Immunofluorescence analysis of B-1 cell ontogeny in the mouse

In order to further understand the developmental aspects ofB-1 cells, we characterized the ontogeny of this B cell populationin the spleen and peritoneal cavity of BALB/c mice. Althoughthere are B-1 cells in the spleen within the first 1–3weeks after birth, they do not at any stage represent the majorityof splenic B cells. Splenic B-1 cells reach peak levels at 9days after birth. The mesenterlc lining that covers the smallintestine of 7-day-old mice contains a population of IgM⁺ Bcells, while at the same age, there are few lymphold cells inthe peritoneal cavity. Between 7 and 8 days after birth thereis an influx of B cells into the peritoneal cavity. At 8 days,the first detectable peritoneal B cells appear to be of theB-1 type based on expression of IL-5 receptor and CD5. However,these peritoneal B-1 cells do not express Mac-1. This antigenIs not expressed by the majority of peritoneal B-1 calls until3 weeks. This study indicates that the majority of early splenicB cells are not B-1 cells and it suggests that the mesenterlctissues surrounding the gut contain B lymphocytes which trafficinto the peritoneal cavity where they then reside.     

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.     

Is the activated T helper stimulus able to induce Ig isotype switching in cultured human B cells?

It Is confirmed that large amounts of IgM, IgG, and IgA areproduced when human B cells are cultured with T cells activatedby immobilized CD3 antibody (CD3 system). IL-2 was essential;lowerlevels of Ig production with different isotype ratios were obtainedif IL-4 or IL-6 replaced IL-2. Depletion of sIgG⁺ or sIgA⁺ cellsfrom the B population to be cultured markedly reduced productionof IgG or IgA. Culturesof B cells selected with the pan-B markersCD19, CD72, or CD21 contained similar levels of Ig of all threeisotypes, whereas B cells selected for sIgM or sIgD expressionproduced IgM but very little IgG or IgA indicating that littleisotype switching was occurring. Production of IgG or IgA fromcells expressing these isotypes was more efficient than productionof IgM from IgM⁺ IgD⁺ cells. These results are considered inthe light of the demonstration by others of the production ofmultiple isotypes from single sIgM⁺-selected B cells. Clonedhuman T cells from a single donor induced production of allthree isotypes, but the proportions varied indicating that thepotent T-B cell interactions inducing B cell activation mayoverride and conceal the operation of isotype specific cellinteractions. Some T clones used at an optimal dose were aseffective untreated as X-irradiated, whereas with other clonesmaximumIg production was not achieved without irradiation.     

IL-5 receptor positive B cells, but not eosinophils, are functionally and numerically influenced in mice carrying the X-linked immune defect

Mouse IL-5 (mIL-5) acts on B cells and eosinophils to inducegrowth and differentiation through the mIL-5 specific receptor(mIL-5R). The functional high-affinity mIL-5R is a heterodimercomposed of and ß chains. We investigated the expressionof mIL-5R and the responsiveness of B cells and eosinophilsto mIL-5 In X-linked immunodeficient (xld) mice. mIL-5R expressionanalyzed by using mAbs specific for and ß chainsrevealed that xld B cells had fewer mIL-5R⁺mIL-5Rß⁺than BALB/c B cells. In particular, a decrease in the numberof peritoneal mIL-5R⁺ B cells among Ly-1 B cells (known as B-1cells) was remarkable. Furthermore, the frequency of precursorsof mIL-5 responsive B cells in xld mice was 100-fold lower thanthat of BALB/c mice. Interestingly, sorted mIL-5R+ peritonealB cells from xld mice displayed a low response to mIL-5. Intraperltonealinjection of mIL-5 into BALB/c mice induced polyclonal IgM productionand an increase in the number of eosinophils. The same regimenfailed to induce an increase in the same parameters in xld mice.However, xld mice showed mIL-5-induced eoslnophilla in peripheralblood to a similar extent as BALB/c mice. Eosinophils from mIL-5-injectedxld mice expressed both and ß chains of mIL-5, andresponded to mlL-5 with prolonged in vitro survival.     

