The generation of memory cells. I. The role of C3 in the generation of B memory cells.

Adult thymectomized, repopulated mice were chronically depleted of circulating C3 by treatment with cobra venom factor after primary immunization with dinitrophenylated haemocyanin (DNP-KLH). This treatment totally abrogated the development of B-cell memory in such mice, as assayed by a co-operative lymphocyte transfer. The failure of memory development appeared to involve impaired precursor proliferation following priming. It was further shown that the localization of DNP-KLH in splenic lymphoid follicles is both antibody and C3-dependent; thymus-deprived mice make sufficient antibody to DNP-KLH to effect follicular localization of the antigen. On the basis of these and earlier observations we suggest that the development of B-memory cells involves the formation of antigen-antibody-C3 complexes on dendritic cells in lymphoid follicles. C3 may serve to stabilize the antigen bridge between dendritic cells and virgin precursors. In complete contrast, C3 depletion had little effect on the functional expression of primed B cells, thus suggesting that only the early stages of B-cell triggering are C3 dependent.     

The generation of memory cells. V. Preferential priming of IgG1 B memory cells by immunization with antigen IgG2 antibody complexes.

We have studied the effects of priming mice with complexes of dinitrophenylated (DNP)-haemocyanin (KLH) and anti-DNP antibody on the generation of DNP-specific B memory cells producing IgG1 or IgG2 antibodies. Immunization with DNP-KLH alone (with or without adjuvant) induced roughly equal proportions of IgG1 and IgG2 memory cells, at all times after priming. In sharp contrast, immunization with DNP-KLH polyclonal anti-DNP antibody complexes induced 80%--90% IgG1 memory cells, especially early after priming. Further studies using conventional and hybridoma anti-DNP antibodies showed that this effect was induced by complexes containing IgG2 antibodies, and not by those made with IgG1 antibodies. The latter induced roughly equal proportions of IgG1 and IgG2 memory cells. Priming for preferential IgG1 memory was not induced by complexes made with (Fab')2 fragments of IgG2a antibody, nor was it seen in T cell-deprived mice immunized with antigen IgG2a complexes. The mechanisms involved in this phenomenon are unknown, but presumably reflect the well established capacity of immune complexes to concentrate in lymphoid follicles, which seem to be sites of B memory-cell generation.     

B-cell subsets responsive to fluorescein-conjugated antigens. III. Differential effect of E. Coli lipopolysaccharide on T-dependent and T-independent responses in vivo.

The effect of E. coli lipopolysaccharide (LPS) on the induction of both hapten-specific immunity and tolerance was studied in an in vivo system utilizing putative T-cell dependent (TD) or T-cell independent (TI) challenge antigens. The administration of LPS 1 day prior to challenge preempted the response of C3D2 mice to a TD antigen (FL-KLH) but had little effect on the response to FL-Ficoll, a TI antigen. LPS did not affect the responsiveness of C3H/HeJ mice, an LPS-unresponsive strain, to either antigen. The reduction of the response to a putative T-dependent antigen by LPS pre-treatment was only temporary since mice challenged 7 days after LPS responded normally in vivo. We also confirmed that LPS administered shortly after a tolerogen prevented FL-specific IgG tolerence induction and produced B-cell priming to a subsequent T-dependent antigenic challenge. LPS, however, did not significantly interfere with tolerance induction in terms of the IgM responce to either challenge antigen. These results suggest that LPS acts either directly or indirectly on a subpopulation of B cells responsive to a TD antigen. Our data further reflect the heterogeneity of B-cell subpopulations responsive to various polyclonal activators.     

The generation of memory cells. II. Generation of B memory cells with preformed antigen-antibody complexes.

Mice were immunized with preformed complexes of dinitrophenylated haemocyanin (DNP-KLH) and anti-DNP or anti-KLH antibodies, and subsequently assayed for the generation of B memory (BM) cells. Complexes formed at equivalence or in slight antigen excess were far more effective than antigen alone in generating memory: as little as 100 ng DNP-KLH-anti-DNP produced substantial B cell priming. Anti-carrier and anti-hapten antibodies were equally effective. Complexes generated memory more rapidly than antigen alone, and the adjuvant effect was not simply due to aggregation of the antigen. Optimal priming by complexes required the integrity of the Fc portion of the antibody: F(ab')2 antibody fragments were less effective. The capacity of complexes to prime BM cells was abrogated by depriving mice of C3. C3 was also required for localization of complexes within splenic lymphoid follicles; complexes made with F(ab')2 localized in follicles, but less efficiently than those made with intact antibody. These results extend earlier findings (Klaus & Humphrey, 1977) and strongly suggest that the generation of BM cells involves the C3-dependent localization of antigen-antibody complexes within lymphoid follicles and strengthen the concept that germinal centres are the birthplace of BM cells.     

