Role of the major histocompatibility complex in T cell activation of B cell subpopulations: antigen-specific and H-2-restricted monoclonal TH cells activate Lyb-5+ B cells through an antigen-nonspecific and H-2-unrestricted effector pathway

Monoclonal T helper (TH) cell populations were employed to study the mechanism of activation of the Lyb-5+ B cell subpopulation in T cell-dependent antibody responses in vitro. It was demonstrated that monoclonal T cell populations were sufficient to help rigorously T-depleted unprimed (B + accessory) cells for direct plaque-forming cell responses to trinitrophenyl- (TNP) conjugated keyhole limpet hemocyanin (KLH). The activation of several lines of cloned (H-2b X H-2k)F1 TH cells was antigen (KLH) specific and H-2 restricted. Individual clones were restricted to H -2b, H-2k, or unique (H-2b X H-2k)F1 encoded determinants. Under the experimental conditions employed, responses mediated by cloned TH cells were found to result in the activation of the Lyb-5+ B cell subpopulation. The activation of Lyb-5+ B cells by cloned TH cells did not require covalent linkage of carrier and hapten, and responses could be stimulated in the presence of free KLH plus TNP conjugated to an irrelevant carrier. The H-2 restriction of TH cell function was shown to reflect a requirement for T cell recognition of determinants expressed by accessory cells, whereas no requirement existed for restricted T cell recognition of B cells. These findings suggest that the help provided by monoclonal TH cells, once activated, was both antigen nonspecific and H-2 unrestricted. Consistent with this interpretation, it was found that the supernatant of antigen-stimulated TH cells provided antigen-nonspecific help to T-depleted spleen cells. Thus, these results demonstrate that the activation of Lyb-5+ B cells by antigen-specific and H-2-restricted monoclonal TH cell populations is itself antigen nonspecific and H-2 unrestricted.     

Identification of a human protein homologous to the mouse Lyb-2 B cell differentiation antigen and sequence of the corresponding cDNA

We have isolated and sequenced cDNA clones encoding the human homolog of the mouse Lyb-2 B cell differentiation Ag. Previous data suggest that Lyb-2 might represent a growth factor or lymphokine receptor. Human Lyb-2 mRNA is expressed in normal human tonsils and bone marrow cells, in the pre-B cell line REH, in three Burkitt lymphoma cell lines, and in some EBV-transformed B cell lines, but not in antibody-secreting myeloma cell lines, T cell lines, or a promyelocytic leukemia cell line. These data indicate that expression of human Lyb-2 is restricted to B lineage cells and turned off in antibody-secreting plasma cells. A polyclonal mouse antiserum was raised against human Lyb-2 and immunoprecipitates a Mr 42,000 protein from REH, Raji, and Daudi cells and from mouse L(tk) cells transfected with the human Lyb-2 cDNA in an expression vector. The human Lyb-2 protein is related to both the asialoglycoprotein receptor and CD23, the B cell-specific FcR for IgE. These data demonstrate that human B cells express a previously undescribed cell surface protein that is homologous to mouse Lyb-2 and has a similar pattern of expression during B cell development.     

Functional characterization of two stromal cell lines that support B lymphopoiesis.

Bone marrow stromal cells have well documented effects on the production of B lymphocytes, but whether or not stromal cell signals are involved in the pre-B to B cell transition is unclear. The potential of two stromal cell lines, S10 and S17, in this process was examined. Initial experiments, using a short term liquid culture, indicated that S10 and S17 stroma efficiently supported the generation of clonable B cells (B lymphocyte CFU) from their immediate precursors in fresh bone marrow. The contribution of macrophages and other accessory cells in those experiments was minimized through use of a colony assay system that permits the direct effects of stromal cell signals on single B cell progenitors to be evaluated. The results indicated that soluble mediators from the S10 and S17 lines could support colony formation from fresh or cultured surface Ig- bone marrow cells. Colonies supported by S17 stroma appeared on day 15 and contained cells that expressed the B220 Ag; surface IgM expression was never observed. S10 supported colonies appeared on day 7 and routinely included surface IgM+ cells. Individual colonies were capable of undergoing additional growth when picked and replated directly onto the different stroma. Those colonies replated onto S10 stroma generated surface IgM expressing cells in up to 60% of experiments, but colonies transferred onto the S17 cell line included B cells only 10% of the time. These data demonstrate that stromal cells alone can provide the signals necessary for generating a surface IgM+ B cell from precursors but that not all stromal cell lines are equally efficient at doing so.     