Many of the IgA producing plasma cells in murine gut are derived from self-replenishing precursors in the peritoneal cavity

Long term B lineage chimeras are used here to study the originof plasma cells in the mouse. Chlmeric mice are constructedby reconstituting lethally irradiated mice with peritoneal cells(PerC) and bone marrow cells from congenic pairs of mice differingIn Igh-C allotype. All conventional B cells in these mice expressthe allotype of the bone marrow donor and nearly all Ly-1 Blineage cells express the allotype of the PerC donor. FACS analysisand immunohistology of these mice shows that virtually all (sig⁺)B cells in peripheral lymphoid organs are derived from the bonemarrow donor. However, despite this overwhelming number of bonemarrow-derived B cells in these animals, immunohistologicalstaining of lymphold organs and gut shows that nearly half ofthe IgM, IgG, and IgA plasma cells derive from the PerC donor.These data demonstrate that the peritoneal cavity contains amajor reservoir of self-replenishing cells that play a significantrole in the mucosal immune response. The possibility that theseare B cells that belong to the Ly-1 B lineage is discussed.     

Generation of precursor,immature, and mature murine B1‐cell lines from c‐myc/bcl‐xL‐overexpressing pre‐BI cells

Deregulated expression of c‐myc and bcl‐xL is long known to generate transformed B cells in humans and mice. We overexpressed these genes to induce in vitro and in vivo differentiation of fetal liver‐derived mouse pre‐BI cells to B1‐lineage pre‐BII‐like, immature and mature B‐cell lines, and to Ig‐secreting cells. In vitro, doxycycline‐controlled c‐myc/bcl‐xL‐overexpressing CD19⁺CD93⁺c‐kikt⁺IgM^? pre‐BI cells differentiate to and survive as CD19⁺CD93⁺c‐kit^?IgM⁺ immature B1 cells. Timed CpG stimulation of these oncogene‐overexpressing pre‐B or immature B1 cells generates either CD19⁺CD93^lowc‐kit^?IgM^?SLC^? pre‐BII‐like or IgM⁺MHCII⁺CD73⁺CD80⁺CD40⁺ mature B1‐cell lines and IgM‐secreting B1 cells in vitro and fixes their state of differentiation. All cell lines are clonable, but a majority of immature and mature B1‐cell clones eventually reach a nonproliferating, surviving G₀‐state. Transplanted in vivo, c‐myc/bcl‐xL‐overexpressing pre‐B cells expand to mature B1 cells, and to IgM‐ and IgA‐secreting plasmablasts and plasma cells. Within 2 months, plasmablasts have expanded most prominently in BM and spleen, indicating that the host selectively expanded development of these transformed plasma cells. The sIgM⁺ B1‐cell lines and clones offer the possibility to study their roles in the development of B1‐Ab repertoires, of B1‐cell‐mediated autoimmune diseases and of B1‐cell malignancies.     

CD4 , CD8 {gamma}/{delta} cells from normal mice respond to a syngeneic B cell lymphoma and can induce its differentiation

Lymph nodes and spleens from normal unimmunized mice containsmall numbers of CD3⁺, CD4^–, CD8^– (double negative,DN) T cells. Of these, approximately one-third express the markerLy-5(8220) in a form previously seen only on normal B cellsand a population of DN T cells found in mice genetically proneto develop autolmmunity. DN T cells proliferate when co-culturedwith a syngeneic surface Ig⁺ lymphoma, CH12. After one cycleof stimulation with CH12 almost all of the responding CD3⁺ DNcells express Ly-5(B220), suggesting that it is an activationmarker for some DN T cells. The CH12 responding population alsocontains cells with two other phenotypes, Thy-1⁺, CD4^–,CD8^–, Ly-5(B220)⁺, sIgM^–, CD3^– and Thy-i⁺,CD4⁺, CD8^–, Ly-5(B220)^–, sIgM^–, CD3⁺. TheLy-5(B220)⁺, CD3^– population is no longer found afterrepeated stimulation. While the relationship between these threepopulations is unknown, DN I cells can proliferate in the absenceof CD4⁺ or CD8⁺ cells and therefore their proliferation is notdependent on the presence of other T cells or lymphokines producedby CD4⁺ or CD8⁺ T cells. Anti-CD3 Immunoprecipitation of CH12-respondlngcells reveals at least seven different receptor proteins ofwhich five can also be precipitated with an anti-(C1/C2) monoclonalantibody. Thus at least three different TCR– heterodimersare expressed by CH12-responding T cells. The Thy-1⁺, CD4^–,CD8^–, Ly-5(B220)⁺ cells can provide help to CH12 cellsfor Ig secretion even in the absence of the nominal antigenfor the B lymphoma cells, in summary, these results demonstratethat in normal mice there is a small population of CD4^–,CD8^–, Ly-5(B220)⁺ T cells with / receptors which can providehelp for a syngeneic B cell lymphoma. Received 9 May 1989, accepted 31 May 1989.     

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.     

