred from immune to nonimmune mice. Therefore, mice primed with SRBC 6 months previously and nonprimed syngeneic mice were joined for parabiosis and were separated from each other at various intervals after joining. These separated mice were subsequently immunized with SRBC. It was found that, after 3 weeks of parabiosis, the nonprimed members reacted upon an injection of SRBC with a bone marrow IgM, IgG, and IgA PFC response as high as did the previously primed members. Furthermore it could be demonstrated by means of cell transfer experiments that, after a period of parabiosis of 3 weeks, the bone marrow and spleen of the normal mice contained about as many memory cells as the bone marrow and spleen of the immune mice. These results suggest that antibody formation in mouse bone marrow is dependent on a population of potentially circulating memory cells.     

Secondary delayed type hypersensitivity to sheep red blood cells in mice: Dependence on long-lived memory cells

Secondary delayed type hypersensitivity (DTH) to sheep red blood cells (SRBC) in mice is a long-lived memory phenomenon which is characterized by the accelerated reappearance of the state of DTH after a booster injection of the antigen. In this paper the nature of the DTH-related T memory cells accounting for secondary DTH was investigated. Parabiosis of primed and nonprimed mice for a period of 4 weeks resulted in an equally large secondary DTH responsiveness in both partners. This ability was maintained in both members for at least 6 months after termination of the parabiosis. These results indicate that (a) DTH-related T memory cells are potentially circulating cells, and (b) the persistence of these memory cells is not dependent on the presence of the antigen which induced their generation. Subcutaneous (sc) injection of intravenously (iv) primed mice with a small dose of antithymocyte serum before boosting did prevent the development of secondary DTH responsiveness in sc boosted mice, but not in iv boosted mice. Treatment of primed mice with vinblastine or azathioprine did not decrease the capacity of adoptive transfer of secondary DTH by means of spleen cells, but passive transfer of secondary DTH was completely abolished by this treatment. These results suggest that (a) SRBC-induced DTH-related T memory cells are nonproliferating, partially sessile, partially recirculating cells, and (b) these memory cells proliferate before they become DTH-related effector cells.     

Lymphotoxin-alpha-dependent spleen microenvironment supports the generation of memory B cells and is required for their subsequent antigen-induced activation

Lymphotoxin alpha-deficient (LTalpha-/-) mice show dramatically reduced IgG responses after either primary or secondary immunizations with sheep red blood cells (SRBC). When splenocytes from SRBC-primed wild-type donor mice were infused into irradiated naive wild-type recipient mice, they generated a robust memory IgG response, but not when infused into LTalpha-/- recipients, indicating that the microenvironment that develops in LTalpha-/- mice is incompetent to support the activation of this memory response. When irradiated wild-type mice were reconstituted with splenocytes from primed LTalpha-/- donors and then challenged with the same immunizing Ag, no memory response was observed, indicating further that memory cells could not be generated in the LTalpha-/- environment. To address which lymphocyte subsets were impaired in the LTalpha-/- mice, we performed reconstitution experiments using a hapten/carrier system and T cells and B cells from different primed donors. There was no detectable defect in either the generation or expression of memory T cells from LTalpha-/- donors. In contrast, B cells were not primed for memory in the microenvironment of LTalpha-/- mice. Additionally, primed wild-type memory B cells could not express a memory IgG response in the LTalpha-/- microenvironment. Thus, splenic white pulp structure, which depends on the expression of LTalpha for its development and maintenance, is needed to support the generation of memory B cells and to permit existing memory B cells to express an isotype switched memory Ig response following antigenic challenge.     

Antigenic markers on mouse lymphoid cells: origin of cells mediating immunologic memory

Specific antisera were used for the purification of thymus dependent and thymus independent or bursa equivalent lymphoid cells in the mouse. Spleen cells from mice immune to sheep erythrocytes, a thymus dependent antigen, or to E. coli 055:B5 lipopolysaccharide, a thymus independent antigen, were treated with anti-θ (C3H) serum or anti-MBLA serum and complement prior to their adoptive transfer into lethally irradiated syngeneic recipients. Syngeneic thymocytes, bone marrow cells, or spleen cells from nonimmune donors were appropriately added to antiserum treated cells prior to transfer. The secondary response to these antigens was assayed in recipient spleens six days after cell transfer. The kinetics of the primary response to SRBC was investigated as to its effect on origin of specific hyper-reactive T or B lymphoid cells.The adoptive response to CPS originated in the B lymphoid cell population. Immunologic memory to CPS was demonstrated in recipients of immune cells, compared to recipients of normal cells, by a five fold increase in antibody forming cells.The IgM and IgG adoptive immune response to high doses of SRBC depended upon an increased number of specifically hyper-reactive T-lymphoid cells to facilitate cooperation between T and B lymphocytes. High doses of SRBC initially stimulated T cell memory but at 42 days after priming an increased number of specifically hyper-reactive B lymphoid cells were present.     

