Immature B cells from human and mouse bone marrow can change their surface light chain expression

The capacity of bone marrow-derived surface immunoglobulin-positive (sIg⁺) human and mouse immature B cells, generated either in vitro or in vivo, to change their light (L) chain expression, has been assayed by the number of cells which change in vitro from one type of L chain to the other type, or to no sIg at all. Immature sIg⁺ B cells were generated in vitro from sIg^? precursor cells from human or mouse bone marrow. The immature sIg⁺ cells expressed RAG-1. Human sIg⁺ cells expressed xfr; and λ L chains in ratios between 1:1 and 3:1, whereas in mouse cells, this ratio ranged from 10:1 to 20:1. Upon reculture of the human and mouse xfr;⁺sIg⁺ cells, about half of them remained xfr;⁺, a quarter became λ⁺, and another quarter became sIg^?. Between 1 and 3% expressed both xfr; and λ chains. Of the human λ⁺ cells, about two-thirds remained λ⁺, only 1 to 2% became xfr;⁺, while the other third became sIg^?. Again, between 1 and 3% expressed both xfr; and λ L chains. These results indicate that expression of sIgM in the B cell membrane does not terminate L chain gene rearrangement, and that some order exists in xfr; versus λ gene rearrangements. Hence, human and mouse xfr;⁺ immature B cells can become λ⁺, but very few of the λ⁺ cells can become xfr;⁺, and both can become sIg^?. Further, human CD10⁺/sIg⁺ xfr;⁺ and λ⁺ cells and mouse B220^low/sIg^low xfr;⁺ cells enriched from bone marrow, i.e. immature B cells differentiated in vivo, changed their Ig phenotype upon in vitro culture, but in lower frequencies. By contrast, human and mouse mature B cells did not change their L chain or Ig phenotype. Hence, at least a part of the sIg⁺ immature B cells in bone marrow retain the capacity to change their L chain and Ig phenotype, and this capacity is lost when they become mature, peripheral B cells.     

Isolation of peritoneal precursors of B-1 cells in the adult mouse

Two weeks of daily peritoneopheresis of adult mice result in the selective depletion of B-1 cells, followed by the appearance of a population of B220⁺IgM^?lymphocytes in the peritoneal cavity. These cells share with bone marrow (BM) pre-B cells expression of λ5, V_preB, and RAG-1 genes and a higher fraction of unrearranged V to DJ heavy (H) chain immunoglobulin (Ig) gene segments, when compared with mature B lymphocytes. Upon transfer to SCID recipients, sorted peritoneal B220⁺IgM^? cells fail to colonize the BM, repopulate very few B cells in the spleen, but entirely reconstitute the B-1 cell compartment in the peritoneal and pleuropericardial cavities. In contrast, parallel transfers of sorted BM B220⁺IgM^? cells result in reconstitution of the BM and spleen B lineage cell compartments, but in no coelomic B cell repopulation. Both types of pre-B cells reconstitute splenic plasma cells of donor origin, but with markedly distinct efficiencies: the ratio of IgM-plasma cell/B cell numbers in the spleens of peritoneal pre-B cell recipients is more than 500-fold higher than that of recipients reconstituted by BM pre-B cells. We take these data to indicate that (1) differentiative commitment to the B-1 cell population occurs before selection events on mature cells; (2) B-1 precursors exist or may be locally produced in the adult mouse; (3) there is a lineage-related differential ability of mature B cells to undergo terminal differentiation to high-rate Ig secretion.     

Stimulation by T Cell Independent Antigens Can Relieve the Arrest of Differentiation of Immature Auto-Reactive B Cells in the Bone Marrow*

