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.     

B-1 cells in the bone marrow are a significant source of natural IgM

Natural IgM antibodies secreted in the absence of antigenic challenge are important contributors to antimicrobial immunity and tissue homeostasis. Early studies identified BM and, to a lesser extent the spleen, as main tissue sources of this spontaneously secreted IgM. However, the responsible B-cell subset has never been identified. Using multicolor flow cytometry, cell sorting and chimeric mice in which B-1 and B-2 cells and their secreted antibodies are distinguished by their Ig-allotype, we unequivocally identify the natural IgM-secreting cells in spleen and, for the first time, in the BM as IgM(+) IgD(lo/-) CD19(hi) CD43(+) CD5(+/-) B-1 cells. The newly identified population of BM B-1 cells shows many of the phenotypic characteristics of splenic B-1 cells but is distinct from B-1 cells in the peritoneal cavity, which generate at best very small amounts of IgM. Antibody-secreting spleen and BM B-1 cells are distinct also from terminally differentiated plasma cells generated from antigen-induced conventional B cells, as they express high levels of surface IgM and CD19 and lack expression of CD138. Overall, these data identify populations of non-terminally differentiated B-1 cells in spleen and BM as the most significant producers of natural IgM.     

Inside the thymus, Mls antigen is exclusively presented by B lymphocytes.

The ability to stimulate an Mls-1 mixed lymphocyte reaction (MLR) is predominantly expressed by low density B lymphocytes in the spleen and peritoneal cavity of normal adult mice, and is absent in splenic B cells 1 month after lethal irradiation and reconstitution from autologous bone marrow. Coreconstitution of these mice with normal syngeneic peritoneal cells restores the stimulatory potential of splenic B cells, but sorted CD5+ or CD5- IgM+ lymphocytes from peritoneum are equally good stimulators, suggesting that functional Mls-1 expression may require long life spans and selection. Bone-marrow-reconstituted DBA/2 mice that fail to express Mls-1 antigens in the periphery nevertheless maintain T-cell receptor V beta 6 and 8.1 deletions among the newly formed T cells. These findings led us to directly investigate the Mls stimulatory ability of purified antigen-presenting cell populations inside the thymus. We report here that thymic B lymphocytes seem to represent the only intrathymic cell population able to stimulate Mls-1 MLR.     

Immunoglobulin-bearing B cells reconstitute and maintain the murine Ly-1 B cell lineage

Previous reconstitution studies in irradiated mice distinguished Ly-1 B from other B cells. The predominant B cell populations in spleen and lymph node are readily reconstituted from progenitors present in adult bone marrow. Ly-1 B cells, in contrast, are reconstituted from cells in peritoneum rather than bone marrow. Further studies, presented here, demonstrate that Ly-1 B populations are reconstituted from peritoneal cells that already express both surface immunoglobulin (IgM) and Ly-1, that is, Ly-1 B. Such cells reconstitute and maintain the Ly-1 B population in irradiated recipients for at least 6 months.     

The effect of an immunoglobulin mu transgene on B cell maturation.

In mu 17.2.25-transgenic (M54) mice the absolute number of surface IgM (sIgM) B cells in lymphoid organs is drastically reduced compared to normal C57BL/6 mice and a high frequency of B cells express the immunoglobulin (Ig) encoded by the transgene rather than endogenous Ig on the surface. To determine the effect of a mu transgene on B cell development, adoptive cell transfers were performed using mu transgenic (M54) bone marrow and fetal liver cells. The data presented support the following conclusions: (a) adult transgenic bone marrow contains functional B cell precursors able to mature and repopulate the spleen and peritoneum of recipient mice. The relative frequency of transgene (sIgMa) and endogenous (sIgMb) surface sIgM-positive B cells reconstituted by transgenic bone marrow in allotype-matched C57BL/6 recipients is the same as in the M54 donors; (b) serum analysis indicates that transgenic bone marrow donor cells can reconstitute B cells in congenic and severe combined immunodeficient (SCID) recipient mice; (c) transgenic fetal liver cells are not a richer source of precursors for B cells expressing endogeneous Ig; (d) in transgenic mice sIgM+ B cells are not restricted to the CD5+ phenotype, however, the relative frequency of sIgMb B cells that are CD5+ is higher in transgenic than normal mice; and (e) bone marrow cells from adult normal and transgenic mice are able to generate CD5+ B lymphocytes in the spleen and peritoneum of allotype-congenic and neonatal SCID recipient mice. The results indicate that the presence of a complete mu heavy chain transgene does not result in a selective developmental block of "conventional" bone marrow-derived pre-B and B cells.     