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.     

Structural and serological studies of lipopolysaccharides of Citrobacter O35 and O38 antigenically related to Salmonella

Abstract Lipopolysaccharide (LPS) was administered into sheep red blood cells (SRBC)-primed mice, and the effect of LPS on SRBC-specific memory cells was investigated. Spleen cells from SRBC-primed mice which were injected with LPS exhibited much lower in vitro secondary plaque-forming cells (PFC) responses to SRBC than those from untreated SRBC-primed mice. The in vitro anti-SRBC response of the spleen cells to LPS was also reduced. The combination experiments of B cells and T cells from SRBC-primed mice which were injected with or without LPS demonstrated that the reduction of immune responses to SRBC after administration of LPS was caused by the defect of SRBC-specific B memory cells, but not T memory cells. B cell type rosette-forming cells (RFC) for SRBC markedly decreased after injection of LPS, while PFC as antibody-forming cells did not increase subsequently. Therefore, the reduction of RFC was not due to their differentiation into PFC. The lymphoid follicles in the spleens from mice injected with LPS were stained positively by in situ nick end labeling specific for fragmented DNA. A large percentage of Ig⁺ spleen cells from SRBC-primed mice which were injected with LPS was also stained positively. The injection of glucocorticoids into SRBC-primed mice induced similar reduction of B memory cells. It was suggested that LPS might induce apoptosis of B memory cells and regulate B cell memory in antigen-nonspecific manner.     

Secondary delayed-type hypersensitivity to sheep red blood cells in mice: a long-lived memory phenomenon

Immunization of mice with sheep red blood cells (SRBC) can induce the capacity to react with a secondary delayed-type hypersensitivity (DTH) immune response upon a booster injection of the antigen. In this paper the kinetics of secondary DTH after intravenous (iv) immunization with various doses of SRBC was studied by means of the foot swelling test. Dose-response studies showed that maximal secondary DTH responsiveness was obtained by iv administration of a priming dose of 3 × 10⁴ SRBC and a booster dose of 3 × 10⁵ SRBC 2 months later. Secondary DTH in such treated mice was characterized by an earlier appearance of the state of DTH, an earlier peak reactivity, and an increased intensity of the DTH response as compared to the primary DTH response. Up to 1 year after priming, a secondary DTH could be elicited, indicating the long-lived character of this memory phenomenon. With increasing intervals between the priming and booster injection, a gradual shift to a later time, of the peak secondary DTH reactivity was found. The capacity of primed mice to react with an increased intensity upon a booster injection could be adoptively transferred into lethally irradiated recipients by means of spleen cells obtained from primed mice. This phenomenon appeared to be highly dependent on Thy 1.2⁺ cells and on the booster dose of SRBC. The DTH reaction, evoked in such recipients, showed a prolonged time course.     

B-cell suppression of antibody response to sheep red blood cells in mice of high- and low-responding genotypes.

CBA and C57B1 mice (high and low responders to sheep red blood cells, respectively) were injected intravenously with syngeneic lymph node, marrow, spleen, or thymus cells together with sheep red blood cells (SRBC), and the production of antibody-forming cells (AFC) was assayed in the spleen. Transfer of lymph node, marrow, spleen, or thymus cells led to a significant enhancement of immune responsiveness in low-responding C57B1 mice. In contrast, transfer of marrow, lymph node, or spleen cells to high-responding CBA mice was accompanied by a decline in AFC production. These effects were magnified if syngeneic cell donors had been primed with SRBC; suppression in CBA mice and stimulation in C57B1 mice were especially pronounced after transfer of SRBC-primed lymphoid cells. Pretreatment of CBA donors with cyclophosphamide in a dose causing selective B-cell depletion completely abrogated the suppression of immune responsiveness. A large dose (10⁷) of syngeneic B cells injected together with SRBC suppressed the accumulation of AFC in both CBA and C57B1 mice. No suppression of immune responsiveness was observed after transfer of intact thymus cells, hydrocortisone-resistant thymocytes, or activated T cells. We conclude that suppression of the immune response to SRBC is induced by B cells. At the same time, there is a possibility that the addition of “excess” B cells acts as a signal, triggering suppressor T cells.     