The pair of μH-chain and kL-chain transgenes encoding the Sp6 TNP/DN A-specific IgM was bred onto the rearrangement-deficient genetic background of RAG-2T mice, and onto the kL-chain expression-deficient background of iEkT mice. Bone marrow of Sp6 transgenic RAG-2T mice contained normal numbers of B220(CD45R)⁺ c-kit⁺ pro/preB-I-like cells and normal numbers of B220(CD45R)⁺TAC⁺ preB-II-like cells. Most strikingly, the numbers of immature sIgM⁺ B cells in the bone marrow were at least five-fold lower than normal, while mature B cells were almost undetectable in bone marrow as well as spleen. Hence, B cell development in these mice appears to be arrested at the transition from preB-II to immature B cells. The contents of bone marrow and spleen of the different precursors, immature and mature B cell compartments in Sp6iEkT mice were found to be similar to those of normal mice except that all sIg⁺ cells expressed μL-chains, of which 40% coexpressed the transgenic kL-chain. It indicates that the repertoire of μL-chain rearrangements and the μL-chains expressed from it suffices to relieve the arrest of differentiation seen in Sp6RAG-2T mice. The T cell-independent antigen TNP-Ficoll elicited within 5 days a response of the Sp6RAG-2T mice to develop to IgM-secreting cells and to fill the serum pool with the Sp6 transgenic IgM to 100 μg/ml, i. e. to normal serum levels of IgM in normal mice. TNP-Ficoll appears to interfere with the arrest of differentiation. Two scenarios for this arrest of differentiation and its relief by the T-independent antigen TNP-Ficoll are discussed.     

IL-2 receptor {alpha} chain (CD25JAC) expression defines a crucial stage in pre-B cell development

The analysis of the expression of the a chain of the IL-2 receptor(CD25.TAC) on the surface of B lineage cells In mouse bone marrowreveals that it is a useful marker to distinguish pre-B-I frompre-B-II cells. CD25 Is not expressed on CD45R(B220)⁺ c-kit⁺CD43⁺ TdT⁺ ₅⁺ C_µ^– slg^– lgH chain locus DJ_H-rearrangedpre-B-I cells of mouse bone marrow. It is expressed on largecycling CD45R(B220)⁺ c-kit⁺ CD43⁺ TdT⁺ ₅⁺ C_µ^– sig^–and on small resting CD45R(B220)⁺ c-kit⁺ CD43^– TdT⁺ ₅⁺C_µ^– sig^– sig- IgH chain locus VHDJH-rearrangedpre-B-II cells. Therefore, the transition from pre-B-I to largepre-B-II cells is marked by the downregulation of c-kit andterminal deoxynucleotldyl transferase (TdT), and by the upregulattonof CD25. SCID, RAG-2T, _µMT and ₆T mutant mice do havenormal, If not elevated numbers of pre-B-I cells but lack allCD25⁺ pre-B-II cells in their bone marrow. The expression ofa transgenic H chain under control of the _µH chain enhancerin RAG-2T bone marrow B lineage precursors allows the developmentof large and small CD25⁺ pre-B-II cells. The results suggestthat the differentiation of pre-B-I to pre-B-II cells in mousebone marrow requires the expression of _µH chains and surrogateL chains in membranes, probably on the surface of precursorB cells.     

Bone Marrow Stroma-Dependent Modulation of CD45R Isoform Expression on Abelson Virus Transformed Pre-B Cells

The CD45 glycoprotein family exhibits cell-lineage-associated structural heterogeneity which is due, in part, to alternative pre-mRNA splicing. The Abelson murine leukemia (A-MuLV) preferentially transforms immature B cells that express a B-cell-specific high molecular weight CD45 isoform, called B220. However, we observed that A-MuLV-transformed cell lines are often B220^- while maintaining high levels of “pan” CD45 expression. In vitro transformation of murine bone marrow revealed that the stromal microenvironment over which A-MuLV-transformed lymphoblasts are grown affected the B220 phenotype of the pre-B cells. Over a period of a few weeks, B220⁺ populations grown over a clonal stromal cell line gradually became B220^-. However, the transition from a B220⁺ to B220^- phenotype was dependent on the lot of fetal calf serum used. In contrast, cells grown over a heterogeneous bone marrow stroma maintained B220⁺ expression for long periods of time. The appearance of B220^- cells in clonal B220⁺ populations indicated that the change in phenotype resulted in part from modulation of B220 expression. B220^- B-cell lines did not express the high molecular weight CD45 RNA species indicating that the B220^- phenotype was due to alternative pre-mRNA splicing. Finally, the shift from B220⁺ to B220^- was not accompanied by changes in the stage of development of the cultures. These observations demonstrate that expression of B220 is not required for the continued proliferation of Abelson-transformed pre-B cells and is regulated by unknown environmental factors.     