Anti-DNP antibody responses to DNP-histone H1 in mice.

Anti-DNP antibody responses to DNP-histone H1 in C3H/HeN mice were not suppressed by in vivo treatment with carrageenan in which many phagocytic macrophages were presumed to be impaired. Rather higher antibody responses to this antigen were observed in athymic nude mice than in heterozygous nude mice. Further, non-adherent spleen-cell and T-cell depleted spleen cells induced in vitro anti-DNP antibody responses to DNP-histone H1 to the same extent as normal spleen cells. These results suggest that anti-DNP antibody responses to DNP-histone H1 are macrophage- and thymus-independent. It was also observed that IgG-type anti-DNP antibodies to DNP-histone H1 were produced although most thymus-independent antigens were shown to induce predominantly IgM type antibodies but little, if any, IgG type antibodies. Furthermore, histone H1 did not show any polyclonal B-cell activator activities in contrast to many other thymus-independent antigens which act as polyclonal B-cell activators.     

Studies on B-cell memory. I. Generation and exhaustion of B-cell memory by thymus-dependent antigen in T-cell depleted mice.

Mice depleted of T cells by adult thymectomy, X-irradiation and reconstitution with syngeneic bone marrow cells, either untreated or treated with anti-Thy-1 serum and complement, were immunized intensively with alum-precipitated bovine serum albumin (AP-BSA) along with or without bacterial lipopolysaccharide (LPS), but no significant anti-BSA antibody response was detected. Priming of the T-cell depleted mice, however, either by a single injection of AP-BSA plus LPS or by multiple injections of AP-BSA without LPS, resulted in the generation of immunological memory. A single injection of AP-BSA without LPS was ineffective. The memory required the aid of syngeneic T cells to be recalled by the challenge with AP-BSA plus LPS. On the other hand, multiple injections of AP-BSA plus LPS did not cause the generation of memory and the response of these mice to the challenge was lower than that of unprimed control mice. These results suggest that (1) the anti-BSA response is highly dependent on the helper function of T cells, (2) the degree of T-cell requirement for the memory generation is very low, and (3) priming with too much strong stimulation in the absence of functional T cells leads to the suppression or abortion of previously generated immunological memory.     

Immunological memory in latent Japanese encephalitis virus infection.

Long term B-cell memory to Japanese encephalitis virus (JEV) in latently infected mice was investigated by adoptive cell transfer. Both IgM and IgG memory were elicited by antigen challenge or cyclophosphamide induced reactivation of virus. A weak antigen-specific IgM response for a brief period and a strong IgG response were detected in Swiss albino mice exposed to secondary infection. A correlation between the secondary IgM antibody and protection against JEV challenge was observed in adoptive transfer experiments. This was abrogated by pretreatment of the serum with 2-mercaptoethanol. Similarly secondary immune splenic T-cells up to day 5 post-reactivation provided protection. These results suggest that a long term antigen-specific IgM and IgG memory was induced by JEV challenge in latently infected mice. Further, the role of IgM antibody and T-cells in the response of mice to secondary JEV infection has been shown.     

Epitope-specific regulation. III. Induction of allotype-restricted suppression for IgG antibody responses to individual epitopes on complex antigens

Maternal antibody to the paternal Igh-1b (IgG_2a) allotype induces chronic suppression for Igh-1b (1b) production in (BALB/c × SJL)F₁ hybrid mice. These mice characteristically remain incapable of producing lb antibody responses until about 3 months of age and then enter a remission phase during which they produce normal 1b antibody responses to antigens introduced initially at this time. Thus young allotype-suppressed mice do not produce lb antibody responses to dinitrophenylated keyhole limpet hemocyanin (DNP-KLH) and 1b anti- KLH. The suppression of 1b antibody responses in young allotype-suppressed mice prevents the expression, rather than the development, of 1b memory for priming antigen epitopes. Furthermore, it not only prevents the expression of such memory cells initially but results in the induction of a continued suppression that specifically prevents their expression after the onset of remission. Thus mice primed with DNP-KLH while allotype suppression is still active develop normal 1b memory for DNP and KLH but nonetheless fail to produce lb anti-DNP and 1b anti-KLH responses, even when restimulated with DNP-KLH during remission. These mice also fail to produce 1b anti-DNP when stimulated with DNP on an unrelated carrier molecule, i.e., with DNP-chicken gamma globulin (CGG). This suppression is both epitope-specific and allotype-specific. That is, although 1b responses to DNP on CGG are suppressed, 1b responses to CGG epitopes on DNP-CGG proceed normally. Furthermore, there is no suppression of other isotype and allotype responses either to DNP or to the CGG epitopes. These data therefore define an Igh-restricted epitope-specific mechanism that can be induced to persistently suppress 1b antibody responses to epitopes introduced initially during active (1b) allotype suppression.     