Activation of natural killer cells by the mAb YTA-1 that recognizes leukocyte function-associated antigen-1

The mAb YTA-1, which brightly stains CD3^–CD16⁺ large granularlymphocytes (LGL)/natural killer (NK) cells and CD8⁺ T cellsby immunofluorescence, is specific for leukocyte function-associatedantigen (LFA)-1. Some mAbs recognizing the LFA-1 chain (CD11a)or LFA-1ß chain (CD18) inhibited the binding of YTA-1to peripheral blood mononuclear cells. YTA-1 mAb could be chemicallycross-linked to 170 and 96 kDa molecules, whose molecular weightscorrespond to those of LFA-1 and ß respectively.YTA-1bound to COS-7 cells co-transfected with CD11a and CD18 cDNAs,but not to untransfected cells. Reactivities of YTA-1 to K562cells transfected with LFA-1 and ß(CD11a/CD18) cDNAsand to CHO cells transfected with Mac-1 (CD11b/CD18) or p150,95 (CD11c/CD18) cDNAs strongly suggest that YTA-1 recognizeseither LFA-1 or an epitope formed by a combination of LFA-1and ß. Treatment of fresh CD3^–CD16⁺ LGL withYTA-1 augmented cytolytic activity and induced proliferation.F(ab')₂ fragments of YTA-1 augmented NK cytotoxicity, indicatingthat the NK activating signal was transmitted through LFA-1without involvement of Fc receptor III. In contrast, the othermAbs against LFA-1 could not activate NK cells. These resultscollectively indicate that YTA-1 recognizes a unique epitopeof LFA-1, which is involved in activation of fresh NK cells.     

Kinetics of B cell subpopulations in peripheral lymphoid tissues: evidence for the presence of phenotypically distinct short-lived and long-lived B cell subsets

A small proportion of the slg⁺ B lymphocytes in peripheral lymphoidorgans [22% in spleen and 6 % in lymph node (LN)] in rat carriesthe Thy-1 antigen. These Thy-1⁺ B cells represent newly formedbone marrow (BM) derived (or Immature) B cells. In this studywe investigated the kinetic behavior of Thy-1⁺ and Thy-1^–B cells in various lymphoid tissues. The renewal rates of theseB cells in young adult rats were determined by continuous administrationof 5-bromo-2-deoxyuridine (BrdU) for up to 6 weeks. At severaltime intervals, Thy-1⁺ and Thy-1^– B cell subpopulationsin BM, blood, spleen and popllteal LN were analyzed for thepresence of incorporated BrdU, using three-color immunocytologyon cytospin preparations. In all tissues studied, the Thy-1⁺B cells were rapidly renewed with a rate that varied between58 % (BM) and 22 % (LN) per day. Virtually all Thy-1+ B cellswere labeled by BrdU within a period of 8 –16 days, indicatingthat all these cells are relatively short-lived. By contrast,the replacement of Thy-1^– B cells in these tissues was30 –40 times lower, and ranged between 2.0 and 0.8 % perday. In absolute numbers we estimate that 57 million Thy-1⁺B cells are renewed per day in the BM whereas only 10 millionThy-1^– B cells are replaced in the pool of long-livedperipheral B cells. This implicates a cell loss of 80 % at thetransition of the Thy-1⁺ to the Thy-1^– B cell stage. Thefew B cells that actually become incorporated into the poolof mature B cells are most probably selected on the basis ofthe specificity of their slg.     

{gamma}{delta} T cells promote CD4 and CD8 expression by SCID thymocytes

Molecular studies of the TCR, which is expressed by a minorsubpopulatlon of T lymphocytes in all vertebrate species, havedefined a subset which expresses a receptor with extreme junctionaldiversity and a second subset, most commonly found in eplthella,which expresses a receptor of very limited diversity. In thedeveloping murine thymus, T cells appear in an ordered sequenceof specific v rearrangements, V3V, 1 on day 14, V2V1 on day17, and subsequently V4V5, V6, or V7. We demonstrate that thetransfer of expanded populations of cells from newborn thymusand cell lines expressing the invariant V3V1 receptor into SCIDmice, which lack T and B cells, results in the appearance ofCD3^–CD4⁺CD8⁺ thymocytes. Thus, one role of the early appearingV3V1 T cells in thymlc development in vivo is to promote CD4and CO8 surface expression on precursor cells.     

