Immune function in aged mice. I. T-cell responsiveness using phytohaemagglutinin as a functional probe.

The loss of cell-mediated immunity with age was assessed by a detailed analysis of the in vitro response of murine lymphocytes to the well-defined probe of T-cell function, PHA (phytohaemagglutinin). The reduced mitogenic activity of lymphoid cells from old mice compared with young mice could not be explained in terms of a shift in kinetics of the responding cells. Removal of macrophages, which are known to exert a regulatory effect on T-cell function, failed to reverse the poor response of old lymphoid cells. Furthermore, no evidence was found for a role of soluble inhibitors released by either lymphocytes or macrophages in the decreased response of old cells. Not only were old cells less efficient in producing such factors, but in addition, they responded less well to them than did young cells. Taken together, these observations implied that the defect in PHA responsiveness of old cells is due to a disturbance in the T cells themselves rather than to any extracellular influences. The total number of T cells, assessed by labelling with anti-Thy-1 serum was comparable in old and young animals. Selective depletion of a subpopulation of PHA-reactive cells was excluded by direct quantitation of PHA-binding cells. Thus, 25% of small lymphocytes from the spleens of old mice bound ¹²⁵I-labelled PHA ([¹²⁵I]PHA) compared with 15% in the case of young mice. To show that the cells binding PHA were those reacting to it, a suicide technique was used. Spleen cells pretreated with [¹²⁵I]PHA failed to respond to subsequent challenge with the specific mitogen, but could mount a normal response to a control (B-cell), mitogen, LPS (lipopolysaccharide). When PHA cultures were carried out in the presence of colchicine, fewer cells from old mice were found to react to the mitogenic signal. In the absence of evidence for depletion of precursor cells, the conclusion was reached that the T-cell defect in old mice is more likely to be qualitative than quantitative, perhaps due to metabolic or structural abnormalities preventing lymphocyte transformation and/or proliferation.     

Frequency analysis of the antibody specificity repertoire of mitogen-reactive B cells and "spontaneously" occurring "background" plaque-forming cells in nude mice

The antibody specificity repertoire of lipopolysaccharide (LPS)-reactive B cells has been determined in the spleens and bone marrow (BM) of C57BL/Ka athymic nude mice using a limiting dilution culture system that allows the growth and development of every LPS-reactive B cell into a clone of IgM-secreting cells. In addition, the numbers of "spontaneously" occurring ("background") IgM-, IgG-, and IgA-secreting cells as well as the "background" IgM antibody specificity repertoire has been assessed in spleens and BM. The frequencies of antigen-specific LPS-reactive B cells of C57BL/Ka nude and thymus-bearing mice showed a great similarity and ranged from 1 in 1000 to 1 in 2500 for sheep red blood cells (SRBC), horse red blood cells (HRBC), and goat red blood cells (GRBC), from 1 in 10 to 1 in 25 for 5-iodo-3-nitrophenyl-coupled (SRBC), from 1 in 15 to 1 in 150 for 4-hydroxy-3,5-dinitrophenyl-coupled SRBC, and from 1 in 70 to 1 in 140 for 2,4,6-trinitrophenyl-coupled SRBC. The specificity repertoire of the "background" IgM-secreting cells differed from that of age-matched thymus-bearing controls and was different in young and old C57BL/Ka nude mice. Within the limitations of having assessed only a minor fraction of the total B-cell antibody specificity repertoire and supposing that nude mice are largely devoid of functional T cells, the data presented suggest that the generation of the specificity repertoire of newly-formed B cells is hardly or not affected by T cells. On the other hand, T cells do affect the expression of the established repertoire, represented by "background" immunoglobulin-secreting cells.     

In situ hybridization analysis of the age-associated decline in IL-2 mRNA expressing murine T cells

Only a small decrease in the number of L3T4- cells was observed in the Con A-stimulated splenocyte cultures of old mice as compared to young, which cannot account for the threefold decrease in IL-2 production. Northern and dot blot analysis of RNA from splenocytes containing equivalent numbers of L3T4+ cells from young and old mice showed that cells from old mice express less IL-2 mRNA after mitogenic stimulation than cells from young mice. Direct analysis by in situ hybridization of stimulated splenocytes from young and old mice then showed approximately a threefold decrease in the percentage of IL-2 mRNA expressing cells in the spleens of old mice as compared to young (8.7 +/- 4.1% old; 28.7 +/- 11.7% young). The average level of expression of IL-2 mRNA was not significantly different between cells from young and old mice; however, there were approximately 40% fewer cells expressing an intermediate to high amount of IL-2 mRNA in old mice as compared to young (26.3% vs 41.8%). These data suggest that the decrease in IL-2 production with age is associated primarily with a decrease in the frequency of IL-2 mRNA-expressing cells in old mice, especially in those cells expressing intermediate to high levels of IL-2 mRNA.     