Biased VH gene expression in murine CD5 B cells results from age-dependent cellular selection.

Flow cytometry-purified, peritoneal and splenic CD5+ and CD5- B cells from neonatal and adult C57BL/6 mice were studied for expression of VH and Vx gene families in RNA colony blot assays, and for frequencies of clones secreting antibodies to bromelain-treated mouse red blood cells (BrMRBC), single-stranded DNA, trimethyl ammonium and bovine gamma-globulin, by limiting dilution. The results show few overall differences between the two B cell subsets, which both manifest ontogenic D-proximal VH preferences that are lost with age. Biased VH11 expression in CD5 B cells is high in adult peritoneum and spleen but absent in newborns. It only partly correlates with the selection of anti-BrMRBC reactivity, which is considerably higher in peritoneum than in spleen. No particular Vx bias was observed in any of the populations studied with the possible exception of Vx22 in peritoneal CD5+ B cells. We conclude that the antibody repertoire expressed by peritoneal CD5+ B cells of adult mice is not the result of a genetic program, but rather the consequence of local, age-dependent cellular selection mechanisms.     

Inside the thymus,Mls antigen is exclusively presented by B lymphocytes

The ability to stimulate an Mls-1 mixed lymphocyte reaction (MLR) is predominantly expressed by low density B lymphocytes in the spleen and peritoneal cavity of normal adult mice, and is absent in splenic B cells 1 month after lethal irradiation and reconstitution from autologous bone marrow. Coreconstitution of these mice with normal syngeneic peritoneal cells restores the stimulatory potential of splenic B cells, but sorted CD5⁺ or CD5⁻ IgM⁺ lymphocytes from peritoneum are equally good stimulators, suggesting that functional Mls-1 expression may require long life spans and selection. Bone-marrow-reconstituted DBA/2 mice that fail to express Mls-1 antigens in the periphery nevertheless maintain T-cell receptor Vβ 6 and 8.1 deletions among the newly formed T cells. These findings led us to directly investigate the Mls stimulatory ability of purified antigen-presenting cell populations inside the thymus. We report here that thymic B lymphocytes seem to represent the only intrathymic cell population able to stimulate Mls-1 MLR.     

Bone marrow cells from young and old New Zealand black mice can reconstitute B lymphocytes in severe combined immunodeficient recipients

The formation of B lymphocytes in young New Zealand Black (NZB) mice proceeds at an accelerated rate, resulting in a deficiency of B lineage progenitors in mice of 15 weeks of age and older. Multiple studies have indicated that intrinsic defects in B lineage cells as well as in the hemopoietic microenvironment in which they develop contribute to these cellular abnormalities. To determine whether the B-cell hyperactivity observed in young mice could be observed in a normal environment, bone marrow cells from young (4 weeks or less) NZB donors were transplanted into Severe Combined Immunodeficient (SCID) mice that have a marked deficiency of lymphocytes but an apparently normal hemopoietic microenvironment. Engraftment of donor lymphoid cells can occur without pretransplant conditioning regimens, thus minimizing the chances of transferring microenvironmental elements. Marrow from young NZB donors reconstituted surface IgM-expressing B cells and CFU-B (B-cell colony-forming unit) in the marrow of SCID mouse recipients to levels comparable to that observed with donor NZB.xid marrow. The latter mice carry the xid gene that ameliorates the defects exhibited by B lineage cells of NZB mice. Both the number of surface IgM-expressing B cells and CFU-B were higher in the spleen of SCID mice that received NZB grafts than marrow cells from donor BALB/c or NZB.xid mice. Marrow from young NZB donors also reconstituted Thy-1, L3T4 and Lyt2-expressing cells in the spleen to levels higher than observed with young NZB.xid donor cells. The transplantation of marrow from 6-month-old NZB donors made it possible to test whether B lineage cells were present in that tissue and could mediate reconstitution in the normal SCID environment. Marrow from old NZB donors did reconstitute B cells in the marrow and spleen of SCID recipients. The level of reconstitution was comparable to that mediated by young BALB/c cells and twice that of old NZB.xid donor cells. The absolute number of splenic CFU-B was also higher in recipients of old NZB marrow as compared to young BALB/c cells. Old NZB.xid donor marrow reconstituted splenic Thy-1, L3T4 and Lyt2 T cells to levels less than observed with NZB donor cells. Analysis of serum Ig in recipients of old NZB cells indicated higher levels of total IgM as compared to mice engrafted with NZB.xid cells, and anti-single stranded DNA antibodies were detected.     

