Ontogeny of B-lymphocyte function. XIV. Maturation of the capacity of B cells to re-express surface immunoglobulin after treatment with anti-immunoglobulin

The maturation of the ability of the B-cell population to re-express surface immunoglobulin (sIg) after its removal by treatment with rabbit anti-mouse immunoglobulin (RAMIg) was studied in LAF1, C57BL/6, and C57L mice. As demonstrated by previous workers, the B-cell population from immature mice failed to re-express sIg after treatment with RAMIg. We have shown that the age at which the B-cell population acquires the capacity to re-express sIg is different in different strains and that the order in which the B-cell population of the different strains acquires the capacity to re-express sIg is different from the order in which their B-cell populations acquire the capacity to produce high-affinity antibodies. This suggests that these represent distinct differentiation events in the development of the B-cell population. In all of the strains studied the maturation of the capacity to re-express sIg occurred in two steps. After the first maturation step the B-cell population was able to re-express sIg after treatment with RAMIg for 1 hr but did not re-express sIg after treatment with RAMIg for 24 hr. After the second maturation step the B-cell population could re-express sIg even after 24 hr treatment with RAMIg. It has been suggested by previous workers that the inability of the immature B-cell population to re-express sIg could represent one of the mechanisms responsible for the development of B-cell self-tolerance. It is suggested here that the existence of a period during which cells become tolerant only upon prolonged exposure to antigen could protect the developing B cells from becoming unresponsive to transiently experienced foreign antigens but still permit them to become tolerant to self antigens which are continuously present.     

Ability of chicken B cells from different compartments to respond to TNP-Ficoll

The responsiveness of chicken B cells from various compartments to T-independent antigens was studied by immune transfers of spleen and bursa cells into immunosuppressed recipients. Bursa cells from 8- to 10-wk-old donors failed to respond to trinitrophenylated Ficoll (TNP-F) even when thymus cells or splenic T cells were added. Spleen cells from the same donors transferred responses, as judged both by anti-TNP plaque-forming cells (PFC) per spleen and serum anti-TNP titers. In contrast, responses to TNP-Brucella abortus (TNP-BA) were transferred at least as well as by bursa as by spleen cells. Rabbit anti-chicken T cell serum plus complement treatment of the spleen cells reduced their ability to transfer responses to sheep erythrocytes, but either did not affect or enhanced serum antibody responses to TNP-BA and TNP-F. In intact animals, responsiveness to i.v. injected TNP-F was found to develop slowly after hatching in the chicken. At the age of 2 and 3 mo, PFC/spleen on day 4 after TNP-F injection were only 20% and 40%, respectively, of the adult response. Thymectomy at hatching further delayed this development, resulting in 12% and 45% of the adult control response at ages of 3 and 4 mo. It is concluded that responsiveness to the TI-2 antigen, TNP-F, develops slower than that to the TI-1 antigen, TNP-BA, and is restricted to the splenic B cell compartment. In addition, this development appears to be faster in the presence rather than in the absence of the thymus. In view of the previously shown effect of thymus on bursa development, these data suggest that the maturation of TI-1 antigen (TNP-F)-respondent chicken B cells requires residence in both the bursa and spleen before the development of responsiveness to such antigens.     

Subpopulations of human thymus cells differing in their capacity to form stable E-rosettes and in their immunologic reactivity.

The majority of human thymus cells from young donors form stable E-rosettes with sheep red blood cells (SRBC) that do not distintegrate after prolonged incubation at 37 degrees C. With advancing age the proportion of thymus cells forming such rosettes decreases gradually. The thymus of a patient receiving prednisone treatment was found to contain only a few cells that formed stable E-rosettes. The minor population of thymus cells that fails to form stable E-rosettes (non-rosetting or NR cells) was isolated and tested for its cell surface markers and immunologic reactivity in vitro. Most of the NR-cells were capable of forming regular E-rosettes with SRBC at room temperature. Like the majority of human thymus cells they were sensitive to the cytotoxic effect of normal constituted less than 0.2% of the original thymus cell suspensions, but about 1 to 3% of the NR-population. Thymus cells from donors over the age of 36 and from a prednisone-treated child responded in vitro to stimulation with either phytohemagglutinin (PHA) or concanavalin A (Con A). Unfractionated thymus cells from children up to the age of 14 failed to react to either PHA or Con A, but their NR-population responded vigorously to both lectins. In contrast to unfractionated thymus cell suspensions from children, the NR fraction showed a significant reactivity in mixed lymphocyte cultures with mitomycin-C treated allogeneic lymphocytes. It is concluded that like the thymus of other species, the human thymus contains a minor population of cortisone-resistant cells endowed with many of the immunologic properties characteristic for periperal T lymphocytes.     