Normal B cell precursors responsive to recombinant murine IL-7 and inhibition of IL-7 activity by transforming growth factor-beta

The ability of stromal cells in bone marrow to support B lymphopoiesis may be partially mediated by secretion of biologically active factors. The first cytokine with lymphopoietic activity to be molecularly cloned from stromal cells, IL-7, was originally identified by its growth-promoting activity on long term cultured lymphocytes. We now report that murine rIL-7 is a potent proliferative stimulus for B cell progenitors isolated from fresh bone marrow. Proliferation was initially most obvious among large precursor cells which bear the B lineage associated Ag, Ly5/220 and BP1. A majority of these also contained cytoplasmic Ig mu H chains. Extended culture with IL-7 resulted in a predominance of immature c mu- lymphocytes. No effect by IL-7 was observed on the proliferation of mature lymphocytes. It also did not induce maturation in a number of early B lineage cell lines, or promote the formation of LPS-responsive, clonable B cells from precursors. When incorporated into semisolid agar medium, IL-7 specifically and rapidly induced the formation of pre-B cell colonies in a linear fashion with respect to numbers of cells cultured from either purified B cell progenitor preparations or unfractionated bone marrow. In both liquid and agar culture conditions, the IL-7 proliferative activity was inhibitable by two related forms of transforming growth factor (TGF) beta, TGF-beta 1 and TGF-beta 2. Taken together, these results indicate that IL-7 is a stimulus for replication of normal B lineage cells at an early stage of differentiation, and its activity can be modulated by other cytokines. IL-7 also provides a means of studying the progeny of a single B cell progenitor, and of enumerating clonable pre-B cells in the absence of colony formation by other cell types in bone marrow.     

The role of recombinant stem cell factor in early B cell development. Synergistic interaction with IL-7.

The cDNA for stem cell factor was recently isolated from Buffalo rat liver cells (BRL-3A) and recombinant rat stem cell factor produced from Escherichia coli (rrSCF164). rrSCF164 synergizes with rhIL-7 to stimulate pre-B clonal growth in agar culture of mouse bone marrow cells, and in this study we have characterized the role of rrSCF164 in B cell development. The combination of rrSCF164 plus rhIL-7 stimulated increased colony numbers compared with the sum of colonies stimulated by rrSCF164 and rhIL-7 alone. Also, increased cell proliferation per colony was stimulated by the combination of rrSCF164 plus rhIL-7 compared with rhIL-7 or rrSCF164 alone. The colonies formed with rrSCF164 plus rhIL-7 and rhIL-7 alone contained exclusively pre-B cells, which expressed B220 Ag and cytoplasmic mu-chain, but were negative for surface Ig expression. Morphological examination of the cells in the colonies showed blast-like characteristics. rrSCF164 alone and in combination with rhIL-7 stimulated generation of B220+ cells in liquid culture of B220- cells, whereas rhIL-7 alone had no stimulatory effect on B220- cells. Both stem cell factor mRNA and bioactivity were detected in a mouse bone marrow-derived stromal cell line, termed OZ-11. We propose that stem cell factor is a stromal-derived factor that synergizes with IL-7 to stimulate the proliferation and differentiation of pro-B cells to pre-B cells, which become responsive to IL-7 alone.     

Expression and ontogeny of CD2 on murine B cells.

Expression of CD2 on developing and mature murine B cells was examined by using an antipeptide antiserum (L50). Most Ig-bearing splenic B cells were found to express CD2. Anti-CD5 and anti-B220 mAb divided the peritoneal B cells into two populations expressing high and low levels of these proteins; both populations were found to express uniform levels of CD2. Abelson murine leukemia virus-transformed pre-B cell lines derived from fetal liver and adult bone marrow were analyzed to delineate the ontogeny of CD2 in the B cell lineage. The results show that onset of CD2 expression correlates with the presence of cytoplasmic mu-chain. Therefore, the earliest CD2+ pre-B cell in the developing B cell population appears to be the classical pre-B cell.     