Isotypes and specificities of immunoglobulins produced by germ-free mice fed chemically defined ultrafiltered "antigen-free" diet

The regulation of the "spontaneously" occurring ("background") Ig synthesis of mice has been studied by determining the numbers of IgM-, IgG- and IgA-secreting cells and a part of the IgM antibody-specificity repertoire in spleen, bone marrow (BM) and mesenteric lymph nodes (MLN) of conventional and "antigen-free" mice. These antigen-free mice were germ-free raised and fed an ultrafiltered solution of chemically defined low molecular weight nutrients, and thus devoid of exogenous antigenic stimulation. The secretion of IgM, IgG and IgA by spleen, BM and MLN cells was assessed in the protein A plaque assay, while specific IgM antibody-secreting cells were detected by plaque assays specific for differently haptenated sheep red blood cells. In general, antigen-free and conventional mice were found to have roughly equal numbers of IgM-secreting cells in spleen and BM. The number of IgG-secreting cells in the spleen of antigen-free mice was the same as in the spleen of conventional mice, but in the BM their number was 3-5-fold decreased. About one half of the antigen-free mice did not have MLN, and in the half which did, 5 times less IgM- and more than 100-fold less IgG-secreting cells were found as compared with conventional mice. The number of IgA-secreting cells in antigen-free mice was drastically decreased in all three organs tested. The antibody-specificity repertoire of the "background" IgM-secreting cells in the spleen and BM of the antigen-free and conventional mice was much alike. This indicates that in antigen-free mice the available antibody repertoires are established independently of exogenous and/or mitogenic stimulation.     

Peritoneal Ly-1 B cells: genetic control, autoantibody production, increased lambda light chain expression

Previous studies demonstrate that Ly-1 B cells and their progenitors are clearly detectable in peritoneum in normal mice. In this publication, we show (a) that peritoneal Ly-1 B cells resemble splenic Ly-1 B cells with respect to surface marker expression and functional activity (autoantibody production); (b) that Ly-1 B frequencies in peritoneum are considerably higher than in spleen; and (c) that genetic mechanisms reduce peritoneal Ly-1 B frequencies to minimal levels in SJL-related mice and to below detectability in CBA/N and other mice with the X-linked immunodeficiency (Xid). In addition, we show that that peritoneal (and perhaps splenic) Ly-1 B populations demonstrate an unique bias in immunoglobulin commitment. That is, they are selectively enriched for cells that express IgM heavy chains in association with lambda light chains. Thus, as a whole, evidence presented here defines the peritoneum as a tightly regulated lymphocyte compartment that normally houses a large population of mature Ly-1 B cells with distinctive functional properties.     

A major peritoneal reservoir of precursors for intestinal IgA plasma cells

Studies presented examine the origin of IgA plasma cells in B lineage chimeric mice constructed by reconstituting lethally irradiated mice with a mixture of syngeneic bone marrow cells and peritoneal cells from Ig heavy chain allotype congenic donors. In these mice, essentially all B cells in spleen and Peyer's patches are derived from the bone marrow donor; however Ly-1 B lineage cells which have been mainly detected in the peritoneum are derived from the peritoneal cell donor. Surprisingly, roughly half of the IgA plasma cells in the lamina propria of the gut are also derived from the peritoneal cell donor, suggesting an important role for peritoneally-derived B cells in the mucosal immune response.     

Can peritoneal B cells be rendered unresponsive?

Ly-1+ B cells have been reported to produce a number of autoantibodies, and to be involved in the selection and regulation of the conventional B cell repertoire. It is not known if these B cells, which are found in high numbers in the peritoneum of normal adult mice, themselves can be regulated. In this study, we evaluated the sensitivity of peritoneal B cells (PBCs) versus conventional splenic B cells to regulation in a model system for tolerance. Normal splenic (conventional) or PBCs (containing both CD5+ and CD5- 'sister' cells) were cultured overnight with either F(ab')2 or intact IgG anti-mouse Ig, washed, and then challenged with fluorescein(FL)-coupled to Brucella abortus (BA), trimethylammonium (TMA)-BA or lipopolysaccharide (LPS), and the IgM responses to the FL and TMA haptens measured. In contrast to spleen cells, which exhibited up to a 90% reduction in anti-FL responsiveness, pretreated PBCs were mostly resistant to this form of tolerance regardless of challenge. The anti-TMA response of PBCs, which reflects the skewed VH11 usage by peritoneal CD5 B cells, was also resistant to tolerance. However, splenic TMA-specific B cells appeared to be sensitive to unresponsiveness induced by anti-Ig. Signaling studies show that PBCs have a blunted initial Ca2+ response, suggesting that the consequence of anti-Ig crosslinking may be defective in these cells. Furthermore, phorbal myristate acetate and/or ionomycin treatment of both PB and splenic B cells led to hyporesponsiveness to LPS challenge. This suggests that PBCs may be defective in a signalling pathway, perhaps involving protein kinase C activation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Peritoneal B-2 cells comprise a distinct B-2 cell population with B-1b-like characteristics