In vitro sensitization to transplantation antigens. VII. Enhanced graft-vs-host activity of in vitro allosensitized lymphoid cells

We have previously demonstrated that C57BL/6J lymphoid cells sensitized in vitro to C3H/He transplantation antigens, present on macrophage monolayers, can transfer an accelerated C3H allograft response to recipient C57 mice. The present report indicates that C57 lymphoid cells sensitized to C3H alloantigens, present on macrophage monolayers, can also mediate a graft-versus-host (GVH) reaction in (C3H × C57) F₁ newborn mice. This GVH reaction is of greater magnitude than that produced by noncultured C57 cells. The magnitude of the augmented GVH reaction produced by cultured C57 cells is dependent on the source of lymphoid cells: lymph node, spleen, and bone marrow cells are consistently more active than cultured thymus cells—the reduced capability of cultured thymus cells to mediate the GVH reaction parallels their reduced ability to transfer allograft immunity. To test whether monolayers, other than macrophages, can sensitize lymphoid cells in vitro we incubated C57 lymphoid cells on C3H-derived L cells. Lymph node cells incubated with L cells demonstrate an increased GVH reaction in newborn mice. The in vitro sensitization of spleen and bone marrow cells on L cells is less consistent. Thymus cannot be sensitized by L cells. Monolayers of L cells are therefore not as efficient a sensitizing source as macrophages.     

T cell regulation of immunoglobulin class expression in the B cell response to TNP-Ficoll: characterization of the T cell responsible for preferential enhancement of the IgG2a response

Syngeneic T cells injected into athymic nu/nu mice cause a preferential enhancement in the amount of IgG2a anti-TNP Ab produced by these mice to TNP-Ficoll. This enhancement appears to be caused by T cell effects on the IgG switching pathway. Through the use of F1----parent chimeras, the helper T cells were shown to affect TNP-Ficoll-responsive B cells in an H-2-unrestricted manner. The ability of T cells to mediate this IgG2a enhancement did not appear to be unique to any particular murine genetic background, because it was observed with T cells and nu/nu mice of C57BL/10, BALB/c, CBA/Ca, and B10.D2 strains. Priming of T cell donors with Ficoll or TNP-Ficoll did not increase the ability of splenic T cells, on a per cell basis, to enhance the IgG2a Ab response to TNP-Ficoll. The T cell population responsible for modulating the isotypic response was found to be sensitive to C-mediated cytotoxicity with both anti-Lyt-2 and anti-Lyt-1 hybridoma Ab. Although T cells from both the thymus and the spleen expressed enhancing activity, splenic T cells were more effective, on a per cell basis, than were thymocytes. The observations suggest that T cells that appear to enhance the switch to IgG2a in TNP-Ficoll-responsive B cells are not effectively primed by the antigen and interact with TNP-Ficoll-activated B cells through an H-2-unrestricted mechanism.     

Ontogeny of B-lymphocyte function: XII. Evidence that the ability to generate memory cells precedes the ability to produce antibody-secreting cells

Using a cell transfer system it was shown that lymphoid cells of B.C-9 mice mature to be able to generate a primary response of high magnitude and high affinity to dinitrophenylated γ-globulin at about 4 weeks of age. Irradiated mice reconstituted with lymphoid cells from B.C-9 or C57BL/6 donors younger than 4 weeks of age fail to produce high-affinity plaque-forming cells and their serum antibody responses were of low magnitude. Nevertheless such recipients give adult-like, high-affinity, high-magnitude secondary responses when assayed at both the plaque-forming cell and serum antibody levels. The antibody produced by the reconstituted mice, in both the primary and secondary responses, was shown to be of donor cell origin in transfers between allotype congenic pairs. Thus, naive, immature lymphoid cells, although not capable of giving rise to high-affinity antibody-secreting cells in a primary response, are capable of giving rise to high-affinity memory B cells which in turn can give an adult-like, high-affinity secondary response following boosting.     

Age-related changes in the cellular immune response of lymph node and thymus cells in long-lived mice.