Influence of aging on intracellular free calcium and proliferation of mouse T-cell subsets from various lymphoid organs.

The influence of aging on T-cell activation and proliferation was examined in lymphocytes derived from peripheral blood, spleen, and lymph nodes of WBB6F1 C57B1/6J x WB/Re) mice. Following activation with anti-CD3 monoclonal antibodies, the greatest age-related changes were seen in CD4+ cells derived from spleens of 27- to 30-month-old mice. These CD4+ lymphocytes showed reduced [Ca2+]i signaling and decreased proliferation in the presence of exogenous interleukin 2. CD8+ cells from spleens of old animals showed reduced [Ca2+]i but not altered proliferation. Both CD4+ and CD8+ cells derived from peripheral blood of old mice showed decreased peak [Ca2+]i, but no defect in cell proliferation. In contrast, age-related deficits in either [Ca2+]i or proliferation were not observed in CD4+ and CD8+ cells from lymph nodes. Additionally, the percentage of CD4+ cells was decreased in all lymphoid organs from old mice, while the percentage of CD8+ cells was similar in lymphoid organs of old and young mice. Old mice had a significant increase in expression of Pgp-1 in CD4+ cells from spleen and peripheral blood and CD8+ cells derived from lymph node. Our studies indicate that there are differential effects of aging in T lymphocytes derived from different lymphoid organs in mice. Among the cell sources and subsets examined, the age-related changes noted in CD4+ cells from mouse peripheral blood were the most similar to those previously observed in the corresponding peripheral blood lymphocyte subset in humans.     

Cloning of B cells from autoimmune MRL-lpr/lpr and MRL.xid mice

The relationship between colony formation (cloning) of B cells and their activation in murine autoimmunity was investigated in MRL-lpr/lpr and MRL.xid mice. Cells from MRL-lpr/lpr mice showed similar requirements for in vitro growth as normal CBA/J and BALB/c cells, with maximal colony formation in the presence of the supporting factors lipopolysaccharide and sheep red blood cells. The frequency of colony-forming cells from MRL-lpr/lpr spleens or hapten-specific B-cell preparations was slightly greater than the two normal control strains, with this difference significant only for a comparison of BALB/c and MRL-lpr/lpr spleens. In contrast, MRL-lpr/lpr mice bearing the xid gene for B-cell immunodeficiency (MRL.xid) had markedly reduced B-cell colony formation. These mice nevertheless expressed anti-DNA antibodies, although at levels reduced from that of MRL-lpr/lpr controls. These results indicate that enhanced in vitro colony formation need not accompany B-cell hyperactivity in murine autoimmune disease and that autoantibody production can occur in mice with impairment in this growth property.     

The decline in murine splenic PHA and LPS responsiveness with age is primarily due to an intrinsic mechanism

Changes in splenic B and T lymphocyte number and mitogenic activity with age were quantitated in (A X C57BL/6)F1 (AB6F1) hybrid mice. Although both the B and T lymphocyte proliferative reactivity to their respective mitogens, lipopolysaccharide (LPS) and phytohemagglutinin (PHA), declined significantly with age, an earlier and more marked reduction was recorded for the T cell response. The decline in B and T lymphocyte mitogenic activity with age could not be correlated with a corresponding reduction in the percentage of splenic B or T lymphocytes. The main focus of this study was to determine if the reduction in T and B lymphocyte mitogenic activity with age results primarily from a mechanism intrinsic to the lymphoid lineage itself or from adverse extracellular factors that increase with age. Bone marrow cells (BMC) derived from individual young and old donor AB6F1 mice were transplanted into the neutral environment of young, lethally irradiated syngeneic recipients. Number and mitogenic activity of splenic T and B lymphocytes were recorded for the original BMC donors as well as for the recipients of the young and old BMC lines 9 mo after the BMC transplants. A predominance of the donor (male) rather than recipient (female) karyotype within the mitogen-responding populations of recipient mice confirmed a donor BMC take. The PHA and LPS response levels exhibited by the old donors were 30% and 70% of those of the young donors, respectively. These differences in PHA and LPS reactivity recorded between young and old donors were maintained between recipients of young and old donor BMC lines. Thus, even under the influence of a young recipient environment, old BMC were incapable of giving rise to mitogen responding cells with a functional competence equivalent to that of their younger counterparts. This finding would lend further support to the theory that an intrinsic mechanism is responsible for the decline in murine mitogenic activity with age.     