Properties of mouse CD40: Cellular distribution of CD40 and B cell activation by monoclonal anti-mouse CD40 antibodies

We describe here the derivation of a rat monoclonal antibody (mAb) against mouse CD40 (designated 3/23), which stains 45–50% of spleen cells of adult mice, approximately 90% of which are B cells. Interestingly, some 5–10% of both CD4⁺ and CD8⁺ T cells in the spleens of (some, but not all) adult, unimmunized mice are also CD40⁺, whereas CD40⁺ cells were not detectable in the thymus, even following collagenase digestion. Some 35–40% of lymphoid cells in the bone marrow of adult mice are CD40⁺ and virtually all of these are B220⁺, and hence of the B cell lineage: triple-color flow cytometry showed that CD40 is expressed at low levels on some 30% of pre-B cells, at intermediate levels on 80% of immature B cells and on essentially all mature B cells in the bone marrow. These results, therefore, suggest that in the mouse CD40 is expressed relatively late during the process of B cell differentiation. The mAb induced marked up-regulation of major histocompatibility complex class II molecules, CD23 and B7.2 antigens on mature B cells. It also stimulated modest levels of DNA synthesis in mature B cells by itself: this was markedly enhanced by suboptimal concentrations of mitogenic (but not non-mitogenic) anti-μ and anti-δ mAb, and moderately enhanced by co-stimulation with interleukin-4. Hyper-cross-linking of CD40 (using biotinylated mAb and avidin) also enhanced the proliferative response to anti-CD40.     

PB76: a novel surface glycoprotein preferentially expressed on mouse pre-B cells and plasma cells detected by the monoclonal antibody G-52

A monoclonal antibody (mAb) G-5-2 was isolated which binds to transformed as well as normal cells of the B lineage but not to cells of the T cell, myeloid lineages nor to fibroblasts. mAb G-5-2 reacts with pre-B and plasma cell-transformed lines, and it preferentially recognizes normal pre-B cells from fetal liver and bone marrow as well as plasma cells from spleen of mice. G-5-2⁺ fetal liver cells isolated by cell sorter express mRNA for μ heavy chain Ig gene and generate in vitro antibody-producing cells when co-cultured with lipopolysaccharide and rat thymocyte filler cells. During development the frequency and staining intensity of G-5-2⁺ cells in fetal liver from normal mice increases from 1% G-5-2⁺ cells at day 14 to～7% positive cells at day 18 of gestation. Several strains or normal mice contain comparable numbers of G-5-2⁺ cells as well as B-220⁺ and BP-1⁺ B cell precursors in the fetal liver. Mice carrying the xid mutation have 3-4-fold less G-5-2⁺ as well as B-220⁺ and BP-1⁺ cells in the fetal liver, suggesting that the effects of the xid mutation may be manifested from early stages of B cell development. Fetal liver cells from mice carrying the scid mutation were found to contain normal numbers of G-5-2⁺ as well as B-220⁺ and BP-1⁺ pre-B cells. These results indicate that differentiation from progenitors to pre-B cells in scid mice may occur normally; the scid mutation would thus appear to affect the process of rearrangement and expression of the Ig genes in the developing pre-B cells. mAb G-5-2 precipitates a 76-kDa glycoprotein from surface-radiolabeled pre-B cells and plasma cells. Taken together, these results indicate that G-5-2 mAb recognizes a novel B cell lineage-specific surface molecule called PB76 which is preferentially expressed by pre-B cells and plasma cells.     

In rat bone marrow Thy-1 antigen is present on cells with membrane immunoglobulin and on precursors of peripheral B lymphocytes.

Rat bone marrow cells carrying Thy-1 antigen were studied morphologically, and tested for their independence of the thymus and their relationship to the B lymphocyte lineage. Using a fluorescence-activated cell sorter to separate Thy-1⁺ and Thy-1^- fractions, it has been confirmed that up to 50 % of all nucleated bone marrow cells are Thy-1⁺, most of which have the morphology of small lymphocytes. Thy-1^-cells were mainly neutrophils and erythroid. Thy-1⁺ cells were found also in the marrow of B rats (rats thymectomized as adults, irradiated and reconstituted with syngeneic bone marrow from thymectomized donors drained of recirculating lymphocytes), though at a lower frequency (roughly half) than of normal rats. In both normal and B rats about 1/4 of the Thy-1⁺ cells also bore lymphocyte surface immunoglobulin (sIg), and these doubly labeled cells accounted for the majority (～ 2/3) of marrow cells carrying large amounts of sIg. Therefore, unlike mice, Thy-1 is not a marker of thymus-dependent lymphocytes in rats. The B precursor activity of marrow fractions was measured in a long-term re-constitution assay counting sIg⁺ cells in the thoracic duct of lethally irradiated recipients. Virtually all the precursors were in the Thy-1⁺ or sIg^- fractions, and were barely detectable among Thy-1^- or sIg⁺ cells. Thus, in the rat peripheral B lymphocytes descend from precursors bearing Thy-1 antigen but lacking sIg.     