Relative capabilities of lymph node and thymus cells from (C57BL/6J) × Balb/cJ) F₁-hybrid mice and from (C57BL/6J × 129J)F₁—hybrid mice 6, 20, and 30 mo of age to respond in the mixed lymphocyte reaction were assessed. Both the direct response and the ability to synergize with partner lymph node or thymus cells were studied. Lymph node cells demonstrated an age-related decline both in responding directly to stimulation by allogeneic cells, and in ability to synergize with syngeneic thymocytes. Thymus cells showed an age-related increase in response to direct stimulation, and no clear-cut age-related change in synergizing capability. In these long-lived mouse strains at least one cause of the age-related decline in cellular immunity relates to developing deficiencies in the recirculating lymphoid pool, probably T₂ cells.     

Effect of age on the capacity of the bone marrow and the spleen cells to generate B lymphocytes

The effect of age on the regeneration of the B cell population was studied by cell transfer methods, using the allotype-congenic mouse strains BALB/c (Igha) and C.B-17 (Ighb) as donors of old and young bone marrow (BM) and spleen cells, and C.AL-20 (Igho) as recipients. This design allowed us to identify the origin of the sIgD+ B cells present in the recipients. It was found that in a simple cell transfer, BM cells or spleen cells of aged donors could reconstitute the peripheral B cell population of irradiated, thymectomized recipients essentially as effectively as could BM or spleen cells from young donors. However, when BM cells from aged donors and from young donors were mixed and were used to reconstitute a single recipient, the cells from the aged donor were less efficient than were the cells from the young donor. We found that sIgD+ B cells of young donor origin predominated in the peripheral B cell population of the recipient at 3 to 6 wk after cell transfer. In the BM of the recipients, however, there was no difference in the incidence of sIgD+ B cells derived from the young and the old donors. When recipients were reconstituted with a mixture of spleen cells from old and young mice, the sIgD+ cells of young donor allotype showed a tendency to predominate in the peripheral B cell population, although this predominance was not statistically significant. Under such competitive conditions, the spleen cells of aged donors were less efficient than the BM of aged donors in reconstituting the sIgD+ B cell population of the recipient's BM, but were more efficient in reconstituting the splenic sIgD+ cells. Thus, a subtle defect in the B cell precursor population of the BM and the spleen of aged mice has been demonstrated. The role of T cells in the generation of sIgD+ cells was also analyzed.     

Humoral factors in very young NZB mice that enhance the maturation of normal B cell precursors. Partial purification and characterization

Soluble factors that enhance maturation of murine B lineage precursor cells in vitro were partially purified from the serum of very young NZB mice and characterized biochemically and biologically. Activity was initially detected by induction of colony-forming activity and surface immunoglobulin (sIg) on normal sIg- marrow cells as well as responsiveness of a pre-B cell line. Pooled sera from 4- to 5-wk-old NZB mice were initially fractionated on Sephacryl S-300 and Sephadex G-100 superfine columns. Fractions with activity (corresponding to m.w. of 15,000 to 45,000) were pooled and further separated. The activity was eluted as a single peak by hydrophobic (phenyl-Sepharose, with 0.8 M (NH4)2SO4) and lentil lectin affinity chromatography but resolved into three distinct peaks in preparative isoelectric focusing (IEF), with pI values of 3.5, 7.8, and 8.4. The latter two merged into a single peak with a pI value of 8.8 when the sample was further treated with neuraminidase before IEF. These three IEF fractions, each of which were enriched at least 1000-fold in specific activity relative to starting serum, were then characterized. Each was stable at pH 2 but sensitive to trypsin, 10 M urea, and heat treatment (56 degrees C for 1 hr). In nonreduced SDS-poly-acrylamide gel electrophoresis, their mobilities corresponded to m.w. of 17,000 for peak I (pI 3.5), 15,000 for peak II (pI 7.8), and 15,000 for peak III (pI 8.4). Interleukin 1, interleukin 2, interleukin 3, colony-stimulating factor for granulocyte and macrophage progenitors, antiviral, or B cell growth factor type I-like activities were not demonstrable. Peaks II and III, but not peak I, induced Ig secretion of anti-stimulated B cells. Peak I was also less effective than peaks II and III in induction of sIg on an established pre-B cell line. However, all fractions were equally effective in enhancing maturation of normal sIg- B lineage cells. Thus, serum from 4- to 5-wk-old NZB mice contains at least two distinct soluble factors that can enhance the maturation of sIg- B lineage cells in vitro. The biologic and biochemical characteristics of these factors appear to differ from those of previously well-defined cytokines.     

