Ontogeny of B-lymphocyte function: XIII. Kinetics of maturation of neonatal B lymphocytes to produce a heterogeneous antibody response to a T-independent antigen

The ontogeny of the capacity of a B-cell population to produce a heterogeneous, relatively high-affinity plaque-forming cell (PFC) response to the T-independent antigen trinitrophenylated-Ficoll (TNP-F) was studied in a cell transfer system. Lethally irradiated mice were reconstituted with liver cells from neonatal donors and were immunized with TNP-F at various times thereafter. In contrast to the results of our previous studies on the ontogeny of the response to T-dependent antigens, it was found that, in the cell transfer recipient, the response of an immature B-cell population to a T-independent antigen matures slowly (21–28 days). Furthermore, this maturation does not appear to require the presence of adult thymus cells as does the maturation of the response of a B-cell population to T-dependent antigens. Thus, it appears that the acquisition of the capacity of a B-cell population to produce a high-affinity, heterogeneous, PFC response to T-dependent and T-independent antigens occurs under different regulatory influences.     

Unresponsiveness to lymphoid-mediated signals at the neonatal follicular dendritic cell precursor level contributes to delayed germinal center induction and limitations of neonatal antibody responses to T-dependent antigens

The factors limiting neonatal and infant IgG Ab responses to T-dependent Ags are only partly known. In this study, we assess how these B cell responses are influenced by the postnatal development of the spleen and lymph node microarchitecture. When BALB/c mice were immunized with alum-adsorbed tetanus toxoid at various stages of their immune development, a major functional maturation step for induction of serum IgG, Ab-secreting cells, and germinal center (GC) responses was identified between the second and the third week of life. This correlated with the development of the follicular dendritic cell (FDC) network, as mature FDC clusters only appeared at 2 wk of age. Adoptive transfer of neonatal splenocytes into adult SCID mice rapidly induced B cell follicles and FDC precursor differentiation into mature FDC, indicating effective recruitment and signaling capacity of neonatal B cells. In contrast, adoptive transfer of adult splenocytes into neonatal SCID mice induced primary B cell follicles without any differentiation of mature FDC and failed to correct limitations of tetanus toxoid-induced GC. Thus, unresponsiveness to lymphoid-mediated signals at the level of neonatal FDC precursors delays FDC maturation and GC induction, thus limiting primary Ab-secreting cell responses to T-dependent Ags in early postnatal life.     

Adaptive transfer of neonatally induced tolerance into normal mice

Neonatally induced tolerance in mice was adoptively transferred with living splenocytes into normal immunocompetent syngeneic adult recipients. It was also transferred passively with antisera from tolerant mice into syngeneic neonatal recipients. Immune splenocytes transferred anamnestic responsiveness rather than tolerance. No free antibody was detected by heat elution from tolerant spleen cells, but it was found in a form complexed with antigen and dissociable with acid. These results together with those reported in the accompanying paper on neonatal tolerance suggest a role for antibody in the induction and maintenance of this model of tolerance.     

Antigen modulation of the secondary immune response. VI. Contribution of cells recruited from precursor pool to expression of memory.

The adoptive transfer system has been used extensively to study the ability of antigen triggered memory cells to become antibody forming cells and/or to proliferate and expand the memory cell population. Selective antigen triggering of the memory cells for low and high affinity antibody formation has also been studied in this way. One of the main counter-arguments to the interpretation of these data is that the presence of antigen in the adoptive host may lead to recruitment of new memory cells from either a host or donor precursor population. In this paper we examined the contribution of both host and donor precursor cells to the total antibody response in adoptive secondary recipients. The following donor-host combinations were used in which the recipients were given 1 mg fluid antigen intravenously: (A) normal (non-immune) donors to normal irradiated recipients; (B) normal donors to carrier primed irradiated recipients; (C) carrier primed donors to normal irradiated recipients; (D) normal donors to carrier primed recipients with challenge and subsequent transfer to additional carrier primed recipients; (E) carrier primed donor to normal recipients to carrier primed recipients; (F) repeat of B and C above with multiple antigen administration; (G) purified immune (DNP-BGG) donor T cells mixed with normal B cells transferred to normal irradiated recipients. In most cases recruitment was seen but this represented less than 4% of the responses seen with immune cells. Thus we conclude that this level of recruitment does not compromise the use of the adoptive transfer system for studying selective antigen triggering of memory 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.     