HLA-DR and H-2E transgenes differentially mediate TCR-specific positive selection

The use of HLA transgenic mice in models of immunity and diseaseassumes that human MHC molecules are able to contribute towardthe positive selection of the mouse TCR repertoire. As an initialstep towards analysis of this we have compared the relativeability of DR/Eß or E/Eß complexes to induceT cell receptor (TCR) positive selection in H-2Ea and HLA-DRAtransgenic mice lacking endogenous E. The results show that,like E/Eß, the hybrid DR/ß complexes arecapable of mediating positive selection of V_ß2⁺;,V_ß6⁺, and V_ß10⁺ cells. However, differenceswere found between the effects of the two transgenes. Thus,while V_ß6⁺ cells were efficiently selected in bothH-2Ea and DRA transgenic mice, positive selection of V_ß10⁺cells was less apparent in the DRA transgenic mice. Variationbetween Ea and DRA transgenic mice is consistent with the notionthat this process is dependent on differential binding of endogenouspeptides to the E/Eß and DR/Eß complexes.Furthermore, contrary to expectations, in neither set of micewas positive selection limited solely to the CD4⁺ subset. Thus,examples were found in which V_ß-specific positiveselection was confined to either the CD4⁺ or CD8⁺ subsets, andothers in which both subpopulations were concomltantly increased.In the case of V_ß2 positive selection, H-2Ea transgenicmice showed expansion of these cells in both the CD4⁺ and CD8⁺subpopulations whlle in DRA transgenic mice this occurred predominantlyin the CD8⁺ subpopulatlon.     

Splenic extrafollicular reactions and BM plasma cells sustain IgM response associated with hypercholesterolemia

Hypercholesterolemia associated with atherosclerotic disease is known to be associated with increased total and oxidized (ox) low‐density lipoprotein (LDL)‐specific IgM antibodies in circulation. However, the B‐cell responses accounting for this increase remain to be elucidated. Here, we observed an association between total IgM and oxLDL‐specific IgM autoantibodies with cholesterol in the plasma of hypercholesterolemic apolipoprotein E deficient (apoE^?/?) mice. Our findings also indicated that oxLDL‐specific IgM autoantibodies production was restricted to the spleen, but not the lymph nodes. Further examination of the spleen revealed that the extrafollicular responses, but not germinal center reactions, were the dominant antibody‐producing pathway. A quiescent population of IgM⁺ plasma cells including oxLDL‐specific IgM antibody secreting cells in BM also sustained the elevated IgM antibodies response in circulation. We determined that IgM⁺ plasma cells in the BM were, at least in part, splenic derived by depleting CD11c⁺ DCs and plasmablasts to disrupt the humoral responses. In addition, lowering hypercholesterolemia reduced IgM response by interfering with extrafollicular and BM responses. By elucidating the mechanism underlying the elevated IgM response observed in hypercholesterolemia, this study provides insight into novel immunotherapeutic avenues.     

B-lineage cell deficits in bone marrow of lpr/lpr mice

Analyses of bone marrow (BM) lymphocytes in C57BL/6 mice homozygousfor the lpr mutation (BS.Ipr) disclosed low numbers of pre-Band B cells, as compared with age-matched control B6 mice. BMdepletion in B6.lpr mice was selective for B-lineage cells,appeared in young adults, and developed markedly with age anddisease progression, contrasting with the peripheral lymphocytehypercellularity. Normalization of pre-B and B cellularity inBM of B6.lprmice was observed after administration of polyclonalIg, that also markedly improved the clinical condition. Isolatedpre-B (B220⁺ IgM^–) cells from B6 or B6.lpr mice, however,showed essentially the same rates of IL-7-dependent proliferationand differentiation to B (lgM⁺) cells in culture, indicatingthat the BM B-lineage deficit is not the result of an intrinsicdefect inB cell generation.     

Suppression of B cell differentiation by ligation of membrane-bound IgM

Using B cells from the transgenic mouse line B6-Sp6 and control littermates, stimulated by lipopolysaccharide (LPS) under novel culture conditions that provide for the response of all B cells, we show here that specific ligation of the surface IgM molecules always results in inhibition of terminal differentiation and immunoglobulin secretion by activated cells, regardless of the ligand. Thus, monoclonal antibodies to (a) the CH region of Ig (anti-μ. and anti-allotype), (b) the Cx region, (c) the V region (anti-idiotype) of surface IgM, as well as (d) multivalent antigen (2,4,6-trinitrophenyl-bovine serum albumin), all show similar effects and dose-response curves. IgD-negative transgenic B cells are equally sensitive to IgM ligation-dependent inhibition, as control (IgD-positive) B cells. The allotype specificity of this inhibition, assessed by using anti-u, allotype reagents to inhibit and assay the responses, suggests that B cells expressing transgenic or endogenous IgM in transgenic B6-Sp6 mice are largely independent populations. These observations establish that anti-IgM antibodies in conjunction with appropriate LPS stimulation, provide a universal model system for functional characterization of B cell responses.     