In situ characterization in freeze-fractured mouse thymuses of lymphoepithelial complexes ultrastructurally similar to isolated thymic nurse cells

Scanning and transmission electron microscopy of the cracked surfaces of cryofractured pre-fixed C57BL/Ka mouse thymus reveals the existence of cell complexes, distinct from the surrounding cell organization, in which groups of lymphocytes are delimited by large cytoplasmic sheets or envelopes. These complexes, located in the subcapsular and cortical regions, display morphological features similar to that of the thymic nurse cells (TNCs), which can be isolated from the mouse or human thymus enzymatically dissociated. They can be considered as dynamic systems able to modify their three-dimensional organization, namely with regard to intrathymic cellular traffic involved in T-lymphocyte maturation.     

The influence of dietary protein insufficiency on the murine thymus. Evidence for an intrathymic pool of progenitor cells capable of thymus regeneration after severe atrophy.

Low protein diets initiated at wearning in Balb/c mice cause a rapid and profound reduction in thymus weight and cellularity. Thymus weight falls to less than that of involuted thymus of adult mice and remains depressed for as long as diets are fed. Although most peripheral T cell functions do not appear to be depressed, suppressor cell activity was not as vigorous in deprived animals despite the presence of functional suppressor populations. Thymus growth was reinitiated promptly when high protein diets were fed to deprived animals. Thymus regeneration appeared to be due to both a resident population of stem cells which persisted in the thymus through the period of deprivation and a second, probably bone-marrow derived, population of stem cells. It is suggested that in normal mice the synchronized growth of the first population produces the characteristic innate growth pattern of the thymus. This is superimposed on the growth of the second population which continuously seeds the thymus and is constantly replaced. Protein deprivation severely restricts the growth of the first and second population, but both maintain their capacity for growth during long periods of protein restriction.     

Cell surface expression of cytotoxic T lymphocyte-defined, AKR/Gross leukemia virus-associated tumor antigens by normal AKR.H-2b splenic B cells

We previously described a system in which H-2Kb-restricted C57BL/6 (B6) cytotoxic T lymphocytes (CTL) could be raised that were specific for tumors, such as the thymic lymphoma AKR.H-2b SL1, that were induced by endogenous AKR/Gross murine leukemia virus and that expressed the Gross cell surface antigen. In this study, certain normal lymphoid cells from AKR.H-2b mice were also found to express target antigens defined by such anti-AKR/Gross virus CTL. AKR.H-2b spleen, but surprisingly not thymus, cells stimulated the production of anti-AKR/Gross virus CTL when employed at either the in vivo priming phase or the in vitro restimulation phase of anti-viral CTL induction. This selective stimulation by spleen vs thymus cells was not dependent on the age of the mice over the range (3 to 28 wk) tested. Both AKR.H-2b spleen and thymus cells, however, were able to stimulate the generation of H-2-restricted B6 anti-AKR minor histocompatibility (H) antigen-specific CTL. Thus, AKR.H-2b spleen cells appeared to display the same sets (minor H and virus-associated) of cell surface antigens recognized by CTL as the AKR.H-2b SL1 tumor, whereas AKR.H-2b thymocytes were selectively missing the virus-associated target antigens, a situation analogous to that of cl. 18-5, a variant subclone of AKR.H-2b SL1 insusceptible to anti-AKR/Gross virus CTL. Like AKR.H-2b thymocytes, neither AKR spleen cells or thymocytes nor B6.GIX + thymocytes were able to stimulate the generation of anti-AKR/Gross virus CTL from primed B6 responder cell populations. In contrast, both T cell-enriched and B cell-enriched preparations derived from AKR.H-2b spleen cells were able to stimulate at the in vitro phase of induction, although B cell-enriched preparations were considerably more efficient. The discordant results obtained with AKR.H-2b spleen cells vs thymocytes were confirmed and extended in experiments in which these cells were employed as target cells to directly assess the cell surface expression of virus-associated, CTL-defined antigens. Thus, AKR.H-2b spleen cells, but not thymocytes, were recognized by anti-AKR/Gross virus CTL when fresh normal cells were tested as unlabeled competitive inhibitors, or when mitogen blasts were tested as labeled targets. Fresh or lipopolysaccharide-stimulated B cell-enriched spleen cells were as efficiently recognized as unseparated spleen cell preparations. Unexpectedly, fresh or Lens culinaris hemagglutinin-stimulated T cell-enriched spleen cell preparations, although susceptible to anti-minor H CTL, were almost as poor as targets for anti-viral CTL as were thymocytes. Together, these results demonstrate the H-2-restricted expression of CTL-defined, endogenous, AKR/Gross virus-associated target antigens by normal AKR.H-2b splenic B cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effector and regulatory cells in autoimmune oophoritis elicited by neonatal thymectomy.