Nematospiroides dubius and Nippostrongylus brasiliensis: delayed type hypersensitivity responses to ovalbumin in the infected mouse

Mice (C57BL) infected with the intestinal nematode Nematospiroides dubius showed depressed delayed type hypersensitivity responses to ovalbumin administered subcutaneously in Freund's complete adjuvant. IgG and IgM responses to this inoculum were unaffected. It is unlikely that the depression arose from impairment of the ear test response because responses to an extract of the adult parasite were measurable and ear testing with lipopolysaccharide yielded normal responses in infected mice. Furthermore, mice immunized on the day of infection responded normally, whilst long term infected mice ear challenged with antigen pulsed macrophages gave depressed responses. The in vitro proliferative responses of cells from the spleens and from the lymph nodes draining the site of immunization were enhanced marginally by N. dubius infection. Furthermore, these cells induced normal or elevated adoptive delayed-type hypersensitivity and IgG responses in irradiated recipients. These findings suggest that N. dubius does not compromise the development of ovalbumin specific T cells involved in a delayed type hypersensitivity response. Evidence for the induction of suppressor cells by N. dubius is discussed, and the findings are compared with results obtained with Nippostrongylus brasiliensis, a parasite which is rejected rapidly from the mouse.     

Requirements for the adoptive transfer of antibody responses to a priming antigen in man

Antibody responses to recall vaccines can be adoptively transferred after marrow transplantation in man. Transfer of responses to priming Ag has not been successful, although this would broaden the range of organisms to which recipients could be protected. To investigate the importance of T cells and Ag in such transfer we primed marrow donors with keyhole limpet hemocyanin (KLH) 1 or 3 wk before marrow harvesting. B cells secreting IgM and IgG anti-KLH antibody were present in donor marrow at both 1 and 3 wk after immunization. After T cell depletion, donor marrow was infused into chemo-irradiated recipients, half of whom were immunized pretransplant with KLH. We found no evidence for the transfer of the IgM component of the response. Clonal expansion of the transferred IgG antibody-secreting cells with a corresponding rise in recipient serum IgG antibody levels was seen only when donors were primed 3 wk before marrow harvest and when the recipients were also immunized. IEF and immunoblotting demonstrated that successful transfer coincided with maturation of the IgG primary response from a polyclonal to an oligoclonal pattern and confirmed that donor oligoclonal bands appeared in the recipient serum. We conclude that the immunization protocols required for the transfer of antibody responses to priming Ag reflect the initial dependence of unprimed B cells on T cell help and on prolonged Ag stimulation. Ag-stimulated primary B cells in T cell-depleted marrow respond only to the noncognate growth and differentiation signals available in the chemo-irradiated recipient after an initial period of clonal selection and expansion in the donor which is both T cell and Ag dependent. Even after this initial selection, continued expansion of antibody-secreting clones in recipients retains an absolute dependence on Ag stimulation. Immunization techniques to protect transplant recipients against organisms such as Pseudomonas and CMV may need to be modified accordingly.     