A rheumatoid factor transgenic mouse model of autoantibody regulation

To address whether B cells expressing a disease-associated autospeclficityare regulated In normal mice, we have established a rheumatoidfactor (RF) transgenic model of autoimmunity, using V genesderived from an IgA anti-lgG2a RF isolated from an autoimmuneMRU//pr mouse. As we wished to study Induction of toleranceduring B cell development, we cloned the V_H gene Into an IgMexpression vector. The RF we chose binds only lgG2a of the ‘a’allotype (lgG2a) but not lgG2a^b allowing us to produce transgenicanimals on IgH^a and lgH^b backgrounds, which either express orlack the self-antigen. Two transgenic lines were studied. Usingmice which lack the self-antigen, we show by fluorescence activatedcell sorting and hybrldoma analysis that the H and L transgenesare expressed to the exclusion of endogenous genes in most splenicB cells.In spite of good allellc exclusion, transgenic micewhich are genetically capable of expressing lgG2a' have reducedbut significant ( {small tilde}50 _µ/tg/ml) serum levels.Nonetheless, the frequency and numbers of transgene-expressingB cells In peripheral lymphold organs of such mice which havethe self-antigen are similar to those which lack It (lgH^b mice).Thus, B cells expressing an antiself lgG2a surface receptorcan develop In this system. Whether such B cells are anerglcor otherwise regulated in autoantlgen-expressing mice Is discussed.     

Brucella abortus induces a novel cytokine gene expression pattern characterized by elevated IL-10 and IFN-{gamma} in CD4+ T cells

Immunization of BALB/c mice with killed Brucella abortus (BA)has previously been shown to Increase serum lgG2a levels andlong-term T cell clones from these mice secrete T_h1-associatedcytokines: IFN- and IL-2 but not IL-4 or IL-5. We analyzed cytokinegene expression following primary immunization with BA to determinewhen CD4⁺ T cells first express cytokine genes and whether specifichypothesized cytokine patterns (e.g. T_h precursor, T_h0) couldbe identified prior to a T_h1-like pattern. Our results demonstrateda highly consistent and novel pattern of T_h 1/T_h2 cytokine geneexpression characterized by elevated IL-10 and IFN- in CD4⁺T cells which rapidly manifests itself and is sustained forat least 10 days after immunization. No elevation in IL-2 cytokinegene expression was observed and treatment of BA-immunlzed micewith blocking anti- IL-2 antibodies had no effect on the cytokinegene expression pattern, although treatment with anti-IFN antibodiesresulted in increased IL-4, IL-5, and IL-9 cytoklne gene expression,In the absence of any change In IFN- or IL-10 as early as 4days after immunization. These results suggest that a wholepathogen may trigger sufficient costimulatory signals to rapidlyinduce effector T cells in the absence of elevated IL-2 andthat IL-10 Is specifically elevated in certain T_h1-like responses.     

Non-tolerant B cells cause autoimmunity in anti-CD8 IgG2a-transgenic mice

Using a pair of γ2a/x immunoglobulin genes, transgenic mice were generated to study tolerance induction in B cells that express IgG2a autoantibodies. The transgenic IgG2a specifically binds CD8 α chains of the CD8.2 allotype expressed on the surface of CD8⁺ T cells, but not CD8 molecules expressed by the CD8.1 allele. Thus, IgG2a transgenic mice expressing the CD8.1 allele were used as controls to monitor B cell development and mice expressing CD8.2 were used to study B cell tolerance. Both types of mice showed transgenic γ2a expression on the surface of B cells. Expression of endogenous heavy chain alleles was strongly inhibited in immature B cell subsets, whereas mature B cells co-expressed transgenic γ2a and endogenous IgM/D. The transgenic x chain expression leads only to partial allelic exclusion of endogenous light chains. B cells that express high levels of transgenic CD8.2-specific IgG2a were identified using soluble CD8-Ig. In CD8.1⁺ and in CD8.2⁺ mice, we found no differences in expression and maturation of transgenic anti-CD8.2 IgG2a⁺ B cells. High levels of serum anti-CD8.2 IgG2a antibodies led to the elimination of CD8⁺ T cells, causing a severe defect in cytotoxic immune responses. These results show that tolerance induction is incomplete in the CD8.2⁺ mice, either because IgG2a⁺ B cells are resistant to censoring mechanisms or because the secreted CD8-specific IgG2a antibodies render the CD8 autoantigen inaccessible to the B cells. This contrasts strongly with the efficient induction of B cell tolerance in mice expressing anti-CD8.2 IgM autoantibodies.