Cellular events in tolerance. VII. Decrease in tolerant spleens of PFC precursors stimulated in vitro by specific antigen or mitogen.

Spleen cells from adult mice rendered tolerant to the fluorescein (FL) hapten (as FL-sheep γ-globulin) were analyzed at limiting dilution for the numbers of precursors stimulatable either by specific antigen (FL-polymerized flagellin; FL-POL) or by a polyclonal B-cell activator (E. coli lipopolysaccharide; LPS). As expected, the number of PFC presursors activated by FL-POL was reduced more than fourfold in the spleens of FL-tolerant mice compared to normal controls. In contrast, LPS was able to trigger equivalent numbers of “FL-specific” PFC precursors in both normal and tolerant spleens. However, the clones stimulated by LPS were predominantly the “low-avidity” precursors in FL-tolerant spleens as shown by plaque inhibition studies. In addition, after FL-gelatin enrichment of normal or tolerant spleen cells, which contain equal numbers of antigen-binding cells, we found that purified cells from tolerant mice were in fact reduced in the numbers of clonable precursors upon LPS stimulation. Two other B-cell mitogens, POL and PPD, also failed to activate PFC precursors from FL-gelatin-purified tolerant spleen cells. Our results suggest that some high-avidity clones may be functionally deleted even in adult B-cell tolerance as previously noted for neonatal tolerance.     

Biological effects of Corynebacterium parvum. IV. Adjuvant and inhibitory activities on B lymphocytes

The PFC response to the thymus-independent antigen SIII (type 3 pneumococcal polysaccharide) was amplified in mice injected 4 days previously with killed Corynebacterium parvum. This adjuvant activity was demonstrable with high (2–50 μg) but not low (0.1–0.5 μg) doses of SIII. Induction of tolerance was unaffected. Depression of the response resulted from simultaneous injection of SIII with either C. parvum or Bordetella pertussis, while prior treatment with the latter was without effect. Responsiveness to SIII was transiently but potently suppressed in spleen cells transferred into lethally irradiated, C. parvum pretreated mice.Although C. parvum is an effective B cell adjuvant, other data imply that it acts indirectly on these lymphocytes. It is argued that both adjuvant and suppressive activities of C. parvum on the B cell response to SIII are most probably mediated by activated macrophages.     

Neonatally induced tolerance to soluble protein antigens. I. Analysis of B-cell and T helper cell function in mice tolerant to bovine serum albumin or fowl gamma-globulin

High dose tolerance to either bovine serum albumin (BSA) or fowl γ-globulin (FGG) was induced in CBA mice by neonatal injection. Tolerance to BSA lasted about 9 weeks, and that to FGG, about 18 weeks. Splenic B-cell function was analyzed using quantitative in vivo assays and in vitro limiting dilution analysis. Tolerogen-specific IgM- and non-IgM-producing B cells are depleted at least threefold in the spleens of tolerant mice. Tolerogen-specific T-helper-cell function was examined by immunization with haptenated antigens. Analysis of the recovery from tolerance indicates that the return to normal function in the tolerogen-specific B-cell and T helper fractions coincides with the return to normal responsiveness by the whole animal.     

Properties of fractionated spleen cells from NZB/W mice.

The unit gravity sedimentation technique was used to separate spleen cells from sevveral strains of mice. Settling patterns (plot of cell number against settling rate) were similar for BALB/c, DBA/2, C3H/He, and NZB/W mice of different ages. In particular, no subpopulation was found by this technique to be missing from the spleens of old NZB/W mice.A number of functional studies performed with the separated cells proved more informative than the settling patterns themselves. Fractions of cells which sedimented at a rate of between about 6 mm/hr and 10 mm/hr were enriched in responsiveness to PHA, Con A, and allogeneic cells. These fractions obtained from old NZB/W mice lacked such activities. However, the active fractions from young NZB/W spleens, which were enriched in θ-bearing cells, could restore the responsiveness of old NZB/W mice to primary immunization with sheep erythrocytes. These studies indicate that functional separation of spleen cells from NZB/W mice is possible and that activities lacking in whole spleens from old NZB/W mice are also lacking in the separate fractions. The ability to restore helper T cell function in old NZB/W mice with active fractions from young NZB/W mice has implications for further study and treatment of their autoimmune disease.     