B-1 and B-2 cells are lymphocyte populations that differ in development, surface marker expression, tissue localization, and function. Though mainly found in the spleen, lymph nodes, and circulation of mice, small numbers of B-2 cells are found in the peritoneal cavity, a site predominantly populated by B-1 cells. Here, we characterized peritoneal B-2 cells, and determined their relationship to B-1 cells. We found that peritoneal B-2 cells appear to be intermediate between splenic B-2 and peritoneal B-1 cells in terms of surface marker expression of B220, CD80, and CD43, expression of several marker genes, and in vitro viability and IgM secretion. Adoptive transfer of peritoneal B-2 cells into severe combined immunodeficiency mice resulted in the acquisition of a phenotype reminiscent of B-1b cells, as shown by up-regulation of Mac-1 and CD43, and down-regulation of CD23. Moreover, adoptively transferred peritoneal B-2 cells recapitulated B-1 cell function by producing natural IgM in recipient mice. These data suggest that peritoneal B-2 cells express some characteristics of B-1b cells and that this similarity increases with additional time in the peritoneal cavity.     

B-1a cells are imprinted by the microenvironment in spleen and peritoneum

B-1a cells are found mainly in the peritoneal cavity of mice but are also present in the spleen. Gene expression profiling defined many genes differentially expressed in B-1a cells from these two sites. To see whether this gene expression pattern was imprinted by the particular microenvironment, peritoneal or spleen cells from recombinant L2 mice mainly consisting of B-1a cells were adoptively transferred into Rag1-/- mice. Re-isolated peritoneal and splenic B-1a cells were analyzed for expression of three indicator genes--vcam-1, adamdec1 and spi-c. The expression of these genes was up-regulated in splenic and down-regulated in peritoneal cells. This particular pattern was observed for peritoneal or splenic donor cells transferred either intraperitoneally or intravenously. Similar results were obtained when levels of surface IgM or frequencies of Mac-1+ B-1 cells were compared after transfer. This suggests that the environment induces the particular genetic program of B-1a cells and argues against an independent ontogeny.     

Recipient age determines the success of intraperitoneal transplantation of peritoneal cavity B cells.

In vivo studies of lymphocyte biology have used intravenous (i.v.) injection as the primary mode of cell transfer, a protocol consistent with the anatomic distribution of most lymphocytes. However, for study of peritoneal cavity B cells, i.v. injection does not correlate with anatomical localization. This report describes the restoration of B-cell function in B lymphocyte-defective X-chromosome-linked immune-defective (XID) mice after intraperitoneal transfer of immunoglobulin heavy chain (Igh)-disparate peritoneal cavity (PerC) cells. In contrast to i.v. transfer, intraperitoneal (i.p.) transfer restored B-cell function in young, but not adult (> 8 weeks), XID mice. When host and donor Igh allotype matched, PerC B-cell engraftment was noted in older recipients; this reconstitution however, was also age-dependent. Migration from the peritoneum to systemic circulation was necessary for serum IgM production as shown by the presence of donor antibody-secreting cells in the host spleen. Host lymphocytes also influenced the success of i.p. transplantation as severe combined immune-deficient mice, regardless of age, exhibited donor serum IgM production. Recipient age, Igh allotype, and immune-deficiency were found to have an impact on the ability of i.p.-transferred PerC B cells to restore B-cell function in XID mice.     

Enhancement of mucosal antibody responses to Salmonella typhimurium and the microbial hapten phosphorylcholine in mice with X-linked immunodeficiency by B-cell precursors from the peritoneal cavity.