Antigraft responses to the H-28c antigen by B6 and B6D2F1 mice

The survival of minor H antigen-bearing skin grafts from donors congenic with C57BL/6 (B6) was compared in B6, B6D2, and AB6 hybrid recipients. In a case singled out for further study, B6 mice were found to reject HW 110 skin (H-28^c antigen) rapidly, whereas B6D2 mice rejected HW110 skin much more slowly and variably. Both major histocompatibility complex (MHC)-linked and non-MHC genes appeared to affect the survival of HW110 strain skin grafts on B6 and B6D2 recipients. Results of several experiments appear to rule out the sharing of H-28° epitopes between donors and recipients as an explanation for the relatively poor response of B6D2 mice to HW 110 skin grafts. Experiments involving bone marrow chimeras produced by the reciprocal exchange of bone marrow between irradiated B6 and B6D2 mice suggest that bone marrow-derived donor cells and non-bone-marrow-derived host cells each contribute to the immune response phenotype with respect to the H-28° antigen. An attempt was made to determine whether B6D2 mice that failed to reject HW110 strain skin grafts possessed suppressor cells specific for the H-28^c antigen. Spleen cells from poorly responsive B6D2 mice failed to suppress the rejection of HW 110 skin grafts when assayed in immunodeficient mice that were provided with cells from immune 136132 donors that were highly responsive to HW110 skin grafts.     

Hepatitis B Virus Core Antigen Binds and Activates Naive Human B Cells In Vivo: Studies with a Human PBL-NOD/SCID Mouse Model

The hepatitis B virus (HBV) core (HBc) antigen (HBcAg) is a highly immunogenic subviral particle. Studies with mice have shown that HBcAg can bind and activate B cells in a T-cell-independent fashion. By using a human peripheral blood leukocyte (hu-PBL)-Nod/LtSz-Prkdc(scid)/Prkdc(scid) (NOD/SCID) mouse model, we show here that HBcAg also activates human B cells in vivo in a T-cell-independent way. HBcAg was capable of inducing the secretion of HBcAg-binding human immunoglobulin M (IgM) in naive human B cells derived from adult human and neonatal (cord blood) donors when these hu-PBL were transferred directly into the spleens of optimally conditioned NOD/SCID mice. No such responses were found in chimeric mice that were given hu-PBL plus HBV e antigen or hu-PBL plus phosphate-buffered saline. In addition, HBcAg activated purified human B cells to produce anti-HBc IgM in the chimeric mice, thus providing evidence that HBcAg behaves as a T-cell-independent antigen in humans. However, HBcAg-activated hu-PBL from naive donors were unable to switch from IgM to IgG production, even after a booster dose of HBcAg. Production of HBcAg-specific IgG could only be induced when hu-PBL from subjects who had recovered from or had an ongoing chronic HBV infection were transferred into NOD/SCID mice. Our data suggest that humans also have a population of naive B cells that can bind HBcAg and is subsequently activated to produce HBcAg-binding IgM.     

Maturation of the lymphoid system. I. Induction of tolerance in neonates with a T-dependent antigen that is an obligate immunogen in adults.

A/J mice displayed a striking ontogenetic difference in the capacity to respond to DNP-Ficoll, a T-independent antigen, and to aggregated human gamma-globulin (AHGG), a T-dependent antigen. Thus, whereas responses to DNP-Ficoll of 4-day-old mice were similar in magnitude to those of adult animals, responses to AHGG did not become pronounced until mice were some 30 to 40 days of age. The inability of young animals to respond to AHGG was reflective of a negative consequence of lymphocyte/antigen interaction, since such mice became specifically unresponsive to subsequent challenges with AHGG. Unresponsiveness induced by neonatal injection of AHGG lasted 50 to 60 days, in contrast to that induced by deaggregated HGG, which persisted some 100 days longer. The unresponsive state induced by injection of neonates with AHGG maintained itself upon adoptive transfer and did not appear to be linked to suppressive factors associated with either serum or lymphoid cells for its maintenance. Finally, AHGG was also shown to be capable of inducing unresponsiveness in neonatal, athymic mice. These results demonstrate that AHGG, the normally immunogenic form of HGG in adult mice, can serve as an effective tolerogen when administered into a neonatal environment.     

Adult murine lymph node cells respond blastogenically to a new differentiation antigen on isologous and autologous B lymphocytes.