Regulation of early precursor B cell proliferation in mouse bone marrow: stimulation by exogenous agents mediated by macrophages in the spleen.

An increase in pre-B cell proliferation and B lymphocyte production in mouse bone marrow has previously been shown to follow the administration of various foreign agents in vivo. The responses of early precursor B cells before the expression of mu chains (pro-B cells) have now been examined, using double immunofluorescence labeling for terminal deoxynucleotidyl transferase (TdT) and B220 glycoprotein as detected by monoclonal antibody 14.8. A single injection of sheep red blood cells (SRBC) was followed by an increase in the number of cells in three defined populations of early precursor B cells lacking mu chains (TdT+ 14.8- cells, TdT+ 14.8+ cells, and 14.8+ mu- cells) as well as cytoplasmic mu-bearing pre-B cells and surface mu-bearing B lymphocytes. An accompanying increase in proliferative activity was indicated by the numbers of 14.8+ mu- cells and pre-B cells which accumulated in metaphase after inducing mitotic arrest with vincristine. These effects were all abrogated either by treating mice with silica to depress macrophage function or by splenectomy. In mice given multiple injections of SRBC for 4 weeks the elevated levels of early precursor B cell production and B cell genesis were sustained. The work demonstrates that the in vivo production of early precursor B cells, putatively including those at the stage of Ig heavy chain gene rearrangement, can be stimulated by exposure to external agents acting indirectly by a silica-sensitive, spleen-dependent mechanism. The findings suggest that the level of pro-B cell proliferation and primary B cell genesis normally taking place in mouse bone marrow may reflect the level of exposure to potential stimulants in the external environment mediated by activation of splenic macrophages. The possibility that abnormally high levels of macrophage activation could predispose to dysregulations of the B cell lineage is raised.     

Functional heterogeneity of the Lyb-5- B cell subpopulation: mutant xid B cells and normal Lyb-5- B cells differ in their responsiveness to phenol-extracted lipopolysaccharide

In the present study, responses stimulated by phenol-extracted lipopolysaccharide (LPS(phenol)) and butanol-extracted LPS (LPS(butanol)) were used to assess the possibility that xid B cells might not be identical to the Lyb-5- B cells present in normal mice. It was found that xid B cells responded well only to LPS(butanol) whereas normal B cells responded well to both LPS(butanol) and LPS(phenol). Thus, LPS(butanol) appeared to be a TI-1 antigen and LPS(phenol) appeared to be a TI-2 antigen. In contrast to classical TI-2 responses, however, responses stimulated by LPS(phenol) did not exhibit a stringent requirement for accessory cells. Furthermore, if LPS(phenol) were a classical TI-2 antigen, it should only activate Lyb-5+ B cells. To determine if the responsiveness of normal B cells to LPS(phenol) were due, at least in part, to the stimulation of normal Lyb-5- B cells, the responsiveness of normal neonatal B cells and normal adult B cells that had been pretreated with anti-Lyb-5.1 + C was assessed. It was found that both normal neonatal B cells and normal adult Lyb-5- B cells did respond well to LPS(phenol). Thus, even though LPS(phenol) does not stimulate xid B cells, these data demonstrate that LPS(phenol) is different from other TI-2 antigens. More importantly, these data also demonstrate that xid B cells and normal Lyb-5- B cells are not identical. It is hypothesized that the normal Lyb-5- B cell subpopulation is heterogeneous, consisting of an Lyb-5(1)- and an Lyb-5(2)-B cell subset with the xid mutation blocking the differentiation of Lyb-5(1)-B cells into Lyb-5(2)-B cells.     