Mechanisms of clonal abortion tolerogenesis. III. Antigen abrogates functional maturation of surface immunoglobulin-negative adult bone marrow lymphocytes.

The fluorescence-activated cell sorter (FACS) was used to fractionate adult CBAmouse bone marrow cells on the basis of their size and the amount of surfaceimmunoglobulin (sIg) expressed. It was noted that a population of viable, small sIg^?cells could generate up to 28% slg⁺ cells within 24 h in tissue culture. The capacity of various cell fractions to respond to T-independent stimulation by antibody productionwas studied. Fractionated cells were placed into microculture at limiting dilution inthe presence of thymus “filler” cells, stimulated with E. coli lipopolysaccharide (LPS)and after 3 to 5 days, assayed for the presence of antibody-forming cell clones to thehapten (4-hydroxy-3-iodo-5-nitrophenyl) acetyl (NIP). Through appropriate statistical means, the number of anti-NIP precursors originally present was calculated. Whilethe sIg⁺ fraction contained approximately 70 cells in 10⁶ capable of immediate development into anti-NIP clones, the slg^? fraction contained very few reactive cells. However, the cloning capacity of this fraction increased markedly following 24 h of maturation in tissue culture. When bone marrow cells were exposed to NIP-coupled human gamma-globulin (NIP-HGG) acting as a B cell tolerogen, some tolerance was induced in vitro reflecting therelative immaturity of these cells. When the FACS-sorted sIg^? cells were similarlytreated, there was virtually complete abrogation of their functional maturation intoclonable anti-NIP precursors. The effect of LPS on cells converting from the sIg^? tosIg⁺ status was investigated. When LPS and NIP-HGG were concomitantly present, about one-third of the cells escaped tolerance induction. LPS thus “rescued” somecells from clonal abortion, but certainly did not totally abolish tolerance induction. The significance of these results for the clonal abortion theory of B lymphocytetolerance is briefly discussed. The conclusion is reached that cells converting from thesIg^? to sIg⁺ status in the normal course of differentiation represent a prime target fortolerance induction.     

Interferon-γ arrests proliferation and causes apoptosis in stromal cell/interleukin-7-dependent normal murine pre-B cell lines and clones in vitro,but does not induce differentiation to surface immunoglobulin-positive B cells

Normal pre-B cells from fetal liver or bone marrow of the mouse proliferate for long periods of time in tissue culture on stromal cells in the presence of interleukin-7 (IL-7). Their IgH loci are partly in germ-line, partly in D_HJ_H-rearranged configuration, while their light chain loci are in germ-line configuration. They express the pre-B cell-specific genes V_preB and λ₅. Proliferation of these pre-B cells is inhibited by interferon (IFN)-γ, with half-maximal inhibition at concentrations between 0.1 and 1 unit/ml. Normal pre-B cells exposed to IFN-γ die by apoptosis, as is evidenced by the disintegration of pre-B cell DNA into oligonucleosomal multimers of 180-200 bp. While the proliferation of pre-B cells from Eμ-bcl-2 transgenic (tg) mice is inhibited by IFN-γ, these cells do not die by apoptosis. IFN-γ does not induce differentiation to more mature B lineage cells. In the absence of IL-7 normal pre-B cells differentiate to V_HD_HJ_H/V_LJ_L-rearranged, surface immunoglobulin-positive B cells expressing the a chain of the IL-2 receptor. They also down-regulate the expression of V_preB and X.5, and lose the capacity to proliferate on stromal cells in the presence of IL-7. In contrast, both normal and Eμ-bcl-2 tg pre-B cells exposed to IFN-γ in the presence of stromal cells and IL-7 fail to differentiate, i.e. do not express surface immunoglobulin, retain expression of V_preB and λ₅, do not express the α chain of the IL-2 receptor, and retain the capacity to proliferate on stromal cells in the presence of IL-7, once IFN-γ is removed. The potential usefulness of a treatment of acute lymphocytic leukemia of the B cell lineage (pre B-ALL) with IFN-γ is discussed.     

