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.     

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.     

Composition of TCR-CD3 complex in human intestinal intraepithelial lymphocytes: lack of Fc{varepsilon}Rl{gamma} chain

Human intestinal intraepithelial lymphocytes (DEL) are a uniquepopulation of predominantly CD8ß⁺ TCRß⁺lymphocytes and, to a lesser extent, TCR⁺ lymphocytes that proliferatepoorly to anti-CD3 mitogenic signals but display significantcytolytic activity. Studies in mouse model systems have shownthat the chain of the high-CD3 affinity receptor for IgE (FcRl)may substitute for the chain in the TCR-CD3 complex of iIEL.This has suggested that the functional properties of these cellsmay be associated with an altered composition of the TCR-CD3complex. We therefore analyzed the TCR-CD3 complex of normalhuman iIEL. One-and two-dimensional non-reducing/reducing SDS-PAGEanalysis of CD3, CD3, CD3, and FcRr chain immunopreclpitatesof cell surface radiolabeled proteins with subunit-specificantibodies revealed a TCR-CD3 complex without associated FcRrchains. Thus, normal human NEL contain a TCR-CD3 complex thatconsists predominantly of , homodimers in association with theß TCR and CD3, and , similar to the majority of peripherallymphocytes. This indicates that the distinct properties ofhuman DEL are not associated with substitutions of the FcRlchain in the TCR-CD3 complex.     

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.     

TNF-{alpha} regulates IL-4-induced Fc{varepsilon}RII/CD23 gene expression and soluble Fc{varepsilon}RII release by human monocytes

We examined the regulatory effects of TNF- on IL-4-induced geneexpression of the low-affinity receptor for IgE (FcRII/CD23)in human monocytes and IL-4-induced soluble FcRII (sFcRII) releasefrom monocytes. IL-4-induced FcRII expression on the surfaceof monocytes was reduced by TNF- as early as 1 day after cultureand the effect of TNF- increased with prolonged culture. Thepresent analysis was designed to examine whether or not TNF-could suppress IL-4-induced FcRII mRNA expression and enhancedIL-4-induced sFcRII release. The addition of TNF- to monocytecultures with IL-4 significantly reduced FcRII expression onthe surface of monocytes and significantly increased sFcRIIrelease from monocytes. Over time, there was an inverse relationshipbetween the disappearance of cell surface FcRII and the appearanceof sFcRII in culture supernatants. FcRII mRNA expression inmonocytes cultured with IL-4 was not affected by TNF- when examinedat 6 h after cultivation. When the cells were cultured withTNF- for more than 24 h, however, TNF- down-regulated IL-4-inducedFcRII mRNA levels. This correlated with the kinetics of down-regulationof IL-4-induced FCRII expression on the surface of monocytesby TNF-. These results suggest that TNF-dependent reductionof IL-4-;induced FcRII expression on the surface of monocytesresulted, at least in part, from the suppression of FcRII mRNAexpression and the enhancement of sFcRII release.     

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.     

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 protein kinase C-{varepsilon} in glucocorticoid-induced apoptosis in thymocytes

Apoptosis is induced in immature thymocytes by physiologicalpeak levels of glucocortlcold hormones, especially in murineand rat cells. Endogenous glucocortlcolds may have some rolein thymic selection. Glucocorticold-lnduced thymocyte apoptosisappears to be dependent on protein kinase C (PKC), since itis inhibited by PKC inhibitors. PKC Is a family of closely relatedenzymes, consisting of Ca²⁺-dependent (PKC-, -ßI,-ßII, and ) and Ca²⁺-Independent (PKC-, -ß,(L), -, -, and -) isozymes. In the present study, we analyzedthe role of PKC in glucocorticold-lnduced apoptosis in murinethymocytes and found that glucocorticold selectively inducesan increase in Ca²⁺ -Independent PKC activity in the paniculatefraction of Immature thymocytes but not in that of mature Tcells. The increase as well as the apoptosis was inhibited byactlnomycln D, cyclohexlmkte, or the glucocortteoid receptorantagonist, RU 38486. Immunoblottlng studies revealed the selectivetranslocatlon of PKC-from the cytosollc fraction to the paniculatefraction upon glucocortlcold treatment. These results suggestthat glucocorticold-lnduced apoptosis in immature thymocytesinvolves glucocorticold receptor-mediated and selective activationof PKC-through de novo synthesis of macromolecules.     

