Monoclonal anti-{lambda}5 antibody FS1 identifies a 130 kDa protein associated with {lambda}5 and Vpre-B on the surface of early pre-B cell lines

mAbs specific for mouse 5 protein were prepared by fusion ofspleen cells from a hamster Immunized with recomblnant 5 proteinsynthesized in bacteria and the mouse myeloma cell line SP2/0-Ag14.Here we report the characteristics of the antibodies producedby the FS1 hybridoma. FS1 antibody stains a variety of mousepre-B cell lines but not B cell lines or T cell lines. The stainingof the pre-B cell lines Awl and C-7 by phycoerythrin (PE)-con]ugatedFS1 (FS1 - PE) can be blocked by prelncubatlon of these cellswith unconjugated FS1 antibody or with affinity purified polyclonal5 specific Ig but not with normal hamster or mouse IgG or withaffinity purified polyclonal antl-Mb-1 Ig. From these experimentswe concluded that FS1 specifically recognizes 5 protein. Weused FS1 -PE to probe for surface (s) 5⁺ cells in normal BALB/cmouse bone marrow. Such cells were undetectable when total bonemarrow or FACS sorted subpopulatlons were analyzed. However,when B220^plus;, CD43⁺, s5⁺ bone marrow cells were cultured for4 days on the stromal cell line FLST2 in the presence of IL-7,s5 expression became apparent. Further expansion of these cellsin IL7 alone augmented the s5 expression to readily detectablelevels. This modulation may indicate that s5 expression on normalbone marrow cells in vivo is transient and that at any givenmoment only a small fraction of bone marrow cells expresseslow levels of 5 protein on the surface. Alternatively the bindingof our FS1 mAb to the s5 molecules on normal bone marrow cellsmay be blocked by other proteins binding to the 8X5 complexin vivo and directly ex vivo. Previous analysis of surface 5associated proteins on early mouse pre-B cell lines using apolyclonal anti-5 rabbit antlserum had suggested that s5 proteinwas associated with a high molecular weight protein. Analysisof 5 and Its associated proteins on early pre-B cell lines usingour FS1 mAb confirmed our previous finding and showed that theearly 5 receptor contains at least three proteins: 5, V_pre-B,and an as yet uncharacteiized protein with a molecular weightof 130,000 designated p130.     

Re-evaluation of the probabilities for productive rearrangements on the {kappa} and{lambda}loci

V-J rearrangements at Ig light chain (IgL) genes occur in restingsmall pre-B cells. In the absence of cell division, the probabilityof productive and rearrangements is proportional to the outputof ⁺ B and ⁺ B cells in bone marrow. The kinetics and probabilityof productive or rearrangements was assessed in three groupsof mice carrying two (wild-type), one or no intact Ig gene,and the following conclusion are drawn, and rearrangementsoccur independently at different kinetics, and rearrangementsare initiated at a time when rearrangements are stopping. Theprobability of productive and rearrangements per chromosomeis calculated to be –60 and –20% respectively. Thus,a gene can attempt rearrangements up to three times per chromosomeduring B cell development. These findings explain that the observedratio of ⁺ B/⁺ B cell production in wild-type mice is 95/5.     

Identification, in mouse macrophages and in serum, of a soluble receptor for the Fc portion of IgG (Fc{gamma}R) encoded by an alternatively spliced transcript of the Fc{gamma}RII gene

Low affinity FcR are a heterogeneous group of glycoproteinswhich exist in transmembrane (TM) as well as in soluble forms.Two membrane isoforms of the murine type II FcR, FcRilb1 andFc;Rilb2, have been described. They result from the translationof alternatively spliced premRNA, FcRilb2 lacking sequencesof the first intracytoplasmic domain (IC1). Soluble forms ofFcR (sFcR) have previously been shown to result from proteolysisof membrane receptors. We report here the identification, inmacrophages, of a mRNA derived from the FCRll gene by splicingexons encoding the TM and IC1 domains, i.e. corresponding toa TM-deleted FcRllb2 mRNA. A soluble protein possibly encodedby this mRNA was identified in macrophage supernatants. In accordancewith FcR nomenclature, we propose to name this new FcRll IsoformFcRllb3. It is the most abundant 8FcR present in serum, as comparedwith 8FcR resulting from cleavage of membrane FcR.     