Lyb-8.2: A new B cell antigen defined and characterized with a monoclonal antibody

A DBA/1 B10.D2-specific monoclonal antibody (CY34) is described which defines a new murine B lymphocyte differentiation antigen designated Lyb-8.2. The ontogeny, strain distribution, and cell-surface density of the antigen were studied by radioimmunoassay and by fluorescence-activated cell sorter (FACS) analysis. Lyb-8.2 appears to be expressed on pre-B cells and on all mature B lymphocytes. Lyb-8.2 molecules immunoprecipitated from surface labeled B10.D2 spleen cells migrated in polyacrylamide gels with an apparent mol. wt. of 95000–105000 daltons and were bound by lentil lectin. The expression of Lyb-8.2 is controlled by a locus on chromosome 7 that is closely linked to Gpi-1 and RP-2. Added Lyb-8.2-specific antibody did not measurably impair B lymphocyte function in several in vitro systems studied.     

A role for Lyb-2 in B cell activation mediated by a B cell stimulatory factor

The Lyb-2 system of the mouse is involved in regulation of a proliferative step in the differentiation of B cells responding to T-dependent antigen. The present study concerns the role of Lyb-2 in an early phase of B cell activation with respect to B cell receptor functions for activation factors. It is shown that interaction of monoclonal anti (alpha)-Lyb-2 antibody with Lyb-2 on the B cell surface induces B cell proliferation by synergistic action with B cell growth factor II-containing factor or interleukin 1. In contrast, alpha-Lyb-2 antibody could not synergize with the Con A-induced culture supernatant of T cell hybridoma FS6-14.13 (FS6) containing B cell stimulatory factor-1 (BSF-1; formerly called BCGF I), and the effect of combining the two was only additive on B cell proliferation. Absorption studies showed that BSF-1 in FS6 could be absorbed by unstimulated B cells, about 95% of which were at Go phase of the cell cycle, but not by thymocytes, and more importantly that alpha-Lyb-2 antibody blocked the absorption in an Lyb-2-specific manner, possibly by competing with BSF-1. It is thus likely that alpha-Lyb-2 antibody may interact with a BSF-1 receptor on B cells or a molecule closely associated with it. Interestingly, alpha-Lyb-2 antibody mimicked the action of BSF-1 in a costimulator assay with affinity-purified goat alpha-mouse IgM antibody, but could not replace all the activities ascribed to BSF-1. Possible mechanisms involved are discussed.     

Cell surface antigens expressed by subsets of pre-B cells and B cells

A large number of monoclonal antibodies, produced by immunizing rats with mouse pre-B cell lines, have been analyzed for their ability to define cell surface antigens expressed by B cells at early stages of differentiation. Whereas many antibodies recognized antigens on pre-B cell lines, only two clones detected cell surface antigens that were distinguished by their restricted distribution among a panel of continuous cell lines and cells from various tissues. Monoclonal antibody clone AA4.1 recognized a cell surface antigen found on all pre-B lymphomas and on one of three B lymphomas tested. This antigen was found on cells at highest frequency in the bone marrow. Adult spleen and fetal liver also have detectable numbers of AA4.1+ cells. Cells that did not express this antigen include plasmacytomas, two of three B lymphomas, T lymphomas, a stem cell line, adult liver, brain, thymus, and lymph node cells. Clone GF1.2 detected an antigen on some pre-B cell lines, one of three B lymphomas tested, and a small fraction of cells from adult bone marrow, spleen, lymph node, and fetal liver. Plasmacytomas, some pre-B lymphomas, two B lymphomas, T lymphomas, adult liver, brain, and thymus cells were negative. In adult bone marrow, AA4.1 bound to all cytoplasmic IgM+ pre-B cells, whereas GF1.2 detected one-half of these cells. Both antibodies recognized approximately 50% of surface IgM+ (sIgM+) bone marrow cells. A small population of bone marrow cells lacking any detectable Ig (surface or cytoplasmic) also reacted with these antibodies. Depletion of AA4.1 or GF1.2 antigen-bearing cells from bone marrow reduced the ability of bone marrow B cells to respond to LPS by 50 to 65%. Experiments with a cloned pre-B lymphoma demonstrate that AA4.1+ pre-B cells become sIgM+ GF1.2+ B cells after activation with LPS. These antibodies recognize cell surface determinants with restricted distribution among the B lymphocyte lineage because they detect antigens displayed by normal and transformed immature B lymphocytes.     