Frequency analysis of functional immunoglobulin C- and V-gene expression by mitogen-reactive B cells in germfree mice fed chemically defined ultra-filtered "antigen-free" diet

The frequencies of lipopolysaccharide (LPS)-reactive B cells and their antibody specificity repertoire have been determined in the spleen and bone marrow (BM) of conventional (CV) and "antigen-free" C3H/HeCr mice of various ages. The antigen-free mice were germfree (GF)-raised and were fed an ultrafiltered solution of chemically defined (CD) low m.w. nutrients, and were thus devoid of exogenous antigenic stimulation. Spleen and BM cells were grown in a limiting dilution culture system that allows the growth and development of every newly formed LPS-reactive B cell into a clone of IgM-secreting cells which are capable of switching to other immunoglobulin (Ig) heavy chain isotypes (C-gene expression). The secretion of IgM and IgG1 was determined in the protein A plaque assay, whereas specific IgM antibody-secreting cells (V-gene expression) were detected in plaque assays specific for various heterologous erythrocytes and sheep red blood cells (SRBC) coupled with a number of different haptens. The absolute frequency of LPS-reactive B cells and their capacity to switch to IgG1-secretion was not significantly different in 8- to 12-wk-old and 52-wk-old GF-CD mice and their age-matched CV controls. Moreover, no differences were observed in the frequencies of antigen-specific B cells within the pool of LPS reactive B cells. These frequencies ranged from 1 in 20 to 1 in 50 for NIP4-SRBC and NNP2-SRBC, from 1 in 100 to 1 in 150 for NIP0.4-SRBC, from 1 in 50 to 1 in 100 for TNP30-SRBC, and from 1 in 1000 to 1 in 2000 for SRBC and horse red blood cells. Within the limitations of having determined the switching capacity of IgM to IgG1 only and having assessed only a minor fraction of the total B cell antibody-specificity repertoire, the data indicate that young and old GF-CD mice, although devoid of exogenous antigenic and/or mitogenic stimulation, generate B cells with a similar switching capacity and a similar IgM antibody specificity repertoire as CV mice.     

Evolution of B-cell clonal expansions with age

B-cell clonal expansions (BCE) in young mice are transient, detectable for less than 4 weeks. In contrast, BCE in old mice persist more than 2 months. The greater persistence of BCE in old mice does not appear to be due to the age of the host as the survival of phenyloxazolone chicken serum albumin-induced BCE in most old mice was shorter than in young mice. This raises the possibility that persistent BCE seen in old mice develop over time from transient BCE present earlier in life. To test this hypothesis, young C57BL/6 mice were immunized with hen egg lysozyme (HEL) during the first year of life. By 28 months of age, the majority of these mice had developed a benign, persistent BCE associated with a HEL-specific serum mIg. We also investigated whether benign, persistent BCE, present in 18-month-old mice, can evolve into B-cell lymphomas. We observed that four of eight C57BL/6 mice that survived to 29 months of age had developed diffuse large cell lymphomas. In three of these mice, this diagnosis was made by microscopic analysis of the lymphoid organs. In one mouse, a macroscopic lymphoma was present that permitted us to demonstrate that the IgH mRNA CDR3 length and sequence in the malignant lymphoma was derived from a persistent BCE present 11 months earlier. Together these observations are consistent with the hypothesis that stepwise accumulation of genetic alterations combined with Darwinian selection underlies the evolution of B cells from transient BCE in young mice into persistent BCE, serum mIg, and B-cell lymphomas observed in older mice.     

Elaboration of lymphotoxin by freshly isolated human T lymphocytes and continuous lymphoid-cell lines.