Cells derived from lymph nodes (LN) of adult CBA mice respond blastogenically to mitomycin-treated autologous, as well as isologous spleen cells. This isogeneic LN-to-spleen (mixed lymphocyte culture) is best obtained when both responder and stimulator cells are derived from donors greater than 10 weeks of age. Responsive cells appear restricted to LN since they could not be detected in adult spleen, marrow, or thymus. LN cells do not require the presence of spleen in order to differentiate into responder cells since those derived from neonatally splenectomized mice are fully active. Stimulator cells appear in the spleen, bear Ig on their surfaces, and can be detected in spleens of irradiated, bone marrow-reconstituted mice. Experiments comparing the responsiveness of adult LN cells and that of neonatal T cells toward mitomycin C-treated lymphoid cells from a variety of sources suggest the presence of two iso-antigens on B lymphocytes. Since both antigens apparently are absent on precursor bone marrow cells and develop with time, they have been classified as murine differentiation antigens 1 and 2 (MDA-1, MDA-2). Whereas both appear in the spleen, only one, MDA-1, is also detectable by this methodology in LN. Both MDA-1 and MDA-2 activate neonatal T cells, but MDA-2 triggers only adult LN. Whereas MDA-2 developed in an x-irradiated, bone marrow-reconstituted spleen, MDA-1 did not over a 9-week interval.     

Cellular aspects of tolerance. V. The in vivo cooperative role of accessory and thymus derived cells in responsiveness and unresponsiveness of SJL mice

Adult (8-week-old) SJL mice reach a relatively low degree of tolerance when injected with aggregate free rabbit γ-globulin (RGG). To analyze this phenomenon, we first examined indirect plaque-forming responses (PFC) in terms of participation of accessory and thymus-derived cells. Double transfer experiments were used; accessory cells were removed from donor cells by filtration over glasswool and their capacity reduced in recipients by 3 day preirradiation or by horse erythrocyte-mediated blockage. Using this type of experimental arrangement we found that the antibody response to RGG required the cooperation of accessory and thymus-derived cells. The induction of tolerance was affected by the presence of accessory cells. Preirradiated secondary recipients were reconstituted with spleen cells from accessory cell-deprived donors which had received thymus and bone marrow cells. In some experiments, the thymus and bone marrow cells were passed over glasswool. The primary recipients were left untreated or were given tolerogen. A more profound state of tolerance (reduction in plaque forming response) was the consequence of the incapacitation or removal of accessory cells. The magnitude of the reduction in PFC was directly related to the completeness of accessory cell removal and incapacitation. Responsiveness could be restored by administration of irradiated spleen cells as a source of accessory cells. The need for thymus-derived (T) cells in the antibody response was demonstrated by double transfer experiments in which the primary recipient was restored with thymus cells alone, bone marrow cells alone, or with a mixture of cell types.     

Unidirectional IgG allotype- and isotype-specific suppressor cells in congeneic mice

By the use of allotypic markers on immunoglobulin molecules of isotypes IgG1 and IgG2a, in transfers of spleen cells between Igh haplotype congeneic partner strains BALB/c (Igha) and CB20 (=BALB/c-Ighb), the expression of donor and recipient lymphocytes could be followed differentially. BALB/c donor's allotype a was produced in nonirradiated CB20 recipients for months. By contrast, CB20 donor's allotype b disappeared in nonirradiated BALB/c recipients shortly after transfer. These BALB/c recipients of CB20 spleen cells ("CB20-primed") developed lymphocytes which were able to suppress the autochthoneous allotype b production of CB20 irradiated or CB20 nu/nu or neonatal F1 (BALB/c female X CB20 male) recipients immediately after transfer. Titers decreased with a half life of about 4 days, resembling that of immunoglobulin molecules. The suppression was restricted to the IgG2a isotype of allotype b. Neither the other isotype IgG1 of allotype b, nor, in the reciprocal transfer experiment, IgG1 or IgG2a of allotype a was affected. Analogous transfers between Igh congeneic partners on a C57B1/6 genomic background revealed the same susceptibility of allotype b-producing cells from C57B1/6 donors toward suppression by C57B1/6-Igha mice as recipients. Allotype suppression, induced by cell transfer, is thus unidirectional in that Igha haplotype mice react against allotype b but not vice versa, and it is isotype-specific, only directed against IgG2a, and not IgG1.     