Studies on primed precursors of effector T cells involved in delayed-type hypersensitivity to sheep red blood cells in mice

The nature of primed precursor T cells (primed pre-TD), capable of differentiating into effector T cells (TD) that mediate delayed-type hypersensitivity (DTH), was investigated in B10 mice which were primed by intravenous (iv) injection of various doses of sheep red blood cells (SRBC). The presence of primed pre-TD was detected by the ability of T cells in the spleens from primed mice, which were treated in vitro with pertussis toxin and then transferred into naive recipient mice, to generate DTH in the recipient mice 14 days after transfer. The primed pre-TD were induced antigen specifically 1 day after mice were primed by iv injection of a suboptimal (10(3)), an optimal (10(5)), or supraoptimal (10(9)) dose of SRBC. They were replaced by TD 4 days after priming in optimally sensitized mice, while they were maintained without generating TD for at least 5 weeks after priming in mice primed with either a suboptimal or a supraoptimal dose of SRBC. They were L3T4-positive and dense cells, fractionated in the high-density layers on a discontinuous Percoll density gradient, and capable of transforming into less dense TD, fractionated in the low-density layers. These results indicate that primed pre-TD, which are induced by an antigen signal and then can be activated by a nonspecific stimulus, are present not only in responsive mice but also in unresponsive mice, suggesting that either the generation of TD from primed pre-TD or primed pre-TD alone is the decisive factor for either responsiveness or unresponsiveness.     

Transition in the character of immunological memory in mice after immunization. II. Memory in T and B cell populations.

Teh immunological memory in antibody response of mice to bovine serum albumin (BSA) was investigated at the level of antibody-producing cells or their precursor B cells and thymus-dependent helper T cells. Spleen cells obtained from mice previously primed with alum-precipitated BSA at various times were transferred to irradiated syngeneic mice. Spleen cells from mice immunized 8 days or 64 days before presented a high degree of adoptive secondary response, whereas the adoptive response of cells from mice immunized 2 days previously was found to be inferior even to that of unprimed spleen cells. Primed spleen cells treated with anti-mouse thymocyte rabbit serum plus complement were supplemented with normal thymus cells and the restoration of the responsiveness was examined. It was suggested that the memory was carried mainly by T cells in the earlier phases of the immunological memory (2 days or 8 days after the primary immunization). On the other hand, the immunological memory in the B-cell population was shown to grow gradually toward the later phase (later than 40 days).     

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.     

Clonal expansion of IgM B memory cells in vitro.

Clonal development of Srbc-primed IgM B cell precursors has been studied in vitro. Cells were cultured in the presence of LPS and Srbc, as well as additional T cells derived from three sources: specific Srbc-activated T cells, allogeneic spleen cells and normal thymus cells. Clones developing in the presence of Srbc-activated T cells reached larger sixes than did those developing in the presence of allogeneic cells, thymus cells, or only those primed T cells indigenous in the primed spleen population. However, in all these experiments, precursors primed as described attained considerably larger clone sixes than did normal unprimed precursors. Two conclusions can be made: (1) primed precursors have a greater capacity to generate progeny pfc in response to LPS + Srbc than do normal precursors and (2) specifically activated T cells appear to play a role in elevating pfc production which occurs in response to LPS and Srbc.     