Antibody response elicited by T-dependent and T-independent antigens in gene targeted {kappa}-deficient mice

Animal models substantially contribute to the understandingof the pathogenesis of various human diseases, including thoseassociated with genetic defects. Our study investigated thecharacteristics of antibody responses elicited by T-dependentand T-independent antigens in mice rendered k-deficterrt bytargeted deletion of the J_kC_k gene segments. It is known thatin normal murine species the k repertoire dominates the antibodyrepertoire (k/ratio = 95:5). Our results indicate that the kgene deletion causes the alternative usage of 1 (93%) and 2(7%) light chains, confirming previous studies demonstratingthat in k-deficlent mice all B cells express IG receptors. Theanti-trinitrophenylbenzene (TUP) response in K–/–mice was compensated for by 1 and 2 bearing Igs. However, isoelectricfocusing analysis of anti-TNP antibodies showed a considerablymore restricted pattern of anti-TNP antibodies in K–/–as compared with antibodies in normal mice. No major differenceswere observed in the affinity for the hapten of or1 or 2 mAbsobtained from 129/Sv and K–/– mice. Furthermore,1 and 2 chains can reconstitute the expression of an Idiotype(460ld) borne on anti-TNP antibodies. The 460ld was detectedboth in polyclonal and monoclonal anti-TNP antibodies obtainedfrom K–/– mice. Our results clearly showed thatthe anti-TNP repertoire is compensated by the repertoire eventhough the latter is clonally restricted in K–/–mice.     

Emigration of selected subsets of {gamma}{delta}+ T cells from the adult murine thymus

Cells bearing the form of the TCR make up only 1–3% ofT cells in the adult murine thymus and peripheral lymphold organs.Evidence from studies of nude mice suggests that the developmentof at least some T cells is thymus dependent; however, untilnow it has not been directly demonstrated that cells are exportedfrom the thymus. In this paper we have used the technique oflabelling thymocytes in vivo with FITC, followed by flow cytometrlcanalysis to trace cells emigrating from the thymus to the spleen.Using this approach we have been able to demonstrate for thefirst time that T cells are exported from the adult murinethymus to the spleen. We also demonstrate that the cells emigratingto the spleen are a selected subset of thymocytes being heatstable antigen positive, Thy-1⁺, and expressing low levels ofCD44 (Pgp-1). In addition, investigation of TCR V; gene usageamong adult ⁺ thymocytes, recent emigrants, and spleen cells,indicated a selective emigration of cells expressing certainVgenes.     

The development of dermatitis infiltrated by {gamma}{delta} T cells in IL-7 transgenic mice

Transgenic mice constitutively expressing IL-7 developed severedermatitis with erythroderma and alopecia. The skin lesionswere characterized by massive infiltration of mononuclear cells.Immunofluorescence staining showed that most of the infiltratingcells were T cells with the majority bearing the TCR otherthan the V5 moiety. Furthermore, the number of T cells hadincreased in the lymphold organs of the dermatitis animals.These findings idicate the strong relationship between the expressionof IL-7 and the development of T cells in vivo and the pathologicalinvolvement of proliferated and/or activated T cells in skindisease.     

TCR isoform containing the Fc receptor {gamma} chain exhibits structural and functional differences from isoform containing CD3{xi}

The structure and function of the TCR-CD3 complex containinga homodimer of the gamma chain of the high affinity receptorfor IgE (FcR) (FcR⁺ TCR) was investigated by transfecting theFcR gene into a CD3^–, CD3^–, FcR^– T cell line.Introduction of FcR, as well as CD3, induced a high expressionof the TCR-CD3 complex on the cell surface. Transfected FCRformed a homodimer and associated firmly with the TCRßdimer but only weakly with the CD3. Stimulation of both FcRand CD3 transfectants by antibodies against TCR or CD3 inducedaccumulation of inositol phosphates, the Ca²⁺ response, IL-2production, and growth inhibition. On the other hand, antigenstimulation of transfectants expressing FcR as well as CD3 inducedIL-2 production, but only the latter exhibited the antigen-inducedgrowth inhibition. In vitro kinase assay suggested that theCD3 dimer but not the FcR dimer associates with the Fyn kinase.These results indicate that the FcR homodlmer Is able to forma functional TCR complex but that the mode of assembly and thesignaling function of FcR⁺ TCR, including its association withtyrosine klnase(s), may differ from the TCR-CD3 complex containingCD3 homodimers (⁺ TCR). This provides an example which illustratesthat different TCR isoforms mediate distinct signals and functions.     

{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.     