B cell deficiency progresses with lineage maturation in nude.X-linked immunodeficient mice B cell deficiency progresses with lineage maturation.

Previously we showed that unlike normal, nude, or X-linked immune deficient (xid) mice, nude.xid mice are deficient in bone marrow pre-B cell targets for Abelson murine leukemia virus transformation. We show that nude.xid bone marrow is deficient in both CD45(B220)+ and CD45(B220)- surface (s)IgM- progenitors that give rise to B cell colonies in Whitlock-Witte cultures. CD45(B220)+ precursors had normal differentiation potential in vitro. CD45(B220)- precursors differentiated into CD45(B220)+ cells at the same rate as normal controls, but acquired sIgM at a much slower rate. These results correlated with the observation that in nude.xid mice the severity of B lineage defects correlates with maturity: a profound (ninefold) deficit of sIgM+, CD45(B220)+ mature B cells, a fivefold deficit in the sIgM-, CD45(B220)+ precursors of short term B cell colonies (colonies forming within 4-5 days in Whitlock-Witte cultures), and a moderate (twofold) decrease in the frequency of sIgM-, CD45(B220)- (less mature) precursors of long term B cell colonies (colonies forming after 14 days of Whitlock-Witte culture. Thus the combination of the nude and xid mutations produces a deficiency in early B cell progenitors and the deficiency becomes more profound with further maturation. Therefore the lack of mature B cells is the result of a cascade effect. Inasmuch as bone marrow progenitors are affected, and these are the source of the vast majority of B cells, most B cells are affected by the xid mutation and the xid defect cannot be attributed to a loss of a fetal lineage of B cells. These results suggest that xid affected cells lack the capacity to progress efficiently through differentiation in the absence of an exogenous factor(s) that is dependent on the product of a normal allele at the nude locus. This product might be supplied in vivo by a T cell or T cell-dependent source and/or epithelial elements such as bone marrow stromal cells all of which are known to be affected by the nude mutation.     

Role of I-A molecules in early stages of B cell maturation.

The role of the Ia molecule in the early phase of B cell development remains controversial. In contrast to previous studies, we have detected minute amounts of Ia (I-A) molecule on early B lineage (B220+IgM-) cells from normal bone marrow, using ELISA. The presence of the I-A molecule even on pro-B cells was deduced from experiments in which a monoclonal anti-I-A antibody completely blocked the generation of pre-B cells from B progenitor (B220-) cells in stromal cell-dependent B cell culture. Inasmuch as this antibody did not inhibit the maturation of pre-B cells to IgM+ B cells in culture, the I-A molecule on early B lineage cells probably plays a role in their maturation. We also examined the role of the I-A molecule in early B cell development, using transgenic mice harboring the antisense DNA to I-A beta-chain gene. The amount of I-A molecule on splenic B cells from the young transgenic mice decreased in the presence of abundant amounts of the antisense RNA. B cell development was perturbed in spleen from the transgenic mice. Stromal cell-dependent B cell cultures from these mice clearly showed that the maturation of B lineage cells was delayed at a very early stage of development (B220- to B220+). We propose that the I-A molecule on early B lineage cells may play an essential role in their maturation.     

V-region directed selection in differentiating B lymphocytes.

We here analyse the repertoire of VH7183 rearrangements isolated from different stages of B cell differentiation in adult mice. The nucleotide sequence analyses of VH7183-D-JH rearrangements derived from large pre-B cells (B220+, mu-), small pre-B cells (B220+, mu-) and mature B cells (B220+, mu+) isolated from adult bone marrow revealed a sequential accumulation, among functional rearrangements, of D segments of the FL16 family and a depletion of D segments using the second and the third reading frame (RF). One member (VH7183.1) of the VH7183 gene family was utilized in 60-80% of the rearrangements of all populations analysed. In neonates the majority of the rearrangements utilizing this gene was found to be functional. In contrast, > 96% of the VH7183 rearrangements isolated from adult spleen were non-functional. These data provide evidence for cellular selection of VH regions acting at different points of the B cell differentiation pathway and at the transition of B cells from the bone marrow to the periphery.     

Ontogeny and distribution of the murine B cell Fc gamma RII.