The observation that approximately half of the B cells in the murine intestinal lamina propria are derived from peritoneal CD5 B-cell precursors raises the question of their contribution to mucosal protection. Using mice with X-linked immunodeficiency which are deficient in CD5+ B cells, we showed that they mount little serum and virtually no intestinal immunoglobulin M (IgM), IgG, and IgA antibody responses following oral inoculation with live Salmonella typhimurium. Nonresponsive Xid mice were reconstituted with responsive CBA/Ca donor cell preparations which were constitutively enriched or depleted of CD5 B-cell precursors. Reconstitution of irradiated Xid mice with CD5 B-cell-deficient bone marrow from CBA/Ca donors marginally improved IgM responses in the intestinal mucosa but had no effect on IgG or IgA in response to oral immunization with live S. typhimurium. Whenever Xid mice were reconstituted with donor cells from the peritoneal cavity, which are enriched for CD5 B-cell precursors, strong IgA and in some cases IgG responses in the intestinal mucosa were stimulated in response to oral immunization. When mucosal and serum antibody responses were compared, the peritoneal donor cells again reinstated maximal serum antibody responses to S. typhimurium. Serum and mucosal responses to the bacterial hapten phosphorylcholine could be induced in Xid mice after immunization with S. typhimurium or hapten-carrier conjugates but only following reconstitution with donor cells containing CD5 B-cell precursors. These observations suggest that different lymphoid compartments are enriched for regulatory or effector cells which vary in their contributions to the mucosal antibody response against epitopes on S. typhimurium.     

Early B cell precursors in long-term bone marrow culture: selective development in the bone marrow of irradiated recipients

The primary site for the growth and differentiation of B cell precursors in irradiated recipient mice was investigated. Bone marrow (BM) cells from lipopolysaccharide (LPS)-responder C57BL/6 mice were transferred into irradiated LPS-nonresponder C57BL/10ScCr mice, and the generation of donor-derived B cells in the recipient was monitored by determining the immunoglobulin-producing cells developed in the LPS-stimulated cultures of recipient's spleen cells as well as BM cells. As previously stated, the transfer of fresh BM cells resulted in the development of LPS-reactive cells both in spleen and BM simultaneously. On the other hand, when long-term cultured BM cells which were shown to be devoid of B cells and pre-B cells were used as the donor cells, the development of LPS-reactive cells was first observed only in BM, and subsequently in both BM and spleen. The failure to detect LPS-reactive cells in the spleen in the early phase, or day 11 after irradiation and reconstitution, was not attributed to the culture condition or the existence of suppressive activity in day 11 spleen cells. These results indicate that B cell precursors lodge only in the BM of irradiated recipients, grow and differentiate in the same place, and then the differentiated progeny migrate to peripheral lymphoid organs.     

Regulatory T cells control autoimmunity following syngeneic bone marrow transplantation

Sublethally irradiated, immunodeficient, C57BL/6 RAG-2 gene-deleted recipient mice reconstituted with T cell-depleted bone marrow (BM) grafts frequently developed diarrhea, lost weight and showed signs of autoimmunity, dying between 4 and 7 weeks after reconstitution. Mice died despite evidence of efficient donor-derived hemato-lymphoid reconstitution, and disease was associated with the presence of IgG anti-nuclear antibodies. Autoimmunity was initiated by T cells, but could be prevented by transfer of naturally arising regulatory T cells. In contrast, lethally irradiated, BM-reconstituted immunocompetent, C57BL/6 mice survived without signs of autoimmunity. Survival of immunocompetent mice was shown to be due to the presence of residual, extra-thymically located, radio-resistant, functional regulatory T cells. The importance of regulatory T cells was further shown by the reduced survival of immunocompetent BM recipients whose CD25+ T cells had been depleted prior to bone marrow transplantation. The implications of these results in the context of syngeneic graft-versus-host disease following BM transplantation are discussed.     

A Major Peritoneal Reservoir of Precursors for Intestinal IgA Plasma Cells

Studies presented examine the origin of IgA plasma cells in B lineage chimeric mice constructed by reconstituting lethally irradiated mice with a mixture of syngeneic bone marrow cells and peritoneal cells from Ig heavy chain allotype congenic donors. In these mice, essentially all B cells in spleen and Peyer's patches are derived from the bone marrow donor; however Ly-1 B lineage cells which have been mainly detected in the peritoneum are derived from the peritoneal cell donor. Surprisingly, roughly half of the IgA plasma cells in the lamina propria of the gut are also derived from the peritoneal cell donor, suggesting an important role for peritoneally-derived B cells in the mucosal immune response.     

A Major Peritoneal Reservoir of Precursors for Intestinal IgA Plasma Cells

Studies presented examine the origin of IgA plasma cells in B lineage chimeric mice constructed by reconstituting lethally irradiated mice with a mixture of syngeneic bone marrow cells and peritoneal cells from Ig heavy chain allotype congenic donors. In these mice, essentially all B cells in spleen and Peyer's patches are derived from the bone marrow donor; however Ly-1 B lineage cells which have been mainly detected in the peritoneum are derived from the peritoneal cell donor. Surprisingly, roughly half of the IgA plasma cells in the lamina propria of the gut are also derived from the peritoneal cell donor, suggesting an important role for peritoneally-derived B cells in the mucosal immune response.