Freshly isolated human T lymphocytes were demonstrated to produce lymphotoxin (LT) after mitogenic stimulation with phytohemagglutinin (PHA). In contrast, freshly isolated B lymphocytes, stimulated with two B-cell mitogens [pokeweed mitogen (PWM) and Staphylococcus protein A (Staph A)] did not produce the lymphokine, although thymidine incorporation was increased in these cells. We also examined a series of nine continuous human lymphoid-cell lines with B-cell markers and observed the spontaneous release of either large or small amounts of cytotoxin, or none at all. Cytotoxin from one of the productive cell-lines (H4218) was compared in detail with that obtained from PHA-stimulated, freshly isolated human lymphocytes. The behavior of the two cytotoxins was found to be identical in respect to migration on polyacrylamide gel, neutralization with rabbit anti-human α-LT serum, ultracentrifugation on 5–30% sucrose gradients, and stability for 15 min at 75 °C. Observation of these identical parameters strongly suggests that the α-LT elaborated by PHA-stimulated, freshly isolated human lymphoid cells is the same as the cytotoxin obtained from the continuous human lymphoid-cell line H4218. Thus α-LT may also be produced in quantity from continuous lymphoid-cell lines by mass tissue-culture techniques, which are more readily applicable to large-scale production than is purification from freshly cultured human lymphoid tissues. Notably, in cultures of freshly isolated human lymphoid cells, only T cells, and not B cells, generated lymphotoxin. However, continuous human lymphoid-cell lines with B-cell markers can also secrete this lymphokine.     

Lymphocyte function in experimental African trypanosomiasis. III. Loss of lymph node cell responsiveness

The relationship between immunosuppression and suppressor cell activity in the lymphoid organs of animals with experimental African trypanosomiasis has been examined further. In the present study we measure the primary in vitro PFC response to SRBC by spleen and lymph node cells from Trypanosoma rhodesiense infected or drug-cured C57BL/6 mice. Passive transfer experiments with this culture system tested for the presence or absence of suppressor cells. We demonstrate that infected mice exhibit immunosuppression in the spleen cell population several weeks before becoming suppressed at the level of the lymph node cell populations. Although suppressor cells are present in immunosuppressed spleen cell populations, suppression of lymph node cell responsiveness was not attributable to suppressor cells detectable withi, lymph nodes. After Berenil treatment of terminally infected mice immunocompetence was restored gradually, first to the lymph node cells and subsequently to the spleen cell population. Recovery of spleen cell responsiveness was attributable to the loss of detectable suppressor cell activity within spleens. These results demonstrate that there is anatomical restriction of the suppressor cell population to trypanosome-infected mouse spleen and that loss of immunocompetence in the lymph nodes may be due to factors unrelated to suppressor cell effects.     

Concanavalin A-mediated in vitro activation of lymphocytes primed against syngeneic SV40-induced tumor-associated antigens in mice into secondary effector cells capable of specifically preventing tumor growth.

Spleen cells from BALB/c mice immunized against a syngeneic SV40-induced tumor, mKSA, prevented specifically the growth of the corresponding tumor in the tumor cell neutralization assay following preincubation for 5 days with mitogenic concentrations of concanavalin A. This reactivity was shown to be T cell dependent, independent of remaining concanavalin A, and was detected at least up to 60 days following in vivo antigenic immunization. A similar reactivity was obtained with mitogenic concentrations of phytohemagglutinin but not with the B-cell mitogen lypopolysaccharide. Since this reactivity was indistinguishable from that obtained upon in vitro secondary antigenic stimulation with SV40-transformed cells, it is suggested that activation of precytotoxic cells against a syngeneic tumor by concanavalin A into cytotoxic cells may be mediated by the same or similar receptors triggered by the stimulating tumor-associated antigens.     

Immunorestoration of old mice by injection of thymus extract: Enhancement of T cell-T cell cooperation in the in vitro antibody response

The effect of injecting a thymus extract (TP-1 or Thy-5) into immunodeficient old mice on the in vitro antibody response of their spleen cells was investigated by techniques suitable for dissecting out T- and B-cell reactivities. The anti-TNP antibody response of HRBC-primed spleen cells from old mice uninjected or injected with TP-1 or Thy-5 was elicited in vitro by TNP-HRBC or TNP-Ficoll. Treatment with TP-1 or Thy-5 was found to induce only a slight increase in the anti-TNP antibody response to both immunogens. The helper activity of HRBC-primed spleen cells from untreated or treated old mice was titrated by adding graded numbers of these primed cells to cultures containing a constant number of normal spleen cells from young mice and the immunogen TNP-HRBC. Under these conditions it was found that both thymus extracts are very effective in restoring T cell-T cell cooperation in the generation of helper cell activity.     