Evidence for a calcium regulated, bidirectional intronic promoter in the murine TCR V{alpha}1 gene

Previous studies of the TCR chain gene have located promoterelements 5' to the start of the various V genes. The only fullycharacterized enhancer for the entire chain gene (V, J andC genes) has been located {small tilde}3 kb from the 3' endof C. We now report the existence of additional regulatory elementslocated in the introns of several murine V genes (V1, V3 andVB6.2.16). In the case of V1, this element appears to be a promoterwith bidirectional activity that is not T cell specific. Interestingly,upstream of the promoter in the antisense strand, an open readingframe has been found that codes for a small molecular weightprotein ({small tilde}60 amino acids) that contains a prollne-richregion and a tyrosine-isoleucine motif that has homology toIgß (the B29 gene product). A rabbit antiserum madeagainst this sequence has confirmed its existence by Westernblot and immunoprecipitation. Thus this V1 intronic promoterhas the potential not only to induce the formation of a truncatedV1 gene product, but also regulates the expression of a smallmolecular weight protein that may be involved in lymphocyteantigen receptor signaling. The activity of this promoter isregulated by changes in intracellular calcium. In the presenceof ionomycin the promoter is down-regulated in the sense directionand its activity is enhanced in the antisense direction. Thisresult suggests that this promoter can act differentially toproduce two very different gene products. The bidirectionalV1 promoter appears to be the first in the Ig superfamily toinduce potentially functional proteins in both directions.     

Cytokine dependence of V{gamma}3 thymocytes: mature but not immature V{gamma}3 cells require endogenous IL-2 and IL-7 to survive evidence for cytokine redundancy

It has previously been described that V3 cells can proliferateextensively in vitro in the presence of different cytokines.Here, the role of cytokines in the maintenance of V3 cells inthe thymus has been determined. Culture of fetal thymocytesin cell suspension for 24 h showed that, whereas immature TCR^lowHSA^highV3cells remained viable, all mature TCR^highHSA_lowV cells died.These cells died by apoptosis since protein synthesis was requiredand flow cytometric analysis as well as DNA gel electrophoresisshowed that the DNA was degraded to oligonucleosomal bands.Addition of IL-2, IL-4 or IL-7 to suspension cultures of fetalthymocytes rescued V3 cells from dying. Addition of IL-1, IL-3,IL-5, IL-6, IL-9, TNE- or IFN- was without effect. Phenotypicanalysis showed that the -chain of the IL-2 receptor (IL-2R)was expressed by part of the immature V3 thymocytes, all matureV3 cells expressed the p-chain of the IL-2 receptor (IL-2RP).Addition of anti-IL-2R mAb to fetal thymic organ culture (FTOC)resulted in a moderate reduction of the cell number of matureV3 thymocytes. Addition of anti-IL-2Ra, anti-IL-4 or anti-IL-7mAb had no effect. The cell number of mature V3 cells was highlyreduced when both anti-IL-2Rp and anti-IL-7 mAb were added toFTOC. These results show that IL-2 and IL-7 are actively involvedin the maintenance of mature V3 cells in the thymus. This cytokinedependence of mature Vthymocytes may explain their selectivelocalization in skin epithelium.     

Soluble human high-affinity receptor for IgE abrogates the IgE-mediated allergic reaction

The high-affinity receptor for IgE (FcRI) has a tetrameric structurecomposed of one, one ß, and two disulfide-linked subunits, of which the subunit binds IgE with high affinity.A recombinant soluble form of the ectodomain of the human FcRIsubunit (rsFcRI) was recently generated by gene engineeringand was verified to bind IgE with an affinity as high as thatof native FcRI on the cell surface. rsFcRI was prepared on alarge scale in order to analyze its biological function. rsFcRIcompletely inhibited IgE binding to the cell surface, resultingin abrogation of the chemical mediator release from RBL-2H3cells. Furthermore it completely abolished the passive cutaneousanaphylaxis (PCA) response by trapping IgE specifically whenitwas administered into rats prior to IgE sensltizatlon. Evenafter IgE sensitizatlon, treatment of rsFcRI substantially reducedthe PCA response. It was finally shown that rsFcRI inhibitedIgE binding to human peripheral blood basophils and the histaminerelease from them. In this paper we address the ability of rsFcRIto specifically prevent the IgE-mediated allergic reaction.     