Transitional B cell subsets in human bone marrow

B cells originate from precursors in the bone marrow, and the first cells which migrate to the peripheral blood have been classified as ‘transitional B cells’. Transitional B cells have been characterized in human blood with stage 1 (T1) and stage 2 (T2) subsets being proposed. In the present study, 27 normal human bone marrow samples were analysed for transitional B cell markers by eight‐colour flow cytometry. T1 transitional B cells (CD45⁺CD19⁺CD10⁺IgM⁺IgD^lo) and T2 transitional B cells (CD45⁺CD19⁺CD10⁺IgM⁺IgD⁺) were identified in normal bone marrow samples at a mean frequency of 3·2 and 3·1% of total B lineage cells, respectively. A majority of the bone marrow transitional B cells were CD24^hiCD38^hi, the phenotype of blood transitional B cells. Consistent with recent peripheral blood data, T2 B cells had a significantly higher CD21 expression compared with T1 B cells (72·4 versus 40·9%) in the bone marrow. These data raise the possibility that transitional B cells are capable of differentiating from T1 to T2 B cells within the bone marrow. Furthermore, transitional cells at either stages 1 or 2 might be capable of migrating out of the bone marrow.     

Impaired Ca2+ mobilization by X-linked agammaglobulinaemia (XLA) B cells in response to ligation of the B cell receptor (BCR)

XLA bone marrow samples were shown to contain B cells expressing IgM, and pre-B cells that express the μ-surrogate light chain (μψLC) complex, albeit at a reduced frequency to that found in normal bone marrow. Antibody ligation of μ heavy chain on these cells and an XLA B cell line did not induce a Ca²⁺ flux, whereas ligation of μ heavy chain on normal bone marrow cells, μψLC⁺ pre-B cell lines and an IgM⁺ B cell line did. The block in XLA B cells was not due to a defect in the basic mechanism of Ca²⁺ flux generation, as the cells responded well to thapsigargin. In addition, the defect did not affect T cells, which were shown to respond to CD3 antibody with a Ca²⁺ flux. Ligation of μ heavy chain on XLA bone marrow cells did, however, activate tyrosine kinases, resulting in tyrosine phosphorylation of a cellular protein with a molecular weight of approximately 115 kD. These results indicate that Btk may be necessary for the generation of the Ca²⁺ flux in response to ligation of μ heavy chain on B cells and μψLC⁺ pre-B.     

Adult bone marrow contains precursors for CD5+ B cells

To determine directly whether B cell precursors of adult origin are capable of generating CD5⁺ B cells, we reconstituted neonatal C3H.SCID mice with adult C57BL/6 bone marrow and analyzed splenic B cells 10 months later. Surface staining and flow cytometry revealed that the B cells were of donor origin and that 30% were CD5⁺. This confirms that in vivo generated CD5⁺ B cells can be adult derived. After anti-IgM (but not lipopolysaccharide) stimulation in vitro, virtually all of the B cells from the bone marrow-reconstituted mice expressed surface CD5. Sequence analysis of expressed V_HDJ_H genes from the CD5⁺ B cells present after anti-IgM stimulation revealed a high frequency of N nucleotide addition in CDR3 regions. The presence of N nucleotides indicates that these sequences were derived from CD5⁺ B cells of adult origin rather than from long-lived fetal precursor B cells present in either the adult bone marrow at the time of transfer or adult spleen. These experiments demonstrate conclusively that adult bone marrow contains precursors for CD5⁺ B cells and that unlike fetal liver-derived precursors these express terminal deoxynucleotidyl transferase.     

Old mice retain bone marrow B1 progenitors, but lose B2 precursors, and exhibit altered immature B cell phenotype and light chain usage

The bone marrow of old adult mice (∼2 years old) has reduced B lymphopoiesis; however, whether the B1 pathway in adult bone marrow is also compromised in senescence is not known. Herein, we show that phenotypic (IgM⁻Lin⁻CD93⁺[AA4.1⁺] CD19⁺B220^low/−) B1 progenitors are retained in old bone marrow even as B2 B cell precursors are reduced. Moreover, B1 progenitors from both young adult and old mice generated new B cells in vitro enriched for CD43 expression, likely due to their activation, and exhibited increased λ light chain usage and diminished levels of κ light chain expression. B1 progenitors were shown to have lower surrogate light chain (λ5) protein levels than did B2 pro-B cells in young mice and these levels decreased in both B1 and B2 precursor pools in old age. These results indicate that the B1 B cell pathway persists during old age in contrast to the B2 pathway. Moreover, B1 B cell progenitors generated new B cells in the adult bone marrow that have distinct surface phenotype and light chain usage. This is associated with decreased surrogate light chain expression, a characteristic held in common by B1 progenitors as well as B2 precursors in old mice.     