Immunological memory in latent Japanese encephalitis virus infection

Long term B-cell memory to Japanese encephalitis virus (JEV) in latently infected mice was investigated by adoptive cell transfer. Both IgM and IgG memory were elicited by antigen challenge or cyclophosphamide induced reactivation of virus. A weak antigen-specific IgM response for a brief period and a strong IgG response were detected in Swiss albino mice exposed to secondary infection. A correlation between the secondary IgM antibody and protection against JEV challenge was observed in adoptive transfer experiments. This was abrogated by pretreatment of the serum with 2-mercaptoethanol. Similarly secondary immune splenic T-cells up to day 5 post-reactivation provided protection. These results suggest that a long term antigen-specific IgM and IgG memory was induced by JEV challenge in latently infected mice. Further, the role of IgM antibody and T-cells in the response of mice to secondary JEV infection has been shown.     

Generation of memory cells. III. Antibody class requirements for the generation of B-memory cells by antigen--antibody complexes.

Immunizing mice with preformed antigen--antibody complexes is a highly effective means of generating B-memory cells. In the present study we have compared the capacity of mouse anti-dinitrophenyl (DNP) IgM, IgG1, IgG2 and IgA antibodies to generate DNP-specific memory, when given in complex with antigen (DNP-KLH: keyhole limpet haemocyanin). Two monoclonal IgM antibodies exerted no adjuvant effect, whereas a monoclonal IgA antibody was effective, IgG2 antibodies were a more powerful adjuvant than IgG1, regardless of whether anti-DNP or anti-KLH antibodies were used. Furthermore, the capacity of DNP-KLH--antibody complexes to localize in splenic lymphoid follicles could be ranked IgG2 greater than IgG1 greater than IgA; IgM complexes did not localize in follicles. These results correlate well with data (presented elsewhere) on the capacity of these different antibodies to activate mouse complement, and confirm that C activation is an essential requirement for both follicular localization of immune complexes, and for the generation of B-memory cells. Although activation of the alternative complement pathway is sufficient to effect both processes, the results with IgG2 antibodies raise the possibility that classical pathway activation may be more effective.     

Non-specific factor replaces T cells in an IgG response to soluble antigens.

The antigen and T-cell requirements for the final stages of proliferation and maturation of DNP-KLH primed and boosted mouse spleen cells into IgG antibody secreting cells have been studied in vitro. The requirement for free antigen ceases after 24-48 h in vitro. The carrier-specific T-cell requirement for triggering of activated B cells by a soluble antigen (DNP-KLH) can be replaced in T cell-depleted cultures by non-antigen specific T cell-replacing factors (TRF). However, if the carrier protein is changed, TRF restores the IgG response of T cell-depleted cultures only if antigen is presented to B cells in particulate form, e.g. on the surface of macrophages, or in the presence of small amounts of antibody against the carrier protein. Thus, direct interaction between soluble protein and B cells is not sufficient to allow TRF to effectively replace specific T cells. Since TRF must be added at the start of culture, the initiation of B-cell maturation into IgG secretion by TRF occurs during B-cell proliferation, and is followed by further proliferation before IgG antibody can be detected.     

The influence of T cells on the initiation and expression of immunological memory.

Nude and normal CBA mice have been used in adoptive transfer experiments to analyse the development of immunological memory. The development of B-cell memory to xenogeneic erythrocyte antigens is to a very large degree dependent on the presence of T cells, with IgG memory being somewhat more dependent than IgM memory. In this system, the expression of B memory, that is the transformation of memory cells to antibody-secreting cells under the inductive influence of antigen, is largely dependent on the presence of T cells. Primed (educated) T cells can have an antigen-specific potentiating effect on unprimed B cells in the presence of antigen.     