Different cytoplasmic structure of the CD3{zeta} family dimer modulates the activation signal and function of T cells

The TCR complex transduces the antigen recognition signal throughcommon activation motifs present in both CD3 chains and , dimerswithin the complex. We have investigated functional roles ofthe cytoplasmic domain in and CD3 for T cell activation inearly and late responses by comparing the signaling capabilityof the TCR complexes containing mutant lacking some or allmotifs, or chain, another family molecule. The results withthe mutant , lacking all motifs indicated that CD3 can transducesignals to cause earty activation events and production of IL-2upon antigen stimulation in the absence of , motifs. However,any one of the ; motifs was required to respond to Thy-1 stimulationand this requirement cannot be replaced by other CD3 chains.Such , motif-dependent responses were also observed in tyrosinephosphoryiation of a 90 kDa protein upon TCR stimulation. Furthermore,we found that the C-terminal unique region of the chain exhibitsinhibitory function in phosphoryiation and Ca2⁺ response uponTCR stimulation as well as IL-2 production upon Thy-1 stimulation.Collectively, the present analyses suggest that two types ofsignals are induced through the TCR-CD3 complex: (I) the commonmotif-dependent signals which are mediated equally through ,dimers and CD3, and (II) , specific motif-dependent signals.Differences in the cytoplasmic domain of , family moleculesmay modulate the cooperation of these two signals, resultingin alteration of T cell functions.     

Molecular cloning and characterization of guinea pig Fc{gamma}RIII: expression but not function is independent of the {gamma} chain of Fc{gamma}FceRI

We have isolated two cDNA clones encoding the guinea pig receptorfor the Fc portion of lgG2 (Fc2R) from a guinea pig peritonealmacrophage cDNA library. Analysis of the predicted amino acidsequence of the one cDNA clone indicated that the guinea pigFc2R Is a type I transmembrane protein and has 72% DNA sequencehomology and 57% protein sequence homology with the human FcRIII.Therefore, we propose that the guinea pig Fc2R Is referred toas guinea pig FcRIII. The most important finding In this reportis that the obtained cDNA directed the cell surface expressionof the Fc2R on COS-7 cells without association with the chainof the high-affinity IgE receptor (FcRly) which is requiredfor human and mouse FcRIII to be expressed on the cell surface.Furthermore, we demonstrated that the endocytosis activity ofFcRIII is dependent upon the association with FcRl, suggestingthat FcRl is Involved in the functions of guinea pig FcRIII.The other clone was found to lack the sequence encoding transmembraneand cytoplasmic domains, suggesting the presence of a solubleform of guinea pig FcRIII. Northern blot analysis and RT-PCRshowed that a transmembrane form of guinea pig FcRIII was expressedin peritoneal macrophages, but not in neutrophils In spite ofthe fact that they express Fc2R, indicating that the Fc2R onneutrophils is a product of a distinct gene.     

{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.     

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.     

Phenotypic and functional properties of murine {gamma}{delta} T cell clones derived from malaria immunized, {alpha}{beta} T cell-deficient mice

Six murine T cell clones expressing TCR were generated frommalaria immunized, ß T celldeficient mice. Phenotypiccharacterization of these clones has revealed that, in contrastto conventional ß T cells, there is a considerabledegree of heterogeneity among these clones with regard to theirsurface markers and their lymphokine profile. One clone wasfound to display significant anti-parasite activity in vivoupon adoptive transfer. We attempted to determine whether theprotective clone differs in one or more key characteristicsfrom the non-protective clones. Although no obvious patternpeculiar to the protective clone was observed, it appears thatmore than one parameter may, in combination, define a distinctprotective phenotype, and thus explain the functional differencebetween the protective and non-protective clones.     

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.