Fidelity and infidelity in commitment to B‐lymphocyte lineage development

Summary: During B‐lymphocyte development in mouse fetal liver and bone marrow, a pre‐B I cell stage is reached in which the cells express B‐lineage‐specific genes, such as CD19, Iga and Igb, and V_preB and l5, which encode the surrogate light (SL) chain. In these pre‐B I cells both alleles of the immunoglobulin heavy (IgH) chain locus are D_H J_H rearranged. Transplantation of pre‐B I cells from wild‐type (e.g. C57Bl/6) mice in histocompatible RAG‐deficient hosts leads to long‐term reconstitution of some of the mature B‐cell compartments and to the establishment of normal IgM levels, a third of the normal serum IgA levels, and IgG levels below the detection limit. Neither T‐lineage nor myeloid cells of donor origin can be detected in the transplanted hosts, indicating that the pre‐B I cells are committed to B‐lineage differentiation. Consequently, the B‐cell‐reconstituted hosts respond to T‐cell‐independent antigens but not to T‐cell‐dependent antigens. Responses to T‐cell‐dependent antigens can be restored in the pre‐B I‐cell‐transplanted, RAG‐deficient hosts by the concomitant transplantation of mature CD4+ T cells. The transplanted wild‐type pre‐B I cells do not home back to the bone marrow and become undetectable shortly after transplantation. B‐lymphocyte development in Pax‐5‐deficient mice becomes arrested at the transition of pre‐B I to pre‐B II cells i.e. at the stage when V_H to D_HJ_H rearrangements occur and when the pre‐B‐cell receptor, complete with H chains and SL chains, is normally formed. T‐lineage and myeloid cell development in these mice is normal. Pre‐B I cells of Pax‐5‐deficient mice have a wild‐type pre‐B I‐cell‐like phenotype: while they do not express Pax‐5‐controlled CD19 gene, and express Iga to a lesser extent, they express Igb, V_preB and l5, and proliferate normally in vitro on stromal cells in the presence of interleukin (IL)‐7. Clones of these pre‐B I cells carry characteristic D_H J_H rearrangements on both IgH chain alleles. However, removal of IL‐7 from the tissue cultures, unlike wild‐type pre‐B I cells, does not induce B‐cell differentiation to surface IgM‐expressing B cells, but induces macrophage differentiation. This differentiation into macrophages requires either the presence of stromal cells or addition of macrophage colony‐stimulating factor (M‐CSF). Addition of M‐CSF followed by granulocyte–macrophage colony‐stimulating factor induces the differentiation to MHC class II‐expressing, antigen‐presenting dendritic cells. In vitro differentiation to granulocytes and osteoclasts can also be observed in the presence of the appropriate cyto­kines. Moreover, transplantation of Pax‐5‐deficient pre‐B I clones into RAG‐deficient hosts, while not allowing B‐cell differentiation, leads to the full reconstitution of the thymus with all stages of CD4–CD8– and CD4+CD8+ thymocytes, to normal positive and negative selection of thymocytes in the thymus, and to the development of normal, reactive mature CD4+ and CD8+ T‐cell compartments in the peripheral lymphoid tissues, all carrying the clone‐specific D_H J_H rearrangements. On the other hand, Iga, Igb, V_preB and l5 are turned off in the thymocytes, demonstrating that the expression of these genes does not commit cells irreversibly to the B lineage. Furthermore, Pax‐5‐deficient pre‐B I cells are long‐term reconstituting cells. They home back to the bone marrow of the RAG‐deficient host, can be reisolated and regrown in tissue culture, and can be retransplanted into a secondary RAG‐deficient host. This again develops thymocytes and mature T cells and allows the transplanted clonal pre‐B I cells to home to the bone marrow.     