Multiple routes to B-cell memory

B-cell memory describes the populations of cells that provide long-term humoral immunity: long-lived antibody-secreting plasma cells that reside mainly in the bone marrow and memory B cells. Interestingly, the memory B-cell population is heterogenous, although the importance of this heterogeneity has been unclear. Recent studies have investigated the formation and function of memory in different settings. In particular, T-independent memory-like cells and T-dependent (TD) IgM memory B cells qualitatively differ from canonical TD class-switched memory B cells; however, these studies suggest that IgM memory cells preserve the memory population over long periods of time. These subsets are evocative of the evolution of the humoral immune response, with memory-like cells appearing before acquisition of germinal centers, suggesting that there are multiple pathways to producing B-cell memory.     

Low molecular weight antigen arrays delete high affinity memory B cells without affecting specific T-cell help

An ongoing, T-cell dependent, secondary antibody response to an epitope can be suppressed in vivo by low molecular weight, soluble polymers, bearing multiple copies of the same epitope. This study illustrates that such suppressive T-cell independent antigen arrays target the epitope-specific, high affinity, memory B cells for long-term functional elimination.Splenocytes from hyperimmune unsuppressed donors, when adoptively transferred into irradiated recipients will readily reconstitute a secondary anti-hapten response after antigenic challenge. No such response was observed with splenocytes transferred from hyperimmune donors suppressed with antigen arrays. The extent of suppression depended on antigen array dose and duration of exposure in the donor animals. The suppressive antigen array carryover from the donors into the recipients was negligible and insufficient to account for the observed suppression.B cells from hyperimmune mice producing high affinity anti-fluorescein antibodies, generated by multiple fluoresceinated ovalbumin (FL-OVA) injections, were helped efficiently by T cells from hyperimmune donors, which were either unsuppressed or suppressed with antigen arrays. Accordingly, help from T cells, specific for the carrier protein remains intact after such suppression. Neither lipopolysaccharide (LPS), nor additional transferred carrier-primed T cells could reverse the unresponsiveness of adoptively transferred splenocytes from suppressed animals. Flow cytometry showed that the number of hapten-specific B cells was markedly reduced after suppression.Collectively, these data show that the long term elimination of an ongoing T-cell dependent antibody response by suppressive exogenous antigen arrays is due to the functional deletion of high affinity, antigen-specific B cells, even in the presence of adequate T-cell help. The long-term nature of such functional deletion strongly suggests physical deletion of the antigen-specific B cell population.     

Functional depletion of T- and B-memory cells and other lymphoid cell subpopulations-during trypanosomiasis.

T. brucei infection in mice causes generalized immunosuppression with multiple changes in the cells of the lymphoid tissue. Loss of B cell responsiveness to antigens and mitogens, and the induction of suppressive T-cells and macrophages, have been previously reported (Hudson, Byner, Freeman & Terry, 1976; Corsini, Clayton, Askonas & Ogilvie, 1977; Jayawardena & Waksman, 1977). In this study, purified B- or T-cell populations from infected mice have been tested functionally in vitro or in vivo by transfer into syngeneic irradiated hosts to separate the cells from trypanosomes or their products. B-memory cells for thymus dependent (DNP-KLH) and thymus independent (DNP-Ficoll) antigens are depleted or lose their potential to respond to the antigen during T. brucei infection. Similarly, purified T-helper cells, and T-cells reactive to allogeneic target cells in mixed lymphocyte reactions are functionally defective. By 16 days of infection all these responses are less than 10% of the normal level. The loss of B-cell function follows the peak parasitaemia and is accompanied by increases in the serum levels of both IgM and IgG. Enhanced Ig production and decline in B-cell potential also occur in T-deprived mice and in CBA/N mice which lack a subset of T-independent B-cells. Cells affecting delayed hypersensitivity reactions retain their activity throughout trypanosome infection and so far provide the only exception to the general decline in immune potential.     

Impaired Antigen-Specific B-Cell Response and Altered Splenic Microstructure in Mice Following Continuous Administration of IL-4 In Vivo

The effect of long term in vivo administration of IL-4 on the induction of antigen-specific B cells, the splenic microenvironment and the yield of antigen-specific antibody producing hybridomas was studied. Immunization with DNP-KLH, followed by 12 weeks continuous IL-4 treatment resulted in increased numbers of total splenic (non-DNP) IgM and IgG AFC (antibody forming cells) on day 5 after booster, whereas the DNP-specific IgG and IgG1 AFC were reduced compared to age-matched control animals not treated with IL-4. In addition, an almost 300-fold increase in non-DNP IgE was found while the IgE anti-DNP response was minimal.
When the splenic cells were used in a fusion protocol, a relative decrease in yield of antigen-specific hybridomas was found in the long term IL-4 treated mice. Immunohistological staining of spleen sections from mice treated with IL-4 up until the time of booster revealed reduced B-cell follicle area and germinal centre numbers. These results show that extensive IL-4 treatment reduced antigen-specific B-cell formation and suggests a reduction in the number of B cells entering the memory B-cell pathway in the spleen.     