(C57BL/6 x A/J)F1 (B6AF1) mice thymectomized between days 1 and 4 of age develop autoimmune oophoritis (D3TX oophoritis) 4 to 6 wk later. Oophoritis can be adoptively transferred to young recipients, and the disease in D3TX mice is prevented by reconstitution with normal adult spleen cells. The present study was further defined the nature of the effector and suppressor cells. Contrary to an earlier report, oophoritis is transferred to syngeneic and not allogeneic recipients. The spleen cells from D3TX mice when stimulated in vitro with Con A, also transfer oophoritis to adult recipients. The effector cells are CD4+: oophoritis transfer is abrogated by CD4 antibody and not by CD8 antibody and C. Spleen cells from D3TX male mice transfer disease less efficiently than female cells, thus endogenous ovarian Ag may be required for activation of effector T cells. T cells from normal adult spleen that suppress D3TX oophoritis also appear to be of CD4+ phenotype. These cells are likely to be derived from adult thymus because adult thymocytes also suppress D3TX disease. We were unable to substantiate the earlier claim that suppressor cells in normal mice are ovarian Ag specific. Thus male and female spleen cells suppress disease with comparable efficiency, and deprivation of endogenous ovarian Ag by neonatal ovariectomy of cell donors had no observable effect on disease suppression.     

Visualization, fate, and pathogenicity of antigen-specific CD8+ T cells in the graft-versus-host reaction.

To follow the fate of alloreactive T cell effectors in graft-vs-host disease, Ld-specific CD8+ T cells from C57BL/6 2C TCR-transgenic donors were transplanted into sublethally irradiated (750 cGy) Ld+ or Ld- recipients. In Ld- C57BL/6 or (BALB/c-dm2 x C57BL/6)F1 recipients, naive 2C T cells engrafted and survived long term, but did not acquire effector function. In Ld+ (BALB/c x C57BL/6)F1 recipients, 2C T cells engrafted, expanded, became cytolytic, destroyed host B cells and double-positive thymocytes, and later disappeared. Despite marked damage to lymphoid and hemopoietic cells by 2C T cells, no significant pathology was detected in other organs, and recipients survived. Ld+ (BALB/c x C57BL/6)F1 recipients died when LPS/endotoxin was administered on day 7 after cell transfer, while Ld- (BALB/c-dm2 x C57BL/6)F1 recipients survived. Our findings show that under certain conditions, a CD8+ T cell population recognizing an extremely limited repertoire of Ags can initiate graft-vs-host disease.     

Tyrosine phosphorylation of CD19 in pre-B and mature B cells.

Cross-linking of B cell surface immunoglobulins (sIg) results in activation of mature B cells and stimulates a molecular signaling mechanism for antigen-specific B cell expansion and differentiation. This signaling pathway is dependent on tyrosine (Tyr) phosphorylation and results in the activation of sIg-associated src family kinases and p72SYK. Rapid Tyr phosphorylation occurs on multiple protein substrates. Here we show that activation of B cells by cross-linking sIg results in an increase in Tyr phosphorylation of the lineage-restricted B cell surface antigen CD19, and show that it is a major substrate of activated Tyr kinase following sIg stimulation. Lower levels of constitutive CD19 Tyr phosphorylation occurred in most sIg+ mature B cell lines examined and in normal dense tonsillar B cells. We also find that when CD19 is Tyr-phosphorylated it becomes competent to interact with SH2 domains suggesting a mechanism whereby, following B cell activation, CD19 could be linked to intracellular signaling pathways. In sIg- pre-B cell lines, CD19 was expressed but was not constitutively phosphorylated on tyrosine. Upon CD19 cross-linking, Tyr phosphorylation of CD19 was induced in sIg- pre-B cell lines. CD19 cross-linking also directly induced Tyr phosphorylation of CD19 and other substrates in mature B cells. The ability of CD19 to signal in the absence of sIg expression may provide important stimulation in pre-B cell development.     