Structure, biosynthesis, and transduction properties of the Human {micro}-{psi}L Complex: similar behavior of preB and intermediate preB-B cells in transducing ability

In human preB cells, the µ chain is associated with asurrogate light chain composed of the VpreB and -like gene products.Using anti-peptide antibodies directed against VpreB and -likeepitopes, we identified the discrete components of the µ-L(pseudo-light) chain complex in various preB cell lines, andin intermediate preB-B cells that co-expressed the L and thex chain. The -like gene product was identified as a single bandat 20 kDa, disulfide linked to the µ chain. VpreB wasdetected at 16 kDa and, depending upon the cell lines, an isoformof this polypeptide was also present at 15 kDa. In addition,-like-VpreB chain complexes not associated with µ wereidentified both in cell lysates and culture supernatants. Pulse-chaseexperiments indicated that VpreB was transiently associatedwith two new polypeptides of molecular weights 17.5 and 36 kDa.Expression of µ-L and co-expression of µ-L and µLat the surface of preB and intermediate preB-B cells respectivelywas detected by cytofiuorimetry. The signal transduction abilityof the complex in both types of cells was shown by measuringthe calcium mobilization and the phosphorylation of tyrosyiresidues upon stimulation by antl-µ. Signal events weresimilar in both cases, but differed from those induced in amature B cell line.This points to a definite function of thepreB cell receptor and suggests that the intermediate preB-Bcell line still lacks some molecular components that conditioninitiation of a mature B cell transduction signal.     

Peripheral lymphoid development and function in TCR mutant mice

We describe the development and function of the peripheral lymphoidsystem of mutant mice rendered deficient in either ßor T cells via targeting of TCR genes In embryonic stem cells.In the spleen of ß T cell-deficient mice, T cellsdo not compensate in numbers for the lack of ß Tcells, but B cells do. ß T cell-deficient mice areunable to mount an antibody response to ovalbumln and do notreject skin allografts. Natural killer cell function is notimpaired in any of the mutant mice. TCR mutant mice will proveuseful in dissecting differential functions of ßand T cells in vivo.     

Predominant expression of invariant V{alpha}14+ TCR {alpha} chain in NK1.1+ T cell populations

A novel T cell subset characterized by cell surface NK1.1⁺ TCRß⁺expression was investigated for its TCR usage, particularlythat of invariant V14 TCR, which was found to be preferentiallyused in peripheral CD4^–CD8^–T cells developed atextrathymic sites. We found that NK₊ ß T cell subsetsaccount for 0.4% in thymocytes, 5% in the splenic T cells and40.5% in the bone marrow T cells. Among these NK⁺ ßT cells, two distinct subsets were detected; cell surface TCRV14⁺and V14^– subpopulations. Almost all of NK⁺ ßthymocytes express V14 mRNA; however, only<20% were positive,while >80% were negative or undetectable for V14 TCR expressionon the cell surface in the thymus. Similarly,50% of NK⁺ ßT cells in spleen and bone marrow are V14⁺; as revealed by FACS.TCR repertoire analysis by nucleotide sequences on inverse PCRproducts demonstrated that most NK⁺ ß T cells expressan invariant TCR encoded by the V14J281 gene with a 1 base N-regionin all tissues. Thus, invariant V14 TCR is uniquely expressedon NK T cells, and can be a marker to distinguish NK, NK T andT cells.     

Involvement of {alpha}1 and {alpha}4 integrins in gut mucosal injury of graft-versus-host disease

We have investigated the involvement of adhesion molecules inthe lymphocyte infiltration associated with acute intestinalgraft-versus-host disease (GVHD) induced by injection of C3Hlymph node cells into irradiated (C3H x DBA/2)F₁ mice. Firstwe analyzed the expression profile of adhesion molecules including1, 2, 4, 5, 6, L and ß7 integrins, CD44 and L-selectinof lymphocytes from lymph nodes and gut mucosa in normal mice.In normal mice, intraepithelial lymphocytes (IEL) and laminapropria lymphocytes (LPL) uniquely showed increased expressionof 1, 2 and ß7 integrins, and decreased expressionof L-selectin compared with that of lymphocytes of the lymphnodes and Peyer's patches. In mice with GVHD, IEL and LPL ofdonor lymph node cells origin underwent phenotyplc changes characterizedby the increased expression of 1, L and ß7 integrins,and the loss of L-selectin. The expression profile of adhesionmolecules on IEL and LPL of GVHD mice resembled that of normalmice except for the lack of 2 integrin. Treatment of GVHD micewith anti-1,-4 or-ß7 integrin antibody alone partiallyprevented the mucosal pathology of intestinal GVHD, whereasonly mice treated with anti-1 showed reduced donor lymphocyticinfiltration into the intestinal mucosa. In contrast, treatmentwith anti-L or anti-CD44 antibody did not affect the intestinalGVHD. Furthermore, dual blockade of both 1 and 4 integrins completelyinhibited the mucosal pathology and donor lymphocyte infiltrationof intestinal GVHD. These results indicate that 1 and 4 integrinsplay an important role in the pathology of intestinal GVHD.