Reaginic antibody formation in the mouse. VII. Induction of suppressor T cells for IgE and IgG antibody responses.

Intravenous injections of urea-denatured ovalbumin (UD-OA) into OA-primed high responder mice suppressed the antibody response not only to the priming antigen but also to subsequent immunization with dinitrophenyl derivatives of OA (DNP-OA). The transfer of normal spleen cells or OA-primed spleen cells into UD-OA-treated animals did not restore the capacity of responding to DNP-OA to form anti-DNP IgE and IgG antibodies. The transfer of splenic T cell fraction from the UD-OA-treated animals into normal syngeneic mice diminished both IgE and IgG antibody responses of the recipients to DNP-OA. The B cell-rich fraction from the same donors failed to affect the anti-hapten antibody response and enhanced anti-cancer (OA) IgG antibody response of the recipients. It was also found that the transfer of T cell-rich fraction of OA-primed spleen cells failed to suppress antibody response of the recipients to DNP-OA. The results indicated that spleen cells of UD-OA-treated mice contained suppressor T cells which are distinct from helper cells. Suppressive activity of T cells in the UD-OA treated animals was specific for OA. The transfer of the T cell-rich fraction failed to suppress anti-DNP antibody response of the recipients to DNP-KLH.     

Ontogeny of B lymphocyte function. XV. A role for thymus cells in the maturation of the capacity of a subpopulation of B cells to re-express surface immunoglobulin after treatment with anti-immunoglobulin

The maturation of the C57BL/6 B cell population to be able to re-express surface immunoglobulin (sIg) after its removal by treatment with rabbit antimouse Ig (RAMIg) was studied in a cell transfer system. It was found that thymus cells were required for the maturation of a subset of the B cell population to be able to re-express sIg. The B cell population of irradiated, thymectomized mice reconstituted with spleen cells from donors under 2 wk of age remained deficient in their ability to re-express sIg even after 4 wk residence in the cell transfer recipient. In contrast, if adult thymus cells were transferred together with the immature B cells, the B cell population matured to be able to re-express sIg after treatment with RAMIg. Approximately one-third of the B cell population appears to require thymus cells for this maturation. The maturation of the thymus cell population to be capable of mediating this maturation of the B cell population occurs in two steps: between 2 and 3 and between 3 and 4 wk of age. This timing corresponds to the age at which the B cell population of C57BL/6 mice normally acquires the capacity to re-express sIg, which we have previously shown to also occur in two steps. Thymus cells from 3-wk-old donors can mediate the first step in B cell maturation to be able to re-express sIg, but cannot mediate the second step in this maturation of the B cell population. Thymus cells from 4-wk-old donors can mediate both steps in the maturation of the B cell population. The results suggest that thymus cells are involved in regulating some aspects of B cell differentiation.     

Regulation of antigen induced changes in cyclic nucleotide levels in NZB/wf1 mice.

Normal C57BL/6J mice respond to the iv injection of antigen with an increase in splenic cAMP at 2 min. NZB/WF₁ mice are predisposed to autoimmune and immunological disorders upon aging. The ability of NZB/WF₁ mice to respond to antigen with an increase in their splenic cAMP level was found to diminish with age. This loss of responsiveness is antigen specific and not due to a loss of adenylate cyclase activity in spleen cells of old NZB/WF₁ mice. The adoptive transfer of spleen cells from unresponsive old mice into responder young mice inhibited the cAMP response to antigen by the recipients. Spleen cells from young responsive mice, on transfer into old nonresponsive NZB/WF₁ recipients, resulted in restoration of the cAMP response to antigen. In both cases, the activity of donor cells was dependent on the transfer of T cells. These results indicate that populations of T cells participate in the regulation of the cAMP response to antigen by NZB/WF₁ mice. The response of old mice could also be restored by treatment with indomethacin, and also the spleen cells of such treated donors failed to suppress the cAMP response of young recipients. Together, the results suggest a role for prostaglandins in regulating the cAMP response to antigen.     