The distribution and expression of the IgG FcRII (Fc gamma RII) on normal murine B cells was examined. Using multicolor flow cytometry, spleens from neonatal mice of increasing age and adult bone marrow were analyzed for expression of the Fc gamma RII. In addition, B cells from peripheral lymphoid organs, as well as panel of B cell tumors, were tested. The results demonstrate that the Fc gamma RII is expressed on all pre-B cells and immature B cells in the neonatal spleen and adult bone marrow, on all mature B cells in peripheral lymphoid organs, and on switched B cells in Peyer's patches. Furthermore, the Fc gamma RII was found to be present on B cell tumors representative of all stages of B cell maturation and differentiation. Taken together, the results indicate that Fc gamma RII is expressed during the entire lifetime of the B cell. In addition, examination of spleen cells from neonatal mice revealed a large number of pre-B cells, phenotypically defined as B220+, IgM-. These pre-B cells were present at birth, peaked in number between 2 and 3 wk of age, and became a minor population by day 30. Further phenotypic analysis of these cells demonstrated the expression of the BLA-1 and BP-1 Ag, and the lack of T cell and NK cell markers, thus confirming their assignment to the B cell lineage. Finally, the Fc gamma RII present on these pre-B cells was shown to be functional, by virtue of its ability to bind aggregated IgG.     

Lyb-5+ B cells can be activated by major histocompatibility complex-restricted as well as unrestricted activation pathways

It has previously been demonstrated that B cells can be activated through two distinct T helper (Th) cell-dependent pathways, one requiring both carrier-hapten linkage and MHC-restricted T-B interaction and the other requiring neither. In addition, it has been shown that different B cell subpopulations exist and that these subpopulations differ in their activation requirements. Previous studies demonstrated that resting B cells containing an Lyb-5+ subpopulation were activated by MHC-unrestricted T cell signals, whereas resting Lyb-5- B cells were activated only through MHC-restricted T-B interaction. It was suggested that this difference resulted from the ability of Lyb-5+ but not Lyb-5-B cells to respond to soluble MHC-unrestricted Th signals. Because Lyb-5+ B cells were responsive in these previous experiments to MHC-unrestricted Th signals, it could not be determined whether Lyb-5+ B cells were also responsive to MHC-restricted Th signals. Consequently, the present study was undertaken to directly address the question of whether Lyb-5+ B cells can be activated under appropriate conditions by MHC-restricted as well as unrestricted T cell-B cell interactions. It was found that unprimed normal B cells (containing Lyb-5+ and Lyb-5-B cells) but not unprimed xid-defective populations (Lyb-5- only) can be activated by cloned KLH-specific and MHC-restricted Th cells in response to either high or low concentrations of TNP-KLH. The IgM response of Lyb-5+-containing B cells to a high concentration of antigen (10 micrograms/ml) was MHC unrestricted, whereas the IgM response of unprimed Lyb-5+ B cells to a low concentration of antigen (0.001 micrograms/ml) was MHC restricted. Thus, unprimed Lyb-5+ B cells can be activated through both MHC-restricted and unrestricted pathways. It was further demonstrated that the activation requirements of Lyb-5+ and Lyb-5- B cells differed even for MHC-restricted B cell activation.     

Enhancement by monoclonal anti-Lyb-2 antibody of antigen-specific B lymphocyte expansion stimulated by TNP-Ficoll and T lymphocyte-derived factors

By using antigen-specific populations of B cells (TNP-ABC) we have demonstrated that the type-2 antigen TNP-Ficoll was capable of initiating B cell proliferation only in the presence of T cell-derived factors. Monoclonal-anti-Lyb-2.1 antibody acted synergistically with a T cell-derived supernatant, as well as with B cell-stimulating factor (BSF-1) to enhance the level of B cell expansion obtained in this in vitro system. This effect of anti-Lyb-2.1 mAB was observed at each day of the antigen-driven B cell expansion and was seen only with B cells purified from strains expressing the Lyb-2.1 allele. The epitope density of hapten on the Ficoll plays a critical role in this process, because Ficoll that is haptenated with low density of hapten was not found to be stimulatory. These results suggest that the Lyb-2 surface molecule influences the antigen-driven B cell growth that is stimulated by type 2 antigens and BSF-1.     