Expression of CD2 on porcine B lymphocytes

Remarkable interspecies differences in CD2 expression on B lymphocytes have been reported in mammals. Human and rat B cells lack CD2, whilst B lymphocytes in mice are CD2⁺. In pigs, B cells have been supposed not to express CD2. We show here, however, that CD2 is present at a low level on a prominent subset of porcine B cells. Moreover, we describe changes in the proportions of CD2⁺ and CD2^? B-cell subsets during ontogeny. Before contact with microflora, the majority of peripheral surface immunoglobulin M⁺ (sIgM⁺) B cells express CD2 and sIgM⁺CD2^? B cells are rare. Shortly after colonization of conventional (CV) piglets with complex intestinal microflora, numerous CD2^?B cells appear in the periphery and their relative number increases with age in both CV and specific pathogen-free (SPF) pigs. However, monoassociation of germ-free (GF) piglets with a single Escherichia coli strain does not result in a significant increase of sIgM⁺CD2^?B cells in the periphery. We suggest that CD2 is down-regulated in porcine B lymphocytes upon activation with microflora in mucosa-associated lymphatic tissues. In bone marrow (BM), we identified putative porcine B-cell precursors. These cells express CD2 at low density and do not bear either the common myelomonocytic antigen or T and B-lymphocyte receptors. Similar to mouse and human pre-B cells, this lymphocyte-sized subset expresses CD25 and class II antigens. CD2 positivity of these cells indicates that CD2 is expressed earlier than sIgM during B lymphopoiesis in pigs.     

T cell specialization at environmental interfaces: T cells from the lung and the female genital tract of lpr and gld mice differ from their splenic and lymph node counterparts

Mice homozygous for lpr and gld accumulate CD4^?CD8^? (double-negative, DN) B220⁺CD5¹⁰Thy-1¹⁰ αβ T cells in the spleen and lymph nodes (LN), while mucosal gut T cells are normal. To study other mucosa-associated T cell populations, we examined T cell subsets separated according to expression of αβ T cell receptor, CD4, CD5, CD8, Thy-1 and B220 in the lung and the female genital tract (FGT) of adult MRL lpr, C3H lpr and C3H gld mice. αβ T cell accumulation was detected in both the FGT and the lungs of lpr and gld mice but, in contrast to the spleen and LN, equal proportions of DN B220⁺ and CD4⁺ of CD8⁺ (single-positive, SP) B220^? T cells were observed in these sites, and the T cells had an increased expression of Thy-1 and CD5. Staining for CD44, L-selectin, and CD45RB revealed a higher percentage of effector/memory T cells in lpr and gld lungs and FGT compared to spleens and LN. CD69 expression suggested chronic activation of DN and SP T cells in lpr and gld lungs and FGT. Thus, we show that FGT and lung resident T cells are affected by lpr and gld mutations, but that their phenotypes are distinct from those of systemic T cells. These data suggest that T cells associated with FGT and lung mucosal tissues represent a separate lineage from systemic T cells, and/or that the abnormal T cells in lpr and gld mice are selected against in mucosal surfaces exposed to environmental antigen.     

DIFFERENTIAL STAT5 ACTIVATION AND PHENOTYPIC MARKER EXPRESSION BY IMMUNE CELLS FOLLOWING LOW LEVELS OF ETHANOL CONSUMPTION IN MICE

ABSTRACT

Ethanol has been recognized as an immunosuppressive agent for many years. Effects of high levels of ethanol consumption on immune functions have been extensively studied, but little is known about the effects of low levels (scuh as 5% ethanol) of ethanol consumption. Herein we report that exposure of mice to 5% ethanol for 4–8 weeks decreases IL-2-augmented splenic NK cell activity, decreases the numbers of NK cells in spleen and liver, decreases the number of granulocytes (Gr-1⁺) in bone marrow and spleen, and decreases the percentages of B cells in liver. In contrast, the percentages of CD4⁺CD8⁺ thymocytes, CD4⁺CD8^? splenocytes, CD4⁺CD8^? liver nonparenchymal cells, CD3⁺ splenocytes, and CD3⁺ bone marrow cells were increased. Furthermore, exposure to 5% ethanol increases STAT5 activation in T cells and liver cells while decreases STAT5 activation in NK cells. Taken together, these findings suggest that low levels of ethanol consumption can differentially modulate immune cells in thymus, spleen, bone marrow and liver, which may be due to differential regulation of STAT5 activation by ethanol.