Immunity to vaginal herpes simplex virus-2 infection in B-cell knockout mice

We investigated the involvement of antibody in protection against vaginal herpes simplex virus type-2 (HSV-2) infection by comparing intact and B-cell knockout (KO) mice. Vaginal immunization of intact mice with attenuated HSV-2 markedly reduced an HSV-2 challenge infection in the vagina. In contrast, immunization of B-cell KO mice produced less immunity against the challenge infection and that immunity occurred in a different pattern. At 20 hr after challenge, immunostaining of virus proteins in the vaginal epithelium and shed virus protein titres in the vaginal secretions were not significantly different between immunized and non-immunized B-cell KO mice and were much greater than in immunized intact mice. At 48 hr after challenge, the vaginal infection in immunized B-cell KO mice was markedly less than at 20 hr but remained approximately sevenfold higher than in intact mice. This pattern of challenge infection in the vagina indicates that B cells, and probably the antibody derived from them, provided significant protection against reinfection in intact mice, especially during the first 20 hr after challenge, while other effector mechanisms became important between 20 and 48 hr after challenge. To determine whether T-cell immunity in immunized B-cell KO mice was equal to that in intact mice, we assessed interferon-gamma (IFN-gamma) secretion by memory T cells in vivo in the vagina at 20 hr after challenge. We found no significant differences in the up-regulation of major histocompatibility complex (MHC) class II antigens in the epithelium, up-regulation of vascular cell adhesion molecule-1 (VCAM-1) in vascular endothelium, or recruitment of T cells to the mucosa, indicating that the memory T-cell response to virus challenge was the same in intact and B-cell KO mice.     

Comparative studies on the actions of antigen and polyclonal B-cell activator in differentiation and proliferation of B-cells and B memory cells.

Using the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) as a polyclonal B-cell activator (PBA) and sheep red blood cells (SRBC) as a T cell-dependent antigen, we compared the ability of PBA and antigen to differentiate (generate antibody-forming cells, AFC) and proliferate (generate immunological memory) virgin B cells and B memory cells. In vitro CPS-K induced the differentiation of IgM virgin B cells, IgM B memory cells and IgG B memory cells to AFC, as well as or better than SRBC. The differentiation of B memory cells to AFC by CPS-K did not require the participation of macrophages or T cells, whereas the action of SRBC depended strictly upon the helper actions of these cells. The responsiveness to CPS-K and SRBC of normal and antigen-primed spleen cells as judged by anti-SRBC PFC responses in vitro was markedly decreased after stimulation of virgin B cells and B memory cells in vivo by CPS-K injection into normal or primed mice but greatly increased after the injection of SRBC. The decrease in the responsiveness to CPS-K of spleen cells from mice treated with CPS-K appeared principally due to exhaustion of the functions of B cells and B memory cells. From the present data it has been concluded that the signals required for the differentiation and proliferation of B cells of B memory cells are different from each other, the signal for differentiation being provided by either antigen (SRBC) or PBA (CPS-K), while the signal for proliferation only by antigen.     

Down-regulation of immune responses to inhaled antigen: studies on the mechanism of induced suppression

Repeated exposure of rats to an aerosol of ovalbumin (OVA) or its dinitrophenylated derivative (DNP-OVA) induced carrier-specific tolerance to subsequent challenge with the same haptenated antigen. Following parenteral challenge with DNP-OVA, both anti-DNP and anti-OVA IgE titres were reduced relative to controls, whereas anti-DNP responses following challenge with DNP-Ascaris were normal. Stimulation of tolerant rats with OVA, together with the polyclonal B-cell mitogen LPS, restored their capacity to respond to the antigen. In contrast to WAG rats, which have previously been shown to develop equivalent tolerance in the IgE an IgG antibody classes (Sedgwick & Holt, 1984), BN rats exposed to an OVA aerosol developed high serum titres of anti-OVA IgG. Following parenteral challenge with DNP-OVA, however, anti-DNP IgG responses in the BNs were markedly reduced relative to unexposed controls, while anti-OVA IgG titres were maintained at a high level. Further strain-dependent differences in T-cell function in tolerized rats appeared in in vivo assays of DTH reactivity and in in vitro antigen-driven lymphocyte proliferation. Both BN and WAG rats displayed diminished in vitro responses, whereas DTH reactions were only suppressed in the latter strain.