IgA responses in xid mice: oral antigen primes Peyer's patch cells for in vitro immune responses and secretory antibody production

Because the gut-associated lymphoreticular tissue (GALT), e.g., Peyer's patches (PP), of X-linked immunodeficient (xid) mice possesses a subpopulation of mature B cells, we have characterized the ability of xid mice to respond to the thymic-dependent antigen sheep erythrocytes (SRBC) given by the oral route. Gastric intubation of SRBC to xid (CBA/N X DBA/2) F1 male or CBA/N mice, followed by the in vitro culture of dissociated PP cells with SRBC, resulted in IgM, IgG1, IgG2, and high IgA anti-SRBC plaque-forming cell (PFC) responses. The addition of unprimed PP but not splenic T cells to splenic xid B cell cultures resulted in IgM anti-SRBC PFC responses, suggesting the importance of GALT T cells for support of the immune responses to SRBC by splenic B cells from xid mice. Furthermore, purified PP T cells from SRBC orally primed xid mice supported in vitro IgA anti-SRBC PFC responses in B cell cultures from either the PP or the spleens of nonprimed xid mice. Higher IgA responses, however, occurred in PP, when compared with splenic B cell cultures. Additional evidence that the GALT of xid mice contains functional IgA precursor cells was provided by the finding that cloned H-2k PP T helper cells (PP Th A) supported IgA responses in PP B cell cultures derived from (CBA/N X C3H/HeN) F1 male (xid) mice. On the other hand, splenic B cells from these xid mice, in the presence of PP Th A cells, did not support in vitro responses. These results suggest that unique subpopulations of T cells occur in the GALT of xid and normal mice; one T cell subpopulation may induce immature B cells to become precursor IgA cells in the PP. A separate GALT T cell subpopulation, e.g., isotype-specific helper T cells, effectively collaborates with mature IgA B cells for the induction of IgA responses to orally administered antigen. When xid mice were gastric intubated with SRBC, followed by i.p. injection of SRBC, good splenic IgA anti-SRBC PFC responses were seen. Salivary and serum IgA antibodies were also detected in these xid mice. Nevertheless, the magnitude of the anti-SRBC response in xid mice was lower than that seen in similarly treated normal mice. These studies indicate that the GALT of both xid and normal mice possess unique populations of T cells that support in vitro responses in xid B cell cultures from either the spleen or the PP, which direct the mature B cell populations present toward IgA isotype-specific responses.(ABSTRACT TRUNCATED AT 400 WORDS)     

Increased activation of antigen-primed or memory B cells by bacterial lipopolysaccharide

Treatment with bacterial lipopolysaccharide elicits the appearance of greater numbers of background antigen-specific plaque-forming cells (PFC) in the spleens of mice previously exposed or primed to subimmunogenic amounts of various non-cross-reacting antigens so as to generate detectable immunological memory. These findings suggest that treatment with lipopolysaccharide results in the activation of increased numbers of antigen-primed or memory B cells in mice previously exposed to antigen.     

An analysis of B cell memory. I. Interaction of helper and suppressor effects in the in vitro expression of IgM memory.

The pfc response of Srbc primed IgM memory cells has been characterized by limiting dilution analysis in vitro, in which LPS was used to maximize the response of spleen cells to Srbc. The analysis suggested that, even under these conditions, expression of B cell memory was not directly assayed and cell collaboration effects were still basic to the system.Two types of cells, as defined by function, appeared necessary to elicit optimal clonal proliferation of IgM B memory cells: firstly, helper T cells were essential for B cell proliferation even with LPS present in culture. Under appropriate conditions, helper activity could be provided by normal thymus cells. Secondly, activated T cells were required for the maximal conversion of normal thymus cells to helper cells. A third activity, T cell-dependent suppression, was observed at high cell doses. The implications of these results and the need for a comprehensive analysis of in vitro conditions for each individual type of experiment is discussed.     

Role of antigen-specific B cells in the induction of SRBC-specific T cell proliferation

In this study, we ask whether antigen presentation can be effected by antigen-activated B cells. Antigen-dependent in vitro proliferation of T cells from mice primed with SRBC or HoRBC occurs in the presence of B cells primed to the relevant antigen. B cells prepared from lymph nodes of mice primed with irrelevant antigens are not effective antigen-presenting cells for RBC-specific T cell proliferation over a wide range of SRBC doses. This is true even when both RBC and the antigen to which the B cells are primed are included in the culture. In contrast, B cells specific for a hapten determinant coupled to SRBC are able to support proliferation of T cells specific for SRBC determinants. We conclude from these data that antigen-specific B cells play a role in the induction of T cell proliferative responses to SRBC and HoRBC antigens. Two models are proposed: either B cells, upon antigen interaction with surface antibody, are able to act as accessory cells to induce Ia-dependent proliferation of immune T cells; or B cells augment the T cell proliferative response by secretion of antibody, leading to opsonization of the antigen for macrophage uptake and presentation.     