Immunological studies of aging. Normal B-cell repertoire in aged mice: studies at a clonal level

As previously reported, old mice produce lower avidity plaque-forming cells (PFC) after immunization with 2,4,6-trinitrophenyl-Ficoll (TNP-F) than do young mice. However, if spleen cells from TNP-F-immunized old mice are incubated with hapten to elute auto-anti-idiotype antibody then high avidity PFC, comparable to those in young mice, are detected. To further evaluate the effect of age on the B-cell repertoire anti-2,4,6-trinitrophenyl-bovine gamma globulin (TNP-BGG) hybridomas were prepared from young (6 to 8 weeks old) and old (18 to 24 months old) mice which had been primed and boosted with TNP-BGG. The monoclonal antibodies (MoAb's) were TNP-specific. Spleens from old and young mice were comparable with respect to the incidence of immunoglobulin-secreting hybridomas obtained, the incidence of TNP-BGG-specific hybridomas obtained, and the isotype distribution of the anti-TNP-BGG hybridomas. The avidities for TNP-BGG of the IgG1 anti-TNP-BGG MoAb's obtained from old and young donors were also comparable. The overall results thus suggest that old and young mice have similar B-cell repertoires and that differences in the antibodies produced are due to regulatory influences.     

B-B cell interactions in the spontaneous activation of B cells in autoimmune NZB mice.

We analyzed the mechanism of spontaneous B cell activation in lupus mice by using anticlass-II antibody in vitro. The in vitro culture of B cells from old NZB mice markedly produced Ig without any stimulation, while B cells from NZW mice did not. The addition of anticlass-II antibody (anti-Iad antibody) to the culture inhibited Ig production of NZB B cells in a concentration-dependent manner. On the other hand, the addition of anticlass-I antibody (anti-H-2Dd antibody) and anticlass-II antibody with different specificity (anti-Iak) gave no effect on the Ig production of NZB B cells. When mitomycin C-treated B cells were added to in vitro culture of responder B cells as a stimulator, Ig production of responder B cells was enhanced in a concentration-dependent manner. However, the enhancing effect of the stimulator B cells was abrogated by the pretreatment with anticlass-II antibody. The stimulator B-cell activity to NZB B cells was marked in NZB B cells, moderate in NZB/W F1 B cells, and weak in NZW B cells. Furthermore, the stimulator B-cell activity with regard to NZB B cells was marked in old female NZB B cells, moderate in old male NZB B cells, and weak in young NZB B cells. The expression of class II antigens on the surface of old female NZB B cells was significantly higher than that of old male NZB and young NZB B cells. These results suggest that in lupus mice the spontaneous B-cell activation is induced by an abnormal B-B cell interaction mediated by class II antigens.     

Differences between virgin and memory IgM-antibody-forming-cell precursor B-cells, and correlations with the heterogeneity present in B-cell populations from unimmunized mice.

Previous in vitro studies showed that a large proportion of DNP-reactive B-cells in the spleens of unimmunized mice, unlike B-cells reactive to fowl gamma globulin (FGG), did not adhere to glass-bead columns. B-cell reactivity was assessed by challenge with dinitrophenylated-polymerized flagellin (DNP-POL) or FGG in the presence of POL, both responses being thymus-independent. Now we have shown that when donor mice are immunized with FGG, a greater proportion of FGG-reactive B-cells becomes non-adherent and the column filtrates give an IgM anti-FGG response. This indicates then that the adherence properties of the IgM-producing antibody-forming-cell (AFC)-precursor differed in virgin and immunized B-cell populations. In vitro testing of the thoracic duct cell (TDC) population of normal and immunized mice revealed parallels between thoracic duct B-cells and the non-adherent splenic B-cell population. Thus while the in vitro anti-DNP response of thoracic duct lymphocytes from normal mice reached levels little below those of spleen cells, the anti-FGG response was much lower. However when TDC were taken from mice immunized with FGG, the anti-FGG responses were much higher.Thus a parallel was demonstrated both in terms of non-adherence to glass and presence in the thoracic duct, of a portion of the DNP-reactive B-cell population in unimmunized mice and the FGG-reactive population in FGG-immunized mice. This suggested that the non-adherent DNP-reactive B-cells in unimmunized mice may represent B-cells with experience of cross-reacting antigens. The significance of this heterogeneity of the B-cell populations reactive to certain antigens in unimmunized mice is discussed with particular reference to recent apparently contradictory findings in regard to B-cell activation.