Transfer into the mouse of mast cells differentiated in vitro

A population of mast cells can be derived in vitro by culturing normal spleen cells from C57BL/6 mice with the factor Interleukin 3. These mast cells share the morphological and histochemical features of mast cells at different stages of maturation. We have labelled these in vitro produced mast cells with 111In-Ox and injected them i. v. into normal syngeneic mice. The localisation of labelled cells has been determined 24 and 96 hours after the injection in the spleen, thymus and lymph-nodes.     

Effect of neonatal spleen and thymus implants on H-Y incompatible skin grafts

The transplantation reactivity of adult splenectomized C57BL females to H-Y incompatible skin grafts was significantly reduced by the implantation of spleen and thymus from foetal or 0-1-day-old syngeneic female donors. The thymus implant served as a possible source of natural suppressor cell precursors and the spleen implant as a microenvironment for their maturation.     

Demonstration of a CD3+ lymphocyte subset in the epidermis of athymic nude mice. Evidence for T cell receptor diversity.

The existence of CD3/TCR-bearing lymphocytes in athymic and thymectomized chimeric mice implies that T cell maturation can occur in the absence of a thymus. Considering the possibility that the epidermis may be one of the organs providing T cell educating stimuli, we attempted to characterize the Thy-1+ epidermal lymphocyte population of athymic mice. Immunohistologic studies of epidermal sheets revealed (1) that Thy-1+ epidermal cells of C57BL/6 nu/nu mice are CD5-, CD4-, and predominantly CD8-, and (2) that a minor subset of these cells displays anti-CD3 epsilon reactivity. Although these CD3+ epidermal cells could hardly be detected at 6 wk of age, they comprised approximately 2% of all Thy-1+ epidermal cells in 12-mo-old athymic mice. Most of these CD3+ cells expressed TCR-gamma/delta, but TCR-alpha/beta+ cells were also present. TCR-gamma/delta+ epidermal T cells of athymic mice preferentially expressed TCR V gamma 2, V gamma 4, and V gamma 5 specificities rather than TCR V gamma 3 as found on DETC of euthymic mice. Using mitogenic stimuli, we have succeeded in establishing cell lines and clones from BALB/c nu/nu and C57BL/6 nu/nu epidermis. Their marker profile corresponds to that seen on resident CD3+ epidermal cells, as well as on a very small subset of CD3+ splenic and lymph node lymphocytes of athymic mice. The ontogenetic relationship, if any, between the epidermal and lymphoid CD3+, CD5-, CD4-, CD8- cells, has yet to be clarified. Cell lines/clones representative of resident CD3+ epidermal cells of nu/nu mice should provide a useful tool in the elucidation of homing patterns and functional properties of extrathymically matured T cells.     

B lymphocyte development in rabbit: progenitor B cells and waning of B lymphopoiesis

In mammals that use gut-associated lymphoid tissues for expansion and somatic diversification of the B cell repertoire, B lymphopoiesis occurs early in ontogeny and does not appear to continue throughout life. In these species, including sheep, rabbit, and cattle, little is known about the pathway of B cell development and the time at which B lymphopoiesis wanes. We examined rabbit bone marrow by immunofluorescence with anti-CD79a and anti-mu and identified both proB and preB cells. The proB cells represent the vast majority of B-lineage cells in the bone marrow at birth and by incorporation of 5-bromo-2'-deoxyuridine, they appear to be a dynamic population. PreB cells reach maximum levels in the bone marrow at 3 wk of age, and B cells begin to accumulate at 7 wk of age. We cloned two VpreB and one lambda5 gene and demonstrated that they are expressed within B-lineage cells in bone marrow. VpreB and lambda5 coimmunoprecipitated with the mu-chain in lysates of 293T cells transfected with VpreB, lambda5, and mu, indicating that VpreB, lambda5, and mu-chains associate in a preB cell receptor-like complex. By 16 wk of age, essentially no proB or preB cells are found in bone marrow and by PCR amplification, B cell recombination excision circles were reduced 200-fold. By 18 mo of age, B cell recombination excision circles were reduced 500- to 1000-fold. We suggest that B cell development in the rabbit occurs primarily through the classical, or ordered, pathway and show that B lymphopoiesis is reduced over 99% by 16 wk of age.