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.     

The role of T cells in IgG production; thymus-dependent antigens induce B cell memory in the absence of T cells.

B cell memory was shown to develop in congenitally athymic (nu/nu) mice after injection with small amounts of thymus-dependent antigens, in particular heterologous serum proteins, such as fown gamma-globulin (FGG) or DNP-bovine-serum albumin (DNP-BSA). Large doses of proteins (10 mg) tended to produce a specific B cell unresponsiveness, although there was still some evidence of B cell priming. The antigen did not have to be in a multivalent form to interact with B cell so as to induce immunologic memory or tolerance. In contrast to the induction of B cell memory, the production of IgG antibody in this system was found to be strongly T cell dependent. Thymus-independent antigens like LPS or POL with pronounced adjuvant effects on IgG production in normal or surgically thymectomized mice, could not replace T cells in allowing an IgG response against thymus-dependent antigens in congenitally athymic mice. However, the action of T cells once activated is likely to be non-antigen-specific, since it was shown that supernatants of antigen-activated-syngeneic T cells stimulated IgG production in cultures of primed B cell populations non-antigen-specifically.     

Suppressor T-cell activity in MRL/Mp-lpr/lpr mice: differential effects on primary and memory antibody responses of MRL/Mp-lpr/lpr and MRL/Mp-+/+ spleen cells to thymus dependent and thymus independent antigens in vitro

We have previously shown that suppressor-T-cell (TS) activity in the spleens of autoimmune MRL/Mp-lpr/lpr (MRL/l) mice is increased after 2 months of age. The TS suppress the in vitro primary IgM response to the thymus-dependent (TD) antigen sheep erythrocytes (SRBC) of B and T cells from young congenic MRL/Mp-+/+ (MRL/n) mice which lack the lymphoproliferation (lpr) gene. The TS are nylon wool nonadherent, Thy 1.2 positive, and radiation sensitive. The studies presented here were done to further characterize the TS and to attempt to determine the mechanism of action of these cells. We found that increased TS activity was also present in the proliferating lymph nodes of old MRL/l mice but not in lymph nodes of young MRL/l or MRL/n mice. The splenic TS equally suppressed the primary IgM SRBC response of both young MRL/l and MRL/n B and T cells, indicating that MRL/l SRBC-specific B and T cells are not resistant to suppression. The IgM response of MRL/n B and T cells to the T-independent (TI) antigen trinitrophenyl conjugated to Brucella abortus (TNP-BA) was not suppressed by the TS, although the IgM response to TNP was suppressed when TNP was coupled to the TD carrier SRBC. The results of kinetics studies of TS expression showed that when the TS were added on Day 0 of culture the SRBC response was suppressed as early as Day 2 of culture; however, when the TS were added on Days 1, 2, or 3 of culture, the suppression was reduced. The TS suppressed the in vitro memory IgG response of spleen cells from MRL/n mice which had been primed with SRBC; the memory IgG responses of spleen cells from MRL/l mice were variably suppressed. Taken together, these results suggest that the TS suppress TH function in early events of antibody production and that some activated B or T cells may be resistant to the effects of the TS. Increased TS activity was not present in the spleens of aged New Zealand Black X NZ White (NZB/W) F1 mice. Possible reasons for the presence of increased TS activity in MRL/l mice and its relation to autoimmune disease is discussed.     

Transition in the Character of Immunological Memory in Mice after Immunization

The immunological memory in antibody response of mice to bovine serum albumin (BSA) was investigated at the level of antibody-producing cells or their precursor B cells and thymus-dependent helper T cells. Spleen cells obtained from mice previously primed with alum-precipitated BSA at various times were transferred to irradiated syngeneic mice. Spleen cells from mice immunized 8 days or 64 days before presented a high degree of adoptive secondary response, whereas the adoptive response of cells from mice immunized 2 days previously was found to be inferior even to that of unprimed spleen cells. Primed spleen cells treated with anti-mouse thymocyte rabbit serum plus complement were supplemented with normal thymus cells and the restoration of the responsiveness was examined. It was suggested that the memory was carried mainly by T cells in the earlier phases of the immunological memory (2 days or 8 days after the primary immunization). On the other hand, the immunological memory in the B-cell population was shown to grow gradually toward the later phase (later than 40 days).