Multiple levels of selection responsive to immunoglobulin light chain and heavy chain structures impede the development of Dmu-expressing B cells

The truncated/V(H)-less mouse H chain Dmu forms precursor B cell receptors with the surrogate L chain complex that promotes allelic exclusion but not other aspects of pre-B cell development, causing most progenitor B cells expressing this H chain to be eliminated at the pre-B cell checkpoint. However, there is evidence that Dmu-lambda1 complexes can be made and are positively selected during fetal life but cannot sustain adult B lymphopoiesis. How surrogate and conventional L chains interpret Dmu's unusual structure and how that affects signaling outcome are unclear. Using nonlymphoid and primary mouse B cells, we show that secretion-competent lambda1 L chains could associate with both full-length H chains and Dmu, whereas secretion-incompetent lambda1 L chains could only do so with full-length H chains. In contrast, Dmu could not form receptors with a panel of kappa L chains irrespective of their secretion properties. This was due to an incompatibility of Dmu with the kappa-joining and constant regions. Finally, the Dmu-lambda1 receptor was less active than the full-length mouse mu-lambda1 receptor in promoting growth under conditions of limiting IL-7. Thus, multiple receptor-dependent mechanisms operating at all stages of B cell development limit the contribution of B cells with Dmu H chain alleles to the repertoire.     

Phenotypic and functional characterization of c-kit expression during intrathymic T cell development.

We have studied the expression and function of c-kit on subsets of mouse thymocytes. c-kit was primarily expressed on subpopulations of CD4-CD8-CD3- triple negative (TN) cells. The strongest c-kit expression was associated with subsets that represent the least mature TN cells, including CD44+CD25- TN, and a subpopulation of CD25+ TN. These cells were also Thy-1lo, H-2Khi TSA-1hi, HSAlo, B220-, Mac-1-, and Gr-1-. Additionally, the recently described pre-TN thymocyte population (CD4loCD3-CD8-) was also c-kit+. CD25+ TN thymocytes proliferated in the presence of IL-7 and stem cell factor (the ligand for c-kit), and this proliferation was completely inhibited in the presence of anti-c-kit. Furthermore, the addition of anti-c-kit to 2-deoxyguanosine-treated fetal thymic lobes undergoing reconstitution with fetal liver-derived precursor cells inhibited their T cell differentiation potential. These observations indicate an important role for c-kit/stem cell factor interactions during early thymocyte development.     

Differential regulation by Ly-5 and Lyb-2 of IgG production induced by lipopolysaccharide and B cell stimulatory factor-1 (IL-4)

We have previously shown that mAb Ly-5 which on B cells recognizes a 220,000-Da (B220) molecule, inhibits LPS-induced IgG responses without affecting IgM or proliferative responses, whereas mAb Lyb-2 which modulates B cell activation processes induced by B cell stimulatory factor-1 (BSF-1) or IL-4, has no effect on LPS-induced B cell responses. In this report we further examined the cellular mechanisms of Ly-5 antibody action and the effect of Lyb-2 antibody in IgG responses induced by LPS and BSF-1. The results presented demonstrated that the inhibitory effect of Ly-5 antibody seems to be restricted to the IgG class and is observed in all IgG subclasses induced by LPS. Limiting dilution analysis showed that the Ly-5 antibody reduces primarily the precursor frequency of IgG-secreting cells and that the effect on the clone size is partial. Lyb-2 antibody, on the other hand, greatly inhibited IgG1 induction initiated by LPS and BSF-1 by the action on processes triggered by BSF-1, although it could not reverse the reduced IgG2b or IgG3 responses. Limiting dilution analysis revealed that Lyb-2 antibody reduces the precursor frequency but not the clone size of BSF-1-induced IgG1-producing cells, supporting our previous proposition that Lyb-2 plays a critical role in the B cell differentiation mediated by BSF-1. Taken together, these results indicate that both Ly-5 and Lyb-2 are important molecules in IgG subclass regulation, each acting on a distinct activation step.