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

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

T cell priming in vivo: a major role for B cells in presenting antigen to T cells in lymph nodes

Previous studies have shown that lymph node (LN) T cells from mice given repeated injections of anti-mu antisera from birth (mu sm) fail to mount secondary T proliferative responses to antigen in vitro after s.c. priming in vivo. This finding raised the possibility that priming of T cells in LN depends on the presence of B cells, Ig+ B lymphocytes being absent in mu sm. In support of this idea, the present paper shows that the priming defect in LN of mu sm can be largely overcome by injecting B cell populations s.c. 1 day before s.c. priming with antigen. Restoration of LN priming was observed with s.c. injection of highly purified populations of small B cells but not with heat-killed or lightly irradiated B cells. Homing studies indicated that approximately 10% of s.c.-injected B cells reached the draining LN. In other studies, irradiated mice injected i.v. with purified T cells manifested poor priming in LN after s.c. injection of antigen. It was reasoned that the LN priming defect in this situation reflected the lack of B cells in irradiated mice, B cells being highly radiosensitive. In support of this notion, it was found that s.c. injection of B cells into irradiated recipients of T cells led to high priming of T cells in LN after s.c. injection of antigen. Although T cells exposed to antigen in B-depleted LN of mu sm and irradiated mice gave negligible T proliferative responses in vitro, low but significant levels of primed T helper function were detected in a sensitive T helper assay in vivo. In light of this finding, our working hypothesis is that the initial induction of T cells to antigen in LN is controlled by resident dendritic cells (or other non-B antigen-presenting cells), the main role of B cells being to control the clonal expansion of activated T cells.     

Ontogeny of B-lymphocyte function with respect to the heterogeneity of antibody affinity-1,2.

Neonatal liver or adult spleen was used as a source of B-lymphocytes in reconstituting lethally irradiated, syngeneic mice. Recipients were all given excess adult, syngeneic thymus cells and were immunized with dinitrophenylated bovine gamma globulin. The distribution of avidities of plaque-forming cells produced by immunized recipients of neonatal liver was highly restricted in comparison with animals reconstituted with adult spleen indicating a restriction of B-lymphocyte heterogeneity in the neonatal mouse.     

Immunoregulation in the rat: ontogeny of B cell responses to types 1, 2, and T-dependent antigens

The development of rat B cells has been examined in neonatal and adult Fischer rats through the use of type 1 (TNP-Brucella abortus), type 2 (TNP-LPS(Ph), TNP-Ficoll) and T cell-dependent (TD) (SRBC) antigens. In vivo splenic PFC responses to TNP-Brucella abortus could be induced in newborn rats and by 12 days of age had reached adult levels. In contrast, the responses to the type 2 and TD antigens were 30% and 70%, respectively, of the adult levels at 30 days of age. Adoptive transfer of the B cells from neonatal and young rats into irradiated adult hosts demonstrated that the kinetics in the development of responses to these antigens (early for type 1, intermediate for TD, and late for type 2) were not due to limiting accessory cell or T cell help in immature rats. In vitro cultures of purified B cells from neonatal and adult rats were responsive to TNP-BA and TNP-LPS(Ph) but not to TNP-Ficoll and SRBC. However, the addition of spleen cell-derived Con A supernatant to the B cell cultures resulted in responses to all four antigens, which arose as a function of B cell age, with kinetics that were identical to those observed in vivo. Fluorescent staining of B cells from rats of various ages for cell surface IgM and analysis on the fluorescence-activated cell sorter (FACS) revealed that all splenic B cells from rats 4 days of age expressed a relatively high level of sIgM, and that a subpopulation that expressed a relatively low level of sIgM increased with age until it represented approximately 50% of the adult splenic B cells. Challenging Con A supernatant-supplemented cultures of FACS-prepared low sIgM+ and high sIgM+ cells revealed that B cells responsive to TNP-Ficoll were confined to the ontogenically late-arising low sIgM+ subpopulation but that B cells responsive to TNP-BA, TNP-LPS(Ph), and SRBC were present in both subpopulations.